16
Research Article Antiaging and Anxiolytic Effects of Combinatory Formulas Based on Four Medicinal Herbs Rui Li, 1 Wingman Chan, 1 Waikin Mat, 1 Yiucheong Ho, 1 Rigil K. Yeung, 1 Shuiying Tsang, 1 and Hong Xue 1,2 1 Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong 2 Applied Genomics Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong Correspondence should be addressed to Hong Xue; [email protected] Received 26 October 2016; Revised 26 January 2017; Accepted 19 February 2017; Published 28 March 2017 Academic Editor: Kuttulebbai N. S. Sirajudeen Copyright © 2017 Rui Li et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e objective of the present study was to search for medicinal-herb combinations based on Radix Bupleurum chinense DC (“B”), Rhizoma Corydalis yanhusuo WT Wang (“Y”), Caulis Polygonum multiflorum unb (“P”), and Flos Albizia julibrissin Durazz (“A”) for antiaging, anxiolytic, and sedative effects. Application of the d-galactose induced accelerated-aging model employing male ICR mice showed that oral administration of some combinations of B, Y, P, and A significantly improved spatial memory in Y-maze test and reduced brain levels of tumor necrosis factor- and interleukin-6 based on immunoassays and oxidative stress marker malondialdehyde, based on the thiobarbituric acid test, and the loss of whiskers, indicating antiaging and antineurodegeneration effects. In addition, some of the combinatory formulas induced anxiolysis measured using the elevated plus-maze test and/or sedative effects measured using the hole-board test. Over the range of dosages examined, all possible combinations of the four herbs were devoid of any significant side effects in the form of altered locomotor activity, decreased muscle coordination, or anterograde amnesia assessed using the photobeam and rotarod and step-through passive avoidance methods, respectively. e results suggest that various combinations of the B, Y, P, and A herbs could be useful as nonsedative, antiaging and/or antineurodegenerative agents, or anxiolytic agents. 1. Introduction e world’s population is aging rapidly, with the over-65 age group outnumbering the below-15 age group in a fast increasing number of countries. Age-related cognitive decline and neurodegeneration are therefore becoming foremost health and social concerns. Physiologically, damage to the structures of the medial temporal lobe including the hip- pocampus contributes to a deficit in spatial memory [1, 2], and oxidative damage by reactive oxygen species (ROS) is commonly associated with aging [3, 4]. As well, proinflam- matory agents such as interleukin-6 (IL-6), C-reactive pro- tein, and tumor necrosis factor-alpha (TNF-) can enhance resistance to infectious disease but also susceptibility in old age to a chronic low-grade inflammation, or “inflammag- ing,” that constitutes a highly significant factor for both morbidity and mortality [5, 6]. Long-lived people, especially centenarians, seem to cope with inflammaging through an “anti-inflammaging” cytokine response that may represent a key to longevity [7]. erefore, treatments that can alleviate spatial memory deficit, relieve oxidative stress, and lower the levels of proinflammatory agents such as TNF- and IL-6 could be important for reducing some of the effects of aging. Since old people are oſten beset as well by such neurophysiological conditions as anxiety and sleeplessness, which are aggravated by the feelings of helplessness and loneliness common to old age, treatments for anxiety and sleep disorders are equally fundamental for the health of the aged. e health problems arising from aging, age-related neurodegenerative diseases, anxiety disorders, and sleep dis- orders require therapeutic agents that can cross the blood- brain barrier and are relatively free of adverse side effects so that they can be administered on a chronic basis. Tra- ditional Chinese Medicine (TCM) employs approximately Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2017, Article ID 4624069, 15 pages https://doi.org/10.1155/2017/4624069

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Page 1: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Research ArticleAntiaging and Anxiolytic Effects of Combinatory FormulasBased on Four Medicinal Herbs

Rui Li1 Wingman Chan1 Waikin Mat1 Yiucheong Ho1 Rigil K Yeung1

Shuiying Tsang1 and Hong Xue12

1Division of Life Science Hong Kong University of Science and Technology Clear Water Bay Hong Kong2Applied Genomics Center Hong Kong University of Science and Technology Clear Water Bay Hong Kong

Correspondence should be addressed to Hong Xue hxueusthk

Received 26 October 2016 Revised 26 January 2017 Accepted 19 February 2017 Published 28 March 2017

Academic Editor Kuttulebbai N S Sirajudeen

Copyright copy 2017 Rui Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The objective of the present study was to search for medicinal-herb combinations based on Radix Bupleurum chinense DC (ldquoBrdquo)RhizomaCorydalis yanhusuoWTWang (ldquoYrdquo) Caulis PolygonummultiflorumThunb (ldquoPrdquo) and FlosAlbizia julibrissinDurazz (ldquoArdquo)for antiaging anxiolytic and sedative effects Application of the d-galactose induced accelerated-aging model employing male ICRmice showed that oral administration of some combinations of B Y P and A significantly improved spatial memory in Y-mazetest and reduced brain levels of tumor necrosis factor-120572 and interleukin-6 based on immunoassays and oxidative stress markermalondialdehyde based on the thiobarbituric acid test and the loss of whiskers indicating antiaging and antineurodegenerationeffects In addition some of the combinatory formulas induced anxiolysis measured using the elevated plus-maze test andorsedative effects measured using the hole-board test Over the range of dosages examined all possible combinations of the four herbswere devoid of any significant side effects in the form of altered locomotor activity decreased muscle coordination or anterogradeamnesia assessed using the photobeam and rotarod and step-through passive avoidance methods respectively The results suggestthat various combinations of the B Y P and A herbs could be useful as nonsedative antiaging andor antineurodegenerative agentsor anxiolytic agents

1 Introduction

The worldrsquos population is aging rapidly with the over-65age group outnumbering the below-15 age group in a fastincreasing number of countries Age-related cognitive declineand neurodegeneration are therefore becoming foremosthealth and social concerns Physiologically damage to thestructures of the medial temporal lobe including the hip-pocampus contributes to a deficit in spatial memory [1 2]and oxidative damage by reactive oxygen species (ROS) iscommonly associated with aging [3 4] As well proinflam-matory agents such as interleukin-6 (IL-6) C-reactive pro-tein and tumor necrosis factor-alpha (TNF-120572) can enhanceresistance to infectious disease but also susceptibility in oldage to a chronic low-grade inflammation or ldquoinflammag-ingrdquo that constitutes a highly significant factor for bothmorbidity and mortality [5 6] Long-lived people especiallycentenarians seem to cope with inflammaging through an

ldquoanti-inflammagingrdquo cytokine response that may represent akey to longevity [7] Therefore treatments that can alleviatespatial memory deficit relieve oxidative stress and lowerthe levels of proinflammatory agents such as TNF-120572 andIL-6 could be important for reducing some of the effectsof aging Since old people are often beset as well by suchneurophysiological conditions as anxiety and sleeplessnesswhich are aggravated by the feelings of helplessness andloneliness common to old age treatments for anxiety andsleep disorders are equally fundamental for the health of theaged

The health problems arising from aging age-relatedneurodegenerative diseases anxiety disorders and sleep dis-orders require therapeutic agents that can cross the blood-brain barrier and are relatively free of adverse side effectsso that they can be administered on a chronic basis Tra-ditional Chinese Medicine (TCM) employs approximately

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 4624069 15 pageshttpsdoiorg10115520174624069

2 Evidence-Based Complementary and Alternative Medicine

500 medicinal herbs and multiherb combinations compris-ing complementary herbs are usually favored over singleherbs [8ndash11] The toxicities of these herbs are also eitherlimited or at least well understood from centuries of usagePreviously our search for an anxiolytic herbal formula hasled to the development of the ldquoBYPArdquo or Erhuhuantengcombination comprising the four Chinese medicinal herbsRadix Bupleurum chinense DC (ldquoBrdquo) Rhizoma Corydalisyanhusuo WT Wang (ldquoYrdquo) Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albizia julibrissin Durazz (ldquoArdquo) [12]The B Y P and A herbs are widely employed in TCM Thecontents of B include saikosaponin A which displays anti-inflammatory anticancer and antioxidative effects [13ndash15]and saikosaponin D which induces immunomodulatory andanticancer effects [16 17] Y contains dl-tetrahydropalmatinewhich induces anxiolytic effects in mice when adminis-tered orally [18] The l-tetrahydropalmatine isomer is adopamine receptor antagonist with potential application totreatment of drug addiction [19ndash22] and amelioration ofanxiety and depression-related symptoms of posttraumaticstress disorder in rats [23] P is employed as a sedative forneurasthenia and insomnia and to activate blood circulationin collaterals for treatment of aching limbs It can inducehypnotic effects when coadministered with pentobarbital[24] A is employed as sedative and tranquilizer It increasespentobarbital-induced sleeping time in a dose dependentmanner [25 26] and exhibits antidepressant effect in theforced swim test [27] The anxiolytic effect of the BYPAformula also clearly indicates that its four constituent herbscontain neuroactive ingredients that can cross the blood-brain barrier Accordingly herbal combinations based on BY P andAhave been examined in the present study regardingtheir potential utility for treating the effects of normalaging age-related degenerative diseases anxiety disordersand sleep disorders

To examine the effects of herbal formulas comprisingcombinations of B Y P and A on aging-related changesthe d-galactose-induced aging model was employed whichis based on the findings that chronic exposure to d-galactosenot only causes shortened lifespan in Drosophila andhousefly [28 29] but also brings about neurodegenerationincreased oxidative stress in the brain and spatial memoryimpairment [30 31] The application of this accelerated-aging model has led to such findings as the ameliorationof cognitive dysfunction and neurodegeneration by R-120572-lipoic acid [30] prevention of cognitive impairment andhippocampus senescence by ginsenoside Rg1 [32] and reduc-tion of elevated brain ROS TNF-120572 and IL-6 by asiaticacid [33] Analysis of anxiety and sedation was carriedout using the elevated plus-maze test and hole-board testrespectively

2 Materials and Methods

21 Chemical Compounds Radioactive [3H]-flunitrazepam(Methyl-[3H] 814 Cimmol) [3H]-muscimol (Methylene-[3H] 255 Cimmol) and [3H]-spiperone (Benzene ring-[3H] 157 Cimmol) were obtained from Perkin-Elmer (Life

Sciences Inc Boston MA) Dilutions were made using50mMTris-Cl pH74 and theywere stored at 4∘CDiazepam(DZ) and GABA were obtained from Sigma (St Louis MOUSA) Diazepam was dissolved in 09 NaCl in the presenceof 1 DMSO for animal testsThese drugs were administeredto mice in a delivery volume of 10mlkg through the oralroute d-galactose malondialdehyde (MDA) and thiobarbi-turic acid (TBA) were obtained from Sigma (St Louis MOUSA) and d-galactose was dissolved in 09NaCl for animaltreatment

22 Animals Male ICR mice from the Animal and PlantCare Facility Hong KongUniversity of Science amp Technology(HKUST) were housed in groups of five to ten with food andwater ad libitum and kept on 0800- to 2000-lightdark cycleIn the experiments the mice were separated randomly intodifferent groups and the number of mice used in each groupin the different experiments is described in Sections 24 2627 210 and 211 All animal experiments were preapprovedby the HKUST Animal Ethics Committee and conducted inaccordance with the Code of Practice for Care and Use ofAnimals for Experimental Purposes approved by the AnimalWelfare Advisory Group the Agriculture Fisheries andConservation Department and the Department of Health ofHong Kong Special Administrative Region (Animal ethicalcode number HKUST 2015029)

23 Preparation of Herbal Extracts To prepare the four-herb BYPA extract 65 g B 45 g Y 45 g P and 45 g A werepowdered and boiled in 2 L of 5 acetic acid for 2 hoursThe supernatant was collected and the residue was reboiledin 2 L of 5 acetic acid for 2 hours The two batches ofsupernatant were pooled together filtered and boiled downto a volume of approximately 100ml prior to drying in ovenat 92∘C Thereupon 300ml 100 ethanol was added to thefinely powdered dried residue and redried at 92∘CThe driedextract was weighed and powdered

To prepare an extract from a subset of the four constituentherbs one or more of the herbs were omitted in performingthe same procedure for example only 65 g B 45 g Y and 45 gP were boiled in 2 L of 5 acetic acid for 2 hours and so on inpreparing the three-herb BYP extract or only 45 g P and 45 gA were boiled in 2 L of 5 acetic acid for 2 hours and so onin preparing the two-herb PA extract

24 d-Galactose-Induced Accelerated-Aging Model 10-week-old ICR male mice were randomly separated into groupsvehicle group (Veh 119899 = 20) d-galactose treated controlgroup (d-gal 119899 = 20) and herbal extract-treated groups(119899 = 12group) Mice were given daily 150mgkg d-galactose by subcutaneous injection accompanied by eitherdaily oral administration of the vehicle (09 NaCl) inthe d-gal group or daily oral administration of one of theBYPA BYP BYA BPA YPA BY BP and YP herbal extracts(120mgkg) for an exposure period of 8 weeks The Vehgroup received daily injection of saline (09 NaCl) andalso daily oral administration of saline (09 NaCl) for 8weeks

Evidence-Based Complementary and Alternative Medicine 3

Veh

D-g

al

BYPA BY

P

BYA

BPA

YPA BPBY YP

0

10

20

30

40

50

60

entr

ies i

nto

nove

l arm lowast

lowast lowast lowastlowast

lowastlowast

(a)

BYP BP

D-g

al

BYA

BPA

YPA BY YPVeh

BYPA

0

10

20

30

40

50

60

70

ti

me s

pent

in n

ovel

arm

lowastlowast lowastlowast

(b)

Figure 1 Spatialmemory impairment in d-galactose-induced agingmice d-galmicewere given daily 150mgkg d-galactose by subcutaneousinjection with or without daily oral coadministration of different combinatorial herbal formulas (120mgkg) for eight weeks prior to the Y-maze test Decreased percentile entry into novel arm (a) or time spent in novel arm (b) comparedwith vehicle-treated group (Veh) representedspatial memory impairment Data are expressed in mean plusmn SD (119901 lt 0001) indicates significant difference between d-gal group and Vehgroup whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant difference between herbal group and d-gal group based on one-way ANOVAtest followed by Newman-Keuls test

25 Y-Maze Test The test a two-trial memory task based ona free choice exploration paradigm in a Y-maze avoids theuse of electric shock or deprivation and the need for learningof any rule Hippocampal damage and chronic stress havebeen shown to cause impaired spatial memory performancein the Y-maze [34 35] In the study illustrated in Figure 1the test was given to each animal at the end of the 8-weekexposure to d-galactose using a Y-maze that consisted ofthree identical arms spaced at an angle of 120∘ to one anotherdesignated respectively as the ldquostart armrdquo ldquoopen armrdquo andldquonovel armrdquo At the start of the test the entrance to thenovel arm was closed off and animal was placed into thestart arm and allowed to explore the start arm and the openarm freely for 10 minutes The animal was removed from themaze for 60minutes before being reintroduced into themazenow with the entrance to the novel arm also opened up sothat the animal could freely explore all three arms for a testperiod of 5 minutes During this test period the number ofentries made by the animal into each of the three arms wascounted and the total time spent by the animal in each ofthe three arms was recorded where any arm entry or armexit was defined by the placement of all four paws inside oroutside the arm Animals with total loss of spatial memorywould enter into the three arms randomly making sim33 ofarm entries into the novel arm and spending sim33 of thetime in the novel arm In contrast animals with either noloss or only partial loss of spatial memory would enter intoand also spend time in the novel arm preferentially as unex-plored territory causing both of these percentages to exceed33

26 Neuroinflammation and Oxidative Stress in Brain Micewere sacrificed by euthanasia at the end of the 8-week d-galactose exposure period (119899 = 6group) and the brain wasremoved and homogenized in phosphate buffer saline pH72 The levels of TNF-120572 and IL-6 in the brain were mea-sured as neuroinflammation markers using ELISA with solid

phase sandwich kits (Invitrogen Corporation Camerillo CAUSA) The minimum detectable levels were 3 pgml for eachof TNF-120572 and IL-6 To assess oxidative stress the level of thelipid peroxidation product MDA a widely employed markerfor oxidative stress in animal tissues was estimated usingthe spectrophotometric assay at 532 nm for reaction betweenTBA and MDA [36 37]

27 Elevated Plus-Maze Test 4ndash6-week-old male ICR micewere randomly separated into groups Vehicle (Veh namely09 NaCl 119899 = 20) 30 60 90 or 120mgkg herbal extract(119899 = 20) or 1 or 3mgkg DZ (119899 = 15) was orally administered35 minutes prior to experiment The test apparatus consistedof four arms 25 times 5 cm each extending from a central 5times 5 cm platform in the shape of a plus sign Two opposingarms were enclosed by 20 cm opaque high walls making upthe closed arms The plus-maze was elevated 40 cm aboveground Each mouse was placed on to the center of the mazewith head facing an open armThe frequency and time spentin the open and closed arms were recorded for a period of5 minutes An arm entry was recorded when all four pawswere inside the arm At the end of each test the apparatuswas thoroughly cleansed and dried before the start of thenext test An increased percentage of time spent in openarms compared with vehicle-treated controls signified ananxiolytic effect [38 39]

To test whether the anxiolytic effect elicited by herbalextract could be blocked by the BZ-site antagonist flumazenil4ndash6-week-old male mice were randomly separated into threegroups At 35 minutes prior to elevated plus-maze test thefirst group (119899 = 25) received vehicle (09NaCl po) whereasthe second group (119899 = 18) and the third group (119899 = 20)received herbal extract (po) At 15 minutes prior to elevatedplus-maze test the first and second groups received vehicle(ip) whereas the third group received 125mgkg flumazenil(ip)

4 Evidence-Based Complementary and Alternative Medicine

28 Hole-Board Test After the elevated plus-maze test thetest mice were subjected to the hole-board test The hole-board apparatus consisted of a wooden box 60 times 60 times 20 cmwith four holes of 3 cm diameter evenly spaced on the floorEach mouse was placed at the center of the hole-board andthe number of head-dips into the holes was recorded for aperiod of 5 minutes A head-dip was scored when the headof the mouse was dipped into the hole to the extent that theeyes of the mouse passed below the hole on the floor Aftereach trial the floor of the apparatus was thoroughly cleansedand dried before the start of the next test Decreased numberof head-dips compared with Veh controls was indicative of asedative effect [40]

29 Locomotor Activity Test Following the elevated plus-maze and hole-board tests the testmicewere further assessedfor locomotor activity by means of the ZIL-2 apparatus(Beijing Institute of Materia Medica) The test apparatuswith dimensions of 60 cm times 60 cm times 12 cm consisted ofa transparent plastic cylindrical container equipped withthree evenly spaced infrared beams with photodetectorsThe number of transitions across the photocell beams wasrecorded automatically over a period of 5 minutes Anincrease or decrease in the number of transitions indicated anincrease or decrease in locomotor activity respectively [41]

210 Rotarod Test 4ndash6-week-old male ICR mice were ran-domly separated into groups Vehicle (Veh namely 09NaCl 119899 = 20) 120mgkg herbal extract (119899 = 15)or 1 or 3mgkg DZ (119899 = 15) was orally administered35 minutes prior to the test The rotarod test for motorcoordination utilized a custom-built apparatus consisting of acylindrical rotarod (25 cm diameter) with a textured surfaceplaced 05m above the ground Prior to vehicle or extractadministration the mice were first trained to stay for 2minon the rotarod revolving at 16 rpm To test the effect of vehicleor extract treatment the time each treated mouse remainedon the rotarod was recorded with cut-off set at 2min suchthat any mouse that remained on the rotarod after 2min wasaccorded a time on rotarod of 2min [42]

211 Step-through Passive Avoidance Test 4ndash6-week-oldmaleICRmicewere randomly separated into groups Vehicle (Vehnamely 09 NaCl 119899 = 20) 120mgkg herbal extract (119899 =15) or 1 or 3mgkg DZ (119899 = 15) was orally administered 35minutes prior to training The apparatus was a two-chamberbox (Chinese Academy of Chinese Medical Science Beijing)with one of the chambers being opaque (darkened) and theother transparent (lighted) In the training trials each mousewas placed into the lighted chamber and the door connectingthe lighted and darkened chambers was opened 10 secondslater Mice that did not enter the darkened chamber within 15seconds were excluded from the experiment After themouseentered the darkened chamber the door was closed and a 2-second electric foot shock of 04mA was delivered throughthe grid floor Ten seconds later the mouse was transferredfrom the darkened chamber back to the home cage After aninterval of 24 hours it was returned to the lighted chamber

The door to the darkened chamber was opened 10 secondslater and the time taken for the mouse to enter the darkenedchamber was recorded as the step-through latency cut-offwas set at 300 seconds such that the step-through latency forany mouse that did not enter the darkened chamber by 300seconds was recorded as 300 seconds Significant decrease instep-through latency indicated anterograde amnesia [43]

212 Preparation of Synaptosomal Membrane Sprague Daw-ley rats (sim200 g) were decapitated and the whole brainswere rapidly removed and frozen in liquid nitrogen Thetissue was thawed in 20 volumes of ice-cold 032M sucroseand homogenized in anULTRA-TURRAX homogenizerThehomogenate was centrifuged at 1000timesg for 10min at 4∘C andthe supernatant was again centrifuged at 20000timesg for 20minutes at 4∘CThe pellet from the second centrifugationwasresuspended in 50mM Tris-Cl pH 74 to a concentration of1mgml and stored atminus80∘Cuntil use For the [3H]-muscimoland [3H]-spiperone binding assays the pellet from the secondcentrifugation was further homogenized stirred on ice with20 volumes of ice-cold double-distilledwater and centrifugedat 48000timesg for 20 minutes at 4∘C this was repeated twicebefore resuspending the final pellet in 50mMTris-Cl pH 74to a concentration of 1mgml

213 Radioligand Binding Assays In these assays 1 nM [3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperonewas incubated with 02mgml synaptosomal membranes anddifferent concentrations of herbal extract in 50mM Tris-Cl pH 74 at either 4∘C for 90min for [3H]-flunitrazepamor [3H]-muscimol or 25∘C for 60min for [3H]-spiperonebinding Subsequently the incubation mixture was placedon a Whatman GFB filter on a Brandel 24-well harvesterand washed with ice-cold 50mM Tris-Cl pH 74 Eachwashed filter was incubated with 4ml scintillation cocktailfor at least 60 minutes before measurement of radioactivityin a Beckman-Coulter LS6500 scintillation counter Half-maximal inhibition concentrations (IC50) were determinedby nonlinear regression analysis performed using Prism 50(Graphpad Software) To assess nonspecific binding 1 nM[3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperone was incubated with synaptosomal membranes and10 120583M diazepam 10 120583M GABA or 10 120583M (+)-butaclamolrespectively in 50mM Tris-Cl pH 74 at either 4∘C for90min for [3H]-flunitrazepam or [3H]-muscimol or 25∘Cfor 60min for [3H]-spiperone under these conditions the[3H]-ligand bound in each instance represented nonspecificbinding

214 Statistical Analysis Results were expressed as mean plusmnstandard deviation (SD) One-way ANOVA test followedby Newman-Keuls test was performed on behavioral dataBinding assay data were expressed as mean plusmn SD of fourindependent incubations each in duplicate samplings Bind-ing affinity was determined by nonlinear regression analysisAll statistical analyses were performed using Prism 50(Graphpad Software)

Evidence-Based Complementary and Alternative Medicine 5

0

2

4

6

8TN

F-al

pha (

pgm

g pr

otei

n)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

lowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowast

(a)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

0

1

2

3

4

IL-6

(pg

mg

prot

ein)

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

0

5

10

15

MD

A (n

mol

mg

prot

ein)

Veh

D-g

al

BYP

BYA

BPA

YPA BY BP YP

BYPA

lowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

(c)

Figure 2 Anti-inflammatory and antioxidative effects in brains of d-galactose-induced aging mice Different groups of mice were treatedas described in Figure 1 The anti-inflammatory effect was evaluated by measurement of the brain levels of TNF-120572 (a) and IL-6 (b) andantioxidant effect was evaluated bymeasurement of the brain level ofMDA (c) Data are expressed inmeanplusmn SD (119901 lt 001) or (119901 lt 0001)indicates significant difference between d-gal group and Veh group whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant differencebetween herbal group and d-gal group based on one-way ANOVA test followed by Newman-Keuls test

3 Results

31 Antiaging Effects in d-Galactose-Induced Mouse AgingModel In this model over an 8-week d-galactose exposureperiod the vehicle group (ldquoVehrdquo) of animals received dailyinjection and oral administration of vehicle (09 NaCl) thed-galactose group (ldquod-galrdquo) received daily injection of d-galactose (150mgkg) and oral administration of vehicle andeach herbal group received daily injection of d-galactose plusoral administration of herbal extract prepared from two ormore of the B Y P and A herbs (see Section 24)

In the Y-maze test the d-gal animals displayed a signif-icant decrease in entries into and time spent in the novelarm of the maze with 119901 lt 0001 indicating a loss of spatialmemory compared with the Veh group (Figures 1(a) and1(b)) Daily oral treatment with a BYPA BYA BY BP orYP extract accompanying d-galactose injection increased thepercentile novel arm entries compared with that of the d-galmice to 119901 lt 005 and oral treatment with BYP increasedthe percentile novel arm entries compared with that of thed-gal mice to 119901 lt 001 Daily oral treatment with BYP orBY also increased the percentile time spent in the novel armcompared with the d-gal mice to 119901 lt 001

The levels of TNF-120572 and IL-6 were increased in the brainsof d-gal mice compared with Veh mice (119901 lt 001) Dailyoral cotreatment with BYA or YP decreased the brain level of

TNF-120572 compared with that of the d-gal mice to 119901 lt 005 andcotreatment with BYPA BYP BY or BP decreased the brainlevel of TNF-120572 compared with that of the d-gal mice moreextensively to 119901 lt 001 (Figure 2(a)) Daily oral cotreatmentwith BYPA decreased the brain level of IL-6 compared withthat of the d-gal mice to 119901 lt 005 and cotreatment withBYP BY BP or YP decreased the brain level of IL-6 comparedwith that of the d-gal mice more extensively to 119901 lt 001(Figure 2(b))

When the level of oxidative stress in the brain wasassessed the marker compound MDA was found to beincreased in the brains of d-galmice comparedwithVehmice(119901 lt 0001) Daily oral cotreatment with BYA BPA or YPeffectively decreased the brain MDA compared with that ofthe d-gal mice to 119901 lt 005 and cotreatment with BYPABYP BY or BP decreased the brain level of MDA comparedwith that of the d-gal mice more markedly to 119901 lt 001(Figure 2(c))

Mice treatedwith d-galactose for 8weeks showed variableextents of loss of whiskers Table 1 shows one particularlysensitive batch of mice where loss of whiskers was displayedby as many as 50 of the d-gal mice In contrast none ofthe mice in the Veh group or in the groups that received d-galactose injections along with an extract from BYPA BYPBYA BPA or YPA displayed any loss of whiskers Thusexposure of sensitive mice to d-galactose inflicted damage to

6 Evidence-Based Complementary and Alternative Medicine

(a)

(I)

(II)

(III)

(IV)

(V)

(VI)

(VII)

(d)

(c)

(b)

Figure 3 Loss of whiskers by d-galactose-induced aging mice (I) Photographs of two Veh mice (IIa IIb) four d-galactose treated mice thatshowed loss of whiskers and (IIc IId) four d-galactose treated mice that did not show loss of whiskers (III) Two of the mice treated withd-galactose and BYPA (IV) two of the mice treated with d-galactose and BYP (V) two of the mice treated with d-galactose and BYA (VI)two of the mice treated with d-galactose and BPA and (VII) two of the mice treated with d-galactose and YPA

facial tissues resulting in loss of whiskers in accord with anearlier report of coarse fur and severe hair loss in d-galactosetreated mice [44] On this basis the findings in Table 1 alsoillustrated in Figure 3 suggest that the protective antiagingeffects of the BYPA BYP BYA BPA and YPA extracts were

not confined to brain tissues but extended significantly tofacial skin as well (119901 = 0014)

32 Anxiolytic and Sedative Effects The single-herb extractsof Y P and A but not that of B displayed anxiolytic effects

Evidence-Based Complementary and Alternative Medicine 7

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

PAYA BY BA BP120 120 120 120 120120

YP60 90 12030

BYPA

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

Veh 31DZ

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast lowast

lowastlowast

Figure 4 Anxiolytic effect of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to anxiolytic effect using the elevated plus-mazetest where increased percentage time spent in open arms compared with Veh represented an anxiolytic effect Data are expressed in mean plusmnSD of the percentage of time spent in open arms lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001) indicates significant difference from Veh groupbased on one-way ANOVA test followed by Newman-Keuls test

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 2: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

2 Evidence-Based Complementary and Alternative Medicine

500 medicinal herbs and multiherb combinations compris-ing complementary herbs are usually favored over singleherbs [8ndash11] The toxicities of these herbs are also eitherlimited or at least well understood from centuries of usagePreviously our search for an anxiolytic herbal formula hasled to the development of the ldquoBYPArdquo or Erhuhuantengcombination comprising the four Chinese medicinal herbsRadix Bupleurum chinense DC (ldquoBrdquo) Rhizoma Corydalisyanhusuo WT Wang (ldquoYrdquo) Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albizia julibrissin Durazz (ldquoArdquo) [12]The B Y P and A herbs are widely employed in TCM Thecontents of B include saikosaponin A which displays anti-inflammatory anticancer and antioxidative effects [13ndash15]and saikosaponin D which induces immunomodulatory andanticancer effects [16 17] Y contains dl-tetrahydropalmatinewhich induces anxiolytic effects in mice when adminis-tered orally [18] The l-tetrahydropalmatine isomer is adopamine receptor antagonist with potential application totreatment of drug addiction [19ndash22] and amelioration ofanxiety and depression-related symptoms of posttraumaticstress disorder in rats [23] P is employed as a sedative forneurasthenia and insomnia and to activate blood circulationin collaterals for treatment of aching limbs It can inducehypnotic effects when coadministered with pentobarbital[24] A is employed as sedative and tranquilizer It increasespentobarbital-induced sleeping time in a dose dependentmanner [25 26] and exhibits antidepressant effect in theforced swim test [27] The anxiolytic effect of the BYPAformula also clearly indicates that its four constituent herbscontain neuroactive ingredients that can cross the blood-brain barrier Accordingly herbal combinations based on BY P andAhave been examined in the present study regardingtheir potential utility for treating the effects of normalaging age-related degenerative diseases anxiety disordersand sleep disorders

To examine the effects of herbal formulas comprisingcombinations of B Y P and A on aging-related changesthe d-galactose-induced aging model was employed whichis based on the findings that chronic exposure to d-galactosenot only causes shortened lifespan in Drosophila andhousefly [28 29] but also brings about neurodegenerationincreased oxidative stress in the brain and spatial memoryimpairment [30 31] The application of this accelerated-aging model has led to such findings as the ameliorationof cognitive dysfunction and neurodegeneration by R-120572-lipoic acid [30] prevention of cognitive impairment andhippocampus senescence by ginsenoside Rg1 [32] and reduc-tion of elevated brain ROS TNF-120572 and IL-6 by asiaticacid [33] Analysis of anxiety and sedation was carriedout using the elevated plus-maze test and hole-board testrespectively

2 Materials and Methods

21 Chemical Compounds Radioactive [3H]-flunitrazepam(Methyl-[3H] 814 Cimmol) [3H]-muscimol (Methylene-[3H] 255 Cimmol) and [3H]-spiperone (Benzene ring-[3H] 157 Cimmol) were obtained from Perkin-Elmer (Life

Sciences Inc Boston MA) Dilutions were made using50mMTris-Cl pH74 and theywere stored at 4∘CDiazepam(DZ) and GABA were obtained from Sigma (St Louis MOUSA) Diazepam was dissolved in 09 NaCl in the presenceof 1 DMSO for animal testsThese drugs were administeredto mice in a delivery volume of 10mlkg through the oralroute d-galactose malondialdehyde (MDA) and thiobarbi-turic acid (TBA) were obtained from Sigma (St Louis MOUSA) and d-galactose was dissolved in 09NaCl for animaltreatment

22 Animals Male ICR mice from the Animal and PlantCare Facility Hong KongUniversity of Science amp Technology(HKUST) were housed in groups of five to ten with food andwater ad libitum and kept on 0800- to 2000-lightdark cycleIn the experiments the mice were separated randomly intodifferent groups and the number of mice used in each groupin the different experiments is described in Sections 24 2627 210 and 211 All animal experiments were preapprovedby the HKUST Animal Ethics Committee and conducted inaccordance with the Code of Practice for Care and Use ofAnimals for Experimental Purposes approved by the AnimalWelfare Advisory Group the Agriculture Fisheries andConservation Department and the Department of Health ofHong Kong Special Administrative Region (Animal ethicalcode number HKUST 2015029)

23 Preparation of Herbal Extracts To prepare the four-herb BYPA extract 65 g B 45 g Y 45 g P and 45 g A werepowdered and boiled in 2 L of 5 acetic acid for 2 hoursThe supernatant was collected and the residue was reboiledin 2 L of 5 acetic acid for 2 hours The two batches ofsupernatant were pooled together filtered and boiled downto a volume of approximately 100ml prior to drying in ovenat 92∘C Thereupon 300ml 100 ethanol was added to thefinely powdered dried residue and redried at 92∘CThe driedextract was weighed and powdered

To prepare an extract from a subset of the four constituentherbs one or more of the herbs were omitted in performingthe same procedure for example only 65 g B 45 g Y and 45 gP were boiled in 2 L of 5 acetic acid for 2 hours and so on inpreparing the three-herb BYP extract or only 45 g P and 45 gA were boiled in 2 L of 5 acetic acid for 2 hours and so onin preparing the two-herb PA extract

24 d-Galactose-Induced Accelerated-Aging Model 10-week-old ICR male mice were randomly separated into groupsvehicle group (Veh 119899 = 20) d-galactose treated controlgroup (d-gal 119899 = 20) and herbal extract-treated groups(119899 = 12group) Mice were given daily 150mgkg d-galactose by subcutaneous injection accompanied by eitherdaily oral administration of the vehicle (09 NaCl) inthe d-gal group or daily oral administration of one of theBYPA BYP BYA BPA YPA BY BP and YP herbal extracts(120mgkg) for an exposure period of 8 weeks The Vehgroup received daily injection of saline (09 NaCl) andalso daily oral administration of saline (09 NaCl) for 8weeks

Evidence-Based Complementary and Alternative Medicine 3

Veh

D-g

al

BYPA BY

P

BYA

BPA

YPA BPBY YP

0

10

20

30

40

50

60

entr

ies i

nto

nove

l arm lowast

lowast lowast lowastlowast

lowastlowast

(a)

BYP BP

D-g

al

BYA

BPA

YPA BY YPVeh

BYPA

0

10

20

30

40

50

60

70

ti

me s

pent

in n

ovel

arm

lowastlowast lowastlowast

(b)

Figure 1 Spatialmemory impairment in d-galactose-induced agingmice d-galmicewere given daily 150mgkg d-galactose by subcutaneousinjection with or without daily oral coadministration of different combinatorial herbal formulas (120mgkg) for eight weeks prior to the Y-maze test Decreased percentile entry into novel arm (a) or time spent in novel arm (b) comparedwith vehicle-treated group (Veh) representedspatial memory impairment Data are expressed in mean plusmn SD (119901 lt 0001) indicates significant difference between d-gal group and Vehgroup whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant difference between herbal group and d-gal group based on one-way ANOVAtest followed by Newman-Keuls test

25 Y-Maze Test The test a two-trial memory task based ona free choice exploration paradigm in a Y-maze avoids theuse of electric shock or deprivation and the need for learningof any rule Hippocampal damage and chronic stress havebeen shown to cause impaired spatial memory performancein the Y-maze [34 35] In the study illustrated in Figure 1the test was given to each animal at the end of the 8-weekexposure to d-galactose using a Y-maze that consisted ofthree identical arms spaced at an angle of 120∘ to one anotherdesignated respectively as the ldquostart armrdquo ldquoopen armrdquo andldquonovel armrdquo At the start of the test the entrance to thenovel arm was closed off and animal was placed into thestart arm and allowed to explore the start arm and the openarm freely for 10 minutes The animal was removed from themaze for 60minutes before being reintroduced into themazenow with the entrance to the novel arm also opened up sothat the animal could freely explore all three arms for a testperiod of 5 minutes During this test period the number ofentries made by the animal into each of the three arms wascounted and the total time spent by the animal in each ofthe three arms was recorded where any arm entry or armexit was defined by the placement of all four paws inside oroutside the arm Animals with total loss of spatial memorywould enter into the three arms randomly making sim33 ofarm entries into the novel arm and spending sim33 of thetime in the novel arm In contrast animals with either noloss or only partial loss of spatial memory would enter intoand also spend time in the novel arm preferentially as unex-plored territory causing both of these percentages to exceed33

26 Neuroinflammation and Oxidative Stress in Brain Micewere sacrificed by euthanasia at the end of the 8-week d-galactose exposure period (119899 = 6group) and the brain wasremoved and homogenized in phosphate buffer saline pH72 The levels of TNF-120572 and IL-6 in the brain were mea-sured as neuroinflammation markers using ELISA with solid

phase sandwich kits (Invitrogen Corporation Camerillo CAUSA) The minimum detectable levels were 3 pgml for eachof TNF-120572 and IL-6 To assess oxidative stress the level of thelipid peroxidation product MDA a widely employed markerfor oxidative stress in animal tissues was estimated usingthe spectrophotometric assay at 532 nm for reaction betweenTBA and MDA [36 37]

27 Elevated Plus-Maze Test 4ndash6-week-old male ICR micewere randomly separated into groups Vehicle (Veh namely09 NaCl 119899 = 20) 30 60 90 or 120mgkg herbal extract(119899 = 20) or 1 or 3mgkg DZ (119899 = 15) was orally administered35 minutes prior to experiment The test apparatus consistedof four arms 25 times 5 cm each extending from a central 5times 5 cm platform in the shape of a plus sign Two opposingarms were enclosed by 20 cm opaque high walls making upthe closed arms The plus-maze was elevated 40 cm aboveground Each mouse was placed on to the center of the mazewith head facing an open armThe frequency and time spentin the open and closed arms were recorded for a period of5 minutes An arm entry was recorded when all four pawswere inside the arm At the end of each test the apparatuswas thoroughly cleansed and dried before the start of thenext test An increased percentage of time spent in openarms compared with vehicle-treated controls signified ananxiolytic effect [38 39]

To test whether the anxiolytic effect elicited by herbalextract could be blocked by the BZ-site antagonist flumazenil4ndash6-week-old male mice were randomly separated into threegroups At 35 minutes prior to elevated plus-maze test thefirst group (119899 = 25) received vehicle (09NaCl po) whereasthe second group (119899 = 18) and the third group (119899 = 20)received herbal extract (po) At 15 minutes prior to elevatedplus-maze test the first and second groups received vehicle(ip) whereas the third group received 125mgkg flumazenil(ip)

4 Evidence-Based Complementary and Alternative Medicine

28 Hole-Board Test After the elevated plus-maze test thetest mice were subjected to the hole-board test The hole-board apparatus consisted of a wooden box 60 times 60 times 20 cmwith four holes of 3 cm diameter evenly spaced on the floorEach mouse was placed at the center of the hole-board andthe number of head-dips into the holes was recorded for aperiod of 5 minutes A head-dip was scored when the headof the mouse was dipped into the hole to the extent that theeyes of the mouse passed below the hole on the floor Aftereach trial the floor of the apparatus was thoroughly cleansedand dried before the start of the next test Decreased numberof head-dips compared with Veh controls was indicative of asedative effect [40]

29 Locomotor Activity Test Following the elevated plus-maze and hole-board tests the testmicewere further assessedfor locomotor activity by means of the ZIL-2 apparatus(Beijing Institute of Materia Medica) The test apparatuswith dimensions of 60 cm times 60 cm times 12 cm consisted ofa transparent plastic cylindrical container equipped withthree evenly spaced infrared beams with photodetectorsThe number of transitions across the photocell beams wasrecorded automatically over a period of 5 minutes Anincrease or decrease in the number of transitions indicated anincrease or decrease in locomotor activity respectively [41]

210 Rotarod Test 4ndash6-week-old male ICR mice were ran-domly separated into groups Vehicle (Veh namely 09NaCl 119899 = 20) 120mgkg herbal extract (119899 = 15)or 1 or 3mgkg DZ (119899 = 15) was orally administered35 minutes prior to the test The rotarod test for motorcoordination utilized a custom-built apparatus consisting of acylindrical rotarod (25 cm diameter) with a textured surfaceplaced 05m above the ground Prior to vehicle or extractadministration the mice were first trained to stay for 2minon the rotarod revolving at 16 rpm To test the effect of vehicleor extract treatment the time each treated mouse remainedon the rotarod was recorded with cut-off set at 2min suchthat any mouse that remained on the rotarod after 2min wasaccorded a time on rotarod of 2min [42]

211 Step-through Passive Avoidance Test 4ndash6-week-oldmaleICRmicewere randomly separated into groups Vehicle (Vehnamely 09 NaCl 119899 = 20) 120mgkg herbal extract (119899 =15) or 1 or 3mgkg DZ (119899 = 15) was orally administered 35minutes prior to training The apparatus was a two-chamberbox (Chinese Academy of Chinese Medical Science Beijing)with one of the chambers being opaque (darkened) and theother transparent (lighted) In the training trials each mousewas placed into the lighted chamber and the door connectingthe lighted and darkened chambers was opened 10 secondslater Mice that did not enter the darkened chamber within 15seconds were excluded from the experiment After themouseentered the darkened chamber the door was closed and a 2-second electric foot shock of 04mA was delivered throughthe grid floor Ten seconds later the mouse was transferredfrom the darkened chamber back to the home cage After aninterval of 24 hours it was returned to the lighted chamber

The door to the darkened chamber was opened 10 secondslater and the time taken for the mouse to enter the darkenedchamber was recorded as the step-through latency cut-offwas set at 300 seconds such that the step-through latency forany mouse that did not enter the darkened chamber by 300seconds was recorded as 300 seconds Significant decrease instep-through latency indicated anterograde amnesia [43]

212 Preparation of Synaptosomal Membrane Sprague Daw-ley rats (sim200 g) were decapitated and the whole brainswere rapidly removed and frozen in liquid nitrogen Thetissue was thawed in 20 volumes of ice-cold 032M sucroseand homogenized in anULTRA-TURRAX homogenizerThehomogenate was centrifuged at 1000timesg for 10min at 4∘C andthe supernatant was again centrifuged at 20000timesg for 20minutes at 4∘CThe pellet from the second centrifugationwasresuspended in 50mM Tris-Cl pH 74 to a concentration of1mgml and stored atminus80∘Cuntil use For the [3H]-muscimoland [3H]-spiperone binding assays the pellet from the secondcentrifugation was further homogenized stirred on ice with20 volumes of ice-cold double-distilledwater and centrifugedat 48000timesg for 20 minutes at 4∘C this was repeated twicebefore resuspending the final pellet in 50mMTris-Cl pH 74to a concentration of 1mgml

213 Radioligand Binding Assays In these assays 1 nM [3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperonewas incubated with 02mgml synaptosomal membranes anddifferent concentrations of herbal extract in 50mM Tris-Cl pH 74 at either 4∘C for 90min for [3H]-flunitrazepamor [3H]-muscimol or 25∘C for 60min for [3H]-spiperonebinding Subsequently the incubation mixture was placedon a Whatman GFB filter on a Brandel 24-well harvesterand washed with ice-cold 50mM Tris-Cl pH 74 Eachwashed filter was incubated with 4ml scintillation cocktailfor at least 60 minutes before measurement of radioactivityin a Beckman-Coulter LS6500 scintillation counter Half-maximal inhibition concentrations (IC50) were determinedby nonlinear regression analysis performed using Prism 50(Graphpad Software) To assess nonspecific binding 1 nM[3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperone was incubated with synaptosomal membranes and10 120583M diazepam 10 120583M GABA or 10 120583M (+)-butaclamolrespectively in 50mM Tris-Cl pH 74 at either 4∘C for90min for [3H]-flunitrazepam or [3H]-muscimol or 25∘Cfor 60min for [3H]-spiperone under these conditions the[3H]-ligand bound in each instance represented nonspecificbinding

214 Statistical Analysis Results were expressed as mean plusmnstandard deviation (SD) One-way ANOVA test followedby Newman-Keuls test was performed on behavioral dataBinding assay data were expressed as mean plusmn SD of fourindependent incubations each in duplicate samplings Bind-ing affinity was determined by nonlinear regression analysisAll statistical analyses were performed using Prism 50(Graphpad Software)

Evidence-Based Complementary and Alternative Medicine 5

0

2

4

6

8TN

F-al

pha (

pgm

g pr

otei

n)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

lowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowast

(a)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

0

1

2

3

4

IL-6

(pg

mg

prot

ein)

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

0

5

10

15

MD

A (n

mol

mg

prot

ein)

Veh

D-g

al

BYP

BYA

BPA

YPA BY BP YP

BYPA

lowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

(c)

Figure 2 Anti-inflammatory and antioxidative effects in brains of d-galactose-induced aging mice Different groups of mice were treatedas described in Figure 1 The anti-inflammatory effect was evaluated by measurement of the brain levels of TNF-120572 (a) and IL-6 (b) andantioxidant effect was evaluated bymeasurement of the brain level ofMDA (c) Data are expressed inmeanplusmn SD (119901 lt 001) or (119901 lt 0001)indicates significant difference between d-gal group and Veh group whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant differencebetween herbal group and d-gal group based on one-way ANOVA test followed by Newman-Keuls test

3 Results

31 Antiaging Effects in d-Galactose-Induced Mouse AgingModel In this model over an 8-week d-galactose exposureperiod the vehicle group (ldquoVehrdquo) of animals received dailyinjection and oral administration of vehicle (09 NaCl) thed-galactose group (ldquod-galrdquo) received daily injection of d-galactose (150mgkg) and oral administration of vehicle andeach herbal group received daily injection of d-galactose plusoral administration of herbal extract prepared from two ormore of the B Y P and A herbs (see Section 24)

In the Y-maze test the d-gal animals displayed a signif-icant decrease in entries into and time spent in the novelarm of the maze with 119901 lt 0001 indicating a loss of spatialmemory compared with the Veh group (Figures 1(a) and1(b)) Daily oral treatment with a BYPA BYA BY BP orYP extract accompanying d-galactose injection increased thepercentile novel arm entries compared with that of the d-galmice to 119901 lt 005 and oral treatment with BYP increasedthe percentile novel arm entries compared with that of thed-gal mice to 119901 lt 001 Daily oral treatment with BYP orBY also increased the percentile time spent in the novel armcompared with the d-gal mice to 119901 lt 001

The levels of TNF-120572 and IL-6 were increased in the brainsof d-gal mice compared with Veh mice (119901 lt 001) Dailyoral cotreatment with BYA or YP decreased the brain level of

TNF-120572 compared with that of the d-gal mice to 119901 lt 005 andcotreatment with BYPA BYP BY or BP decreased the brainlevel of TNF-120572 compared with that of the d-gal mice moreextensively to 119901 lt 001 (Figure 2(a)) Daily oral cotreatmentwith BYPA decreased the brain level of IL-6 compared withthat of the d-gal mice to 119901 lt 005 and cotreatment withBYP BY BP or YP decreased the brain level of IL-6 comparedwith that of the d-gal mice more extensively to 119901 lt 001(Figure 2(b))

When the level of oxidative stress in the brain wasassessed the marker compound MDA was found to beincreased in the brains of d-galmice comparedwithVehmice(119901 lt 0001) Daily oral cotreatment with BYA BPA or YPeffectively decreased the brain MDA compared with that ofthe d-gal mice to 119901 lt 005 and cotreatment with BYPABYP BY or BP decreased the brain level of MDA comparedwith that of the d-gal mice more markedly to 119901 lt 001(Figure 2(c))

Mice treatedwith d-galactose for 8weeks showed variableextents of loss of whiskers Table 1 shows one particularlysensitive batch of mice where loss of whiskers was displayedby as many as 50 of the d-gal mice In contrast none ofthe mice in the Veh group or in the groups that received d-galactose injections along with an extract from BYPA BYPBYA BPA or YPA displayed any loss of whiskers Thusexposure of sensitive mice to d-galactose inflicted damage to

6 Evidence-Based Complementary and Alternative Medicine

(a)

(I)

(II)

(III)

(IV)

(V)

(VI)

(VII)

(d)

(c)

(b)

Figure 3 Loss of whiskers by d-galactose-induced aging mice (I) Photographs of two Veh mice (IIa IIb) four d-galactose treated mice thatshowed loss of whiskers and (IIc IId) four d-galactose treated mice that did not show loss of whiskers (III) Two of the mice treated withd-galactose and BYPA (IV) two of the mice treated with d-galactose and BYP (V) two of the mice treated with d-galactose and BYA (VI)two of the mice treated with d-galactose and BPA and (VII) two of the mice treated with d-galactose and YPA

facial tissues resulting in loss of whiskers in accord with anearlier report of coarse fur and severe hair loss in d-galactosetreated mice [44] On this basis the findings in Table 1 alsoillustrated in Figure 3 suggest that the protective antiagingeffects of the BYPA BYP BYA BPA and YPA extracts were

not confined to brain tissues but extended significantly tofacial skin as well (119901 = 0014)

32 Anxiolytic and Sedative Effects The single-herb extractsof Y P and A but not that of B displayed anxiolytic effects

Evidence-Based Complementary and Alternative Medicine 7

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

PAYA BY BA BP120 120 120 120 120120

YP60 90 12030

BYPA

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

Veh 31DZ

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast lowast

lowastlowast

Figure 4 Anxiolytic effect of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to anxiolytic effect using the elevated plus-mazetest where increased percentage time spent in open arms compared with Veh represented an anxiolytic effect Data are expressed in mean plusmnSD of the percentage of time spent in open arms lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001) indicates significant difference from Veh groupbased on one-way ANOVA test followed by Newman-Keuls test

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Behavioural Neurology

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Evidence-Based Complementary and Alternative Medicine 3

Veh

D-g

al

BYPA BY

P

BYA

BPA

YPA BPBY YP

0

10

20

30

40

50

60

entr

ies i

nto

nove

l arm lowast

lowast lowast lowastlowast

lowastlowast

(a)

BYP BP

D-g

al

BYA

BPA

YPA BY YPVeh

BYPA

0

10

20

30

40

50

60

70

ti

me s

pent

in n

ovel

arm

lowastlowast lowastlowast

(b)

Figure 1 Spatialmemory impairment in d-galactose-induced agingmice d-galmicewere given daily 150mgkg d-galactose by subcutaneousinjection with or without daily oral coadministration of different combinatorial herbal formulas (120mgkg) for eight weeks prior to the Y-maze test Decreased percentile entry into novel arm (a) or time spent in novel arm (b) comparedwith vehicle-treated group (Veh) representedspatial memory impairment Data are expressed in mean plusmn SD (119901 lt 0001) indicates significant difference between d-gal group and Vehgroup whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant difference between herbal group and d-gal group based on one-way ANOVAtest followed by Newman-Keuls test

25 Y-Maze Test The test a two-trial memory task based ona free choice exploration paradigm in a Y-maze avoids theuse of electric shock or deprivation and the need for learningof any rule Hippocampal damage and chronic stress havebeen shown to cause impaired spatial memory performancein the Y-maze [34 35] In the study illustrated in Figure 1the test was given to each animal at the end of the 8-weekexposure to d-galactose using a Y-maze that consisted ofthree identical arms spaced at an angle of 120∘ to one anotherdesignated respectively as the ldquostart armrdquo ldquoopen armrdquo andldquonovel armrdquo At the start of the test the entrance to thenovel arm was closed off and animal was placed into thestart arm and allowed to explore the start arm and the openarm freely for 10 minutes The animal was removed from themaze for 60minutes before being reintroduced into themazenow with the entrance to the novel arm also opened up sothat the animal could freely explore all three arms for a testperiod of 5 minutes During this test period the number ofentries made by the animal into each of the three arms wascounted and the total time spent by the animal in each ofthe three arms was recorded where any arm entry or armexit was defined by the placement of all four paws inside oroutside the arm Animals with total loss of spatial memorywould enter into the three arms randomly making sim33 ofarm entries into the novel arm and spending sim33 of thetime in the novel arm In contrast animals with either noloss or only partial loss of spatial memory would enter intoand also spend time in the novel arm preferentially as unex-plored territory causing both of these percentages to exceed33

26 Neuroinflammation and Oxidative Stress in Brain Micewere sacrificed by euthanasia at the end of the 8-week d-galactose exposure period (119899 = 6group) and the brain wasremoved and homogenized in phosphate buffer saline pH72 The levels of TNF-120572 and IL-6 in the brain were mea-sured as neuroinflammation markers using ELISA with solid

phase sandwich kits (Invitrogen Corporation Camerillo CAUSA) The minimum detectable levels were 3 pgml for eachof TNF-120572 and IL-6 To assess oxidative stress the level of thelipid peroxidation product MDA a widely employed markerfor oxidative stress in animal tissues was estimated usingthe spectrophotometric assay at 532 nm for reaction betweenTBA and MDA [36 37]

27 Elevated Plus-Maze Test 4ndash6-week-old male ICR micewere randomly separated into groups Vehicle (Veh namely09 NaCl 119899 = 20) 30 60 90 or 120mgkg herbal extract(119899 = 20) or 1 or 3mgkg DZ (119899 = 15) was orally administered35 minutes prior to experiment The test apparatus consistedof four arms 25 times 5 cm each extending from a central 5times 5 cm platform in the shape of a plus sign Two opposingarms were enclosed by 20 cm opaque high walls making upthe closed arms The plus-maze was elevated 40 cm aboveground Each mouse was placed on to the center of the mazewith head facing an open armThe frequency and time spentin the open and closed arms were recorded for a period of5 minutes An arm entry was recorded when all four pawswere inside the arm At the end of each test the apparatuswas thoroughly cleansed and dried before the start of thenext test An increased percentage of time spent in openarms compared with vehicle-treated controls signified ananxiolytic effect [38 39]

To test whether the anxiolytic effect elicited by herbalextract could be blocked by the BZ-site antagonist flumazenil4ndash6-week-old male mice were randomly separated into threegroups At 35 minutes prior to elevated plus-maze test thefirst group (119899 = 25) received vehicle (09NaCl po) whereasthe second group (119899 = 18) and the third group (119899 = 20)received herbal extract (po) At 15 minutes prior to elevatedplus-maze test the first and second groups received vehicle(ip) whereas the third group received 125mgkg flumazenil(ip)

4 Evidence-Based Complementary and Alternative Medicine

28 Hole-Board Test After the elevated plus-maze test thetest mice were subjected to the hole-board test The hole-board apparatus consisted of a wooden box 60 times 60 times 20 cmwith four holes of 3 cm diameter evenly spaced on the floorEach mouse was placed at the center of the hole-board andthe number of head-dips into the holes was recorded for aperiod of 5 minutes A head-dip was scored when the headof the mouse was dipped into the hole to the extent that theeyes of the mouse passed below the hole on the floor Aftereach trial the floor of the apparatus was thoroughly cleansedand dried before the start of the next test Decreased numberof head-dips compared with Veh controls was indicative of asedative effect [40]

29 Locomotor Activity Test Following the elevated plus-maze and hole-board tests the testmicewere further assessedfor locomotor activity by means of the ZIL-2 apparatus(Beijing Institute of Materia Medica) The test apparatuswith dimensions of 60 cm times 60 cm times 12 cm consisted ofa transparent plastic cylindrical container equipped withthree evenly spaced infrared beams with photodetectorsThe number of transitions across the photocell beams wasrecorded automatically over a period of 5 minutes Anincrease or decrease in the number of transitions indicated anincrease or decrease in locomotor activity respectively [41]

210 Rotarod Test 4ndash6-week-old male ICR mice were ran-domly separated into groups Vehicle (Veh namely 09NaCl 119899 = 20) 120mgkg herbal extract (119899 = 15)or 1 or 3mgkg DZ (119899 = 15) was orally administered35 minutes prior to the test The rotarod test for motorcoordination utilized a custom-built apparatus consisting of acylindrical rotarod (25 cm diameter) with a textured surfaceplaced 05m above the ground Prior to vehicle or extractadministration the mice were first trained to stay for 2minon the rotarod revolving at 16 rpm To test the effect of vehicleor extract treatment the time each treated mouse remainedon the rotarod was recorded with cut-off set at 2min suchthat any mouse that remained on the rotarod after 2min wasaccorded a time on rotarod of 2min [42]

211 Step-through Passive Avoidance Test 4ndash6-week-oldmaleICRmicewere randomly separated into groups Vehicle (Vehnamely 09 NaCl 119899 = 20) 120mgkg herbal extract (119899 =15) or 1 or 3mgkg DZ (119899 = 15) was orally administered 35minutes prior to training The apparatus was a two-chamberbox (Chinese Academy of Chinese Medical Science Beijing)with one of the chambers being opaque (darkened) and theother transparent (lighted) In the training trials each mousewas placed into the lighted chamber and the door connectingthe lighted and darkened chambers was opened 10 secondslater Mice that did not enter the darkened chamber within 15seconds were excluded from the experiment After themouseentered the darkened chamber the door was closed and a 2-second electric foot shock of 04mA was delivered throughthe grid floor Ten seconds later the mouse was transferredfrom the darkened chamber back to the home cage After aninterval of 24 hours it was returned to the lighted chamber

The door to the darkened chamber was opened 10 secondslater and the time taken for the mouse to enter the darkenedchamber was recorded as the step-through latency cut-offwas set at 300 seconds such that the step-through latency forany mouse that did not enter the darkened chamber by 300seconds was recorded as 300 seconds Significant decrease instep-through latency indicated anterograde amnesia [43]

212 Preparation of Synaptosomal Membrane Sprague Daw-ley rats (sim200 g) were decapitated and the whole brainswere rapidly removed and frozen in liquid nitrogen Thetissue was thawed in 20 volumes of ice-cold 032M sucroseand homogenized in anULTRA-TURRAX homogenizerThehomogenate was centrifuged at 1000timesg for 10min at 4∘C andthe supernatant was again centrifuged at 20000timesg for 20minutes at 4∘CThe pellet from the second centrifugationwasresuspended in 50mM Tris-Cl pH 74 to a concentration of1mgml and stored atminus80∘Cuntil use For the [3H]-muscimoland [3H]-spiperone binding assays the pellet from the secondcentrifugation was further homogenized stirred on ice with20 volumes of ice-cold double-distilledwater and centrifugedat 48000timesg for 20 minutes at 4∘C this was repeated twicebefore resuspending the final pellet in 50mMTris-Cl pH 74to a concentration of 1mgml

213 Radioligand Binding Assays In these assays 1 nM [3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperonewas incubated with 02mgml synaptosomal membranes anddifferent concentrations of herbal extract in 50mM Tris-Cl pH 74 at either 4∘C for 90min for [3H]-flunitrazepamor [3H]-muscimol or 25∘C for 60min for [3H]-spiperonebinding Subsequently the incubation mixture was placedon a Whatman GFB filter on a Brandel 24-well harvesterand washed with ice-cold 50mM Tris-Cl pH 74 Eachwashed filter was incubated with 4ml scintillation cocktailfor at least 60 minutes before measurement of radioactivityin a Beckman-Coulter LS6500 scintillation counter Half-maximal inhibition concentrations (IC50) were determinedby nonlinear regression analysis performed using Prism 50(Graphpad Software) To assess nonspecific binding 1 nM[3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperone was incubated with synaptosomal membranes and10 120583M diazepam 10 120583M GABA or 10 120583M (+)-butaclamolrespectively in 50mM Tris-Cl pH 74 at either 4∘C for90min for [3H]-flunitrazepam or [3H]-muscimol or 25∘Cfor 60min for [3H]-spiperone under these conditions the[3H]-ligand bound in each instance represented nonspecificbinding

214 Statistical Analysis Results were expressed as mean plusmnstandard deviation (SD) One-way ANOVA test followedby Newman-Keuls test was performed on behavioral dataBinding assay data were expressed as mean plusmn SD of fourindependent incubations each in duplicate samplings Bind-ing affinity was determined by nonlinear regression analysisAll statistical analyses were performed using Prism 50(Graphpad Software)

Evidence-Based Complementary and Alternative Medicine 5

0

2

4

6

8TN

F-al

pha (

pgm

g pr

otei

n)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

lowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowast

(a)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

0

1

2

3

4

IL-6

(pg

mg

prot

ein)

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

0

5

10

15

MD

A (n

mol

mg

prot

ein)

Veh

D-g

al

BYP

BYA

BPA

YPA BY BP YP

BYPA

lowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

(c)

Figure 2 Anti-inflammatory and antioxidative effects in brains of d-galactose-induced aging mice Different groups of mice were treatedas described in Figure 1 The anti-inflammatory effect was evaluated by measurement of the brain levels of TNF-120572 (a) and IL-6 (b) andantioxidant effect was evaluated bymeasurement of the brain level ofMDA (c) Data are expressed inmeanplusmn SD (119901 lt 001) or (119901 lt 0001)indicates significant difference between d-gal group and Veh group whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant differencebetween herbal group and d-gal group based on one-way ANOVA test followed by Newman-Keuls test

3 Results

31 Antiaging Effects in d-Galactose-Induced Mouse AgingModel In this model over an 8-week d-galactose exposureperiod the vehicle group (ldquoVehrdquo) of animals received dailyinjection and oral administration of vehicle (09 NaCl) thed-galactose group (ldquod-galrdquo) received daily injection of d-galactose (150mgkg) and oral administration of vehicle andeach herbal group received daily injection of d-galactose plusoral administration of herbal extract prepared from two ormore of the B Y P and A herbs (see Section 24)

In the Y-maze test the d-gal animals displayed a signif-icant decrease in entries into and time spent in the novelarm of the maze with 119901 lt 0001 indicating a loss of spatialmemory compared with the Veh group (Figures 1(a) and1(b)) Daily oral treatment with a BYPA BYA BY BP orYP extract accompanying d-galactose injection increased thepercentile novel arm entries compared with that of the d-galmice to 119901 lt 005 and oral treatment with BYP increasedthe percentile novel arm entries compared with that of thed-gal mice to 119901 lt 001 Daily oral treatment with BYP orBY also increased the percentile time spent in the novel armcompared with the d-gal mice to 119901 lt 001

The levels of TNF-120572 and IL-6 were increased in the brainsof d-gal mice compared with Veh mice (119901 lt 001) Dailyoral cotreatment with BYA or YP decreased the brain level of

TNF-120572 compared with that of the d-gal mice to 119901 lt 005 andcotreatment with BYPA BYP BY or BP decreased the brainlevel of TNF-120572 compared with that of the d-gal mice moreextensively to 119901 lt 001 (Figure 2(a)) Daily oral cotreatmentwith BYPA decreased the brain level of IL-6 compared withthat of the d-gal mice to 119901 lt 005 and cotreatment withBYP BY BP or YP decreased the brain level of IL-6 comparedwith that of the d-gal mice more extensively to 119901 lt 001(Figure 2(b))

When the level of oxidative stress in the brain wasassessed the marker compound MDA was found to beincreased in the brains of d-galmice comparedwithVehmice(119901 lt 0001) Daily oral cotreatment with BYA BPA or YPeffectively decreased the brain MDA compared with that ofthe d-gal mice to 119901 lt 005 and cotreatment with BYPABYP BY or BP decreased the brain level of MDA comparedwith that of the d-gal mice more markedly to 119901 lt 001(Figure 2(c))

Mice treatedwith d-galactose for 8weeks showed variableextents of loss of whiskers Table 1 shows one particularlysensitive batch of mice where loss of whiskers was displayedby as many as 50 of the d-gal mice In contrast none ofthe mice in the Veh group or in the groups that received d-galactose injections along with an extract from BYPA BYPBYA BPA or YPA displayed any loss of whiskers Thusexposure of sensitive mice to d-galactose inflicted damage to

6 Evidence-Based Complementary and Alternative Medicine

(a)

(I)

(II)

(III)

(IV)

(V)

(VI)

(VII)

(d)

(c)

(b)

Figure 3 Loss of whiskers by d-galactose-induced aging mice (I) Photographs of two Veh mice (IIa IIb) four d-galactose treated mice thatshowed loss of whiskers and (IIc IId) four d-galactose treated mice that did not show loss of whiskers (III) Two of the mice treated withd-galactose and BYPA (IV) two of the mice treated with d-galactose and BYP (V) two of the mice treated with d-galactose and BYA (VI)two of the mice treated with d-galactose and BPA and (VII) two of the mice treated with d-galactose and YPA

facial tissues resulting in loss of whiskers in accord with anearlier report of coarse fur and severe hair loss in d-galactosetreated mice [44] On this basis the findings in Table 1 alsoillustrated in Figure 3 suggest that the protective antiagingeffects of the BYPA BYP BYA BPA and YPA extracts were

not confined to brain tissues but extended significantly tofacial skin as well (119901 = 0014)

32 Anxiolytic and Sedative Effects The single-herb extractsof Y P and A but not that of B displayed anxiolytic effects

Evidence-Based Complementary and Alternative Medicine 7

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

PAYA BY BA BP120 120 120 120 120120

YP60 90 12030

BYPA

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

Veh 31DZ

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast lowast

lowastlowast

Figure 4 Anxiolytic effect of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to anxiolytic effect using the elevated plus-mazetest where increased percentage time spent in open arms compared with Veh represented an anxiolytic effect Data are expressed in mean plusmnSD of the percentage of time spent in open arms lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001) indicates significant difference from Veh groupbased on one-way ANOVA test followed by Newman-Keuls test

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

4 Evidence-Based Complementary and Alternative Medicine

28 Hole-Board Test After the elevated plus-maze test thetest mice were subjected to the hole-board test The hole-board apparatus consisted of a wooden box 60 times 60 times 20 cmwith four holes of 3 cm diameter evenly spaced on the floorEach mouse was placed at the center of the hole-board andthe number of head-dips into the holes was recorded for aperiod of 5 minutes A head-dip was scored when the headof the mouse was dipped into the hole to the extent that theeyes of the mouse passed below the hole on the floor Aftereach trial the floor of the apparatus was thoroughly cleansedand dried before the start of the next test Decreased numberof head-dips compared with Veh controls was indicative of asedative effect [40]

29 Locomotor Activity Test Following the elevated plus-maze and hole-board tests the testmicewere further assessedfor locomotor activity by means of the ZIL-2 apparatus(Beijing Institute of Materia Medica) The test apparatuswith dimensions of 60 cm times 60 cm times 12 cm consisted ofa transparent plastic cylindrical container equipped withthree evenly spaced infrared beams with photodetectorsThe number of transitions across the photocell beams wasrecorded automatically over a period of 5 minutes Anincrease or decrease in the number of transitions indicated anincrease or decrease in locomotor activity respectively [41]

210 Rotarod Test 4ndash6-week-old male ICR mice were ran-domly separated into groups Vehicle (Veh namely 09NaCl 119899 = 20) 120mgkg herbal extract (119899 = 15)or 1 or 3mgkg DZ (119899 = 15) was orally administered35 minutes prior to the test The rotarod test for motorcoordination utilized a custom-built apparatus consisting of acylindrical rotarod (25 cm diameter) with a textured surfaceplaced 05m above the ground Prior to vehicle or extractadministration the mice were first trained to stay for 2minon the rotarod revolving at 16 rpm To test the effect of vehicleor extract treatment the time each treated mouse remainedon the rotarod was recorded with cut-off set at 2min suchthat any mouse that remained on the rotarod after 2min wasaccorded a time on rotarod of 2min [42]

211 Step-through Passive Avoidance Test 4ndash6-week-oldmaleICRmicewere randomly separated into groups Vehicle (Vehnamely 09 NaCl 119899 = 20) 120mgkg herbal extract (119899 =15) or 1 or 3mgkg DZ (119899 = 15) was orally administered 35minutes prior to training The apparatus was a two-chamberbox (Chinese Academy of Chinese Medical Science Beijing)with one of the chambers being opaque (darkened) and theother transparent (lighted) In the training trials each mousewas placed into the lighted chamber and the door connectingthe lighted and darkened chambers was opened 10 secondslater Mice that did not enter the darkened chamber within 15seconds were excluded from the experiment After themouseentered the darkened chamber the door was closed and a 2-second electric foot shock of 04mA was delivered throughthe grid floor Ten seconds later the mouse was transferredfrom the darkened chamber back to the home cage After aninterval of 24 hours it was returned to the lighted chamber

The door to the darkened chamber was opened 10 secondslater and the time taken for the mouse to enter the darkenedchamber was recorded as the step-through latency cut-offwas set at 300 seconds such that the step-through latency forany mouse that did not enter the darkened chamber by 300seconds was recorded as 300 seconds Significant decrease instep-through latency indicated anterograde amnesia [43]

212 Preparation of Synaptosomal Membrane Sprague Daw-ley rats (sim200 g) were decapitated and the whole brainswere rapidly removed and frozen in liquid nitrogen Thetissue was thawed in 20 volumes of ice-cold 032M sucroseand homogenized in anULTRA-TURRAX homogenizerThehomogenate was centrifuged at 1000timesg for 10min at 4∘C andthe supernatant was again centrifuged at 20000timesg for 20minutes at 4∘CThe pellet from the second centrifugationwasresuspended in 50mM Tris-Cl pH 74 to a concentration of1mgml and stored atminus80∘Cuntil use For the [3H]-muscimoland [3H]-spiperone binding assays the pellet from the secondcentrifugation was further homogenized stirred on ice with20 volumes of ice-cold double-distilledwater and centrifugedat 48000timesg for 20 minutes at 4∘C this was repeated twicebefore resuspending the final pellet in 50mMTris-Cl pH 74to a concentration of 1mgml

213 Radioligand Binding Assays In these assays 1 nM [3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperonewas incubated with 02mgml synaptosomal membranes anddifferent concentrations of herbal extract in 50mM Tris-Cl pH 74 at either 4∘C for 90min for [3H]-flunitrazepamor [3H]-muscimol or 25∘C for 60min for [3H]-spiperonebinding Subsequently the incubation mixture was placedon a Whatman GFB filter on a Brandel 24-well harvesterand washed with ice-cold 50mM Tris-Cl pH 74 Eachwashed filter was incubated with 4ml scintillation cocktailfor at least 60 minutes before measurement of radioactivityin a Beckman-Coulter LS6500 scintillation counter Half-maximal inhibition concentrations (IC50) were determinedby nonlinear regression analysis performed using Prism 50(Graphpad Software) To assess nonspecific binding 1 nM[3H]-flunitrazepam 3 nM [3H]-muscimol or 1 nM [3H]-spiperone was incubated with synaptosomal membranes and10 120583M diazepam 10 120583M GABA or 10 120583M (+)-butaclamolrespectively in 50mM Tris-Cl pH 74 at either 4∘C for90min for [3H]-flunitrazepam or [3H]-muscimol or 25∘Cfor 60min for [3H]-spiperone under these conditions the[3H]-ligand bound in each instance represented nonspecificbinding

214 Statistical Analysis Results were expressed as mean plusmnstandard deviation (SD) One-way ANOVA test followedby Newman-Keuls test was performed on behavioral dataBinding assay data were expressed as mean plusmn SD of fourindependent incubations each in duplicate samplings Bind-ing affinity was determined by nonlinear regression analysisAll statistical analyses were performed using Prism 50(Graphpad Software)

Evidence-Based Complementary and Alternative Medicine 5

0

2

4

6

8TN

F-al

pha (

pgm

g pr

otei

n)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

lowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowast

(a)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

0

1

2

3

4

IL-6

(pg

mg

prot

ein)

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

0

5

10

15

MD

A (n

mol

mg

prot

ein)

Veh

D-g

al

BYP

BYA

BPA

YPA BY BP YP

BYPA

lowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

(c)

Figure 2 Anti-inflammatory and antioxidative effects in brains of d-galactose-induced aging mice Different groups of mice were treatedas described in Figure 1 The anti-inflammatory effect was evaluated by measurement of the brain levels of TNF-120572 (a) and IL-6 (b) andantioxidant effect was evaluated bymeasurement of the brain level ofMDA (c) Data are expressed inmeanplusmn SD (119901 lt 001) or (119901 lt 0001)indicates significant difference between d-gal group and Veh group whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant differencebetween herbal group and d-gal group based on one-way ANOVA test followed by Newman-Keuls test

3 Results

31 Antiaging Effects in d-Galactose-Induced Mouse AgingModel In this model over an 8-week d-galactose exposureperiod the vehicle group (ldquoVehrdquo) of animals received dailyinjection and oral administration of vehicle (09 NaCl) thed-galactose group (ldquod-galrdquo) received daily injection of d-galactose (150mgkg) and oral administration of vehicle andeach herbal group received daily injection of d-galactose plusoral administration of herbal extract prepared from two ormore of the B Y P and A herbs (see Section 24)

In the Y-maze test the d-gal animals displayed a signif-icant decrease in entries into and time spent in the novelarm of the maze with 119901 lt 0001 indicating a loss of spatialmemory compared with the Veh group (Figures 1(a) and1(b)) Daily oral treatment with a BYPA BYA BY BP orYP extract accompanying d-galactose injection increased thepercentile novel arm entries compared with that of the d-galmice to 119901 lt 005 and oral treatment with BYP increasedthe percentile novel arm entries compared with that of thed-gal mice to 119901 lt 001 Daily oral treatment with BYP orBY also increased the percentile time spent in the novel armcompared with the d-gal mice to 119901 lt 001

The levels of TNF-120572 and IL-6 were increased in the brainsof d-gal mice compared with Veh mice (119901 lt 001) Dailyoral cotreatment with BYA or YP decreased the brain level of

TNF-120572 compared with that of the d-gal mice to 119901 lt 005 andcotreatment with BYPA BYP BY or BP decreased the brainlevel of TNF-120572 compared with that of the d-gal mice moreextensively to 119901 lt 001 (Figure 2(a)) Daily oral cotreatmentwith BYPA decreased the brain level of IL-6 compared withthat of the d-gal mice to 119901 lt 005 and cotreatment withBYP BY BP or YP decreased the brain level of IL-6 comparedwith that of the d-gal mice more extensively to 119901 lt 001(Figure 2(b))

When the level of oxidative stress in the brain wasassessed the marker compound MDA was found to beincreased in the brains of d-galmice comparedwithVehmice(119901 lt 0001) Daily oral cotreatment with BYA BPA or YPeffectively decreased the brain MDA compared with that ofthe d-gal mice to 119901 lt 005 and cotreatment with BYPABYP BY or BP decreased the brain level of MDA comparedwith that of the d-gal mice more markedly to 119901 lt 001(Figure 2(c))

Mice treatedwith d-galactose for 8weeks showed variableextents of loss of whiskers Table 1 shows one particularlysensitive batch of mice where loss of whiskers was displayedby as many as 50 of the d-gal mice In contrast none ofthe mice in the Veh group or in the groups that received d-galactose injections along with an extract from BYPA BYPBYA BPA or YPA displayed any loss of whiskers Thusexposure of sensitive mice to d-galactose inflicted damage to

6 Evidence-Based Complementary and Alternative Medicine

(a)

(I)

(II)

(III)

(IV)

(V)

(VI)

(VII)

(d)

(c)

(b)

Figure 3 Loss of whiskers by d-galactose-induced aging mice (I) Photographs of two Veh mice (IIa IIb) four d-galactose treated mice thatshowed loss of whiskers and (IIc IId) four d-galactose treated mice that did not show loss of whiskers (III) Two of the mice treated withd-galactose and BYPA (IV) two of the mice treated with d-galactose and BYP (V) two of the mice treated with d-galactose and BYA (VI)two of the mice treated with d-galactose and BPA and (VII) two of the mice treated with d-galactose and YPA

facial tissues resulting in loss of whiskers in accord with anearlier report of coarse fur and severe hair loss in d-galactosetreated mice [44] On this basis the findings in Table 1 alsoillustrated in Figure 3 suggest that the protective antiagingeffects of the BYPA BYP BYA BPA and YPA extracts were

not confined to brain tissues but extended significantly tofacial skin as well (119901 = 0014)

32 Anxiolytic and Sedative Effects The single-herb extractsof Y P and A but not that of B displayed anxiolytic effects

Evidence-Based Complementary and Alternative Medicine 7

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

PAYA BY BA BP120 120 120 120 120120

YP60 90 12030

BYPA

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

Veh 31DZ

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast lowast

lowastlowast

Figure 4 Anxiolytic effect of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to anxiolytic effect using the elevated plus-mazetest where increased percentage time spent in open arms compared with Veh represented an anxiolytic effect Data are expressed in mean plusmnSD of the percentage of time spent in open arms lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001) indicates significant difference from Veh groupbased on one-way ANOVA test followed by Newman-Keuls test

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Evidence-Based Complementary and Alternative Medicine 5

0

2

4

6

8TN

F-al

pha (

pgm

g pr

otei

n)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

lowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowast

(a)

D-g

al

BYPA BY

P

BYA

BPA

YPA BY BP YPVeh

0

1

2

3

4

IL-6

(pg

mg

prot

ein)

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

0

5

10

15

MD

A (n

mol

mg

prot

ein)

Veh

D-g

al

BYP

BYA

BPA

YPA BY BP YP

BYPA

lowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

(c)

Figure 2 Anti-inflammatory and antioxidative effects in brains of d-galactose-induced aging mice Different groups of mice were treatedas described in Figure 1 The anti-inflammatory effect was evaluated by measurement of the brain levels of TNF-120572 (a) and IL-6 (b) andantioxidant effect was evaluated bymeasurement of the brain level ofMDA (c) Data are expressed inmeanplusmn SD (119901 lt 001) or (119901 lt 0001)indicates significant difference between d-gal group and Veh group whereas lowast(119901 lt 005) or lowastlowast(119901 lt 001) indicates significant differencebetween herbal group and d-gal group based on one-way ANOVA test followed by Newman-Keuls test

3 Results

31 Antiaging Effects in d-Galactose-Induced Mouse AgingModel In this model over an 8-week d-galactose exposureperiod the vehicle group (ldquoVehrdquo) of animals received dailyinjection and oral administration of vehicle (09 NaCl) thed-galactose group (ldquod-galrdquo) received daily injection of d-galactose (150mgkg) and oral administration of vehicle andeach herbal group received daily injection of d-galactose plusoral administration of herbal extract prepared from two ormore of the B Y P and A herbs (see Section 24)

In the Y-maze test the d-gal animals displayed a signif-icant decrease in entries into and time spent in the novelarm of the maze with 119901 lt 0001 indicating a loss of spatialmemory compared with the Veh group (Figures 1(a) and1(b)) Daily oral treatment with a BYPA BYA BY BP orYP extract accompanying d-galactose injection increased thepercentile novel arm entries compared with that of the d-galmice to 119901 lt 005 and oral treatment with BYP increasedthe percentile novel arm entries compared with that of thed-gal mice to 119901 lt 001 Daily oral treatment with BYP orBY also increased the percentile time spent in the novel armcompared with the d-gal mice to 119901 lt 001

The levels of TNF-120572 and IL-6 were increased in the brainsof d-gal mice compared with Veh mice (119901 lt 001) Dailyoral cotreatment with BYA or YP decreased the brain level of

TNF-120572 compared with that of the d-gal mice to 119901 lt 005 andcotreatment with BYPA BYP BY or BP decreased the brainlevel of TNF-120572 compared with that of the d-gal mice moreextensively to 119901 lt 001 (Figure 2(a)) Daily oral cotreatmentwith BYPA decreased the brain level of IL-6 compared withthat of the d-gal mice to 119901 lt 005 and cotreatment withBYP BY BP or YP decreased the brain level of IL-6 comparedwith that of the d-gal mice more extensively to 119901 lt 001(Figure 2(b))

When the level of oxidative stress in the brain wasassessed the marker compound MDA was found to beincreased in the brains of d-galmice comparedwithVehmice(119901 lt 0001) Daily oral cotreatment with BYA BPA or YPeffectively decreased the brain MDA compared with that ofthe d-gal mice to 119901 lt 005 and cotreatment with BYPABYP BY or BP decreased the brain level of MDA comparedwith that of the d-gal mice more markedly to 119901 lt 001(Figure 2(c))

Mice treatedwith d-galactose for 8weeks showed variableextents of loss of whiskers Table 1 shows one particularlysensitive batch of mice where loss of whiskers was displayedby as many as 50 of the d-gal mice In contrast none ofthe mice in the Veh group or in the groups that received d-galactose injections along with an extract from BYPA BYPBYA BPA or YPA displayed any loss of whiskers Thusexposure of sensitive mice to d-galactose inflicted damage to

6 Evidence-Based Complementary and Alternative Medicine

(a)

(I)

(II)

(III)

(IV)

(V)

(VI)

(VII)

(d)

(c)

(b)

Figure 3 Loss of whiskers by d-galactose-induced aging mice (I) Photographs of two Veh mice (IIa IIb) four d-galactose treated mice thatshowed loss of whiskers and (IIc IId) four d-galactose treated mice that did not show loss of whiskers (III) Two of the mice treated withd-galactose and BYPA (IV) two of the mice treated with d-galactose and BYP (V) two of the mice treated with d-galactose and BYA (VI)two of the mice treated with d-galactose and BPA and (VII) two of the mice treated with d-galactose and YPA

facial tissues resulting in loss of whiskers in accord with anearlier report of coarse fur and severe hair loss in d-galactosetreated mice [44] On this basis the findings in Table 1 alsoillustrated in Figure 3 suggest that the protective antiagingeffects of the BYPA BYP BYA BPA and YPA extracts were

not confined to brain tissues but extended significantly tofacial skin as well (119901 = 0014)

32 Anxiolytic and Sedative Effects The single-herb extractsof Y P and A but not that of B displayed anxiolytic effects

Evidence-Based Complementary and Alternative Medicine 7

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

PAYA BY BA BP120 120 120 120 120120

YP60 90 12030

BYPA

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

Veh 31DZ

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast lowast

lowastlowast

Figure 4 Anxiolytic effect of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to anxiolytic effect using the elevated plus-mazetest where increased percentage time spent in open arms compared with Veh represented an anxiolytic effect Data are expressed in mean plusmnSD of the percentage of time spent in open arms lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001) indicates significant difference from Veh groupbased on one-way ANOVA test followed by Newman-Keuls test

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

6 Evidence-Based Complementary and Alternative Medicine

(a)

(I)

(II)

(III)

(IV)

(V)

(VI)

(VII)

(d)

(c)

(b)

Figure 3 Loss of whiskers by d-galactose-induced aging mice (I) Photographs of two Veh mice (IIa IIb) four d-galactose treated mice thatshowed loss of whiskers and (IIc IId) four d-galactose treated mice that did not show loss of whiskers (III) Two of the mice treated withd-galactose and BYPA (IV) two of the mice treated with d-galactose and BYP (V) two of the mice treated with d-galactose and BYA (VI)two of the mice treated with d-galactose and BPA and (VII) two of the mice treated with d-galactose and YPA

facial tissues resulting in loss of whiskers in accord with anearlier report of coarse fur and severe hair loss in d-galactosetreated mice [44] On this basis the findings in Table 1 alsoillustrated in Figure 3 suggest that the protective antiagingeffects of the BYPA BYP BYA BPA and YPA extracts were

not confined to brain tissues but extended significantly tofacial skin as well (119901 = 0014)

32 Anxiolytic and Sedative Effects The single-herb extractsof Y P and A but not that of B displayed anxiolytic effects

Evidence-Based Complementary and Alternative Medicine 7

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

PAYA BY BA BP120 120 120 120 120120

YP60 90 12030

BYPA

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

Veh 31DZ

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast lowast

lowastlowast

Figure 4 Anxiolytic effect of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to anxiolytic effect using the elevated plus-mazetest where increased percentage time spent in open arms compared with Veh represented an anxiolytic effect Data are expressed in mean plusmnSD of the percentage of time spent in open arms lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001) indicates significant difference from Veh groupbased on one-way ANOVA test followed by Newman-Keuls test

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Evidence-Based Complementary and Alternative Medicine 7

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

PAYA BY BA BP120 120 120 120 120120

YP60 90 12030

BYPA

0

10

20

30

40

50

60

70

80

ti

me s

pent

in o

pen

arm

s

Veh 31DZ

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast lowast

lowastlowast

Figure 4 Anxiolytic effect of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to anxiolytic effect using the elevated plus-mazetest where increased percentage time spent in open arms compared with Veh represented an anxiolytic effect Data are expressed in mean plusmnSD of the percentage of time spent in open arms lowast(119901 lt 005) lowastlowast(119901 lt 001) or lowastlowastlowast(119901 lt 0001) indicates significant difference from Veh groupbased on one-way ANOVA test followed by Newman-Keuls test

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

8 Evidence-Based Complementary and Alternative Medicine

PAYA BY BA BP

0

10

20

30

40N

umbe

r of h

ead-

dips

0

10

20

30

40

Num

ber o

f hea

d-di

ps

0

10

20

30

40

Num

ber o

f hea

d-di

ps

60 90 12030B

60 90 12030Y

60 90 12030P

60 90 12030A

60 90 12030BYP

60 90 12030BYA

60 90 12030BPA

60 90 12030YPA

Veh 31DZ

60 90 12030BYPA

120 120 120 120 120120 YP

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Figure 5 Sedative effects of different combinations of B Y P and A herbs The performances of mice treated with saline vehicle (Veh)diazepam (DZ) and different single-herb or multiherb extracts were assessed with respect to sedative effect using the hole-board test wherereduced head-dips represented a sedative effect Data are expressed in mean plusmn SD of the number of head-dips lowast(119901 lt 005) lowastlowast(119901 lt 001) orlowastlowastlowast(119901 lt 0001) indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

as evidenced by significant increase in percentage of timespent in open arms in the elevated plus-maze test startingfrom an oral dose of 60mgkg The B- P- and A-treatedmice displayed sedative behavior as evidenced by significantdecrease in head-dips in the hole-board test starting from60mgkg in the case of P- and A-treated mice as well asstarting from the even lower dose of 30mgkg in the case ofB-treatedmice In contrast the Y-treatedmice did not displayany sedation up to 120mgkg (Figures 4 and 5)

The two-herb BA and PA extracts induced significantanxiolytic effects and only PA but not BA induced significantsedative effectsThe three-herb extracts BYA andYPAyieldedsignificant anxiolytic effect as well as sedative effect whereas

BPA gave rise to significant anxiolytic effect but no sedativeeffect up to 120mgkg BYP did not give rise to any significantanxiolytic or sedative effect As reported earlier [12] the four-herb BYPA combination induced significant anxiolysis but nosedative effect up to 120mgkg (Figures 4 and 5)

33 Locomotor Activity Motor Coordination and AnterogradeAmnesia There was no significant change in any of the one-two- three- or four-herb extract-treated mice at 120mgkgwhereas 3mgkg diazepam induced a significant reduction(119901 lt 0001) in locomotor activity (Figure 6(a)) There wasalso no significant change in the time mice managed to stayon the rotarod (Figure 6(b)) or in the step-through latency

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 9: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Evidence-Based Complementary and Alternative Medicine 9

0

100

200

300

400Lo

com

otor

activ

ity

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

(a)

020406080

100120140

Tim

e on

rota

rod

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400St

ep-th

roug

h lat

ency

(s)

BYPA YP

A BP BABYBPA

BYP PAYPBYA YA B P

Veh AY31

DZ

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Absence of side effects in herbal extract-treatedmice (a) Locomotor activity evaluated using the locomotor activity test (b) musclecoordination evaluated using the rotarod test and (c)memory impairment evaluated using the step-through passive avoidance test All herbalextracts were tested at the dose of 120mgkg and DZ was tested at 1mgkg and 3mgkg Data are expressed in mean plusmn SD lowastlowastlowast(119901 lt 0001)indicates significant difference from Veh group based on one-way ANOVA test followed by Newman-Keuls test

Table 1 Prevention of loss of whiskers in d-galactose-induced agingmice

Treatment group Loss of whiskers ( mice)0 +

Veh 100 0d-gallowast 50 50d-gal + BYPA 100 0d-gal + BYP 100 0d-gal + BYA 100 0d-gal + BPA 100 0d-gal + YPA 100 0119899 = 12micegroup ldquo0rdquo no loss of whiskers ldquo+rdquo loss of whiskerslowastBased on two-sided Fisherrsquos exact test the d-gal group with 612 whiskerloss differed significantly from the Veh group with 012 whisker loss (119901 =0014) from each of the BYPA BYP BYA BPA and YPA extract-treatedgroups showing 012 whisker loss (119901 = 0014) and from a combined herbal-treated group with 060 whisker loss (119901 = 59 times 10minus6) Photographs ofrepresentative mice from the different treatment groups are illustrated inFigure 3

in the step-through passive avoidance test (Figure 6(c))following administration of any of the herbal extracts testedat 120mgkg whereas diazepam at either 1mgkg or 3mgkgreduced both the time on the rotarod and the length of step-through latency (119901 lt 0001) These findings indicated thatnone of the herbal extracts tested induced any significant sideeffect in terms of altered level of locomotor activity reducedmotor coordination on the rotarod or anterograde amnesiain the step-through avoidance test

34 Interactions with Neuroreceptors The B Y A and BYPAextracts all inhibited the binding of the GABAA receptorbenzodiazepine- (BZ-) site agonist [3H]-flunitrazepam to ratcerebral cortex membranes with half-inhibition concentra-tion (IC50) values within the same order of magnitude How-ever cooperative binding was observed between P-extractand [3H]-flunitrazepam (Figure 7(a)) While the competitivebinding of ingredients in Y A or BYPA to the BZ-site wasconsistent with the BZ-site playing a role in the anxiolyticeffects of Y A and BYPA (Figure 4) the competitive bindingof B ingredients to the BZ-site but lack of anxiolysis by Bup to 120mgkg as well as the cooperative binding of P tothe BZ-site was suggestive of complex interactions of theingredients of B or P with GABAA receptors On the otherhand the sensitivity of Y- P- and A-induced anxiolysismeasured based on increase in time spent in open arms in theelevated plus-maze to inhibition by the BZ-site antagonistflumazenil (Figure 8) was consistent with participation ofthe BZ-site in anxiolysis by Y P and A In Figure 7(b)extracts of the single herbs B Y P A and the four-herb BYPAall inhibited the binding of the GABAA receptor GABA-site ligand [3H]-muscimol to the membranes However theIC50 value of 009mgml for BYPA was more than an orderof magnitude lower than the IC50 values of 550mgml157mgml 349mgml and 102mgml for B Y P and Arespectively clearly pointing to the presence of synergismbetween the constituents of B Y P and A with respect to[3H]-muscimol binding to the GABA-site Extracts B Y andBYPA also inhibited membrane binding of the dopamineD2 receptor ligand [3H]-spiperone (Figure 7(c)) furtherdemonstrating the breadth of interactions of the B Y P andAingredients with neuroreceptors that could contribute to the

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 10: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

10 Evidence-Based Complementary and Alternative Medicine

050

100150200250300350400450

[H]-

fluni

traz

epam

bin

ding

()

10 2 3minus2minus3 minus1minus4

log[Extract] (mgml)

(a)

0

50

100

150

[H]-

mus

cim

ol b

indi

ng (

)

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(b)

0

50

100

150

[H]-

spip

eron

e bin

ding

()

minus3 minus2 minus1minus4 1 20log[Extract] (mgml)

(c)

Figure 7 Effects on ligand binding to membrane neuroreceptors Effects of extracts of BYPA (◼ black) B (998771 green) Y (998787 light blue) P (Qdark blue) andA (e yellow) (a) Binding of [3H]-flunitrazepamyielding IC50 values of 137plusmn005mgml 105plusmn010mgml 141plusmn006mgmland 060 plusmn 012mgml for BYPA B Y and A respectively and EC50 of 401 plusmn 039mgml for P (b) Binding of [3H]-muscimol yielding IC50values of 009 plusmn 006mgml 550 plusmn 008mgml 157 plusmn 012mgml 349 plusmn 014mgml and 102 plusmn 007mgml respectively for BYPA B Y Pand A and (c) binding of [3H]-spiperone yielding IC50 values of 171plusmn025mgml 426plusmn009mgml and 114plusmn007mgml for BYPA B andY with little inhibition by P or A Data represent mean plusmn SEM Total [3H]-ligand binding recorded in each of panels (a)ndash(c) represented thesum of specific and nonspecific bindings (see Section 213) with specific binding constituting more than 90 75 and 60 of total bindingrespectively

synergisms and antagonisms between these four medicinalherbs

4 Discussion

Based on the d-galactose-induced accelerated-aging modelthe present study showed that extracts of various mixturesof the B Y P and A herbs especially the BYP BYA BY BPYP and BYPA extracts brought about significant protectionagainst one or more aging effects including improvement ofspatial memory (Figure 1) reduction in neuroinflammationin terms of the brain levels of proinflammatory cytokinesTNF-120572 and IL-6 or reduction in oxidative stress in thebrain in terms of the level of MDA (Figure 2) Notablyspatial memory deficit and enhanced neuroinflammation areencountered not only in normal aging but also in Alzheimerrsquosdisease (AD) [45ndash48] and increased oxidative stress in the

brain is common to normal aging AD and Parkinsonrsquosdisease (PD) [3 4 49ndash51] In this regard it has been suggestedthat combination therapy consisting of a drug targetingthe amyloid-beta (A120573) andor tau protein together with amedication modulating neuroinflammation may delay theprogression of AD [52] Accordingly the present findingssuggest that the BYP BYA BY BP YP and BYPA extractsrepresent potentially useful agents for the prevention andortreatment of the effects of normal aging AD and PD Theseextracts as in the case of all other extracts comprising two ormore of the B Y P andAherbs were devoid of significant sideeffects with respect to altered locomotor activity decreasedmotor coordination on the rotarod and anterograde amnesiain the passive step-through avoidance test at 120mgkg(Figure 6) The BYPA BYP BYA BPA and YPA extractsalso have been found to protect facial tissues against theloss of whiskers (Figure 3) In addition the BA BPA and

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 11: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

tim

e spe

nt in

ope

n ar

m

B + FBVeh(a)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

YVeh Y + F

lowastlowastlowast

(b)

0

20

40

60

ti

me s

pent

in o

pen

arm

PVeh P + F

lowastlowastlowast

(c)

0

20

40

60

80

ti

me s

pent

in o

pen

arm

A + FAVeh

lowastlowastlowast

(d)

Figure 8 Anxiolytic effects of single herbs blocked by the GABAA receptor BZ-site antagonist flumazenil Mice were treated with salinevehicle (Veh) single-herb extract (120mgkg of oral B Y P or A) with or without flumazenil (namely F 125mgkg ip) Anxiolytic effectwas evaluated based on time spent in open arms Data represent mean plusmn SD (119901 lt 0001) indicates significant difference between testgroup receiving single herb without flumazenil and Veh group whereas lowastlowastlowast(119901 lt 0001) indicates significant difference between test groupreceiving single herb with flumazenil and test group receiving single herb without flumazenil based on one-way ANOVA test followed byNewman-Keuls test

BYPA extracts can provide anxiolysis and the PA BYA andYPA extracts can provide both anxiolysis and sedation forthe treatment of sleep disorders (Figures 4 and 5) All theextracts derived from B Y P andor A showed little evidenceof adverse side effects regarding altered level of locomotoractivity reducedmotor coordination or anterograde amnesia(Figure 6)

Multiherb formulas of Chinese medicine are typicallycombination therapies that comprise complementary ldquoJunrdquo(Sovereign) ldquoChenrdquo (Minister) ldquoZuordquo (Assistant) and ldquoShirdquo(Courier) herbs with the aim of multitargeting and opti-mizing therapeutic efficacy and reducing adverse side effectsthrough mutual antagonisms and synergisms between thecomponent herbs [8ndash11] In the present study examples ofpronounced synergisms and antagonisms with respect toanxiolysis and antagonisms with respect to sedative effects

were observed among component herbs (Figures 9(a) and9(b)) The BZ-site of GABAA receptors has been impli-cated in both anxiolysis and sedation [53 54] Furthermoreevidence indicated that aging is accompanied by reducedglutamic acid decarboxylase activity and GABA synthesis inhippocampal interneurons resulting in enhanced neuronalexcitability contributing to memory impairment [55ndash57]and GABAA receptor modulators have been employed toimprove both spatial memory and working memory [58 59]TNF-120572 could increase mitochondrial ROS production [6061] and ROS also could trigger proinflammatory cytokineexpression via activation ofNF-120581B [62]Therefore the param-eters of anxiolysis sedation spatial memory performancebrain MDA TNF-120572 and IL-6 levels could be intercorrelatedalthough the exact relationships among them yet awaitdelineation

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 12: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

12 Evidence-Based Complementary and Alternative Medicine

BA YP BYPA

minus10

0

10

20

30

ti

me s

pent

in o

pen

arm

s(m

inus

veh

icle

cont

rol)

(a)

BP BA BYPAminus20

minus10

0

10

20

30

Num

ber o

f hea

d-di

ps(m

inus

veh

icle

cont

rol)

(b)

Figure 9 Synergisms and antagonisms between herbs (a) Examples of synergism or antagonism with respect to anxiolytic effects measuredbased on time spent in open arms in elevated plus-maze test The paired columns for herbal combinations BA and BYPA show in eachinstance that the anxiolytic effect induced by the combination (red column on the right) exceeded the sum of effects induced by the individualcomponent herbs (composite column on the left B in green Y in light blue P in dark blue and A in yellow) in contrast the paired columnsfor YP show that the anxiolytic effects induced by its component herbs Y and Pwere abolished by antagonisms within the YP combination (b)Examples of antagonism with respect to sedative effects measured based on number of head-dips in the hole-board test The paired columnsfor herbal combinations BP BA and BYPA show that the sedative effects induced by the component herbs in each instance (composite columnon the left) were completely abolished by antagonisms within each combination Sedative effects were estimated based on head-dips in thehole-board testThe anxiolytic or sedative effects represented by the combination columns were those indicated in Figures 4 and 5 for a dosageof 120mgkg The individual effects of single herbs represented in the composite column were derived from the dose-response relationshipsfor the single-herb data in Figures 4 and 5 the total dosage in each composite column was 120mgkg and the contributions of individualherbs were estimated based on their weight ratios in the combination and interpolations of their individual dose-response curves

Importantly although each of the B P and A herbs whenadministered alone at 30mgkg or 60mg upwards gave riseto significant sedation none of the BYP BPA BY BP BAYP YA and BYPA combinations induced significant sedationat 120mgkg (Figure 5) Since sedative drugs are difficult toadminister over prolonged periods the nonsedative propertyof BYP BY BP YP and BYPA on account of antagonismsbetween their component herbs usefully facilitates theirchronic application as potential antiaging or antineurodegen-eration agents

5 Conclusions

In conclusion oral administrations of various two- three-and four-herb combinations of Radix Bupleurum chinenseDC (ldquoBrdquo) Rhizoma Corydalis yanhusuo WT Wang (ldquoYrdquo)Caulis Polygonum multiflorumThunb (ldquoPrdquo) and Flos Albiziajulibrissin Durazz (ldquoArdquo) have been found to alleviate spatialmemory deficit and decrease the brain levels of TNF-120572 IL-6 and MDA andor loss of whiskers in the d-galactose-induced accelerated-aging mouse model In addition some

of the combinations induced anxiolytic effects unaccompa-nied by sedative effects sedative effects unaccompanied byanxiolytic effects or both anxiolytic and sedative effects allwithout any significant side effects with respect to locomotoractivity motor coordination or anterograde amnesia Theseresults therefore suggest that the two- three- and four-herb combinations of these four medicinal herbs couldprovide potentially useful oral-active therapeutic agents ornutraceuticals for the prevention andor treatment of nor-mal aging Alzheimerrsquos disease Parkinsonrsquos disease anxietydisorders andor sleep disorders The findings also suggestthat the ingredients of these four medicinal herbs furnish apromising source for the isolation and identification of single-compound agents useful for the prevention andor treatmentand usage as experimental probes into the etiology andpathophysiology of clinical conditions important to normalaging or age-related neurodegeneration Furthermore sincevarious combinations of B Y P and A could protect againstaccelerated aging both in the brain and in facial tissuescomprehensive investigations are called for to determine thescope and magnitude of the antiaging effects exerted by

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 13: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Evidence-Based Complementary and Alternative Medicine 13

varied combinations of these four herbs on the changes indifferent human tissues and organs in the course of normaland pathological aging

Conflicts of Interest

A US patent on the findings described in this paper has beenfiled by PharmacoGenetics Limited of the Hong Kong Uni-versity of Science andTechnology Entrepreneurship Programof which Hong Xue is the inventor All the authors confirmthat the grant from Innovation andTechnology Fund ofHongKongGovernment (SERATProject no SP49302) whichwasreceived does not reflect any conflict of interest

Authorsrsquo Contributions

Hong Xue conceived the study Rui Li Wingman ChanWaikinMat YiucheongHo andRigil K Yeungperformed theexperiments Rui Li and Wingman Chan analyzed the dataand Rui Li Shuiying Tsang and Hong Xue wrote the paperAll the authors discussed the results and commented on themanuscript This finalized manuscript has been approved byall the authors

Acknowledgments

The authors are grateful for support of Innovation and Tech-nology Fund of Hong Kong Government (SERAT Project noSP49302) and to Ms Peggy Lee for expert assistance

References

[1] C A Barnes ldquoAging and the physiology of spatial memoryrdquoNeurobiology of Aging vol 9 pp 563ndash568 1988

[2] O Von Bohlen Und Halbach C Zacher P Gass and KUnsicker ldquoAge-related alterations in hippocampal spines anddeficiencies in spatial memory in micerdquo Journal of NeuroscienceResearch vol 83 no 4 pp 525ndash531 2006

[3] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[4] G Barja ldquoFree radicals and agingrdquo Trends in Neurosciences vol27 no 10 pp 595ndash600 2004

[5] C Franceschi M Capri D Monti et al ldquoInflammaging andanti-inflammaging a systemic perspective on aging andlongevity emerged from studies in humansrdquo Mechanisms ofAgeing and Development vol 128 no 1 pp 92ndash105 2007

[6] C Franceschi and J Campisi ldquoChronic inflammation (Inflam-maging) and its potential contribution to age-associated dis-easesrdquo Journals of Gerontology Series A Biological Sciences andMedical Sciences vol 69 pp S4ndashS9 2014

[7] P L Minciullo A Catalano G Mandraffino et al ldquoInflammag-ing and anti-inflammaging the role of cytokines in extremelongevityrdquo Archivum Immunologiae et Therapiae Experimen-talis vol 64 no 2 pp 111ndash126 2016

[8] L Wang G-B Zhou P Liu et al ldquoDissection of mechanismsof Chinese medicinal formula Realgar-Indigo naturalis as aneffective treatment for promyelocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 12 pp 4826ndash4831 2008

[9] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[10] J Qiu ldquolsquoBack to the futurersquo for Chinese herbal medicinesrdquoNature Reviews Drug Discovery vol 6 no 7 pp 506ndash507 2007

[11] L Wu Y Wang Z Li B Zhang Y Cheng and X Fan ldquoIden-tifying roles of ldquo Jun-Chen-Zuo-Shirdquo component herbs ofQiShenYiQi formula in treating acute myocardial ischemia bynetwork pharmacologyrdquo Chinese Medicine vol 9 no 1 article24 2014

[12] H Xue and J T Wong ldquoThe Erhuhuanteng Chinese herbaldecoctionrdquo China Patent No ZL 2004100697873 2004

[13] J-C Chen N-W Chang J-G Chung and K-C Chen ldquoSai-kosaponin-A induces apoptotic mechanism in human breastMDA-MB-231 and MCF-7 cancer cellsrdquo American Journal ofChinese Medicine vol 31 no 3 pp 363ndash377 2003

[14] Y Sun T-T Cai X-B Zhou and Q Xu ldquoSaikosaponin ainhibits the proliferation and activation of T cells throughcell cycle arrest and induction of apoptosisrdquo InternationalImmunopharmacology vol 9 no 7-8 pp 978ndash983 2009

[15] S-J Wu Y-H Lin C-C Chu Y-H Tsai and J C-J ChaoldquoCurcumin or saikosaponin a improves hepatic antioxidantcapacity and protects against CCl4-induced liver injury in ratsrdquoJournal of Medicinal Food vol 11 no 2 pp 224ndash229 2008

[16] V K W Wong M M Zhang H Zhou et al ldquoSaikosaponin-d enhances the anticancer potency of TNF- via overcoming itsundesirable response of activating NF-Kappa B signalling incancer cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2013 Article ID 745295 14 pages 2013

[17] Y-L Hsu P-L Kuo L-C Chiang and C-C Lin ldquoInvolvementof p53 nuclear factor 120581b and FasFas ligand in induction ofapoptosis and cell cycle arrest by saikosaponin d in humanhepatoma cell linesrdquo Cancer Letters vol 213 no 2 pp 213ndash2212004

[18] W C Leung H Zheng M Huen S L Law and HXue ldquoAnxiolytic-like action of orally administered dl-tetra-hydropalmatine in elevated plus-mazerdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 27 no 5 pp775ndash779 2003

[19] S-X Xu L-P Yu Y-R Han Y Chen and G-Z Jin ldquoEffects oftetrahydroprotoberberines on dopamine receptor subtypes inbrainrdquo Acta Pharmacologica Sinica vol 10 no 2 pp 104ndash1101989

[20] J R Mantsch S-J Li R Risinger et al ldquoLevo-tetrahydro-palmatine attenuates cocaine self-administration and cocaine-induced reinstatement in ratsrdquo Psychopharmacology vol 192no 4 pp 581ndash591 2007

[21] Y-L Liu L-D Yan P-L Zhou C-F Wu and Z-H GongldquoLevo-tetrahydropalmatine attenuates oxycodone-inducedconditioned place preference in ratsrdquo European Journal ofPharmacology vol 602 no 2-3 pp 321ndash327 2009

[22] Z Yang Y-C Shao S-J Li et al ldquoMedication of l-tetra-hydropalmatine significantly ameliorates opiate craving andincreases the abstinence rate in heroin users a pilot studyrdquoActaPharmacologica Sinica vol 29 no 7 pp 781ndash788 2008

[23] B Lee B Sur M Yeom I Shim H Lee and D H HahmldquoL-tetrahydropalmatine ameliorates development of anxietyand depression-related symptoms induced by single prolongedstress in ratsrdquo Biomolecules amp Therapeutics vol 22 no 3 pp213ndash222 2014

[24] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 14: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

14 Evidence-Based Complementary and Alternative Medicine

[25] W Ji WWang andW Liu ldquoEffects of n-butanol extract of FlosAlbiziae on sleep time and acute toxicity in micerdquo Chinese ArchTraditional Chinese Medicine vol 25 no 2 pp 242ndash244 2007

[26] T H Kang S J Jeong N Y Kim R Higuchi and Y C KimldquoSedative activity of two flavonol glycosides isolated from theflowers of Albizzia julibrissin Durazzrdquo Journal of Ethnopharma-cology vol 71 no 1-2 pp 321ndash323 2000

[27] Z Li M Zhang Z Mao and G Fan ldquoStudies on fraction withantidepressant activity from the flower of Albizzia JulibrissinDurazzrdquo LiShiZhenMedicine andMateria Medica Research vol8 article 013 2006

[28] R G Jordens M D Berry C Gillott and A A BoultonldquoProlongation of life in an experimental model of aging inDrosophila melanogasterrdquo Neurochemical Research vol 24 no2 pp 227ndash233 1999

[29] X Cui L Wang P Zuo et al ldquoD-Galactose-caused life short-ening in Drosophila melanogaster and Musca domestica isassociatedwith oxidative stressrdquoBiogerontology vol 5 no 5 pp317ndash325 2004

[30] X Cui P Zuo Q Zhang et al ldquoChronic systemic D-galactoseexposure induces memory loss neurodegeneration and oxida-tive damage in mice protective effects of R-120572-lipoic acidrdquoJournal of Neuroscience Research vol 84 no 3 pp 647ndash6542006

[31] C F Chen S Y Lang P P Zuo N Yang X Q Wang and CXia ldquoEffects of d-galactose on the expression of hippocampalperipheral-type benzodiazepine receptor and spatial memoryperformances in ratsrdquo Psychoneuroendocrinology vol 31 no 7pp 805ndash811 2006

[32] J Zhu XMu J Zeng et al ldquoGinsenoside Rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of D-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[33] P-C Chao M-C Yin and M-C Mong ldquoAnti-apoptotic andanti-glycative effects of asiatic acid in the brain of D-galactosetreatedmicerdquoFood andFunction vol 6 no 2 pp 542ndash548 2015

[34] F Dellu A Contarino H Simon G F Koob and L H GoldldquoGenetic differences in response to novelty and spatial memoryusing a two-trial recognition task in micerdquo Neurobiology ofLearning and Memory vol 73 no 1 pp 31ndash48 2000

[35] C D Conrad L A M Galea Y Kuroda and B S McEwenldquoChronic stress impairs rat spatial memory on the Y maze andthis effect is blocked by tianeptine pretreatmentrdquo BehavioralNeuroscience vol 110 no 6 pp 1321ndash1334 1996

[36] W A Pryor ldquoOn the detection of lipid hydroperoxides inbiological samplesrdquo Free Radical Biology and Medicine vol 7no 2 pp 177ndash178 1989

[37] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[38] M S Y Huen K-M Hui J W C Leung et al ldquoNaturallyoccurring 21015840-hydroxyl-substituted flavonoids as high-affinitybenzodiazepine site ligandsrdquo Biochemical Pharmacology vol66 no 12 pp 2397ndash2407 2003

[39] D Treit J Menard and C Royan ldquoAnxiogenic stimuli in theelevated plus-mazerdquo Pharmacology Biochemistry and Behaviorvol 44 no 2 pp 463ndash469 1993

[40] S E File and S Pellow ldquoThe effects of triazolobenzodiazepinesin two animal tests of anxiety and in the holeboardrdquo BritishJournal of Pharmacology vol 86 no 3 pp 729ndash735 1985

[41] K M Hui M S Y Huen H Y Wang et al ldquoAnxiolytic effectof wogonin a benzodiazepine receptor ligand isolated fromScutellaria baicalensis Georgirdquo Biochemical Pharmacology vol64 no 9 pp 1415ndash1424 2002

[42] T Karl R Pabst and S Von Horsten ldquoBehavioral phenotypingof mice in pharmacological and toxicological researchrdquo Experi-mental and Toxicologic Pathology vol 55 no 1 pp 69ndash83 2003

[43] F Nazari-Serenjeh A Rezayof and M-R Zarrindast ldquoFunc-tional correlation between GABAergic and dopaminergic sys-tems of dorsal hippocampus and ventral tegmental area inpassive avoidance learning in ratsrdquo Neuroscience vol 196 pp104ndash114 2011

[44] F An G D Yang J M Tian and S H Wang ldquoAntioxidanteffects of the orientin and vitexin in Trollius chinensis Bungein D-galactose-aged micerdquoNeural Regeneration Research vol 7no 33 pp 2565ndash2575 2012

[45] H Tanila ldquoWading pools fading memories-place navigation intransgenic mouse models of Alzheimerrsquos diseaserdquo Frontiers inAging Neuroscience vol 4 article 11 2012

[46] M T Heneka M J Carson J E Khoury et al ldquoNeuroinflam-mation in Alzheimerrsquos diseaserdquo The Lancet Neurology vol 14no 4 pp 388ndash405 2015

[47] E Tarkowski N Andreasen A Tarkowski and K BlennowldquoIntrathecal inflammation precedes development ofAlzheimerrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 9 pp 1200ndash1205 2003

[48] J C Breitner L D Baker T J Montine et al ldquoExtended resultsof the Alzheimerrsquos disease anti-inflammatory prevention trialrdquoAlzheimerrsquos and Dementia vol 7 no 4 pp 402ndash411 2011

[49] G Perry A D Cash and M A Smith ldquoAlzheimer disease andoxidative stressrdquo Journal of Biomedicine and Biotechnology vol2002 no 3 pp 120ndash123 2002

[50] C Henchcliffe and F M Beal ldquoMitochondrial biology andoxidative stress in Parkinson disease pathogenesisrdquo NatureClinical Practice Neurology vol 4 no 11 pp 600ndash609 2008

[51] J C Fernandez-Checa A Fernandez A Morales M MarıC-R Garcıa-Ruiz and A Colell ldquoOxidative stress and alteredmitochondrial function in neurodegenerative diseases lessonsfrom mouse modelsrdquo CNS and Neurological DisordersmdashDrugTargets vol 9 no 4 pp 439ndash454 2010

[52] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[53] W E Haefely J R Martin J G Richards and P SchochldquoThemultiplicity of actions of benzodiazepine receptor ligandsrdquoCanadian Journal of Psychiatry vol 38 supplement 4 pp S102ndashS108 1993

[54] F Wang Z Xu L Ren S Y Tsang and H Xue ldquoGABA119860 recep-tor subtype selectivity underlying selective anxiolytic effect ofbaicalinrdquoNeuropharmacology vol 55 no 7 pp 1231ndash1237 2008

[55] J A McQuail C J Frazier and J L Bizon ldquoMolecular aspectsof age-related cognitive decline the role of GABA signalingrdquoTrends in Molecular Medicine vol 21 no 7 pp 450ndash460 2015

[56] A M A Ylinen R Miettinen A Pitkanen A I Gulyas T FFreund and P J Riekkinen ldquoEnhanced GABAergic inhibitionpreserves hippocampal structure and function in a model ofepilepsyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 17 pp 7650ndash7653 1991

[57] J-L Zhou Q Zhao and G L Holmes ldquoEffect of levetiracetamon visual-spatial memory following status epilepticusrdquo EpilepsyResearch vol 73 no 1 pp 65ndash74 2007

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 15: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Evidence-Based Complementary and Alternative Medicine 15

[58] M T Koh S Rosenzweig-Lipson and M Gallagher ldquoSelectiveGABAA1205725 positive allosteric modulators improve cognitivefunction in aged rats withmemory impairmentrdquoNeuropharma-cology vol 64 pp 145ndash152 2013

[59] F Wang Z Xu C T Yuen et al ldquo621015840-Dihydroxyflavone asubtype-selective partial inverse agonist of GABAA receptorbenzodiazepine siterdquo Neuropharmacology vol 53 no 4 pp574ndash582 2007

[60] V Goossens J Grooten K De Vos and W Fiers ldquoDirect evi-dence for tumor necrosis factor-inducedmitochondrial reactiveoxygen intermediates and their involvement in cytotoxicityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 18 pp 8115ndash8119 1995

[61] N Baregamian J Song C E Bailey J Papaconstantinou BM Evers and D H Chung ldquoTumor necrosis factor-120572 andapoptosis signal-regulating kinase 1 control reactive oxygenspecies release mitochondrial autophagy and c-Jun N-terminalkinasep38 phosphorylation during necrotizing enterocolitisrdquoOxidativeMedicine and Cellular Longevity vol 2 no 5 pp 297ndash306 2009

[62] M J Morgan and Z Liu ldquoCrosstalk of reactive oxygen speciesand NF-120581B signalingrdquo Cell Research vol 21 no 1 pp 103ndash1152010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 16: Antiaging and Anxiolytic Effects of Combinatory Formulas ...downloads.hindawi.com/journals/ecam/2017/4624069.pdf · Antiaging and Anxiolytic Effects of Combinatory Formulas Based

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom