ResearchArticle - pdfs.semanticscholar.org€¦ · Radix Notoginseng, a widely used traditional Chinese medicine known as Sanqi or Tianqi in China, is the root of Panax notoginseng

Research ArticleFraction n-Butanol of Radix Notoginseng ProtectsPC12 Cells from A12057325ndash35-Induced Cytotoxicity andAlleviates Cognitive Deficits in SAMP8 Mice by AttenuatingOxidative Stress and A120573 Accumulation

Jin-Lan Huang12 Ying-Qin Feng3 Li-Ru Bai2 Mei-Chun Qin4 Zhe-Hao Xu4

Zhao-Rong Liu2 Wen-Bing Chen2 Deng-PanWu12 and Zhen-Guo Zhong4

1 Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy Pharmacy School Xuzhou Medical UniversityXuzhou Jiangsu 221004 China2Department of Pharmacology Pharmacy School Xuzhou Medical University Xuzhou Jiangsu 221004 China3Department of Pharmacy Liuzhou Traditional Chinese Medical Hospital Liuzhou Guangxi 545001 China4Scientific Research Center of Traditional Chinese Medicine Guangxi University of Chinese MedicineNanning Guangxi 530200 China

Correspondence should be addressed to Deng-Pan Wu dengpanwuxzhmueducn and Zhen-Guo Zhong gxtcmuzzg163com

Received 1 June 2017 Revised 14 August 2017 Accepted 23 August 2017 Published 3 October 2017

Academic Editor Krishnadas Nandakumar

Copyright copy 2017 Jin-Lan Huang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chinese medicine has been used for Alzheimerrsquos disease (AD) treatment for thousands of years with more effective and fewer sideeffects Therefore developing effective potential candidates from Chinese medicine against AD would be considered as critical andefficient therapy for AD treatment This study was designed to evaluate the neuronal protective effect of fraction n-butanol (NB)of Radix Notoginseng on A120573

25ndash35-induced PC12 cells explore the effect of the tested fraction on spatial learning and memory andcharacterize the impacts of fraction NB on antioxidant enzymes A120573 production and APP and BACE1 expressions The resultsrevealed that fraction NB could promote proliferation of PC12 cells and protect and rescue PC12 cells from A120573

25ndash35-induced celldeathMoreover fractionNB could improve spatial learning andmemory impairments of senescence-accelerated prone8 (SAMP8)mice and attenuate oxidative stress and reduce the production of A120573 by inhibiting the expressions of APP and BACE1 in the brainsof SAMP8 mice The result of single dose acute toxicity assay showed that fraction NB had a mild toxicity in vivo The pronouncedactions against AD and in vivo low toxicity of fraction NB suggest that fraction NB may be a useful alternative to the current ADtreatment

1 Introduction

Alzheimerrsquos disease (AD) the most prevalent cause ofdementia worldwide is defined neuropathologically by thedeposition of amyloid-120573 peptides (A120573) into senile plaquesin the extracellular space neuronal cell death induced byoxidative stress and intracellular formation of neurofibrillarytangles The senile plaque is mainly composed of A120573 withlength of 40 or 42 residues A120573 is generated from 120573-amyloidprecursor protein (APP) firstly cleaved by120573-secretase (120573-APP

cleaving enzyme 1 BACE1) and then by 120574-secretase Abnor-mal accumulation of extracellular A120573 in amyloid plaquescauses neuronal damage impairment of cognitive functionand ultimately the development of dementia [1] Further-more growing evidence suggests that oxidative stress playsimportant roles in A120573 deposition and cognitive dysfunctionof AD It has been established that A120573 induces oxidative stressand oxidative stress in turn enhances the A120573 productionand aggregation which promotes neuronal degenerationand death resulting in cognitive function impairment [2]

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 8469754 10 pageshttpsdoiorg10115520178469754

2 Evidence-Based Complementary and Alternative Medicine

Antioxidant treatments in the early stage of AD pathogenesiswere able to alleviate the cognitive function impairment andreduce A120573 accumulation in AD [3] Accordingly identifica-tion of potential protective candidates to attenuate oxidativestress and eliminate excessive accumulation of A120573 and ulti-mately improve cognitive function impairment is consideredas an important strategy for AD treatment

Radix Notoginseng a widely used traditional Chinesemedicine known as Sanqi or Tianqi in China is the rootof Panax notoginseng (Burk) F H Chen belonging to thegenus Panax family Araliaceae Studies have reported thatRadix Notoginseng has plenty of pharmacological proper-ties including antioxidant antiatherosclerotic antitumourand haemostatic activities [4] Recent studies have demon-strated that fraction n-butanol (NB) of Radix Notoginsenghas antiproliferative effects on human colorectal cancercells [5] inhibitory effects on osteoclastogenesis in LPS-activated RAW2647 cells [6] and anti-inflammatory effectson collagen-induced arthritic mice [7] However it is unclearwhether fraction NB has antagonistic effect on AD

We and others reported that fraction NB extracted fromRadix Notoginseng contained a large proportion of ginseno-side Rb1 Rg1 and notoginsenoside R1 [5 8] It has beenestablished that ginsenoside Rb1 Rg1 and notoginsenosideR1 have abilities to suppress oxidative stress and attenuateA120573-induced neuronal damage [9ndash11] Thus it is reasonableto hypothesize that fraction NB might have an effect againstAD

Therefore this study was designed to evaluate the neu-ronal protective effect of fraction NB extracted from RadixNotoginseng on A120573

25ndash35-induced PC12 cells Moreover 3-month-old senescence-accelerated prone8 (SAMP8) micewere intragastrically given fraction NB for 8 consecutiveweeks to explore the effect of the tested fraction on spatiallearning and memory and characterize the impacts of frac-tion NB on antioxidant enzymes A120573 production and APPandBACE1 expressions thereby providing a new opportunityfor research in regard to the pharmaceutical prevention andtreatment of AD

2 Material and Methods

21 Chemicals Radix Notoginseng were collected in Yulincity Guangxi Zhuang Autonomous Region China and ver-ified by Professor Wei songji from Guangxi University ofChinese Medicine The extract condition of fraction NB wasperformed according to our previous study [8] Briefly air-dried and powdered Radix Notoginseng roots (45 kg) wereconducted with 95 and 60 ethanol (each 4000mL) bypercolating at room temperature for a week to give totalethanol extract (1400 g) The extract was dissolved in waterand then extracted with petroleum ether (PE) acetic ether(AE) and then water-saturated n-butanol (NB) to obtainfractions PE (244 g) AE (2130 g) and NB (6000 g)

22 Reagents Methyl thiazolyl tetrazolium (MTT) wasobtained from Amresco (Ohio USA) Huperzine A (HupA) was from Zhejiang Zhenyuan Pharmaceutical Co Ltd

(Zhejiang China) The SOD and GSH-PX activity detectionkits were purchased from Nanjing Jiancheng BioengineeringInstitute (Nanjing China) TIANScript RT Kit was fromTiangen Biotech Co Ltd (Beijing China) A120573

25ndash35 was fromGibco (NewYork USA) All other reagents were from Sigma-Aldrich unless stated otherwise

23 Cell Culture PC12 cell line purchased from ShanghaiBiological Institute was suspended in RMPI-1640 (GibcoUSA) containing 5 fetal bovine serum (FBS) (HycloneUSA) 10 horse serum (Gibco USA) 100 120583gmL strepto-mycin and 100UmL penicillin in 5 CO

2at 37∘C

24 Animals Drug Treatment and Tissue Preparation Ithas been established that increased oxidative stress and A120573immunoreactive granular structures were found in SAMP8mice as young as 2 months of age Remarkable memorydeficits apparent reduction in the activity of antioxidantenzymes such as superoxide dismutase (SOD) and glu-tathione peroxidase (GSH-PX) and increased amyloidplaque deposition were found in 5-month-old SAMP8 mice[12]Thus sixty 3-month-old SAMP8 and twelve senescence-accelerated-resistant (SAMR1) (3 months old control strainof SAMP8) pathogen- and virus-free mice were obtainedfrom Tianjin University of Traditional Chinese Medicine(Tianjin China) SAMP8 mice were randomly divided intofive groups model group fraction NB high-dosage fractionNB middle-dosage and fraction NB low-dosage groups andHup A group Since Hup A has been widely used for thetreatment of AD owing to its roles in inhibiting acetyl-cholinesterase (AChE) activity reducing A120573 accumulationalleviating oxidative stress and improving cognitive function[13 14] thusHupAwas considered as a positive control in thestudy SAMR1mice were considered as the control groupThehigh- middle- and low-dosage groups were intragastricallygiven 300 150 and 75mgkg of fraction NB respectively perday while Hup A group was treated with 03mgkg Hup A bygavage every day for 2 months The middle dose of fractionNB was confirmed according to the dose of Radix Noto-ginseng used in clinics and the dose ofHupAwas determinedon the basis of our previous study [15] The same volume ofdistilled water was given to the model and control groupsAfter examination of learning and memory the mice wereethically sacrificed and the brains were excised for ELISAassay activity detection and real-time PCR assay Animalcare and experimental procedures were implemented accord-ing to the document ldquoGuidance Suggestions for Caring forLaboratory Animalsrdquo produced by the Ministry of Scienceand Technology of China in 2006

25 MTT Assay PC12 cells were seeded into 96-well platesat a density of 104 cellsmL for 24 h For the detection of cellproliferation cells were treated with fractions PE AE andNB at the concentrations of 10 120583gmL 20120583gmL 40 120583gmLand 80120583gmL for 72 h respectively To evaluate the protectiveeffect of fraction NB the cells were treated with fraction NBat the concentrations of 10 120583gmL 20120583gmL 40 120583gmL and80 120583gmL for 24 h before exposure to 10 120583M A120573

25ndash35 and

Evidence-Based Complementary and Alternative Medicine 3

the cells were then incubated with A12057325ndash35 and different

concentrations of fraction NB for another 72 h For assessingthe therapeutic effect of fraction NB the cells were incubatedwith 10 120583M A120573

25ndash35 for 24 h After the incubation cells wereexposed to fraction NB at the indicated concentrations for72 h After treatments MTT assay was performed accordingto our previous study [16]

26 Single Dose Acute Toxicity A single dose acute toxicitystudy was performed to assess the acute toxicity of fractionNB The toxicity protocol was described previously [1718] and approved by the State Food and Drug Admin-istration of China Briefly healthy Kunming-strain mice(119899 = 20 18ndash22 g male) from Guangxi University of Chi-nese Medicine were intragastrically given once fraction NBat 1945 gkg Untreated mice were used as a negative con-trol A bolus dose of more than 1945 gkg of fraction NBwas not attempted due to the limited solubility of frac-tion NB in the vehicle solution After administrationmice overall health was monitored and body weight wasdetected on days 7 and 14 On day 14 fraction NB-treatedmice and untreated mice were ethically sacrificed after anes-thesia The organs including liver heart kidney and spleenwere excised and weighted The relative organ weight wascalculated Relative organ weight = organ weight (g)bodyweight (g) times 100 Moreover the maximal tolerant dosewhich is the dose of Radix Notoginseng confirmed by themaximal dosage of fraction NB was calculated and the clin-ical daily dose of Radix Notoginseng was determined basedon the dosage used in clinics Sincemaximal tolerantmultiple(MTM) is one of the parameters widely used to assess theacute toxicity of new drugs of traditional Chinese medicine[17ndash19] MTMwas calculated to assess the toxicity of fractionNB according to the following formula [18 19]

MTM

=maximal tolerant dose (g)

average body weight of mouse (20 g)

divideclinical daily dose of Radix Notoginseng (g)

average human body weight (60 kg)

(1)

27 MorrisWater Maze Test The capacity for spatial learningand memory was carried out using Morris water maze(MWM) test as previously described [20] The capacity ofspatial learning was examined by place navigation test Themice were subjected to four consecutive daily training trialsBefore the trials themice were subjected to a watermaze taskin a swimming pool for 2 minutes in the absence of theplatform to adapt to the environment In each trial themice were randomly placed to the water at one of four startlocations and trained to locate the hidden platform with amaximum trial time of 60 seconds and an interval of approx-imately 30 seconds The mice were allowed to stay on theplatform for 3 seconds before starting the next trial fromanother location The platform was located in the sameposition for all trials The time to reach the platform (escapelatency) and the percentage of time in the target quadrant

were recorded On day 5 a spatial probe test was employedto detect the capacity of spatial memory In the test the micewere subjected to a water pool without the hidden platformand started at the start location in the quadrant oppositeto the quadrant where the former platform was placed Thepercentage of time spent in the former platform quadrant wasrecorded

28 SOD and GSH-PX Activity Detection SOD and GSH-PXactivities weremeasured in accordance with the activity assaykit instructions of SOD and GSH-PX respectively Actionbuffers were mixed with the supernatants of tissue samplesand incubated at 37∘C A visible spectrophotometer wasperformed to detect the optical densities of the mixtures at550 nm and 405 nm for SOD and GSH-PX activities respec-tively Experimental data for the activities were expressed asthe units per mg protein sample

29 Enzyme-Linked Immunosorbent Assay (ELISA) Afterthe behavioral tests the mice were sacrificed The brainswere homogenized (1 10 wv) in cold normal saline and thesupernatants were decanted for the measurement of total A120573level using a commercially available high-sensitive ELISA kit(CUSABIO Biotech Co Ltd China) following manufacturerinstructions Each sample was measured in duplicate Dataare represented as nanogram (ng) per mL

210 mRNA Extraction Reverse Transcription and Real-Time PCR 2120583g total RNA 4 120583L 5 times RT Buffer 1 120583L RTEnzyme Mix 1 120583L Primer Mix and RNase-free water werecontained in the reaction system of reverse transcriptionReal-time PCR reaction was performed according to ourprevious study [15] The primer sequences were as fol-lows BACE1 (307 bp) sense (51015840-CTGTTATGGGAGCCG-TCATC-31015840) antisense (51015840-GCAAAGTCATCGTGCTGGT-31015840) APP (314 bp) sense (51015840-GGAAGCAGCCAATGAGAG-AC-31015840) antisense (51015840-AGAGCAGGGACAGAGACTGG-31015840)ACTB (263 bp) sense (GAGACCTTCAACACCCCAGC)antisense (ATGTCACGCACGATTTCCC)The relative geneexpression was calculated by 2ndashΔΔCp method where ΔΔCp =ΔCptreatment minus ΔCpcontrol and ΔCp = Cptarget gene minus Cp119860

CT119861gene

[15 21]

211 Statistical Analysis Experimental data in each groupwere presented as mean plusmn SD Analysis of variance wasperformed with SPSS software for windows 130 119875 lt 005was considered statistically significant

3 Results

31 Effects of Fractions Isolated from Radix Notoginseng onProliferation in PC12 Cells Firstly for assessing the effects ofthe fractions isolated from Radix Notoginseng on cell prolif-eration we treated PC12 cells with different concentrationsof fraction PE AE or NB extracted from Radix Notoginsengfor 72 h MTT assay was performed to assess the effect ofthe fractions on proliferation of PC12 cells As shown in


Figure 1(a) the increased proliferative ability was observed infractions AE- and NB-treated cells while a sharp reductionin the proliferation was shown in fraction PE-treated cellsIt should be noted that fraction NB-treated cells showeda higher proliferative ability than fraction AE-treated cells(Figure 1(a)) indicating that fraction NB may have potentialto promote PC12 cells proliferation

32 Effect of Fraction NB on A12057325ndash35 Induced Cytotoxicityin PC12 Cells Plenty of studies showed that A120573-inducedcytotoxicity in PC12 cells was a common and reliable cellulartoxicitymodel for AD related studies in vitro [22] To evaluatethe protective effect of fractionNB PC12 cells were pretreatedwith fraction NB for 24 h prior to A120573

25ndash35 incubation andthen treated with the tested fraction for 72 h Results ofMTT assay showed that exposure of cells to 10 120583M A120573

25ndash35for 24 h led to a significant decrease in cell viability (Fig-ure 1(b)) while pretreatment with 40120583gmL and 80 120583gmLfraction NB significantly suppressed A120573

25ndash35-induced celldeath (Figure 1(b)) In order to clarify whether fraction NBcould rescue the cells from A120573

25ndash35-induced cytotoxicityPC12 cells were incubated with A120573

25ndash35 for 24 h and thenposttreated with fraction NB at indicated concentrations for72 h The results indicated that fraction NB at 40 120583gmL and80 120583gmL could remarkably attenuate A120573

25ndash35-induced celldeath (Figure 1(c)) These results demonstrate that fractionNB holds the potential not only to protect but also to rescuePC12 cells from A120573

25ndash35-induced cell death

33 Assessment of Acute Toxicity of Fraction NB Using SingleDoseAcuteAssay Due to the limited solubility of fractionNBin the vehicle solution a dose of 1945 gkg was consideredas a maximal dose In order to assess the acute toxicity offraction NB at this dosage mice were intragastrically given abolus dose of 1945 gkg of fraction NB As shown in Figure 2the body weights of fraction NB-treated and control micegained from 2006 plusmn 126 g and 2032 plusmn 113 g to 2849 plusmn171 g and 2956 plusmn 190 g respectively and the body weightof fraction NB-treated group on days 1 7 and 14 did nothave significant difference compared to the control groupMoreover the changes of relative organweights of liver heartkidney and spleen in fractionNB-treated group did not showany difference when compared with the control group (Fig-ure 2) Additionally the acute toxicity of fraction NB wasalso estimated using MTM parameter MTM is the multipleof maximal tolerant dose of mice divided by clinical dailydose of humans as described in inMaterial andMethods It isgenerally believed that the tested drug is considered safe if themaximal tolerant dose of mice is more than 100 times as highas the clinical daily dose of humans (MTM ge 100) [18 19]Theresult showed that the maximal tolerant dose of mice is 972times as high as the clinical daily dose of humans (MTM =972) suggesting a low toxicity of fraction NB Together theabove results indicate that fraction NB has a mild toxicity invivo

34 Effect of Fraction NB on Learning and Memory Abilitiesof SAMP8 Mice in the Morris Water Maze The cognitive

function was evaluated using MWM test In the placenavigation test the average escape latency and percentage oftime in the target quadrant of four-day training were cal-culated As shown in Figure 3 the mean escape latency offour days of learning in control group and fraction NB-treated groups was significantly shorter than that in themodel group while the percentages of quadrant timeincreased in the control group and fraction NB-treatedgroups compared to the model group In the probe trial ofMWM study the percentage of time spent in the target quad-rant of each mouse was recorded to evaluate the learning andconsolidation of the location of target quadrant during thefour days of training As illustrated in Figure 3 the percentageof quadrant time in the model group significantly decreasedwhen compared to the control group Nevertheless afterSAMP8 mice were treated with different concentrations offraction NB the percentage of quadrant time markedlyincreased (Figure 3) These results suggest that fraction NBcan improve spatial learning and memory impairment ofSAMP8 mice

35 Effect of FractionNB on SOD andGSH-PXActivities in theBrains of SAMP8 Mice To investigate whether fraction NBcould attenuate oxidative stress in the brains of SAMP8micethe activities of antioxidant enzymes such as SOD and GSH-PX were detected using activity detection kit As illustratedin Figures 4(b) and 4(c) the activities of SOD and GSH-PX were remarkably lower in the model group than in thecontrol group After SAMP8micewere treatedwith high- andmiddle-dosage fraction NB the activities of SOD and GSH-PX were significantly increased These results demonstratethat fraction NB has the ability to attenuate oxidative stressin the brains of SAMP8 mice

36 Effect of Fraction NB on A120573 Accumulation in SAMP8Mice Brains To determine whether fractionNB had an effecton A120573 production ELISA assay was used to measure thelevel of A120573 in the brains of SAMP8 mice after the mice wereintragastrically administrated with fraction NB As shown inFigure 4(a) the levels of A120573 in the control and fraction NBhigh- and middle-dosage groups were significantly lowerthan that in the model group indicating that fraction NBsuppresses the accumulation of A120573 in the brains of SAMP8mice

37 Effect of Fraction NB on APP and BACE1 mRNA Expres-sions Since A120573 is generated from APP through cleavages byBACE1 a rate-limiting enzyme of A120573 production [23] weexamined the effects of fraction NB on mRNA levels of APPandBACE1 in the brains of SAMP8mice As shown in Figures4(d) and 4(e) themRNA levels of APP andBACE1 in the con-trol group were significantly lower than in the model groupAfter SAMP8 mice were treated with high- and middle-dosage fraction NB APP mRNA expression was remarkablydecreased Meanwhile high-dosage fraction NB markedlysuppressed the BACE1 mRNA expression of SAMP8 miceThese results suggest that fraction NB treatment has thecapability to inhibit APP and BACE1 mRNA expressions


00

01

02

03

04

05

06

Fraction PEFraction AEFraction NB

OD

val

ues

Control 80402010Fractions (gmL)

lowast lowast

lowast

lowast

lowast lowast

lowast

lowastlowast

(a)

OD

val

ues

00

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10 804020Control

lowast lowast

Fraction NB (gmL)25ndash35

(b)

OD

val

ues

00

02

04

06

08

10 804020

lowastlowast

ControlFraction NB (gmL)

25ndash35

(c)

Figure 1 Effect of fraction NB on proliferation and A12057325ndash35 induced cytotoxicity in PC12 cells (a) Effects of the fractions isolated from Radix

Notoginseng on proliferation of PC12 cells (b) Cells were pretreated with fraction NB (10ndash80120583gmL) for 24 h before being incubated with10 120583M A120573

25ndash35 and OD values were measured using the MTT assay (c) Cells were with 10 120583M A12057325ndash35 for 24 h and then posttreated with

10ndash80120583gmL fraction NB for 72 h and OD values were measured using the MTT assay The data in different groups were expressed as themean plusmn SD from 3 experiments Statistical difference between groups was assessed by 119905-test using the SPSS 130 software 119875 lt 005 versusthe control group lowast119875 lt 005 lowastlowast119875 lt 001 versus A12057325ndash35-treated group

4 Discussion

Alzheimerrsquos disease (AD) the most prevalent cause ofdementia worldwide affects millions of people every yearUnfortunately up to now there is no effective treatment forthe disease Available therapies only offer small symptomaticrelief without preventing the progression of the disease andwith considerable side effects Chinese herbal medicine hasbeen used for AD treatment for thousands of years withmoreeffective and fewer side effectsTherefore developing effectivepotential candidates from Chinese herbal medicine againstAD would be considered as critical and efficient therapy forAD treatment In the present study we extracted fractionsPE AE and NB from Radix Notoginseng and evaluated their

effects on proliferation of PC12 cells using MTT assay Itwas observed that fraction NB had a pronounced ability topromote proliferation of PC12 cells (Figure 1(a)) Moreoverthe effects of fraction NB on A12057325ndash35-induced cytotoxicity inPC12 cells were examined Consistent with other reports weobserved that the viability of cells exposed to A120573

25ndash35 wasmarkedly inhibited when compared to the controls (Fig-ure 1(b)) Pretreatment or posttreatment with fraction NBsignificantly suppressed A120573

25ndash35-induced cytotoxicity (Fig-ure 1(c)) indicating that fraction NB holds the potential notonly to protect but also to rescue PC12 cells from A120573


The acute toxicity of fractionNBwas assessed using singledose acute toxicity assay approved by the State Food andDrug


18

20

22

24

26

28

30

32

34

ControlFraction NB

Day 1 Day 7 Day 14

Body

wei

ght (

g)

(a)

Relat

ive o

rgan

wei

ght

0

10

20

30

40

50

60

HeartLiver

SpleenKidney

Control Fraction NB

(b)

Figure 2 Assessment of acute toxicity of fraction NB using single dose acute assay (a) Body weights of fraction NB-treated and control miceMice were given fraction NB at a single dose of 1945 gkg Body weight of each mouse was recorded on days 1 7 and 14 after treatment (b)Relative organ weights of liver heart spleen and kidney On day 14 after treatment mice were ethically sacrificed and heart liver spleen andkidney were excised Relative organ weights of liver heart spleen and kidney were calculated Data in each experiment represent mean plusmn SDfrom 20 independent samples Statistically significant differences were calculated by one-way ANOVA using the SPSS 130 software

Administration of China After mice were intragastricallygiven a single dose of 1945 gkg a maximal dose due to thelimited solubility the body weights of fraction NB-treatedand control mice gained from 2006plusmn126 g and 2032plusmn113 gto 2849 plusmn 171 g and 2956 plusmn 190 g respectively and thechanges of body weight of fraction NB-treated group on days1 7 and 14 did not show significant difference compared tothe control group (Figure 2) Meanwhile the relative organweights of liver heart kidney and spleen in fraction NB-treated group showed no significant difference compared tothe control group (Figure 2) In addition since MTM ge 100 isadopted as the standard for the drug safety [18 19] the resultthat the maximal tolerant dose of mice is 972 times as highas the clinical daily dose of humans (MTM = 972) indicates alow toxicity of fraction NB Taken together the above resultssuggest that fraction NB has a mild toxicity in vivo

ADpatients present progressive decline in cognitive func-tion and personality impairment In the early stage of AD thepatients manifest short-term memory loss and in advancedstage the patients manifest confusion aggression moodswings long-term memory loss and social isolation [24] Inorder to assess the effect of fraction NB on spatial learningand memory function of SAMP8 all mice underwent MWMtest after 8 weeks of drug administration The results fromthe place navigation test revealed that the escape latency offractionNB-treatedmice was decreased while percentages oftime in the target quadrant in fraction NB-treated groupswere remarkably increased when compared to the modelgroup (Figure 3) The findings of the probe trial showedthat treatment with fraction NB significantly increased thepercentage of quadrant time (Figure 3)These results indicate

that fraction NB possesses the ability to improve cognitivedysfunction in SAMP8 mice

It has been established that oxidative stress plays impor-tant role in early AD stage [25] Oxidative imbalance andresultant neuronal damage may promote neuronal degen-eration and death resulting in cognitive deficits [25] Theimpaired antioxidant enzymes such as SOD and GSH-PXwere found in postmortem brain tissue of AD patients [26]In the study the activities of SOD and GSH-PX in thebrains of SAMP8 mice were examined the results of whichshowed that a significant decrease in the activities of SOD andGSH-PX was found in the brains of SAMP8 mice whencompared to the control SAMR1 mice (Figures 4(b) and4(c)) These results are consistent with the trend of the datapublished in literatures [27] Importantly fraction NB treat-ment remarkably increased the activities of SOD and GSH-PX in the brains of SAMP8 mice demonstrating an antioxi-dant capacity of fraction NB

It is established that APP can be cleaved by 120573- and 120574-secretases leading to the generation of A120573 and BACE1 is the120573-secretase responsible for A120573 production [28] Overpro-duction of A120573 in the brains of AD patients triggers thecomplicated pathological changes ultimately leading to cog-nitive dysfunction in AD [29] Our study demonstrates thatfraction NB could significantly reduce the production of A120573by inhibiting mRNA expressions of APP and BACE1 (Figures4(a) 4(d) and 4(e)) indicating that inhibition of APP andBACE1 mRNA expressions may be one of the mechanisms offraction NB precluding A120573 generation However whetherfractionNBhas effects on 120574-secretase needs to be investigatedin further study


Esca

pe la

tenc

y (s

)

Con

trol

Mod

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Hig

h-do

se

Mid

dle-

dose

Low

-dos

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Hup

erzi

ne A

0

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lowastlowastlowastlowast lowastlowast

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(a)

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f qua

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(b)

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f qua

dran

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Con

trol

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Hig

h-do

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dle-

dose

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-dos

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Hup

erzi

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lowastlowast

lowast

(c)

Figure 3 Effect of fraction NB on learning and memory abilities of SAMP8 in the MWM test After treatment with different concentrationsof fraction NB the learning and memory abilities of SAMP8 mice were detected using MWM ((a) and (b)) The average escape latency andpercentage of time in the target quadrant of four-day training respectively (c) The percentage of time spent in the target quadrant of eachmouse on day 5 Data in each experiment represent mean plusmn SD from 10ndash12 independent samples Statistically significant differences werecalculated by one-way ANOVA using the SPSS 130 software 119875 lt 005 119875 lt 001 versus the control group lowast119875 lt 005 lowastlowast119875 lt 001 versusthe model group

There are reports showing that oxidative stress con-tributes to A120573 generation either by downregulating the low-density lipoprotein receptor-related protein 1 (LRP-1) andupregulating the receptor for advanced glycation end prod-ucts (RAGE) or by increasing the activity of 120573- and 120574-secretases [30] A120573 in return activate free radical genera-tion by the nicotinamide adenine dinucleotide phosphate(NADPH) oxidase complex leading to increased productionof ROS [31] Overproduction of ROS and abnormal A120573 depo-sition in the brains of AD patients ultimately cause neuronalapoptosis giving rise to cognitive impairment [2] There-fore pharmacologic strategies designed to suppress bothoxidative stress and A120573 generation may be more beneficial

to the prevention and treatment of AD The findings thatfraction NB could attenuate oxidative stress and reduceA120573 deposition thereby improving cognitive dysfunction ofSAMP8 mice demonstrated that fraction NB may be apromising candidate for AD treatment

5 Conclusion

Our results reported here demonstrate that fraction NB hasa prominent ability to promote proliferation and protectA12057325ndash35-induced neurotoxicity in PC12 cells and alleviates

cognitive impairment in SAMP8 mice through attenuatingoxidative stress and A120573 accumulation by inhibiting APP and


A

(ng

mL)

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(Um

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(Um

g pr

ot)

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(d)

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BACE

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RNA

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lACT

B

00

10

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14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)

Figure 4 (a) Effect of fraction NB on A120573 accumulation in SAMP8 mice brains After treatment with fraction NB the level of A120573 in thebrains of SAMP8 mice was detected using ELISA assay ((b) and (c)) Effect of fraction NB on SOD and GSH-PX activities in the brains ofSAMP8 mice After treatment with fraction NB the activities of SOD and GSH-PX were measured using activity detection kit ((d) and (e))Effect of fraction NB on APP and BACE1 mRNA expressions Data in each experiment represent mean plusmn SD from 4-5 independent samplesStatistically significant differences were calculated by one-way ANOVA using the SPSS 130 software 119875 lt 005 119875 lt 001 versus the controlgroup lowast119875 lt 005 lowastlowast119875 lt 001 versus the model group


BACE1 expressions It is noteworthy that high dose of fractionNB has shown almost equally positive outcomes as comparedto Hup A which may result from the similar mechanismof action of fraction NB to Hup A [13 14 32] Thus thepronounced actions of fraction NB against AD and its lowtoxicity in vivo suggest that fraction NB may be a usefulalternative to AD treatment It should be noted however thatthe treatment of fraction NB was initiated in 3-month-oldSAMP8 mice prior to the onset of AD-like pathology andfunctional impairments Consequently whether fraction NBcould relieve impairments after the onset of AD symptomsandwhether fractionNB candelay the onset or has preventiveeffects on AD should be investigated in further study

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Jin-Lan Huang Ying-Qin Feng and Li-Ru Bai contributedequally to this project

Acknowledgments

This project was supported by the National Natural ScienceFoundation of China under Grants 81460598 and 81660644Initializing Fund of XuzhouMedical University under GrantsD2014017 and D2014010 Opening Foundation of JiangsuKey Laboratory of Anesthesiology under Grant KJS1404and Director Fund of Jiangsu Key Laboratory of New DrugResearch and Clinical Pharmacy under Grant ZR-XY201402

References

[1] J Cui X Wang X Li et al ldquoTargeting the 120574-120573-secretaseinteraction reduces 120573-amyloid generation and amelioratesAlzheimerrsquos disease-related pathogenesisrdquo Cell Discovery vol 1p 15021 2015

[2] Z Cai B Zhao and A Ratka ldquoOxidative stress and beta-amyloid protein in Alzheimerrsquos diseaserdquo NeuroMolecularMedicine vol 13 no 4 pp 223ndash250 2011

[3] M E S Ferreira A S de Vasconcelos T da Costa Vilhena et alldquoOxidative stress in Alzheimerrsquos disease should we keep tryingantioxidant therapiesrdquo Cellular and Molecular Neurobiologyvol 3 no 5 pp 595ndash614 2015

[4] T Wang R Guo G Zhou et al ldquoTraditional uses botanyphytochemistry pharmacology and toxicology of Panax noto-ginseng (Burk) F H Chen a reviewrdquo Journal of Ethnopharma-cology vol 188 pp 234ndash258 2016

[5] C Z Wang J T Xie A Fishbein et al ldquoAntiproliferative effectsof different plant parts of Panax notoginseng on SW480 humancolorectal cancer cellsrdquo Phytotherapy Research vol 23 no 1 pp6ndash13 2009

[6] Y-J Jang M-E Kim and S-Y Ko ldquoN-Butanol extracts ofPanax notoginseng suppress LPS-induced MMP-2 expressionin periodontal ligament fibroblasts and inhibit osteoclastogen-esis by suppressing MAPK in LPS-activated RAW2647 cellsrdquoArchives of Oral Biology vol 56 no 11 pp 1319ndash1327 2011

[7] S Chang Y Choi J Park et al ldquoAnti-inflammatory effects ofBT-201 an n-butanol extract of Panax notoginseng observedin vitro and in a collagen-induced arthritis modelrdquo ClinicalNutrition vol 26 no 6 pp 785ndash791 2007

[8] R M Lu Z Q Huang B Li J H Wei and Z G ZhongldquoChemical constituents of radix notogisengrdquo Chinese Journal ofExperimental Traditional Medical Formulae pp 62ndash64 2016

[9] J Liu J He L Huang L Dou S Wu and Q L YuanldquoNeuroprotective effects of ginsenoside Rb1 on hippocampalneuronal injury and neurite outgrowthrdquo Neural RegenerationResearch vol 9 no 9 pp 943ndash950 2014

[10] Y Zhou H Q Li and L Lu ldquoGinsenoside Rg1 provides neu-roprotection against blood brain barrier disruption and neuro-logical injury in a rat model of cerebral ischemiareperfusionthrough downregulation of aquaporin 4 expressionrdquo Phy-tomedicine vol 21 no 7 pp 998ndash1003 2014

[11] X Meng G Sun J Ye H Xu H Wang and X Sun ldquoNoto-ginsenoside R1-mediated neuroprotection involves estrogenreceptor-dependent crosstalk between Akt and ERK12 path-ways A novel mechanism of Nrf2ARE signaling activationrdquoFree Radical Research vol 48 no 4 pp 445ndash460 2014

[12] I Akiguchi M Pallas H Budka et al ldquoSAMP8 mice as a neu-ropathological model of accelerated brain aging and dementiaToshio Takedarsquos legacy and future directionsrdquo Neuropathology2017

[13] H-Y Zhang ldquoNew insights into huperzine A for the treatmentof Alzheimerrsquos diseaserdquo Acta Pharmacologica Sinica vol 33 no9 pp 1170ndash1175 2012

[14] G Yang Y Wang J Tian and J-P Liu ldquoHuperzine A forAlzheimerrsquos disease a systematic review and meta-analysis ofrandomized clinical trialsrdquo PLoS ONE vol 8 no 9 Article IDe74916 2013

[15] J-L Huang X Jing X Tian et al ldquoNeuroprotective propertiesof panax notoginseng saponins via preventing oxidative stressinjury in SAMP8 micerdquo Evidence-based Complementary andAlternative Medicine vol 2017 Article ID 8713561 7 pages 2017

[16] D-P Wu T-Y Lin J-Y Lv et al ldquoCestrum nocturnumflower extracts attenuate proliferation and induce apoptosis inmalignant cells through inducing DNA damage and inhibitingtopoisomerase II activityrdquo Evidence-based Complementary andAlternativeMedicine vol 2017 Article ID 1456786 8 pages 2017

[17] L Xia L X Wang and J Z Mou ldquoToxic effects of radixpolygoni multifori praeparata and fructus tribuli in micerdquo inEvaluation and Analysis of Drug-Use in Hosptials of China pp34-35 2010

[18] J Pan H M Chui Y Yu and W S Li ldquoExperimental study ofacute toxicity and hypoxia tolerance of Jushenrdquo Pharmacy andClinics of Chinese Materia Medica pp 26-27 2014

[19] X L Jing G H Jiang J Zhang M F Zhu Q Chen Y Lianet al ldquoStudies of acute toxicity and anti-inflammatory actionof herba Xanthiirdquo Journal of Chengdu University of TraditionalChinese Medicine pp 34-35 2015

[20] R Morris ldquoDevelopments of a water-maze procedure forstudying spatial learning in the ratrdquo Journal of NeuroscienceMethods vol 11 no 1 pp 47ndash60 1984

[21] H Y Ahn Y J Chung B S Kim et al ldquoClinical significanceof the BRAF V600E mutation in multifocal papillary thyroidcarcinoma in Koreardquo Surgery vol 155 no 4 pp 689ndash695 2014

[22] Y-H Wang and G-H Du ldquoGinsenoside Rg1 inhibits 120573-secretase activity in vitro and protects against A120573-inducedcytotoxicity in PC12 cellsrdquo Journal of Asian Natural ProductsResearch vol 11 no 7 pp 604ndash612 2009


[23] M E Kennedy A W Stamford X Chen et al ldquoThe BACE1inhibitor verubecestat (MK-8931) reduces CNS b-Amyloid inanimal models and in Alzheimerrsquos disease patientsrdquo ScienceTranslationalMedicine vol 8 no 363 Article ID 363ra150 2016

[24] J-Q Li L Tan H-F Wang et al ldquoRisk factors for predictingprogression from mild cognitive impairment to Alzheimerrsquosdisease a systematic review and meta-analysis of cohort stud-iesrdquo Journal of Neurology Neurosurgery and Psychiatry vol 87no 5 pp 476ndash484 2016

[25] Z Chen andC Zhong ldquoOxidative stress inAlzheimerrsquos diseaserdquoNeuroscience Bulletin vol 30 no 2 pp 271ndash281 2014

[26] R A Omar Y-J Chyan A C Andorn B Poeggeler N KRobakis andMA Pappolla ldquoIncreased expression but reducedactivity of antioxidant enzymes in Alzheimerrsquos diseaserdquo Journalof Alzheimerrsquos Disease vol 1 no 3 pp 139ndash145 1999

[27] D L Marcus CThomas C Rodriguez et al ldquoIncreased perox-idation and reduced antioxidant enzyme activity in AlzheimerrsquosdiseaserdquoExperimentalNeurology vol 150 no 1 pp 40ndash44 1998

[28] A Kimura S Hata and T Suzuki ldquoAlternative selection of siteAPP-cleaving enzyme 1 (BACE1) cleavage sites in amyloid 120573-protein precursor (APP) harboring protective and pathogenicmutations within the A120573 sequencerdquo Journal of Biological Chem-istry vol 291 no 46 pp 24041ndash24053 2016

[29] J Delrieu P J Ousset T Voisin and B Vellas ldquoAmyloid betapeptide immunotherapy in Alzheimer diseaserdquo Revue Neu-rologique vol 170 no 12 pp 739ndash748 2014

[30] V Srikanth A Maczurek T Phan et al ldquoAdvanced glycationendproducts and their receptor RAGE in Alzheimerrsquos diseaserdquoNeurobiology of Aging vol 32 no 5 pp 763ndash777 2011

[31] S I Mota R O Costa I L Ferreira et al ldquoOxidative stressinvolving changes in Nrf2 and ER stress in early stages ofAlzheimerrsquos diseaserdquo Biochimica et Biophysica Acta - MolecularBasis of Disease vol 1852 no 7 pp 1428ndash1441 2015

[32] Q Shi J Fu D Ge et al ldquoHuperzine A ameliorates cognitivedeficits and oxidative stress in the hippocampus of rats exposedto acute hypobaric hypoxiardquo Neurochemical Research vol 37no 9 pp 2042ndash2052 2012

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Antioxidant treatments in the early stage of AD pathogenesiswere able to alleviate the cognitive function impairment andreduce A120573 accumulation in AD [3] Accordingly identifica-tion of potential protective candidates to attenuate oxidativestress and eliminate excessive accumulation of A120573 and ulti-mately improve cognitive function impairment is consideredas an important strategy for AD treatment

Radix Notoginseng a widely used traditional Chinesemedicine known as Sanqi or Tianqi in China is the rootof Panax notoginseng (Burk) F H Chen belonging to thegenus Panax family Araliaceae Studies have reported thatRadix Notoginseng has plenty of pharmacological proper-ties including antioxidant antiatherosclerotic antitumourand haemostatic activities [4] Recent studies have demon-strated that fraction n-butanol (NB) of Radix Notoginsenghas antiproliferative effects on human colorectal cancercells [5] inhibitory effects on osteoclastogenesis in LPS-activated RAW2647 cells [6] and anti-inflammatory effectson collagen-induced arthritic mice [7] However it is unclearwhether fraction NB has antagonistic effect on AD

We and others reported that fraction NB extracted fromRadix Notoginseng contained a large proportion of ginseno-side Rb1 Rg1 and notoginsenoside R1 [5 8] It has beenestablished that ginsenoside Rb1 Rg1 and notoginsenosideR1 have abilities to suppress oxidative stress and attenuateA120573-induced neuronal damage [9ndash11] Thus it is reasonableto hypothesize that fraction NB might have an effect againstAD

Therefore this study was designed to evaluate the neu-ronal protective effect of fraction NB extracted from RadixNotoginseng on A120573

25ndash35-induced PC12 cells Moreover 3-month-old senescence-accelerated prone8 (SAMP8) micewere intragastrically given fraction NB for 8 consecutiveweeks to explore the effect of the tested fraction on spatiallearning and memory and characterize the impacts of frac-tion NB on antioxidant enzymes A120573 production and APPandBACE1 expressions thereby providing a new opportunityfor research in regard to the pharmaceutical prevention andtreatment of AD

2 Material and Methods

21 Chemicals Radix Notoginseng were collected in Yulincity Guangxi Zhuang Autonomous Region China and ver-ified by Professor Wei songji from Guangxi University ofChinese Medicine The extract condition of fraction NB wasperformed according to our previous study [8] Briefly air-dried and powdered Radix Notoginseng roots (45 kg) wereconducted with 95 and 60 ethanol (each 4000mL) bypercolating at room temperature for a week to give totalethanol extract (1400 g) The extract was dissolved in waterand then extracted with petroleum ether (PE) acetic ether(AE) and then water-saturated n-butanol (NB) to obtainfractions PE (244 g) AE (2130 g) and NB (6000 g)

22 Reagents Methyl thiazolyl tetrazolium (MTT) wasobtained from Amresco (Ohio USA) Huperzine A (HupA) was from Zhejiang Zhenyuan Pharmaceutical Co Ltd

(Zhejiang China) The SOD and GSH-PX activity detectionkits were purchased from Nanjing Jiancheng BioengineeringInstitute (Nanjing China) TIANScript RT Kit was fromTiangen Biotech Co Ltd (Beijing China) A120573

25ndash35 was fromGibco (NewYork USA) All other reagents were from Sigma-Aldrich unless stated otherwise

23 Cell Culture PC12 cell line purchased from ShanghaiBiological Institute was suspended in RMPI-1640 (GibcoUSA) containing 5 fetal bovine serum (FBS) (HycloneUSA) 10 horse serum (Gibco USA) 100 120583gmL strepto-mycin and 100UmL penicillin in 5 CO

2at 37∘C

24 Animals Drug Treatment and Tissue Preparation Ithas been established that increased oxidative stress and A120573immunoreactive granular structures were found in SAMP8mice as young as 2 months of age Remarkable memorydeficits apparent reduction in the activity of antioxidantenzymes such as superoxide dismutase (SOD) and glu-tathione peroxidase (GSH-PX) and increased amyloidplaque deposition were found in 5-month-old SAMP8 mice[12]Thus sixty 3-month-old SAMP8 and twelve senescence-accelerated-resistant (SAMR1) (3 months old control strainof SAMP8) pathogen- and virus-free mice were obtainedfrom Tianjin University of Traditional Chinese Medicine(Tianjin China) SAMP8 mice were randomly divided intofive groups model group fraction NB high-dosage fractionNB middle-dosage and fraction NB low-dosage groups andHup A group Since Hup A has been widely used for thetreatment of AD owing to its roles in inhibiting acetyl-cholinesterase (AChE) activity reducing A120573 accumulationalleviating oxidative stress and improving cognitive function[13 14] thusHupAwas considered as a positive control in thestudy SAMR1mice were considered as the control groupThehigh- middle- and low-dosage groups were intragastricallygiven 300 150 and 75mgkg of fraction NB respectively perday while Hup A group was treated with 03mgkg Hup A bygavage every day for 2 months The middle dose of fractionNB was confirmed according to the dose of Radix Noto-ginseng used in clinics and the dose ofHupAwas determinedon the basis of our previous study [15] The same volume ofdistilled water was given to the model and control groupsAfter examination of learning and memory the mice wereethically sacrificed and the brains were excised for ELISAassay activity detection and real-time PCR assay Animalcare and experimental procedures were implemented accord-ing to the document ldquoGuidance Suggestions for Caring forLaboratory Animalsrdquo produced by the Ministry of Scienceand Technology of China in 2006

25 MTT Assay PC12 cells were seeded into 96-well platesat a density of 104 cellsmL for 24 h For the detection of cellproliferation cells were treated with fractions PE AE andNB at the concentrations of 10 120583gmL 20120583gmL 40 120583gmLand 80120583gmL for 72 h respectively To evaluate the protectiveeffect of fraction NB the cells were treated with fraction NBat the concentrations of 10 120583gmL 20120583gmL 40 120583gmL and80 120583gmL for 24 h before exposure to 10 120583M A120573

25ndash35 and






MTM





(1)






CT119861gene

[15 21]


3 Results



















00

01

02

03

04

05

06


OD

val

ues


lowast lowast

lowast

lowast

lowast lowast

lowast

lowastlowast

(a)

OD

val

ues

00

01

02

03

04

05

06

07

10 804020Control

lowast lowast


(b)

OD

val

ues

00

02

04

06

08

10 804020

lowastlowast


25ndash35

(c)





4 Discussion








18

20

22

24

26

28

30

32

34

ControlFraction NB

Day 1 Day 7 Day 14

Body

wei

ght (

g)

(a)

Relat

ive o

rgan

wei

ght

0

10

20

30

40

50

60

HeartLiver

SpleenKidney

Control Fraction NB

(b)








Esca

pe la

tenc

y (s

)

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

0

10

20

30

40

50

60


lowastlowast

(a)

Perc

enta

ge o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


lowastlowast

(b)

Per

cent

age o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowast

(c)




5 Conclusion




A

(ng

mL)

00

02

04

06

08

10

12

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowastlowast

(a)

SOD

(Um

g pr

ot)

0

5

10

15

20

25

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(b)

GSH

-PX

(Um

g pr

ot)

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(c)

02

04

06

08

APP

mRN

A le

velA

CTB

00

10

12

14

16

18

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowast

lowast lowastlowast

(d)

02

04

06

08

BACE

1 m

RNA

leve

lACT

B

00

10

12

14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















MTM





(1)






CT119861gene

[15 21]


3 Results



















00

01

02

03

04

05

06


OD

val

ues


lowast lowast

lowast

lowast

lowast lowast

lowast

lowastlowast

(a)

OD

val

ues

00

01

02

03

04

05

06

07

10 804020Control

lowast lowast


(b)

OD

val

ues

00

02

04

06

08

10 804020

lowastlowast


25ndash35

(c)





4 Discussion








18

20

22

24

26

28

30

32

34

ControlFraction NB

Day 1 Day 7 Day 14

Body

wei

ght (

g)

(a)

Relat

ive o

rgan

wei

ght

0

10

20

30

40

50

60

HeartLiver

SpleenKidney

Control Fraction NB

(b)








Esca

pe la

tenc

y (s

)

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

0

10

20

30

40

50

60


lowastlowast

(a)

Perc

enta

ge o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


lowastlowast

(b)

Per

cent

age o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowast

(c)




5 Conclusion




A

(ng

mL)

00

02

04

06

08

10

12

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowastlowast

(a)

SOD

(Um

g pr

ot)

0

5

10

15

20

25

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(b)

GSH

-PX

(Um

g pr

ot)

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(c)

02

04

06

08

APP

mRN

A le

velA

CTB

00

10

12

14

16

18

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowast

lowast lowastlowast

(d)

02

04

06

08

BACE

1 m

RNA

leve

lACT

B

00

10

12

14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































00

01

02

03

04

05

06


OD

val

ues


lowast lowast

lowast

lowast

lowast lowast

lowast

lowastlowast

(a)

OD

val

ues

00

01

02

03

04

05

06

07

10 804020Control

lowast lowast


(b)

OD

val

ues

00

02

04

06

08

10 804020

lowastlowast


25ndash35

(c)





4 Discussion








18

20

22

24

26

28

30

32

34

ControlFraction NB

Day 1 Day 7 Day 14

Body

wei

ght (

g)

(a)

Relat

ive o

rgan

wei

ght

0

10

20

30

40

50

60

HeartLiver

SpleenKidney

Control Fraction NB

(b)








Esca

pe la

tenc

y (s

)

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

0

10

20

30

40

50

60


lowastlowast

(a)

Perc

enta

ge o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


lowastlowast

(b)

Per

cent

age o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowast

(c)




5 Conclusion




A

(ng

mL)

00

02

04

06

08

10

12

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowastlowast

(a)

SOD

(Um

g pr

ot)

0

5

10

15

20

25

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(b)

GSH

-PX

(Um

g pr

ot)

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(c)

02

04

06

08

APP

mRN

A le

velA

CTB

00

10

12

14

16

18

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowast

lowast lowastlowast

(d)

02

04

06

08

BACE

1 m

RNA

leve

lACT

B

00

10

12

14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

01

02

03

04

05

06


OD

val

ues


lowast lowast

lowast

lowast

lowast lowast

lowast

lowastlowast

(a)

OD

val

ues

00

01

02

03

04

05

06

07

10 804020Control

lowast lowast


(b)

OD

val

ues

00

02

04

06

08

10 804020

lowastlowast


25ndash35

(c)





4 Discussion








18

20

22

24

26

28

30

32

34

ControlFraction NB

Day 1 Day 7 Day 14

Body

wei

ght (

g)

(a)

Relat

ive o

rgan

wei

ght

0

10

20

30

40

50

60

HeartLiver

SpleenKidney

Control Fraction NB

(b)








Esca

pe la

tenc

y (s

)

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

0

10

20

30

40

50

60


lowastlowast

(a)

Perc

enta

ge o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


lowastlowast

(b)

Per

cent

age o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowast

(c)




5 Conclusion




A

(ng

mL)

00

02

04

06

08

10

12

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowastlowast

(a)

SOD

(Um

g pr

ot)

0

5

10

15

20

25

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(b)

GSH

-PX

(Um

g pr

ot)

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(c)

02

04

06

08

APP

mRN

A le

velA

CTB

00

10

12

14

16

18

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowast

lowast lowastlowast

(d)

02

04

06

08

BACE

1 m

RNA

leve

lACT

B

00

10

12

14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















18

20

22

24

26

28

30

32

34

ControlFraction NB

Day 1 Day 7 Day 14

Body

wei

ght (

g)

(a)

Relat

ive o

rgan

wei

ght

0

10

20

30

40

50

60

HeartLiver

SpleenKidney

Control Fraction NB

(b)








Esca

pe la

tenc

y (s

)

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

0

10

20

30

40

50

60


lowastlowast

(a)

Perc

enta

ge o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


lowastlowast

(b)

Per

cent

age o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowast

(c)




5 Conclusion




A

(ng

mL)

00

02

04

06

08

10

12

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowastlowast

(a)

SOD

(Um

g pr

ot)

0

5

10

15

20

25

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(b)

GSH

-PX

(Um

g pr

ot)

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(c)

02

04

06

08

APP

mRN

A le

velA

CTB

00

10

12

14

16

18

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowast

lowast lowastlowast

(d)

02

04

06

08

BACE

1 m

RNA

leve

lACT

B

00

10

12

14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Esca

pe la

tenc

y (s

)

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

0

10

20

30

40

50

60


lowastlowast

(a)

Perc

enta

ge o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


lowastlowast

(b)

Per

cent

age o

f qua

dran

t tim

e

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowast

(c)




5 Conclusion




A

(ng

mL)

00

02

04

06

08

10

12

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowastlowast

(a)

SOD

(Um

g pr

ot)

0

5

10

15

20

25

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(b)

GSH

-PX

(Um

g pr

ot)

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(c)

02

04

06

08

APP

mRN

A le

velA

CTB

00

10

12

14

16

18

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowast

lowast lowastlowast

(d)

02

04

06

08

BACE

1 m

RNA

leve

lACT

B

00

10

12

14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















A

(ng

mL)

00

02

04

06

08

10

12

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

lowastlowast

(a)

SOD

(Um

g pr

ot)

0

5

10

15

20

25

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(b)

GSH

-PX

(Um

g pr

ot)

0

10

20

30

40

50

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A


(c)

02

04

06

08

APP

mRN

A le

velA

CTB

00

10

12

14

16

18

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowast

lowast lowastlowast

(d)

02

04

06

08

BACE

1 m

RNA

leve

lACT

B

00

10

12

14

Con

trol

Mod

el

Hig

h-do

se

Mid

dle-

dose

Low

-dos

e

Hup

erzi

ne A

lowastlowast

(e)








Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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ResearchArticle - pdfs.semanticscholar.org€¦ · Radix Notoginseng, a widely used traditional Chinese medicine known as Sanqi or Tianqi in China, is the root of Panax notoginseng