Research ArticleAntioxidant and Anti-Inflammatory Activity Determination ofOne Hundred Kinds of Pure Chemical Compounds Using Offlineand Online Screening HPLC Assay

Kwang Jin Lee You Chang Oh Won Kyung Cho and Jin Yeul Ma

Korean Institute of Oriental Medicine (KIOM) KMApplication Center 70 Cheomdan-ro Dong-gu Daegu 701-300 Republic of Korea

Correspondence should be addressed to Jin Yeul Ma jymakiomrekr

Received 21 April 2015 Revised 3 July 2015 Accepted 30 July 2015

Academic Editor Abbas A Mahdi

Copyright copy 2015 Kwang Jin Lee 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

This study investigated the antioxidant activity of one hundred kinds of pure chemical compounds foundwithin a number of naturalsubstances and oriental medicinal herbs (OMH) Three different methods were used to evaluate the antioxidant activity of DPPHradical-scavenging activity ABTS radical-scavenging activity and online screeningHPLC-ABTS assaysThe results indicated that 17compounds exhibited better inhibitory activity against ABTS radical thanDPPH radicalThe IC

50rate of amore practical substance

is determined and the ABTS assay IC50values of gallic acid hydrate (+)-catechin hydrate caffeic acid rutin hydrate hyperoside

quercetin and kaempferol compounds were 103 plusmn 025 312 plusmn 051 159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015120583gmL respectivelyThe ABTS assay is more sensitive to identifying the antioxidant activity since it has faster reaction kineticsand a heightened response to antioxidants In addition there was a very small margin of error between the results of the offline-ABTS assay and those of the online screeningHPLC-ABTS assayWe also evaluated the effects of 17 compounds on theNO secretionin LPS-stimulated RAW 2647 cells and also investigated the cytotoxicity of 17 compounds using a cell counting kit (CCK) in orderto determine the optimal concentration that would provide an effective anti-inflammatory action with minimum toxicity Theseresults will be compiled into a database and this method can be a powerful preselection tool for compounds intended to be studiedfor their potential bioactivity and antioxidant activity related to their radical-scavenging capacity

1 Introduction

Natural substances and oriental medicinal herbs (OMH)have been traditionally administered to treat or preventvarious diseases in Asia including Korea China and Japan[1] Generally OMH have very effective anticancer anti-inflammatory and antivirus properties [2] and researchershave reported that these natural substances also exhibitantioxidant activity In addition their long historical clinicalpractice and reliable therapeutic efficacymake them excellentsources to discover natural bioactive compounds [3] OMHhave received extensive attention for their use as drugsfunctional foods and cosmetic materials [1 4] An extractionsolvent composed of water and ethanol is commonly used toextract the bioactive compounds fromOMHwith subsequentboiling and distillation to obtain useful components [5]The chemical constituents of OMH have been shown to

be composed of natural products including triterpenessteroids alkaloids flavonoids and polysaccharides [3 6]During our investigation on the potential antioxidant activityof the commonly known phytochemical one hundred kindsof pure compounds were identified Reactive oxygen species(ROS) which originate from oxygen are naturally producedby some enzymes as part of the metabolism within thecytoplasm [7 8] However excess ROS have a fatal effect onoxygen toxicity and cellular dysfunction In addition excessROS have also been linked to maladies such as cancer strokeParkinsonrsquos disease heart disease arteriosclerosis infectionageing and autoimmune disease [8 9] Many studies havebeen carried out on the antioxidant activity that eliminatesROS to obtain more conclusive information [10 11] andOMH have been reported to contain these kinds of antiox-idants ABTS DPPH and lipid peroxidation inhibition Thecorresponding target compounds were used to identify the

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 165457 13 pageshttpdxdoiorg1011552015165457

2 Evidence-Based Complementary and Alternative Medicine

antioxidant activity especially using DPPH or ABTS radicaltechnique [12] Recently sensitive online HPLC methods(online HPLC-DPPH and online HPLC-ABTS assays) havebeen developed to analyse free radical-scavenging activity[5 13] An online systemhas been introduced to rapidly deter-mine the antioxidant activity of each component in the givencompounds and online screening HPLC postcolumn assayinvolving DPPH or ABTS radical techniques has been devel-oped to provide a new analysis screening technology methodwith which the bioactive compounds can be spectrophoto-metrically monitored due to the decrease in absorbance at515 or 734 nm [14]This newmethod was successfully appliedto screen and identify the natural bioactivity of complexmixtures especially for OMH [15] In this study we conductDPPH and ABTS assays to screen for the antioxidant activityof one hundred kinds of pure chemical compounds so theIC50

rate of a more practical substance is determined Wealso evaluated the cytotoxicity of 17 compounds including(1) (+)-catechin hydrate (2) calycosin (3) caffeic acid (4)curcumin (5) eugenol (6) ferulic acid (7) gallic acid hydrate(8) hyperoside (9) kaempferol (10) magnolol (11) quercetin(12) quercetin 3-beta-D-glucoside (13) quercitrin hydrate(14) rutin hydrate (15) sinapic acid (16) vanillylacetone and(17) L-(+)-ascorbic acid by using a CCK assay to determinethe optimal concentration that would be effective for anti-inflammatory activity with a minimum toxicity [9 16] Inaddition the results of an online HPLC-ABTS assay of someof the compoundswere compared and analysed to find amorepractical approach toward the use of online screening HPLC-ABTS assays to quickly pinpoint peaks in chromatograms thatcorrespond to bioactive compounds

2 Experimental

21 Reagents and Materials One hundred kinds of purechemical compounds were purchased from KFDA (Korea)Daejung (Korea) Sigma (USA) Chem Faces (China) TCI(Japan) ChromaDex (USA) Fluka (USA) Wako (Japan)GlycoSyn (New Zealand USA) Santa Cruz Biotech (USA)China Lang Chem Inc (China) and RD Chemical (USA)The following reagents were used for radical-scavengingassays ABTS (221015840-azino-bis(3-ethylbenzothiazoline-6-sul-fonic acid)) DPPH (22-diphenyl-1-picrylhydrazyl) potas-sium persulfate and trifluoroacetic acid (TFA) were pur-chased from Sigma (USA) The HPLC-grade methanol andacetonitrile were purchased from J T Baker (USA)The tripledistilled water was filtered with a 02120583m membrane filter(Advantec Tokyo Japan) before analysis Materials for cellculture were obtained from Lonza (Basel Switzerland) LPSbovine serum albumin (BSA) and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) were purchasedfrom Sigma (St Louis MO USA) Antibodies for ELISAwere obtained from eBioscience (San Diego CA USA) Thechemical structures of potentially selected compounds areshown in Figure 1

22 Standard Sample Preparation The high purity standardsample (higher than gt95) was prepared by dissolving

2mg of each standard chemical in 20mL of methanol andadjusting the concentration to 100 120583gmL

23 Offline DPPH Assay for Antioxidant Activity EvaluationThe DPPH radical cation method [17] was modified to eval-uate the free radical-scavenging effect of one hundred purechemical compounds The DPPH reagent was DPPH (8mg)dissolved in MeOH (100mL) for a solution concentrationof 80 120583LmL To determine the scavenging activity 100 120583LDPPH reagent was mixed with 100120583L of sample in a 96-well microplate and was incubated at room temperaturefor 30min After incubation the absorbance was measured514 nm using an ELISA reader (TECAN Groding Austria)and 100 methanol was used as a control The DPPHscavenging effect was measured using the following formula

Radical scavenging ()

= [

(119860)control minus (119860)sample

(119860)control]times 100

(1)

The IC50DPPH values (the concentration of sample required

for inhibition of 50 of DPPH radicals) were obtainedthrough extrapolation from regression analysis The antiox-idant was evaluated based on this IC

50value

24 Offline-ABTS Assay for Antioxidant Activity EvaluationThe ABTS radical cation method [17] was modified toevaluate the free radical-scavenging effect of one hundredpure chemical compounds The ABTS reagent was preparedby mixing 5mL of 7mM ABTS with 88120583L of 140mMpotassium persulfate The mixture was then kept in the darkat room temperature for 16 h to allow free radical generationand was then diluted with water (1 44 vv) To determinethe scavenging activity 100 120583L ABTS reagent was mixedwith 100 120583L of sample in a 96-well microplate and wasincubated at room temperature for 6min After incubationthe absorbance was measured 734 nm using an ELISA reader(TECAN Groding Austria) and 100methanol was used asa control The ABTS scavenging effect was measured usingthe following formula

Radical scavenging ()

= [

(119860)control minus (119860)sample

(119860)control]times 100

(2)

The IC50ABTS values (the concentration of sample required

for inhibition of 50 of ABTS radicals) were obtainedthrough extrapolation from regression analysis The antiox-idant activity was evaluated based on this IC

50value

25 Online Screening HPLC-ABTS Analysis The onlineradical-scavenging activity of one hundred kinds of purestandard compounds was determined using the ABTS assaymodifying the methods used by Stewart et al [18] A 2mMABTS stock solution containing 35mMpotassium persulfatewas prepared and was kept in the dark at room temperaturefor 16 h to allow the completion of radical generation and

Evidence-Based Complementary and Alternative Medicine 3

MW 17012Gallic acid hydrate

MW 302236Quercetin

MW 18016Caffeic acid

MW 28624Kaempferol

MW 46438Hyperoside

MW 61052Rutin hydrate

OHO

HO O

OH

OH

O

OH

OH

HO

O

OH

HO

HOCOOH

OHO

HO

OH

OH

OH

O

OHO

HO

OH

OH

O

C9H8O4 C15H10O7 C7H6O5 C15H10O6

C15H14O6 C21H20O12 C27H30O16

MW 29027(+)-Catechin hydrate

O

OH

OH

O

OH

OH

O

OH

OH

HO

OO

OHHO

HOO

H3C middot3H2O

OHO

HO

OH

OH

OH middotXH2O

OH

OH

HO

HOOC

middotH2O

Figure 1 Chemical structure of the superior antioxidant activity compounds

Regents

Sample injection

ColumnPump

DADdetector

Pump

Passive splitReaction loop

detectorVWD

Regents

Figure 2 Schematic of online screening HPLC-ABTS system

was then diluted with water (1 29 vv) Each pure samplewas injected into a Dionex Ultimate 3000 HPLC system(Thromo Scientific) The chromatographic columns used inthis experiment are commercially available this is obtainedfrom RS-tech (046 times 25 cm 5 120583m C

18 Daejeon Korea)

The injection volume was 10 120583L and the flow rate of themobile phase was 10mLmin The wavelength of the UVdetector was fixed at 203 254 and 320 nm The compositionof the mobile phases was as follows A watertrifluoroaceticacid = 99901 vol and B acetonitrile 100 The run timewas 70min and the solvent program was the linear gradientmethod (90 10ndash60 40 A B vol) Figure 2 is a schematicshowing the online coupling of HPLC to a DAD (diode arraydetector) and the continuous flow ABTS assay Online HPLCthen arrived at a ldquoTrdquo piece whereABTSwas addedTheABTS

flow rate was 05mLmin delivered by a Dionex Ultimate3000 Pump After mixing through a 1mL loop which wasmaintained at 40∘C the absorbance was measured by a VISdetector at 734 nm Data were analyzed using Chromeleon 7software

26 Cell Culture and Drug Treatment RAW 2647 cellswere obtained from the American Type Culture Collection(ATCC Manassas VA USA) and grown in RPMI 1640medium containing 10 FBS and 100UmL of antibioticssulfate The cells were incubated in humidified 5 CO

2

atmosphere at 37∘C To stimulate the cells the medium waschangedwith freshRPMI 1640mediumandLPS (200 ngmL)[18 19]was added in the presence or absence of 17 compounds(1 5 and 10 120583gmL) for 24 h

4 Evidence-Based Complementary and Alternative Medicine

27 Cell Viability Assay Cytotoxicity was analyzed usingCCK (Dojindo Japan) 17 compounds were added to the cellsand incubated for 24 hours at 37∘C with 5 CO

2 10 120583L CCK

solutionswere added to eachwell and the cells were incubatedfor another 1 h Then the optical density was read at 450 nmusing an ELISA reader (Infinite M200 Tecan MannedorfSwitzerland)

28 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated with17 compounds and stimulated with LPS for 24 hours Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

29 Inflammatory Cytokine Determination Cells were seed-ed at a density of 5 times 105 cellsmL in 24-well culture plates andpretreated with three concentrations of 17 compounds for 1hour before LPS stimulation ELISA plates (Nunc RoskildeDenmark) were coated overnight at 4∘C with capture anti-body diluted in coating buffer (01M carbonate pH 95) andthen washed five times with phosphate-buffered saline (PBS)containing 005 Tween 20The nonspecific protein-bindingsites were blocked with assay diluent buffer (PBS containing10 FBS pH 70) for more than 1 hour Promptly samplesand standards were added to the wells After overnight ofincubation at 4∘C theworking detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 hour Subsequently substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in darkness before the reaction was stopped withstop solution (2N H

3PO4) The optical density was read at

450 nm [19]

210 Statistical Analysis The results are expressed as meanplusmn SD values for the number (119899 = 3 times) of experimentsStatistical significance was compared for each treated groupwith the control and determined by Studentrsquos 119905-tests Eachexperiment was repeated at least three times to yield com-parable results Values with 119901 lt 001 and 119901 lt 0001 wereconsidered significant

3 Result and Discussion

Several researches have revealed that a variety of natural andchemical compounds from natural substance crops fruitsvegetables and oriental medicinal herbs (OMH) have shownhigh antioxidant activity after the extraction and purificationprocesses [3] In addition variousmethods have been used todetermine the antioxidant activity of natural substance cropsfoods and plant products [1 4] The present study used threedifferent methods to evaluate the antioxidant activity con-sisting of DPPH radical-scavenging activity ABTS radical-scavenging activity and online screeningHPLC-ABTS assaysTherefore this work documented for the first time a

comparison of the antioxidant activities of one hundred kindsof pure chemical compounds

31 Offline DPPH and ABTS Assay Antioxidant activityreportedly has an effect on various different bioactivities(whitening anti-inflammation and high blood pressure)The antioxidant activity of natural substances and OMHhas been widely studied and thus this study identifies theantioxidant activity of standard substances that have origi-nated from various OMH in terms of their DPPH radical-scavenging activity and ABTS radical-scavenging activityTheDPPHandABTS radical-scavenging assays offer a redox-functioned proton ion for unstable free radicals and playa critical role in stabilizing detrimental free radicals in thehuman body This is generally achieved by taking advantageof the fact that unstable violet DPPH and ABTS free radicalstransform to stable yellow DPPH free radicals by acceptinga hydrogen ion from antioxidants In terms of the antiox-idant activity the ability to eliminate hydroxyl radicals orsuperoxide radicals through a physiologic action or throughoxidation is evaluated and a high index indicates a strongantioxidant activity Table 1 provides the results of the DPPHand ABTS radical scavenging in 100 ppm for one hundredkinds of pure standard compounds used in this study 17 com-pounds among the one hundred kinds of pure standard com-pounds ((1) (+)-catechin hydrate (2) calycosin (3) caffeicacid (4) curcumin (5) eugenol (6) ferulic acid (7) gallic acidhydrate (8) hyperoside (9) kaempferol (10) magnolol (11)quercetin (12) quercetin 3-beta-D-glucoside (13) quercitrin(14) rutin hydrate (15) sinapic acid (16) vanillylacetoneand (17) L-(+)-ascorbic acid) have an antioxidant activity ofover 90 Table 2 shows the IC

50rate of compounds with

a strong antioxidant activity The ABTS radical-scavengingmeasurement method which is commonly used to evaluatethe antioxidant activity takes advantage of the fact that ABTSfree radicals become stable by accepting a hydrogen ion fromthe antioxidant losing their blue colors Moreover in theABTS assay as well as in the DPPH assay when antioxidantactivity occurs the ability to eliminate hydroxyl radicals orsuperoxide radicals through physiologic action or oxidationis evaluated with a high index indicating a strong antioxidantactivity Each of the DPPH and ABTS are compounds thathave a proton free radical with a characteristic absorptionthat decreases significantly upon exposure to proton radicalscavengers DPPH and ABTS radical-scavenging throughantioxidant activity are well known to be attributable to theirhydrogen-donating ability (Tables 1 and 2)The concentrationof these compounds required to inhibit 50 of the radical-scavenging effect (IC

50) has been determined by testing a

series of concentrations In particular the sample with (+)-catechin hydrate caffeic acid eugenol gallic acid hydratehyperoside quercetin vanillylacetone and L-(+)-ascorbicacid compounds showed the strongest activity In additionthe 17 compounds showed better inhibitory activity againstABTS radical than the DPPH radicals That is the ABTSassay is more sensitive in identifying antioxidant activitybecause of the faster reaction kinetics and its response toantioxidants is higher Consequently this study shows thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Free radical-scavenging capacities of antioxidant activity available measured with DPPH and ABTS assay on microwell plate

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS1 Albiflorin Wako 20813 minus015 plusmn 039 014 plusmn 5272 Alisol A Wako 20379 minus047 plusmn 071 1292 plusmn 0863 Alisol B Wako 21155 minus166 plusmn 023 1278 plusmn 1354 Amygdalin KFDA 21861 minus085 plusmn 067 1231 plusmn 0035 Anthraquinone Wako 48028 012 plusmn 070 086 plusmn 5126 Atractylenolide iii Chem Faces 40271 minus207 plusmn 053 134 plusmn 8297 Aucubin Wako 28874 minus164 plusmn 079 122 plusmn 9748 Baicalein KFDA 37004 9584 plusmn 015 9937 plusmn 0129 Benzoic acid Sigma 81887 188 plusmn 042 380 plusmn 02610 Berberine Chem Faces 29730 064 plusmn 043 8468 plusmn 25511 Berberine HCl KFDA 26895 minus030 plusmn 030 874 plusmn 83812 Caffeic acid Sigma 55506 9591 plusmn 016 9966 plusmn 02413 Calycosin Chem Faces 35179 6451 plusmn 059 9919 plusmn 00514 Catalpol Wako 27599 minus255 plusmn 047 112 plusmn 96215 Chrysin Sigma 39333 045 plusmn 053 9913 plusmn 00616 Cimifugin Chem Faces 32646 minus089 plusmn 021 1439 plusmn 18117 Cinnamyl alcohol Sigma 74527 minus003 plusmn 037 1518 plusmn 07518 cis-Inositol Sigma 55506 015 plusmn 026 054 plusmn 12219 Costunolide Sigma 43044 286 plusmn 017 2013 plusmn 87420 Crocin Sigma 10236 2443 plusmn 128 4773 plusmn 06721 Curcumin Sigma 27146 9750 plusmn 063 9997 plusmn 01622 (+)-Catechin hydrate TCI 34451 9450 plusmn 016 9915 plusmn 00623 18-Dihydroxy-3-methylanthraquinone Sigma 39333 minus094 plusmn 054 209 plusmn 26024 D-(minus)-Lactic acid Sigma 111012 251 plusmn 240 2829 plusmn 07425 D-(+)-Chiro-inositol Sigma 55506 049 plusmn 033 021 plusmn 04526 Daidzein Wako 39333 052 plusmn 061 9949 plusmn 04927 Decursin KFDA 30454 minus301 plusmn 091 097 plusmn 27528 Decursinol Chem Faces 40607 minus159 plusmn 103 1512 plusmn 18129 Dioscin Sigma 11507 minus293 plusmn 130 043 plusmn 27530 Diosgenin Sigma 24118 minus138 plusmn 070 196 plusmn 92431 D-Pinitol Sigma 51499 minus227 plusmn 033 1272 plusmn 18532 67-Dimethylesculetin RD Chemical 48499 minus274 plusmn 054 131 plusmn 02133 (minus)-Epicatechin Sigma 34451 9451 plusmn 041 9961 plusmn 01634 (minus)-Epigallocatechin gallate Sigma 21816 9569 plusmn 014 9951 plusmn 02435 Eleutheroside B Wako 26855 minus204 plusmn 102 197 plusmn 28536 Emodin TCI 37004 220 plusmn 116 9127 plusmn 13937 Ephedrine-HCl KFDA 49581 minus211 plusmn 152 000 plusmn 01238 Ergosterol Chem Faces 25211 minus165 plusmn 134 055 plusmn 94639 Eugenol Sigma 60901 9372 plusmn 012 9991 plusmn 05540 Evodiamine KFDA 32964 421 plusmn 155 4076 plusmn 20941 Ferulic acid Sigma 51499 9512 plusmn 024 9896 plusmn 02742 Gallic acid hydrate TCI 58782 9556 plusmn 003 10130 plusmn 21243 Geniposide Chem Faces 25749 minus145 plusmn 090 122 plusmn 94944 Genistein TCI 37004 minus152 plusmn 030 9850 plusmn 04845 Genistin Wako 23128 223 plusmn 064 10065 plusmn 00346 Geraniol Sigma 11249 minus243 plusmn 167 1494 plusmn 095

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS47 Glabridin Wako 30829 4220 plusmn 088 10058 plusmn 00448 Glimepiride Sigma 20382 minus133 plusmn 063 784 plusmn 23049 Glycyrrhetic acid TCI 21246 066 plusmn 065 1023 plusmn 06250 Glycyrrhizin TCI 12152 159 plusmn 196 1236 plusmn 11551 Gomisin A KFDA 24012 180 plusmn 028 174 plusmn 02252 Gomisin N KFDA 24971 045 plusmn 051 380 plusmn 02153 Hesperidin KFDA 20223 3184 plusmn 037 10021 plusmn 00154 Hyperoside Chem Faces 21534 9316 plusmn 025 9962 plusmn 02055 21015840-Hydroxy-41015840-methoxy-acetophenone Sigma 60176 minus281 plusmn 028 9973 plusmn 10456 Icariin TCI 14778 382 plusmn 108 1460 plusmn 01257 Imperatorin Chem Faces 36999 minus071 plusmn 231 074 plusmn 951

58 Isoimperatorin Santa CruzBiotech 36999 027 plusmn 022 306 plusmn 217

59 Jujuboside A Chem Faces 8283 minus163 plusmn 035 026 plusmn 97460 Kaempferol Chem Faces 34936 9502 plusmn 022 9984 plusmn 04161 Liquiritigenin Chem Faces 39024 1226 plusmn 086 012 plusmn 06862 Loganin KFDA 25616 262 plusmn 128 580 plusmn 06263 Magnolol KFDA 37547 5450 plusmn 012 7774 plusmn 10664 Mevinolin Sigma 24719 minus026 plusmn 007 278 plusmn 069

65 Morroniside China LangChem Inc 24608 711 plusmn 058 1962 plusmn 183

66 Naringin Sigma 17226 305 plusmn 037 10036 plusmn 00567 Nodakenin Chem Faces 24486 068 plusmn 045 1570 plusmn 19268 Oleanolic acid Wako 21896 minus037 plusmn 054 000 plusmn 01269 Ononin Sigma 23234 223 plusmn 158 2291 plusmn 18970 Oxymatrine Chem Faces 37826 minus163 plusmn 017 072 plusmn 88571 Oxypeucedanin Chem Faces 34931 minus011 plusmn 027 1910 plusmn 23872 Paeoniflorin TCI 20813 429 plusmn 143 2203 plusmn 09173 Paeonol Sigma 60179 099 plusmn 221 1979 plusmn 23574 Palmatine chloride hydrate Sigma 25782 257 plusmn 242 8415 plusmn 24375 Palmatine Chem Faces 29205 012 plusmn 023 6805 plusmn 36376 p-Coumaric acid Sigma 60916 889 plusmn 004 3852 plusmn 08477 Poncirin KFDA 16819 minus144 plusmn 127 8420 plusmn 36678 Puerarin Wako 24017 989 plusmn 016 10062 plusmn 00679 Quercetin Sigma 33086 9602 plusmn 008 10018 plusmn 00680 Quercetin 3-120573-D-glucoside Sigma 21534 9443 plusmn 002 9994 plusmn 00681 Quercitrin hydrate Sigma 22303 9332 plusmn 004 9912 plusmn 01482 Rutaecarpine KFDA 34804 minus141 plusmn 060 9723 plusmn 06383 Rutin hydrate Sigma 16379 9357 plusmn 013 10010 plusmn 00284 Saikosaponin a KFDA 12804 minus067 plusmn 079 1617 plusmn 16285 Salicylaldehyde Sigma 81887 207 plusmn 004 10036 plusmn 00586 Schisandrin KFDA 23121 minus239 plusmn 089 1029 plusmn 24387 Sennoside A KFDA 11591 minus309 plusmn 064 8786 plusmn 32188 Sequoyitol GlycoSyn 51499 minus320 plusmn 137 1028 plusmn 20789 Sinapic acid Fluka 44599 9484 plusmn 033 9999 plusmn 02390 Spinosin Chem Faces 16433 minus096 plusmn 226 8016 plusmn 22091 Tetrandrine Fluka 16058 6083 plusmn 211 10028 plusmn 006

Evidence-Based Complementary and Alternative Medicine 7

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS92 trans-Cinnamaldehyde Sigma 75666 283 plusmn 069 1900 plusmn 13793 trans-Cinnamic acid Sigma 67495 019 plusmn 015 002 plusmn 94594 Uric acid Sigma 59485 4037 plusmn 197 9833 plusmn 03195 Vanillylacetone Sigma 51485 9378 plusmn 006 9992 plusmn 02496 Wogonin KFDA 35179 102 plusmn 051 10057 plusmn 01397 Wogonoside Chem Faces 21721 1084 plusmn 010 9862 plusmn 03298 Ziyuglycoside I Chem Faces 13038 minus171 plusmn 100 001 plusmn 98599 Z-Ligustilide Chem Faces 53129 192 plusmn 076 6869 plusmn 214100 Ascorbic acid (Vitamin C) Daejung 56779 9956 plusmn 089 9989 plusmn 004

Table 2 Antioxidant activity of 17 compounds with offline DPPHand ABTS IC50 assay

Number NameRadical- scavenging IC50

(120583gmL)DPPH ABTS

1 (+)-Catechin hydrate 525 plusmn 031 312 plusmn 0512 Calycosin 6188 plusmn 119 3321 plusmn 3593 Caffeic acid 450 plusmn 030 159 plusmn 0064 Curcumin 889 plusmn 024 499 plusmn 0455 Eugenol 522 plusmn 025 322 plusmn 0456 Ferulic acid 949 plusmn 021 199 plusmn 0127 Gallic acid hydrate 156 plusmn 038 103 plusmn 0258 Hyperoside 544 plusmn 036 354 plusmn 0399 Kaempferol 778 plusmn 030 370 plusmn 01510 Magnolol 8557 plusmn 140 837 plusmn 05611 Quercetin 266 plusmn 024 189 plusmn 03312 Quercetin 3-120573-D-glucoside 705 plusmn 059 359 plusmn 08913 Quercitrin hydrate 755 plusmn 077 423 plusmn 08414 Rutin hydrate 972 plusmn 106 468 plusmn 12415 Sinapic acid 826 plusmn 041 536 plusmn 08516 Vanillylacetone 569 plusmn 000 345 plusmn 00517 Ascorbic acid (Vitamin C) 365 plusmn 023 265 plusmn 046

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015 120583gmL respectively

32 Online HPLC-ABTS Assay Analysis The most popularapproach utilises a relatively stable coloured radical solutionof DPPH or ABTS which is added postcolumn to the HPLCflow Drug food functional material and plant and OMHsamples are evaluated for their antioxidant capacities accord-ing to a variety of antioxidant activity test methods suchas those for ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20] The HPLC analysesreact postcolumn with the ABTS and the reduction isdetected as a negative peak by a VIS absorbance detector at

734 nm The ABTS radical is much more water soluble thanDPPH [13] so ABTS better shows the details of an onlineHPLC-ABTS assay system that analysed the 17 given com-pounds (Figures 3(a) and 3(b)) Combined UV (positive sig-nals) andABTS quenching (negative signals) chromatogramsof the 17 compounds ((1) gallic acid hydrate 119877

119905 546min

(2) (+)-catechin hydrate 119877119905 926min (3) caffeic acid 119877

119905

1112min (4) ferulic acid 119877119905 1587min (5) rutin hydrate 119877

119905

1599min (6) sinapic acid 119877119905 1615min (7) hyperoside 119877

119905

1649min (8) quercetin 3-beta-D-glucoside119877119905 1682min (9)

vanillylacetone 119877119905 1792min (10) quercitrin 119877

119905 1890min

(11) calycosin 119877119905 2381min (12) quercetin 119877

119905 2431min (13)

kaempferol 119877119905 2837min (14) eugenol 119877

119905 2898min (15)

curcumin 119877119905 4020min (16) magnolol 119877

119905 4398min and

(17) L-(+)-ascorbic acid) (not detected L-(+)-ascorbic acid)(each concentration 100 ppm) are presented in Figure 3(a) Ofthese seven compounds that showed excellent activity werefurther analysed Several eluted substances were detected inthe 7 compounds including (1) gallic acid hydrate (210 nm)(2) (+)-catechin hydrate (210 nm) (3) caffeic acid (320 nm)(4) rutin hydrate (210 nm) (5) hyperoside (210 nm) (6)quercetin (210 nm) and (7) kaempferol (254 nm) which areobserved as a positive signal on theUVdetector (210 254 and320 nm) The retention times (119877

119905) of (1) gallic acid hydrate

(119877119905562min) (2) (+)-catechin hydrate (119877

119905946min) (3)

caffeic acid (1198771199051112min) (4) rutin hydrate (119877

1199051586min) (5)

hyperoside (1198771199051626min) (6) quercetin (119877

1199052358min) and

(7) kaempferol (1198771199052830min) are reported in Figure 3(b)The

other compounds exhibited a hydrogen-donating capacity(negative peak) towards the ABTS radical at the appliedconcentrationThese results therefore reveal that this methodcan be applied for quick screening of antioxidant activity ormore precisely of radical-scavenging activity (Table 3) Thiswork confirms the feasibility of assessing the bioactivity ofspecific phytochemicals by using an online screening HPLC-ABTS assay This method was successfully applied to screenand identify the antioxidant activity of natural substancesand OMH complex mixtures [5 15] The results show theshape of the chromatogram by the competitive adsorptionand desorption In addition the screening methods for therapid activity can provide useful information in basic researchon natural products chemistry and isolation analysis It isconsidered that the data will only be valuable in engineering

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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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

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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

Journal of

ObesityJournal of

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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

2 Evidence-Based Complementary and Alternative Medicine

antioxidant activity especially using DPPH or ABTS radicaltechnique [12] Recently sensitive online HPLC methods(online HPLC-DPPH and online HPLC-ABTS assays) havebeen developed to analyse free radical-scavenging activity[5 13] An online systemhas been introduced to rapidly deter-mine the antioxidant activity of each component in the givencompounds and online screening HPLC postcolumn assayinvolving DPPH or ABTS radical techniques has been devel-oped to provide a new analysis screening technology methodwith which the bioactive compounds can be spectrophoto-metrically monitored due to the decrease in absorbance at515 or 734 nm [14]This newmethod was successfully appliedto screen and identify the natural bioactivity of complexmixtures especially for OMH [15] In this study we conductDPPH and ABTS assays to screen for the antioxidant activityof one hundred kinds of pure chemical compounds so theIC50

rate of a more practical substance is determined Wealso evaluated the cytotoxicity of 17 compounds including(1) (+)-catechin hydrate (2) calycosin (3) caffeic acid (4)curcumin (5) eugenol (6) ferulic acid (7) gallic acid hydrate(8) hyperoside (9) kaempferol (10) magnolol (11) quercetin(12) quercetin 3-beta-D-glucoside (13) quercitrin hydrate(14) rutin hydrate (15) sinapic acid (16) vanillylacetone and(17) L-(+)-ascorbic acid by using a CCK assay to determinethe optimal concentration that would be effective for anti-inflammatory activity with a minimum toxicity [9 16] Inaddition the results of an online HPLC-ABTS assay of someof the compoundswere compared and analysed to find amorepractical approach toward the use of online screening HPLC-ABTS assays to quickly pinpoint peaks in chromatograms thatcorrespond to bioactive compounds

2 Experimental

21 Reagents and Materials One hundred kinds of purechemical compounds were purchased from KFDA (Korea)Daejung (Korea) Sigma (USA) Chem Faces (China) TCI(Japan) ChromaDex (USA) Fluka (USA) Wako (Japan)GlycoSyn (New Zealand USA) Santa Cruz Biotech (USA)China Lang Chem Inc (China) and RD Chemical (USA)The following reagents were used for radical-scavengingassays ABTS (221015840-azino-bis(3-ethylbenzothiazoline-6-sul-fonic acid)) DPPH (22-diphenyl-1-picrylhydrazyl) potas-sium persulfate and trifluoroacetic acid (TFA) were pur-chased from Sigma (USA) The HPLC-grade methanol andacetonitrile were purchased from J T Baker (USA)The tripledistilled water was filtered with a 02120583m membrane filter(Advantec Tokyo Japan) before analysis Materials for cellculture were obtained from Lonza (Basel Switzerland) LPSbovine serum albumin (BSA) and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) were purchasedfrom Sigma (St Louis MO USA) Antibodies for ELISAwere obtained from eBioscience (San Diego CA USA) Thechemical structures of potentially selected compounds areshown in Figure 1

22 Standard Sample Preparation The high purity standardsample (higher than gt95) was prepared by dissolving

2mg of each standard chemical in 20mL of methanol andadjusting the concentration to 100 120583gmL

23 Offline DPPH Assay for Antioxidant Activity EvaluationThe DPPH radical cation method [17] was modified to eval-uate the free radical-scavenging effect of one hundred purechemical compounds The DPPH reagent was DPPH (8mg)dissolved in MeOH (100mL) for a solution concentrationof 80 120583LmL To determine the scavenging activity 100 120583LDPPH reagent was mixed with 100120583L of sample in a 96-well microplate and was incubated at room temperaturefor 30min After incubation the absorbance was measured514 nm using an ELISA reader (TECAN Groding Austria)and 100 methanol was used as a control The DPPHscavenging effect was measured using the following formula

Radical scavenging ()

= [

(119860)control minus (119860)sample

(119860)control]times 100

(1)

The IC50DPPH values (the concentration of sample required

for inhibition of 50 of DPPH radicals) were obtainedthrough extrapolation from regression analysis The antiox-idant was evaluated based on this IC

50value

24 Offline-ABTS Assay for Antioxidant Activity EvaluationThe ABTS radical cation method [17] was modified toevaluate the free radical-scavenging effect of one hundredpure chemical compounds The ABTS reagent was preparedby mixing 5mL of 7mM ABTS with 88120583L of 140mMpotassium persulfate The mixture was then kept in the darkat room temperature for 16 h to allow free radical generationand was then diluted with water (1 44 vv) To determinethe scavenging activity 100 120583L ABTS reagent was mixedwith 100 120583L of sample in a 96-well microplate and wasincubated at room temperature for 6min After incubationthe absorbance was measured 734 nm using an ELISA reader(TECAN Groding Austria) and 100methanol was used asa control The ABTS scavenging effect was measured usingthe following formula

Radical scavenging ()

= [

(119860)control minus (119860)sample

(119860)control]times 100

(2)

The IC50ABTS values (the concentration of sample required

for inhibition of 50 of ABTS radicals) were obtainedthrough extrapolation from regression analysis The antiox-idant activity was evaluated based on this IC

50value

25 Online Screening HPLC-ABTS Analysis The onlineradical-scavenging activity of one hundred kinds of purestandard compounds was determined using the ABTS assaymodifying the methods used by Stewart et al [18] A 2mMABTS stock solution containing 35mMpotassium persulfatewas prepared and was kept in the dark at room temperaturefor 16 h to allow the completion of radical generation and

Evidence-Based Complementary and Alternative Medicine 3

MW 17012Gallic acid hydrate

MW 302236Quercetin

MW 18016Caffeic acid

MW 28624Kaempferol

MW 46438Hyperoside

MW 61052Rutin hydrate

OHO

HO O

OH

OH

O

OH

OH

HO

O

OH

HO

HOCOOH

OHO

HO

OH

OH

OH

O

OHO

HO

OH

OH

O

C9H8O4 C15H10O7 C7H6O5 C15H10O6

C15H14O6 C21H20O12 C27H30O16

MW 29027(+)-Catechin hydrate

O

OH

OH

O

OH

OH

O

OH

OH

HO

OO

OHHO

HOO

H3C middot3H2O

OHO

HO

OH

OH

OH middotXH2O

OH

OH

HO

HOOC

middotH2O

Figure 1 Chemical structure of the superior antioxidant activity compounds

Regents

Sample injection

ColumnPump

DADdetector

Pump

Passive splitReaction loop

detectorVWD

Regents

Figure 2 Schematic of online screening HPLC-ABTS system

was then diluted with water (1 29 vv) Each pure samplewas injected into a Dionex Ultimate 3000 HPLC system(Thromo Scientific) The chromatographic columns used inthis experiment are commercially available this is obtainedfrom RS-tech (046 times 25 cm 5 120583m C

18 Daejeon Korea)

The injection volume was 10 120583L and the flow rate of themobile phase was 10mLmin The wavelength of the UVdetector was fixed at 203 254 and 320 nm The compositionof the mobile phases was as follows A watertrifluoroaceticacid = 99901 vol and B acetonitrile 100 The run timewas 70min and the solvent program was the linear gradientmethod (90 10ndash60 40 A B vol) Figure 2 is a schematicshowing the online coupling of HPLC to a DAD (diode arraydetector) and the continuous flow ABTS assay Online HPLCthen arrived at a ldquoTrdquo piece whereABTSwas addedTheABTS

flow rate was 05mLmin delivered by a Dionex Ultimate3000 Pump After mixing through a 1mL loop which wasmaintained at 40∘C the absorbance was measured by a VISdetector at 734 nm Data were analyzed using Chromeleon 7software

26 Cell Culture and Drug Treatment RAW 2647 cellswere obtained from the American Type Culture Collection(ATCC Manassas VA USA) and grown in RPMI 1640medium containing 10 FBS and 100UmL of antibioticssulfate The cells were incubated in humidified 5 CO

2

atmosphere at 37∘C To stimulate the cells the medium waschangedwith freshRPMI 1640mediumandLPS (200 ngmL)[18 19]was added in the presence or absence of 17 compounds(1 5 and 10 120583gmL) for 24 h

4 Evidence-Based Complementary and Alternative Medicine

27 Cell Viability Assay Cytotoxicity was analyzed usingCCK (Dojindo Japan) 17 compounds were added to the cellsand incubated for 24 hours at 37∘C with 5 CO

2 10 120583L CCK

solutionswere added to eachwell and the cells were incubatedfor another 1 h Then the optical density was read at 450 nmusing an ELISA reader (Infinite M200 Tecan MannedorfSwitzerland)

28 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated with17 compounds and stimulated with LPS for 24 hours Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

29 Inflammatory Cytokine Determination Cells were seed-ed at a density of 5 times 105 cellsmL in 24-well culture plates andpretreated with three concentrations of 17 compounds for 1hour before LPS stimulation ELISA plates (Nunc RoskildeDenmark) were coated overnight at 4∘C with capture anti-body diluted in coating buffer (01M carbonate pH 95) andthen washed five times with phosphate-buffered saline (PBS)containing 005 Tween 20The nonspecific protein-bindingsites were blocked with assay diluent buffer (PBS containing10 FBS pH 70) for more than 1 hour Promptly samplesand standards were added to the wells After overnight ofincubation at 4∘C theworking detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 hour Subsequently substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in darkness before the reaction was stopped withstop solution (2N H

3PO4) The optical density was read at

450 nm [19]

210 Statistical Analysis The results are expressed as meanplusmn SD values for the number (119899 = 3 times) of experimentsStatistical significance was compared for each treated groupwith the control and determined by Studentrsquos 119905-tests Eachexperiment was repeated at least three times to yield com-parable results Values with 119901 lt 001 and 119901 lt 0001 wereconsidered significant

3 Result and Discussion

Several researches have revealed that a variety of natural andchemical compounds from natural substance crops fruitsvegetables and oriental medicinal herbs (OMH) have shownhigh antioxidant activity after the extraction and purificationprocesses [3] In addition variousmethods have been used todetermine the antioxidant activity of natural substance cropsfoods and plant products [1 4] The present study used threedifferent methods to evaluate the antioxidant activity con-sisting of DPPH radical-scavenging activity ABTS radical-scavenging activity and online screeningHPLC-ABTS assaysTherefore this work documented for the first time a

comparison of the antioxidant activities of one hundred kindsof pure chemical compounds

31 Offline DPPH and ABTS Assay Antioxidant activityreportedly has an effect on various different bioactivities(whitening anti-inflammation and high blood pressure)The antioxidant activity of natural substances and OMHhas been widely studied and thus this study identifies theantioxidant activity of standard substances that have origi-nated from various OMH in terms of their DPPH radical-scavenging activity and ABTS radical-scavenging activityTheDPPHandABTS radical-scavenging assays offer a redox-functioned proton ion for unstable free radicals and playa critical role in stabilizing detrimental free radicals in thehuman body This is generally achieved by taking advantageof the fact that unstable violet DPPH and ABTS free radicalstransform to stable yellow DPPH free radicals by acceptinga hydrogen ion from antioxidants In terms of the antiox-idant activity the ability to eliminate hydroxyl radicals orsuperoxide radicals through a physiologic action or throughoxidation is evaluated and a high index indicates a strongantioxidant activity Table 1 provides the results of the DPPHand ABTS radical scavenging in 100 ppm for one hundredkinds of pure standard compounds used in this study 17 com-pounds among the one hundred kinds of pure standard com-pounds ((1) (+)-catechin hydrate (2) calycosin (3) caffeicacid (4) curcumin (5) eugenol (6) ferulic acid (7) gallic acidhydrate (8) hyperoside (9) kaempferol (10) magnolol (11)quercetin (12) quercetin 3-beta-D-glucoside (13) quercitrin(14) rutin hydrate (15) sinapic acid (16) vanillylacetoneand (17) L-(+)-ascorbic acid) have an antioxidant activity ofover 90 Table 2 shows the IC

50rate of compounds with

a strong antioxidant activity The ABTS radical-scavengingmeasurement method which is commonly used to evaluatethe antioxidant activity takes advantage of the fact that ABTSfree radicals become stable by accepting a hydrogen ion fromthe antioxidant losing their blue colors Moreover in theABTS assay as well as in the DPPH assay when antioxidantactivity occurs the ability to eliminate hydroxyl radicals orsuperoxide radicals through physiologic action or oxidationis evaluated with a high index indicating a strong antioxidantactivity Each of the DPPH and ABTS are compounds thathave a proton free radical with a characteristic absorptionthat decreases significantly upon exposure to proton radicalscavengers DPPH and ABTS radical-scavenging throughantioxidant activity are well known to be attributable to theirhydrogen-donating ability (Tables 1 and 2)The concentrationof these compounds required to inhibit 50 of the radical-scavenging effect (IC

50) has been determined by testing a

series of concentrations In particular the sample with (+)-catechin hydrate caffeic acid eugenol gallic acid hydratehyperoside quercetin vanillylacetone and L-(+)-ascorbicacid compounds showed the strongest activity In additionthe 17 compounds showed better inhibitory activity againstABTS radical than the DPPH radicals That is the ABTSassay is more sensitive in identifying antioxidant activitybecause of the faster reaction kinetics and its response toantioxidants is higher Consequently this study shows thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Free radical-scavenging capacities of antioxidant activity available measured with DPPH and ABTS assay on microwell plate

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS1 Albiflorin Wako 20813 minus015 plusmn 039 014 plusmn 5272 Alisol A Wako 20379 minus047 plusmn 071 1292 plusmn 0863 Alisol B Wako 21155 minus166 plusmn 023 1278 plusmn 1354 Amygdalin KFDA 21861 minus085 plusmn 067 1231 plusmn 0035 Anthraquinone Wako 48028 012 plusmn 070 086 plusmn 5126 Atractylenolide iii Chem Faces 40271 minus207 plusmn 053 134 plusmn 8297 Aucubin Wako 28874 minus164 plusmn 079 122 plusmn 9748 Baicalein KFDA 37004 9584 plusmn 015 9937 plusmn 0129 Benzoic acid Sigma 81887 188 plusmn 042 380 plusmn 02610 Berberine Chem Faces 29730 064 plusmn 043 8468 plusmn 25511 Berberine HCl KFDA 26895 minus030 plusmn 030 874 plusmn 83812 Caffeic acid Sigma 55506 9591 plusmn 016 9966 plusmn 02413 Calycosin Chem Faces 35179 6451 plusmn 059 9919 plusmn 00514 Catalpol Wako 27599 minus255 plusmn 047 112 plusmn 96215 Chrysin Sigma 39333 045 plusmn 053 9913 plusmn 00616 Cimifugin Chem Faces 32646 minus089 plusmn 021 1439 plusmn 18117 Cinnamyl alcohol Sigma 74527 minus003 plusmn 037 1518 plusmn 07518 cis-Inositol Sigma 55506 015 plusmn 026 054 plusmn 12219 Costunolide Sigma 43044 286 plusmn 017 2013 plusmn 87420 Crocin Sigma 10236 2443 plusmn 128 4773 plusmn 06721 Curcumin Sigma 27146 9750 plusmn 063 9997 plusmn 01622 (+)-Catechin hydrate TCI 34451 9450 plusmn 016 9915 plusmn 00623 18-Dihydroxy-3-methylanthraquinone Sigma 39333 minus094 plusmn 054 209 plusmn 26024 D-(minus)-Lactic acid Sigma 111012 251 plusmn 240 2829 plusmn 07425 D-(+)-Chiro-inositol Sigma 55506 049 plusmn 033 021 plusmn 04526 Daidzein Wako 39333 052 plusmn 061 9949 plusmn 04927 Decursin KFDA 30454 minus301 plusmn 091 097 plusmn 27528 Decursinol Chem Faces 40607 minus159 plusmn 103 1512 plusmn 18129 Dioscin Sigma 11507 minus293 plusmn 130 043 plusmn 27530 Diosgenin Sigma 24118 minus138 plusmn 070 196 plusmn 92431 D-Pinitol Sigma 51499 minus227 plusmn 033 1272 plusmn 18532 67-Dimethylesculetin RD Chemical 48499 minus274 plusmn 054 131 plusmn 02133 (minus)-Epicatechin Sigma 34451 9451 plusmn 041 9961 plusmn 01634 (minus)-Epigallocatechin gallate Sigma 21816 9569 plusmn 014 9951 plusmn 02435 Eleutheroside B Wako 26855 minus204 plusmn 102 197 plusmn 28536 Emodin TCI 37004 220 plusmn 116 9127 plusmn 13937 Ephedrine-HCl KFDA 49581 minus211 plusmn 152 000 plusmn 01238 Ergosterol Chem Faces 25211 minus165 plusmn 134 055 plusmn 94639 Eugenol Sigma 60901 9372 plusmn 012 9991 plusmn 05540 Evodiamine KFDA 32964 421 plusmn 155 4076 plusmn 20941 Ferulic acid Sigma 51499 9512 plusmn 024 9896 plusmn 02742 Gallic acid hydrate TCI 58782 9556 plusmn 003 10130 plusmn 21243 Geniposide Chem Faces 25749 minus145 plusmn 090 122 plusmn 94944 Genistein TCI 37004 minus152 plusmn 030 9850 plusmn 04845 Genistin Wako 23128 223 plusmn 064 10065 plusmn 00346 Geraniol Sigma 11249 minus243 plusmn 167 1494 plusmn 095

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS47 Glabridin Wako 30829 4220 plusmn 088 10058 plusmn 00448 Glimepiride Sigma 20382 minus133 plusmn 063 784 plusmn 23049 Glycyrrhetic acid TCI 21246 066 plusmn 065 1023 plusmn 06250 Glycyrrhizin TCI 12152 159 plusmn 196 1236 plusmn 11551 Gomisin A KFDA 24012 180 plusmn 028 174 plusmn 02252 Gomisin N KFDA 24971 045 plusmn 051 380 plusmn 02153 Hesperidin KFDA 20223 3184 plusmn 037 10021 plusmn 00154 Hyperoside Chem Faces 21534 9316 plusmn 025 9962 plusmn 02055 21015840-Hydroxy-41015840-methoxy-acetophenone Sigma 60176 minus281 plusmn 028 9973 plusmn 10456 Icariin TCI 14778 382 plusmn 108 1460 plusmn 01257 Imperatorin Chem Faces 36999 minus071 plusmn 231 074 plusmn 951

58 Isoimperatorin Santa CruzBiotech 36999 027 plusmn 022 306 plusmn 217

59 Jujuboside A Chem Faces 8283 minus163 plusmn 035 026 plusmn 97460 Kaempferol Chem Faces 34936 9502 plusmn 022 9984 plusmn 04161 Liquiritigenin Chem Faces 39024 1226 plusmn 086 012 plusmn 06862 Loganin KFDA 25616 262 plusmn 128 580 plusmn 06263 Magnolol KFDA 37547 5450 plusmn 012 7774 plusmn 10664 Mevinolin Sigma 24719 minus026 plusmn 007 278 plusmn 069

65 Morroniside China LangChem Inc 24608 711 plusmn 058 1962 plusmn 183

66 Naringin Sigma 17226 305 plusmn 037 10036 plusmn 00567 Nodakenin Chem Faces 24486 068 plusmn 045 1570 plusmn 19268 Oleanolic acid Wako 21896 minus037 plusmn 054 000 plusmn 01269 Ononin Sigma 23234 223 plusmn 158 2291 plusmn 18970 Oxymatrine Chem Faces 37826 minus163 plusmn 017 072 plusmn 88571 Oxypeucedanin Chem Faces 34931 minus011 plusmn 027 1910 plusmn 23872 Paeoniflorin TCI 20813 429 plusmn 143 2203 plusmn 09173 Paeonol Sigma 60179 099 plusmn 221 1979 plusmn 23574 Palmatine chloride hydrate Sigma 25782 257 plusmn 242 8415 plusmn 24375 Palmatine Chem Faces 29205 012 plusmn 023 6805 plusmn 36376 p-Coumaric acid Sigma 60916 889 plusmn 004 3852 plusmn 08477 Poncirin KFDA 16819 minus144 plusmn 127 8420 plusmn 36678 Puerarin Wako 24017 989 plusmn 016 10062 plusmn 00679 Quercetin Sigma 33086 9602 plusmn 008 10018 plusmn 00680 Quercetin 3-120573-D-glucoside Sigma 21534 9443 plusmn 002 9994 plusmn 00681 Quercitrin hydrate Sigma 22303 9332 plusmn 004 9912 plusmn 01482 Rutaecarpine KFDA 34804 minus141 plusmn 060 9723 plusmn 06383 Rutin hydrate Sigma 16379 9357 plusmn 013 10010 plusmn 00284 Saikosaponin a KFDA 12804 minus067 plusmn 079 1617 plusmn 16285 Salicylaldehyde Sigma 81887 207 plusmn 004 10036 plusmn 00586 Schisandrin KFDA 23121 minus239 plusmn 089 1029 plusmn 24387 Sennoside A KFDA 11591 minus309 plusmn 064 8786 plusmn 32188 Sequoyitol GlycoSyn 51499 minus320 plusmn 137 1028 plusmn 20789 Sinapic acid Fluka 44599 9484 plusmn 033 9999 plusmn 02390 Spinosin Chem Faces 16433 minus096 plusmn 226 8016 plusmn 22091 Tetrandrine Fluka 16058 6083 plusmn 211 10028 plusmn 006

Evidence-Based Complementary and Alternative Medicine 7

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS92 trans-Cinnamaldehyde Sigma 75666 283 plusmn 069 1900 plusmn 13793 trans-Cinnamic acid Sigma 67495 019 plusmn 015 002 plusmn 94594 Uric acid Sigma 59485 4037 plusmn 197 9833 plusmn 03195 Vanillylacetone Sigma 51485 9378 plusmn 006 9992 plusmn 02496 Wogonin KFDA 35179 102 plusmn 051 10057 plusmn 01397 Wogonoside Chem Faces 21721 1084 plusmn 010 9862 plusmn 03298 Ziyuglycoside I Chem Faces 13038 minus171 plusmn 100 001 plusmn 98599 Z-Ligustilide Chem Faces 53129 192 plusmn 076 6869 plusmn 214100 Ascorbic acid (Vitamin C) Daejung 56779 9956 plusmn 089 9989 plusmn 004

Table 2 Antioxidant activity of 17 compounds with offline DPPHand ABTS IC50 assay

Number NameRadical- scavenging IC50

(120583gmL)DPPH ABTS

1 (+)-Catechin hydrate 525 plusmn 031 312 plusmn 0512 Calycosin 6188 plusmn 119 3321 plusmn 3593 Caffeic acid 450 plusmn 030 159 plusmn 0064 Curcumin 889 plusmn 024 499 plusmn 0455 Eugenol 522 plusmn 025 322 plusmn 0456 Ferulic acid 949 plusmn 021 199 plusmn 0127 Gallic acid hydrate 156 plusmn 038 103 plusmn 0258 Hyperoside 544 plusmn 036 354 plusmn 0399 Kaempferol 778 plusmn 030 370 plusmn 01510 Magnolol 8557 plusmn 140 837 plusmn 05611 Quercetin 266 plusmn 024 189 plusmn 03312 Quercetin 3-120573-D-glucoside 705 plusmn 059 359 plusmn 08913 Quercitrin hydrate 755 plusmn 077 423 plusmn 08414 Rutin hydrate 972 plusmn 106 468 plusmn 12415 Sinapic acid 826 plusmn 041 536 plusmn 08516 Vanillylacetone 569 plusmn 000 345 plusmn 00517 Ascorbic acid (Vitamin C) 365 plusmn 023 265 plusmn 046

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015 120583gmL respectively

32 Online HPLC-ABTS Assay Analysis The most popularapproach utilises a relatively stable coloured radical solutionof DPPH or ABTS which is added postcolumn to the HPLCflow Drug food functional material and plant and OMHsamples are evaluated for their antioxidant capacities accord-ing to a variety of antioxidant activity test methods suchas those for ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20] The HPLC analysesreact postcolumn with the ABTS and the reduction isdetected as a negative peak by a VIS absorbance detector at

734 nm The ABTS radical is much more water soluble thanDPPH [13] so ABTS better shows the details of an onlineHPLC-ABTS assay system that analysed the 17 given com-pounds (Figures 3(a) and 3(b)) Combined UV (positive sig-nals) andABTS quenching (negative signals) chromatogramsof the 17 compounds ((1) gallic acid hydrate 119877

119905 546min

(2) (+)-catechin hydrate 119877119905 926min (3) caffeic acid 119877

119905

1112min (4) ferulic acid 119877119905 1587min (5) rutin hydrate 119877

119905

1599min (6) sinapic acid 119877119905 1615min (7) hyperoside 119877

119905

1649min (8) quercetin 3-beta-D-glucoside119877119905 1682min (9)

vanillylacetone 119877119905 1792min (10) quercitrin 119877

119905 1890min

(11) calycosin 119877119905 2381min (12) quercetin 119877

119905 2431min (13)

kaempferol 119877119905 2837min (14) eugenol 119877

119905 2898min (15)

curcumin 119877119905 4020min (16) magnolol 119877

119905 4398min and

(17) L-(+)-ascorbic acid) (not detected L-(+)-ascorbic acid)(each concentration 100 ppm) are presented in Figure 3(a) Ofthese seven compounds that showed excellent activity werefurther analysed Several eluted substances were detected inthe 7 compounds including (1) gallic acid hydrate (210 nm)(2) (+)-catechin hydrate (210 nm) (3) caffeic acid (320 nm)(4) rutin hydrate (210 nm) (5) hyperoside (210 nm) (6)quercetin (210 nm) and (7) kaempferol (254 nm) which areobserved as a positive signal on theUVdetector (210 254 and320 nm) The retention times (119877

119905) of (1) gallic acid hydrate

(119877119905562min) (2) (+)-catechin hydrate (119877

119905946min) (3)

caffeic acid (1198771199051112min) (4) rutin hydrate (119877

1199051586min) (5)

hyperoside (1198771199051626min) (6) quercetin (119877

1199052358min) and

(7) kaempferol (1198771199052830min) are reported in Figure 3(b)The

other compounds exhibited a hydrogen-donating capacity(negative peak) towards the ABTS radical at the appliedconcentrationThese results therefore reveal that this methodcan be applied for quick screening of antioxidant activity ormore precisely of radical-scavenging activity (Table 3) Thiswork confirms the feasibility of assessing the bioactivity ofspecific phytochemicals by using an online screening HPLC-ABTS assay This method was successfully applied to screenand identify the antioxidant activity of natural substancesand OMH complex mixtures [5 15] The results show theshape of the chromatogram by the competitive adsorptionand desorption In addition the screening methods for therapid activity can provide useful information in basic researchon natural products chemistry and isolation analysis It isconsidered that the data will only be valuable in engineering

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

Evidence-Based Complementary and Alternative Medicine 3

MW 17012Gallic acid hydrate

MW 302236Quercetin

MW 18016Caffeic acid

MW 28624Kaempferol

MW 46438Hyperoside

MW 61052Rutin hydrate

OHO

HO O

OH

OH

O

OH

OH

HO

O

OH

HO

HOCOOH

OHO

HO

OH

OH

OH

O

OHO

HO

OH

OH

O

C9H8O4 C15H10O7 C7H6O5 C15H10O6

C15H14O6 C21H20O12 C27H30O16

MW 29027(+)-Catechin hydrate

O

OH

OH

O

OH

OH

O

OH

OH

HO

OO

OHHO

HOO

H3C middot3H2O

OHO

HO

OH

OH

OH middotXH2O

OH

OH

HO

HOOC

middotH2O

Figure 1 Chemical structure of the superior antioxidant activity compounds

Regents

Sample injection

ColumnPump

DADdetector

Pump

Passive splitReaction loop

detectorVWD

Regents

Figure 2 Schematic of online screening HPLC-ABTS system

was then diluted with water (1 29 vv) Each pure samplewas injected into a Dionex Ultimate 3000 HPLC system(Thromo Scientific) The chromatographic columns used inthis experiment are commercially available this is obtainedfrom RS-tech (046 times 25 cm 5 120583m C

18 Daejeon Korea)

The injection volume was 10 120583L and the flow rate of themobile phase was 10mLmin The wavelength of the UVdetector was fixed at 203 254 and 320 nm The compositionof the mobile phases was as follows A watertrifluoroaceticacid = 99901 vol and B acetonitrile 100 The run timewas 70min and the solvent program was the linear gradientmethod (90 10ndash60 40 A B vol) Figure 2 is a schematicshowing the online coupling of HPLC to a DAD (diode arraydetector) and the continuous flow ABTS assay Online HPLCthen arrived at a ldquoTrdquo piece whereABTSwas addedTheABTS

flow rate was 05mLmin delivered by a Dionex Ultimate3000 Pump After mixing through a 1mL loop which wasmaintained at 40∘C the absorbance was measured by a VISdetector at 734 nm Data were analyzed using Chromeleon 7software

26 Cell Culture and Drug Treatment RAW 2647 cellswere obtained from the American Type Culture Collection(ATCC Manassas VA USA) and grown in RPMI 1640medium containing 10 FBS and 100UmL of antibioticssulfate The cells were incubated in humidified 5 CO

2

atmosphere at 37∘C To stimulate the cells the medium waschangedwith freshRPMI 1640mediumandLPS (200 ngmL)[18 19]was added in the presence or absence of 17 compounds(1 5 and 10 120583gmL) for 24 h

4 Evidence-Based Complementary and Alternative Medicine

27 Cell Viability Assay Cytotoxicity was analyzed usingCCK (Dojindo Japan) 17 compounds were added to the cellsand incubated for 24 hours at 37∘C with 5 CO

2 10 120583L CCK

solutionswere added to eachwell and the cells were incubatedfor another 1 h Then the optical density was read at 450 nmusing an ELISA reader (Infinite M200 Tecan MannedorfSwitzerland)

28 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated with17 compounds and stimulated with LPS for 24 hours Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

29 Inflammatory Cytokine Determination Cells were seed-ed at a density of 5 times 105 cellsmL in 24-well culture plates andpretreated with three concentrations of 17 compounds for 1hour before LPS stimulation ELISA plates (Nunc RoskildeDenmark) were coated overnight at 4∘C with capture anti-body diluted in coating buffer (01M carbonate pH 95) andthen washed five times with phosphate-buffered saline (PBS)containing 005 Tween 20The nonspecific protein-bindingsites were blocked with assay diluent buffer (PBS containing10 FBS pH 70) for more than 1 hour Promptly samplesand standards were added to the wells After overnight ofincubation at 4∘C theworking detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 hour Subsequently substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in darkness before the reaction was stopped withstop solution (2N H

3PO4) The optical density was read at

450 nm [19]

210 Statistical Analysis The results are expressed as meanplusmn SD values for the number (119899 = 3 times) of experimentsStatistical significance was compared for each treated groupwith the control and determined by Studentrsquos 119905-tests Eachexperiment was repeated at least three times to yield com-parable results Values with 119901 lt 001 and 119901 lt 0001 wereconsidered significant

3 Result and Discussion

Several researches have revealed that a variety of natural andchemical compounds from natural substance crops fruitsvegetables and oriental medicinal herbs (OMH) have shownhigh antioxidant activity after the extraction and purificationprocesses [3] In addition variousmethods have been used todetermine the antioxidant activity of natural substance cropsfoods and plant products [1 4] The present study used threedifferent methods to evaluate the antioxidant activity con-sisting of DPPH radical-scavenging activity ABTS radical-scavenging activity and online screeningHPLC-ABTS assaysTherefore this work documented for the first time a

comparison of the antioxidant activities of one hundred kindsof pure chemical compounds

31 Offline DPPH and ABTS Assay Antioxidant activityreportedly has an effect on various different bioactivities(whitening anti-inflammation and high blood pressure)The antioxidant activity of natural substances and OMHhas been widely studied and thus this study identifies theantioxidant activity of standard substances that have origi-nated from various OMH in terms of their DPPH radical-scavenging activity and ABTS radical-scavenging activityTheDPPHandABTS radical-scavenging assays offer a redox-functioned proton ion for unstable free radicals and playa critical role in stabilizing detrimental free radicals in thehuman body This is generally achieved by taking advantageof the fact that unstable violet DPPH and ABTS free radicalstransform to stable yellow DPPH free radicals by acceptinga hydrogen ion from antioxidants In terms of the antiox-idant activity the ability to eliminate hydroxyl radicals orsuperoxide radicals through a physiologic action or throughoxidation is evaluated and a high index indicates a strongantioxidant activity Table 1 provides the results of the DPPHand ABTS radical scavenging in 100 ppm for one hundredkinds of pure standard compounds used in this study 17 com-pounds among the one hundred kinds of pure standard com-pounds ((1) (+)-catechin hydrate (2) calycosin (3) caffeicacid (4) curcumin (5) eugenol (6) ferulic acid (7) gallic acidhydrate (8) hyperoside (9) kaempferol (10) magnolol (11)quercetin (12) quercetin 3-beta-D-glucoside (13) quercitrin(14) rutin hydrate (15) sinapic acid (16) vanillylacetoneand (17) L-(+)-ascorbic acid) have an antioxidant activity ofover 90 Table 2 shows the IC

50rate of compounds with

a strong antioxidant activity The ABTS radical-scavengingmeasurement method which is commonly used to evaluatethe antioxidant activity takes advantage of the fact that ABTSfree radicals become stable by accepting a hydrogen ion fromthe antioxidant losing their blue colors Moreover in theABTS assay as well as in the DPPH assay when antioxidantactivity occurs the ability to eliminate hydroxyl radicals orsuperoxide radicals through physiologic action or oxidationis evaluated with a high index indicating a strong antioxidantactivity Each of the DPPH and ABTS are compounds thathave a proton free radical with a characteristic absorptionthat decreases significantly upon exposure to proton radicalscavengers DPPH and ABTS radical-scavenging throughantioxidant activity are well known to be attributable to theirhydrogen-donating ability (Tables 1 and 2)The concentrationof these compounds required to inhibit 50 of the radical-scavenging effect (IC

50) has been determined by testing a

series of concentrations In particular the sample with (+)-catechin hydrate caffeic acid eugenol gallic acid hydratehyperoside quercetin vanillylacetone and L-(+)-ascorbicacid compounds showed the strongest activity In additionthe 17 compounds showed better inhibitory activity againstABTS radical than the DPPH radicals That is the ABTSassay is more sensitive in identifying antioxidant activitybecause of the faster reaction kinetics and its response toantioxidants is higher Consequently this study shows thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Free radical-scavenging capacities of antioxidant activity available measured with DPPH and ABTS assay on microwell plate

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS1 Albiflorin Wako 20813 minus015 plusmn 039 014 plusmn 5272 Alisol A Wako 20379 minus047 plusmn 071 1292 plusmn 0863 Alisol B Wako 21155 minus166 plusmn 023 1278 plusmn 1354 Amygdalin KFDA 21861 minus085 plusmn 067 1231 plusmn 0035 Anthraquinone Wako 48028 012 plusmn 070 086 plusmn 5126 Atractylenolide iii Chem Faces 40271 minus207 plusmn 053 134 plusmn 8297 Aucubin Wako 28874 minus164 plusmn 079 122 plusmn 9748 Baicalein KFDA 37004 9584 plusmn 015 9937 plusmn 0129 Benzoic acid Sigma 81887 188 plusmn 042 380 plusmn 02610 Berberine Chem Faces 29730 064 plusmn 043 8468 plusmn 25511 Berberine HCl KFDA 26895 minus030 plusmn 030 874 plusmn 83812 Caffeic acid Sigma 55506 9591 plusmn 016 9966 plusmn 02413 Calycosin Chem Faces 35179 6451 plusmn 059 9919 plusmn 00514 Catalpol Wako 27599 minus255 plusmn 047 112 plusmn 96215 Chrysin Sigma 39333 045 plusmn 053 9913 plusmn 00616 Cimifugin Chem Faces 32646 minus089 plusmn 021 1439 plusmn 18117 Cinnamyl alcohol Sigma 74527 minus003 plusmn 037 1518 plusmn 07518 cis-Inositol Sigma 55506 015 plusmn 026 054 plusmn 12219 Costunolide Sigma 43044 286 plusmn 017 2013 plusmn 87420 Crocin Sigma 10236 2443 plusmn 128 4773 plusmn 06721 Curcumin Sigma 27146 9750 plusmn 063 9997 plusmn 01622 (+)-Catechin hydrate TCI 34451 9450 plusmn 016 9915 plusmn 00623 18-Dihydroxy-3-methylanthraquinone Sigma 39333 minus094 plusmn 054 209 plusmn 26024 D-(minus)-Lactic acid Sigma 111012 251 plusmn 240 2829 plusmn 07425 D-(+)-Chiro-inositol Sigma 55506 049 plusmn 033 021 plusmn 04526 Daidzein Wako 39333 052 plusmn 061 9949 plusmn 04927 Decursin KFDA 30454 minus301 plusmn 091 097 plusmn 27528 Decursinol Chem Faces 40607 minus159 plusmn 103 1512 plusmn 18129 Dioscin Sigma 11507 minus293 plusmn 130 043 plusmn 27530 Diosgenin Sigma 24118 minus138 plusmn 070 196 plusmn 92431 D-Pinitol Sigma 51499 minus227 plusmn 033 1272 plusmn 18532 67-Dimethylesculetin RD Chemical 48499 minus274 plusmn 054 131 plusmn 02133 (minus)-Epicatechin Sigma 34451 9451 plusmn 041 9961 plusmn 01634 (minus)-Epigallocatechin gallate Sigma 21816 9569 plusmn 014 9951 plusmn 02435 Eleutheroside B Wako 26855 minus204 plusmn 102 197 plusmn 28536 Emodin TCI 37004 220 plusmn 116 9127 plusmn 13937 Ephedrine-HCl KFDA 49581 minus211 plusmn 152 000 plusmn 01238 Ergosterol Chem Faces 25211 minus165 plusmn 134 055 plusmn 94639 Eugenol Sigma 60901 9372 plusmn 012 9991 plusmn 05540 Evodiamine KFDA 32964 421 plusmn 155 4076 plusmn 20941 Ferulic acid Sigma 51499 9512 plusmn 024 9896 plusmn 02742 Gallic acid hydrate TCI 58782 9556 plusmn 003 10130 plusmn 21243 Geniposide Chem Faces 25749 minus145 plusmn 090 122 plusmn 94944 Genistein TCI 37004 minus152 plusmn 030 9850 plusmn 04845 Genistin Wako 23128 223 plusmn 064 10065 plusmn 00346 Geraniol Sigma 11249 minus243 plusmn 167 1494 plusmn 095

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS47 Glabridin Wako 30829 4220 plusmn 088 10058 plusmn 00448 Glimepiride Sigma 20382 minus133 plusmn 063 784 plusmn 23049 Glycyrrhetic acid TCI 21246 066 plusmn 065 1023 plusmn 06250 Glycyrrhizin TCI 12152 159 plusmn 196 1236 plusmn 11551 Gomisin A KFDA 24012 180 plusmn 028 174 plusmn 02252 Gomisin N KFDA 24971 045 plusmn 051 380 plusmn 02153 Hesperidin KFDA 20223 3184 plusmn 037 10021 plusmn 00154 Hyperoside Chem Faces 21534 9316 plusmn 025 9962 plusmn 02055 21015840-Hydroxy-41015840-methoxy-acetophenone Sigma 60176 minus281 plusmn 028 9973 plusmn 10456 Icariin TCI 14778 382 plusmn 108 1460 plusmn 01257 Imperatorin Chem Faces 36999 minus071 plusmn 231 074 plusmn 951

58 Isoimperatorin Santa CruzBiotech 36999 027 plusmn 022 306 plusmn 217

59 Jujuboside A Chem Faces 8283 minus163 plusmn 035 026 plusmn 97460 Kaempferol Chem Faces 34936 9502 plusmn 022 9984 plusmn 04161 Liquiritigenin Chem Faces 39024 1226 plusmn 086 012 plusmn 06862 Loganin KFDA 25616 262 plusmn 128 580 plusmn 06263 Magnolol KFDA 37547 5450 plusmn 012 7774 plusmn 10664 Mevinolin Sigma 24719 minus026 plusmn 007 278 plusmn 069

65 Morroniside China LangChem Inc 24608 711 plusmn 058 1962 plusmn 183

66 Naringin Sigma 17226 305 plusmn 037 10036 plusmn 00567 Nodakenin Chem Faces 24486 068 plusmn 045 1570 plusmn 19268 Oleanolic acid Wako 21896 minus037 plusmn 054 000 plusmn 01269 Ononin Sigma 23234 223 plusmn 158 2291 plusmn 18970 Oxymatrine Chem Faces 37826 minus163 plusmn 017 072 plusmn 88571 Oxypeucedanin Chem Faces 34931 minus011 plusmn 027 1910 plusmn 23872 Paeoniflorin TCI 20813 429 plusmn 143 2203 plusmn 09173 Paeonol Sigma 60179 099 plusmn 221 1979 plusmn 23574 Palmatine chloride hydrate Sigma 25782 257 plusmn 242 8415 plusmn 24375 Palmatine Chem Faces 29205 012 plusmn 023 6805 plusmn 36376 p-Coumaric acid Sigma 60916 889 plusmn 004 3852 plusmn 08477 Poncirin KFDA 16819 minus144 plusmn 127 8420 plusmn 36678 Puerarin Wako 24017 989 plusmn 016 10062 plusmn 00679 Quercetin Sigma 33086 9602 plusmn 008 10018 plusmn 00680 Quercetin 3-120573-D-glucoside Sigma 21534 9443 plusmn 002 9994 plusmn 00681 Quercitrin hydrate Sigma 22303 9332 plusmn 004 9912 plusmn 01482 Rutaecarpine KFDA 34804 minus141 plusmn 060 9723 plusmn 06383 Rutin hydrate Sigma 16379 9357 plusmn 013 10010 plusmn 00284 Saikosaponin a KFDA 12804 minus067 plusmn 079 1617 plusmn 16285 Salicylaldehyde Sigma 81887 207 plusmn 004 10036 plusmn 00586 Schisandrin KFDA 23121 minus239 plusmn 089 1029 plusmn 24387 Sennoside A KFDA 11591 minus309 plusmn 064 8786 plusmn 32188 Sequoyitol GlycoSyn 51499 minus320 plusmn 137 1028 plusmn 20789 Sinapic acid Fluka 44599 9484 plusmn 033 9999 plusmn 02390 Spinosin Chem Faces 16433 minus096 plusmn 226 8016 plusmn 22091 Tetrandrine Fluka 16058 6083 plusmn 211 10028 plusmn 006

Evidence-Based Complementary and Alternative Medicine 7

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS92 trans-Cinnamaldehyde Sigma 75666 283 plusmn 069 1900 plusmn 13793 trans-Cinnamic acid Sigma 67495 019 plusmn 015 002 plusmn 94594 Uric acid Sigma 59485 4037 plusmn 197 9833 plusmn 03195 Vanillylacetone Sigma 51485 9378 plusmn 006 9992 plusmn 02496 Wogonin KFDA 35179 102 plusmn 051 10057 plusmn 01397 Wogonoside Chem Faces 21721 1084 plusmn 010 9862 plusmn 03298 Ziyuglycoside I Chem Faces 13038 minus171 plusmn 100 001 plusmn 98599 Z-Ligustilide Chem Faces 53129 192 plusmn 076 6869 plusmn 214100 Ascorbic acid (Vitamin C) Daejung 56779 9956 plusmn 089 9989 plusmn 004

Table 2 Antioxidant activity of 17 compounds with offline DPPHand ABTS IC50 assay

Number NameRadical- scavenging IC50

(120583gmL)DPPH ABTS

1 (+)-Catechin hydrate 525 plusmn 031 312 plusmn 0512 Calycosin 6188 plusmn 119 3321 plusmn 3593 Caffeic acid 450 plusmn 030 159 plusmn 0064 Curcumin 889 plusmn 024 499 plusmn 0455 Eugenol 522 plusmn 025 322 plusmn 0456 Ferulic acid 949 plusmn 021 199 plusmn 0127 Gallic acid hydrate 156 plusmn 038 103 plusmn 0258 Hyperoside 544 plusmn 036 354 plusmn 0399 Kaempferol 778 plusmn 030 370 plusmn 01510 Magnolol 8557 plusmn 140 837 plusmn 05611 Quercetin 266 plusmn 024 189 plusmn 03312 Quercetin 3-120573-D-glucoside 705 plusmn 059 359 plusmn 08913 Quercitrin hydrate 755 plusmn 077 423 plusmn 08414 Rutin hydrate 972 plusmn 106 468 plusmn 12415 Sinapic acid 826 plusmn 041 536 plusmn 08516 Vanillylacetone 569 plusmn 000 345 plusmn 00517 Ascorbic acid (Vitamin C) 365 plusmn 023 265 plusmn 046

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015 120583gmL respectively

32 Online HPLC-ABTS Assay Analysis The most popularapproach utilises a relatively stable coloured radical solutionof DPPH or ABTS which is added postcolumn to the HPLCflow Drug food functional material and plant and OMHsamples are evaluated for their antioxidant capacities accord-ing to a variety of antioxidant activity test methods suchas those for ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20] The HPLC analysesreact postcolumn with the ABTS and the reduction isdetected as a negative peak by a VIS absorbance detector at

734 nm The ABTS radical is much more water soluble thanDPPH [13] so ABTS better shows the details of an onlineHPLC-ABTS assay system that analysed the 17 given com-pounds (Figures 3(a) and 3(b)) Combined UV (positive sig-nals) andABTS quenching (negative signals) chromatogramsof the 17 compounds ((1) gallic acid hydrate 119877

119905 546min

(2) (+)-catechin hydrate 119877119905 926min (3) caffeic acid 119877

119905

1112min (4) ferulic acid 119877119905 1587min (5) rutin hydrate 119877

119905

1599min (6) sinapic acid 119877119905 1615min (7) hyperoside 119877

119905

1649min (8) quercetin 3-beta-D-glucoside119877119905 1682min (9)

vanillylacetone 119877119905 1792min (10) quercitrin 119877

119905 1890min

(11) calycosin 119877119905 2381min (12) quercetin 119877

119905 2431min (13)

kaempferol 119877119905 2837min (14) eugenol 119877

119905 2898min (15)

curcumin 119877119905 4020min (16) magnolol 119877

119905 4398min and

(17) L-(+)-ascorbic acid) (not detected L-(+)-ascorbic acid)(each concentration 100 ppm) are presented in Figure 3(a) Ofthese seven compounds that showed excellent activity werefurther analysed Several eluted substances were detected inthe 7 compounds including (1) gallic acid hydrate (210 nm)(2) (+)-catechin hydrate (210 nm) (3) caffeic acid (320 nm)(4) rutin hydrate (210 nm) (5) hyperoside (210 nm) (6)quercetin (210 nm) and (7) kaempferol (254 nm) which areobserved as a positive signal on theUVdetector (210 254 and320 nm) The retention times (119877

119905) of (1) gallic acid hydrate

(119877119905562min) (2) (+)-catechin hydrate (119877

119905946min) (3)

caffeic acid (1198771199051112min) (4) rutin hydrate (119877

1199051586min) (5)

hyperoside (1198771199051626min) (6) quercetin (119877

1199052358min) and

(7) kaempferol (1198771199052830min) are reported in Figure 3(b)The

other compounds exhibited a hydrogen-donating capacity(negative peak) towards the ABTS radical at the appliedconcentrationThese results therefore reveal that this methodcan be applied for quick screening of antioxidant activity ormore precisely of radical-scavenging activity (Table 3) Thiswork confirms the feasibility of assessing the bioactivity ofspecific phytochemicals by using an online screening HPLC-ABTS assay This method was successfully applied to screenand identify the antioxidant activity of natural substancesand OMH complex mixtures [5 15] The results show theshape of the chromatogram by the competitive adsorptionand desorption In addition the screening methods for therapid activity can provide useful information in basic researchon natural products chemistry and isolation analysis It isconsidered that the data will only be valuable in engineering

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

27 Cell Viability Assay Cytotoxicity was analyzed usingCCK (Dojindo Japan) 17 compounds were added to the cellsand incubated for 24 hours at 37∘C with 5 CO

2 10 120583L CCK

solutionswere added to eachwell and the cells were incubatedfor another 1 h Then the optical density was read at 450 nmusing an ELISA reader (Infinite M200 Tecan MannedorfSwitzerland)

28 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated with17 compounds and stimulated with LPS for 24 hours Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

29 Inflammatory Cytokine Determination Cells were seed-ed at a density of 5 times 105 cellsmL in 24-well culture plates andpretreated with three concentrations of 17 compounds for 1hour before LPS stimulation ELISA plates (Nunc RoskildeDenmark) were coated overnight at 4∘C with capture anti-body diluted in coating buffer (01M carbonate pH 95) andthen washed five times with phosphate-buffered saline (PBS)containing 005 Tween 20The nonspecific protein-bindingsites were blocked with assay diluent buffer (PBS containing10 FBS pH 70) for more than 1 hour Promptly samplesand standards were added to the wells After overnight ofincubation at 4∘C theworking detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 hour Subsequently substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in darkness before the reaction was stopped withstop solution (2N H

3PO4) The optical density was read at

450 nm [19]

210 Statistical Analysis The results are expressed as meanplusmn SD values for the number (119899 = 3 times) of experimentsStatistical significance was compared for each treated groupwith the control and determined by Studentrsquos 119905-tests Eachexperiment was repeated at least three times to yield com-parable results Values with 119901 lt 001 and 119901 lt 0001 wereconsidered significant

3 Result and Discussion

Several researches have revealed that a variety of natural andchemical compounds from natural substance crops fruitsvegetables and oriental medicinal herbs (OMH) have shownhigh antioxidant activity after the extraction and purificationprocesses [3] In addition variousmethods have been used todetermine the antioxidant activity of natural substance cropsfoods and plant products [1 4] The present study used threedifferent methods to evaluate the antioxidant activity con-sisting of DPPH radical-scavenging activity ABTS radical-scavenging activity and online screeningHPLC-ABTS assaysTherefore this work documented for the first time a

comparison of the antioxidant activities of one hundred kindsof pure chemical compounds

31 Offline DPPH and ABTS Assay Antioxidant activityreportedly has an effect on various different bioactivities(whitening anti-inflammation and high blood pressure)The antioxidant activity of natural substances and OMHhas been widely studied and thus this study identifies theantioxidant activity of standard substances that have origi-nated from various OMH in terms of their DPPH radical-scavenging activity and ABTS radical-scavenging activityTheDPPHandABTS radical-scavenging assays offer a redox-functioned proton ion for unstable free radicals and playa critical role in stabilizing detrimental free radicals in thehuman body This is generally achieved by taking advantageof the fact that unstable violet DPPH and ABTS free radicalstransform to stable yellow DPPH free radicals by acceptinga hydrogen ion from antioxidants In terms of the antiox-idant activity the ability to eliminate hydroxyl radicals orsuperoxide radicals through a physiologic action or throughoxidation is evaluated and a high index indicates a strongantioxidant activity Table 1 provides the results of the DPPHand ABTS radical scavenging in 100 ppm for one hundredkinds of pure standard compounds used in this study 17 com-pounds among the one hundred kinds of pure standard com-pounds ((1) (+)-catechin hydrate (2) calycosin (3) caffeicacid (4) curcumin (5) eugenol (6) ferulic acid (7) gallic acidhydrate (8) hyperoside (9) kaempferol (10) magnolol (11)quercetin (12) quercetin 3-beta-D-glucoside (13) quercitrin(14) rutin hydrate (15) sinapic acid (16) vanillylacetoneand (17) L-(+)-ascorbic acid) have an antioxidant activity ofover 90 Table 2 shows the IC

50rate of compounds with

a strong antioxidant activity The ABTS radical-scavengingmeasurement method which is commonly used to evaluatethe antioxidant activity takes advantage of the fact that ABTSfree radicals become stable by accepting a hydrogen ion fromthe antioxidant losing their blue colors Moreover in theABTS assay as well as in the DPPH assay when antioxidantactivity occurs the ability to eliminate hydroxyl radicals orsuperoxide radicals through physiologic action or oxidationis evaluated with a high index indicating a strong antioxidantactivity Each of the DPPH and ABTS are compounds thathave a proton free radical with a characteristic absorptionthat decreases significantly upon exposure to proton radicalscavengers DPPH and ABTS radical-scavenging throughantioxidant activity are well known to be attributable to theirhydrogen-donating ability (Tables 1 and 2)The concentrationof these compounds required to inhibit 50 of the radical-scavenging effect (IC

50) has been determined by testing a

series of concentrations In particular the sample with (+)-catechin hydrate caffeic acid eugenol gallic acid hydratehyperoside quercetin vanillylacetone and L-(+)-ascorbicacid compounds showed the strongest activity In additionthe 17 compounds showed better inhibitory activity againstABTS radical than the DPPH radicals That is the ABTSassay is more sensitive in identifying antioxidant activitybecause of the faster reaction kinetics and its response toantioxidants is higher Consequently this study shows thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Free radical-scavenging capacities of antioxidant activity available measured with DPPH and ABTS assay on microwell plate

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS1 Albiflorin Wako 20813 minus015 plusmn 039 014 plusmn 5272 Alisol A Wako 20379 minus047 plusmn 071 1292 plusmn 0863 Alisol B Wako 21155 minus166 plusmn 023 1278 plusmn 1354 Amygdalin KFDA 21861 minus085 plusmn 067 1231 plusmn 0035 Anthraquinone Wako 48028 012 plusmn 070 086 plusmn 5126 Atractylenolide iii Chem Faces 40271 minus207 plusmn 053 134 plusmn 8297 Aucubin Wako 28874 minus164 plusmn 079 122 plusmn 9748 Baicalein KFDA 37004 9584 plusmn 015 9937 plusmn 0129 Benzoic acid Sigma 81887 188 plusmn 042 380 plusmn 02610 Berberine Chem Faces 29730 064 plusmn 043 8468 plusmn 25511 Berberine HCl KFDA 26895 minus030 plusmn 030 874 plusmn 83812 Caffeic acid Sigma 55506 9591 plusmn 016 9966 plusmn 02413 Calycosin Chem Faces 35179 6451 plusmn 059 9919 plusmn 00514 Catalpol Wako 27599 minus255 plusmn 047 112 plusmn 96215 Chrysin Sigma 39333 045 plusmn 053 9913 plusmn 00616 Cimifugin Chem Faces 32646 minus089 plusmn 021 1439 plusmn 18117 Cinnamyl alcohol Sigma 74527 minus003 plusmn 037 1518 plusmn 07518 cis-Inositol Sigma 55506 015 plusmn 026 054 plusmn 12219 Costunolide Sigma 43044 286 plusmn 017 2013 plusmn 87420 Crocin Sigma 10236 2443 plusmn 128 4773 plusmn 06721 Curcumin Sigma 27146 9750 plusmn 063 9997 plusmn 01622 (+)-Catechin hydrate TCI 34451 9450 plusmn 016 9915 plusmn 00623 18-Dihydroxy-3-methylanthraquinone Sigma 39333 minus094 plusmn 054 209 plusmn 26024 D-(minus)-Lactic acid Sigma 111012 251 plusmn 240 2829 plusmn 07425 D-(+)-Chiro-inositol Sigma 55506 049 plusmn 033 021 plusmn 04526 Daidzein Wako 39333 052 plusmn 061 9949 plusmn 04927 Decursin KFDA 30454 minus301 plusmn 091 097 plusmn 27528 Decursinol Chem Faces 40607 minus159 plusmn 103 1512 plusmn 18129 Dioscin Sigma 11507 minus293 plusmn 130 043 plusmn 27530 Diosgenin Sigma 24118 minus138 plusmn 070 196 plusmn 92431 D-Pinitol Sigma 51499 minus227 plusmn 033 1272 plusmn 18532 67-Dimethylesculetin RD Chemical 48499 minus274 plusmn 054 131 plusmn 02133 (minus)-Epicatechin Sigma 34451 9451 plusmn 041 9961 plusmn 01634 (minus)-Epigallocatechin gallate Sigma 21816 9569 plusmn 014 9951 plusmn 02435 Eleutheroside B Wako 26855 minus204 plusmn 102 197 plusmn 28536 Emodin TCI 37004 220 plusmn 116 9127 plusmn 13937 Ephedrine-HCl KFDA 49581 minus211 plusmn 152 000 plusmn 01238 Ergosterol Chem Faces 25211 minus165 plusmn 134 055 plusmn 94639 Eugenol Sigma 60901 9372 plusmn 012 9991 plusmn 05540 Evodiamine KFDA 32964 421 plusmn 155 4076 plusmn 20941 Ferulic acid Sigma 51499 9512 plusmn 024 9896 plusmn 02742 Gallic acid hydrate TCI 58782 9556 plusmn 003 10130 plusmn 21243 Geniposide Chem Faces 25749 minus145 plusmn 090 122 plusmn 94944 Genistein TCI 37004 minus152 plusmn 030 9850 plusmn 04845 Genistin Wako 23128 223 plusmn 064 10065 plusmn 00346 Geraniol Sigma 11249 minus243 plusmn 167 1494 plusmn 095

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS47 Glabridin Wako 30829 4220 plusmn 088 10058 plusmn 00448 Glimepiride Sigma 20382 minus133 plusmn 063 784 plusmn 23049 Glycyrrhetic acid TCI 21246 066 plusmn 065 1023 plusmn 06250 Glycyrrhizin TCI 12152 159 plusmn 196 1236 plusmn 11551 Gomisin A KFDA 24012 180 plusmn 028 174 plusmn 02252 Gomisin N KFDA 24971 045 plusmn 051 380 plusmn 02153 Hesperidin KFDA 20223 3184 plusmn 037 10021 plusmn 00154 Hyperoside Chem Faces 21534 9316 plusmn 025 9962 plusmn 02055 21015840-Hydroxy-41015840-methoxy-acetophenone Sigma 60176 minus281 plusmn 028 9973 plusmn 10456 Icariin TCI 14778 382 plusmn 108 1460 plusmn 01257 Imperatorin Chem Faces 36999 minus071 plusmn 231 074 plusmn 951

58 Isoimperatorin Santa CruzBiotech 36999 027 plusmn 022 306 plusmn 217

59 Jujuboside A Chem Faces 8283 minus163 plusmn 035 026 plusmn 97460 Kaempferol Chem Faces 34936 9502 plusmn 022 9984 plusmn 04161 Liquiritigenin Chem Faces 39024 1226 plusmn 086 012 plusmn 06862 Loganin KFDA 25616 262 plusmn 128 580 plusmn 06263 Magnolol KFDA 37547 5450 plusmn 012 7774 plusmn 10664 Mevinolin Sigma 24719 minus026 plusmn 007 278 plusmn 069

65 Morroniside China LangChem Inc 24608 711 plusmn 058 1962 plusmn 183

66 Naringin Sigma 17226 305 plusmn 037 10036 plusmn 00567 Nodakenin Chem Faces 24486 068 plusmn 045 1570 plusmn 19268 Oleanolic acid Wako 21896 minus037 plusmn 054 000 plusmn 01269 Ononin Sigma 23234 223 plusmn 158 2291 plusmn 18970 Oxymatrine Chem Faces 37826 minus163 plusmn 017 072 plusmn 88571 Oxypeucedanin Chem Faces 34931 minus011 plusmn 027 1910 plusmn 23872 Paeoniflorin TCI 20813 429 plusmn 143 2203 plusmn 09173 Paeonol Sigma 60179 099 plusmn 221 1979 plusmn 23574 Palmatine chloride hydrate Sigma 25782 257 plusmn 242 8415 plusmn 24375 Palmatine Chem Faces 29205 012 plusmn 023 6805 plusmn 36376 p-Coumaric acid Sigma 60916 889 plusmn 004 3852 plusmn 08477 Poncirin KFDA 16819 minus144 plusmn 127 8420 plusmn 36678 Puerarin Wako 24017 989 plusmn 016 10062 plusmn 00679 Quercetin Sigma 33086 9602 plusmn 008 10018 plusmn 00680 Quercetin 3-120573-D-glucoside Sigma 21534 9443 plusmn 002 9994 plusmn 00681 Quercitrin hydrate Sigma 22303 9332 plusmn 004 9912 plusmn 01482 Rutaecarpine KFDA 34804 minus141 plusmn 060 9723 plusmn 06383 Rutin hydrate Sigma 16379 9357 plusmn 013 10010 plusmn 00284 Saikosaponin a KFDA 12804 minus067 plusmn 079 1617 plusmn 16285 Salicylaldehyde Sigma 81887 207 plusmn 004 10036 plusmn 00586 Schisandrin KFDA 23121 minus239 plusmn 089 1029 plusmn 24387 Sennoside A KFDA 11591 minus309 plusmn 064 8786 plusmn 32188 Sequoyitol GlycoSyn 51499 minus320 plusmn 137 1028 plusmn 20789 Sinapic acid Fluka 44599 9484 plusmn 033 9999 plusmn 02390 Spinosin Chem Faces 16433 minus096 plusmn 226 8016 plusmn 22091 Tetrandrine Fluka 16058 6083 plusmn 211 10028 plusmn 006

Evidence-Based Complementary and Alternative Medicine 7

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS92 trans-Cinnamaldehyde Sigma 75666 283 plusmn 069 1900 plusmn 13793 trans-Cinnamic acid Sigma 67495 019 plusmn 015 002 plusmn 94594 Uric acid Sigma 59485 4037 plusmn 197 9833 plusmn 03195 Vanillylacetone Sigma 51485 9378 plusmn 006 9992 plusmn 02496 Wogonin KFDA 35179 102 plusmn 051 10057 plusmn 01397 Wogonoside Chem Faces 21721 1084 plusmn 010 9862 plusmn 03298 Ziyuglycoside I Chem Faces 13038 minus171 plusmn 100 001 plusmn 98599 Z-Ligustilide Chem Faces 53129 192 plusmn 076 6869 plusmn 214100 Ascorbic acid (Vitamin C) Daejung 56779 9956 plusmn 089 9989 plusmn 004

Table 2 Antioxidant activity of 17 compounds with offline DPPHand ABTS IC50 assay

Number NameRadical- scavenging IC50

(120583gmL)DPPH ABTS

1 (+)-Catechin hydrate 525 plusmn 031 312 plusmn 0512 Calycosin 6188 plusmn 119 3321 plusmn 3593 Caffeic acid 450 plusmn 030 159 plusmn 0064 Curcumin 889 plusmn 024 499 plusmn 0455 Eugenol 522 plusmn 025 322 plusmn 0456 Ferulic acid 949 plusmn 021 199 plusmn 0127 Gallic acid hydrate 156 plusmn 038 103 plusmn 0258 Hyperoside 544 plusmn 036 354 plusmn 0399 Kaempferol 778 plusmn 030 370 plusmn 01510 Magnolol 8557 plusmn 140 837 plusmn 05611 Quercetin 266 plusmn 024 189 plusmn 03312 Quercetin 3-120573-D-glucoside 705 plusmn 059 359 plusmn 08913 Quercitrin hydrate 755 plusmn 077 423 plusmn 08414 Rutin hydrate 972 plusmn 106 468 plusmn 12415 Sinapic acid 826 plusmn 041 536 plusmn 08516 Vanillylacetone 569 plusmn 000 345 plusmn 00517 Ascorbic acid (Vitamin C) 365 plusmn 023 265 plusmn 046

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015 120583gmL respectively

32 Online HPLC-ABTS Assay Analysis The most popularapproach utilises a relatively stable coloured radical solutionof DPPH or ABTS which is added postcolumn to the HPLCflow Drug food functional material and plant and OMHsamples are evaluated for their antioxidant capacities accord-ing to a variety of antioxidant activity test methods suchas those for ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20] The HPLC analysesreact postcolumn with the ABTS and the reduction isdetected as a negative peak by a VIS absorbance detector at

734 nm The ABTS radical is much more water soluble thanDPPH [13] so ABTS better shows the details of an onlineHPLC-ABTS assay system that analysed the 17 given com-pounds (Figures 3(a) and 3(b)) Combined UV (positive sig-nals) andABTS quenching (negative signals) chromatogramsof the 17 compounds ((1) gallic acid hydrate 119877

119905 546min

(2) (+)-catechin hydrate 119877119905 926min (3) caffeic acid 119877

119905

1112min (4) ferulic acid 119877119905 1587min (5) rutin hydrate 119877

119905

1599min (6) sinapic acid 119877119905 1615min (7) hyperoside 119877

119905

1649min (8) quercetin 3-beta-D-glucoside119877119905 1682min (9)

vanillylacetone 119877119905 1792min (10) quercitrin 119877

119905 1890min

(11) calycosin 119877119905 2381min (12) quercetin 119877

119905 2431min (13)

kaempferol 119877119905 2837min (14) eugenol 119877

119905 2898min (15)

curcumin 119877119905 4020min (16) magnolol 119877

119905 4398min and

(17) L-(+)-ascorbic acid) (not detected L-(+)-ascorbic acid)(each concentration 100 ppm) are presented in Figure 3(a) Ofthese seven compounds that showed excellent activity werefurther analysed Several eluted substances were detected inthe 7 compounds including (1) gallic acid hydrate (210 nm)(2) (+)-catechin hydrate (210 nm) (3) caffeic acid (320 nm)(4) rutin hydrate (210 nm) (5) hyperoside (210 nm) (6)quercetin (210 nm) and (7) kaempferol (254 nm) which areobserved as a positive signal on theUVdetector (210 254 and320 nm) The retention times (119877

119905) of (1) gallic acid hydrate

(119877119905562min) (2) (+)-catechin hydrate (119877

119905946min) (3)

caffeic acid (1198771199051112min) (4) rutin hydrate (119877

1199051586min) (5)

hyperoside (1198771199051626min) (6) quercetin (119877

1199052358min) and

(7) kaempferol (1198771199052830min) are reported in Figure 3(b)The

other compounds exhibited a hydrogen-donating capacity(negative peak) towards the ABTS radical at the appliedconcentrationThese results therefore reveal that this methodcan be applied for quick screening of antioxidant activity ormore precisely of radical-scavenging activity (Table 3) Thiswork confirms the feasibility of assessing the bioactivity ofspecific phytochemicals by using an online screening HPLC-ABTS assay This method was successfully applied to screenand identify the antioxidant activity of natural substancesand OMH complex mixtures [5 15] The results show theshape of the chromatogram by the competitive adsorptionand desorption In addition the screening methods for therapid activity can provide useful information in basic researchon natural products chemistry and isolation analysis It isconsidered that the data will only be valuable in engineering

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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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

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

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Free radical-scavenging capacities of antioxidant activity available measured with DPPH and ABTS assay on microwell plate

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS1 Albiflorin Wako 20813 minus015 plusmn 039 014 plusmn 5272 Alisol A Wako 20379 minus047 plusmn 071 1292 plusmn 0863 Alisol B Wako 21155 minus166 plusmn 023 1278 plusmn 1354 Amygdalin KFDA 21861 minus085 plusmn 067 1231 plusmn 0035 Anthraquinone Wako 48028 012 plusmn 070 086 plusmn 5126 Atractylenolide iii Chem Faces 40271 minus207 plusmn 053 134 plusmn 8297 Aucubin Wako 28874 minus164 plusmn 079 122 plusmn 9748 Baicalein KFDA 37004 9584 plusmn 015 9937 plusmn 0129 Benzoic acid Sigma 81887 188 plusmn 042 380 plusmn 02610 Berberine Chem Faces 29730 064 plusmn 043 8468 plusmn 25511 Berberine HCl KFDA 26895 minus030 plusmn 030 874 plusmn 83812 Caffeic acid Sigma 55506 9591 plusmn 016 9966 plusmn 02413 Calycosin Chem Faces 35179 6451 plusmn 059 9919 plusmn 00514 Catalpol Wako 27599 minus255 plusmn 047 112 plusmn 96215 Chrysin Sigma 39333 045 plusmn 053 9913 plusmn 00616 Cimifugin Chem Faces 32646 minus089 plusmn 021 1439 plusmn 18117 Cinnamyl alcohol Sigma 74527 minus003 plusmn 037 1518 plusmn 07518 cis-Inositol Sigma 55506 015 plusmn 026 054 plusmn 12219 Costunolide Sigma 43044 286 plusmn 017 2013 plusmn 87420 Crocin Sigma 10236 2443 plusmn 128 4773 plusmn 06721 Curcumin Sigma 27146 9750 plusmn 063 9997 plusmn 01622 (+)-Catechin hydrate TCI 34451 9450 plusmn 016 9915 plusmn 00623 18-Dihydroxy-3-methylanthraquinone Sigma 39333 minus094 plusmn 054 209 plusmn 26024 D-(minus)-Lactic acid Sigma 111012 251 plusmn 240 2829 plusmn 07425 D-(+)-Chiro-inositol Sigma 55506 049 plusmn 033 021 plusmn 04526 Daidzein Wako 39333 052 plusmn 061 9949 plusmn 04927 Decursin KFDA 30454 minus301 plusmn 091 097 plusmn 27528 Decursinol Chem Faces 40607 minus159 plusmn 103 1512 plusmn 18129 Dioscin Sigma 11507 minus293 plusmn 130 043 plusmn 27530 Diosgenin Sigma 24118 minus138 plusmn 070 196 plusmn 92431 D-Pinitol Sigma 51499 minus227 plusmn 033 1272 plusmn 18532 67-Dimethylesculetin RD Chemical 48499 minus274 plusmn 054 131 plusmn 02133 (minus)-Epicatechin Sigma 34451 9451 plusmn 041 9961 plusmn 01634 (minus)-Epigallocatechin gallate Sigma 21816 9569 plusmn 014 9951 plusmn 02435 Eleutheroside B Wako 26855 minus204 plusmn 102 197 plusmn 28536 Emodin TCI 37004 220 plusmn 116 9127 plusmn 13937 Ephedrine-HCl KFDA 49581 minus211 plusmn 152 000 plusmn 01238 Ergosterol Chem Faces 25211 minus165 plusmn 134 055 plusmn 94639 Eugenol Sigma 60901 9372 plusmn 012 9991 plusmn 05540 Evodiamine KFDA 32964 421 plusmn 155 4076 plusmn 20941 Ferulic acid Sigma 51499 9512 plusmn 024 9896 plusmn 02742 Gallic acid hydrate TCI 58782 9556 plusmn 003 10130 plusmn 21243 Geniposide Chem Faces 25749 minus145 plusmn 090 122 plusmn 94944 Genistein TCI 37004 minus152 plusmn 030 9850 plusmn 04845 Genistin Wako 23128 223 plusmn 064 10065 plusmn 00346 Geraniol Sigma 11249 minus243 plusmn 167 1494 plusmn 095

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS47 Glabridin Wako 30829 4220 plusmn 088 10058 plusmn 00448 Glimepiride Sigma 20382 minus133 plusmn 063 784 plusmn 23049 Glycyrrhetic acid TCI 21246 066 plusmn 065 1023 plusmn 06250 Glycyrrhizin TCI 12152 159 plusmn 196 1236 plusmn 11551 Gomisin A KFDA 24012 180 plusmn 028 174 plusmn 02252 Gomisin N KFDA 24971 045 plusmn 051 380 plusmn 02153 Hesperidin KFDA 20223 3184 plusmn 037 10021 plusmn 00154 Hyperoside Chem Faces 21534 9316 plusmn 025 9962 plusmn 02055 21015840-Hydroxy-41015840-methoxy-acetophenone Sigma 60176 minus281 plusmn 028 9973 plusmn 10456 Icariin TCI 14778 382 plusmn 108 1460 plusmn 01257 Imperatorin Chem Faces 36999 minus071 plusmn 231 074 plusmn 951

58 Isoimperatorin Santa CruzBiotech 36999 027 plusmn 022 306 plusmn 217

59 Jujuboside A Chem Faces 8283 minus163 plusmn 035 026 plusmn 97460 Kaempferol Chem Faces 34936 9502 plusmn 022 9984 plusmn 04161 Liquiritigenin Chem Faces 39024 1226 plusmn 086 012 plusmn 06862 Loganin KFDA 25616 262 plusmn 128 580 plusmn 06263 Magnolol KFDA 37547 5450 plusmn 012 7774 plusmn 10664 Mevinolin Sigma 24719 minus026 plusmn 007 278 plusmn 069

65 Morroniside China LangChem Inc 24608 711 plusmn 058 1962 plusmn 183

66 Naringin Sigma 17226 305 plusmn 037 10036 plusmn 00567 Nodakenin Chem Faces 24486 068 plusmn 045 1570 plusmn 19268 Oleanolic acid Wako 21896 minus037 plusmn 054 000 plusmn 01269 Ononin Sigma 23234 223 plusmn 158 2291 plusmn 18970 Oxymatrine Chem Faces 37826 minus163 plusmn 017 072 plusmn 88571 Oxypeucedanin Chem Faces 34931 minus011 plusmn 027 1910 plusmn 23872 Paeoniflorin TCI 20813 429 plusmn 143 2203 plusmn 09173 Paeonol Sigma 60179 099 plusmn 221 1979 plusmn 23574 Palmatine chloride hydrate Sigma 25782 257 plusmn 242 8415 plusmn 24375 Palmatine Chem Faces 29205 012 plusmn 023 6805 plusmn 36376 p-Coumaric acid Sigma 60916 889 plusmn 004 3852 plusmn 08477 Poncirin KFDA 16819 minus144 plusmn 127 8420 plusmn 36678 Puerarin Wako 24017 989 plusmn 016 10062 plusmn 00679 Quercetin Sigma 33086 9602 plusmn 008 10018 plusmn 00680 Quercetin 3-120573-D-glucoside Sigma 21534 9443 plusmn 002 9994 plusmn 00681 Quercitrin hydrate Sigma 22303 9332 plusmn 004 9912 plusmn 01482 Rutaecarpine KFDA 34804 minus141 plusmn 060 9723 plusmn 06383 Rutin hydrate Sigma 16379 9357 plusmn 013 10010 plusmn 00284 Saikosaponin a KFDA 12804 minus067 plusmn 079 1617 plusmn 16285 Salicylaldehyde Sigma 81887 207 plusmn 004 10036 plusmn 00586 Schisandrin KFDA 23121 minus239 plusmn 089 1029 plusmn 24387 Sennoside A KFDA 11591 minus309 plusmn 064 8786 plusmn 32188 Sequoyitol GlycoSyn 51499 minus320 plusmn 137 1028 plusmn 20789 Sinapic acid Fluka 44599 9484 plusmn 033 9999 plusmn 02390 Spinosin Chem Faces 16433 minus096 plusmn 226 8016 plusmn 22091 Tetrandrine Fluka 16058 6083 plusmn 211 10028 plusmn 006

Evidence-Based Complementary and Alternative Medicine 7

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS92 trans-Cinnamaldehyde Sigma 75666 283 plusmn 069 1900 plusmn 13793 trans-Cinnamic acid Sigma 67495 019 plusmn 015 002 plusmn 94594 Uric acid Sigma 59485 4037 plusmn 197 9833 plusmn 03195 Vanillylacetone Sigma 51485 9378 plusmn 006 9992 plusmn 02496 Wogonin KFDA 35179 102 plusmn 051 10057 plusmn 01397 Wogonoside Chem Faces 21721 1084 plusmn 010 9862 plusmn 03298 Ziyuglycoside I Chem Faces 13038 minus171 plusmn 100 001 plusmn 98599 Z-Ligustilide Chem Faces 53129 192 plusmn 076 6869 plusmn 214100 Ascorbic acid (Vitamin C) Daejung 56779 9956 plusmn 089 9989 plusmn 004

Table 2 Antioxidant activity of 17 compounds with offline DPPHand ABTS IC50 assay

Number NameRadical- scavenging IC50

(120583gmL)DPPH ABTS

1 (+)-Catechin hydrate 525 plusmn 031 312 plusmn 0512 Calycosin 6188 plusmn 119 3321 plusmn 3593 Caffeic acid 450 plusmn 030 159 plusmn 0064 Curcumin 889 plusmn 024 499 plusmn 0455 Eugenol 522 plusmn 025 322 plusmn 0456 Ferulic acid 949 plusmn 021 199 plusmn 0127 Gallic acid hydrate 156 plusmn 038 103 plusmn 0258 Hyperoside 544 plusmn 036 354 plusmn 0399 Kaempferol 778 plusmn 030 370 plusmn 01510 Magnolol 8557 plusmn 140 837 plusmn 05611 Quercetin 266 plusmn 024 189 plusmn 03312 Quercetin 3-120573-D-glucoside 705 plusmn 059 359 plusmn 08913 Quercitrin hydrate 755 plusmn 077 423 plusmn 08414 Rutin hydrate 972 plusmn 106 468 plusmn 12415 Sinapic acid 826 plusmn 041 536 plusmn 08516 Vanillylacetone 569 plusmn 000 345 plusmn 00517 Ascorbic acid (Vitamin C) 365 plusmn 023 265 plusmn 046

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015 120583gmL respectively

32 Online HPLC-ABTS Assay Analysis The most popularapproach utilises a relatively stable coloured radical solutionof DPPH or ABTS which is added postcolumn to the HPLCflow Drug food functional material and plant and OMHsamples are evaluated for their antioxidant capacities accord-ing to a variety of antioxidant activity test methods suchas those for ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20] The HPLC analysesreact postcolumn with the ABTS and the reduction isdetected as a negative peak by a VIS absorbance detector at

734 nm The ABTS radical is much more water soluble thanDPPH [13] so ABTS better shows the details of an onlineHPLC-ABTS assay system that analysed the 17 given com-pounds (Figures 3(a) and 3(b)) Combined UV (positive sig-nals) andABTS quenching (negative signals) chromatogramsof the 17 compounds ((1) gallic acid hydrate 119877

119905 546min

(2) (+)-catechin hydrate 119877119905 926min (3) caffeic acid 119877

119905

1112min (4) ferulic acid 119877119905 1587min (5) rutin hydrate 119877

119905

1599min (6) sinapic acid 119877119905 1615min (7) hyperoside 119877

119905

1649min (8) quercetin 3-beta-D-glucoside119877119905 1682min (9)

vanillylacetone 119877119905 1792min (10) quercitrin 119877

119905 1890min

(11) calycosin 119877119905 2381min (12) quercetin 119877

119905 2431min (13)

kaempferol 119877119905 2837min (14) eugenol 119877

119905 2898min (15)

curcumin 119877119905 4020min (16) magnolol 119877

119905 4398min and

(17) L-(+)-ascorbic acid) (not detected L-(+)-ascorbic acid)(each concentration 100 ppm) are presented in Figure 3(a) Ofthese seven compounds that showed excellent activity werefurther analysed Several eluted substances were detected inthe 7 compounds including (1) gallic acid hydrate (210 nm)(2) (+)-catechin hydrate (210 nm) (3) caffeic acid (320 nm)(4) rutin hydrate (210 nm) (5) hyperoside (210 nm) (6)quercetin (210 nm) and (7) kaempferol (254 nm) which areobserved as a positive signal on theUVdetector (210 254 and320 nm) The retention times (119877

119905) of (1) gallic acid hydrate

(119877119905562min) (2) (+)-catechin hydrate (119877

119905946min) (3)

caffeic acid (1198771199051112min) (4) rutin hydrate (119877

1199051586min) (5)

hyperoside (1198771199051626min) (6) quercetin (119877

1199052358min) and

(7) kaempferol (1198771199052830min) are reported in Figure 3(b)The

other compounds exhibited a hydrogen-donating capacity(negative peak) towards the ABTS radical at the appliedconcentrationThese results therefore reveal that this methodcan be applied for quick screening of antioxidant activity ormore precisely of radical-scavenging activity (Table 3) Thiswork confirms the feasibility of assessing the bioactivity ofspecific phytochemicals by using an online screening HPLC-ABTS assay This method was successfully applied to screenand identify the antioxidant activity of natural substancesand OMH complex mixtures [5 15] The results show theshape of the chromatogram by the competitive adsorptionand desorption In addition the screening methods for therapid activity can provide useful information in basic researchon natural products chemistry and isolation analysis It isconsidered that the data will only be valuable in engineering

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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of

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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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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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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS47 Glabridin Wako 30829 4220 plusmn 088 10058 plusmn 00448 Glimepiride Sigma 20382 minus133 plusmn 063 784 plusmn 23049 Glycyrrhetic acid TCI 21246 066 plusmn 065 1023 plusmn 06250 Glycyrrhizin TCI 12152 159 plusmn 196 1236 plusmn 11551 Gomisin A KFDA 24012 180 plusmn 028 174 plusmn 02252 Gomisin N KFDA 24971 045 plusmn 051 380 plusmn 02153 Hesperidin KFDA 20223 3184 plusmn 037 10021 plusmn 00154 Hyperoside Chem Faces 21534 9316 plusmn 025 9962 plusmn 02055 21015840-Hydroxy-41015840-methoxy-acetophenone Sigma 60176 minus281 plusmn 028 9973 plusmn 10456 Icariin TCI 14778 382 plusmn 108 1460 plusmn 01257 Imperatorin Chem Faces 36999 minus071 plusmn 231 074 plusmn 951

58 Isoimperatorin Santa CruzBiotech 36999 027 plusmn 022 306 plusmn 217

59 Jujuboside A Chem Faces 8283 minus163 plusmn 035 026 plusmn 97460 Kaempferol Chem Faces 34936 9502 plusmn 022 9984 plusmn 04161 Liquiritigenin Chem Faces 39024 1226 plusmn 086 012 plusmn 06862 Loganin KFDA 25616 262 plusmn 128 580 plusmn 06263 Magnolol KFDA 37547 5450 plusmn 012 7774 plusmn 10664 Mevinolin Sigma 24719 minus026 plusmn 007 278 plusmn 069

65 Morroniside China LangChem Inc 24608 711 plusmn 058 1962 plusmn 183

66 Naringin Sigma 17226 305 plusmn 037 10036 plusmn 00567 Nodakenin Chem Faces 24486 068 plusmn 045 1570 plusmn 19268 Oleanolic acid Wako 21896 minus037 plusmn 054 000 plusmn 01269 Ononin Sigma 23234 223 plusmn 158 2291 plusmn 18970 Oxymatrine Chem Faces 37826 minus163 plusmn 017 072 plusmn 88571 Oxypeucedanin Chem Faces 34931 minus011 plusmn 027 1910 plusmn 23872 Paeoniflorin TCI 20813 429 plusmn 143 2203 plusmn 09173 Paeonol Sigma 60179 099 plusmn 221 1979 plusmn 23574 Palmatine chloride hydrate Sigma 25782 257 plusmn 242 8415 plusmn 24375 Palmatine Chem Faces 29205 012 plusmn 023 6805 plusmn 36376 p-Coumaric acid Sigma 60916 889 plusmn 004 3852 plusmn 08477 Poncirin KFDA 16819 minus144 plusmn 127 8420 plusmn 36678 Puerarin Wako 24017 989 plusmn 016 10062 plusmn 00679 Quercetin Sigma 33086 9602 plusmn 008 10018 plusmn 00680 Quercetin 3-120573-D-glucoside Sigma 21534 9443 plusmn 002 9994 plusmn 00681 Quercitrin hydrate Sigma 22303 9332 plusmn 004 9912 plusmn 01482 Rutaecarpine KFDA 34804 minus141 plusmn 060 9723 plusmn 06383 Rutin hydrate Sigma 16379 9357 plusmn 013 10010 plusmn 00284 Saikosaponin a KFDA 12804 minus067 plusmn 079 1617 plusmn 16285 Salicylaldehyde Sigma 81887 207 plusmn 004 10036 plusmn 00586 Schisandrin KFDA 23121 minus239 plusmn 089 1029 plusmn 24387 Sennoside A KFDA 11591 minus309 plusmn 064 8786 plusmn 32188 Sequoyitol GlycoSyn 51499 minus320 plusmn 137 1028 plusmn 20789 Sinapic acid Fluka 44599 9484 plusmn 033 9999 plusmn 02390 Spinosin Chem Faces 16433 minus096 plusmn 226 8016 plusmn 22091 Tetrandrine Fluka 16058 6083 plusmn 211 10028 plusmn 006

Evidence-Based Complementary and Alternative Medicine 7

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS92 trans-Cinnamaldehyde Sigma 75666 283 plusmn 069 1900 plusmn 13793 trans-Cinnamic acid Sigma 67495 019 plusmn 015 002 plusmn 94594 Uric acid Sigma 59485 4037 plusmn 197 9833 plusmn 03195 Vanillylacetone Sigma 51485 9378 plusmn 006 9992 plusmn 02496 Wogonin KFDA 35179 102 plusmn 051 10057 plusmn 01397 Wogonoside Chem Faces 21721 1084 plusmn 010 9862 plusmn 03298 Ziyuglycoside I Chem Faces 13038 minus171 plusmn 100 001 plusmn 98599 Z-Ligustilide Chem Faces 53129 192 plusmn 076 6869 plusmn 214100 Ascorbic acid (Vitamin C) Daejung 56779 9956 plusmn 089 9989 plusmn 004

Table 2 Antioxidant activity of 17 compounds with offline DPPHand ABTS IC50 assay

Number NameRadical- scavenging IC50

(120583gmL)DPPH ABTS

1 (+)-Catechin hydrate 525 plusmn 031 312 plusmn 0512 Calycosin 6188 plusmn 119 3321 plusmn 3593 Caffeic acid 450 plusmn 030 159 plusmn 0064 Curcumin 889 plusmn 024 499 plusmn 0455 Eugenol 522 plusmn 025 322 plusmn 0456 Ferulic acid 949 plusmn 021 199 plusmn 0127 Gallic acid hydrate 156 plusmn 038 103 plusmn 0258 Hyperoside 544 plusmn 036 354 plusmn 0399 Kaempferol 778 plusmn 030 370 plusmn 01510 Magnolol 8557 plusmn 140 837 plusmn 05611 Quercetin 266 plusmn 024 189 plusmn 03312 Quercetin 3-120573-D-glucoside 705 plusmn 059 359 plusmn 08913 Quercitrin hydrate 755 plusmn 077 423 plusmn 08414 Rutin hydrate 972 plusmn 106 468 plusmn 12415 Sinapic acid 826 plusmn 041 536 plusmn 08516 Vanillylacetone 569 plusmn 000 345 plusmn 00517 Ascorbic acid (Vitamin C) 365 plusmn 023 265 plusmn 046

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015 120583gmL respectively

32 Online HPLC-ABTS Assay Analysis The most popularapproach utilises a relatively stable coloured radical solutionof DPPH or ABTS which is added postcolumn to the HPLCflow Drug food functional material and plant and OMHsamples are evaluated for their antioxidant capacities accord-ing to a variety of antioxidant activity test methods suchas those for ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20] The HPLC analysesreact postcolumn with the ABTS and the reduction isdetected as a negative peak by a VIS absorbance detector at

734 nm The ABTS radical is much more water soluble thanDPPH [13] so ABTS better shows the details of an onlineHPLC-ABTS assay system that analysed the 17 given com-pounds (Figures 3(a) and 3(b)) Combined UV (positive sig-nals) andABTS quenching (negative signals) chromatogramsof the 17 compounds ((1) gallic acid hydrate 119877

119905 546min

(2) (+)-catechin hydrate 119877119905 926min (3) caffeic acid 119877

119905

1112min (4) ferulic acid 119877119905 1587min (5) rutin hydrate 119877

119905

1599min (6) sinapic acid 119877119905 1615min (7) hyperoside 119877

119905

1649min (8) quercetin 3-beta-D-glucoside119877119905 1682min (9)

vanillylacetone 119877119905 1792min (10) quercitrin 119877

119905 1890min

(11) calycosin 119877119905 2381min (12) quercetin 119877

119905 2431min (13)

kaempferol 119877119905 2837min (14) eugenol 119877

119905 2898min (15)

curcumin 119877119905 4020min (16) magnolol 119877

119905 4398min and

(17) L-(+)-ascorbic acid) (not detected L-(+)-ascorbic acid)(each concentration 100 ppm) are presented in Figure 3(a) Ofthese seven compounds that showed excellent activity werefurther analysed Several eluted substances were detected inthe 7 compounds including (1) gallic acid hydrate (210 nm)(2) (+)-catechin hydrate (210 nm) (3) caffeic acid (320 nm)(4) rutin hydrate (210 nm) (5) hyperoside (210 nm) (6)quercetin (210 nm) and (7) kaempferol (254 nm) which areobserved as a positive signal on theUVdetector (210 254 and320 nm) The retention times (119877

119905) of (1) gallic acid hydrate

(119877119905562min) (2) (+)-catechin hydrate (119877

119905946min) (3)

caffeic acid (1198771199051112min) (4) rutin hydrate (119877

1199051586min) (5)

hyperoside (1198771199051626min) (6) quercetin (119877

1199052358min) and

(7) kaempferol (1198771199052830min) are reported in Figure 3(b)The

other compounds exhibited a hydrogen-donating capacity(negative peak) towards the ABTS radical at the appliedconcentrationThese results therefore reveal that this methodcan be applied for quick screening of antioxidant activity ormore precisely of radical-scavenging activity (Table 3) Thiswork confirms the feasibility of assessing the bioactivity ofspecific phytochemicals by using an online screening HPLC-ABTS assay This method was successfully applied to screenand identify the antioxidant activity of natural substancesand OMH complex mixtures [5 15] The results show theshape of the chromatogram by the competitive adsorptionand desorption In addition the screening methods for therapid activity can provide useful information in basic researchon natural products chemistry and isolation analysis It isconsidered that the data will only be valuable in engineering

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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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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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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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Table 1 Continued

Number Compounds names Compoundspurchased Concentration 120583M (120583molL) Radical scavenging ()

DPPH ABTS92 trans-Cinnamaldehyde Sigma 75666 283 plusmn 069 1900 plusmn 13793 trans-Cinnamic acid Sigma 67495 019 plusmn 015 002 plusmn 94594 Uric acid Sigma 59485 4037 plusmn 197 9833 plusmn 03195 Vanillylacetone Sigma 51485 9378 plusmn 006 9992 plusmn 02496 Wogonin KFDA 35179 102 plusmn 051 10057 plusmn 01397 Wogonoside Chem Faces 21721 1084 plusmn 010 9862 plusmn 03298 Ziyuglycoside I Chem Faces 13038 minus171 plusmn 100 001 plusmn 98599 Z-Ligustilide Chem Faces 53129 192 plusmn 076 6869 plusmn 214100 Ascorbic acid (Vitamin C) Daejung 56779 9956 plusmn 089 9989 plusmn 004

Table 2 Antioxidant activity of 17 compounds with offline DPPHand ABTS IC50 assay

Number NameRadical- scavenging IC50

(120583gmL)DPPH ABTS

1 (+)-Catechin hydrate 525 plusmn 031 312 plusmn 0512 Calycosin 6188 plusmn 119 3321 plusmn 3593 Caffeic acid 450 plusmn 030 159 plusmn 0064 Curcumin 889 plusmn 024 499 plusmn 0455 Eugenol 522 plusmn 025 322 plusmn 0456 Ferulic acid 949 plusmn 021 199 plusmn 0127 Gallic acid hydrate 156 plusmn 038 103 plusmn 0258 Hyperoside 544 plusmn 036 354 plusmn 0399 Kaempferol 778 plusmn 030 370 plusmn 01510 Magnolol 8557 plusmn 140 837 plusmn 05611 Quercetin 266 plusmn 024 189 plusmn 03312 Quercetin 3-120573-D-glucoside 705 plusmn 059 359 plusmn 08913 Quercitrin hydrate 755 plusmn 077 423 plusmn 08414 Rutin hydrate 972 plusmn 106 468 plusmn 12415 Sinapic acid 826 plusmn 041 536 plusmn 08516 Vanillylacetone 569 plusmn 000 345 plusmn 00517 Ascorbic acid (Vitamin C) 365 plusmn 023 265 plusmn 046

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and 370 plusmn015 120583gmL respectively

32 Online HPLC-ABTS Assay Analysis The most popularapproach utilises a relatively stable coloured radical solutionof DPPH or ABTS which is added postcolumn to the HPLCflow Drug food functional material and plant and OMHsamples are evaluated for their antioxidant capacities accord-ing to a variety of antioxidant activity test methods suchas those for ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging [20] The HPLC analysesreact postcolumn with the ABTS and the reduction isdetected as a negative peak by a VIS absorbance detector at

734 nm The ABTS radical is much more water soluble thanDPPH [13] so ABTS better shows the details of an onlineHPLC-ABTS assay system that analysed the 17 given com-pounds (Figures 3(a) and 3(b)) Combined UV (positive sig-nals) andABTS quenching (negative signals) chromatogramsof the 17 compounds ((1) gallic acid hydrate 119877

119905 546min

(2) (+)-catechin hydrate 119877119905 926min (3) caffeic acid 119877

119905

1112min (4) ferulic acid 119877119905 1587min (5) rutin hydrate 119877

119905

1599min (6) sinapic acid 119877119905 1615min (7) hyperoside 119877

119905

1649min (8) quercetin 3-beta-D-glucoside119877119905 1682min (9)

vanillylacetone 119877119905 1792min (10) quercitrin 119877

119905 1890min

(11) calycosin 119877119905 2381min (12) quercetin 119877

119905 2431min (13)

kaempferol 119877119905 2837min (14) eugenol 119877

119905 2898min (15)

curcumin 119877119905 4020min (16) magnolol 119877

119905 4398min and

(17) L-(+)-ascorbic acid) (not detected L-(+)-ascorbic acid)(each concentration 100 ppm) are presented in Figure 3(a) Ofthese seven compounds that showed excellent activity werefurther analysed Several eluted substances were detected inthe 7 compounds including (1) gallic acid hydrate (210 nm)(2) (+)-catechin hydrate (210 nm) (3) caffeic acid (320 nm)(4) rutin hydrate (210 nm) (5) hyperoside (210 nm) (6)quercetin (210 nm) and (7) kaempferol (254 nm) which areobserved as a positive signal on theUVdetector (210 254 and320 nm) The retention times (119877

119905) of (1) gallic acid hydrate

(119877119905562min) (2) (+)-catechin hydrate (119877

119905946min) (3)

caffeic acid (1198771199051112min) (4) rutin hydrate (119877

1199051586min) (5)

hyperoside (1198771199051626min) (6) quercetin (119877

1199052358min) and

(7) kaempferol (1198771199052830min) are reported in Figure 3(b)The

other compounds exhibited a hydrogen-donating capacity(negative peak) towards the ABTS radical at the appliedconcentrationThese results therefore reveal that this methodcan be applied for quick screening of antioxidant activity ormore precisely of radical-scavenging activity (Table 3) Thiswork confirms the feasibility of assessing the bioactivity ofspecific phytochemicals by using an online screening HPLC-ABTS assay This method was successfully applied to screenand identify the antioxidant activity of natural substancesand OMH complex mixtures [5 15] The results show theshape of the chromatogram by the competitive adsorptionand desorption In addition the screening methods for therapid activity can provide useful information in basic researchon natural products chemistry and isolation analysis It isconsidered that the data will only be valuable in engineering

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

8 Evidence-Based Complementary and Alternative Medicine

Table 3 Simultaneous identification of antioxidant activity with online screening HPLC-ABTS assay

Compounds UV wavelength (nm) Retention time (min) Peak area (mAu) PositiveSD

NegativeSDPositive (average) Negative (average)

Gallic acid hydrate 210 5623 72116 51624 0054 0405(+)-Catechin hydrate 210 9463 75974 57981 0076 0328Caffeic acid 320 11123 108475 57808 0048 0433Rutin hydrate 210 15860 51185 13241 0393 0023Hyperoside 210 16263 80346 15631 0017 0213Quercetin 210 23583 109672 22155 0101 0067Kaempferol 254 28303 56806 30651 0143 0071

0 5 10 15 20 25 30 35 40 45 50 55 60minus100minus80minus60minus40minus20

020

Inte

nsity

(mAU

)In

tens

ity (m

AU)

Time (min)

Time (min)0 5 10 15 20 25 30 35 40 45 50 55 60

0100200300400500

12

11

1410 1615

1392

68

73

1

4 + 5

17 compound mixture 280nm

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60minus400minus300minus200minus100

0100

Inte

nsity

(mAU

)

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 600

100200300400500

Kaem

pfer

ol

Que

rcet

in

Hyp

eros

ide

Rutin

hyd

rate Ca

ffeic

acid

Gal

lic ac

id h

ydra

te

Time (min)In

tens

ity (m

AU) 7 compound mixture 280nm

(+)-

Cate

chin

hyd

rate

(b)

Figure 3 Identification antioxidant activity of online screening HPLC-ABTS assay ((a) simultaneous analysis of 17 compounds (1) gallicacid hydrate (2) (+)-catechin hydrate (3) caffeic acid (4) ferulic acid (5) rutin hydrate (6) sinapic acid (7) hyperoside (8) quercetin 3-120573-D-glucoside (9) vanillylacetone (10) quercitrin hydrate (11) calycosin (12) quercetin (13) kaempferol (14) eugenol (15) curcumin (16)magnolol and (17) ascorbic acid (not detected) (b) simultaneous analysis of 7 compounds)

and also very useful as functional materials and pharmaceu-tical materials in commercial processes

33 Anti-Inflammatory Activity Screening

331 Effect of 17 Compounds on RAW2647 Cell Viability Weevaluated the cytotoxicity of the 17 compounds by using CCKto determine the optimal concentration that would be effec-tive in providing anti-inflammatory activity with a minimumtoxicity As shown in Figure 4(a) kaempferol quercetinand curcumin show toxicity at a concentration of 10 120583gmLAlso quercetin 3-beta-D-glucoside has a strong toxicity onmacrophage viability at 5120583gmL or more Vanillylacetonehyperoside gallic acid hydrate sinapic acid rutin hydrateferulic acid (+)-catechin hydrate ascorbic acid calycosincaffeic acid magnolol quercitrin hydrate and eugenol didnot affect cell viability up to 10 120583gmL indicating that these13 compounds are not toxic to cells

332 Effect of the 17 Compounds on NO Production inLPS-Stimulated RAW 2647 Macrophages We evaluated theeffects of 17 compounds on NO secretion in LPS-stimulatedRAW 2647 cells The cells were pretreated with 17 com-pounds at concentrations of 1 5 and 10 120583gmL prior to LPSstimulation and NO production was also measured Weemployed 10 120583Mdexamethasone as a positive control since itis widely used as an anti-inflammatory agent As shown inFigure 4(b) vanillylacetone gallic acid hydrate kaempferolquercetin magnolol and curcumin exhibit a strong inhibi-tory effect on NO secretion upon LPS stimulation The inhi-bitory effects of 10 120583gmL kaempferol quercetin and cur-cumin on NO production were a result of their cytotox-icity However kaempferol quercetin and curcumin exerteffective inhibition at concentrations of 1 and 5 120583gmLIn particular magnolol strongly inhibited NO productionin a dose-dependent manner without toxicity Hyperosidesinapic acid rutin hydrate ferulic acid (+)-catechin hydrate

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

Evidence-Based Complementary and Alternative Medicine 9

020406080

100120140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

0

20

40

60

80

100

120

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

0

20

40

60

80

100

120

140

1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Cel

l via

bilit

y (

)C

ell v

iabi

lity

()

Cel

l via

bilit

y (

)

Con

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(120583gmL)

(120583gmL)

(120583gmL)

(+)-

Cate

chin

hyd

rate

Que

rcet

in3

-120573-

D-g

luco

side

(a)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

Nitr

ic o

xide

(120583M

)

0

20

40

60

80

100

120

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

(120583gmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowast lowastlowastlowastlowast lowastlowast lowastlowast

(120583gmL)

LPS (200ngmL)

LPS (200ngmL)

LPS (200ngmL)

Nitr

ic o

xide

(120583M

)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(b)

Figure 4 Effect of 17 compounds on (a) cell viability andLPS-induced (b)NOproduction inRAW2647 cells RAW2647 cells were pretreatedwith 17 compounds for 1 hour before incubation with LPS for 24 hours (a) Cytotoxicity was evaluated by a CCK (b)The culture supernatantwas analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SE values of triplicatedetermination from independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparing with LPS-stimulation value

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

10 Evidence-Based Complementary and Alternative Medicine

020406080

100120140160180200

10 1 5 10 1 5

(a)

10 1 5 10 1 5 10 1 5 10lowastlowast

lowastlowast

lowastlowast

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

LPS (200ngmL)

(120583gmL)

TNF-120572

(pg

mL)

020406080

100120140160180200

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

(b)

LPS (200ngmL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)0

20406080

100120140

180160

IL-6

(pg

mL)

020406080

100120140160

IL-6

(pg

mL)

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

lowastlowast

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

(120583gmL)

TNF-120572

(pg

mL)

LPS (200ngmL)

020406080

100120140

IL-6

(pg

mL)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowast lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

Figure 5 Continued

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

Evidence-Based Complementary and Alternative Medicine 11

0

20

40

60

80

100

120

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Vani

llyla

ceto

ne

Hyp

eros

ide

Gal

lic ac

id h

ydra

te

Sina

pic a

cid

Rutin

hyd

rate

Feru

lic ac

id

(120583gmL)

LPS (200ngmL)

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10(120583gmL)

Con

LP

SD

ex

Kaem

pfer

ol

Asc

orbi

c aci

d

Caly

cosin

Que

rcet

in

Caffe

ic ac

id

(+)-

Cate

chin

hyd

rate

LPS (200ngmL)

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

(c)

LPS (200ngmL)

10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10

Con

LP

SD

ex

Mag

nolo

l

Curc

umin

Que

rcitr

in h

ydra

te

Euge

nol

Que

rcet

in3

-120573-

D-g

luco

side

(120583gmL)

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

0

20

40

60

80

100

120

IL-1120573

(pg

mL)

Figure 5 Effect of 17 compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokine inmacrophages Cells were pretreated with17 compounds for 1 hour before being incubated with LPS for 24 hours Production of cytokines was measured by ELISA Data shows meanplusmn SE values of triplicate determinations from three independent experiments lowast119901 lt 001 and lowastlowast119901 lt 0001 were calculated from comparingwith LPS-stimulation value

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

12 Evidence-Based Complementary and Alternative Medicine

ascorbic acid calycosin caffeic acid quercitrin hydrate euge-nol and quercetin 3-beta-D-glucoside do not show remark-able suppressive effects

333 Effect of the 17 Compounds on LPS-Induced Inflam-matory Cytokines Production Next we investigated theinhibitory effect of the 17 compounds on the production ofinflammatory cytokines including TNF-120572 IL-6 and IL-1120573which are the other parameters of the inflammation Gallicacid hydrate exerts an inhibitory effect on theTNF-120572 cytokineproduction at all concentrations in a dose-dependent man-ner In addition 5120583gmL kaempferol and 10 120583gmLmagnololshow a strong inhibitory effect (Figure 5(a)) As shown inFigure 5(b) vanillylacetone gallic acid hydrate kaempferoland quercetin significantly inhibited IL-6 cytokine secretionin a statistically significant dose-dependent manner Inaddition all of the compounds showed an inhibitory effect onIL-1120573 cytokine production Gallic acid hydrate kaempferolquercetin and magnolol were especially strong inhibitorsof IL-1120573 cytokine production in a dose-dependent manner(Figure 5(c))

4 Conclusions

This study provides a comparison of the free radical scav-engers in the one hundred kinds of pure chemical com-pounds through an offline DPPH radical-scavenging activityassay ABTS radical-scavenging activity assay and an onlinescreening HPLC-ABTS assay Here the IC

50rate of a more

practical substance is determined The results indicate thattheABTS assay IC

50values of gallic acid hydrate (+)-catechin

hydrate caffeic acid rutin hydrate hyperoside quercetinand kaempferol compounds were 103 plusmn 025 312 plusmn 051159 plusmn 006 468 plusmn 124 354 plusmn 039 189 plusmn 033 and370 plusmn 015 120583gmL respectively This testing methodologyprovided a useful tool to focus efforts on chemically activeradical-scavenging compounds with high kinetic rates andallowed quick gathering of useful information related to themolecular compounds in terms of their antioxidant activitypotential In addition there was a very small margin oferror between the results of the offline-ABTS assay andthose of the online screening HPLC-ABTS assay We alsoevaluated the effects of 17 compounds on NO secretionin LPS-stimulated RAW 2647 cells and the cytotoxicityof the 17 compounds using CCK to determine the opti-mal concentration that would be effective to provide anti-inflammatory activity with a minimum toxicityThese resultswill be compiled into a database and this method cantherefore be a powerful preselection tool for compoundsintended to be studied for their potential bioactivity andantioxidant activity related to their radical-scavenging capa-city

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was achieved at KM Application Center KIOMThe authors also acknowledge the support from the Study onDrug Efficacy Enhancement Using Bioconversion for HerbalMedicines (K15280) project

References

[1] K Ishimaru K Nishikawa T Omoto I Asai K Yoshihiraand K Shimomura ldquoTwo flavone 21015840-glucosides from Scutellariabaicalensisrdquo Phytochemistry vol 40 no 1 pp 279ndash281 1995

[2] H G Park S Y Yoon J Y Choi et al ldquoAnticonvulsant effect ofwogonin isolated from Scutellaria baicalensisrdquoEuropean Journalof Pharmacology vol 574 no 2-3 pp 112ndash119 2007

[3] J H Lee B W Lee B Kim et al ldquoChanges in phenolic com-pounds (Isoflavones and Phenolic acids) and antioxidant prop-erties in high-protein soybean (Glycine max L cv Saedanbaek)for different roasting conditionsrdquo Journal of the Korean Societyfor Applied Biological Chemistry vol 56 no 5 pp 605ndash612 2013

[4] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines a reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[5] K J Lee S D Choi and J Y Ma ldquoPhytochemical analysisof curcumin from turmeric by RP-HPLCrdquo Asian Journal ofChemistry vol 25 no 2 pp 995ndash998 2013

[6] L-L Hu Q-Y Ma S-Z Huang et al ldquoTwo new phenoliccompounds from the fruiting bodies of Ganoderma tropicumrdquoBulletin of the Korean Chemical Society vol 34 no 3 pp 884ndash886 2013

[7] B Hazra M D Sarma and U Sanyal ldquoSeparation methods ofquinonoid constituents of plants used in Oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[8] O Adegoke and P B C Forbes ldquoChallenges and advances inquantum dot fluorescent probes to detect reactive oxygen andnitrogen species a reviewrdquoAnalytica Chimica Acta vol 862 pp1ndash13 2015

[9] P S Melo A P Massarioli C Denny et al ldquoWinery by-pro-ducts extraction optimization phenolic composition and cyto-toxic evaluation to act as a new source of scavenging of reactiveoxygen speciesrdquo Food Chemistry vol 181 pp 160ndash169 2015

[10] K S Kim S H Lee Y S Lee et al ldquoAnti-oxidant activitiesof the extracts from the herbs of Artemisia apiaceardquo Journal ofEthnopharmacology vol 85 no 1 pp 69ndash72 2003

[11] Z Gao K Huang X Yang and H Xu ldquoFree radical scavengingand antioxidant activities of flavonoids extracted from theradix of Scutellaria baicalensisGeorgirdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1472 no 3 pp 643ndash650 1999

[12] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] S-Y Shi Y-P Zhang X-Y Jiang et al ldquoCoupling HPLC toon-line post-column (bio)chemical assays for high-resolutionscreening of bioactive compounds from complex mixturesrdquoTrends in Analytical Chemistry vol 28 no 7 pp 865ndash877 2009

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

Evidence-Based Complementary and Alternative Medicine 13

[15] K J Lee N-Y Song Y C Oh W-K Cho and J Y Ma ldquoIsola-tion and bioactivity analysis of ethyl acetate extract from Acertegmentosum using in vitro assay and on-line screening HPLC-ABTS+ systemrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 150509 15 pages 2014

[16] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflam-matory effects of ethanol extract from Acer tegmentosumrdquoJournal of Ethnopharmacology vol 128 no 1 pp 139ndash147 2010

[17] N Pellegrini M Ying and C Rice-Evans ldquoScreening of dietarycarotenoids and carotenoid-rich fruits extract for antioxidantactivities applying 221015840-azobis (3-ethylbenzothine-6-surfonicacid) radical cation decolorization assayrdquoMethods in Enzymol-ogy vol 299 pp 384ndash389 1999

[18] A J Stewart W Mullen and A Crozier ldquoOn-line high-per-formance liquid chromatography analysis of the antioxidantactivity of phenolic compounds in green and black teardquoMolec-ular Nutrition amp Food Research vol 49 no 1 pp 52ndash60 2005

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fructus waterextract inhibits pro-inflammatory mediators in lipopolysac-charide-stimulated macrophagesrdquo Journal of Medicinal Foodvol 10 no 3 pp 460ndash466 2007

[20] A A KaracelikM Kucuk Z Iskefiyeli et al ldquoAntioxidant com-ponents of Viburnum opulus L determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methodsrdquoFood Chemistry vol 175 pp 106ndash114 2015

Submit your manuscripts athttpwwwhindawicom

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

Submit your manuscripts athttpwwwhindawicom

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