Bot513-02

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httpslidepdfcomreaderfullbot513-02 110

Botanical Studies (2010) 51 293-302

6These two authors contributed equally to this workCorresponding author E-mail yschangmailcmuedutw

Tel +886-4-22030380 Fax +886-4-22083362

INTRODUCTION

Diabetes mellitus is a common disease with many

complications such as atherosclerosis cardiac dysfunctionretinopathy neuropathy and nephropathy (Sowers et al2001) α-glucosidase (EC 32120) catalyzes the nal stepin the digestive process of carbohydrates Its inhibitors canretard the uptake of dietary carbohydrates and suppress

post pr andi al hy pe rglycemi a and could be us eful fo rtreating diabetic andor obese patients (Toeller 1994)α-Glucosidase inhibitors such as acarbose miglitol and

voglibose are known to reduce postprandial hyperglycemia primarily by interfering with the carbohydrate digestiveenzymes and by delaying glucose absorption Aldose

reductase (EC11121 AR) is the first enzyme in the polyol pathway it catalyzes the reduction of D-glucosefrom the aldehyde form into D-sorbitol with concomitantconversion of NADPH to NADP + (Kador et al 1985a b)It is generally accepted that this polyol pathway plays animportant role in the development of some degenerativecomplications of diabetes The elevated blood glucoselevel characteristic of diabetes mellitus causes signi cantfluxes of glucose through the polyol pathway in tissuessuch as nerves retina lens and kidneys where glucoseuptake is independent of insulin (Chihiro 1998) ThusAR inhibitors have attracted attentions in therapeuticresearches of diabetic complications

Activities of antioxidants α -Glucosidase inhibitors andaldose reductase inhibitors of the aqueous extracts offour Flemingia species in Taiwan

Po-Chow HSIEH 16 Guan-Jhong HUANG 16 Yu-Ling HO 2 Yaw-Huei LIN 3 Shyh-Shyun HUANG 1Ying-Chen CHIANG 1 Mu-Chuan TSENG 4 and Yuan-Shiun CHANG 15

1 Institute of Chinese Pharmaceutical Sciences College of Pharmacy China Medical University Taichung 40402 Taiwan2 Department of Nursing Hungkuang University Taichung 433 Taiwan3 Institute of Plant and Microbial Biology Academia Sinica Nankang Taipei 115 Taiwan4 Bureau of Food and Drug Analysis Department of Health Taipei 11561 Taiwan5Chinese Crude Drug Pharmacy China Medical University Hospital Taichung 40402 Taiwan

(Received October 9 2009 Accepted December 11 2009)

ABSTRACT The aim of this study was to examine the possible antioxidant and antidiabetic effects of

the aqueous extracts of four Flemingia species in Taiwan A number of methods were employed for thisinvestigation including ABTS radical monocation scavenging FRAP (ferric reducing antioxidant power)method DPPH (1 1-diphenyl-2-picrylhydrazyl) radical scavenging total polyphenol content total avonoidcontent total flavonol content and inhibition of α-glucosidase and aldose reductase methods The resultsshowed that the aqueous extract of Flemingia macrophylla (WFM) had the strongest antioxidant activity incomparison with the other extracts We also found that WFM had higher contents of polyphenol compoundsflavonoids and flavonols than the other extracts The correlation coefficient (R 2) values of TEAC (troloxequivalent antioxidant capacity) and FRAP showed high correlations (R 2=083) The R 2 values of TEAC andtotal polyphenol content showed a higher correlation (R 2=066) The R 2 values of TEAC and total avonoidcontent for the aqueous extracts was 094 The antidiabetic activities of the four Flemingia species werestudied in vitro using α-glucosidase and aldose reductase (AR) inhibitory methods WFM had the highestinhibitory activities on α-glucosidase and aldose reductase with IC 50 (concentration with 50 inhibition)of 15392 plusmn 020 μgmL and 7936 plusmn 320 μgmL respectively The positive control (genistein) had higherinhibitory activities on α-glucosidase and aldose reductase (IC 50 1665 plusmn 092 μgmL and 4562 plusmn 216 μgmL) respectively In LC-MS-MS analyses the chromatograms of WFM with the highest antioxidant andantidiabetic activity were established Genistein might be an important bioactive compound in WFM extractThis experiment suggests that WFM might serve as a good resource for future development of antioxidant andantidiabetic drugs

Keywords Aldose reductase Antioxidant α-glucosidase Flemingia Free radical Polyphenol

BIOCHEMISTRY

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294 Botanical Studies Vol 51 2010

The inhibitory effects of plant phytochemicalsincluding polyphenols against carbohydrate hydrolyzingenzymes contribute to the lowering of postprandialhyperglycemia in diabetic management as observed invivo (Griffiths and Moseley 1980) Further evidencethat polyphenolic compounds is linked to prevention ofdiabetic complications stems from in vivo studies with

diabetic rats polyphenolic compounds in plant materialsare capable of reducing oxidative stress by scavengingreactive oxygen species and preventing cell damage(Fukuda et al 2004) The polyphenolic compounds inedible plants are currently regarded as natural antioxidantsand their antioxidant activities are important for humanhealth (Sabu et al 2002)

Plants constitute a rich source of bioactive chemicals(Kador et al 1985a b Williamson et al 1992) Sincemany plants are largely free from adverse effects andhave excellent pharmacological actions they could

possibly lead to the development of new classes of saferantidiabetic agents or diabetic complication resolvingagents In addition some flavonoids and polyphenolsas well as sugar derivatives are found to be effective ininhibiting α-glucosidase and aldose reductase (Haraguchiet al 1996 Lee and Kim 2001) Therefore much efforthas been focused on plants to produce potentially useful

products such as commercial α-glucosidase inhibitors andaldose reductase inhibitors or lead compounds

The Fl emingi a genus known as lsquoI-Tiao-Gungrsquo inChinese is distributed in tropical areas The traditionalusages of the roots of Flemingia species have been forthe treatment of rheumatism arthropathy leucorrheamenalgia menopausal syndrome chronic nephritis and

improvement of bone mineral density (Li et al 2008)Only few studies have confirmed the pharmacologicalactivity of members in the Flemingia genus For exampleit was reported that the extract of the root of F philip-

pinensis ( F prostrata (FP)) exhibited anti-oxidative anti-inflammatory estrogenic and anti-estrogenic activities(Li et al 2008) The stems of F macrophylla have beenused in traditional medicine as an antirheumatic and anti-in ammatory agent and for improving blood circulationFurthermore its flavonoids have inhibitory effects on Aβ-induced neurotoxicity (Shiao et al 2005)

The objectives of this work were to investigate theantioxidant and antidiabetic properties of the aqueous ex-tracts of Flemingia macrophylla (Willd) Kuntze ex Prain(FM) Flemingia prostrata Roxb (FP) Flemingia lineata (L) Roxb (FL) and Flemingia strobilifera (L) R Br ExAit (FS) by comparing them with chemical compoundssuch as glutathione (GSH) or genistein and to nd out thelevels of their inhibitory activities on α-glucosidase andaldose reductase through a series of in vitro tests

MATERIALS AND METHODS

MaterialsGSH potassium peroxodisulfate (K 2S2O8) DPPH Tris

(hydroxylmethyl) aminomethane potassium ferricyanide(K 3Fe(CN) 6) TCA ferric chloride (FeCl 3) aluminumchloride hexahydrate (AlCl 3middot6H 2O) 22rsquo-azinobis-(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) sodium

bica rbo na te (Na HC O 3) sodium phosphate dibasic(Na 2HPO 4) sodium phosphate monobasic (NaH 2PO 4)genistein standard and other chemicals were purchased

from Sigma Chemical Co (St Louis MO USA) Folin-Ciocalteu solution and 95 ethanol were purchased fromMerck Co (Santa Ana CA USA) Plant materials werecollected from Taichung Nantou and Hsinchu countiesin Taiwan They were identified and authenticated byDr Chao-Lin Kuo Associate professor and ChairmanDepartment of Chinese Medicine Recourses ChinaMedical University Taichung Taiwan

Preparing aqueous extracts of plant materialsDried herb roots (100 g for each species) were boiled

with 1 L deioned water for 1 hour Filtration and collectionof the extracts were done three times The resultingdecoction (about 1 L) was evaporated to 10 mL and driedin vacuum at 50ordmC The dried extract was weighted anddissolved in distilled water (stock 4 mgmL) and storedin -20ordmC for the usage in later steps For each sample theyield was calculated in percentage by dividing the quantityof dry mass obtained after extraction by the dry weight ofthe herb used (100 g)

Determining antioxidant activities by ABTS middot+ scavenging ability

The ABTS middot+ scavenging ability was determinedaccording to the method of Chang et al (2007a b)

Aqueous solution of ABTS (7 mM) was oxidized with potassium peroxodisulfate (245 mM) for 16 h in the darkat room temperature The ABTS middot+ solution was dilutedwith 95 ethanol to an absorbance of 075 plusmn 005 at734 nm (Beckman UV-Vis spectrophotometer ModelDU640B) For each sample an aliquot (20 μL) of sample(125 μgmL) was mixed with 180 μL ABTS middot+ solutionand then the absorbance was read at 734 nm after 1 minTrolox was used as a reference standard A standard curvewas constructed for Trolox at 0 15625 3125 625125 250 500 μM concentrations TEAC was expressedin millimolar concentration of trolox solution with theantioxidant equivalent to a 1000 ppm solution of thesample under investigation

Ferric reducing antioxidant power assayThe ferric reducing antioxidant power (FRAP) assay of

the crude extracts was carried out according to the methodof Huang et al (2008) This assay measured the changein absorbance at 593 nm due to the action of electrondonating antioxidants changing the colorless oxidizedFe 3+ into blue colored Fe 2+-tripyridyltriazine compoundTo prepare the FRAP reagent a mixture of 01 M acetate

buffer (pH 36) 10 mM 2 4 6-tris(2-pyridyl)- s-triazine(TPTZ) and 20 mM ferric chloride (1011 vvv) was

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HSIEH et al mdash Antioxidants and antidiabetic effects of four Flemingia species 295

made For each sample an aliquot (10 μL) of samplesolution (125 μgmL concentration) was mixed with 300μL FRAP reagent and the absorbance was read at 593nm after 15 min A standard curve was constructed forFeSO 47H 2O at 0 3125 625 125 250 500 1000 μgmL concentrations In the FRAP assay the antioxidantefficiencies of the samples were calculated according to

the reaction signal given by an Fe2+

solution of knownconcentration which represented an one-electronexchange reaction The results were corrected for dilutionand expressed in μmol Fe 2+mg

Determining antioxidant activity by DPPHradical scavenging ability

The effects of crude extracts and positive controls(GSH and genistein) on DPPH radicals were estimatedaccording to the method of Huang et al (2007) Aliquotsof crude extracts (20 μL) at various concentrations wereeach mixed with 100 mM Tris-HCl buffer (80 μL pH74) and then with 100 μL of DPPH in ethanol to a nalconcentration of 250 μM All the mixtures were shakenvigorously and left to stand at room temperature for 20min in the dark The absorbance of the reaction solutionswere measured spectrophotometrically at 517 nm TheDPPH decolorizations of the samples were calculated in

percentage according to the equation decolorization= [1- (ABS sample ABS control )] times 100 IC 50 value was theeffective concentration in which DPPH 50 of radicalswere scavenged and was obtained by interpolation withlinear regression analysis A lower IC 50 value indicated agreater antioxidant activity

Determination of total polyphenol contentThe total polyphenol content of the crude extracts

were determined according to the method of Huang et al(2005) For each sample 20 μL of the extract (125 μgmL) was added to 200 μL distilled water and 40 μL ofFolin-Ciocalteu reagent The mixture was allowed to standat room temperature for 5 min and then 40 μL of 20sodium carbonate was added to the mixture The resulting

blue complex was measured at 680 nm Catechin was usedas a standard for the calibration curve The polyphenolcontent was calibrated using the calibration curve

based linear equation The total polyphenol content wasexpressed as mg catechin equivalentg dry weight The dryweight indicated was the sample dry weight

Determination of total avonoid contentThe total flavonoid contents of the crude extracts

were determined according to the method of Huang etal (2004a b) For each sample an aliquot of 15 mLextract was added to an equal volume of 2 AlCl 36H 2O(2 g in 100 mL methanol) solution The mixtures werevigorously shaken and the absorbances at 430 nm wereread after 10 min of incubation Rutin was used as thestandard for the calibration curve The total flavonoidcontent was calibrated using the linear equation based

on the calibration curve The total avonoid content wasexpressed as mg rutin equivalentg dry weight The dryweight indicated was the sample dry weight

Determination of total avonol contentThe total flavonol content of the crude extracts was

determined according to the method of Chang et al(2007a b) For each extract an aliquot of 200 μL wasadded to 1 mL of 01 p-dimethylaminocinnamaldehyde(DMACA) in methanolHCl (31 vv) All the mixtureswere vigorously shaken and the absorbances were readafter 10 min of incubation at 640 nm Catechin was usedas a standard for the calibration curve The total avonolcontent was calibrated using the linear equation basedon the calibration curve The total flavonol content wasexpressed as mg catechin equivalentg dry weight The dryweight indicated was the sample dry weight

Inhibition assay for alpha-glucosidase activity

The alpha-glucosidase inhibitory effects of the aqueousextracts of the four Fl emingia species were assayedaccording to the procedure described previously byMatsui et al (2001) with minor modifications Brieflythe enzyme reaction was performed using p-Nitrophenyl-alpha-D-glucopyranoside (PNP-glycoside) as a substratein 01 M piperazine- N Nacute-bis (2-ethanesulfonic acid) (PIPES) buffer pH 68 The PNP-glycoside (20 mM) was

premixed with samples at various concentrations Eachmixture was added to an enzyme solution (001 unit) tomake 05 ml of nal volume The reaction was terminated

by adding 1 ml of 064 N -(1-naphthyl) ethylenediaminesolution (pH 107) Enzymatic activity was quanti ed by

measuring the p-nitrophenol released from PNP-glycosideat 405 nm wave length All reactions were carried out at37degC for 30 min with three replications Acarbose wasused as a positive control One set of mixtures preparedwith an equivalent volume of PIPES buffer instead oftested samples was used as control The concentrationof the extracts required to inhibit 50 of α-glucosidaseactivity under the assay conditions was de ned as the IC 50 value

Measuring aldose reductase activity in vitro Crude AR was prepared as in the following steps lenses

were removed from Sprague-Dawley (SD) rats weighing250-280 g and were kept frozen until use A homogenateof rat lens was prepared in accordance with the methoddescribed by Hayman and Kinoshita (1965) A partially

puri ed enzyme with a speci c activity of 65 Umg wasroutinely used in the evaluations of enzyme inhibitionThe partially purified material was separated into 10mL aliquots and stored at -40degC The AR activity wasspectrophotometrically assayed by measuring the decreasein NADPH absorption at 340 nm over a 4 min periodusing DL-glyceraldehyde as a substrate Each 10 mLcuvette containing equal units of enzyme 01M sodium

phosphate buffer (pH 62) and 03 mM NADPH either with

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or without 10 mM substrate and inhibitor was prepared(Lim et al 2006) One set of mixtures prepared with anequivalent volume of sodium phosphate buffer instead oftested samples was used as control The concentration ofthe extracts required to inhibit 50 of ΑΡ activity underthe assay conditions was de ned as the IC 50 value

Analyses of genistein and WFM by LC-MS-MS Moderate amounts of WFM were weighed and

dissolved in water At first the solutions were filteredthrough 045 μm PVDF filters The LC-MS-MS (Waters2695 separations module detector Waters 996 photodiodearray detector with a ES-D609 mass spectrometer)analysis was carried out under the following conditionsthe Waters Cosmosil 5C18-AR-II column (5 μm 46 times 150mm) was used with 025 methanol for mobile phase Aacetonitrile was used as mobile phase B and water wasused as mobile phase C The ratio of ABC was 202060and the gradient elution was ran at a ow rate of 05 mLmin The injection volume was 10 μL and a wavelengthof 254 nm was used for detection Pure genistein was alsoanalyzed using LC-MS-MS under the same conditions andthe retention time was used to identify the genistein in thesamples

Statistical analysesExperimental results were presented as the mean plusmn

standard deviation (SD) of three parallel measurementsThe statistical analyses were performed by one-wayANOVA followed by Dunnettrsquos t test The difference wasconsidered to be statistically signi cant when the p valuewas less than 005

RESULTS AND DISCUSSION

Extraction yieldsThe yields in the aqueous extracts of the four Flemingia

species were given in Table 1 The yield percentages ofaqueous extracts (code as W) in decreasing order wereas follows WFL (1257) gt WFS (1167) gt WFM(1065) gt WFP (988)

Antioxidant activity estimated by ABTS andFRAP assay

ABTS assay is often used in evaluating the totalantioxidant power of single compound and complexmixtures of various plants (Huang et al 2004a b Huanget al 2006) In this assay ABTS radical monocationswere generated directly from the stable form of potassium

peroxodisulfate s Rad icals wer e gener ated bef ore theantioxidants were added to prevent interference ofcompounds which could have affected radical formationThis modification made the assay less susceptible toartifacts and prevented overestimation of antioxidant

power (Sanchez-Moreno 2002) Antiox idant sampleswere added to the reaction medium when the absorbance

became stab le and then the antioxidant activi ty was

measured in terms of decolorizationResults of the ABTS assay were expressed in TEAC

value A higher TEAC value meant that the sample hada stronger antioxidant activity TEAC values of the four

Flemingia species were determined from the calibrationcurve as shown in Table 2 Antioxidant activities of theaqueous extracts of the four Flemingia species were inthe following decreasing order WFM (113 plusmn 005 mMmg extract) gt aqueous extract of Flemingia prostrata (WFP) (048 plusmn 002 mMmg extract) gt aqueous extract of

Flamingia lineate (WFL) (036 plusmn 001 mMmg extract)gt aqueous extract of Fl am in gi a st robi li fe ra (WFS)(032 plusmn 001 mM mg extract) The antioxidant potencyof genistein (positive control) was 031plusmn 001 mMmgextract

The FRAP values of the aqueous extracts of the four Flemingia species were in the following order WFM(153 plusmn 002 μmol Fe 2+mg extract) gt WFL (077 plusmn 003μmol Fe 2+mg extract) gt WFS (042 plusmn 001 μmol Fe 2+mgextract) gt WFP (040 plusmn 0 02 μmol Fe 2+mg extract) Theantioxidant potency of genistein was 023 plusmn 002 μmol

Fe2+

mg extracts (Table 2) Thus it showed that WFM hadthe highest activity

Both FRAP and TEAC assays were used to estimatethe total antioxidant power because they were quick andsimple to perform and the reactions were reproducibleand linearly related to the molar concentration of theantioxidants (Benzie et al 1999) FRAP assay wasinitially developed to assay the plasma antioxidantcapacity however it can also be used to measure theantioxidant capacity of a wide range of biological samples

pur e co mp ou nd s frui ts wi ne s an d an ima l tis sue s(Katalinic et al 2006)

Table 2 Total antioxidant activity assessed by TEAC andFRAP

Species and positivecontrol

TEAC (mMmgextract)

FRAP (μmol Fe 2+mgextract)

WFL 036 plusmn 001 077 plusmn 003

WFM 113 plusmn 005 153 plusmn 002

WFP 048 plusmn 002 040 plusmn 002

WFS 032 plusmn 001 042 plusmn 001

Genistein 031 plusmn 001 023 plusmn 002

All values represent means plusmn SD of triplicate tests ( n = 3)

Table 1 Extraction yields from the aqueous extracts of four Flemingia species

Sample Aqueous extract yield ( ww) a

WFL 1257

WFM 1065

WFP 988

WFS 1167aDried weight basis

8102019 Bot513-02



The correlation coefficients (R 2) of the FRAP andTEAC assays conducted on the aqueous extracts of thefour Flemingia species were shown in Figure 1 The R 2 values of FRAP and TEAC assay showed high correlation(R 2=083)

Scavenging activity against 1 1-diphenyl-2-

picrylhydrazyl radicalsThe relatively stable organic DPPH radicals are widely

used in model systems to investigate the scavengingactivities of several natural compounds such as phenolicsand anthocyanins or crude mixtures A DPPH radical isscavenged by antioxidants through the donation of a protonto form the reduced DPPH The color changes from purpleto yellow after reduction which could be quantified byits decrease of absorbance at wavelength 517 nm Radicalscavenging activity increases when the percentage of freeradical inhibition increases Table 3 shows the IC 50 valuesfor the radical-scavenging activities of the four Flemingia species GSH and genistein using the DPPH colorimetricmethod It was found that WFM had the lowest IC 50 value(3634 plusmn 122 μgmL) followed by WFL (18734 plusmn 128)WFS (19797 plusmn 141 μgmL) WFP (20476 plusmn 057 μgmL)The four extracts showed signi cant differences ( plt005)in radical-scavenging activity As demonstrated by theabove results the most active sample was WFM howeverits antioxidant capacity was still stronger than GSH andgenistein positive controls (7177 plusmn 139 μgmL and 368 plusmn535 μgmL) in DPPH assay

Total polyphenol flavonoid and flavonolcontent

The total polyphenol avonoid and avonol contentsof the four Fl emingi a species are shown in Table 4The total polyphenol content was expressed as μg ofcatechin equivalent per milligram of dry weight The total

polyphenol contents of the extracts of the four Flemingia species ranged from 4546 to 19773 μg CEmg and

decreased in the following order WFM gt WFL gt WFP gtWFS WFM had the highest polyphenolic content

The total flavonoid contents were expressed as μg ofrutin equivalent per milligram of dry weight The totalflavonoid contents of the extracts of the four Flemingia species ranged from 053 to 075 μg REmg and decreasedin the following order WFM gt WFS gt WFP gt WFLWFM had the highest avonoid content

The total flavonol contents were expressed as μg ofcatechin equivalent per milligram of dry weight The totalflavonol contents of the extracts of the four Flemingia species ranged from 205 to 276 μg CEmg and decreasedin the following order WFM gt WFL gt WFS gt WFPWFM had the highest avonols

Both flavonoid and flavonol are polyphenoliccompounds Polyphenolic compounds have importantroles in stabilizing lipid oxidation and are associated withantioxidant activities (Yen et al 1993) The phenoliccompounds may contribute directly to antioxidativeactions (Duh et al 1999) It is suggested that 10 g of

polyphenolic compounds from a daily diet rich in fruitsand vegetables has inhibitory effects on mutagenesisand carcinogenesis in humans (Tanaka et al 1998)The antioxidative activities observed could be ascribed

both to the different mechanisms exer ted by various phenolic compounds and to the synergi st ic effects ofdifferent compounds The antioxidant assay used in

Table 3 IC 50 values of the aqueous extracts of four Flemingia species in DPPH radical scavenging activity

Species and positive controls IC 50 (microgmL) a

WFL 18734 plusmn 128

WFM 3634 plusmn 122

WFP 20476 plusmn 057

WFS 19797 plusmn 141GSH 7177 plusmn 139

Genistein 368 plusmn 535aValues represent means plusmn SD of three parallel measurements

Figure 1 Correlation coef cients (R 2) of TEAC and FRAP inthe aqueous extracts of the four Flemingia species

Table 4 Contents of phytochemicals extracted from theaqueous extracts of four Flemingia species

Species Total phenols a

(micro g CEmg)Total avonoids b

(micro g REmg)Total avonols a

(micro g CEmg)

WFL 13942 plusmn 159 053 plusmn 002 211 plusmn 010

WFM 19773 plusmn 105 075 plusmn 004 276 plusmn 064WFP 12500 plusmn 058 058 plusmn 001 205 plusmn 004

WFS 4546 plusmn 018 074 plusmn 004 208 plusmn 004

All data are expressed as means plusmn SD of triplicate tests ( n =3) ( p lt 005)

aData expressed in microg catechin equivalentmg dry weight (microgCEmg)

bData expressed in microg rutin equivalentmg dry weight (microg rutinmg)

8102019 Bot513-02



this study measured the oxidative products at the earlyand final stages of oxidation The antioxidants haddifferent functional properties such as reactive oxygenspecies scavenging eg quercetin rutin and catechin(Liu et al 2008) free radical generation inhibitionsand chain-breaking activity for example p -coumaricacids (Laranjinha et al 1995) and metal chelation (Van-

Acker et al 1998) These antioxidative compounds areusually phenolic compounds that are effective in donating protons such as tocopherols avonoids and other organicacids However the active contents responsible for theantioxidative activities of the four Flemingia species arestill unclear Therefore further work must be performed toisolate and identify these components

Relationship between the total antioxidantpower and the total polyphenol avonoid andavonol content

The correlation coef cients (R 2) of the total antioxidant power (TEAC) and the total polyphenol TEAC and thetotal flavonoid and TEAC and the total flavonol of theaqueous extracts were shown in Figure 2 The R 2 valueof TEAC and the total polyphenol content of the water(Figure 2A) extracts was 066 Similarly R 2 value ofTEAC and the total flavonoid content of the aqueous(Figure 2B) extracts was 026 R 2 value of TEAC andtotal avonol content of the aqueous (Figure 2C) extractswas 094 Among the above 3 statistics we could see thatthere were high correlations between TEAC and the total

polyphenol and also TEAC and the total avonol

Inhibitiory assay for alpha-glucosidase activity

The α-glucosidase inhibitory activity of the aqueousextracts of the four Flemingia species are shown in Table5 The effectiveness of enzymatic inhibition of the aqueousextracts of the four Flemingia species were determined bycalculating IC 50 The lower the value the higher the qualityof enzymatic inhibition The IC 50 of the four Flemingia species in inhibiting α-glucosidase ranged from 15392 to146860 μgmL and its effectiveness was ranged as in thefollowing increasing order WFM gt WFL gt WFP gt WFSWFM had the highest α-glucosidase inhibitory activity(IC 50 = 15392 plusmn 020 μgmL) The positive controlsagainst α-glucosidase inhibitory activity were genistein

(IC 50 = 1665 plusmn 092 μgmL) and acarbose (IC 50 = 259604plusmn 056 μgmL)

The IC 50 of positive control for alpha-glucosidaseinhibitor (acarbose) is found much higher in the presentassay which is similar to many previous literatures Littlearticles had discussed it in detail (Youn et al 2004 Shindeet al 2008) When compared to acarbose as the control

only mammalian enzyme was inhibited This was expectedsince acarbose has been shown to be a potent inhibitor of

Figure 2 Correlation coefficients (R 2) of TEAC and total polyphenol (A) avonoid (B) and avonol (C) contents in theaqueous extracts of the four Flemingia species

Table 5 IC 50 values of the aqueous extracts of four Flemingia species in α-glucosidase inhibition



WFM 15392 plusmn 020


WFS 146860 plusmn 210

Acarbose 259604 plusmn 056


8102019 Bot513-02



mammalian sucrase and maltase and inactive against yeastand bacterial forms (Kim et al 2004)

Polyphenolic compounds in plants have long beenrecognized to inhibit the activities of digestive enzymes

because of their ability to bind with proteins (Grif ths andMoseley 1980) Various in vitro assays have shown thatmany plant polyphenols possess carbohydrate hydrolyzing

enzyme inhibitory activities These compounds includegreen tea polyphenols which inhibit the activities ofα-glucosidase and sucrase (Hara and Honda 1992)sweet potato polyphenols which inhibit the activities ofα-glucosidase (Matsui et al 2001) and berry polyphenolswhich inhibit the activities of α-glucosidase and α-amylase(McDougall and Stewart 2005)

Genistein belongs to the isoflavonoid family Most previous studies have focused on the pharmacologicalactivities of genistein as a tyrosine kinase inhibitorand its chemoprotectant activities against cancers andcardiovascular disease Recently Dong-Sun et al also have

reported that genistein could be a potent α-glucosidaseinhibitor (Lee and Lee 2001)

Measurement of aldose reductase activity invitro

AR the principal enzyme of the polyol pathway has been shown to play an important role in the complicationsassociated with diabetes The AR inhibitory activity ofaqueous extracts of the four Flemingia species are shownin Table 6 The IC 50 of the extracts of the four Flemingia species AR inhibitory activities ranged from 7936 μgmL to 17241 μgmL and increased as in the followingorder WFM gt WFS gt WFLgt WFP WFM had the highestAR inhibitory activity (IC 50 = 7936 plusmn 320 μgmL) The

positive control in the AR inhibitory activity assay wasgenistein (IC 50 = 4562 plusmn 216 μgmL)

Many natural compounds have been tested for ARinhibitory activities Medicinal plants are particularlylikely to be non-toxic and may be useful for the preventionand treatment of diabetes-related complications (Preetet al 2006) In addition to its antioxidant propertiesgenistein has an inhibitory effect on the formation of

advanced glycation end products (Jang et al 2006)Further evidence that genistein can inhibit diabetic related

Figure 3 (A) High performance liquid chromatogtraphic profile of genistein in WFM and genistein standard (B) Daughter ionspectrum of major peaks in WFM (Rt = 1688) and genistein standard (Rt = 1695)

8102019 Bot513-02



problems stems from studies with type 2 diabetic animalsgenistein has been shown to decrease blood glucose andglycated hemoglobin levels (HbA1 IC) and increase theglucagoninsulin ratio (Lee 2006)

It has been well-acknowledged that plant-derivedextracts and phytochemicals are potential alternativesto synthetic inhibitors against AR and α-glucosidase

(Lee 2006) Currently AR inhibitor and α-glucosidaseinhibitor compounds isolated from plants are classi ed asditerpene- triterpene- and avonoid-related compoundsIn this study the active component isolated from WFMagainst aldose reductase and α-glucosidase was identi edas genistein even though the inhibitory responses variedwith concentrations

Compositional analyses of genistein and WFMby LC-MS-MS

To verify whether genistein was present in WFM or not both WFM and genistein standard were separated through

HPLC column separately under the same conditions A prominent peak appeared in WFM (Rt = 1688) whichwas equivalent to the genistein peak with Rt = 1695(Figure 3A) In order to con rm the identity of genisteinin WFM the two peaks were then subject to ESIMSMSanalyses The daughter ion spectrum of the major peakin WFM (Rt = 1688) was found to be identical to that ofthe genistein standard (Figure 3B) LC-MS-MS analysescon rmed the existence of genistein in WFM

In conclusion the results from in vitro experimentsincluding ABTS radical monocation scavenging FRAPmethod (Table 2) DPPH radical scavenging (Table 3)total polyphenol content total flavonoid content andtotal avonol content (Table 4) α-glucosidase inhibition(Table 5) AR inhibition (Table 6) and LC-MS-MS assay(Figure 3) demonstrated that the phytochemicals in theaqueous extracts of the four Flemingia species mighthave significant antioxidant and anti-diabetic activitiesdirectly related to the total amount of polyphenols andflavonols Hence the four Flemingia species could beused as easy accessible sources of natural antioxidants in

pharmaceutical and medical industries For this reasonfurther work should be performed to isolate and identifythe antioxidative or anti-diabetic components of the FM

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Chang HC GJ Huang DC Agrawal CL Kuo CR Wuand HS Tsay 2007a Antioxidant activities and polyphenol

contents of six folk medicinal ferns used as ldquoGusuiburdquo BotStud 48 397-406

Chang HY YL Ho MJ Sheu WH Pen SH Wu GJHuang and YS Chang 2007b Antioxidants and free radi -cal scavenging activity of Phellinus merrillii extracts BotStud 48 407-417

Chihiro NY 1998 Aldose reductase in glucose toxicity a po -tential target for the prevention of diabetic complicationsPharmacol Rev 50 21-34

Duh PD YY Tu and GC Yen 1999 Antioxidant activity ofwater extract of Harng Jyur ( Chyrsanthemum morifolium Ramat) Lebensmittel-Wissenschaft und Technologie 32

269-277Fukuda T H Ito and T Yoshida 2004 Effect of the walnut

polyphenol fraction on oxidative stress in type 2 diabetesmice Biofactors 21 251-253

Grif ths DW and G Moseley 1980 The effect of diets con -taining eld beans of high or low polyphenolic content onthe activity of digestive enzymes in the intestines of rats JSci Food Agric 31 255-259

Hara Y and M Honda 1992 Inhibition of rat small intestinalsucrase and alpha-glucosidase activities by tea polyphenolBiosci Biotechnol Biochem 57 123-124

Haraguchi H I Ohmi S Sakai and A Fukuda 1996 Effect of Polygonum hydropiper sulfated flavonoids on lens aldosereductase and related enzymes J Nat Prod 59 443-445

Hayman S and JH Kinoshita 1965 Isolation and properties oflens aldose reductase J Biol Chem 240 877-882

Huang DJ HJ Chen WC Hou CD Lin and YH Lin2004a Active recombinant thioredoxin h protein with anti-oxidant activities from sweet potato ( Ipomoea batatas [L]Lam lsquoTainong 57rsquo) storage roots J Agric Food Chem 524720-4724

Huang DJ CD Lin HJ Chen and YH Lin 2004b Antioxi -dant and antiproliferative activities of sweet potato ( Ipom-oea batatas [L] Lam lsquoTainong 57rsquo) constituents Bot BullAcad Sin 45 179-186

Huang DJ CD Lin HJ Chen and YH Lin 2005 Antioxi -dative and antiproliferative activities of water spinach ( Ipo-moea aquatica ) constituents Bot Bull Acad Sin 46 99-106

Huang DJ HJ Chen WC Hou and YH Lin 2006 Sweet potato ( Ipomoea batatas [L] Lam lsquoTainong 57rsquo) storageroots mucilage with antioxidant activities in vitro FoodChem 98 774-781

Huang GJ HJ Chen YS Chang MJ Sheu and YH Lin2007 Recombinant sporamin and its synthesized peptideswith antioxidant activities in vitro Bot Stud 48 133-140

Table 6 IC 50 values of the aqueous extracts of four Flemingia species in aldose reductase inhibition



WFM 7936 plusmn 320



Genistein 4562 plusmn 216

a Values represent means plusmn SD of three parallel measurements

8102019 Bot513-02



Huang SS GJ Huang YL Ho YH Lin HJ Hung TNChang MJ Chan JJ Chen and YS Chang 2008 Antiox -idant and antiproliferative activities of the four Hydrocotyle species from Taiwan Bot Stud 49 311-322

Jang DS JM Kim YM Lee YS Kim H Kim and JSKim 2006 Puerariafuran a new inhibitor of advancedglycation end products (AGEs) isolated from the roots of

Pueraria lobata Chem Pharm Bull 5 1315-1317Kador PF WG Robison and JH Kinoshita 1985a The phar -

macology of aldose reductase inhibitors Annu Rev Phar -macol Toxicol 25 691-714

Kador PJ JH Konishita and NE Sharpless 1985b Aldosereductase inhibitors a potential new class of agents for the

pharmacological control of certain diabetic complications JMed Chem 28 841-849

Katalinic V M Milos T Kulisic and M Jukic 2006 Screen -ing of 70 medicinal plant extracts for antioxidant capacityand total phenols Food Chem 94 550-557

Kim YM MH Wang and HI Rhee 2004 A novelα-glucosidase inhibitor from pine bark Carbohydr Res339 715-717

Laranjinha J O Vieira V Madeira and L Almeida 1995Two related phenolic antioxidants with opposite effects onvitamin E content in low density lipoproteins oxidized byferrylmyoglobin consumption versus regeneration ArchBiochem Biophys 323 373-381

Lee DS and SH Lee 2001 Genistein a soy iso avone is a potent α-glucosidase inhibitor FEBS Lett 501 84-86

Lee HS 2005 Cuminaldehyde Aldose reductase andα-glucosidase inhibitor derived from Cuminum cyminum L Seeds J Agric Food Chem 53 2446-2450

Lee HS and MK Kim 2001 Rat intestinal R-glucosidase andlens aldose reductase inhibitory activities of grain extractsFood Sci Biotechnol 10 172-177

Lee JS 2006 Effects of soy protein and genistein on bloodglucose antioxidant enzyme activities and lipid pro le instreptozotocin-induced diabetic rats Life Sci 79 1578-1584

Li H M Yang J Miao and X Ma 2008 Prenylated iso a -vones from Flemingia philippinensis Magn Reson Chem46 1203-1207

Lim SS YJ Jung SK Hyun YS Lee and JS Choi 2006Rat lens aldose reductase inhibitory constituents of Nelum-bo nucifera stamens Phytother Res 20 825-830

Liu CL YS Chen JH Yang and BH Chiang 2008 Antiox -idant activity of tartary ( Fagopyrum tataricum (L) Gaertn)and common ( Fagopyrum esculentum Moench) buckwheatsprouts J Agric Food Chem 56 173-178

Matsui T T Ueda K Sugita N Terahara and K Matsumoto2001 Alpha-glucosidase inhibitors action of natural acyl -ated anthocyanins Survey of natural pigments with potentinhibitory activity J Agric Food Chem 49 1948-1951

McDougall GJ and D Stewart 2005 The inhibitory effectsof berry polyphenols on digestive enzymes Biofactors 23189-195

Preet A MR Siddiqui A Taha J Badhai ME Hussain PKYadava and NZ Baquer 2006 Long-term effect of Trigo-nella foenum-graecum and its combination with sodiumorthovanadate in preventing histopathological and biochem-

ical abnormalities in diabetic rat ocular tissues Mol CellBiochem 289 137-147

Sabu MC K Smitha and R Kuttan 2002 Anti-diabetic activ -ity of green tea polyphenols and their role in reducing oxi-dative stress in experimental diabetes J Ethnopharmacol 83 109-116

Sanchez-Moreno C 2002 Methods used to evaluate the freeradical scavenging activity in foods and biological systemsFood Sci Technol Int 8 121-137

Shiao YJ CN Wang WY Wang and YL Lin 2005 Neuro - protective avonoids from Flemingia macrophylla PlantaMed 71 835-840

Shinde J T Taldone M Barletta N Kunaparaju B Hu SKumar J Placido and SW Zito 2008 α-Glucosidaseinhibitory activity of Syzygium cumini (Linn) Skeels seedkernel in vitro and in Goto-Kakizaki (GK) rats Carbohy -drate Research 343 1278-1281

Sowers JR M Epstein and ED Frohlich 2001 Hypertensionand cardiovascular disease an update Hypertension 37 1053-1105

Tanaka M CW Kuei Y Nagashima and T Taguchi 1998Application of antioxidative maillrad reaction productsfrom histidine and glucose to sardine products Nippon Sui-san Gakkai Shil 54 1409-1414

Toeller M 1994 α-Glucosidase inhibitors in diabetes ef cacyin NIDDM subjects Eur J Clin Invest 24(Suppl 3) 3l-35

Van-Acker SABE GP Van-Balen DJ Vanden-Berg ABast and SABE Vander-Vijgh 1998 Influence of ironchelation on the antioxidant activity of flavonoids Bio -chem Pharmacol 56 935-943

Williamson J C Kilo and RG Tilton 1992 Mechanism ofglucose and diabetes-induced vascular dysfunction In NRuderman J Brownlee and J Williamson (eds) Hypergly -cemia Diabetes and Vascular Disease American Physiol -ogy Society New York pp 107-132

Yen GC PD Duh and CL Tsai 1993 Relationship betweenantioxidant activity and maturity of peanut hulls J AgricFood Chem 41 67-70

Youn JY HY Park and KH Cho 2004 Anti-hyperglycemicactivity of Commelina communis L inhibition of a -glucosi-dase Diabetes Res Clin Pract 66S S149-S155

8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-

TEAC FRAP (R 2) (R 2 083) TEAC

(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-

50 15392 plusmn 020 μgmL 7936 plusmn 320 μgmL genisteinα- (IC 50 =1665 plusmn 092 μgmL 4562 plusmn 216 μgmL)

LC-MS-MS

genistein

α-

8102019 Bot513-02



The inhibitory effects of plant phytochemicalsincluding polyphenols against carbohydrate hydrolyzingenzymes contribute to the lowering of postprandialhyperglycemia in diabetic management as observed invivo (Griffiths and Moseley 1980) Further evidencethat polyphenolic compounds is linked to prevention ofdiabetic complications stems from in vivo studies with

diabetic rats polyphenolic compounds in plant materialsare capable of reducing oxidative stress by scavengingreactive oxygen species and preventing cell damage(Fukuda et al 2004) The polyphenolic compounds inedible plants are currently regarded as natural antioxidantsand their antioxidant activities are important for humanhealth (Sabu et al 2002)

Plants constitute a rich source of bioactive chemicals(Kador et al 1985a b Williamson et al 1992) Sincemany plants are largely free from adverse effects andhave excellent pharmacological actions they could

possibly lead to the development of new classes of saferantidiabetic agents or diabetic complication resolvingagents In addition some flavonoids and polyphenolsas well as sugar derivatives are found to be effective ininhibiting α-glucosidase and aldose reductase (Haraguchiet al 1996 Lee and Kim 2001) Therefore much efforthas been focused on plants to produce potentially useful

products such as commercial α-glucosidase inhibitors andaldose reductase inhibitors or lead compounds

The Fl emingi a genus known as lsquoI-Tiao-Gungrsquo inChinese is distributed in tropical areas The traditionalusages of the roots of Flemingia species have been forthe treatment of rheumatism arthropathy leucorrheamenalgia menopausal syndrome chronic nephritis and

improvement of bone mineral density (Li et al 2008)Only few studies have confirmed the pharmacologicalactivity of members in the Flemingia genus For exampleit was reported that the extract of the root of F philip-

pinensis ( F prostrata (FP)) exhibited anti-oxidative anti-inflammatory estrogenic and anti-estrogenic activities(Li et al 2008) The stems of F macrophylla have beenused in traditional medicine as an antirheumatic and anti-in ammatory agent and for improving blood circulationFurthermore its flavonoids have inhibitory effects on Aβ-induced neurotoxicity (Shiao et al 2005)

The objectives of this work were to investigate theantioxidant and antidiabetic properties of the aqueous ex-tracts of Flemingia macrophylla (Willd) Kuntze ex Prain(FM) Flemingia prostrata Roxb (FP) Flemingia lineata (L) Roxb (FL) and Flemingia strobilifera (L) R Br ExAit (FS) by comparing them with chemical compoundssuch as glutathione (GSH) or genistein and to nd out thelevels of their inhibitory activities on α-glucosidase andaldose reductase through a series of in vitro tests

MATERIALS AND METHODS

MaterialsGSH potassium peroxodisulfate (K 2S2O8) DPPH Tris

(hydroxylmethyl) aminomethane potassium ferricyanide(K 3Fe(CN) 6) TCA ferric chloride (FeCl 3) aluminumchloride hexahydrate (AlCl 3middot6H 2O) 22rsquo-azinobis-(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) sodium

bica rbo na te (Na HC O 3) sodium phosphate dibasic(Na 2HPO 4) sodium phosphate monobasic (NaH 2PO 4)genistein standard and other chemicals were purchased

from Sigma Chemical Co (St Louis MO USA) Folin-Ciocalteu solution and 95 ethanol were purchased fromMerck Co (Santa Ana CA USA) Plant materials werecollected from Taichung Nantou and Hsinchu countiesin Taiwan They were identified and authenticated byDr Chao-Lin Kuo Associate professor and ChairmanDepartment of Chinese Medicine Recourses ChinaMedical University Taichung Taiwan

Preparing aqueous extracts of plant materialsDried herb roots (100 g for each species) were boiled

with 1 L deioned water for 1 hour Filtration and collectionof the extracts were done three times The resultingdecoction (about 1 L) was evaporated to 10 mL and driedin vacuum at 50ordmC The dried extract was weighted anddissolved in distilled water (stock 4 mgmL) and storedin -20ordmC for the usage in later steps For each sample theyield was calculated in percentage by dividing the quantityof dry mass obtained after extraction by the dry weight ofthe herb used (100 g)

Determining antioxidant activities by ABTS middot+ scavenging ability

The ABTS middot+ scavenging ability was determinedaccording to the method of Chang et al (2007a b)

Aqueous solution of ABTS (7 mM) was oxidized with potassium peroxodisulfate (245 mM) for 16 h in the darkat room temperature The ABTS middot+ solution was dilutedwith 95 ethanol to an absorbance of 075 plusmn 005 at734 nm (Beckman UV-Vis spectrophotometer ModelDU640B) For each sample an aliquot (20 μL) of sample(125 μgmL) was mixed with 180 μL ABTS middot+ solutionand then the absorbance was read at 734 nm after 1 minTrolox was used as a reference standard A standard curvewas constructed for Trolox at 0 15625 3125 625125 250 500 μM concentrations TEAC was expressedin millimolar concentration of trolox solution with theantioxidant equivalent to a 1000 ppm solution of thesample under investigation

Ferric reducing antioxidant power assayThe ferric reducing antioxidant power (FRAP) assay of

the crude extracts was carried out according to the methodof Huang et al (2008) This assay measured the changein absorbance at 593 nm due to the action of electrondonating antioxidants changing the colorless oxidizedFe 3+ into blue colored Fe 2+-tripyridyltriazine compoundTo prepare the FRAP reagent a mixture of 01 M acetate

buffer (pH 36) 10 mM 2 4 6-tris(2-pyridyl)- s-triazine(TPTZ) and 20 mM ferric chloride (1011 vvv) was

8102019 Bot513-02



























8102019 Bot513-02





















Fe2+






TEAC (mMmgextract)










WFL 1257

WFM 1065

WFP 988


8102019 Bot513-02



















WFM 3634 plusmn 122









(micro g CEmg)







8102019 Bot513-02






















8102019 Bot513-02















8102019 Bot513-02











LITERATURE CITED





















WFM 7936 plusmn 320





8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02



























8102019 Bot513-02





















Fe2+






TEAC (mMmgextract)










WFL 1257

WFM 1065

WFP 988


8102019 Bot513-02



















WFM 3634 plusmn 122









(micro g CEmg)







8102019 Bot513-02






















8102019 Bot513-02















8102019 Bot513-02











LITERATURE CITED





















WFM 7936 plusmn 320





8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02





















Fe2+






TEAC (mMmgextract)










WFL 1257

WFM 1065

WFP 988


8102019 Bot513-02



















WFM 3634 plusmn 122









(micro g CEmg)







8102019 Bot513-02






















8102019 Bot513-02















8102019 Bot513-02











LITERATURE CITED





















WFM 7936 plusmn 320





8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02



















WFM 3634 plusmn 122









(micro g CEmg)







8102019 Bot513-02






















8102019 Bot513-02















8102019 Bot513-02











LITERATURE CITED





















WFM 7936 plusmn 320





8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02






















8102019 Bot513-02















8102019 Bot513-02











LITERATURE CITED





















WFM 7936 plusmn 320





8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02















8102019 Bot513-02











LITERATURE CITED





















WFM 7936 plusmn 320





8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02











LITERATURE CITED





















WFM 7936 plusmn 320





8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02


































8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

8102019 Bot513-02



α-

1 1 2 3 1 1 4 15

1 2 3 4 5

ABTS FRAP DPPH

α-


(R 2) (R 2=066) TEAC R 2

R 2=094 α-

α-


LC-MS-MS

genistein

α-

Bot513-02

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Transcript of Bot513-02