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Pueraria mirifica leaves, an alternative potential isoflavonoid source

Jutarmas Jungsukcharoen1, Binar Asrining Dhiani2, Wichai Cherdshewasart3,Nawaporn Vinayavekhin4, Polkit Sangvanich4 and Chuenchit Boonchird2,*

1Faculty of Science, Program in Biotechnology, Chulalongkorn University, Bangkok, Thailand; 2Faculty of Science,Department of Biotechnology, Mahidol University, Bangkok, Thailand; 3Faculty of Science, Department of Biology,Chulalongkorn University, Bangkok, Thailand; 4Faculty of Science, Department of Chemistry, ChulalongkornUniversity, Bangkok, Thailand

Received October 8, 2013; accepted January 24, 2014

http://dx.doi.org/10.1080/09168451.2014.910091

We investigated the major leaf isoflavonoidcontents of Pueraria mirifica from three differentcultivars (PM-III, PM-IV, and PM-V) using reverseRP-HPLC analysis. The proportions and net levelsof puerarin, daidzin, genistin, and daidzein in P.mirifica leaves were found to depend on the plantcultivar and to correlate with cultivation tempera-ture and rainfall amount. The crude leaf-extractswere tested using the Yeast Estrogen Screen (YES)assay with both human estrogen receptors (hERand hER). Their estrogenic activity was higherwhen determined by the YES system containinghER than that with hER and was also higherwhen the snq2 than the wildtype yeast wasemployed. The results open the possibility of select-ing and cultivating certain P. mirifica cultivars at afarm scale to produce a sufficient supply of leafmaterial to act as a starting source for the commer-cial scale extraction of these major isoflavonoids.

Key words: Pueraria mirifica; isoflavonoid; YESassay; estrogenic activity

Isoflavonoids, one of the common types of phytoes-trogens, are found in relative abundance in many formsof edible legume seeds1,2) legume-derived foods3,4) anddietary supplements.5) They are of increasing interest tothe food industry due to their potential health benefits,including for the potential chemoprevention of can-cers.6) The recent establishment of several phytoestro-gen databases illustrates the growing interest of thefood industry in phytoestrogens from natural sourcesand their bioactivities.7)

Soy isoflavonoids have been well studied, with mostof the results being obtained from daidzein and geni-stein,8) which appear to harbor anti-cancer properties.9)

However, a new source of isoflavonoids would promote

the alternative production and consumption of suchchemicals from those new plant sources and relieve thepressure on the currently limited amount of availableplant material and risks associated with sole large scalemonoculture production10) or the in vitro culture ofplants.11)

Pueraria mirifica Airy Shaw et Suvatabhandu is aThai indigenous legume herb with a long-term folkmedicinal consumption among Thai women for meno-pausal treatment. The tuberous materials of the plantrevealed strong estrogenic effects in the MCF-7 prolif-eration/antiproliferation,12) uterotrophic13,14) and YeastEstrogen Screen (YES)15) test assays. Among the phy-toestrogen-rich plant materials, the tubers of kudzu(Pueraria lobata) are widely used in traditional Chi-nese, Japanese, and Korean medicines,16) whereas intraditional Thai medicines the related P. mirifica is usedand has been extensively studied. P. lobata has beenanalyzed for the purpose of the potential developmentof the plant products or chemicals for the benefit of thefood industry,17) whereas P. mirifica has been subjectedto long-term studies to establish products for meno-pausal treatment.18,19) At present, these two plants areused as the main botanical ingredients in cosmetic anddietary supplement products.The estrogenic activity in P. mirifica was found to be

stronger than that in P. lobata in the MCF-7 antiprolif-eration, uterotrophic, and ovariectomized ratassays.20,21) However, the analysis of P. mirifica tuberscollected from wild plant populations over a vast areain Thailand in comparison with that of P. lobata col-lected from China revealed that neither daidzin norgenistin, which are the isoflavonoid glycosides derivedfrom daidzein and genistein, respectively, were themajor isoflavonoids in these plants, but rather puerarinwas.22) Accordingly, it is of interest to find out whichisoflavonoids or other phytoestrogens, if any, might beresponsible for the beneficial effects observed in these

*Corresponding author. Email: chuenchit.boo@mahidol.ac.thAbbreviations: RP-HPLC, reverse phase high-performance liquid chromatography; P. mirifica, Pueraria mirifica; YES, yeast estrogen screen; hER,human estrogen receptors; E2, 17-estradiol; RT, room temperature; DMSO, dimethyl sulfoxide; SDA, medium supplemented with adenine; RP,Relative Potency; SNQ2, Sensitivity to 4-NitroQuinoline-N-oxide; oNPG, ortho-nitrophenyl--galactoside; SEM, standard error mean; LOD, limitof detection; NA, not applicable.

Reproduced from Bioscience, Biotechnology, and Biochemistry 78: 917926 (2014). Chuenchit Boonchird: Participant of the 10th UM, 1982-1983.

yamamotoTable of Contents

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assays, whilst the estrogen-like activity and long-termfolk medical use make P. mirifica an interesting candi-date as a potential phytoestrogen source for the devel-opment of commercialized products. However, thebasic knowledge related to phytoestrogens, and espe-cially the control of isoflavonoid synthetic pathwaysand transport/storage within the plant, is still largelyunknown in this species.

In this study, we present the content levels for thefive major isoflavonoids (puerarin, daidzin, genistin,daidzein, and genistein) from the leaves of three differ-ent farm-grown P. mirifica cultivars (PM-III, PM-IV,and PM-V) together with their estrogenic activity, asanalyzed by the YES assay involving the human estro-gen receptors hER and hER. The use of leaves asthe isoflavonoid source has the potential advantageover tubers in that the mature tubers need at least threeyears cultivation to reach maturity, while the plantleaves can be harvested monthly for 9 (cultivar PM-V)to 11 (cultivars PM-III and PM-IV) months a year.Thus, this research may open the possibility of intro-ducing a new more economically viable plant sourcematerial for natural isoflavonoid supply.

Materials and methodsChemicals, biochemicals, and yeast strains. The

isoflavonoid standards puerarin, genistin, daidzein, gen-istein, and 17-estradiol (E2) were purchased fromSigma (St. Louis, MO, USA), whereas daidzin waspurchased from FlukaBiochemika (Buchs, Switzerland).The organic solvents for extraction (analytical grade)and chromatography (HPLC grade) were purchasedfrom Merck (Germany). Water of over 16MM/cm, acomponent of the mobile phase of HPLC, was preparedby Maxima Ultra Pure Water Systems (ELGA). TheSaccharomyces cerevisiae expression strain Y190(Clontech) (MATa, ura352, his3-200, ade2101,trp1901, leu23, 112, gal4 gal80, URA3::GALUAS-lacZ, cyhr2, LYS2::GALUAS-HIS3) containing the lacZreporter gene fused to GALUAS in the chromosomeswas used as the host yeast. For the YES-hER assay,wildtype Y190 was transformed with the plasmidpGBT9-hERLBD, encoding for amino acid residues331595 of the hER fused to GAL4DBD, andpGAD424-hTIF2, encoding for amino acid residues6241287 of the transcriptional intermediary factor(TIF2) fused to GAL4AD. For the YES-hER assay,Y190 was transformed with pGBT9-hERLBD, encod-ing for amino acid residues 213477 of the hER fused to GAL4DBD, and pGAD424-hSRC1, encodingfor amino acid residues 2311094 of steroid receptorco-activator 1 (SRC1) fused to GAL4AD.15) In addi-tion, the YES-hERsnq2 and YES-hERsnq2systems were constructed from the YES-hER andYES-hER systems as above except using theY190-snq2 as the host strain in place of the wildtypeY190 so as to increase the sensitivity of YES system.

Plant material. The fresh mature leaves of the P.mirifica cultivars (PM-III, PM-IV, and PM-V) were cul-tivated in a field trial in the Banpong District ofRatchaburi Province (E9952/N1337), Thailand, and

were collected every month from March 2007 to Febru-ary 2008. The leaf morphology was distinctive betweeneach of the three cultivars, as previously reported.23)

The monthly record of daily mean temperatures andrainfall in the Ratchaburi Province was provided by theMeteorological Department, Ministry of Informationand Communication Technology, Thailand and isshown in Fig. 1.

Extraction and isolation. The collected leaf sam-ples were cleaned, dried in a hot air oven at 80 C for72 h, and subsequently ground into powder and filteredthrough a 120-mesh-size sieve. A 50 g aliquot of theleaf powder was extracted with methanol by vigorousshaking (3 500 mL, 2 h each, RT). After settling, thesupernatant was filtered (Whatman filter paper No.4,Whatman, USA), and the pooled filtrates then evapo-rated in vacuo (Buchi, Germany) at 37 C. The residualmaterial was dissolved in 50 mL deionized water andthen partitioned in 1:1 (v/v) chloroform (3 times) toremove the chlorophyll, harvesting the aqueous phasethat was then mixed at 1:1 (v/v) ratio with n-butanol (3times). After phase separation the butanol phase washarvested and evaporated in vacuo at 45 C. The finalresidue, referred to, as the crude leaf-extract was storedat 4 C.

Reverse phase HPLC analysis. The reverse phaseHPLC system, a Waters Auto Sampler (Waters-717),controller (Waters-600) and photodiode array detector(Waters-2996), utilized a reversed phase C18 column(250 4.6 mm) filled with 5 m ODS2 (Waters Spheri-sorb, Ireland), pre-filtered with a Waters Spherisorb S5ODS2 (4.6 10 mm) guard cartridge. The filter set wasMillipore membrane (0.45 m, 13 mm for the sampleand 47 mm for the mobile phase) of the HA type foraqueous solutions and HV type for organic solvents.The chromatography manager software was operatedwith a personal computer. The crude leaf-extract (1 g)was dissolved in methanol (1 mL) with the aid of soni-cation (30 min, RT), and then sequentially filteredthrough Whatman No.1 filter paper and a 0.45 m (13mm diameter) PTFE filter membrane. The isoflavonoidanalysis was then performed by reverse phase HPLC aspreviously described16) with modification. Elution wasperformed with a linear gradient from 100:0 to 55:45(v/v) 0.1% (v/v) acetic acid: acetonitrile as the mobilephase at a flow rate of 1 mL/min for 50 min, while theeluate was monitored at OD254. The five major isofl-avonoid standards were mixed at appropriate propor-tions for use in chromatogram peak area calibrationcurves, and the comparison of their retention time withthe test samples was performed for identification, andthe peak area was derived for quantification using theEmpower program. The analyses of samples were runin triplicate.

YES assay. The methodology was described in pre-vious study.15) A stock solution of each crude leaf-extract was freshly prepared as serial dilutions(502,000 g/mL) in DMSO. Standard compounds: E2,

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genistein, and genistin were also prepared in DMSO indose ranges of 1016104 M, 108104 M, and 108103 M, respectively, by using DMSO as a negative con-trol. The highest concentration of DMSO in each dilu-tion was 1% (v/v). The YES assay was performed asdescribed elsewhere18) with modification as follows.Yeasts were grown in synthetic dextrose (SD) minimalmedium supplemented with adenine (SDA; 0.67% (w/v)yeast nitrogen base without amino acids, 2% (w/v) glu-cose, and 0.002% (w/v) adenine sulfate) with vigorousshaking overnight (30 C). Then 50 L of the overnightculture was mixed with 200 L of fresh SDA mediumand 2.5 L of either crude leaf-extract (dissolved inDMSO to the desired concentration), DMSO alone(negative control), or E2 (dissolved in DMSO to thedesired concentration; positive control) in a 1.5-mLmicrotube and incubated at 30 C for 4 h with shaking at200 rpm. Thereafter, a 150 L aliquot of the cultured cellsolution was transferred into a 96-well microplate formeasurement of the cell density at an OD660. Anotheraliquot (100 L) of the cell suspension was centrifuged(10,000 g, 5 min) to pellet the cells that were thenresuspended in 200 L of Z-buffer (0.2 mg/mL Zymoly-ase 100T in 0.1M sodium phosphate pH 7.0, 10mMKCl, 1 mM MgSO4, and 3.5 mM-mercaptoethanol)and incubated for 15 min at 30 C. After centrifugation(10,000 g, 5 min) the cell lysate (supernatant) wasrecovered and incubated with 40 L of substrate solution(4 mg/mL oNPG) in 0.1 M sodium phosphate buffer pH7.0), for 30 min at 30 C.24) The reaction was stopped bythe addition of 100 L of 1M Na2CO3, and the celldebris centrifugally removed (8,600 g for 5 min).Finally, 150 L of the supernatant was transferred into a96-well microplate for measurement of the absorbance atOD420 and OD550 to follow the formation of theortho-nitrophenol (o-NP) product. One unit (U) of-galactosidase was defined in terms of Miller U asfollows:

Miller U 1000 OD420 1:75OD550=OD660TV ;

where OD420 represents the absorbance of the oNPproduct, OD550 was the scatter from cell debris, which

when multiplied by 1.75 approximated the scatterobserved at OD420, OD660 was the cell density at thestart of the assay, T was the reaction time (min), and Vthe culture volume used in the assay (mL).The data for -galactosidase activity (U) and the

concentration of tested samples were fitted usingthe four parameters logistic dose-response model of theGraphPad Prism software version 4 (GraphPad Soft-ware Inc., USA), as a Sigmoidal dose-response (vari-able slope), and the EC50 value calculated. TheRelative Potency25) of each crude leaf-extract was cal-culated to allow direct comparison between samples,and was calculated by dividing the obtained EC50 (g/ml) for E2 by the EC50 of the test sample, and thenmultiplying the value by 100. Thus, the RP value of E2was always 100 as the reference standard.

Statistical analysis. Statistical calculations werecarried out with the Statistical Packages for Social Sci-ence (SPSS) software version 17.0 for Windows (SPSSInc., USA). Data are shown as the mean 1 S.E.M.and differences between means, for the isoflavonoidcontents of the crude leaf-extracts, -galactosidaseactivities, EC50, and RP, were tested for statistical sig-nificance by the unpaired T-test, Pearson correlationanalysis, and Duncans analysis of variance, acceptingsignificance at the level of p < 0.05 and p < 0.01.

ResultsPlant harvestingThe three cultivars of P. mirifica produced abundant

numbers and masses of leaves for harvesting everymonth, except when no or too few leaves were presentto harvest, which was in April for the PM-III cultivar,March for the PM-IV cultivar, and February, March,and April for the PM-V cultivar.

Crude leaf-extract yieldThe different yields of the leaf crude extract obtained

for each of the three P. mirifica cultivars over theassayed year are summarized in Table 1 with highest

Fig. 1. The mean monthly temperature (C) and rainfall amount39) at Ratchaburi province, including the field trial area, during the study period(March 2007 to February 2008).Data are shown as the mean S.E.M.

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mean temperature in April and rainfall amount in May.Within each month a significant variation in the crudeleaf-extract yield between the cultivars was evidentalthough cultivar PM-III typically had the highestmonthly yields (in 7/12 months) and the highest aver-age yearly yield, whilst PM-V had the lowest monthly(in 7/12 months) and yearly yield.

HPLC analysisThe five standard isoflavonoids were clearly sepa-

rated and distinct when analyzed by RP-HPLC with theC18 column and monitoring the eluent at OD254(Fig. 2). The HPLC resolution of the crude leaf-extractof each P. mirifica cultivar was complicated, but resolu-tion of the isoflavonoids was still clear with genisteinbeing absent in all three cultivars in all months ofgrowth. A representative example of HPLC tracesderived from samples harvested during January 2008 isshown in Fig. 3.

The calculated amount of each isoflavonoid found ineach P. mirifica cultivar over the 12-month cultivationperiod (Fig. 4) revealed the highest mean total isoflavo-noid content across all three cultivars was found inJune (41.7 4.0 mg/100 g leaf powder) followed bySeptember (37.9 4.3 mg/100 g leaf powder) but thisvaried between the individual cultivars. Cultivar PM-Vhad the highest total isoflavonoid contents in Septem-ber (61.6 4.0 mg/100 g powder), but for PM-III theoptimal month was June, while for PM-IV it wasequally optimal in April and August. Daidzin in thePM-V cultivar was the most prevalent of the isoflavo-noids, where the highest concentration was found inJune (50.6 3.7 mg/100 g powder), however, levels ofdaidzin in the other two cultivars (PM-III and PM-IV)were significantly lower in this and most other months.In contrast, genistin levels were very low in the PM-Vcultivar except in July (13.0 0.1 mg/100 g powder),but was the isoflavonoid with the highest concentrationin the PM-III and PM-IV cultivars for seven and fivemonths, respectively, and with its levels being secondto those of daidzin in several other months. In contrast,puerarin and daidzein remained at relatively low con-centrations over all 12 months in all three cultivars.The monthly rainfall level strongly correlated with,

and so potentially had a significant impact upon thegenistin content in the leaves each month (p < 0.05),whilst the temperature likewise strongly correlated withthe puerarin, daidzin, and daidzein contents (p < 0.01)in the PM-III cultivar (Table S1 in supplementary infor-mation). In contrast, in the PM-V cultivar the rainfallstrongly correlated with the puerarin content (p < 0.01)and the temperature with the daidzin content (p < 0.01).Note, however, that in all four of the above correlationsan actual causation has yet to be established.

YES assayBecause the YES assay is a suitable and simple

method for the in vitro screening of estrogenic activitythat reduces the required time and provides an easymeans for monitoring compared with the in vivo cancercell test, we employed this assay in this study using theS. cerevisiae wildtype and snq2Y190 yeast strains,

Table 1. The leaf crude extract yields obtained (g/100 g leaf pow-der) from each the three different P. mirifica cultivars in each monthover the 12-month cultivation period (March 2007 to February 2008).

Month

Crude leaf extract of P. mirifica (g/100 g leafpowder) (Means S.E.M.)

PM-III PM-IV PM-V

March 2007 1.41 0.44a,b NA NAApril 2007 NA 1.54 0.12a,b NAMay 2007 2.06 0.36a,b,c 2.41 0.11b 1.96 0.08b,c,d

June 2007 2.70 1.04b,c 2.11 0.42b 2.67 0.09d

July 2007 3.37 0.20c 2.52 0.94b 2.34 0.08d

August 2007 1.26 0.13a,b 2.75 0.35b 2.82 0.03d

September 2007 2.44 0.31b,c 1.80 0.24b 2.28 0.38c,d

October 2007 3.25 0.61c 1.67 0.43b 2.44 0.45d

November 2007 2.24 0.23a,b,c 1.55 0.26a,b 0.94 0.94a,b,c

December 2007 2.59 1.06b,c 2.18 0.10b 0.83 0.83a,b

January 2008 2.69 0.29b,c 2.78 0.27b 1.27 0.84a

February 2008 2.17 0.54a,b,c 2.63 0.45b NATotal 2.23 0.18 2.02 0.15 1.46 0.20

Notes: NA = not applicable because the plants shed their leaves for waterpreservation during this month.Data are shown as the mean SEM, derived from nine independent repeats.

a,b,c,drepresent groups of samples with statistically significant differences(p < 0.05) in each column as determined by Duncans analysis.

Fig. 2. HPLC Fingerprints of the five synthetic isoflavonoid standards of puerarin (25 mg/mL, RT = 14.35 min), daidzin (30 mg/mL, RT = 15.93min), genistin (30 mg/mL, RT = 18.96 min), daidzein (25 mg/mL, RT = 26.49 min), and genistein (50 mg/mL, RT = 34.64 min).Profiles shown are representative of those seen from at least 27 independent trials.

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each expressing part of either the hER or hER recep-tor. If the compound binds to ligand binding domain(LBD) of hER fused to DNA binding domain (DB) ofGAL4, it will turn on -galactosidase, harboringGALUAS. The readout is then achieved by performing a-galactosidase assay through the conversion of oNPGto oNP, which is monitored by absorbance of the oNPat 420 nm and compared to that for E2 as a positivecontrol and reference standard.15)

For the standardization of the YES-hER assay withthe wildtype and snq2 yeast strains for the evaluationof estrogenic activity, the EC50 values of E2 in theYES-hER and YES-hER assays (Table 2) were foundto agree fairly well (1.2-fold lower activity) with thatpreviously reported for YES-hER (2.25 1010 M), butwas some 51.3-fold less active than that reported beforefor YES-hER (2.3 1010 M). In this study here, theYES-hER exhibited a 44.2-fold higher estrogenic activ-ity than the YES-hER system, whilst the

YES-hERsnq2 assay exhibited a 283.6-fold higherestrogenic activity than that in the YES-hERsnq2 sys-tem. When the SNQ2 deletion was employed, the estro-genic activity was significantly increased in both thehER and hER based assays (Table 2). Thus, for E2detection the sensitivity was 16.2- and 2.5-fold higherfor the YES-hERsnq2 and YES-hERsnq2 systemscompared to the YES-hER and YES-hER assays,respectively, while for genistein and genistin the sensi-tivity increased by 1.2- and 4.5-fold when evaluated byYES-hERsnq2 compared to YES-hER, respectively,and 11.3- and 8.0-fold higher, respectively, for the YES-hERsnq2 system compared to the YES-hER one. Incontrast to E2, genistein and genistin both showed ahigher estrogenic activity against the hER-based sys-tems than in the hER ones, for both the wildtype andsnq2 yeast strains (Table 2). However, the sensitivity ofgenistein and genistin detection in the YES-hERsnq2assay was significantly lower than that of E2 by about13.1- and 1887-fold, respectively. The estrogenic activityof puerarin, daidzein, and daidzin could not be detectedusing any of the four YES systems within the range ofconcentrations used in this assay (108103 M), and sothey were excluded from the sensitivity assessment.The evaluation of the estrogenic activity of the

P. mirifica crude leaf-extracts revealed that the highestestrogenic activity was always detected with the ERsystems as opposed to the ER ones, and with the snq2yeast strain compared to the wildtype, and so the YES-hER snq2 assay was the most sensitive for all thecultivars (Fig. 5). With respect to the three P. mirificacultures individually, in the PM-III cultivar the highestestrogenic activity detected by the wildtype YES-hERand YES-hER was in September (RP of ~102 and104, respectively), and this declined steadily eachmonth thereafter to a minimum in August. (This and thesubsequent analysis of course assume that this 12-monthperiod from March 2007 to February 2008 represents thepattern seen each year, which remains to be established).A broadly similar trend was observed when assayed bythe YES-hERsnq2 system, but in contrast, whenassayed by the YES-hERsnq2 system, no such clearmonthly pattern over the 12-month period was evidentwith instead the highest activity being seen in Novemberfollowed by that in August (Fig. 5).In some contrast to the PM-III cultivar, in the PM-

IV cultivar, the highest estrogenic activity was detectedin leaves harvested during August when assayed bythree of the four yeast assay systems, but the YES-hER system gave a slightly higher estrogenic activityon leaves harvested during October than in September.Moreover, there was no clear monthly decline from thismaximum over the 12-month period, but rather ittended to decline to a false minimum in November, riseto a smaller peak by February before then decliningagain to the minimum level at April, and then rise tothe maximum level in September or October as above.The highest estrogenic activity of the crude leaf-

extracts from the PM-V cultivar was found in leaveswhich were harvested in January 2008 or May 2007for the YES-hER assay, and in September, July, andNovember for the YES-hER, YES-hERsnq2, andYES-hERsnq2 assays, respectively (Fig. 5), butwithout any clear pattern of monthly decline. Indeed,

Fig. 3. HPLC fingerprints of the crude leaf-extract of P. mirificacultivar (A) PM-III, (B) PM-IV, and (C) PM-V, derived from samplesharvested during January 2008.The profiles shown are representative of those seen from at least 27

independent trials, and from leaves harvested in the other months.

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the monthly variation in estrogen levels determined byboth the YES-hERsnq2 and YES-hERsnq2 assayswere slight, except for a minimum in December(Fig. 5). However, the estrogenic activity of all plantsamples was lower than that of E2, the positive control(Fig. 5), and was not correlated with the isoflavonoidcontents (analysis not shown), suggesting that theremight be other isoflavonoids, or complex interactions

between them, or as-yet-undetermined compoundsresponsible for this activity.

Discussion

The three cultivars of P. mirifica (PM-III, PM-IV,and PM-V) produced abundant numbers and masses of

Fig. 4. Major isoflavonoid profile (mg/100 g powder) of the crude leaf-extracts from the three different cultivars of P. mirifica Harvested in differ-ent months.(A) PM-III, (B) PM-IV, and (C) PM-V. Data are shown as the mean S.E.M.

Table 2. The estrogenic activity, as the EC50 Value and derived relative potency25) using E2 as the reference standard, of the standard genistein

and genistin and reference E2, as determined by the four different YES systems.

Systems

E2 Genistein Genistin

EC50 (M) RPa EC50 (M) RP

a EC50 (M) RPa

YES-hER 2.67 1010 100 2.80 106* 9.53 103 1.37 104 1.95 104

YES-hER 1.18 108 100 6.91 107* 1.71 7.09 105* 1.66 102

YES-hERsnq2 1.65 1011 100 2.33 106* 7.08 104 3.05 105 5.41 105

YES-hERsnq2 4.68 109 100 6.11 108* 7.66 8.83 106* 5.30 102

Notes: Relative Potency (RP) = (EC50 of E2/EC50 of leaf extract) 100. Abbreviation; E2: 17-estradiol. Data are shown as the mean value derived from nine independent repeats.*Significantly different from E2 at p 0.05.aData are shown as the mean SEM derived from nine independent repeat.

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leaves for harvesting every month, except for themonth(s) when there were no or too few leaves to har-vest (April in the PM-III cultivar, March in the PM-IV

cultivar, and February, March, and April in the PM-Vcultivar). During February to April, the decrease inrainfall amount and increase in temperature might cause

Fig. 5. Relative potency values of the crude leaf-extracts from the three different cultivars of P. mirifica harvested in different months.(A) PM-III, (B) PM-IV, and (C) PM-V. RP values, relative to the EC50 of the E2 standard, were derived from the estrogen-like activity EC50 val-

ues as ascertained using the wild type (YES) and snq2 (YES-snq2) Y190 yeast strains with hER or hER. Abbreviation; E2: 17-estradiol.Data are shown as the mean S.E.M. Means with a different lower case letter above them are significantly different (p < 0.05).

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water-limited environment. Plants then become waterstressed and dropped their leaves.26)

HPLC analysisThe crude leaf-extracts derived from the three P. mi-

rifica cultivars varied in their yield and relative compo-sition of the isoflavonoids between the differentcultivars and cultivation month during the 12-monthperiod assayed. Whether this temporal monthly patternis representative of typical years remains to be estab-lished. However, the study also confirmed the existenceof a significant level of accumulated isoflavonoids inthe plant leaves, including puerarin, daidzin, genistin,and daidzein. Determination of the isoflavonoid levelsrevealed significant cultivar-dependent difference, i.e.difference in the isoflavonoid levels among the threecultivars grown in the same field trial and harvested atthe same time, as well as between each month. Daidzinand genistin were found to have the highest averageannual isoflavonoid accumulation in the leaf samples(genistin for PM-III and daidzin for PM-IV and PM-V)with puerarin also being found in all analyzed plantsamples (cultivars and harvest months), but at lowerlevels. In contrast, daidzein was not found in all threecultivars in March to August, plus not in cultivar PM-IV in October, PM-III in November and PM-V inNovember to January. Moreover, genistein was notfound in any of the cultivars in any month of the year.Overall, the chemovariety at the isoflavonoid level inthe three P. mirifica cultivars, which have distinct dif-ferences in their botanical characteristics, were con-firmed. The net yield of total isoflavonoidin P. mirificaleaf (41.68 4.01 mg/ 100 g powder) still lower than intuber (80.67 4.11 mg/100 g powder) about twotimes.27) However, in terms of plant cultivation, leavesharvesting is more worthwhile than tuber collectionbecause tubers spend at least three years to growmaturely whereas leaves need only three months andcan be collected quarterly. For the comparison of isofl-avonoid yield between tuber and leaf harvested peryear in general, the isoflavonoid yield harvested in leafis higher than tuber 6.20 times per year.

The isoflavonoid pattern in P. mirifica leaves over a 12-month cultivation period remains to be ascertained if thispattern holds for other years and so is a real trend or not.However, with that caveat in mind, the puerarin, daidzin,genistin, and daidzein levels in the crude leaf-extractswere found to be strongly, but differentially correlatedwith either the rainfall amount or the average temperatureduring the 12-month cultivation period assayed within thesame plant cultivar in different months (seasons). In addi-tion, while the plant genetics per se (as morphologicallydistinct cultivars in this study) also strongly correlated tothe differences in the isoflavonoid types and levels foundin the leaves over this 12-month assay period. The extrac-tion method employed in this study was efficient toextract a broad range of polar isoflavones e.g. daidzin,daidzein, genistin, and genistein, and the isolated compo-nents showed 9899% purity.28) Therefore, the non-detectable genistein in leave crude extract was not fromthis extraction method. Interestingly, genistein, the isofl-avonoid not detected in this study in the crude leaf-extracts, has been found previously in the tubers of these

three P. mirifica cultivars, although in smaller amountscompared with that of puerarin, genistin, daidzin, anddaidzein.27) The differences in the synthesis and/or accu-mulation of isoflavonoids between leaf and tuberous tis-sues is probably that genistein is an aglycosidiccompound synthesized in the leaf, which is not stableenough for transportation or storage in tuberous root, it isconverted into genistin, (by glucose conjugation) toincrease both its stability (through the protection of reac-tive nucleophilic groups) and water solubility.29,30) It ispossible that genistin is then cleaved to the aglycosidicform after being transported into the tubers, accountingfor the detection of genistin in the leaves but genistein inthe tubers. Indeed, glycoside hydrolase has been reportedin roots of Medicago truncatula, another legume plant inthe same family.31) This enzyme can catalyze the hydroly-sis of the glycosidic linkage in genistin to convert it togenistein. In contrast, a proteomic analysis of soybeanleaves did not report the finding of any enzymes with gly-coside hydrolase activity,32) and so the actual case for P.mirifica remains to be resolved.

YES assayGenistein and genistin showed a higher estrogenic

activity in the YES-hER than the YES-hER assaysystem, in agreement with that previously reported.15)

The sensitivity of all tested compounds determined byYES-hERsnq2 and YES-hERsnq2 was higherthan the corresponding wildtype Y190 yeast strains(YES-hER and YES-hER), which correlated wellwith a previous study.33) In wildtype Y190 yeaststrains, anti-estrogenic activity could be determined byusing tamoxifen against both E2

34) and P. mirifica tubercrude extract.15) Furthermore, the anti-estrogenic activ-ity of medicinal plants was also demonstrated in differ-ence yeast strains.35) Snq2 is a member of yeast ATP-binding cassette (ABC) transporter family,36) which isan exporter of multiple cytotoxic and steroid com-pounds in S. cerevisiae. Deletion of SNQ2 gene wasshown to increase the intracellular steroid level.37) Dou-ble deletion of PDR5, another member of the yeastABC transporter family, and Snq2 increased the uptakeof several compounds,38) while deletion of three yeastABC transporters (PDR5, SNQ2, and YOR1) furtherincreased the detection sensitivity for phytoestrogensby hER.39) Therefore, deletion of the SNQ2 genecauses an increased sensitivity to a wide range of com-pounds because the snq2 cells are unable or less ableto efflux these compounds out of the cell. It is worth-while to examine anti-estrogenic activity in plant leavesby using YES-hERsnq2 and YES-hERsnq2.Although overall investigation shown that isoflavonoidcontents in some month were not correlated with estro-genic activity that not obviously difference in YES-hERsnq2, it was probably that the estrogenic activityassay employed crude extract which might containother compounds exerting estrogenic activity, for exam-ple, deoxymiroestrol that belong to phytoestrogengroup as same as isoflavonoid.40)

There are growing interests in at least two majorclasses of isoflavonoids, namely the glycoside andaglycone forms.41) The latter have been demonstrated

146 147

to be active in the MCF-7 cell activity42) and YES43)

assays, as well as in correlation analysis with its anti-oxidant activity.44) However, the glycoside isoflavo-noids, including daidzin and genistin, can bemetabolized into the more active aglycone form afteroral ingestion from either normal intestinal flora25) orwithin the liver from microsome-associated enzymes.45)

Natural daidzin and genistin could, therefore, be anindirect, yet rich, source of aglycones for human con-sumption.

The results of this study suggest that P. mirificaleaves might be a novel source for the industrial scaleextraction of isoflavonoids. Although there has been anincreasing interest in isoflavonoid production fromplant tissue grown in vitro from soy,11) P. lobata10) andP. mirifica,46) so as to make the production independentof the influence of variation from changes in the physi-cal environment, this in vitro approach is often moreexpensive than a conventional large scale productionmethod using farm grown plants, which for example,can produce an abundant mass of leaves.

Supplemental material

The supplemental material for this paper is availableat http://dx.doi.org/10.1080/09168451.2014.910091.

Acknowledgments

Wichai Cherdshewasart and Jutarmas Jungsukcha-roen were supported by the Higher Education ResearchPromotion and National Research University Project ofThailand, Office of the Higher Education Commission(FW 0663I); The Thailand Research Fund(DBG5180025); the Science for Locale Project underChulalongkorn University Centenary Academic Devel-opment Plan (2008-2012). Chuenchit Boonchird waspartially supported by the Faculty of Science, MahidolUniversity. Binar Asrining Dhiani received supportfrom DIKTI Overseas Scholarships Ministry ofNational Education Republic of Indonesia.

References

[1] Yoshiki Y, Takagi S, Watanabe M, Okubo K. Fractionation ofsoybean functional glycosides from soy-waste based on thechemical reaction of soyasaponin g. Food Chem. 2005;93:591597.

[2] Zhao SH, Zhang LP, Gao P, Shao ZY. Isolation and character-isation of the isoflavones from sprouted chickpea seeds. FoodChem. 2009;114:869873.

[3] Kuhnle GGC, DellAquila C, Aspinall SM, Runswick SA,Joosen AMCP, Mulligan AA, Bingham SA. Phytoestrogen con-tent of fruits and vegetables commonly consumed in the UKbased on LC-MS and 13C-labelled standards. Food Chem.2009;116:542554.

[4] Kuhnle GGC, DellAquila C, Runswick SA, Bingham SA. Vari-ability of phytoestrogen content in foods from different sources.Food Chem. 2008;113:11841187.

[5] Zafra-Gmez A, Garballo A, Garca-Ayuso LE, Morales JC.Improved sample treatment and chromatographic method for thedetermination of isoflavones in supplemented foods. Food Chem.2010;123:872877.

[6] Cornwell T, Cohick W, Raskin I. Dietary phytoestrogens andhealth. Phytochemistry. 2004;65:9951016.

[7] Schwartz H, Sontag G, Plumb J. Inventory of phytoestrogen dat-abases. Food Chem. 2009;113:736747.

[8] Kim GN, Song JH, Kim ES, Choi HT, Jang HD. Isoflavone con-tent and apoptotic effect in HT-29 cancer cells of a soy germextract. Food Chem. 2012;130:404407.

[9] Kirakosyan A, Kaufman PB, Warber S, Bolling S, Chang SC,Duke JA. Quantification of major isoflavonoids and L-canavaninein several organs of kudzu vine (Pueraria montana) and in starchsamples derived from kudzu roots. Plant. Sci. 2003;164:883888.

[10] Matkowski A. In vitro isoflavonoid production in callus fromdifferent organs of Pueraria lobata (Wild.) Ohwi. J. Plant Phys-iol. 2004;161:343346.

[11] Gueven A, Knorr D. Isoflavonoid production by soy plant callussuspension culture. J. Food Eng. 2011;103:237243.

[12] Cherdshewasart W, Traisup V, Picha P. Determination of theestrogenic activity of wild phytoestrogen-rich Pueraria mirificaby MCF-7 proliferation assay. J. Reprod. Develop. 2008;54:6367.

[13] Cherdshewasart W, Kitsamai Y, Malaivijitnond S. Evaluation ofthe estrogenic activity of the wild Pueraria mirifica by vaginalcornification assay. J. Reprod. Develop. 2007;53:385393.

[14] Cherdshewasart W, Sriwatcharakul S, Malaivijitnond S. Varianceof estrogenic activity of the phytoestrogen-rich plant. Maturitas.2008;61:350357.

[15] Boonchird C, Mahapanichkul T, Cherdshewasart W. Differentialbinding with ER and ER of the phytoestrogen-rich plant Pue-raria mirifica. Braz. J. Med. Biol. Res. 2010;43:195200.

[16] Wong KH, Li GQ, Li KM, Razmovski-Naumovski V, Chan K.Kudzu root: Traditional uses and potential medicinal benefits indiabetes and cardiovascular diseases. J. Ethnopharmacol.2011;134:584607.

[17] Wang LZ, Yang B, Du XQ, Yi C. Optimisation of supercriticalfluid extraction of flavonoids from Pueraria lobata. Food Chem.2008;108:737741.

[18] Manonai J, Chittacharoen A, Theppisai U, Theppisai H. Effectof Pueraria mirifica on vaginal health. Menopause.2007;14:919924.

[19] Manonai J, Chittacharoen A, Udomsubpayakul U, Theppisai H,Theppisai U. Effects and safety of Pueraria mirifica on lipidprofiles and biochemical markers of bone turnover rates inhealthy postmenopausal women. Menopause. 2008;15:530535.

[20] Kim HY, Hong JH, Kim DS, Kang KJ, Han SB, Lee EJ, ChungRW, Song KH, Sho KA, Kwack SJ, Kim SS, Park KL, Lee SK,Kim MC, Kim CM, Song IS. Isoflavone content and estrogenactivity in arrowroot Puerariae Radix. Food Sci. Biotechnol.2003;12:2935.

[21] Malaivijitnond S, Chansri K, Kijkuokul P, Urasopon N,Cherdshewasart W. Using vaginal cytology to assess the estro-genic activity of phytoestrogen-rich herb. J. Ethnopharmacol.2006;107:354360.

[22] Cherdshewasart W, Subtang S, Dahlan W. Major isoflavonoidcontents of the phytoestrogen rich-herb Pueraria mirifica incomparison with Pueraria lobata. J. Pharm. Biomed. Anal.2007;43:428434.

[23] Cherdshewasart W, Sriwatcharakul S. Major isoflavonoid con-tents of the 1-year-cultivated phytoestrogen-rich herb, Puerariamirifica. Biosci. Biotechnol. Biochem. 2007;71:25272533.

[24] Malaivijitnond S, Chansri K, Cherdshewasart W, Jareonporn S,Trisomboon H. Effects of Pueraria mirifica, an herb containingphytoestrogens, on reproduction in rodents and monkeys. J. Exp.Zool. Comp. Exp. Biol. 2006;305:152152.

[25] Villeneuve DL, Blankenship AL, Giesy JP. Derivation and appli-cation of relative potency estimates based on in vitro bioassayresults. Environ. Toxicol. Chem. 2000;19:28352843.

[26] Taiz L, Zeiger E, editors. Plant Physiology. 3rd Ed. Sunderland:Sinauer Associates; 2002. p. 591594.

[27] Cherdshewasart W, Subtang S, Dahlan W. Major isoflavonoidcontents of the phytoestrogen rich-herb Pueraria mirifica incomparison with Pueraria lobata. J. Pharm. Biomed. Anal.2007;43:428434.

148 148

[28] Yang F, Ma Y, Ito Y. Separation and purification of isoflavonesfrom a crude soybean extract by high-speed counter-currentchromatography. J. Chromatogr. A. 2001;928:163170.

[29] Gachon CMM, Langlois-Meurinne M, Saindrenan P. Plant sec-ondary metabolism glycosyltransferases: the emerging functionalanalysis. Trends Plant Sci. 2005;10:542549.

[30] Malaivijitnond S. Medical applications of phytoestrogens fromthe Thai herb Pueraria mirifica. Front Med. 2012;6(1):821.

[31] Schenkluhn L, Hohnjec N, Niehaus K, Schmitz U, Colditz F. J.Proteomics. 2010;79:759768.

[32] Xu C, Garrett WM, Sullivan J, Caperna TJ, Natarajan S. Separa-tion and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.Phytochemistry. 2006;67:24312440.

[33] Sievernich A, Wildt L, Lichtenberg-Frat H. In vitro bioactivityof 17-estradiol. J. Steroid Biochem. 2004;92:455463.

[34] Mahapanichkul T. Evaluation of estrogenic activity of KwaoKrua plants using yeast estrogen screen (YES) method [MasterThesis]. Mahidol University; 2006. p. 6769. ISBN 974-04-7491-8.

[35] Kim IG, Kang SC, Kim KC, Choung ES, Zee OP. Screening ofestrogenic and antiestrogenic activities from medicinal plants.Environ. Toxicol. Pharmacol. 2008;25:7582.

[36] Decottignies A, Lambert L, Catty P, Degand H, Epping EA,Moye-Rowley WS, Balzi E, Goffeau A. Identification and char-acterization of SNQ2, a new multidrug ATP binding cassettetransporter of the yeast plasma-membrane. J. Biol. Chem.1995;270:1815018157.

[37] Mah Y, Parle-McDermott A, Nourani A, Delahodde A,Lamprecht A, Kuchler K. The ATP-binding cassette multidrugtransporter Snq2 of Saccharomyces cerevisiae: A novel target forthe transcription factors Pdr1 and Pdr3. Mol. Microbiol.1996;20:109117.

[38] Mitterbauer R, Weindorfer H, Safaie N, Krska R, Lemmens M,Ruckenbauer P, Kuchler K, Adam G. A sensitive and inexpen-sive yeast bioassay for the mycotoxin zearalenone and othercompounds with estrogenic activity. Appl. Environ. Microb.2003;69:805811.

[39] Hasenbrink G, Wildt L, Ludwig J, Lichtenberg-Frate H.Estrogenic effects of natural and synthetic compounds assessedin Saccharomyces cerevisiae. Febs J. 2006;273:8080.

[40] Chansakaow S, Ishikawa T, Seki H, Sekine K, Okada M,Chaichantipyuth C. Identification of deoxymiroestrol as the actualrejuvenating principle of Kwao Keur, Pueraria mirifica. The knownmiroestrol may be an artifact. J. Nat. Prod. 2000;63:173175.

[41] Haron H, Ismail A, Azlan A, Shahar S. Daidzein and genesteincontents in tempeh and selected soy products. Food Chem.2009;115:13501356.

[42] Matsumura A, Ghosh A, Pope GS, Darbre PD. Comparativestudy of oestrogenic properties of eight phytoestrogens in MCF7human breast cancer cells. J. Steroid Biochem. 2005;94:431443.

[43] Lin CC, Tsai YL, Ho CT, Teng SC. Determination of the differ-ential estrogenicity of isoflavonoids by E2-ER-ERE-dependentgene expression in recombinant yeast and MCF-7 human breastcancer cells. Food Chem. 2008;108:719726.

[44] Cherdshewasart W, Sutjit W. Correlation of antioxidant activityand major isoflavonoid contents of the phytoestrogen-rich Pue-raria mirifica and Pueraria lobata tubers. Phytomedicine.2008;15:3843.

[45] Cherdshewasart W, Sriwatcharakul S. Metabolic activation pro-motes estrogenic activity of the phytoestrogen-rich plant. Maturi-tas. 2008;59:128136.

[46] Udomsuk L, Jarukamjorn K, Tanaka H, Putalun W. Improvedisoflavonoid production in Pueraria candollei hairy root culturesusing elicitation. Biotechnol. Lett. 2011;33:369374.

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