Fitoterapia 94 (2014) 62–69

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Fitoterapia

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Isoflavone content and estrogenic activity of different batchesof red clover (Trifolium pratense L.) extracts: An in vitro studyin MCF-7 cells

Paola Spagnuolo a, Emanuela Rasini a, Alessandra Luini a, Massimiliano Legnaro a,Marcello Luzzani b, Enrico Casareto b, Massimiliano Carreri b, Silvano Paracchini b,Franca Marino a, Marco Cosentino a,⁎a Center for Research in Medical Pharmacology, University of Insubria, Varese, Italyb Linnea SA, Riazzino, TI Switzerland

a r t i c l e i n f o

⁎ Corresponding author at: Center for Research in MUniversity of Insubria, Via Ottorino Rossi n. 9, 211Tel.: +39 0332 217410/397410; fax: +39 0332 21

E-mail address: marco.cosentino@uninsubria.it (M

0367-326X/$ – see front matter © 2014 Elsevier B.V.http://dx.doi.org/10.1016/j.fitote.2014.01.027

a b s t r a c t

Article history:Received 16 October 2013Accepted in revised form 27 January 2014Available online 5 February 2014

The estrogenicity of different batches of red clover (Trifolium pratense L., Fabaceae; RCL) extracts andits relationship with the isoflavone content were assessed bymeasuring MCF-7 cell proliferation byflow cytometry and propidium iodide staining. RCL extracts were compared to estradiol (E2) and tothe main RCL isoflavones biochanin A, daidzein, genistein and formononetin. Isoflavone content inthe extracts was assayed by HPLC.E2 and isoflavones increased MCF-7 proliferation in a concentration-dependent fashion, with thefollowing potency order: E2 NNN genistein N biochanin A = daidzein N formononetin. Extractsincreased MCF-7 proliferation with different potencies, which in four out of five extractscorrelated with the ratios 5,7-dihydroxyisoflavones/7-hydroxyisoflavones. The efficacy of allextracts increased with decreasing genistein contents. A solution containing themain isoflavonesat the average concentration of RCL extracts increased MCF-7 proliferation with higher potencyand steeper concentration–response curve. The effects of E2, of RCL extracts and of the isoflavonesolution were inhibited by the estrogen receptor antagonist 4-hydroxytamoxifen.Flow cytometric analysis of MCF-7 proliferation is a suitable bioassay for the estrogenicity ofRCL extracts, thus expanding the characterization of individual batches beyond assessment ofchemical composition and contributing to improved standardization of quality and activity.

© 2014 Elsevier B.V. All rights reserved.

Chemical compounds studied in this article:Biochanin A (PubChem CID: 5280373)Daidzein (PubChem CID: 5281708)Genistein (PubChem CID: 5280961)Formononetin (PubChem CID: 5280378)Estradiol (PubChem CID: 5757)Tamoxifen (PubChem CID: 2733526)4-Hydroxytamoxifen (PubChem CID: 449459)

Keywords:Red cloverTrifolium pratenseEstradiolBiochanin ADaidzeinGenisteinFormononetinEstrogenicityMCF-7

1. Introduction

Red clover (Trifoliumpratense L., Fabaceae; RCL) is a perennialplant rich in phytoestrogens (mainly biochanin A, daidzein,

edical Pharmacology,00 Varese, VA, Italy.7409/397409.. Cosentino).

All rights reserved.

genistein and formononetin) (Fig. 1),whichhas been extensivelyused in traditional and folk medicine. Scientific evidencecurrently supports the use of RCL extracts to treat menopausalsymptoms, as an alternative to hormone replacement therapy,for hyperlipidemia, and to prevent osteoporosis, as well as forbenign prostatic hypertrophy and possibly prostate cancer [1].Most of the established uses of RCL extracts are based on theirestrogenic activity, which has been extensively documented inseveral in vitro and in vivo models [2–4].

Fig. 1. Chemical structure of 17 β-estradiol and the main isoflavones in red clover.

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A critical issue with botanical and herbal preparations istheir chemical standardization and its relationship with theresulting biological activity [5]. RCL extracts are usuallystandardized to their isoflavone content. Recommended dosesof RCL usually yield a minimum of 40 mg total isoflavones perserving; however there is no agreement regarding the com-position in individual isoflavones, and extensive variations ofisoflavone content and profiles have been documented amongcommercial RCL products [6]. Fluctuations in individualisoflavone content may indeed result from differences in ex-traction procedure as well as from natural seasonal variationand variations originating from agricultural conditions [7]. Noinformation exists so far regarding the consequences of diffe-rences in the relative content of individual isoflavones on thebiological activity of RCL extracts, although it has been reportedthat differences in isoflavone content may impact on the bio-availability of individual isoflavones [6].

The aim of the present study was to assess the estrogenicityof different batches of RCL extracts and to assess their possiblerelationship with their content in isoflavones. To this end, wemeasured the in vitro effect of each extract on the proliferation ofthe estrogen-dependent MCF-7 cells, which express estrogenreceptors and exhibit a strong and reproducible proliferativeresponse to 17β-estradiol (E2) as well as to all the compoundswhich act through such receptors [8]. The use of these cells inbioassays for estrogenicity testing is well established (see e.g. 9),

and we previously used MCF-7 cells in our laboratory to assessthe estrogenicity of the lignan 7-hydroxymatairesinol extractedfrom the heartwood of Norway spruce (Picea abies) [10]. In thisstudy, the effects of RCL extracts on MCF-7 cells proliferationwere compared with the responses induced by E2 and by theisoflavones biochanin A, daidzein, genistein and formononetin,which are the reference compounds for the preparation ofstandardized RCL extracts according to the U.S. Pharmaco-peia [11]. Biochanin A, daidzein, genistein and formononetinwere tested alone and in a fixed combination which reproducedthe average isoflavone profile of RCL extracts. Results showedthat the concentration–response relationships of the variousextracts differ for their potencies,which correlatedwith the ratio5,7-dihydroxyisoflavones to 7-hydroxyisoflavones but not withindividual isoflavones. The concentration–response relationshipof isoflavones in fixed combination was different from thoseof RCL extracts, suggesting that other factors, besides therelative concentrations of biochanin A, daidzein, genistein andformononetin, contribute to the biological activity of the extracts.

2. Materials and methods

2.1. Test substances

Five batches of RCL (dark green amorphous powder),prepared between 2009 and 2011 and identified in alphabetical

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order from A to E, were provided by Linnea SA (Riazzino(Locarno), CH) and stored at room temperature. 17β-estradiol(E2), tamoxifen, 4-hydroxytamoxifen, biochanin A, daidzein,genistein and formononetin were obtained from Sigma(St. Louis, MO, USA). Stock solutions of RCL extracts wereprepared at the concentration of 10 mg/ml in dimethylsulfoxide(DMSO) and stored at 4 °C. Stock solutions of E2, isoflavones andtamoxifen were prepared at the concentration of 1 × 10−2 M inDMSO and stored at −20 °C. Finally, stock solutions of4-hydroxytamoxifen were prepared at the concentration of1 × 10−2 M in ethanol and stored at−80 °C. All stock solutionswere serially diluted into culture medium.

2.2. Cells

The human breast cancer MCF-7 cell line was obtainedfrom the European Collection of Cell Cultures (ECACC, PortonDown, UK). Cells were routinely cultured in phenol red-freeDulbecco's modified Eagle's medium (DMEM, EuroClone,Milan, Italy) supplemented with 10% FBS (EuroClone), 2 mML-glutamine (EuroClone) and 100 U/ml penicillin/streptomycin(EuroClone) at 37 °C in a moist atmosphere of 5% CO2. Beforetreatments cells were transferred to phenol red-free DMEMsupplemented with 20% charcoal/dextran-treated fetal bovineserum (FBS, Thermo Scientific, EuroClone), 2 mM L-glutamine(EuroClone) and 100 U/ml penicillin/streptomycin (EuroClone).Subconfluent cultures were then split 1:3 using trypsine/EDTAand plated in 75 cm2 flasks. In each experimental session, cellswere seeded in 6-well plates in the absence or presence of thevarious agents for 24 h. In preliminary experiments, the effectsof vehicles at concentrations equivalent to the highest con-centrations used was always negligible and non-significant(% variation of MCF-7 cells in the S phase of the cell cycle thepresence of DMSO 1:1000 v/v was 0.22 ± 0.81 (n = 6) and inthe presence of ethanol 1:1000 v/v it was −0.44 ± 0.31(n = 6); P N 0.05 in both cases). In experiments with tamox-ifen or 4-hydroxytamoxifen, these substances were added15 min before the addition of E2, isoflavones or RCL extracts. Ineach experimental session, a well containing cells without anytreatmentwas always included as baseline control, while awellcontaining cells treated with E2 1 × 10−10 M was included toallow for possible inter-session variability.

2.3. Flow cytometric analysis of cell proliferation

Cell proliferation was determined by staining DNAwith propidium iodide (PI, Sigma) according to a previouslydescribed method with modifications [10]. To this end,MCF-7 cells were harvested, washed with PBS by centrifuga-tion at 600 g for 5 min, and fixed/permeabilized with 1.5 mlof 70% ice-cold ethanol for 3.5 h at 4 °C. After washing,pellets were resuspended in 50 μl of ribonuclease A solution(RNAse, Sigma) at the final concentration of 100 μg/ml andincubated at 37 °C for 30 min. Cell suspensions were thenadded 0.6 ml PBS containing 50 μg/ml PI and incubated atroom temperature in the dark for 45 min. Flow cytometricanalysis of DNA content was carried out using a BD FACSCantoII flow cytometer (Becton Dickinson Italy, Milan, Italy) withFACSDiva software (version 6.1.3). Fluorescence signal of PI(FL3) was collected on a linear scale using a 670-LP filter.At least 25,000 events/sample were acquired and gated on a

standard bi-parametric dot plot (doublet discriminator plot)FL3-A vs FL3-H to distinguish single cells from doubletsor aggregates. Each DNA histogram was analysed withFlowJo software (version 8.3.2) that fits cell cycle data usingmathematical models to define the percentages (%) of cells inG0/G1, S and G2/M phases of the cell cycle. A typical cell cycleanalysis is shown in Fig. 2. For the purpose of the present study,the percentage of cells in the S phase of the cell cycle, i.e. thepart of the cell cycle inwhichDNA is replicated, was taken as anindex of cell proliferation.

2.4. Determination of isoflavones by HPLC

The four main isoflavones (biochanin A, daidzein, genisteinand formononetin) were detected at 254 nm, using the USP 36method for “powdered red clover extract”. The system wascalibrated for formononetin standard, obtained from USP. Thecolumn was a reversed phase, dimensions 4.6 mm × 250 mm,end-capped silica, 5 μm size, and packing L1. Analytical HPLCwas performed on an Agilent 1200 series, consisting of adegasser, a quaternary low pressure gradient pump (PU 2089plus, Jasco), a programmable autosampler (AS 2057 plus, Jasco),a column heater (CO 2060 plus, Jasco), an interface (LC-NET II/ADC, Jasco) and an UV detector (UV 2070 plus, Jasco). Sampleelaboration, gradient profile and eluent composition were madeaccording to USP 36 NF 31. The flow rate was 1 ml/min and theoven temperature was hold at 45 °C. Other isoflavones(coumestrol, glycitein, ononin, pratensein, prunetin, pseudo-baptigenin, sissotrin and trifoside) were detected at 254 nm,according to an established method [12]. In the case ofcoumestrol, a fluorimetric detector (FP 2020 plus, Jasco; Ex:249 nm, Em: 419 nm) was used instead of the UV detector, inorder to reducematrix interference and tomaximize coumestrolresponse. Sample elaboration, gradient profile and eluent com-position were made according to the reference method [12]. Inpreliminary analysis, the identity of all the isoflavones wasverified by liquid chromatography–mass spectrometry, by use ofa mass detector LCQ DECA (Thermo Fisher Scientific Inc., USA)with the following ESI/MS conditions: spray voltage: 5.00 kV,capillary temperature: 220 °C, capillary voltage: 41 V, and massrange: 50–1000 Da.

2.5. Experimental design and analysis of the data

Experiments were performed in order to obtain completeconcentration–response relationships for each substance.Concentration–response relationships were analyzed bynonlinear regression using GraphPad Prism version 6.00for Windows (GraphPad Software, La Jolla California USA,www.graphpad.com).

Sigmoidal concentration–response curves with variableslope were fitted to the data obtained with E2, isoflavonesand RCL extracts. The function used (Eq. (1)) is described as:

Y ¼ aþ b−að Þ= 1þ 10∧ Log EC50ð Þ−Xð Þ � nð Þ� �

ð1Þ

where Y is the response, while X corresponds to theconcentration of the tested agent. Parameter a equals thebaseline and b is the plateau of the curve (i.e. the maximalresponse, Emax). Log(EC50) is the Log of the concentration

Fig. 2. Typical MCF-7 cell cycle analysis. A: identification and gating of single cells by standard bi-parametric dot plot (doublet discriminator plot) FL3-A vs FL3-H; B:histogram of cell distribution according to DNA content (as assessed by PI staining); C: fitting of cell cycle data by use of FlowJo softare. SeeMethods for further details.

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value when the response is halfway between baseline andplateau, that is the effective concentration, 50% (i.e. theconcentration which elicits 50% of the maximal response).Parameter n is the Hill coefficient, which describes thesteepness of the curve and may be related to the cooperativityof the ligand–receptor binding (the higher the value, the morecooperative the binding). The mean values of EC50, Emax andHill coefficient were finally calculated together with 95%confidence interval (C.I.).

In experiments with the ER antagonists tamoxifen and4-hydroxytamoxifen, data from concentration–responsecurves for the inhibition of the effects of E2 or of RCL extractswere fitted to sigmoidal concentration–response curves withstandard slope (n = 1). The function used (Eq. (2)) was:

Y ¼ aþ b−að Þ= 1þ 10∧ X− Log IC50ð Þð Þ� �

ð2Þ

where Y is the response, while X corresponds to the con-centration of the tested antagonist. Parameter a equals thebaseline and b is the plateau of the curve (with negative values,since an inhibitory effect is measured and the curve goesdownhill). Log(IC50) is the Log of the concentration valuewhenthe response is halfway between baseline and plateau, that isthe inhibitory concentration, 50% (i.e. the concentration whichinhibits 50% of the agonist response). The mean values of IC50together with 95% C.I. were finally calculated.

Correlations between mean EC50 and Emax of RCL extractsand their content in isoflavones were analyzed by linear

regression and the Pearson correlation coefficient was calcu-lated by use of the same statistical software.

3. Results

3.1. Isoflavone content of RCL extracts

The isoflavone content of the various batches of RCL extracts,together with their identification codes, is shown in Table 1. Atypical HPLC assay of the four main isoflavones biochanin A,daidzein, genistein and formononetin in RCL extracts is shownin Fig. 3.

3.2. Effect of E2 and isoflavones

E2 and isoflavones increased the percentage of MCF-7 cellsin the S phase of the cell cycle in a concentration-dependentfashion (Fig. 4). Mean EC50, Emax and Hill coefficients with 95%C.I. for E2 and isoflavones are shown in Table 2.

In agreement with previous observations [10], E2 displayedthe highest potency in the pM–nM concentration range, whileisoflavones were active in the nM–μM range, however withefficacieswhichwere not significantly different from that of E2.

The order of potency was: E2 NNN genistein N biochaninA = daidzein N formononetin. The Hill coefficients of theconcentration–response curves of E2 and all isoflavones werenot significantly different from 1 (Table 2).

Table 1Isoflavone content of RCL extracts expressed as weight %.

RCL extract A B C D E

Biochanin A 20.12 18.60 17.40 22.10 26.47Coumestrol 0.00 0.00 0.00 0.00 0.00Daidzein 0.13 0.16 0.18 0.13 0.13Formononetin 22.48 20.60 24.10 13.90 15.65Genistein 0.45 0.71 0.53 0.53 0.31Glycitein 0.28 0.38 0.38 0.28 0.26Ononin 0.80 0.79 0.74 0.42 0.66Pratensein 0.76 0.85 0.77 1.14 1.11Prunetin 0.65 0.92 0.74 1.49 0.93Pseudobaptigenin 0.52 0.63 0.57 0.52 0.60Sissotrin 1.35 1.64 0.96 1.47 1.75Trifoside 0.07 0.45 0.09 0.52 0.42Ratio (B + G)/(D + F)⁎ 0.91 0.93 0.741 1.613 1.697

⁎ Ratio of 5,7-dihydroxyisoflavones to 7-hydroxyisoflavones (biochanin A +genistein)/(daidzein + formononetin) according to [12].

Fig. 4. Effect of E2 and isoflavones on ΔS phase in cultured MCF-7 cells. Eachpoint is the mean ± SEM of at least 5 experiments. Data expressed as % ofthe effect of E2 0.1 nM.

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3.3. Effect of RCL extracts

All the tested RCL extracts increased MCF-7 cell prolifer-ation with similar efficacy however with a significantlydifferent potency (Fig. 5). Mean EC50, Emax and Hill coeffi-cients with 95% C.I. for all RCL extracts are shown in Table 3.

RCL extracts did not differ between each other according totheir respective mean Emax, however, in comparison to E2, RCLextracts A and E had significantly higher Emax as indicated bythe absence of overlapping between 95% C.I. of their Emax andthe Emax of E2 (P b 0.05 in both cases).

The order of potency of RCL extracts was: A =B ≥ C ≥ D = E. The Hill coefficients of the concentration–response curves of all RCL extracts were not significantlydifferent from 1, with the only exception of RCL extract A,which however had the higher limit of 95% C.I. very close to 1(0.99) (Table 3).

There were no significant correlations between meanEC50 or Emax of the various RCL extracts and their content ineither individual or total isoflavones, with the only excep-tion of a slightly significant inverse correlation betweenmean Emax and the content of genistein (Pearson correlationcoefficient = −0.883; P = 0.047). The mean EC50 of RCL

Fig. 3. Typical HPLC assay of the isoflavones daidzein (peak 1), genistein (pea

extracts A, B, D and E (but not C) had a direct and highlysignificant correlation with the respective ratios of5,7-dihydroxyisoflavones to 7-hydroxyisoflavones (Pearsoncorrelation coefficient = 0.999; P = 0.001).

3.4. Effect of a mix solution of isoflavones

To compare the effects of RCL extracts with those of theisoflavones they contained, first a solution containing a totalisoflavone concentration of 4 mg/ml (i.e. the expected averageconcentration obtained in the stock solutions of RCL extracts)was prepared in DMSO. The relative amount of isoflavonesin the solution was (%): biochanin A, 55.3; daidzein, 0.2;formononetin, 43.5; and genistein, 1.0. The isoflavone solutionincreased the proliferation of MCF-7 cells with a mean Emax

(expressed as % of the effect of E2 0.1 nM) of 109.7 (95% C.I.:105.2–114.2) which was not significantly different from theEmax of E2 and, like the Emax of E2, and was significantly lowerthan the Emax of RCL extracts A and E (P b 0.05 in both cases).

k 2), biochanin A (peak 3) and formononetin (peak 4) in RCL extracts.

Table 2EC50, Emax and the Hill coefficient values of the concentration–response curves of E2 and isoflavones on ΔS phase in cultured MCF-7 cells. Data expressed as % ofthe effect of E2 0.1 nM.

EC50 (M) Emax (ΔS phase)% of E2 0.1 nM

Hill coefficient

Mean 95% C.I. Mean 95% C.I. Mean 95% C.I.

Estradiol 5.94 × 10−12 4.49–7.85 105.4 99.22–111.6 1.23 0.76–1.70Biochanin A 1.58 × 10−7 0.96–2.61 107.3 93.0–121.6 1.09 0.64–1.54Daidzein 1.09 × 10−7 0.77–1.54 103.6 93.7–113.5 1.28 0.75–1.81Formononetin 7.08 × 10−7 5.18–9.68 117.5 105.2–129.8 1.05 0.76–1.34Genistein 4.19 × 10−8 3.11–5.65 112.1 104.3–119.9 1.17 0.81–1.53

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The EC50 of the isoflavone solution was 2.13 × 10−8 gisoflavones/ml (95% C.I.: 1.85–2.44 × 10−8 g isoflavones/ml).For comparison, the EC50 values (with 95% C.I.) of RCL extractsexpressed in terms of 10−8 g isoflavones/ml (according to theirrespective total isoflavone content as reported in Table 1) were:A, 3.32 (2.44–4.52); B, 3.41 (2.60–4.47); C, 5.76 (4.19–7.92); D,6.12 (4.08–9.19); and E, 7.69 (5.24–11.26), which were allsignificantly higher than the EC50 of the isoflavone solution. TheHill coefficient of the concentration–response curve of theisoflavone solution was 2.18 (95% C.I.: 1.35–3.01) and was thussignificantly different from 1 aswell as from the Hill coefficientsof the concentration-response curves of all the RCL extracts,with the only exception of a slight overlapping between itslower 95%C.I. and the higher 95%C.I. of theHill coefficient of RCLextract B (1.35 vs 1.37) (Table 3).

3.5. Effect of tamoxifen and 4-hydroxytamoxifen

In preliminary experiments, the ability of tamoxifen and4-hydroxytamoxifen to affect MCF-7 cell proliferation wasassessed. Both compounds at low concentrations significantlyincreased cell proliferation. Tamoxifen 1 × 10−7 M and4-hydroxytamoxifen 1 × 10−9 M increased the percentageof cells in the S phase of the cell cycle by respectively 2.6 ±

Fig. 5. Effect of RCL extracts on ΔS phase in cultured MCF-7 cells. Each pointis the mean ± SEM of at least 5 experiments. Data expressed as % of theeffect of E2 0.1 nM.

0.5% (n = 6, P b 0.001 vs control) and 1.4 ± 0.5 (n = 6,P = 0.013 vs control). The effect of 4-hydroxytamoxifen inthe 1 × 10−8–1 × 10−6 M concentration range was onaverage in the range ±0.1%, being therefore negligible. Theeffect of tamoxifen 1 × 10−6 was 0.8 ± 0.4 (n = 6), whichwas not significantly different from control. Both tamoxifenand 4-hydroxytamoxifen 1 × 10−5 M decreased MCF-7 cellproliferation by 0.9 ± 0.6% (n = 6, P = 0.011 vs control)and 1.1 ± 0.3 (n = 6, P = 0.075 vs control) respectively.Accordingly, 4-hydroxytamoxifen was selected for subse-quent experiments with E2 and RCL extracts.

3.6. Effect of 4-hydroxytamoxifen on E2 and RCL extracts

In the presence of 4-hydroxytamoxifen, the effect of E21 × 10−10 M on MCF-7 cell proliferation was completelyinhibited, with a mean IC50 (with 95% C.I.) of 6.10 (3.44–10.83) × 10−9 M.

An addition of 4-hydroxytamoxifen also completelyinhibited the effects of RCL extracts at the concentration of5 × 10−6 g/ml, as well as the effect of the isoflavone solutionat the concentration of 2 × 10−6 g isoflavones/ml (which isabout the average concentration of isoflavones in RCL extracts5 × 10−6 g/ml). The IC50 of 4-hydroxytamoxifen towards thevarious RCL extracts and towards the isoflavone solution wasnot significantly different, as shown in Table 4.

4. Discussion

The results of the present study indicate that theestrogenic activity of RCL extracts could be accurately andreproducibly assessed in cultured MCF-7 cells. RCL extractsinduced MCF-7 cell proliferation to a similar or even higherextent in comparison to the reference compound E2. The

Table 3EC50, Emax and the Hill coefficient values of the concentration–responsecurves of RCL extracts on ΔS phase in cultured MCF-7 cells. Data expressedas % of the effect of E2 0.1 nM.

RCLextract

EC50 (10−8 g/ml) Emax (ΔS phase)% of E2 0.1 nM

Hill coefficient

Mean 95% CI Mean 95% CI Mean 95% C.I.

A 7.69 5.65–10.47 126.9 118.4–135.5 0.81 0.63–0.99B 8.50 6.49–11.15 119.8 111.3–128.2 1.07 0.76–1.37C 13.63 9.91–18.74 117.7 109.1–126.3 0.93 0.70–1.17D 16.70 11.12–25.08 117.7 106.5–128.9 0.95 0.66–1.24E 18.06 12.33–26.45 126.6 115.2–138.1 0.98 0.69–1.26

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effect of RCL extracts was completely prevented by theestrogen receptor antagonist 4-hydroxytamoxifen, suggest-ing that it is likely completely dependent upon estrogenreceptor stimulation.

The main purpose of the study was however to assess anypossible differences in the estrogenicity of distinct batches of RCLextracts and their relationship with the content in isoflavones.RCL extracts exhibited different pharmacological potencies. Theorder of potency of RCL extracts was A = B ≥ C ≥ D = E andthe mean EC50 of RCL extracts A, B, D and E (but not C) had adirect and high correlation with the respective ratios of5,7-dihydroxyisoflavones to 7-hydroxyisoflavones, which is therecommended analytical reference for the preparation ofstandardized RCL extracts according to the U.S. Pharmacopeia[11]. In the present experiments the two RCL extracts with thelower ratio displayed higher potencies (about twofold that of theRCL extracts with higher ratio). Such a relationship is unlikely todepend on individual isoflavones, as no correlation was foundbetween the pharmacological profile of RCL extracts and therespective individual or total isoflavone content. Lower ratio5,7-dihydroxyisoflavones to 7-hydroxyisoflavones in theseextracts is mainly dependent upon lower content in biochaninA and higher content in formononetin. However biochanin A perse is about 3.5 times more potent than formononetin, thesedifferences are unlikely to account for the different potenciesof the extracts. The direct relationship between the potencyof RCL extracts and the ratio 5,7-dihydroxyisoflavones to7-hydroxyisoflavones needs to be confirmed by testing moreRCL extracts with different compositions, also in view of thedeviation from this relationship exhibited by RCL extract C.

The only other correlation between the pharmacologicalactivity of RCL extracts and their chemical composition wasan inverse relationship between the maximal efficacy (Emax)and their respective content of genistein. Several lines ofevidence suggest that genistein may have antiproliferativeactivities (see e.g. 13). In the present experiments howevergenistein per se exhibited only stimulatory effects on MCF-7cell proliferation, moreover with the highest potency amongthe various isoflavones tested. It is therefore unlikely that theinverse correlation between mean Emax of RCL extracts andgenistein content depends upon the intrinsic pharmacolog-ical properties of genistein.

The complex relationship between RCL extract composi-tion and their estrogenic properties is also indicated bythe profound differences observed between their effectsand those of a solution containing biochanin A, daidzein,formononetin and genistein at concentrations similar to

Table 4IC50 of the concentration–response curves of 4-hydroxytamoxifen on the effectof RCL extracts 5 × 10−6 g/ml and of the isoflavone solution 2 × 10−6 gisoflavones/ml on ΔS phase in cultured MCF-7 cells.

RCL extract IC50 (M)

Mean 95% CI

A 8.53 × 10−8 4.59–15.86B 7.46 × 10−8 3.96–14.06C 5.00 × 10−8 2.46–10.15D 3.97 × 10−8 1.61–9.76E 5.59 × 10−8 2.93–10.65Isoflavone solution 5.90 × 10−8 2.83–12.29

those occurring in solutions obtained from RCL extracts.Such a solution had higher potency, and in particular the Hillcoefficient of its concentration–response curve was greaterthan unity, while the Hill coefficients of RCL extracts werealways around one. Hill coefficients greater that one are ofteninterpreted in terms of positive cooperativity in the relation-ship between receptor occupancy and response [14]. Wheth-er positive cooperativity actually occurred in the action of theisoflavone solution cannot be established on the basis of thepresent results; nonetheless it may be of interest that eventhe Hill coefficients of individual isoflavones were all aroundone, thus suggesting that complex interactions amongdifferent isoflavones occur when they are simultaneouslyadministered. A role for theRCLphytocomplex in themodulationof the pharmacological activity of its individual components isthus suggested by the present results.

In conclusion, the present study supports the use of MCF-7cells as a reference model to assess the estrogenicity of RCLextracts. In particular, differences in the pharmacologicalprofile or various batches of RCL extracts could be identifiedthrough the flow cytometric analysis of the cell cycle afterstaining of the cellswith PI. It has been suggested that, ideally, abotanical formulation should be standardized, both chemicallyand biologically, by a combination of analytical techniques andbioassays [15]. Flow cytometric analysis of MCF-7 cell prolifer-ation can represent a suitable bioassay to assess the estrogenicprofile of RCL extracts, thus expanding the characterization ofindividual batches beyond the mere assessment of theirchemical composition and contributing to a better standardi-zation of their quality and activity.

Conflict of interest

Marcello Luzzani, Enrico Casareto, andMassimiliano Carreriare employees, and Silvano Paracchini is advisor of Linnea SA(CH). All the other authors declare no conflict of interest.

Acknowledgments

The present study was supported in part by a grant fromFondazione Cariplo (Project RE-D DRUG TRAI-N 2010-1373:Multidisciplinary approaches in research and developmentof innovative drugs: project for an international collabora-tive training network). PS received a two-year fellowshipgrant from Regione Lombardia (Project RE-D DRUG TRAI-NPhytomedicine Platform).

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