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					                                                                   General Endocrinology

  A HPLC EVALUATION OF GENISTEIN – AN ESTROGEN-
MIMETIC PHYTOESTROGEN – IN GLYCINE MAX (SOY) BEANS

              A. Dragomirescu*, 1, A. Muresan2, E. Alexa3, M. Andoni4

1Department   of Dermatopharmacy and Cosmetology, Faculty of Pharmacy, 2Department
of Pathology, Faculty of Medicine,“Victor Babeº” University of Medicine and Pharmacy,
3Banat’s University of Agricultural Science, 4Department of Inorganic Chemistry, Faculty

          of Pharmacy, “Victor Babeº” University of Medicine and Pharmacy

      Abstract
      Background. Phytoestrogens are a heterogenic chemical category in which are
included: isoflavones (genistein, daitzein, glycitein, biochianin A, formononetin, equol),
some lignans from cereals, cumestrans, some components of volatile oils (anetol, scareol),
some steroid saponins (diosgenin) and some phytosterols (beta-sitosterol, stigmasterol).
Genistein is the most discussed phytoestrogenic substance, because it is very well
represented in the botanical sources - especially in Fabaceae (Leguminosae) species, and
mainly in soybeans.
      Aim. The actual study evaluates the quantity of genistein in soybeans
      Methods. We used three extraction techniques: extraction with hydrolysis, extraction
without hydrolysis and, finally, extraction with hydrolysis and clean-up and HPLC
identification.
      Results The experimental results indicated the content of genistein /g soya using the
extraction techniques described.
      Conclusions. Our study shows that extraction without hydrolysis followed by HPLC
chromatographic method detects the genistein from vegetal sources and offer a good
method, proposed by us for the drug industry.
      Key words: genistein, estrogen-mimetic substance, Fabaceae, soybean, HPLC.


                                   INTRODUCTION

      Phytoestrogens are sterolic and non-sterolic substances, synthesized by plants
for their own defense (fungicides, UV filters), which act in animal’s organism like
beta-estrogen receptor ligand. The first observations of those estrogens effect was
happening: Australians sheep fed with Trifolium partensis stopped their own
reproduction, because this Fabaceae species acted like an “oral contraceptive” for
*Correspondence to: A. Dragomirescu, Department of. Dermatopharmacy and Cosmetology,
University of Medicine and Pharmacy, 2 Piata Eftimie Murgu, Timisoara, Romania, tel (40)
0726.503.896 Email: ancadrag65@yahoo.com
                                      Acta Endocrinologica (Buc), vol. V, no. 1, p. 41-47, 2009

                                                                                            41
                                   A. Dragomirescu et al.

sheep. Epidemiological studies showed that asiatic populations are more protected
than others for some estrogen-dependent cancers, and that fact is correlated with the
soy ingestion (1,2).
      Phytoestrogens are a heterogenic chemical category in which are included:
isoflavones (genistein, daitzein, glycitein, biochianin A, formononetin, equol),
some lignans from cereals, cumestrans, some components of volatile oils (anetol,
scareol), some steroid saponins (diosgenin) and some phytosterols (beta-sitosterol,
stigmasterol) (2).
      Genistein is the most discussed phytoestrogenic substance, because it is very
well represented in the botanic sources – specially in Fabaceae (Leguminosae)
species, also because it is one of the most pharmaceutically active phytoestrogens
and because it has a long history of research and of controversial results (3).
Genistein is an isoflavone with chemical structure:4´,5,7–three-hydroxy-isoflavone,
having estrogen-mimetic properties and estrogens-modulant effect, by its ability of
binding the estrogen receptor β. The substance is in course of pharmaceutical
evaluation also because of its ability of acting like a tyrosine-kinase inhibitor. This
enzyme is attached to different membrane receptors, including those which are
“growth factors” (like EGF and TGF) in some tissues (mostly epithelial tissues and
glandular tissues). By this ability, genistein is considered an anticancer agent,
because factors like EGF and TGF are implicated in the mitogenesis process,
including the aberrant mitogenesis (4,5).
      In spite of the fact that genistein does not have a steroid structure, it has a




                     Figure 1. Chemical structure of genistein.


spatial structure similar to estradiol. For this reason, genistein can competitively
bind the estrogen receptor. From the chemical point of view, genistein is included
in the same class of other estrogenic isoflavones (daidzein, glycitein, biocianin A,
equol), but it is the only one which had an inhibitory tyrosine-kinase activity. This
property recommends genistein as the most efficient isoflavone in therapeutics (2).
      Genistein is usually extracted from vegetable sources like: Glycine max,
Medicago sativa, Trifolium pratense, Glycyrrhiza glaxbra, Glycyrrhiza uralensis,
and also from a lot of other Fabaceae species.
      Until now, the pharmacological activity of genistein has already been proved
in the following fields (5-9):
      - the prevention of the estrogen-dependent cancers;

42
                          Quantification of genistein in soybeans

     - bone matrix synthesis (prevention of menopausal osteoporosis);
     - prevention of atherosclerosis, including: decreasing the level of LDL, decreasing
the LDL oxidation, decreasing the neoformation in the atheromatosis damages.
     - protection for aging skin, by two mechanisms: estrogenic and anti-oxidant.
     On the chemical and research market, genistein exists as a product in different
purity variants, also, on the pharmaceutical market genistein exists more or less
pure in differently soy extraction products. The vegetables extracts will substitute
more and more the chemical estrogens medication. More than a simply estrogen
drug, genistein is considered now a selective estrogen receptor modulator (SERM).
     For all those reasons, the most used in alimentation Fabaceae species: Glycine
max, deserves an evaluation from this point of view: genistein quantification.


                       MATERIALS AND METHODS

     Genistein (4,5,7-three-hydroxyisoflavone) was purchased from LC Laboratories
PKC Pharmaceuticals Inc. Ethanol, methanol, dichloromethane were analytical grade
for high performance liquid chromatography (HPLC). All other reagents were
analytical grade. The Nucleosil C-18 column (15 x 0.46), 5 µm particle size was
purchased from Teknokroma, serial no. N 33055. Mini catrigde column Bakerbond spe
Octadecyl (C18) were obtained from Baker Analysed, lot G 48558. The domestic soy
beans that we analyzed were selected from the Banat agriculture cultures.
     Instruments
     A Bischoff LC equipped with a binary gradient pump, 10 µL injection loop,
Gynkotek spectrophotometric detector and Hewlett Packard 3392 A Integrator, was
used for genistein analysis. A transonic T 460 ultrasonic bad from Bender and
Hobein were used to sonication samples.
     Standard solutions
     A pre-weighed amount of genistein standard was dissolved in 100 mL methanol
to form stock solutions 1200 µg/mL. Stock solution was diluted with methanol to
prepare standard solutions and internal standard (IS) solutions 12 µg/mL.
     Extraction
     Genistein was analysed without hydrolysis and after acid hydrolysis, as aglycone.
     Extraction without hydrolysis. Each analysis was performed in duplicate: one
with internal standard and one without internal standard. 10 g of soy beans powder
were extracted with 50 mL methanol 80% and sonicated for 30 min. One mL of IS
solution (12 µg) was added to one sample. Genistein was extracted for 24 hours at
ambient temperature, the sample was centrifuged and the supernatant volume was
adjusted to 50 mL with methanol (11).
     Extraction with hydrolysis. Each analysis was performed in duplicate: one
with internal standard and one without internal standard. 10 g of soy beans powder
were extracted with 10 mL solution HCl 10 M and 40 mL ethanol containing 0.05%
butylhydroxytoluene and then sonicated for 30 min. One mL of IS solution (12 µg)

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                                        A. Dragomirescu et al.

was added to one sample. Hydrolysis was carried out by reflux in a boiling water
bath for 3 h. The tube was adjusted to 50mL with methanol (11).
     Clean-up. Extraction solutions were cleaned up using C18 catridges. The
column was preconditioned with 20 mL water and 2 mL methanol. One mL of each
sample solution was diluted with 10 mL water and applied to column. The genistein
was eluted with 2 mL methanol (11).
     HPLC conditions. Apparatus, Bischoff LC equipped with binary gradient
pump, 10 µL injection loop, Gynkotek spectrophotometric detector and Hewlett
Packard 3392 A Integrator. Column: nucleosil C-18 column. HPLC separation of
genistein is made in good conditions using methylene chloride: methanol (94:6, v/v)
as mobile phase.


                                            RESULTS

     The experimental results indicated 4.20 µg genistein/ g soya fresh weight
respectively 4.46 µg genistein/ g dry substance (moisture of soya 4.8%) in the case
of extraction without hydrolysis and 3.75 µg genistein/ g soya fresh weight
respectively 4.46 µg genistein/ g dry substance when extraction is made with
hydrolysis. With clean up the genistein content is 2.45 µg genistein/ g soya fresh
weight, meaning a recovery rate of 65.33%, comparing to the genistein quantity
extracted by hydrolysis, without clean-up.
           Table 1. Retention time, detection limit and linear range of genistein by HPLC
       Isoflavone       Retention time Detection limit                     Linear range
                        (min)*
                                       µg/ml       µg/g fresh weight       µg/ml          R2
        Genistein        5.75            0.03       0.15                   0.03-30     0.993
     HPLC conditions: Bischoff LC equipped with a binary gradient pump, 10 µl injection loop,
     Gynkotek spectrophotometric detector and Hewlett Packard 3392 A Integrator.
     Column: NUCLEOSIL C-18 column (15 x 0.46), 5 µm particle size.
     Mobile phase: acetonitril:methanol:water:phosphoric acid (30:15:55:0.1), flow rate 1.5 ml/min
     (linear gradient program). Spectrophotometric detector, wavelength 254 nm.
          Table 2. The content of genistein in Soya (Glycine max)
      Fabacea species         Treatment                      Genistein
                                                             µg/g fresh weight     µg/g s.u.
      Soya (Glycine max)      Without hydrolysis             4.20                  4.46
      Soya (Glycine max)      With hydrolysis                3.75                  3.98
      Soya (Glycine max)      with hydrolysis and clean up   2.45                  2.60

          Table 3. Recovery of standard solution (12 µg genistein) with and without hydrolysis
        Fabacea species        Recovery (%)
                               Without hydrolysis   With hydrolysis    Clean-up on the C18
                                                                       cartridge
        Soya (Glycine max)     81,2                 78.33              65,33


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                         Quantification of genistein in soybeans




                                         a                                            b




                                             Figure 2a. HPLC chromatogram of genistein
                                             standard*.
                                             Figure 2b. HPLC chromatogram of soya
                                             extract without hydrolysis*.
                                             Figure 2c HPLC chromatogram of soya extract
                                        c    with hydrolysis*.

     The HPLC chromatograms are given in Figures 2a-c. The retention time,
detection limit and linear range of genistein by HPLC are given in Table 1.
The retention time, using HPLC conditions described in the text, is 5.75 min and the
detection limit 0.03 µg/ml, respectively 0.15 µg/g fresh weight. The results
obtained, the recovery coefficient with and without hydrolysis and C18 cartridges
clean-up are given in Tables 2-3. Recovery tests were performed both with and
without hydrolysis for soya (Table 3). Recovery tests were: 81.2% with hydrolysis
and 78.33% without hydrolysis for soya bean.

                                                                                           45
                                       A. Dragomirescu et al.

                                        DISCUSSION

      Our study shows that the highest genistein concentrations were obtained for
the extraction without hydrolysis. In one of our previous studies, we determined the
concentration of genistein in other two Fabaceae (Leguminosae) species:
Phaseoleus vulgaris beans and Lens culinaris. Genistein determined in Phaseoleus
vulgaris was in a limit of: 0.435 - 0.34 µg/g (in function of extraction type),
meaning 10 times less than for soy beans and for Lens culinaris only 0.20 - <0.15
µg/g, meaning an insignificant concentration. So alimentation with soy beans is
offering an important quantity of genistein, which is significantly grater than for the
nutrition based on other Leguminosae.
      Fabaceae family is one of the richest plant sources in estrogen-mimetic
isoflavones. Those substances (genistein, daidzein, glycitein, equol) are acting by
binding the estrogen receptor-beta in human and animal organisms. This benefit
(used to protect against the menopausal dysfunctions) is added to other nutritional
benefits of Fabaceae family (like the great content in essentials AA, for soy).
      This observation can be correlated with the epidemiological studies of
estrogen-like effects of soy alimentation, in Asiatic people. A good prevention of
menopausal effect is proposed to be realized with an alimentation based on soy
(12,13). However, some possible adverse effects must be considered. So, it should
be pointed the possibility of an androgenic dysfunction in children and adolescents,
if a unilateral soy beans alimentation is used. Additionally, apoptosis is suspected
to be induced by the genistein abuse, mainly in the nervous system (14,15).
      The method described in this paper can be used in drug industry, to obtain the
dietary supplements.
      In conclusion, this study evaluated the quantity of genistein in soybeans, using
three extractions techniques: with hydrolysis, without hydrolysis, and extraction
with hydrolysis and clean-up. For all three methods we determined the quantity of
genistein in fresh and dry substances. The richest concentration in genistein was
obtained for extraction without hydrolysis: 4.20 µg/g, respectively 4.46µg/g. By
HPLC we can determined genistein in Fabaceae species with content higher than 1
µg genistein/g sample, respectively soya (Glycine max) extracts.


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                               Quantification of genistein in soybeans
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