Determination of Daidzein and Genistein in Soy Milk in Iran by Using HPLC Analysis Method
The HPLC system separated completely isofavonoids such
as daidzein (15.2 min) and genstein (17.3 min). Initially, the concentrations
of major isoflavone Genistein and Daidzein in the tested soy milk were
determined. Commercial soy milk samples were analyzed for isoflavones
and two major isoflavones detected: genistein 25.86 (mg L-1)
±0.66 SD and daidzein 8.25 (mg L-1) ±1.13 SD.
Concentrations of genstein in soy milk were higher than daidzein. The
results obtained in this study can serve as a basis for estimating amount
of soy milk can be consumed by people as related to its main isoflavone
The health benefits of soybean products have been well documented. Epidemiological
studies have indicated that the consumption of soybeans products may prevent
certain cancers such as breast cancer, prostate cancer, colon cancer,
(Alekel et al., 1998; Anthony et al., 1996), reduce the
risk of hyperlipidemia, cardiovascular disease and osteoporosis (Arjmandi
et al., 1996; Barnes et al., 1994), have a beneficial role
in chronic renal disease (Fico et al., 2000; Ranich et al.,
2001), lower plasma cholesterol (Franke et al., 1995; Ho et
al., 2000), exhibit an antiatherosclerotic activity (Hillis and Isoi,
1965; Huff et al., 1982) and decrease the risk of coronary heart
disease (Lucas et al., 2001). The commonly studied phytoestrogens
are isoflavones (Fig. 1). Genistein and daidzein possess
antioxidant, anticarcinogenic and anti-osteoporosis activities both
in vivo and in vitro. The intake of genistein and daidzein
has been shown to provide protection again oxidative modification of Low-Density
Lipoprotein (LDL) particles in human volunteers (Tikkanen et al.,
1998). When incubated in human plasma, some genistein and daidzein were
incorporated into LDL particles (Kerry and Abbey, 1998) and incorporation
of esterified genistein and daidzein increased resistance of LDL to coppermediated
oxidation in vitro (Meng et al., 1999). Consumption of soybean
isoflavones was also found to be protective against DNA oxidation in human
lymphocytes (Mitchell and Collins, 1999). In addition, genistein and daidzein
also exhibited, in vitro, strong antioxidant potency in liposomes
challenged with UV exposure, peroxyl and hydroxyl free radicals (Record
et al., 1995; Tissier et al., 2007). Isoflavones including
daidzein and genistein are found almost exclusively in soybeans, which
contain daidzin and genistin as the main glucosides. Thus, isoflavones
are useful as food supplements for the purposes of enhancing human health
and preventing the above diseases. Daidzin and genistin are hydrolyzed
to daidzein and genistein, respectively, by b-glucosidase in the gastrointestinal
system (Piskula et al., 1999; Rucinska et al., 2007; Atteritano
et al., 2008). However, recent investigations have shown that flavonoid
glycosides, including genistin, phloretin and quercetin, can be partially
absorbed without previous hydrolysis of glucose moieties (Paganga and
Rice-Evans, 1997; Ho et al., 2002). In addition, most of the genistein
and daidzein is not present in form of aglycone but instead in the form
of glucuronide and sulfate conjugates in blood (Hendrich, 2002). As shown
in Fig. 1, they have a structure similar to the hormone
estrogen and share some of its physiological properties. For these reasons,
isoflavones are sometimes called phytoestrogens (Markham et al.,
1978; Piskula et al., 1999). Therefore, isoflavones may not only
have a variety of desirable physiological effects on the body, but they
may also act as endocrine disruptors. Exposure to these phytoestrogens
may pose a developmental hazard to infants, because soy products are becoming
increasingly popular as infant foods. Though an accurate assessment of
the evaluation of these contradictory health effects is difficult, knowing
the existence and amount of isoflavones in foods is important. Even though
there are a lot of publication exist regarding the concentrations of isoflavones
in soymilk, since the product is not general in Persian diet. The aim
of this study was to determine the concentrations of daidzein and genistein
the major isoflavones of soy milk in Iran.
||Chemical structures of isoflavones and 17 β-estradiol
MATERIALS AND METHODS
This study, was carried out from 2006 to 2007 at the Women Research Center,
Alzahra University, Iran.
Soy milk samples: Soymilk, supplied by MaxSoy Co. (Iran), The
composition of this specific soy milk (as stated by manufacturer) was
12.5%. protein, 1.25% Carbohydrates (2.5 g L-1, sugars), 5.8%
fat and 5% fiber, with pH 6.6. All samples used were of the same batch
as stated by the manufacturer. All samples were stored at 4°C until
use. Each soy milk samples were belong to different soy bean varieties,
which are imported from Argentina to Iran.
Standards: Genistein and Daidzein were purchased from Sigma. Isoflavone
standard solutions were prepared based on UV maximum absorbance and molar
extinction coefficient (Ollis, 1962). The purity of the standards was
based on the percentage peak area according to the Beckman System Gold
software and final concentrations were adjusted on the basis on these
purities. Stock solutions were stored at -15°C and thoroughly warmed
and redissolved at room temperature for 2 h to ensure complete solubility.
Isoflavone analysis: Isoflavones were extracted from soy milk
samples by the addition of 8 mL of methanol to a 2 mL milk sample and
shaking at 25°C for 2 h. Following incubation the mixture was transferred
to centrifuge tubes and centrifugated for 20 min at 9000 rpm. The supernatant
was then carefully filtered (0.2 μm) into Eppendorf tubes and analyzed
The HPLC system consists of Waters liquid chromatograph (Milford, MA,
USA) equipped with a 600E multisolvent delivery system, an in-line degasser,
a manual injection with 20 μL loop (Rheodyne 7125) and Waters 2487
dual λ absorbance detector. Empower® software was
used for controlling the analytical system and data processing.
The liquid chromatographic method used for the determination of AA consisted
of a gradient elution procedure with UV-Visible detection at 254 nm. Separations
were carried out on a 5 μm RP C18 column of 250x4.6 mm (Spherical,
Optimal® ODS-H, Capital HPLC, UK) fitted with a 5 μm
RP C18 guard column of 20x4.6 mm (Spherical, Optimal® ODS-H,
Capital HPLC, UK). The mobile phase employed was: A (80 mM tetra ethyl
ammonium phosphate pH 2.5) and B (Acetonitril:A 70:30) of 0.5% NaH2PO4
that 50% A increased to 80% in 20 min. Flow rate of the mobile phase was
1.1 mL min-1 and an injection volume of 20 μL was used
in quantitative analysis. The temperature of analytical column was kept
constant at 25°C.
HPLC-grade methanol was purchased from Labscan Analytical Sciences (Bangkok,
Thailand) and ammonium acetate from Sigma. All reagents used in isoflavone
extraction and HPLC analysis were filtered through a 0.5 μm FH membrane
(Millipore, Bedford, MA).
RESULTS AND DISCUSSION
The overall goal of this study was to evaluate the concentrations of
isoflavones in soy milk. Analytes were routinely identified by retention
times in various HPLC systems and by diode-array absorption patterns (Fig.
2-5). The HPLC system separated completely isofavonoids such as daidzein
(15.2 min) and genstein (17.3 min). Initially, the concentrations of major
isoflavone Genistein and Daidzein in the tested soy milk were determined.
Commercial soy milk samples were analyzed for isoflavones and two major
isoflavones detected: genistein 25.86 (mg L-1) ±0.66
SD and daidzein 8.25 (mg L-1) ±1.13 SD. Concentrations
of genstein in soy milk were higher than daidzein ( Fig.
2-5). This profile of : genistein and daidzein derivatives is in agreement
with earlier reports, showing that the genistin and daidzin concentrations
in soy milk range from 24 to 130 mg L-1 and 12 to 94 mg L-1,
respectively (Choi et al., 2000; Fukutake et al., 1996;
Wang et al., 1990; Murphy et al., 1999).
||HPLC Chromatogram of Genistein (RT= 17.3 min) and Daidzein
(RT= 15.329 min) standard calibration curves
||HPLC Chromatogram of soy milk sample one, Genistein
(RT = 17.124 min) and Daidzein (RT = 15.156 min)
||HPLC Chromatogram of Soy Milk sample two, Genistein
(RT = 17.171 min) and Daidzein (RT= 15.202 min)
||HPLC Chromatogram of soy milk sample three, Genistein
(RT = 17.195 min) and Daidzein (RT = 15.218 min)
||Concentrations of genistein (RT = 17.3 min) and daidzein
(RT = 15.2 min) on soy milk samples
|Genistein (mg L-1) = 25.86±0.66, Daidzein
(mg L-1) = 8.25±1.13
Table 1 shows the recoveries of 2 isoflavones in soymilk
samples. Figure 2-5 show the HPLC Chromatogram of genistein
and daidzein standard calibration curves and also HPLC Chromatogram of
genistein and daidzein on three different soy milk samples. The recoveries
for all the isoflavones ranged from 65 to 91%, which were a bit higher
than those (60-90%) reported in the literature (Coward et al.,
1993; Murphy, 1981), probably because of difference in extraction solvents
and methods used. In conclusion, the data indicate that there weren`t
significant differences between isoflavones content of three different
soy milk samples. There results obtained in this study can serve as a
basis for estimating amount of soy milk can be consumed by people as related
to its main isoflavone content.
The authors would like to appreciate, Women Research Center, Alzahra
University, for the use of laboratory facilities and wish to extend their
thanks to authorities of the Maxsoy Co, involved in this study.
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