Isoflavones and Anti-oxidant Activities of Soybeans in Thailand
Soybeans are industrial crops and widely cultivated in central and northern Thailand. Isoflavones have considerable attention because of their beneficial health effects and anti-oxidant properties. The present study aimed to investigate isoflavone levels in form of aglycones (genistein and daidzein) and anti-oxidant activities among 13 soybean cultivars in central and northern Thailand. Three soybean cultivars in central region consisted of Sukhothai 1 (SK1), Sukhothai 2 (SK2) and Srisamrong 1 (SS1) and ten soybean cultivars in northern Thailand were Chiang Mai 1 (CM1), Chiang Mai 2 (CM2), Chiang Mai 3 (CM3), Chiang Mai 6(CM6), Chiang Mai 60 (CM60), SOR JOR 1 (SJ1), SOR JOR 2 (SJ2), SOR JOR 3 (SJ3), SOR JOR 4 (SJ4) and SOR JOR 5 (SJ5). Isoflavones were analysed by solid phase extraction method and anti-oxidant activities were determined using DPPH scavenging assay. The results were found that genistein was the predominant isoflavones in soybeans. Isoflavone levels and % DPPH scavenging activities among soybeans in northern cultivars were significantly higher than those in central cultivars (p=0.000). Soybean cultivars in northern region had an average of 104.08±45.25 μg g-1 for genistein, 76.78±36.91 μg g-1 for daidzein and 25.72±3.67% for DPPH scavenging activities. Soybean cultivars in central region had an average of 18.81±9.10 μg g-1 for genistein, 17.46±12.35 μg g-1 for daidzein and 14.07±1.10% for DPPH scavenging activities. The remarkable findings were that the correlation coefficient of % DPPH scavenging activities with genistein (r = 0.706, p = 0.000) was higher than those with daidzein (r = 0.497, p = 0.000). Present results therefore suggested that genistein showed the most potent anti-oxidant and high bio-availability as a promising candidate for the prevention of cancers and other diseases.
Received: July 16, 2012;
Accepted: September 19, 2012;
Published: December 03, 2012
Soybeans (Glycine max) are industrial crops and widely cultivated in
Thailand with 25,257,600 square meters cultivated area. They are composed of
macronutrients such as protein (36-46%), carbohydrate (30%) and lipid (18%)
which mainly consist of polyunsaturated fatty acids such as linoleic acid (Kim
and Kim, 2005; Cederroth and Nef, 2009). They also
contain micronutrients which include isoflavones, saponins, phytates, vitamins
and minerals. Among soybean micronutrients, isoflavones have considerable attention
because of their biological properties including estrogenic and anti-oxidant
activities (Mitchell et al., 1998; Patel
et al., 2001; Magee et al., 2004;
Lee et al., 2005; Cederroth
and Nef, 2009). Epidemiological and clinical studies have shown that dietary
soy isoflavones is associated with beneficial health effects, such as reduction
of menopause symptom, prevention of coronary heart disease and prevention of
breast and reproductive cancers form (Ozasa et al.,
2004; Hedelin et al., 2006; Bandera
et al., 2011; Bolanos-Diaz et al., 2011;
Setchell et al., 2011; Ollberding
et al., 2012). It have been suggested that some of the effect were
related to the anti-oxidant activities of isoflavones.
Although, there are various soybean cultivars cultivated in Thailand for long times, there is little data regarding their isoflavone contents and anti-oxidant properties in each cultivar. Therefore, the aim of the present study was to investigate isoflavone levels in aglycone forms (genistein and daidzein) and anti-oxidant activities among 13 soybean cultivars in Central and Northern Thailand.
MATERIALS AND METHODS
Cultivars of soybeans and sampling: Between December 2011 and May 2012, thirteen soybean cultivars were took a random for analyzing isoflavones and anti-oxidant activities. Cultivar name, locality of origin and breed certification of 13 soybean cultivars are shown in Table 1. Three soybean cultivars in central Thailand were Sukhothai 1 (SK1), Sukhothai 2 (SK2) and Srisamrong 1 (SS1). Ten soy bean cultivars in northern Thailand were Chiang Mai 1(CM1), Chiang Mai 2 (CM2), Chiang Mai 3 (CM3), Chiang Mai 6(CM6), Chiang Mai 60 (CM60), SOR JOR 1 (SJ1), SOR JOR 2 (SJ2), SOR JOR 3 (SJ3), SOR JOR 4 (SJ4) and SOR JOR 5 (SJ5). One kilogram of each foundation seed was brought from Crops Research Center in Chiang Mai and Sukhothai province. Each cultivar was stepwise sampling until the final sample as 250 g and then ground into powder. The powdered were mixed and took a random 5 samples in each cultivar for analysis. All samples were stored in freezer at -20°C throughout the period of the experiment.
Analysis of isoflavones: Standards of genistein and daidzein were obtained
from Sigma-Aldrich, USA. All solvents for extraction were HPLC grades (J.T.
Baker, USA). Sample extraction and analysis was modified from the method of
Nakamura et al. (2000). Recovery was 118.9% for
genistein and 110% for daidzein. The quantitation limit was 0.95 μg g-1
for genistein and 0.88 μg g-1 for daidzein. The intrabatch coefficient
of variation (% CV) was 10.68% for genistein and 10.06% for daidzein and interbatch
% CV was 10.18% for genistein and 17.78% for daidzein.
Determination of anti-oxidant activities: DPPH scavenging assay was
measured by modified method of Katsube et al. (2004).
One gram of the powdered were mixed with 10 mL of 70%(v/v) ethanol and extracted
for 12 h. The extracts were then filtered with filter paper (Whatman no.1) and
stored at 4°C until analysis. The extract was diluted with water stepwise
and 10 μL of dilution was pipetted into a 96-well plate. One hundred and
eighty-five microliters of DPPH (2,2-diphenyl-2-picrylhydrazyl) solution dissolved
in a 50% ethanol solution was added to each well and the plate was shaken for
5 min at room temperature. The absorbance was read at 550 nm by Microplate Reader
(MultiRead 400, Anthos). The experiment was carried out in triplicate:
Statistical analysis: All results were expressed as Mean±standard
deviation (SD). Data were analyzed by two independent sample test (Mann-Whitney
U test) and Spearman Rank correlation coefficient (r) using SPSS 11.5. Statistical
significance was set at p<0.05.
The highest level of genistein was found in CM1 with mean of 159.14±60.71 μg g-1. The second highest level was that in CM2 (150.85±38.56 μg g-1), followed by SJ3 (128.21±18.39 μg g-1). The highest level of daidzein was found in SJ4 with mean of 97.12±10.08 μg g-1. The second highest level was that in SJ1 (96.40±53.86 μg g-1), followed by CM2 (89.89±12.99 μg g-1). Genistein levels in most strain samples (except CM3, CM6, SJ1 and SJ5) were higher than daidzein. The mean ratio between genistein and daidzein was 1.49 (ranged from 0.31-8.96) (Table 2). Comparing with isoflavones of soybean cultivars in central and northern regions, the levels in northern cultivars (104.08±45.25 μg g-1 for genistein and 76.78±36.91 μg g-1 for daidzein) were significantly higher than those in central cultivars (18.81±9.10 μg g-1 for genistein and 17.46±12.35 μg g-1 for daidzein) (p = 0.000) (Table 3).
The highest % scavenging activities was found in CM1 with mean of 29.4±1.95%.
The second highest scavenging activities was that in SJ2 (29.2±1.3%),
followed by SJ3 and CM2 (27.6±1.82 and 27.6±2.7), respectively
|| Isoflavone levels (μg g-1) and DPPH scavenging
activities in 13 soybean cultivars
|Values are Mean±SD, n = 5
||Isoflavone levels (μg g-1) and DPPH scavenging
activities, compared between soybeans of central and northern regions of
|**Statistically different at 0.01
|| Spearman Rank correlation coefficient between isoflavones
and % DPPH scavenging activities in soybeans
|**Statistical correlation significant at 0.01, n = 65
Comparing with % scavenging activities of soybeans cultivated in central and
northern regions, % scavenging activities in northern cultivars (25.72±3.67%)
were significantly higher than those in central cultivars (14.07±1.10%)
(p = 0.000) (Table 3).
Genistein levels were positively associated with daidzein (r = 0.666, p = 0.000) and positively associated with % scavenging activities (r = 0.706, p = 0.000). Daidzein levels were also positively associated with % scavenging activities (r = 0.497, p = 0.000) (Table 4).
In our study, genistein was the predominant isoflavones in soybeans and the
results were in agreement with several studies (Franke et
al., 1994, 1999; Shao et
The remarkable findings were that isoflavones and anti-oxidant activities in northern cultivars were significantly higher than those in central cultivars. Especially, CM1 from northern cultivar had the highest genistein and anti-oxidant activities. The results therefore suggest to cultivating CM1 for more production of isoflavones and anti-oxidant activities.
Several studies have reported that isoflavone contents were affected by genetic
factor, geographical location and environmental conditions during seed development
(Wang and Murphy, 1994; Tsukamoto
et al., 1995; Seguin et al., 2004;
Vamerali et al., 2012). Central and northern
Thailand are differences in geographical and environmental conditions which
include temperature, precipitation, soil fertility, soil moisture and light
level. Temperature seems to be a major factor affecting isoflavone synthesis.
In Thailand, an average temperature in northern region (26°C) is lower than
in central region (28°C). Soybeans are subtropical plants that require root
zone temperature in range of 25-30°C for optimal cultivation (Jones
and Tisdale, 1921). Several studies have reported that low temperature increased
the activity of enzymes of phenylpropanoid and flavonoid pathways and temperature
higher than 24°C during seed development reduced isoflavone contents (Tsukamoto
et al., 1995; Carrao-Panizzi et al., 1999;
Janas et al., 2002; Posmyk
et al., 2005). Water supply is also expected to be increased isoflavone
contents (Caldwell et al., 2005). However, the
study of Vamerali et al. (2012) reported that
water supply increased protein and yield but effect on isoflavones was negligible.
Genistein and daidzein levels were positively associated with % scavenging
activities (r = 0.706, r = 0.497, p = 0.000, respectively). It was due to their
ability to reduce free radical formation and to scavenge free radicals (Pietta,
2000; Djuric et al., 2001). The remarkable
findings were that the correlation coefficient of % scavenging activities with
genistein was higher than those with daidzein. A number of studies have reported
that genistein showed the most potent anti-oxidant (Wei et
al., 1995; Ruiz-Larrea et al., 1997;
Rimbach et al., 2003). It was also mentioned
that the number and position of hydroxyl groups were factors for anti-oxidants
activities and the c-4 position was crucial (Wei et
al., 1995; Arora et al., 1998). Therefore,
it could be hypothesized that high amounts and relatively high anti-oxidants
of genistein is a promising candidate for the prevention of human cancers and
In conclusion, the important factors affecting isoflavone contents in soybeans were geographical and environmental conditions. The high association between genistein and anti-oxidant activities in soybean indicated that genistein showed the most potent anti-oxidant and high bio-availability as a promising candidate for the prevention of cancers and other diseases.
The study was funded by University of Phayao Grant 2011. We are grateful to Crops Research Centers in Chiang Mai and Sukhothai provinces for giving foundation soybeans and thankful to Emeritus Prof. Dr. Maitree Suttajit and Assoc. Prof. Dr. Nuwat Visavarungroj for their guidance. We are also grateful to School of Science, School of Medical Science and School of Medicine, University of Phayao for assisting laboratories.
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