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Correlation of antioxidant activity and major isoflavonoid contents of the phytoestrogen-rich Pueraria mirifica and Pueraria lobata tubers
ARTICLE IN PRESS
Phytomedicine 15 (2008) 38–43 www.elsevier.de/phymed
Correlation of antioxidant activity and major isoflavonoid contents of the phytoestrogen-rich Pueraria mirifica and Pueraria lobata tubers W. Cherdshewasarta,, W. Sutjitb a
Department of Biology, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand Biotechnology Program, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand
b
Abstract The antioxidant activity of wild Pueraria mirifica collected from 28 of the 76 provinces of Thailand and Pueraria lobata collected from China were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. P. mirifica tuberous extracts showed weak antioxidant activity in comparison with a-tocopherol. Six plant samples exhibited stronger antioxidant activity than the mean value of the P. mirifica population. In addition, the mean value of the P. mirifica population indicated significantly lower antioxidant activity than P. lobata. The analysis of the antioxidant activity of isoflavonoids revealed that puerarin and daidzein exhibited the same level of antioxidant activity as a-tocopherol. The results showed convincingly that puerarin and daidzein in the plant tubers may play an important role in antioxidant activity. The correlation analysis between antioxidant activity and major isoflavonoid contents of plant tubers indicated a significant correlation only with puerarin and a significant lack of correlation with daidzin, daidzein and genistein. r 2007 Elsevier GmbH. All rights reserved. Keywords: Pueraria mirifica; Pueraria lobata; Phytoestrogens; Isoflavonoids; Antioxidant activity; DPPH assay
Introduction Pueraria mirifica Airy Shaw et Suvatabhandu (synonym: Pueraria candollei Wall. Ex Benth var mirifica (Airy Shaw & Suvat.) Nyomdham), family Leguminosae (Kashemsanta et al., 1952) is a Thai indigenous plant with a long record of traditional medicinal consumption among menopausal women for purposes of rejuvenation and estrogen replacement (Suntara, 1931). The plant tubers contain phytoestrogens, including miroestrol (Tayler et al., 1960), deoxymiroestrol (Chansakaow et al., 2000a) and isoflavonoids (Ingham et al., 1989; Chansakaow et al., 2000b). Recently, it was found that isoflavonoids comprise a majority of the tuberous Corresponding author. Tel.: +662 2185379; fax: +662 2185386.
E-mail address: [email protected] (W. Cherdshewasart). 0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.07.058
chemical ingredients, with a great diversity among samples collected from 28 provinces in Thailand (Cherdshewasart et al., 2007b). The key chemicals, as well as crude extract, were tested in cells, animals and humans, and exhibited estrogenic effects (Cain, 1960; Jones et al., 1961; Benson et al., 1961; Chansakaow et al., 2000a; Muangman and Cherdshewasart, 2001; Malaivijitnond et al., 2004a, b, 2006, Jaroenporn et al., 2006; Trisomboon et al., 2006a, b; Urasopon et al., 2007; Trisomboon et al., 2007; Cherdshewasart et al., 2007a) with metabolic activation of the plant chemicals (Lee et al., 2002) and without adverse effects on animals and humans (Cherdshewasart, 2003). P. mirifica extract showed a biphasic response to MCF-7 cells with a strong binding with estrogen at high dose in competition with estrogen receptors (Cherdshewasart et al., 2004a). High concentrations of the plant extract had an
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anti-proliferation effect on HeLa cells (Cherdshewasart et al., 2004b). Pueraria lobata or Kudzu, a tuberous plant found in China, Korea and Japan, contains high amounts of isoflavonoids, especially puerarin and daidzein (Kaufman et al., 1997; Kirakosyan et al., 2003). It is popular for consumption in the treatment of hypertension (Qicheng, 1980) and alcoholism (Lin et al., 1996; Lin and Li, 1998) with antioxidant (Guerra et al., 2000) and has shown anti-dipsotropic activities (Keung and Vallee, 1998). At present, the two plants are used as the main botanical ingredients in cosmetics and dietary supplement products. Antioxidant properties are one of the most important claims for food ingredients, dietary supplements, cosmetics and anticancer natural products. Phytoestrogenrich plants have established antioxidant activity. Genistein and daidzein isolated from soybean seeds showed stronger antioxidant activity than their glycosides, but are ineffective antioxidants as compared to a-tocopherol (Lee et al., 2005). Red clover extract, a rich source of isoflavones, exhibited stronger antioxidant activity than soy (Kroyer, 2004). We therefore set up an antioxidant activity test using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay to screen for highly antioxidant plants among wild P. mirifica collected from 28 of the 76 provinces of Thailand in comparison with P. lobata collected from China. This study will help identify plants with the highest antioxidant activity in a chemical assay so they can be selected for further study. Correlation analysis of isoflavonoid contents and antioxidant activity was also performed to aid understanding of the in vivo antioxidant role of such isoflavonoids.
Materials and methods Plant material The tuberous roots of P. mirifica were collected from 28 of the 76 provinces of Thailand. Identification, voucher specimens and the preparation of powder and EtOH extraction of the plant powder from the tuberous roots of P. mirifica and P. lobata have been described previously (Cherdshewasart et al., 2004a, Cherdshewasart et al., 2007b).
Antioxidant activity test P. mirifica and P. lobata EtOH extracts (6 mg each) were dissolved in 1 ml absolute ethanol (Merck) and kept as stock solutions. The plant extracts were adapted to concentrations of 75, 112.5, 150, 187.5, 225 and 300 mg/ml in an aliquot volume of 50 ml. The stable free radical used was DPPH (Fluka Biochemika, Buschs, Switzerland), freshly prepared at 200 mM in absolute
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ethanol. a-Tocopherol (Fluka Biochemika, Buschs, Switzerland) was adjusted into a serial concentration of 0, 3.12, 6.25, 12.5, 25, 37.5 and 50 mg/ml in absolute ethanol and used as a positive control. Puerarin, genistin, daidzein and genistein (Sigma, St. Louis, MO, USA) and diadzin (Fluka Biochemika, Buschs, Switzerland) were prepared at 104–109 M in ethanol and treated as a parallel experiment. The antioxidant activity was analyzed according to the procedure of DPPH assay with modification (Blois, 1958). The results were indicated as the concentration required effecting a 50% inhibition in terms of decreasing signal peak height (IC50 value) in a microtiter plate reader. DPPH (950 ml) solution and 50 ml sample solution or a-tocopherol were mixed to establish the final volume of 1 ml. The mixture was shaken vigorously and incubated at 37 1C in the dark at room temperature for 30 min. Two hundred microliters of the mixed solution was transferred to a 96-well microtiter plate. The absorbance of the samples was measured at 517 nm against DPPH blank in three independent experiments. The antioxidant activity of the plant extracts was compared with the mean value7SEM of the antioxidant activity of a-tocopherol, the P. mirifica population and P. lobata.
Statistical analysis The results were presented as the mean7SEM of three independent experiments. ANOVA was used for the analysis of the test results (LSD test) and factorial analysis and Duncan analysis of variance at the significance levels of po0.05 and po0.05 or po0.01, respectively, were considered to indicate significance. The ED50 values at 95% confidence limits of the extracts were calculated by Probit analysis. All statistical analyses were performed using SPSS version 10.0 (SPSS Inc.).
Results None of these isoflavonoids exhibited stronger antioxidant activity than a-tocopherol. It was noticed that puerarin and daidzein exhibited antioxidant activity as strong as that of a-tocopherol. Genistin exhibited the weakest antioxidant activity (Table 1). The antioxidant activity of the P. mirifica population and P. lobata is shown in Table 2. The antioxidant activity of the P. mirifica population varied among samples as determined by Duncan analysis. Six plant samples exhibited stronger antioxidant activity than the mean IC50 value of the P. mirifica population (IC50 value ¼ 2904.52 mg/ml). The IC50 value of P. lobata (IC50 value ¼ 2482.00 mg/ml) indicated stronger activity than the mean value for the P. mirifica population. All
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Table 1. Antioxidant activity (IC50 values in mg/ml) of isoflavonoids as compared with a-tocopherol Ranking no.
Isoflavonoid
IC50 value (mg/ml)
1 2 3 4 5 Positive control
Daidzein Puerarin Daidzin Genistein Genistin a-Tocopherol
82.5874.53a 93.2679.14a 108.2679.94*a,b 155.75725.19*b 362.10725.46*c 72.3376.48a
*Significant difference (po0.05) as compared with a-tocopherol. The difference at the level of a,b,c is verified by Duncan’s test.
Table 2. Antioxidant activity (IC50 value in mg/ml) of the Thai Pueraria mirifica population in comparison with Chinese Pueraria lobata Ranking no.
Source (Province)
IC50 value (mg/ml)
Uthai Thani 2470.38737.81*a Nong Bua Lam Phu 2489.98277.62*a,b Phetchaburi 2492.61783.02*a,b Phitsanulok 2526.47746.92*a,b,c Phetchabun 2563.71786.35*a,b,c Ratchaburi 2612.14734.75*a,b,c Chiang Mai 2648.18722.01a,b,c,d Nakhon Sawan 2656.94794.96a,b,c,d,e Lamphun 2680.74783.70a,b,c,d,e Nan 2726.04781.03a,b,c,d,e,f Chiang Rai 2817.63740.06a,b,c,d,e,f,g Sukhothai 2889.48770.73a,b,c,d,e,f,g Kanchanaburi 2893.51715.05a,b,c,d,e,f,g Lop Buri 2900.25723.03a,b,c,d,e,f,g Phrae 2915.64718.91b,c,d,e,f,g Chumphon 2928.93793.89c,d,e,f,g Nakhon Ratchasima 3042.79731.20d,e,f,g,h Sakon Nakhon 3070.63715.95d,e,f,g,h Phayoa 3076.20713.01e,f,g,h Uttharadith 3117.09714.34f,g,h Lampang 3182.16788.66g,h Tak 3192.05723.17g,h Chaiyaphum 3197.86719.83g,h Mae Hong Son 3205.37751.14g,h Prachuap Khiri 3205.841714.92g,h Khan 26 Prachin Buri 3209.307102.50g,h 27 Saraburi 3234.581741.55g,h 28 Kamphaeng Phet 3376.97769.96g,h Mean7SEM 2904.527 30.37a,b,c,d,e,f,g Pueraria lobata 2482.00766.11*a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
*Significant difference (po0.05) as compared with the mean of the Pueraria mirifica population. The difference at the level of a,b,c,d,e,f,g,h is verified by Duncan’s test.
plants showed weak antioxidant activity in comparison with the positive control, a-tocopherol (IC50 value of 72.33 mg/ml).
The correlation analysis between antioxidant activity and major isoflavonoid contents of the plant tubers showed significant correlation with puerarin (po0.05). The plant antioxidant activity showed significant lack of correlation with daidzin, daidzein and genistein (po0.01). Genistin and total isoflavonoid contents of the plant were not correlated with antioxidant activity (Table 3).
Discussion The DPPH assay is a conventional method for antioxidant activity analysis with a high level of sensitivity. The assay is used for screening the antioxidant activity of phytoestrogens (Mitchell et al., 1998) and flavonoids (Okawa et al., 2001). The same analytical method was adapted to screen for high antioxidant activity of the Thai P. mirifica population in comparison with Chinese P. lobata. The Thai P. mirifica population shows lower antioxidant activity than P. lobata and is considered to have weak antioxidant activity in comparison with the positive control, a-tocopherol. Isoflavonoids are major ingredients of P. mirifica tubers (Cherdshewasart et al., 2007b). Correlation analysis revealed that a higher amount of puerarin is correlated with higher antioxidant activity, and a higher amount of daidzin, daidzein or genistein is correlated with lower antioxidant activity. In the test with individual isoflavonoids against a-tocopherol, puerarin (IC50 value ¼ 93.2679.14 mg/ml) and daidzein (IC50 value ¼ 82.5874.53 mg/ml) exhibited no significant difference in antioxidant activity as compared with a-tocopherol (IC50 value of 72.3376.48 mg/ml). The two analyses partially confirm the in vitro test of antioxidant activity of the two chemicals, in which only puerarin was found to be significantly correlated with antioxidant activity of the tuberous samples. Nevertheless, a plant with a high amount of puerarin, namely, Puerariae radix (synonym of P. lobata), showed lower estrogenic activity than the Thai P. mirifica with a lower amount of puerarin. It is concluded that the content of puerarin is not responsible for estrogenic activity (Kim et al., 2003). P. mirifica led to vaginal cornification in ovariectomized rats while P. lobata did not (Malaivijitnond et al., 2006). There is also no correlation between the plant antioxidant activity and antiproliferation effect on some cancer cells, e.g., MCF-7 as in the previous study, because the P. mirifica extract showed a proliferation effect on MCF-7 at low doses but P. lobata did not. Nevertheless, the two plants both showed antiproliferation effects at high doses (Cherdshewasart et al., 2004a). Puerarin is a glycoside and makes up most of the isoflavonoid content in the plant tubers. In this antioxidant assay, there was no addition of S9 mixture
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Table 3.
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Correlation analysis of tuberous major isoflavonoids and antioxidant activity
Pearson correlation Sig. (2-tailed) N
Puerarin
Daidzin
Genistin
Daidzein
Genistein
Total isoflavonoids
.166* .029 174
.261** .001 174
.128 .092 174
.465** .000 174
.308** .000 174
.108 .157 174
*Correlation is significant at po0.05. **Correlation is significant at po0.01.
or drug metabolizing enzymes from liver cells. Hence, glycosides are not cleaved into the active form of aglycosides. Although daidzein shows the most potent antioxidant activity among the isoflavonoids in this study, the chemical is found in small amounts in vivo in the P. mirifica population, with a mean value of 6.1270.40 mg/100 g powder in comparison with puerarin at 23.0171.80 mg/100 g powder. Daidzein thus plays a smaller role in vivo in the plant antioxidant activity. More puerarin and daidzein are found in P. lobata with amounts of 32.8570.72 and 10.347 0.79 mg/100 g powder, respectively (Cherdshewasart et al., 2007b). These figures may explain why P. lobata exhibits stronger antioxidant activity than the Thai P. mirifica population. Furthermore, this may be the explanation of why the two test plants exhibited low antioxidant activity despite containing high amounts of puerarin. Isoflavonoids are relatively poor hydrogen donors and thus could not exhibit strong antioxidant activity. Genistein could react as a moderate inhibitor for both in vivo and in vitro oxidation (Wei et al., 1995), similar to that of daidzein in the analysis with DNA damage assay (Foti et al., 2005), and exhibited the highest antioxidant activity among the analyzed isoflavonoids in the analysis with electron spin resonance spectroscopy, the ferric-reducing ability of plasma (FRAP) assay, Trolox equivalent antioxidant activity (TEAC) and tests of ability to inhibit lipid peroxidation in vitamin-E deficient liver microsomes (Mitchell et al., 1998). It is present in the lowest amount in vivo in the P. mirifica population (1.1970.10 mg/100 g powder) and P. lobata (0.8170.08 mg/100 g powder), however. Therefore, it could not exhibit a significant level of antioxidant activity in this test. A study of the antioxidant efficacy of phytoestrogens in chemical and biological systems revealed that the source, age of tubers and time of collection may have a great influence on this kind of analysis. P. lobata collected from Ghang-Zhou, China, in 2003 (Jiang et al., 2005) showed strong antioxidant activity (IC50 value ¼ 194.076.9 mg/ml). The plant sample used in this study was collected in the same province of Ghang-Zhou in the summer of 2000 (Cherdshewasart et al., 2004a) but showed weak antioxidant activity (IC50 value ¼ 2482.00766.11 mg/ml). Ghang-Zhou is a vast area and genetic diversity among the plants may influence the bioactivity of the plant
tubers. Furthermore, different aqueous extraction protocols may also result in obtaining active ingredients from the plant tissue in different amounts. The cited results used water extraction while the sample used in this experiment was obtained from alcoholic extraction. The present study demonstrated that the two phytoestrogen-rich plants, the Thai P. mirifica population and Chinese P. lobata, exhibited weak antioxidant activity in comparison with the strong antioxidant standard, a-tocopherol. This finding revealed that the high estrogenic activity of the plants, especially P. mirifica, is not related to antioxidant activity. Furthermore, some isoflavonoids exhibited correlation with antioxidants in the in vitro test but not in vivo or in situ because the plant populations per se contained varied amounts of major isoflavonoid contents.
Acknowledgments The authors wish to thank the Graduate Studies, Chulalongkorn University and the National Center for Genetic Engineering and Biotechnology, Ministry of Science and Technology, for research grants.
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Phytomedicine 15 (2008) 38–43 www.elsevier.de/phymed
Correlation of antioxidant activity and major isoflavonoid contents of the phytoestrogen-rich Pueraria mirifica and Pueraria lobata tubers W. Cherdshewasarta,, W. Sutjitb a
Department of Biology, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand Biotechnology Program, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand
b
Abstract The antioxidant activity of wild Pueraria mirifica collected from 28 of the 76 provinces of Thailand and Pueraria lobata collected from China were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. P. mirifica tuberous extracts showed weak antioxidant activity in comparison with a-tocopherol. Six plant samples exhibited stronger antioxidant activity than the mean value of the P. mirifica population. In addition, the mean value of the P. mirifica population indicated significantly lower antioxidant activity than P. lobata. The analysis of the antioxidant activity of isoflavonoids revealed that puerarin and daidzein exhibited the same level of antioxidant activity as a-tocopherol. The results showed convincingly that puerarin and daidzein in the plant tubers may play an important role in antioxidant activity. The correlation analysis between antioxidant activity and major isoflavonoid contents of plant tubers indicated a significant correlation only with puerarin and a significant lack of correlation with daidzin, daidzein and genistein. r 2007 Elsevier GmbH. All rights reserved. Keywords: Pueraria mirifica; Pueraria lobata; Phytoestrogens; Isoflavonoids; Antioxidant activity; DPPH assay
Introduction Pueraria mirifica Airy Shaw et Suvatabhandu (synonym: Pueraria candollei Wall. Ex Benth var mirifica (Airy Shaw & Suvat.) Nyomdham), family Leguminosae (Kashemsanta et al., 1952) is a Thai indigenous plant with a long record of traditional medicinal consumption among menopausal women for purposes of rejuvenation and estrogen replacement (Suntara, 1931). The plant tubers contain phytoestrogens, including miroestrol (Tayler et al., 1960), deoxymiroestrol (Chansakaow et al., 2000a) and isoflavonoids (Ingham et al., 1989; Chansakaow et al., 2000b). Recently, it was found that isoflavonoids comprise a majority of the tuberous Corresponding author. Tel.: +662 2185379; fax: +662 2185386.
E-mail address: [email protected] (W. Cherdshewasart). 0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.07.058
chemical ingredients, with a great diversity among samples collected from 28 provinces in Thailand (Cherdshewasart et al., 2007b). The key chemicals, as well as crude extract, were tested in cells, animals and humans, and exhibited estrogenic effects (Cain, 1960; Jones et al., 1961; Benson et al., 1961; Chansakaow et al., 2000a; Muangman and Cherdshewasart, 2001; Malaivijitnond et al., 2004a, b, 2006, Jaroenporn et al., 2006; Trisomboon et al., 2006a, b; Urasopon et al., 2007; Trisomboon et al., 2007; Cherdshewasart et al., 2007a) with metabolic activation of the plant chemicals (Lee et al., 2002) and without adverse effects on animals and humans (Cherdshewasart, 2003). P. mirifica extract showed a biphasic response to MCF-7 cells with a strong binding with estrogen at high dose in competition with estrogen receptors (Cherdshewasart et al., 2004a). High concentrations of the plant extract had an
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anti-proliferation effect on HeLa cells (Cherdshewasart et al., 2004b). Pueraria lobata or Kudzu, a tuberous plant found in China, Korea and Japan, contains high amounts of isoflavonoids, especially puerarin and daidzein (Kaufman et al., 1997; Kirakosyan et al., 2003). It is popular for consumption in the treatment of hypertension (Qicheng, 1980) and alcoholism (Lin et al., 1996; Lin and Li, 1998) with antioxidant (Guerra et al., 2000) and has shown anti-dipsotropic activities (Keung and Vallee, 1998). At present, the two plants are used as the main botanical ingredients in cosmetics and dietary supplement products. Antioxidant properties are one of the most important claims for food ingredients, dietary supplements, cosmetics and anticancer natural products. Phytoestrogenrich plants have established antioxidant activity. Genistein and daidzein isolated from soybean seeds showed stronger antioxidant activity than their glycosides, but are ineffective antioxidants as compared to a-tocopherol (Lee et al., 2005). Red clover extract, a rich source of isoflavones, exhibited stronger antioxidant activity than soy (Kroyer, 2004). We therefore set up an antioxidant activity test using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay to screen for highly antioxidant plants among wild P. mirifica collected from 28 of the 76 provinces of Thailand in comparison with P. lobata collected from China. This study will help identify plants with the highest antioxidant activity in a chemical assay so they can be selected for further study. Correlation analysis of isoflavonoid contents and antioxidant activity was also performed to aid understanding of the in vivo antioxidant role of such isoflavonoids.
Materials and methods Plant material The tuberous roots of P. mirifica were collected from 28 of the 76 provinces of Thailand. Identification, voucher specimens and the preparation of powder and EtOH extraction of the plant powder from the tuberous roots of P. mirifica and P. lobata have been described previously (Cherdshewasart et al., 2004a, Cherdshewasart et al., 2007b).
Antioxidant activity test P. mirifica and P. lobata EtOH extracts (6 mg each) were dissolved in 1 ml absolute ethanol (Merck) and kept as stock solutions. The plant extracts were adapted to concentrations of 75, 112.5, 150, 187.5, 225 and 300 mg/ml in an aliquot volume of 50 ml. The stable free radical used was DPPH (Fluka Biochemika, Buschs, Switzerland), freshly prepared at 200 mM in absolute
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ethanol. a-Tocopherol (Fluka Biochemika, Buschs, Switzerland) was adjusted into a serial concentration of 0, 3.12, 6.25, 12.5, 25, 37.5 and 50 mg/ml in absolute ethanol and used as a positive control. Puerarin, genistin, daidzein and genistein (Sigma, St. Louis, MO, USA) and diadzin (Fluka Biochemika, Buschs, Switzerland) were prepared at 104–109 M in ethanol and treated as a parallel experiment. The antioxidant activity was analyzed according to the procedure of DPPH assay with modification (Blois, 1958). The results were indicated as the concentration required effecting a 50% inhibition in terms of decreasing signal peak height (IC50 value) in a microtiter plate reader. DPPH (950 ml) solution and 50 ml sample solution or a-tocopherol were mixed to establish the final volume of 1 ml. The mixture was shaken vigorously and incubated at 37 1C in the dark at room temperature for 30 min. Two hundred microliters of the mixed solution was transferred to a 96-well microtiter plate. The absorbance of the samples was measured at 517 nm against DPPH blank in three independent experiments. The antioxidant activity of the plant extracts was compared with the mean value7SEM of the antioxidant activity of a-tocopherol, the P. mirifica population and P. lobata.
Statistical analysis The results were presented as the mean7SEM of three independent experiments. ANOVA was used for the analysis of the test results (LSD test) and factorial analysis and Duncan analysis of variance at the significance levels of po0.05 and po0.05 or po0.01, respectively, were considered to indicate significance. The ED50 values at 95% confidence limits of the extracts were calculated by Probit analysis. All statistical analyses were performed using SPSS version 10.0 (SPSS Inc.).
Results None of these isoflavonoids exhibited stronger antioxidant activity than a-tocopherol. It was noticed that puerarin and daidzein exhibited antioxidant activity as strong as that of a-tocopherol. Genistin exhibited the weakest antioxidant activity (Table 1). The antioxidant activity of the P. mirifica population and P. lobata is shown in Table 2. The antioxidant activity of the P. mirifica population varied among samples as determined by Duncan analysis. Six plant samples exhibited stronger antioxidant activity than the mean IC50 value of the P. mirifica population (IC50 value ¼ 2904.52 mg/ml). The IC50 value of P. lobata (IC50 value ¼ 2482.00 mg/ml) indicated stronger activity than the mean value for the P. mirifica population. All
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Table 1. Antioxidant activity (IC50 values in mg/ml) of isoflavonoids as compared with a-tocopherol Ranking no.
Isoflavonoid
IC50 value (mg/ml)
1 2 3 4 5 Positive control
Daidzein Puerarin Daidzin Genistein Genistin a-Tocopherol
82.5874.53a 93.2679.14a 108.2679.94*a,b 155.75725.19*b 362.10725.46*c 72.3376.48a
*Significant difference (po0.05) as compared with a-tocopherol. The difference at the level of a,b,c is verified by Duncan’s test.
Table 2. Antioxidant activity (IC50 value in mg/ml) of the Thai Pueraria mirifica population in comparison with Chinese Pueraria lobata Ranking no.
Source (Province)
IC50 value (mg/ml)
Uthai Thani 2470.38737.81*a Nong Bua Lam Phu 2489.98277.62*a,b Phetchaburi 2492.61783.02*a,b Phitsanulok 2526.47746.92*a,b,c Phetchabun 2563.71786.35*a,b,c Ratchaburi 2612.14734.75*a,b,c Chiang Mai 2648.18722.01a,b,c,d Nakhon Sawan 2656.94794.96a,b,c,d,e Lamphun 2680.74783.70a,b,c,d,e Nan 2726.04781.03a,b,c,d,e,f Chiang Rai 2817.63740.06a,b,c,d,e,f,g Sukhothai 2889.48770.73a,b,c,d,e,f,g Kanchanaburi 2893.51715.05a,b,c,d,e,f,g Lop Buri 2900.25723.03a,b,c,d,e,f,g Phrae 2915.64718.91b,c,d,e,f,g Chumphon 2928.93793.89c,d,e,f,g Nakhon Ratchasima 3042.79731.20d,e,f,g,h Sakon Nakhon 3070.63715.95d,e,f,g,h Phayoa 3076.20713.01e,f,g,h Uttharadith 3117.09714.34f,g,h Lampang 3182.16788.66g,h Tak 3192.05723.17g,h Chaiyaphum 3197.86719.83g,h Mae Hong Son 3205.37751.14g,h Prachuap Khiri 3205.841714.92g,h Khan 26 Prachin Buri 3209.307102.50g,h 27 Saraburi 3234.581741.55g,h 28 Kamphaeng Phet 3376.97769.96g,h Mean7SEM 2904.527 30.37a,b,c,d,e,f,g Pueraria lobata 2482.00766.11*a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
*Significant difference (po0.05) as compared with the mean of the Pueraria mirifica population. The difference at the level of a,b,c,d,e,f,g,h is verified by Duncan’s test.
plants showed weak antioxidant activity in comparison with the positive control, a-tocopherol (IC50 value of 72.33 mg/ml).
The correlation analysis between antioxidant activity and major isoflavonoid contents of the plant tubers showed significant correlation with puerarin (po0.05). The plant antioxidant activity showed significant lack of correlation with daidzin, daidzein and genistein (po0.01). Genistin and total isoflavonoid contents of the plant were not correlated with antioxidant activity (Table 3).
Discussion The DPPH assay is a conventional method for antioxidant activity analysis with a high level of sensitivity. The assay is used for screening the antioxidant activity of phytoestrogens (Mitchell et al., 1998) and flavonoids (Okawa et al., 2001). The same analytical method was adapted to screen for high antioxidant activity of the Thai P. mirifica population in comparison with Chinese P. lobata. The Thai P. mirifica population shows lower antioxidant activity than P. lobata and is considered to have weak antioxidant activity in comparison with the positive control, a-tocopherol. Isoflavonoids are major ingredients of P. mirifica tubers (Cherdshewasart et al., 2007b). Correlation analysis revealed that a higher amount of puerarin is correlated with higher antioxidant activity, and a higher amount of daidzin, daidzein or genistein is correlated with lower antioxidant activity. In the test with individual isoflavonoids against a-tocopherol, puerarin (IC50 value ¼ 93.2679.14 mg/ml) and daidzein (IC50 value ¼ 82.5874.53 mg/ml) exhibited no significant difference in antioxidant activity as compared with a-tocopherol (IC50 value of 72.3376.48 mg/ml). The two analyses partially confirm the in vitro test of antioxidant activity of the two chemicals, in which only puerarin was found to be significantly correlated with antioxidant activity of the tuberous samples. Nevertheless, a plant with a high amount of puerarin, namely, Puerariae radix (synonym of P. lobata), showed lower estrogenic activity than the Thai P. mirifica with a lower amount of puerarin. It is concluded that the content of puerarin is not responsible for estrogenic activity (Kim et al., 2003). P. mirifica led to vaginal cornification in ovariectomized rats while P. lobata did not (Malaivijitnond et al., 2006). There is also no correlation between the plant antioxidant activity and antiproliferation effect on some cancer cells, e.g., MCF-7 as in the previous study, because the P. mirifica extract showed a proliferation effect on MCF-7 at low doses but P. lobata did not. Nevertheless, the two plants both showed antiproliferation effects at high doses (Cherdshewasart et al., 2004a). Puerarin is a glycoside and makes up most of the isoflavonoid content in the plant tubers. In this antioxidant assay, there was no addition of S9 mixture
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Table 3.
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Correlation analysis of tuberous major isoflavonoids and antioxidant activity
Pearson correlation Sig. (2-tailed) N
Puerarin
Daidzin
Genistin
Daidzein
Genistein
Total isoflavonoids
.166* .029 174
.261** .001 174
.128 .092 174
.465** .000 174
.308** .000 174
.108 .157 174
*Correlation is significant at po0.05. **Correlation is significant at po0.01.
or drug metabolizing enzymes from liver cells. Hence, glycosides are not cleaved into the active form of aglycosides. Although daidzein shows the most potent antioxidant activity among the isoflavonoids in this study, the chemical is found in small amounts in vivo in the P. mirifica population, with a mean value of 6.1270.40 mg/100 g powder in comparison with puerarin at 23.0171.80 mg/100 g powder. Daidzein thus plays a smaller role in vivo in the plant antioxidant activity. More puerarin and daidzein are found in P. lobata with amounts of 32.8570.72 and 10.347 0.79 mg/100 g powder, respectively (Cherdshewasart et al., 2007b). These figures may explain why P. lobata exhibits stronger antioxidant activity than the Thai P. mirifica population. Furthermore, this may be the explanation of why the two test plants exhibited low antioxidant activity despite containing high amounts of puerarin. Isoflavonoids are relatively poor hydrogen donors and thus could not exhibit strong antioxidant activity. Genistein could react as a moderate inhibitor for both in vivo and in vitro oxidation (Wei et al., 1995), similar to that of daidzein in the analysis with DNA damage assay (Foti et al., 2005), and exhibited the highest antioxidant activity among the analyzed isoflavonoids in the analysis with electron spin resonance spectroscopy, the ferric-reducing ability of plasma (FRAP) assay, Trolox equivalent antioxidant activity (TEAC) and tests of ability to inhibit lipid peroxidation in vitamin-E deficient liver microsomes (Mitchell et al., 1998). It is present in the lowest amount in vivo in the P. mirifica population (1.1970.10 mg/100 g powder) and P. lobata (0.8170.08 mg/100 g powder), however. Therefore, it could not exhibit a significant level of antioxidant activity in this test. A study of the antioxidant efficacy of phytoestrogens in chemical and biological systems revealed that the source, age of tubers and time of collection may have a great influence on this kind of analysis. P. lobata collected from Ghang-Zhou, China, in 2003 (Jiang et al., 2005) showed strong antioxidant activity (IC50 value ¼ 194.076.9 mg/ml). The plant sample used in this study was collected in the same province of Ghang-Zhou in the summer of 2000 (Cherdshewasart et al., 2004a) but showed weak antioxidant activity (IC50 value ¼ 2482.00766.11 mg/ml). Ghang-Zhou is a vast area and genetic diversity among the plants may influence the bioactivity of the plant
tubers. Furthermore, different aqueous extraction protocols may also result in obtaining active ingredients from the plant tissue in different amounts. The cited results used water extraction while the sample used in this experiment was obtained from alcoholic extraction. The present study demonstrated that the two phytoestrogen-rich plants, the Thai P. mirifica population and Chinese P. lobata, exhibited weak antioxidant activity in comparison with the strong antioxidant standard, a-tocopherol. This finding revealed that the high estrogenic activity of the plants, especially P. mirifica, is not related to antioxidant activity. Furthermore, some isoflavonoids exhibited correlation with antioxidants in the in vitro test but not in vivo or in situ because the plant populations per se contained varied amounts of major isoflavonoid contents.
Acknowledgments The authors wish to thank the Graduate Studies, Chulalongkorn University and the National Center for Genetic Engineering and Biotechnology, Ministry of Science and Technology, for research grants.
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