Correlation of antioxidant activity and major isoflavonoid contents of the phytoestrogen-rich Pueraria mirifica and Pueraria lobata tubers.Abstract The antioxidant antioxidant, substance that prevents or slows the breakdown of another substance by oxygen. Synthetic and natural antioxidants are used to slow the deterioration of gasoline and rubber, and such antioxidants as vitamin C (ascorbic acid), butylated hydroxytoluene activity of wild Pueraria mirifica collected from 28 of the 76 provinces of Thailand <onlyinclude> Thailand is divided into 75 provinces (Thai: จังหวัด, changwat, singular and plural), which are grouped into 5 groups of provinces - sometimes the East and Central are and Pueraria lobata Pueraria lobata, n See kudzu. Pueraria lobata a coarse, perennial leguminous vine. Has woody stems but very palatable and nutritious foliage equal in value to alfalfa. Called also kudzu. collected from China were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH DPPH 2,2-Diphenyl-1-Picrylhydrazyl (EPR spectroscopy) DPPH Don't Post Porn Here DPPH Direct Productive Person Hours ) assay. P. mirifica tuberous tuberous /tu·ber·ous/ (too?ber-us) covered with tubers; knobby. See also under sclerosis. tu·ber·ous or tu·ber·ose adj. 1. Producing or bearing tubers. 2. extracts showed weak antioxidant activity in comparison with [alpha]-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 [alpha]-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. [c] 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 Rejuvenation Aeson in extreme old age, restored to youth by Medea. [Rom. Myth.: LLEI, I: 322] apples of perpetual youth by tasting the golden apples kept by Idhunn, the gods preserved their youth. [Scand. Myth. 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 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 bi·pha·sic adj. Having two distinct phases: a biphasic waveform; a biphasic response to a stimulus. 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 anti-proliferation effect on HeLa cells (Cherdshewasart et al., 2004b). Pueraria lobata or Kudzu kudzu (k  d`z ), plant of the family Leguminosae (pulse family), native to Japan. , 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 soybean, soya bean, or soy pea, leguminous plant (Glycine max, G. soja, or Soja max) of the family Leguminosae (pulse family), native to tropical and warm temperate regions of Asia, where it has been seeds showed stronger antioxidant activity than their glycosides, but are ineffective antioxidants as compared to [alpha]-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 (6mg 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 [micro]g/ml in an aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) volume of 50 [micro]l. The stable free radical used was DPPH (Fluka Biochemika, Buschs, Switzerland), freshly prepared at 200 [micro]M in absolute ethanol. [alpha]-Tocopherol (Fluka Biochemika, Buschs, Switzerland) was adjusted into a serial concentration of 0, 3.12, 6.25, 12.5, 25, 37.5 and 50 [micro]g/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 [10.sup.-4]-[10.sup.-9]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 ([IC.sub.50] value) in a microtiter plate reader. DPPH (950 [micro]l) solution and 50 [micro]l sample solution or [alpha]-tocopherol were mixed to establish the final volume of 1 ml. The mixture was shaken vigorously and incubated at 37 [degrees]C 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 absorbance /ab·sor·bance/ (-sor´bans) 1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol . 2. 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 value [+ or -] SEM of the antioxidant activity of [alpha]-tocopherol, the P. mirifica population and P. lobata. Statistical analysis The results were presented as the mean [+ or -] SEM of three independent experiments. ANOVA anova see analysis of variance. ANOVA Analysis of variance, see there was used for the analysis of the test results (LSD LSD or lysergic acid diethylamide (lī'sûr`jĭk, dī'ĕth`ələmĭd, dī'ĕthəlăm`ĭd), alkaloid synthesized from lysergic acid, which is found in the fungus ergot ( test) and factorial factorial For any whole number, the product of all the counting numbers up to and including itself. It is indicated with an exclamation point: 4! (read “four factorial”) is 1 × 2 × 3 × 4 = 24. analysis and Duncan analysis of variance at the significance levels of p<0.05 and p<0.05 or p<0.01, respectively, were considered to indicate significance. The [ED.sub.50] values at 95% confidence limits of the extracts were calculated by Probit In probability theory and statistics, the probit function is the inverse cumulative distribution function (CDF), or quantile function associated with the standard normal distribution. analysis. All statistical analyses were performed using SPSS A statistical package from SPSS, Inc., Chicago (www.spss.com) that runs on PCs, most mainframes and minis and is used extensively in marketing research. It provides over 50 statistical processes, including regression analysis, correlation and analysis of variance. version 10.0 (SPSS Inc.). Results None of these isoflavonoids exhibited stronger antioxidant activity than [alpha]-tocopherol. It was noticed that puerarin and daidzein exhibited antioxidant activity as strong as that of [alpha]-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 [IC.sub.50] value of the P. mirifica population ([IC.sub.50] value = 2904.52 [micro]g/ml). The [IC.sub.50] value of P. lobata ([IC.sub.50] value = 2482.00 [micro]g/ml) indicated stronger activity than the mean value for the P. mirifica population. All plants showed weak antioxidant activity in comparison with the positive control, [alpha]-tocopherol ([IC.sub.50] value of 72.33 [micro]g/ml). The correlation analysis between antioxidant activity and major isoflavonoid contents of the plant tubers showed significant correlation with puerarin (p<0.05). The plant antioxidant activity showed significant lack of correlation with daidzin, daidzein and genistein (p<0.01). 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 flavonoids, n.pl common plant pigment compounds that act as antioxidants, enhance the effects of vitamin C, and strengthen connective tissue around capillaries. (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, [alpha]-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 [alpha]-tocopherol, puerarin ([IC.sub.50] value = 93.26 [+ or -] 9.14 [micro]g/ml) and daidzein ([IC.sub.50] value = 82.58 [+ or -] 4.53 [micro]g/ml) exhibited no significant difference in antioxidant activity as compared with [alpha]-tocopherol ([IC.sub.50] value of 72.33 [+ or -] 6.48 [micro]/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 The base value in a numbering system. For example, in the decimal numbering system, the radix is 10. (mathematics) radix - The ratio, R, between the weights of adjacent digits in positional representation of numbers. (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 cornification /cor·ni·fi·ca·tion/ (kor?ni-fi-ka´shun) 1. keratinization. 2. conversion of epithelium to the stratified squamous type. cor·ni·fi·ca·tion n. 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 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 6.12 [+ or -] 0.40 mg/100 g powder in comparison with puerarin at 23.01 [+ or -] 1.80 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 32.85 [+ or -] 0.72 and 10.34 [+ or -] 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 DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. damage assay (Foti et al., 2005), and exhibited the highest antioxidant activity among the analyzed isoflavonoids in the analysis with electron spin resonance electron spin resonance (ESR) or electron paramagnetic resonance (EPR) Technique of spectroscopic analysis (see spectroscopy) used to identify paramagnetic substances (see spectroscopy, the ferric-reducing ability of plasma (FRAP) assay, Trolox equivalent antioxidant activity (TEAC TEAC Tetraethylammonium Chloride TEAC Theological Education for the Anglican Communion TEAC Technology Education Association of California TEAC Turbine Engine Analysis Check TEAC Timber Export Advisory Committee TEAC Training & Education Advisory Committee ) 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 (1.19 [+ or -] 0.10 mg/100 g powder) and P. lobata (0.81 [+ or -] 0.08 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 ([IC.sub.50] value = 194.0 [+ or -] 6.9 [micro]g/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 ([IC.sub.50] value = 2482.00 [+ or -] 66.11 [micro]g/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, [alpha]-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 In place. When something is "in situ," it is in its original location. because the plant populations per se contained varied amounts of major isoflavonoid contents. 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Table 1. Antioxidant activity ([IC.sub.50] values in [micro]g/ml) of
isoflavonoids as compared with [alpha]-tocopherol
Ranking no. Isoflavonoid [IC.sub.50] value ([micro]g/ml)
1 Daidzein 82.58 [+ or -] 4.53[.sup.a]
2 Puerarin 93.26 [+ or -] 9.14[.sup.a]
3 Daidzin 108.26 [+ or -] 9.94*[.sup.a,b]
4 Genistein 155.75 [+ or -] 25.19*[.sup.b]
5 Genistin 362.10 [+ or -] 25.46*[.sup.c]
Positive control [alpha]-Tocopherol 72.33 [+ or -] 6.48[.sup.a]
*Significant difference (p<0.05) as compared with [alpha]-tocopherol.
The difference at the level of [.sup.(a,b,c)] is verified by Duncan's
test.
Table 2. Antioxidant activity ([IC.sub.50] value in [micro]g/ml) of the
Thai Pueraria mirifica population in comparison with Chinese Pueraria
lobata
Source
Ranking no. (Province) [IC.sub.50] value ([micro]g/ml)
1 Uthai Thani 2470.38 [+ or -] 37.81*[.sup.a]
2 Nong Bua Lam 2489.982 [+ or -] 7.62*[.sup.a,b]
Phu
3 Phetchaburi 2492.61 [+ or -] 83.02*[.sup.a,b]
4 Phitsanulok 2526.47 [+ or -] 46.92*[.sup.a,b,c]
5 Phetchabun 2563.71 [+ or -] 86.35*[.sup.a,b,c]
6 Ratchaburi 2612.14 [+ or -] 34.75*[.sup.a,b,c]
7 Chiang Mai 2648.18 [+ or -] 22.01[.sup.a,b,c,d]
8 Nakhon Sawan 2656.94 [+ or -] 94.96[.sup.a,b,c,d,e]
9 Lamphun 2680.74 [+ or -] 83.70[.sup.a,b,c,d,e]
10 Nan 2726.04 [+ or -]
81.03[.sup.a,b,c,d,e,f]
11 Chiang Rai 2817.63 [+ or -]
40.06[.sup.a,b,c,d,e,f,g]
12 Sukhothai 2889.48 [+ or -]
70.73[.sup.a,b,c,d,e,f,g]
13 Kanchanaburi 2893.51 [+ or -]
15.05[.sup.a,b,c,d,e,f,g]
14 Lop Buri 2900.25 [+ or -]
23.03[.sup.a,b,c,d,e,f,g]
15 Phrae 2915.64 [+ or -]
18.91[.sup.b,c,d,e,f,g]
16 Chumphon 2928.93 [+ or -] 93.89[.sup.c,d,e,f,g]
17 Nakhon 3042.79 [+ or -] 31.20[.sup.d,e,f,g,h]
Ratchasima
18 Sakon Nakhon 3070.63 [+ or -] 15.95[.sup.d,e,f,g,h]
19 Phayoa 3076.20 [+ or -] 13.01[.sup.e,f,g,h]
20 Uttharadith 3117.09 [+ or -] 14.34[.sup.f,g,h]
21 Lampang 3182.16 [+ or -] 88.66[.sup.g,h]
22 Tak 3192.05 [+ or -] 23.17[.sup.g,h]
23 Chaiyaphum 3197.86 [+ or -] 19.83[.sup.g,h]
24 Mae Hong Son 3205.37 [+ or -] 51.14[.sup.g,h]
25 Prachuap Khiri 3205.841 [+ or -] 14.92[.sup.g,h]
Khan
26 Prachin Buri 3209.30 [+ or -] 102.50[.sup.g,h]
27 Saraburi 3234.581 [+ or -] 41.55[.sup.g,h]
28 Kamphaeng Phet 3376.97 [+ or -] 69.96[.sup.g,h]
Mean [+ or -] 2904.52 [+ or -]
SEM 30.37[.sup.a,b,c,d,e,f,g]
Pueraria lobata 2482.00 [+ or -] 66.11*[.sup.a]
*Significant difference (p<0.05) as compared with the mean of the
Pueraria mirifica population. The difference at the level
of [.sup.(a,b,c,d,e,f,g,h)] is verified by Duncan's test.
Table 3. Correlation analysis of tuberous major isoflavonoids and
antioxidant activity
Puerarin Daidzin Genistin Daidzein Genistein
Pearson correlation .166* -.261** -.128 -.465** -.308**
Sig. (2-tailed) .029 .001 .092 .000 .000
N 174 174 174 174 174
Total isoflavonoids
Pearson correlation -.108
Sig. (2-tailed) .157
N 174
*Correlation is significant at p<0.05.
**Correlation is significant at p<0.01.
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