Published online 1 March 2007
Published in Crop Sci 47:717-719 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP PHYSIOLOGY & METABOLISM
Production of Isoflavones in Seeds and Seedlings of Different Peanut Genotypes
Ara Kirakosyana,
Peter B. Kaufmana,*,
James A. Dukeb,
E. Mitchell Seymoura,
Sara Warbera and
Steven F. Bollinga
a Univ. of Michigan Integrative Medicine Program (MIM), Univ. of Michigan, Ann Arbor, MI 48109
b Herbal Vineyard, 8210 Murphy Rd., Fulton, MD 20759
* Corresponding author (pbk{at}umich.edu).
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ABSTRACT
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The present study compares concentrations of medicinally important isoflavones in seeds versus seedlings of 20 selected peanut genotypes derived from widely different geographic sources and compares the total isoflavone concentrations in peanut (Arachis hypogaea L.) with those present in soybean [Glycine max (L.) Merr.] and kudzu [Pueraria montana (Lour.) Merr.]. Results of analyses of 20 different peanut genotypes showed that wide variation occurs in isoflavone concentrations both in peanut seeds and peanut seedlings; that peanut seedlings possess 0.8-fold to 27-fold higher concentrations of isoflavones than peanut seeds; that peanut seeds and seedlings possess little or no genistein; that peanut seeds contain one-half the concentrations of isoflavones as soybean seeds, and soybean seedlings contain four times higher concentrations of isoflavones than peanut seedlings; and that kudzu seeds and seedlings, in contrast, contain significantly higher concentrations of isoflavones than either peanut or soybean seeds and seedlings. In conclusion, because peanut seedlings and sprouts are better sources of isoflavones than peanut seeds, we recommend that the former be grown as a vegetable so as to use them in our diet as a good source of isoflavones. Furthermore, the wide variation in isoflavone concentrations in different peanut genotypes, as shown in this study, could be exploited by plant breeders as an easy and reasonable production strategy.
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INTRODUCTION
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MANY EDIBLE legumes in the bean family (Fabaceae) are important sources of isoflavone secondary metabolites and of soluble dietary protein (Dr. Duke's Phytochemical and Ethnobotanical Database, www.ars-grin.gov/duke [verified 8 Feb. 2007]; Kaufman et al., 1997; Kirakosyan et al., 2004). In plants, isoflavones act as important signal and receptor molecules in establishment of N fixing bacteria in legumes and may act as phytoalexins in deterring fungal and bacterial pathogen attack. In humans, they can act by inhibiting DNA topoisomerase and tyrosine kinase (genistein), block cell proliferation associated with breast cancer and prostate cancer (genistein), prevent osteoporosis (attributed to the isoflavones in soybean products), and reduce one's appetite for alcohol (daidzein) (Cseke et al., 2006; Kaufman et al., 1997, 2005; Keung, 2003).
Isoflavones are synthesized as one group of end-products (isoflavones) in the phenylpropanoid biosynthetic pathway (Cseke et al., 2006). They occur in highest levels in the roots, developing seedlings, and seeds of leguminous plants, but are found in lower amounts in leaves, stems, roots, and flowers of older plants. In seeds, they are stored primarily as glucosyl conjugates (e.g., daidzin and genistin). As seeds of legumes germinate, the stored isoflavone conjugates get hydrolyzed by ß-glucosidases to aglycones such as genistein and daidzein; this is accompanied by renewed synthesis of these compounds (Kaufman et al., 1997).
In 1997, we published an extensive survey of isoflavone concentrations in seeds and seedlings of over 79 different taxa of edible legumes (Kaufman et al., 1997). Based on this survey, we concluded that edible legume seedlings contain, on average, one order of magnitude higher concentrations of isoflavones than the seeds, and that seedling roots constituted a better source of these compounds than the developing seedling shoots. Furthermore, a number of edible legumes {e.g., scurfy pea [Cullen corylifolium (L.) Medik.], fava or broad bean (Vicia faba L.), and kudzu had higher concentrations of isoflavones than soybean}. However, peanut or groundnut was not included in this survey. This is unfortunate because peanut seeds are a highly prized, low cost item in the diets of peoples worldwide (excluding those who are allergic to peanuts). The peanut seeds, processed as a meal, are a rich source of isoflavone conjugates and minor glycosides (Singleton et al., 2001; Singleton and Stikeleather, 2002).
In the present study, we compare concentrations of different isoflavones (genistein, genistin, daidzein, and daidzin) in seeds versus seedlings of 20 selected peanut genotypes derived from widely different geographic sources. We also compare these isoflavone concentrations in peanuts with those present in seeds and seedlings of two other legumes, soybean and kudzu. Our working hypothesis is that peanut seedlings will have higher concentrations of the isoflavones studied than peanut seeds, and that isoflavone concentrations in peanut seeds and seedlings will be less than in other edible legumes such as soybean and kudzu.
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MATERIALS AND METHODS
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Selection of Peanut Genotypes
Because genetic background is an important consideration in the examination of types and amounts of isoflavones in edible legume seeds and seedlings (Kaufman et al., 2005), we selected 20 different genotypes of peanut for the present study (Table 1). This peanut seed germplasm was obtained from the USDA Plant Genetic Resources Conservation Unit University of Georgia, Griffin, GA. Seeds of the 20 genotypes were obtained from plants grown under the same environmental conditions at Griffin. Twelve seeds of each of the 20 genotypes of peanut derived from the above source were placed in a –80°C freezer for subsequent extraction and HPLC analysis of isoflavones. The rest of the seeds were kept at 4°C until use for seedling production.
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Table 1. Accession numbers, USDA PI numbers, geographical sources, and genotypes of Arachis hypogaea L. selected for use in this investigation.
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Plant Culture Conditions
Dry seeds of the 20 different peanut cultivars, obtained from the same source as above, were planted to a depth of two times the diameter of the seeds in perlite in a single plastic tray (30-cm width by 60-cm length by 6-cm depth) provided with holes for drainage, 12 seeds per cultivar. The seeds were planted in horizontal rows across the width of the tray, the rows of seeds being 3 cm apart, center to center. They were germinated and the seedlings were grown in a Precision Scientific dual program illuminated incubator (No. 818, Precision Scientific, Winchester, VA) at 25°C under continuous illumination (24 h per day) with two cool white 40 W fluorescent lamps (Ace Hardware Corp., Oak Brook, IL; F40 Universal, 1220-mm length, mounted in the incubator door) providing 600 µmol m–2 s–1 irradiance (photosynthetically active radiation) at the level of the plants. They were watered every 2 d, using tap water. When plants had fully expanded first sets of true leaves and emerging later-formed leaves at the shoot tips (18-d-old seedlings), plants were harvested, washed free of perlite adhering to the roots, and placed in a –80°C freezer for subsequent extraction and HPLC analysis of isoflavones. The experiment under the above conditions was repeated three times.
Extraction and HPLC Analysis of Isoflavones
All plant materials (seeds, roots, and shoots of seedlings) were freeze-dried for 48 h with a Labconco lyophilizer (Model 4.5, Labconco Corp., Kansas City, MO). Dry samples were then powdered in a mortar with pestle. Triplicate samples, 0.5 g each, of the fine powder for each experiment were prepared. These samples were extracted with 10 mL of 80% methanol at 60°C for 12 h, and an aliquot (10 µL) of the extract was analyzed by HPLC. The HPLC conditions were as follows: a Phenomenex Luna column (Torrance, CA); 5-µm pore size, C-18, 150 by 4.60 mm), flow rate of 1 mL min–1, Solvent A = water + 0.1% trifluoroacetic acid (TFA); Solvent B = acetonitrile + 0.1% TFA; HPLC running conditions consisted of a gradient of 5% B to 100% B during a 30-min period; column temperature, 40°C. During the next 5 min, the gradient was reversed, progressing from 100 to 5% with solvent B. Fifteen minutes were then required to recycle back to the initial conditions.
A 10-µL aliquot of sample was injected onto a Shimadzu 10 AD HPLC system with a SPDM-10AV photodiode array detector (Shimadzu, Columbia, MD). Detection was set at 280 nm. Based on our unpublished data, there is no difference in the quantification of isoflavones at 280- and 254-nm wavelengths. The quantitative analysis of each compound in the extracts was analyzed by comparison with the corresponding authentic samples of daidzein, daidzin, genistein, and genistin which were obtained from Sigma-Aldrich Chemical Company (Milwaukee, WI). Each peak was identified by the retention time and the characteristic UV spectrum.
Statistical Analysis of Data
Experiments were repeated three times, and the data were analyzed statistically. All results are given as mean ± standard deviation (SD).
Statistical analyses were performed using the SPSS statistical package (SPSS Inc., Chicago; version 10.0 for Windows). Data were tested at significance levels of P < 0.05 by one-way ANOVA. The subsequent multiple comparisons among means were examined based on the Least Significant Difference (LSD) at the 0.05 probability level.
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RESULTS
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Wide Variation Occurs in Isoflavone Concentrations in Peanut Seeds and Seedlings
Data in Tables 2 and 3 indicate a wide variation in concentrations for each of the isoflavones analyzed for both seeds and seedlings, respectively. In seeds (Table 2), total isoflavones vary from 0.026 to 0.731 mg g–l dry weight. This equals a 28-fold difference. More than half of this 28-fold range depends on a single genotype. This genotype (no. 15) should therefore be the most useful one for breeding purposes. In seedlings (Table 3), total isoflavones vary from 0.253 to 1.113 mg g–l dry weight. This equals a fourfold difference. Such wide variation in isoflavone concentrations has also been reported in different genotypes of fava bean (Kirakosyan et al., 2004) and soybean (Kirakosyan et al., 2006).
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Table 3. Comparison of isoflavone concentrations in peanut seedlings of 20 different genotypes (mean ± SD, n = 3).
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Peanut Seedlings Possess Much Higher Levels of Isoflavones than Peanut Seeds
Results presented in Tables 2 and 3 also indicate that compared to seeds, peanut seedlings possess 0.8-fold to 27-fold higher concentrations of isoflavones, depending on the cultivar. This confirms our earlier studies on seeds versus seedling isoflavone concentrations in other edible legumes (Kaufman et al., 2005).
Peanut Seeds and Seedlings Possess Little or No Genistein
Contrary to our earlier findings with other edible legumes (Kaufman et al., 1997), in the study here, we find that peanut seeds and seedlings contain either no, or only trace amounts of genistein (Tables 2 and 3). However, genistein's glucosyl conjugate, genistin, occurs at expected concentrations in both seeds and seedlings, with higher concentrations occurring in the peanut seedlings as compared with peanut seeds.
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DISCUSSION
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Generally, seedlings and sprouts of peanut are better sources of isoflavones than peanut seeds, based on results presented in Tables 2 and 3. An exception concerns genotype no. 15. It contains less isoflavones in the seedlings than in the seeds (the seedlings contain 84% of the concentration present in the seeds). Based on this observation, the breeding opportunity for this genotype is unique to increase seed isoflavone concentration.
The same generalization concerning isoflavone concentrations can be said for resveratrol in peanut seeds (2.3–4.5 mg g–l dry weight) and seedlings (11.7–25.7 mg g–l dry weight; Wang et al., 2005; see also Tokusoglu et al., 2005). This is relevant because resveratrol is synthesized in the same phenylpropanoid pathway as isoflavones (Cseke et al., 2006). Based on this evidence, there is no reason why peanut seedlings or sprouts cannot be grown as a vegetable, so as to use them in our diet as a good source of isoflavones and of resveratrol. They are used as such in Chinese cuisine. They are also fed to hogs for pork production.
Based on previous studies on kudzu (Kirakosyan et al., 2003) and soybean (Kirakosyan et al., 2006), and the present studies on peanut, we can compare total concentrations of four isoflavones, genistein, daidzein, genistin, and daidzin, as shown in Table 4. Kudzu seeds and seedlings are clearly a much richer source of these isoflavones than either soybean or peanut. Peanut seeds have approximately 1/15th the concentrations of total isoflavones as kudzu seeds, while kudzu seedlings have only seven times higher concentrations than peanut seedlings. Peanut seeds have approximately one-half the concentrations of total isoflavones as soybean seeds, while soybean seedlings have four times higher concentrations than peanut seedlings. So, from a dietary point of view, kudzu is the preferred source of isoflavones. Because of palatability problems with kudzu, it might be best prepared as a powdered formulation to be taken in capsule form. For peanut and soybean seeds, and probably seedlings, palatability is no problem. However, they would have to be ingested in higher amounts to obtain the concentrations of isoflavones equivalent to those present in kudzu seeds and seedlings.
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Table 4. Comparison of mean total isoflavone concentrations in kudzu, soybean, and peanut seeds and seedlings. Isoflavones included in these totals are genistein, daidzein, genistin, and daidzin (mean ± SD, n = 3).
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The wide variation in isoflavone concentrations in different genotypes of peanut confirms what we also discovered for isoflavones in different genotypes of fava bean (Kirakosyan et al., 2004; Kaufman et al., 2005) and soybean (Kirakosyan et al., 2006). Such wide variation in isoflavone concentrations in seeds and seedlings of different peanut genotypes is of importance to peanut growers, who are concerned about the quality of their product, and to peanut plant breeders, who can exploit such variation as an easy and reasonable production strategy (Upadhyaya et al., 2002).
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ACKNOWLEDGMENTS
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Support for this study was provided by the University of Michigan Integrative Medicine program (UMIM).
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication May 12, 2006.
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REFERENCES
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- Kaufman, P.B., J.A. Duke, H. Brielmann, J. Boik, and J.E. Hoyt. 1997. A comparative survey of leguminous plants as sources of the isoflavones, genistein and daidzein: Implications for human health and nutrition. J. Altern. Complement. Med. 3:7–12.[Medline]
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- Kirakosyan, A., P. Kaufman, R.L. Nelson, M.J. Kasperbauer, J.A. Duke, E. Seymour, S.C. Chang, S. Warber, and S. Bolling. 2006. Isoflavone levels in five soybean (Glycine max) genotypes are altered by phytochrome-mediated light treatments. J. Agric. Food Chem. 54:54–58.[CrossRef][ISI][Medline]
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ABSTRACT
INTRODUCTION
