Relative Changes in Tocopherols, Isoflavones, Total Phenolic Content, and Antioxidative Activity in Soybean Seeds at Different Reproductive Stages VINEET KUMAR,* ,† ANITA RANI, † AMIT KUMAR DIXIT, † DEEPAK BHATNAGAR, § AND G. S. CHAUHAN † National Research Centre for Soybean (Indian Council of Agricultural Research), Khandwa Road, Indore, M.P., India 452 017, and School of Biochemistry, Devi Ahilya Vishva Vidyalaya, Khandwa Road, Indore, M.P., India 452 017 Immature seeds of soybean are becoming increasingly popular as a snack/vegetable to harness the health benefits of soybean. They are shelled from the immature pods picked from the mother plant at different reproductive stages. Information concerning changes in antioxidant constituents and antioxidant capacity during reproductive phases of soybean seeds is scarce. The aim of the present study was to determine whether immature seeds picked at different reproductive stages differ in tocopherol, isoflavone, total phenolic contents, free radical scavenging activity, and total antioxidative capacity. Seeds shelled from the soybean pods picked at three reproductive stages (R5, R6, and R7) as well as at full maturity were subjected to high-performance liquid chromatography analyses for tocopherol and isoflavone contents. Significantly higher values (P < 0.05) were observed for tocopherols and isoflavones in immature seeds picked at late reproductive stages. At the first reproductive stage, that is, R5 stage, δ-tocopherol was the predominant form of tocopherol, whereas in subsequent reproductive stages as well as at complete maturity stage, the γ-isomer contributed maximum proportion to the total tocopherol content. Genistein was, in general, the major form of isoflavone at all reproductive stages. Reduction in free radical scavenging activity, total antioxidant capacity, and total phenolic content in late-picked seeds concomitant with increased concentration of tocopherol and isoflavone isomers was observed. The results show that bioactive constituents other than isoflavones and tocopherols may decline with the advancement of maturity. KEYWORDS: Soybean; immature seeds; tocopherols; isoflavones; DPPH free radical scavenging activity; FRAP INTRODUCTION Soy-based foods have emerged as one of the most economical and easily available “functional foods” worldwide. Tocopherols and isoflavones, the two major biomolecules with nutraceutical value, are in high concentrations in soybean seeds. Tocopherols as free radical scavengers diminish the risk of cancer, cardio- vascular diseases, neurodegenerative diseases such as Alzhe- imer’s and Parkinson’s, and enhance the immune system (1, 2). Soy isoflavones as antioxidants have been shown to give protective effect against cardiovascular diseases and type-2 diabetes (3, 4), and their estrogen-like properties have been associated with prevention of hormone-dependent cancers, alleviation of menopausal symptoms, and the enhancement of bone mineral density in women (5, 6). Soybean seeds possess all four naturally occurring tocopherols, namely, R-, -, γ-, and δ, which differ in their ability to quench free radicals due to variation in the number and position of methyl substituents on the chromanol ring as shown in Figure 1. As a result, R-tocopherol possesses maximum antioxidative activity, whereas relative activities for the -, γ-, and δ-isomers are 50, 10, and * Author to whom correspondence should be addressed (fax 917312470520; e-mail vineetksahni@yahoo.com). † National Research Centre for Soybean (Indian Council of Agri- cultural Research). § Devi Ahilya Vishva Vidyalaya. Figure 1. Structure of tocopherol isomers. J. Agric. Food Chem. 2009, 57, 2705–2710 2705 10.1021/jf803122a CCC: $40.75  2009 American Chemical Society Published on Web 03/03/2009