Isoflavone Characterization and Antioxidant Activity of Ohio Soybeans

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Isoflavone Characterization and Antioxidant Activity of Ohio Soybeans
  Isoflavone Characterization and Antioxidant Activity of OhioSoybeans J AE H WAN  L EE , †,‡ M ARJORY  R ENITA , † R ONALD  J. F IORITTO , § S TEVE  K. S T . M ARTIN , § S TEVEN  J. S CHWARTZ , † AND  Y AEL  V ODOVOTZ * ,† Department of Food Science and Technology, Parker Food Science and Technology Building,The Ohio State University, 2015 Fyffe Court, and Department of Horticulture and Crop Science,Howlett Hall, The Ohio State University, 2001 Fyffe Court, Columbus, Ohio 43210 Seventeen Ohio soybeans were screened for isoflavone content and antioxidant activity. Isoflavonecontent was determined by C 18  reversed phase high-performance liquid chromatography coupledwith a photodiode array detector. Antioxidant activities of soybean extracts were measured using2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and photochemiluminescence (PCL) methods. Thehighest and lowest total isoflavone contents were 11.75 and 4.20  µ mol/g soy, respectively, while theaverage was 7.12  µ mol/g soy. Antioxidant activities of soybean extracts ranged from 7.51 to 12.18  µ mol butylated hydroxytoluene (BHT) equivalent/g soy using the DPPH method. Lipid and watersoluble antioxidant activities of soybean extracts ranged from 2.40 to 4.44  µ mol Trolox equivalent/gsoy and from 174.24 to 430.86  µ mol ascorbic acid equivalent/g soy, respectively, using the PCLmethod. KEYWORDS: Soybean cultivars; isoflavones; antioxidant activities INTRODUCTION Consumption of soybeans and soy products has been associ-ated with reducing the risks of various cancers, such as prostateand mammary, and several chronic inflammatory diseases ( 1 ).The health-promoting activity associated with soy consumptionis attributed, in part, to the presence of isoflavones. Researchershave postulated that the purported health benefit may be due toisoflavone estrogenic activity or perhaps antioxidant activity ( 2 , 3 ). The structural similarities of isoflavones to naturally occur-ring estrogens may protect hormone-dependent cancers bymodulating activity of estrogen ( 2 ).As compared to Asian diets where soy foods include tofu,miso, natto, and whole soybeans, the Western style diet islacking in acceptable products containing large amounts of soy( 4 ). One strategy to increase the use of soy is to incorporatesoy-based ingredients into traditional products in the Westerndiet. Selecting for soybean cultivars containing high isoflavonecontent and/or other health-promoting compounds such asantioxidants may enhance the health benefit effects of foodproducts.Isoflavone content in soybeans and in soy products is reportedto range from 1  µ g/g in soy sauces to 540  µ g/g in tempeh, withsoymilk and tofu having the highest isoflavone content ( 5 ).Isoflavones found in soybeans are in the aglycone,   -glucoside,6-O ′′ -malonyl-   -glucoside, or 6-O ′′ -acetyl-   -glucoside forms.Raw soybeans contain mostly glucoside forms of isoflavone andlow percentage of aglycone forms. Isoflavone content in soybeanis influenced by many factors, including genotypes, crop years,crop locations, storage period, and genotype  ×  environmentinteractions ( 6  - 8  ).Antioxidant properties, especially radical scavenging activi-ties, are important due to the deleterious role of free radicals infoods and in biological systems. Excessive formation of freeradicals accelerates the oxidation of lipids in foods and decreasesfood quality and consumer acceptance ( 9 ). Free radicals havealso been associated with the aging process and age-relateddiseases ( 10 ). Superoxide anion, which is a reduced form of molecular oxygen, has been implicated in the initiating oxidationreactions associated with aging ( 10 ). Superoxide anion playsan important role in formation of other reactive oxygen speciessuch as hydrogen peroxide, hydroxyl radical, and singlet oxygen,which induce oxidative damage in lipids, proteins, and DNA( 11 ).In this study, antioxidant activities of soybeans were deter-mined using a DPPH (2,2-diphenyl-1-picryl-hydrazyl) methodand a photochemiluminescence (PCL) method. DPPH has beenwidely used to evaluate the free radical scavenging effectivenessof various flavonoids and polyphenols in food systems ( 12 ,  13 ).PCL measures the luminescence from luminol, a photosensitizer,that generates superoxide anion when exposed to UV light.Antiradical substances react with the superoxide anion, and theremaining luminescence is detected ( 14 ,  15 ). The PCL methodhas been used to assess antioxidant activity in beverages andherbs such as sage, oregano, and evening primrose. PCL is * To whom correspondence should be addressed. Tel: 614-247-7696.Fax: 614-292-0218. E-mail: † Department of Food Science and Technology. ‡ Current address: Department of Food Science and Technology, SeoulNational University of Technology, Seoul, Korea. § Department of Horticulture and Crop Science. J. Agric. Food Chem.  2004,  52,  2647 − 2651  2647 10.1021/jf035426m CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 04/10/2004  approximately 100 - 1000 times more sensitive than conventionalchemical methods using anion radical compounds ( 16  ,  17  ).Soybean is one of the important crops in Ohio and the leadinggrain crop in the area planted ( 18  ). However, systematiccharacterizations of isoflavone content and antioxidant activitiesin Ohio soybean cultivars have not been reported.The objectives of this study were to screen 17 Ohio soybeancultivars for their isoflavone content and antioxidant activitiesand thereby identify the soybean cultivars with high isoflavonecontent and/or high antioxidant activities for further processinginto food ingredients of soy-based functional foods. MATERIALS AND METHODS Materials.  Seventeen soybean cultivars and experimental lines,including Ohio FG1, Ohio FG3, Ohio FG4, Ohio FG5, HS96-3145,HS96-3850, HF01-0019, HS0-3274, HF9667-2-4, HF9662-2-15, HF99-019, HF02-0218, HC95-1503, HS93-4118, Dilworth, Dwight, and Pana,were obtained from the Department of Horticulture of The Ohio StateUniversity. Most of the soybean cultivars were developed in the OhioAgricultural Research and Development Center (OARDC) of The OhioState University and grown and harvested in 2002 at various sites inOhio. These genotypes were chosen to represent a diversity of bothfood and grain type materials. Pedigrees, use type, and locations of production are shown in  Table 1 . All 12 standard isoflavones, includingdaidzein, glycitein, genistein, daidzin, glycitin, genistin, malonyldaidzin, malonyl glycitin, malonyl genistin, acetyl daidzin, acetylglycitin, and acetyl genistin, were purchased from LC Laboratories(Woburn, MA). DPPH, butylated hydroxytoluene (BHT), and for-mononetin were purchased from Aldrich (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade methanol, acetoni-trile, HCl, and acetic acid were purchased from Fisher Scientific(Fairlawn, NJ). Soy Meal Preparation.  Soybeans were dehulled and ground usinga coffee grinder (Black & Decker, Trumbull, CO) to make the soymeal with a particle size less than 0.1 cm in length. Soy meals werestored at  - 20  ° C until use. Isoflavone Analysis.  Isofla V  one Extraction.  Soy meal (0.5 g) of eachcultivar was mixed with 100 mmol/L HCl (2 mL), acetonitrile (7 mL),and deionized water (3 mL) in a 50 mL centrifuge bottle (NalgeCompany, Rochester, NY). Sample bottles were vortexed for 1 minand shaken with a multiwrist shaker (Lab-line Instruments, Inc., MelrosePark, IL) on setting 9 out of 10 scale for 2 h at room temperature beforecentrifuging at 4500 rpm for 30 min (Sorvall, Kendro Laboratory, CO).An aliquot (1 mL) of supernatant was transferred to a 10 mL glassbottle and dried under nitrogen gas flow at room temperature. Driedsamples were stored at  - 20  ° C in the dark until use ( 19 ). Duplicatesof each soybean cultivar were extracted. The reliability of the extractionmethods was assessed by extracting after addition of known concentra-tion of formononetin to HF99-019, HF01-0019, and Dwight soybeancultivars as an internal standard and glycitein in 80% MeOH, which isthe lowest isoflavone in soybean, to HS93-4118, Ohio FG4, and OhioFG3 cultivars and determining its recovery. Recovery percentages forformononetin and glycitein were 98.3  (  5.3 and 97.4  (  5.9%,respectively ( n  )  6).  HPLC Analysis.  A Waters model 2690 HPLC equipped with aWaters 2996 photodiode array detector (PDA) (Waters Associated,Milford, MA) was used to separate, identify, and quantify isoflavones( 20 ). Separation of isoflavones was achieved using a 4  µ m WatersNovapak C 18  reversed phase HPLC column (150 mm × 3.9 mm I.D.)with a Novapak C 18  stationary phase guard column and a 0.5  µ m filterfrom Vydac (Hesperia, CA). One milliliter of 100% methanol was usedto resolubilize the samples for injection. The mixture was vortexedand passed through a 0.2  µ m syringe filter (Alltech Associates Inc.,Deerfield, IL) prior to HPLC injection. The mobile phase consisted of 1% (v/v) acetic acid in water (solvent A) and 100% acetonitrile (solventB) at a flow rate of 0.6 mL/min. The sample injection volume was 10  µ L, and components were eluted using the following solvent gradient:from 0 to 5 min, solvent A was 85%; from 5 to 44 min, solvent A wasdecreased from 85 to 65%; from 44 to 45 min, solvent A was increasedfrom 65 to 85%; finally, solvent A was reequilibrated at 85% for 5min. Between each injection, a mixture of 85% solvent A and 15%solvent B was run for 20 min ( 21 ). The spectra were collected between240 and 400 nm by PDA, and compounds in the eluate were detectedat 260 nm.  Isofla V  one Identification.  All 12 isoflavones, including daidzein,glycitein, genistein, daidzin, glycitin, genistin, malonyl daidzin, malonylglycitin, malonyl genistin, acetyl daidzin, acetyl glycitin, and acetylgenistin, were identified by a combination of the retention time in HPLCchromatograms and UV spectra pattern of pure standard isoflavonecompounds ( 7  ,  22 ,  23 ). Calibration Cur  V  e Preparation and Quantification of Isofla V  ones. Approximately 1 mg of crystalline standard compounds of daidzein,glycitein, genistein, daidzin, glycitin, genistin, malonyl daidzin, malonylgenistin, acetyl daidzin, acetyl glycitin, and acetyl genistin was dissolvedin 80% methanol in water (100 mL) to prepare the stock solutions.The stock solutions were placed in the refrigerator overnight to ensurethe complete solubility of isoflavone. Each stock solution of isoflavoneswas serially diluted with 80% methanol in water. The concentration of working solutions was determined using the Beer - Lambert Law withUV absorbance reading in the range of 240 - 360 nm and their molarextinction coefficients in 80% methanol using a UV - vis spectropho-tometer (Hewlett-Packard 8453) ( 23 ,  24 ). Each isoflavone standardsolution was injected into the HPLC, and the peak areas weredetermined. The relationship between HPLC peak area and concentra-tion of isoflavones from the UV - vis spectrophotometer was calculatedand used for the quantification of the isoflavones. The concentrationof malonyl glycitin was calculated based on acetyl glycitin standard.Isoflavone content in this study was expressed in  µ mol/ g soy ( 23 ).The correlation coefficient ( r  ) of all standard curves for isoflavonestandard compounds was over  + 0.99. Antioxidant Activities.  DPPH Method.  The free radical scavengingactivities of soybean cultivars were determined using a modificationof Ozcelik et al. ( 13 ). The DPPH method was used to measure theantioxidant activities for food systems. Dried sample extracts wereresolubilized in 1 mL of 100% methanol and filtered with a 0.2  µ mdisk filter. DPPH was dissolved in 100% methanol to a concentrationof 0.5 mM. The 3.75 mL of 0.5 mM DPPH was mixed with 0.25 mLof sample extract in methanol. The absorbance changes of the DPPHmixtures were measured at 30 min at 517 nm.The free radical scavenging activity of these samples was expressedas an equivalent of that of BHT, a well-known radical scavenging Table 1.  Genotypes and Planting Locations of 17 Soybean CultivarsUsed in This Study a  cultivar or line pedigree use planting locationDilworth Chapman  ×  Probst grain Hoytville, OHDwight 1 Jack  ×  A86-303014 grain Hoytville, OHHC95-1503 DPL 3478  ×  Sprite 87 grain Cygnet, OHHF01-0019 HF92-080  ×  HS93-6169 food Wooster, OHHF02-0218 ORC 9508  ×  IA 2016 food Hoytville, OHHF9667-2-15 General  ×  GXR 9648 grain Crotton, OHHF9667-2-4 General  ×  GXR 9648 grain Crotton, OHHF99-019 IA 2022  ×  Archer grain Hoytville, OHHS93-4118 2 IA 2007  ×  DSR 304 grain Hoytville, OHHS96-3145 HS89-8843  ×  Ohio FG1 food Wooster, OHHS96-3850 HS89-2966  ×  HS89-8843 food Wooster, OHOhio FG4 Ohio FG1  ×  HS89-3078 food Wooster, OHOhio FG5 Ohio FG1  ×  HS89-3078 food Wooster, OHHS0-3274 HS93-4118  ×  Savoy grain Hoytville, OHOhio FG1 3 LS301  ×  HS84-6247 food Hoytville, OHOhio FG3 HS89-8843  ×  Ohio FG1 food Hoytville, OHPana 4 Jack  ×  Asgrow A3205 grain Hoytville, OH a  References for the registration of some selected cultivars are as follows: (1)Nickell, C. D.; Noel, G. R.; Cary, T. R.; Thomas, D. J.  Crop Sci.  1998 ,  38  , 1398.(2) St. Martin, S. K.; Mills, G. R.; Fioritto, R. J.; Schmitthenner, A. F.; Cooper, R.L.  Crop Sci.  2001 ,  41 , 591. (3) St. Martin, S. K.; Calip-DuBois, A. J.; Fioritto, R.J.; Schmitthenner, A. F.; Min, D. B.; Tang, T.-S.; Yu, Y. M.; Cooper, R. L.; Martin,R. J.  Crop Sci.  1996 ,  36  , 813. (4) Nickell, C. D.; Noel, G. R.; Cary, T. R.; Thomas,D. J.  Crop Sci.  1998 ,  38  , 1398. 2648  J. Agric. Food Chem.,  Vol. 52, No. 9, 2004 Lee et al.  compound used in food systems. A standard curve of the scavengingactivity of BHT on DPPH was obtained by measuring the absorbanceat 517 nm at 30 min of 3.75 mL of 0.5 mM DPPH mixed with 0.25mL of 0.05, 0.075, 0.1, 0.25, and 0.5 mM BHT in 100% methanol.The free radical scavenging activities of soybean cultivars wereexpressed as BHT equivalent as a reference compound. The advantageof using a BHT equivalent expression is to minimize the effects of analysis conditions such as the volume ratio of DPPH and sample orconcentration of DPPH. PCL Method.  A PCL detection method using a Photochem (Analytik Jena AG, Jena, Germany) system was used to measure antioxidantactivities, especially superoxide anion scavenging activity of soybeanextracts ( 16  ). The PCL method was used to test the antioxidant activitiesfor biological systems. Sample extracts were resolubilized in 1 mL of 100% methanol and filtered through a 0.2  µ m disk filter. The antioxidantactivity of these samples was measured using “ACL” and “ACW” kitsprovided, and the procedures were followed as described by themanufacturer. ACL and ACW kits measure integral antioxidativeactivity of lipid and water soluble substances, respectively. Theantioxidant activities of these samples from ACL and ACW kits werereported as Trolox equivalence and ascorbic acid equivalence/g soy,respectively. Statistical Analysis.  Isoflavone analyses of each cultivar wererepeated in quadruplicate. All antioxidant analysis was repeated induplicate or triplicate. The data were analyzed statistically by analysisof variance and  t  -test using StatView (BrainPower Inc., Calabasa, CA).A  P  value  <  0.05 was considered significant. RESULTS AND DISCUSSION Isoflavone Analysis of Soybean Cultivars.  A typical HPLCchromatogram of isoflavones in soybean is shown in  Figure 1 .Genistein, daidzein, and glycitein as well as their   -glucosides(genistin, daidzin, glycitin), 6-O ′′ -acetyl-   -glucosides (acetyl-genistin, acetyldaidzin, acetylglycitin), 6-O ′′ -malonyl-   -gluco-sides (malonylgenistin, malonyldaidzin, malonylglycitin), in-ternal standard were successfully separated and identifiedusing the applied HPLC conditions. The average coefficient of variations for all 12 isoflavone analyses in each soybean cultivarwas less than 5% ( n  )  4).Isoflavone content in 17 soybean cultivars grown in Ohio isshown in  Table 2 . There were significant differences inisoflavone content that included all aglycones and their glucosideconjugates as well as total daidzein, total genistein, totalglycitein, and total isoflavone ( P  <  0.05). The highest totalisoflavone content was 11.75  µ mol/g soy in HF99-019 soybeancultivar, and the lowest was 4.20  µ mol/g soy in HF9667-2-4soybean cultivar, while the average of total isoflavones in the17 soybean cultivars was 7.12  µ mol/g soy. Among the isofla-vones, malonyl genistin content was the highest, followed bymalonyl daidzin and genistin, and glycitein was the lowest,which agrees with the previous reports ( 7  ,  8  ). Total genisteincontent was the highest, followed by total daidzein and totalglycitein, which was 64.7, 32.9, and 2.4% of total isoflavones,respectively.Wang and Murphy ( 6  ) reported that genotypes and plantingyears had greater effects on isoflavone content in soybeans thanplanting locations. Hoeck et al. ( 7  ) suggested that isoflavonecontent seems to be a quantitative trait and thus soybeancultivars containing high isoflavone content can be bred.Therefore, on the basis of the results of this study, HF99-019,Dwight, HS93-4118, and Ohio FG1 cultivars could be recom-mended as potential cultivars with higher isoflavone content.If the identified soybean cultivars are planted in the samelocations and grown in the same environmental conditions, theplanting location effects on the total isoflavone content couldbe minimized. Figure 1.  Representative HPLC chromatogram of isoflavones with aninternal standard in soybean. Table 2.  Isoflavone Contents of 17 Soybean Cultivars Grown in the State of Ohio a   µ mol/g of wet wt base soycultivar DE DI ADI b  MDI GE GI AGI b  MGI GY GYI AGYI MGYI TDE TGYE TGE TIDilworth 0.048a 0.345a 0.390a 2.449a 0.032a 0.513a 0.039a 4.995a tr 0.016a 0.034a 0.050a 3.235a 0.100a 5.581a 8.915aDwight 0.047a 0.413b 0.455b 2.922b 0.049b 0.643b 0.043b 5.880b tr 0.025a 0.025b 0.094b 3.838b 0.145b 6.617b 10.602bHC95-1503 0.054b 0.260c 0.289c 1.000c 0.078c 0.648b 0.032c 3.332c tr 0.012a 0.024b 0.069c 1.605c 0.105a 4.091c 5.803cHF01-0019 0.054b 0.198d 0.217d 0.780d 0.094d 0.479a 0.030c 2.345d tr 0.025b 0.033a 0.057a 1.251d 0.116a 2.947d 4.317dHF02-0218 0.045ac 0.388b 0.440b 2.000e 0.035e 0.712c 0.036d 5.041a tr 0.030b 0.020b 0.096e 2.875e 0.148b 5.826a 8.849aHF9662-2-15 0.022d 0.148e 0.297c 0.875d 0.033d 0.394d 0.045e 3.357c tr 0.014a 0.019c 0.063c 1.344d 0.097a 3.831c 5.272eHF9667-2-4 0.015e 0.147e 0.215d 0.573f 0.019f 0.477a 0.068f 2.594d tr 0.009a 0.023bc 0.023a 0.952f 0.093c 3.160d 4.205dHF99-019 0.105f 0.607f 0.492e 3.758g 0.063g 0.759c 0.035d 5.817b tr 0.025ab 0.028b 0.058ac 4.964g 0.112a 6.675b 11.753fHS93-4118 0.040g 0.434b 0.291c 1.773h 0.037h 0.558a 0.029g 3.110c tr 0.301c 0.030d 0.507d 2.539h 0.839d 3.735c 7.115gHS96-3145 0.048a 0.337a 0.292c 0.957c 0.075i 0.769e 0.033h 3.859e tr 0.042d 0.038e 0.047a 1.635c 0.128e 4.738e 6.503hHS96-3850 0.040g 0.275c 0.302c 0.899c 0.057j 0.603b 0.030g 2.767f tr 0.026e 0.037d 0.106b 1.517c 0.170f 3.458d 5.147eOhio FG4 0.056b 0.563g 0.366f 1.599i 0.056j 0.937f 0.037h 3.669e tr 0.026ab 0.032e 0.050a 2.585h 0.109de 4.700e 7.395gOhio FG5 0.055b 0.634f 0.353f 1.738h 0.052j 0.952f 0.039a 3.763d tr 0.045e 0.039f 0.132e 2.782e 0.218g 4.808e 7.809gHS0-3274 0.042g 0.335h 0.324h 1.685hi 0.041k 0.617b 0.034i 3.989e tr 0.017ab 0.026b 0.060ac 2.389h 0.103a 4.682e 7.175gOhio FG1 0.092h 0.588fg 0.363f 1.504i 0.103l 1.011g 0.035i 3.542c tr 0.055f 0.024f 0.088bc 2.549h 0.168fh 4.693e 7.411gOhio FG3 0.058h 0.424b 0.260c 1.174j 0.073i 0.835h 0.033h 3.184c tr 0.041d 0.023f 0.079bc 1.918i 0.143f 4.127c 6.188hPana 0.043g 0.146e 0.337h 1.356k 0.052b 0.371d 0.030c 4.200g tr 0.012a 0.014c 0.059ac 1.884i 0.086ac 4.655e 6.626haverage 0.051 0.367 0.334 1.591 0.056 0.664 0.037 3.850 tr 0.042 0.028 0.098 2.345 0.170 4.607 7.123 a  Abbreviations: DE, daidzein; DI, daidzin; ADI, acetyl daidzin; MDI, malonyl daidzin; GE, genistein; GI, genistin; AGI, acetyl genistin; MGI, malonyl genistin; GY,glycitein; GYI, glycitin; AGYI, acetyl glycitin; MGYI, malonyl glycitin; TDE, total daidzein; TGYE, total glycitein; TGE, total genistein; TI, total isoflavone; tr, trace amount; TI,sum of TDE, TGYE, and TGE. Different letters are significant among cultivars ( P   < 0.05).  b  Compounds coelute with unknowns. Isoflavone and Antioxidants in Soybeans  J. Agric. Food Chem.,  Vol. 52, No. 9, 2004  2649  Antioxidant Activities.  DPPH Method.  The antioxidantactivities of the 17 soybean cultivars are shown in  Table 3 .There are significant differences in free radical scavengingactivities among soybean cultivars ( P < 0.05). Soybean cultivarsDwight and Dilworth had the highest and lowest free radicalscavenging activities, which were 12.18 and 7.51  µ mol BHTequivalent/g soy, respectively, while the average free radicalscavenging activity of 17 soybean cultivars was 10.13  µ molBHT equivalent/g soy.Free radical scavenging activities of extracts from soybeansand soy products on DPPH have been reported as relativeinhibition percentages ( 25 ,  26  ) or seed coat weight for a 50%decrease in absorbance at 520 nm ( 27  ). Soybean extracts werefound to possess free radical scavenging activities, which wereinfluenced by genetic and environmental difference ( 25 ).Processed soy products such as tofu had approximately 50% of free radical scavenging activity as compared to that of rawsoybeans, which indicates that some processing methods affectfree radical scavenging activity in soybeans ( 26  ).Free radical scavenging activities of soybean extracts (10.13  µ mol BHT equivalent/g soy) are relatively lower than those of other plant materials such as 50% ethanolic extracts of fermentedred bean extracts (approximately 34  µ mol BHT equivalent) ( 28  )and of raw red bean extracts (approximately 53  µ mol BHTequivalent) ( 29 ). This variability may be due to differences inspecies or in extraction methods. PCL Method.  Antioxidant activities measured using the PCLmethods are shown in  Table 3 . There were differences in bothlipid soluble and water soluble antioxidant activities amongsoybean cultivars ( P < 0.05). Soybean cultivars HF01-0019 andHS0-3274 had the highest and the lowest lipid soluble antioxi-dant activities, corresponding to 4.58 and 2.40  µ mol of Trolox,respectively, and the average lipid soluble antioxidant activityof tested soybean cultivars was 3.43  µ mol Trolox equivalent/gsoy. The highest and the lowest water soluble antioxidantactivities were 430.86 and 174.24  µ mol ascorbic acid equiva-lent/g soy in soybean cultivars HF01-0019 and Pana, respec-tively, and the average water soluble antioxidant activity was255.65  µ mol ascorbic acid equivalent/g soy. Soybean extractsresolubilized in methanol had higher water soluble antioxidantactivities than lipid soluble antioxidant activities ( P  <  0.05),which may be due to solubility differences of the differentcompounds.This study is the first report on antioxidant activities of soybean extracts using the PCL detection system. Because of the various antioxidant test methods available, direct comparisonof antioxidant activities of soybean extract with those of otherfruits, beverages, and herbs is not always possible. For example,Wang et al. ( 30 ) reported total antioxidant capacity of fruitsand vegetables, including strawberry, plum, orange, and tomato,as 15.36, 9.49, 7.50, and 1.89  µ mol Trolox equivalent/g fruit,respectively, using the oxygen radical absorbance capacitymethod. These values are approximately 4.4, 2.7, 2.2, and 0.5times, respectively, of the lipid soluble antioxidant activities of average soybean extracts. Toit et al. ( 31 ) reported green tea androsemary had 2.23 and 1.03 mmol ascorbic acid equivalent/gdry weight, respectively, using DPPH methods. These valuesare approximately nine and four times higher than water solubleantioxidant activities of average soybean extracts, respectively.Kim et al. ( 32 ) reported the antioxidant activities of fresh applesas 7.7 - 11.6  µ mol ascorbic acid equivalent/g apple dependingon the test systems, which is approximately 22 - 33 times lessthan water soluble antioxidant activities of average soybeanextracts.Antioxidant activities of isoflavones have been known todepend on the concentrations and structures of isoflavones. Forexample, the glucose linkage to the aglycone reduced theantioxidant activities of isoflavones approximately 50 - 100 times( 33 ). In this study, less than 2.1% (0.15  µ mol out of 7.12  µ mol/gsoy) of isoflavones in soybean are in aglycone form, which maycause the relatively low antioxidant activities of soybeanextracts. It has been reported that at least part of the antioxidantactivities of soybean extracts may arise from other polyphenolicand flavonoid compounds ( 34 ,  35 ). CONCLUSION Seventeen Ohio soybean cultivars were screened for isofla-vone content and antioxidant activity. Soybean cultivars HF99-019, Dwight, HS93-4118, Ohio FG1, and HF01-0019 wereidentified as possible food ingredients due to their highisoflavone content and/or high antioxidant activities. Soybeancultivar HF99-019 had the highest total isoflavone, totalgenistein, total daidzein, daidzein, acetyl daidzin, and malonyldaidzin content. Soybean cultivar Dwight had the highest DPPHscavenging activities and the highest malonyl genistin content.Soybean cultivar HS93-4118 had the highest total glycitein,acetyl glycitin, and malonyl glycitin content and the secondhighest DPPH scavenging activities. Soybean cultivar Ohio FG1showed the highest genistein and genistin content, and HF01-0019 had the highest lipid and water soluble antioxidantactivities determined by the PCL method among tested samples.These identified soybean cultivars, after further processing intofood ingredients, may enhance the health benefits of soy-containing foods. LITERATURE CITED (1) Hendrich, S.; Wang, G. J.; Lin, H. K.; Xu, X.; Tew, B. Y.; Wang,H. J.; Murphy, P. A. Isoflavone metabolism and bioavailability.In  Antioxidant Status, Diet, Nutrition, and Health ; Papas, A. M.,Ed.; CRC Press: Boca Raton, FL, 1999; pp 211 - 230.(2) Hendrich, S.; Wang, G. J.; Xu, X.; Tew, B. Y.; Wang, H. J.;Murphy, P. A. Human bioavailability of soy bean isoflavones:influences of diet, dose, time, and gut microflora.  ACS Symp.Ser.  1998 ,  701 , 150 - 156. Table 3.  Antioxidant Activities of 17 Ohio Soybean Cultivars antioxidant activitiescultivarDPPHmethod a  , d  PCL methodlipid-soluble b  , d  water-soluble c  , d  Dilworth 7.51a 3.19 211.02Dwight 12.18b 4.44 260.96HC95-1503 10.82c 4.17 353.46HF01-0019 9.25d 4.58 430.86HF02-0218 9.61de 3.55 223.08HF9662-2-15 9.54de 3.43 203.28HF9667-2-4 10.14ef 3.43 190.00HF99-019 9.45de 3.42 232.38HS93-4118 11.79b 3.10 183.96HS96-3145 10.05ef 2.85 320.24HS96-3850 10.46fg 3.07 300.08Ohio FG4 10.59g 3.07 274.44Ohio FG5 10.77g 3.84 338.12HS0 − 3274 9.73e 2.40 192.24Ohio FG1 9.58de 3.76 320.40Ohio FG3 10.31fg 3.02 191.28Pana 10.42fg 3.00 174.24average 10.13 3.43 255.65 a  unit:  µ mol BHT equivalent/g soy,  b  unit:  µ mol Trolox equivalent/g soy,  c  unit:  µ mol ascorbic acid equivalent/g soy.  d  Coefficient of variations of each antioxidantanalysis was less than 5%. Different letters are significant among varieties (P<0.05). 2650  J. Agric. 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Antioxidant action and potential antidiabetic propertiesof an isoflavonoid-containing soybean phytochemical extract(SPE).  Phytother. Res.  1999 ,  13 , 601 - 608. Received for review December 4, 2003. Revised manuscript receivedFebruary 6, 2004. Accepted February 13, 2004. This research wassupported by a research grant provided by OARDC. JF035426M Isoflavone and Antioxidants in Soybeans  J. Agric. Food Chem.,  Vol. 52, No. 9, 2004  2651
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