394 Journal of Health Science, 47(4) 394–406 (2001) Content and Composition of tivities1) and reported to be protective against can- cer, cardiovascular diseases and osteoporosis.3–9) Isoflavonoids in Mature or Much research has been reported about the content Immature Beans and Bean of isoflavonoids in soybeans and soybean-derived processed foods.10–23) In contrast, there are few re- Sprouts Consumed in Japan ports about the isoflavonoid content in beans other than soybeans.11,12,18,23) Yumiko Nakamura,* Akiko Kaihara, Japanese people are reported to ingest Kimihiko Yoshii, Yukari Tsumura, isoflavonoids mainly through the consumption of Susumu Ishimitsu, and Yasuhide Tonogai soybeans and its derived processed foods.20) Re- cently, we estimated that the Japanese daily intake Division of Food Chemistry, National Institute of Health Sci- of isoflavonoids from soybeans and soybean-based ences, Osaka Branch, 1–1–43 Hoenzaka, Chuo-ku, Osaka 540– processed foods is 27.80 mg per day (daidzein 0006, Japan (Received January 9, 2001; Accepted April 6, 2001) 12.02 mg, glycitein 2.30 mg and genistein 13.48 mg).24) However, isoflavonoid intake from the The content of 9 types of isoflavonoids (daidzein, consumption of immature beans, sprouts and beans glycitein, genistein, formononetin, biochanin A, other than soybeans has not been elucidated. Here coumestrol, daidzin, glycitin and genistin) in 34 do- we have measured the content of isoflavonoids in mestic or imported raw beans including soybeans, 7 mature and immature beans and bean sprouts con- immature beans and 5 bean sprouts consumed in Ja- sumed in Japan, and have compared the content and pan were systematically analyzed. Each isoflavonoid composition variation between different types and was analyzed in total after acid hydrolysis to the agly- different growth stages of the beans. cone, and intact individual isoflavonoids were also analyzed without hydrolysis. After the sample clean up, daidzein, glycitein, genistein, formononetin, MATERIALS AND METHODS biochanin A, daidzin, glycitin and genistin were deter- mined by HPLC with a diode array detector and Materials —–— Genistein, formononetin and coumestrol was determined by spectrofluorimetry. The biochanin A were purchased from Extrasynthèse content and composition of isoflavonoids varied greatly (Genay, France). Daidzein, daidzin, genistin, between soybean sprouts, immature soybeans and glycitein and glycitin were from Fujicco Co., Ltd. mature beans of the same type but of different source. (Kobe, Japan). Coumestrol was obtained from Fluka Isoflavonoid content was highest in mature soybeans. The composition of isoflavonoids differed in each Chemie AG (Bucks, Switzerland). Flavone, 2,6-di- growth stage of soybeans. In other beans, the largest t-butyl-4-methylphenol (BHT) and dimethyl sulfox- content of isoflavonoids was found in mature ide (DMSO) were from Wako Pure Chemical Indus- chickpeas, but this value was less than 1/27 of the tries, Ltd. (Osaka, Japan). The chemical structures isoflavonoid content in mature soybeans. Thus, the of these isoflavonoids and flavone are shown in contribution of beans other than soybeans to the daily Fig. 1. Acetonitrile, ethanol, n-hexane and metha- intake of isoflavonoids in a Japanese diet is negligible. nol were analytical grade for high performance liq- uid chromatography (HPLC). All other reagents were Key words —–— isoflavonoid, bean, acid hydrolysis, high analytical grade. The water used was Milli-Q grade performance liquid chromatography, diode array detec- or equivalent. tion, spectrofluorometric detection A YMC-pack ODS-AM-303 column (4.6 mm i.d. × 250 mm, 5 µm particle size) was purchased INTRODUCTION from YMC Co., Ltd. (Kyoto, Japan). Mini-cartridge R column Sep-pak plus C18 cartridges were obtained Isoflavonoids, which are found in legumes such from Waters Corporation (Milford, MA, U.S.A.). as soybeans,1,2) have both sterile and estrogenic ac- The domestic and imported mature beans, im- mature beans and sprouts that we analyzed are foods *To whom correspondence should be addressed: Division of commonly consumed in Japan. The imported ma- Food Chemistry, National Institute of Health Sciences, Osaka ture beans were obtained at a port in Hyogo Prefec- Branch, 1–1–43 Hoenzaka, Chuo-ku, Osaka 540–0006, Japan. ture and others were purchased in retail stores in Tel.: +81-6-6941-1533; Fax: +81-6-6942-0716; E-mail: [email protected] Osaka Prefecture. No. 4 395 Fig. 1. Chemical Structures of 9 Isoflavonoids and Flavone (Internal Standard) Instruments —–— A Shimadu LC-10 series amount of DMSO and diluted with methanol to form equipped with binary gradient pump LC-10AD, 10 ml of stock solutions (547–1319 µg/ml). An ap- 100 µl injection loop, autosampler SIL-10A, column propriate amount of each isoflavonoid stock solu- oven CTO-10A set at 40°C, diode array detector tion was then diluted with methanol to make (DAD) SPD-M10AVP, fluorospecrometric detector isoflavonoid standard solutions. Flavone stock so- RF-10A and system controller SCL-10A (Kyoto, lution was diluted with methanol 5 times to prepare Japan) was used for isoflavonoid analysis. a 210 µg/ml internal standard (IS) solution. Standard solutions —–— A pre-weighed amount of Extraction —–— Edible portions of the bean samples each isoflavonoid standard was dissolved in a small were analyzed. Intact individual isoflavonoids were 396 Vol. 47 (2001) analyzed without hydrolysis, and the total content B: 0% (0 min) → 20% (5 min) → 100% (50–60 min) of each isoflavonoid was analyzed as the aglycone → 0% (61 min); flow rate, 1.0 ml/min; DAD moni- after acid hydrolysis by the method of Franke et al.11) toring wavelength, 260 nm for daidzein, daidzin, with a previously described modification.24) Each genistein, genistin, glycitein, glycitin, biochanin A, analysis was performed in triplicate. For the analy- formononetin, 280 nm for flavone, 343 nm for sis of peanuts, lipids were removed before coumestrol; spectrofluorometric detector for isoflavonoid analysis by extraction with 50 ml of n- coumestrol, excitation and emission wavelength, hexane overnight at an ambient temperature. 344 nm and 413 nm; injection volume, 10 µl. (A) Intact isoflavonoids (sample solution A): Recovery Tests —–— A recovery test was performed Ground mature bean powder (1.0 g), homogenized for mature soybean #1, mature kidney bean #2, im- immature beans (2.0 g) or homogenized bean sprouts mature green peas #4 and soybean sprout (moyashi) (3.0 g) were placed in centrifuge tubes. One ml of #1 by adding 1 ml of the appropriate concentration IS solution (flavone 210 µg) and 50 ml of 80% of the isoflavonoid standard solution and 1 ml of IS methanol were added to the centrifugal tubes, which solution to each sample. were then sonicated for 30 min. Isoflavonoids were extracted for 24 hours at ambient temperature, the tube was centrifuged at 1000 × g for 20 min at 5°C RESULTS AND DISCUSSION and the volume was adjusted to 50 ml with metha- nol to form sample solution A. HPLC Analysis of Standard Solution (B) Total isoflavonoids (sample solution B): The HPLC chromatogram of the standard solu- Ground mature bean powder (1.0 g), homogenized tion is shown in Fig. 2. All nine phytoestrogens and immature beans (2.0 g) or homogenized bean sprouts flavone could be determined individually. Previ- (3.0 g) were placed in centrifuge tubes. One ml of ously, we used STR ODS-II (4.6 mm i.d. × 250 mm, IS solution (flavone 210 µg/ml), 10 ml of 10 M HCl 5 µm particle size) column to determine iso- solution and 40 ml of ethanol containing 0.05% BHT flavonoids in soybeans and soybean-derived pro- were added to the centrifugal tube, which was then cessed foods.24) However, there is not good separa- sonicated for 30 min. Hydrolysis was carried out by tion of coumestrol from genistein or biochanin A reflux in a boiling water bath for 3 hr.11) The tube from flavone with the STR ODS-II column. There- was cooled and centrifuged at 1000 × g for 15 min fore, as some beans contain biochanin A,11) we used at 5°C, and the volume was adjusted to 50 ml with the YMC-pack ODS-AM-303 column in this study. methanol to form sample solution B. The coefficients of variations of the relative re- Clean Up (Test Solution) —–— Sample solutions A tention time and peak area of the nine isoflavonoids and B prepared above were cleaned up using Sep- and flavone (10.4–26.1 µg/ml) using DAD were R 17) pak plus C18 cartridges. One ml of each sample 0.01–0.04% and 0.41–1.15%, respectively; those of solution was diluted with 10 ml of water and ap- coumestrol using fluorospectrometric detection were R plied to a Sep-pak plus C18 cartridge column pre- 0.07% and 0.66%, respectively (data not shown). conditioned with methanol and water. The column The relative retention time (flavone set as 1.000), was washed with 20 ml of water followed by 2 ml detection limits and linear dynamic range are shown of 20% methanol, and then isoflavonoids and fla- in Table 1. The detection limits (S/N = 5) of the nine vone were eluted with exactly 2 ml of methanol to isoflavonoids and coumestrol were 44.8–260.8 ng/ml form the test solution. (0.108–0.973 nmol/ml) and 26.1 ng/ml (0.097 nmol/ HPLC Analysis —–— The content of isoflavonoids ml) by DAD and fluorospectrometric detection, re- in each test solution was determined by HPLC us- spectively. The linear dynamic ranges of the stan- ing flavone as an internal standard.11) Coumestrol dard solution were more than 1,000-fold as shown was determined by fluorospectrometry, and other in Table 1. The sensitivity of coumestrol detection isoflavonoids and flavone were determined by DAD. by fluorospectrometry was 10 times higher than by HPLC conditions were as follows.