Bioavailability and health effects of Isoflavones from Bangladeshi soybean and lentils in postmenopausal women

Abstract

Menopause is the transitional event of female life creating a considerable degree of clinical, psychological as well as social problem. Hormone Replacement Therapy (HRT) was thought to be a cornerstone in the management of menopause, but evidences accumulated in the recent past have raised serious questions regarding its safety and usability. Isoflavones are phytoestrogens present in natural sourcesand they resemble estradiol in structure and manner of action. Genistein and daidzein which has been reported to be the most biologically active dietary isoflavones attract great deal of interest in today’s researches. These two isoflavones bind weakly to estrogen receptorα and more strongly to estrogen receptor β, and as this binding is tissue-specific, they possess organ-specific estrogenic and antiestrogeniceffects and they do not have such side effects. In this context, isoflavones are getting increasingly more attention for therapeutic (as an alternate of HRT) interventions. A high intake of dietary isoflavoneshas been suggested to account for the lower rates of climacteric complaints, cardiovascular diseases, breast and endometrial cancers, and osteoporosis-related fractures. In this study it was planned to identify the isoflavones present in soybean[Glycinemax(L.) Merr.], mung dal [Vignaradiata(L.) R. Wilczek] and masoor dal [Lensculinaris (Medik.)]; to quantify theisoflavones in above three foods and to measure the bioavailability of isoflavones in Bangladeshi postmenopausal women. In the analytical part of the study, soybean seeds were collected from Jessore, the Southern agricultural area of Bangladesh. Mung and masoordals were purchased from a local supermarket of Dhaka city.Dry soybean, mung andmasoordalswere dried again, ground into powder (200 mesh) by grinder machine and kept in air-tight containersin a refrigerator until analysis was carried out.Standard genistein and daidzein were purchased from Sigma-Aldrich and were preserved at 4ºC and at-20 ºC, respectively. An amount of 350 mL soy-milk was prepared from the 100g powder bean following a standard procedure and kept in refrigerator. The milk was transferred into four different flasks, frozen in a methanol freezer and dried into powderby a freeze-dryer.A definite amount of soy-milk powder (7 g) was weighed and transferred into a round bottomed flaskand refluxed with n-hexane in a boiling water bath to free oil from the soy-milk powder. The oil free soy-milk powder was extracted with EtOAc. The supernatants were pooled, filtered through filter paper and evaporated to dryness using rotary vacuum evaporator. The resulting dry extract was dissolved in 1 mL of LC grade acetonitrile (ACN). It was then filtered through milipore filter (0.22 µM) and taken in sample vial(2 mL)before analysis in LC-PDA. Masoor and mungdals sample were dried in an oven at 105ºC and ground into powder.A definite amount (25 g) of masoorand mungdals powder was weighed and transferred into a round bottomed flaskand refluxed with n-hexane in a boiling water bath to remove oil/fatty materials from the powder. The fat free mung and masoordals powder were extracted with EtOAc. The supernatants were pooled, filtered through filter paper and evaporated to dryness using rotary vacuum evaporator. The resulting dry extract was dissolved in 1 mL of LC grade ACN. It was then filtered through milipore filter (0.22 µM) and taken in sample vial (2 mL) before analysis in LC-PDA. The separation of isoflavones was performed by LC-PDA on C18 column using mobile phase, ACN: H2O (75: 25) with a flow rate of 0.5 mL/min, wavelength: 268 nm, loop size 20 µL and running time was 10 minutes. Genistein and daidzein were identified in the oil free soy-milk, mung and masoordals with respect to retention time of certified standard genistein and daidzein. Quantification of the isoflavones was done using external calibration curve of the two certified samples which were linear (r2 were 0.999 & 0.997 for genistein and daidzein, respectively). Limit of Detection (LOD) (S/N ratio; 3:1) and Limit of Quantification (LOQ) (S/N ratio; 10:1) were found to be 0.0045 & 0.0135 ppm and 0.25 & 0.75ppm for genistein and daidzein, respectively. Amount of total isoflavones, genistein and daidzeinwere found to be in the soy-milk (80.05μg, 43.81μg and 36.25μg per 100g dry weight powder), in the masoor dal (74.99 µg, 37.33 μg, 37.66 μg per 100g dry weight powder) and in the mung dal (71.66 μg, 44.00 μg and 27.66 μgper 100g dry weight powder) respectively. In the bioavailability part of the study, a total of 16 healthy postmenopausal women (age, mean ±SD, 52.5±5.8 years) were included in the study with the later defined by at least 2year from the time of last menses. The subjects were asked to abstain from foods containing isoflavones at least for 1 week before and during the study. After an overnight fast, each individual was given 350mL of soy-milk, masoor, and mungdal soupswas given to every individual woman which was made from 100g soybean seed, masoor, andmungdalspowder respectively, delivering amounts of daidzein (36.25, 37.66 and 27.66µg respectively) and genistein (43.81, 37.33 and 44.00 µg respectively)as a single bolus. The study was carried out under the department of Gynecology and Obstetrics, Bangladesh Institute of Health Sciences (BIHS) hospital. For study purpose, a postmenopausal woman was admitted in the BIHS hospital for 2 days. Blood samples (5 mL) werecollected by venipuncture, before (baseline) and then 2, 4, 6, 8, 24, 36 and 48 hafter consuming the prepared foods (soups and milk). Blood was drawn via a catheter for the more frequent samplings.The blood samples were centrifuged at 1200 x gand the serum (~2 mL) separated and immediately frozen at -20°C.The study subjects were given mung dal, masoor dal soups and soy-milk subsequently at the interval of 2 week. This 2 week was chosen as washout period. The same batch of soybean,masoor dal, andmung dalwas used throughout the study. Serum sample was extracted and cleaned up using solid-phase extraction (SPEC18Cartridge). The cartridge was conditioned with water (1 mL x 3) followed methanol (1 mL x 3) and then water again. Serum sample was thaw and was passed through the conditioned SPE cartridge, then aqueous 5% methanol (800 µL).The isoflavones were eluted in ethyl acetate-acetonitrile mixture (1:1; 400µL x 2).The concentrations of daidzein and genistein in serum were measured by LC following the procedure which was applied in food analysis. The mean Tmax for peak serum genistein concentrations of soy-milk, masoor, and mungwere obtained 6.0±1.85, 6.0±1.51, and 6.8±1.78 h respectively after administration of the single-bolus oral dose, with a mean Cmax1.62±0.87, 4.03±3.91, and 5.15±3.02 µg/mL respectively. The AUC of the serum concentration was 7.41±4.16, 16±15.12 [median (range), 8.76 (2.18-58.34)], and 17.81±9.94 µg/mL in soy-milk, masoor, and mungdal soups respectively. The results from the study lead to the following conclusions: a) Two isoflavonesdaidzein and genistein were identified in soybean [Glycine max (L.) Merr.], mung dal [Vignaradiata(L.) R. Wilczek] and masoor dal [Lens culinaris (Medik.)]; b) Daidzein, and genisteinwere found to be 36.25 µg/100 gdry weight, and 43.81 µg/100 gdry weight in soybean; 37.66 µg/100gdry weight, and 37.33 µg/100 g dry weightin masoor dal; 27.66 µg/100 gdry weight, and 44.00 µg/100 gdry weight in mung dal respectively; c) The estimated mean of total isoflavones in locally produced soybean, masoor and mungdals was found to be 80.05µg/100 gdry weight, 74.99 µg/100 gdry weightand 71.66 µg/100 g dry weightrespectively; d) The assessed mean of maximum concentration of isoflavonegenistein was found to be 1.62 µg/mL for soy-milk, 4.03 µg/mL for masoor dal soup and 5.15 µg/mL for mung dal soup; f) The calculated mean of area under the curve (AUC) of isoflavonegenistein was found to be 7.41 µg/mL for soy-milk, 15.77 µg/mL for masoor dal soup and 17.81 µg/mL for mung dal soup; g) The average time to maximum concentration (Tmax) of soy-milk, masoor and mung dal soups was 6 hour.