In this study, the pharmacokinetics of plasma daidzein and genistein were evaluated in 12 postmenopausal Thai women after a single dose of orally administered soy beverage and soy extract capsules. These women were enrolled from a pool of volunteers after they had been screened for medical history, BMI, serum follicle-stimulating hormone concentration and blood tests. Since the design of this study was similar to that of the bioequivalence testing, 12 subjects were enrolled according to the minimum number of subjects stipulated by the Canadian and European guidelines for bioequivalence testing.
The soy extract product available in Thailand was not selected as the study preparation because only minimum isoflavone content was labeled without any details about the proportion of daidzin and genistin content. The Canadian soy extract preparation was used as an alternative because the exact total of isoflavone content (18.2 mg/capsule) as well as daidzin and genistin content (9.1 mg/capsule, each) were declared. Nonetheless, our quantification of isoflavones in this preparation revealed that the amount of daidzin and genistin in each capsule was 3.90 ± 0.04 and 11.29 ± 0.17, respectively. Therefore, the orally administered dose in this study was calculated according to our quantification, but not by the amount declared.
Initially, we tried to measure the total isoflavone contents in each preparation by using the acid hydrolysis method [14
]. Briefly, either 1 g of soy flour or powder from the soy extract capsules was dispersed in a mixture of 10 ml of 10 M HCl and 40 ml of 96% ethanol (containing 0.05% butylated hydroxy toluene, BHT) followed by refluxing at 100°C for 2 h. The mixture was cooled to room temperature and the ethanol lost during the refluxing was replaced. One ml of this mixture was centrifuged. Ten μl of clear supernatant was diluted 100 times and determined by the HPLC method. The principle of this assay is to hydrolyze β-glycoside conjugates of isoflavones to aglycones, and the detection of total aglycones reflects the total isoflavones. Unfortunately, we failed to measure total isoflavones as aglycones after acid hydrolysis. The recovery of daidzein and genistein was very low (approximately 20–30%) as compared to the other published data, even though we had tried to vary many factors such as the concentration of HCl, duration of refluxing time, temperature during reflux, amount of BHT added, etc. However, since isoflavones are present predominantly as β-glycoside conjugates (e.g. daidzin and genistin) in most commercially available soy products such as soybean or soy flour (with the exception of fermented soy products) [14
], we used a specific HPLC condition for measuring daidzin and genistin, and another condition for measuring aglycones (daidzein and genistein) without acid hydrolysis. Isoflavones in other forms (malonyl glycoside and acetyl glycoside conjugates) were not determined because their commercial standards were not available. Glycitein and its derivatives were also not determined, due to a much smaller amount found in soybeans [16
Since both soy preparations consist of different proportions of daidzin : genistin (approximately 1:1 for soy flour, 1:3 for soy extract capsules), the appropriate amount of daidzin content in each preparation was therefore considered first for pharmacokinetic comparison. In this study, each volunteer was assigned to receive 2 capsules of soy extract (daidzin : genistin = 7.79 : 22.57 mg) to compare with soy beverage prepared from 15 g of soy flour (9.27 : 10.51 mg). These dosages caused an approximately equal amount of daidzin between the two preparations, whereas, the genistin content in soy extract capsules was approximately two-fold higher than that of soy beverage. The oral administration of these dosages resulted in the plasma concentrations of daidzein and genistein being high enough and convenient for measurement by the HPLC method. In addition, preparing soy beverage from 15 g of soy flour in 300 ml of water was practical and created an acceptable concentration for consumption.
From previous studies, the bioavailability of isoflavones was investigated and compared among various soy food and beverages. So far, there has been no study that compares bioavailability of isoflavones from soy extract versus natural soy food or beverages. Our purpose was to investigate pharmacokinetics of daidzein and genistein after ingestion of soy beverage compared to soy extract capsules in postmenopausal Thai women. Daidzein and genistein contents in soy food can vary and depend on the raw material and processing conditions used to produce a particular food product. Furthermore, in each type of soy food, there are different forms of isoflavones in differing amounts. However, based on the equivalent dose of isoflavones, the administration of different soy food has shown no difference in isoflavone bioavailability [17
]. In our study, the bioavailability, adjusted for dosage (determined by AUC0–32
/dose and AUC0-
/dose) of genistein after ingestion of both soy preparations, was not significantly different. In contrast, the bioavailability of daidzein following ingestion of soy extract capsules was significantly greater than that following ingestion of soy beverage. This might be the result of at least 2 possibilities. Firstly, the food matrix of soy flour may alter the bioavailability of daidzein. Dietary factors such as fiber and carbohydrate have been associated with differences in the metabolism of daidzein to equol [18
]. Urinary recovery of equol is higher following the ingestion of tempeh when compared with homogeneous soymilk and textured vegetable protein. This suggests that the combination of a food matrix might protect daidzein from degradation until it reaches the large intestine where it can be metabolized to equol by the microflora [20
]. Secondly, in this study, we only determined the isoflavone contents in both soy preparations as the forms of β-glycoside conjugates (daidzin and genistin) as well as aglycones (daidzein and genistein). However, soy flour and soy extract capsules might have contained some malonyl glycoside and acetyl glycoside conjugates of isoflavones, which were not measured in this study. We postulate that the proportions of malonyl glycoside and acetyl glycoside conjugates of daidzein in soy extract capsules may be greater than those in soy flour, resulting in better bioavailability after these conjugates are converted and absorbed as daidzein from the gastrointestinal tract.
The mean Tmax
of daidzein and genistein from both soy preparations in this study was shorter than the values of 8–11 h (after ingestion of β-glycoside conjugates) as reported in previous studies [21
]. This difference might result from variation in age, race, uptake rates, hydrolysis of glycosides by gut bacteria or gut wall enzymes, further metabolism (for example glucuronides within the liver), etc. The second peak demonstrated in the plasma concentration-time curves of daidzein and genistein possibly resulted from enterohepatic recirculation of the glucuronide and sulfate conjugates of isoflavones excreted in bile [22
]. The elimination t1/2
of daidzein and genistein in this study was comparable to the values of 6–8 h from other studies [20
It has been suggested that a daily intake of soy isoflavone extract containing 50/50 mg of genistin and daidzin [23
] or 76 mg of isoflavones [24
] can significantly decrease hot flushes in the group treated with soy products over the placebo. A meta-analysis of 38 clinical trials, which examined the relationship between soy protein intake and serum lipids, have shown that the consumption of soy in men and women is associated with a significant decrease in serum cholesterol, LDL and triglyceride levels [25
]. In a randomized, double-blind, placebo-controlled trial, which examined the effects of dietary soy supplements containing 118 mg of isoflavones on the lipid profiles of men and postmenopausal women with relatively normal cholesterol levels, the LDL/HDL ratio decreases in the isoflavone treatment groups without any change in total cholesterol [26
]. In addition, it has been found that those postmenopausal women with greatest phytoestrogen consumption have the highest bone mineral density (BMD) at the hip and spine. Subjects with the highest intake of isoflavones also have significantly lower levels of serum PTH, osteocalcin and urinary N-telopeptide [27
]. Besides, isoflavone supplementation (61.8 mg of isoflavones) for four weeks shows potentially beneficial effects on bone metabolism and serum lipids in perimenopausal women in a randomized controlled trial [28
]. Another trial has demonstrated that continuous dietary intake of isoflavones (37.3 mg/day) for ten weeks may inhibit postmenopausal osteoporosis [29
]. Therefore, in our opinion, the total dose of isoflavones that benefits menopausal health is up to approximately 100 mg/day. Since our study revealed that the bioavailability of genistein from soy beverage and soy extract capsules was similar, and the bioavailability of daidzein was slightly (although statistically significant) lower than that of soy extract capsules, the amount of soy beverage, which provides the isoflavone bioavailability equivalent to soy isoflavone capsules containing 50/50 mg of daidzein and genistein, should be equal to approximately 5 cups/day (15 g of soy flour/cup). If one consumes other soy food or prepares a soy beverage in a higher concentration, the daily volume of consumption would be reduced. This inexpensive soy beverage is an appropriate alternative food supplementation compared to the more expensive soy extract capsules and HRT for postmenopausal Thai women, and is in line with Thailand's present socioeconomic status. However, the different proportion of daidzein and genistein in various soy preparations might affect beneficial outcomes for postmenopausal women. In this aspect, clinical studies should be investigated further.