LC-ESI-MS/MS method for the simultaneous determination of isoformononetin, daidzein, and equol in rat plasma: Application to a preclinical pharmacokinetic study.

Isoformononetin (methoxy isoflavone) is a potent osteogenic isoflavone abundantly present in Butea monosperma, Pisum sativum, Mung bean, Machaerium villosum, Medicago sativa, and Glycine max. In the current study, an LC-ESI-MS/MS method for the simultaneous evaluation of isoformononetin (IFN), daidzein (DZN) and equol (EQL) was developed and validated in rat plasma using biochanin A as an internal standard. IFN, DZN, and EQL separation was achieved by using acetonitrile and acetic acid (0.1%) in the ratio of 90:10 (% v/v) as mobile phase under isocratic conditions at a flow rate of 0.6 mL/min on Atlantis C18 (4.6 × 250 mm, 5.0 µm) column. The achieved method was linear within the concentration range of 0.5-500 ng/mL. The method was effectively applied to investigate the permeability, protein binding estimation and pharmacokinetics studies of IFN in rats. The PAMPA permeability of IFN was found to be high at pH 4.0 and 7.0. The protein binding was found to be about 91% of IFN. The oral bioavailability of IFN was found to be poor (21.6%). IFN was found to have a moderate clearance (2.9 L/h/kg) and a large apparent volume of distribution (12.1 L/kg). The plasma half-life (t1/2) and maximum attainable concentration (Cmax) of IFN at systemic circulation was found to be 1.9 ±â€¯0.6 h and 269.3 ±â€¯0.4 after oral administration.

Safety and feasibility of estrogen receptor-ß targeted phytoSERM formulation for menopausal symptoms: phase 1b/2a randomized clinical trial.

OBJECTIVE: PhytoSERM is a formulation of genistein, daidzein, and S-equol that has an 83-fold selective affinity for estrogen receptor-ß (ERß); and may enhance neuron function and estrogenic mechanisms in the brain without having peripheral estrogenic activity. METHODS: We conducted an overarching, two-stage, dose-ranging, double-blinded, randomized, placebo-controlled trial of 12 weeks duration comparing 50 and 100 mg/d of phytoSERM with placebo for noncognitively impaired, perimenopausal women aged 45 to 60, with intact uteri and ovaries, with at least one cognitive complaint, and one vasomotor-related symptom. Primary objectives were to assess safety and tolerability of a 50 and 100 mg daily dose; and, secondly, to evaluate potential indicators of efficacy on cognition and vasomotor symptoms over 4 and 12 weeks, and using an embedded, 4-week, 2-period, placebo-controlled crossover trial for a subset of participants. RESULTS: Seventy-one women were randomized to treatment; 70 were evaluated at 4 weeks; 12 were entered into the crossover study; 5 did not complete 12 weeks. Reasons for discontinuation were withdrawal of consent (n = 1) and lost to follow-up (n = 4). Adverse events occurred in 16.7% (n = 4) placebo, 39.1% (n = 9) 50 mg/d, and 29.2% (n = 7) 100 mg/d treated participants; 85% were mild and none was severe. Vaginal bleeding occurred in 0, placebo; 1, 50 mg; and 3, 100 mg/d participants. CONCLUSIONS: The phytoSERM formulation was well tolerated at 50 and 100 mg daily doses. Based on safety outcomes, vaginal bleeding at the 100 mg dose, and vasomotor symptoms and cognitive outcomes at 12 weeks, a daily dose of 50 mg was considered preferable for a phase 2 efficacy trial.

Equol producers can have low leptin levels among prediabetic and diabetic females.

OBJECTIVES: Soy isoflavones have received great attention for their beneficial effects on health and disease, i.e., in patients with diabetes. Equol is a biologically active isoflavone-related metabolite with interindividual differences in its production. The current study investigated the relationship between an equol-producing state and the levels of adipocytokine markers in a prediabetic and diabetic population. SUBJECTS AND METHODS: A total of 79 subjects (34 males/45 females) in a prediabetic or diabetic state recruited from the general population were examined regarding their ability to produce equol using urine samples. Clinical data, such as age, smoking as well as anthropometric and biochemical variables, including body mass index (BMI), lipids, insulin, glucose, hemoglobin A1c, leptin and adiponectin, were recorded. RESULTS: Equol producers exhibited lower leptin and leptin/BMI than non-producers among females. Simple correlation tests and stepwise multiple regression analyses revealed a significant inverse correlation between the leptin/BMI and equol-production. This relationship was not found in males. CONCLUSIONS: Female equol producers can have favorable metabolic traits in relation to leptin metabolism in this population. Further studies are needed to confirm these results.

Pharmacokinetics of equol, a soy isoflavone metabolite, changes with the form of equol (dietary versus intestinal production) in ovariectomized rats.

Recent findings indicate that soy isoflavones and their metabolites may play a role in mitigating postmenopausal bone loss. Equol, a metabolite of the soy isoflavone daidzein produced by intestinal bacteria, has shown some potential, but only 30-50% of the U.S. population is capable of converting dietary daidzein to equol. There are limited data on the pharmacokinetics of dietary racemic equol and its metabolites. This study was conducted to assess the levels of equol and its conjugates in plasma for a 24 h period resulting from oral administration of dietary daidzein and racemic equol in ovariectomized Sprague-Dawley rats. Plasma samples were analyzed for conjugated and free forms of equol using LC-MS/MS. The maximum plasma concentration (C(max)) and time to reach it (t(max)) for total equol (conjugated and unconjugated) were 8815 ± 2988 nmol/L and 2.17 ± 2.91 h and 3682 ± 2675 nmol/L and 20.67 ± 4.67 h, for dietary equol and daidzein, respectively. Although the majority of equol metabolites present were glucuronide conjugates (≥99%), there were low levels of equol monosulfate present. The changes in equol metabolism, specifically equol conjugates, due to the form of equol may play a role in the potential health benefits of equol.

Elucidation of the metabolic pathway of S-equol in rat, monkey and man.

S-equol is a selective estrogen receptor ß (ERß) agonist which is produced in certain individuals after ingestion of its precursor daidzein, an isoflavone present in soy. S-equol is thought to provide certain health benefits, including reduced menopausal symptoms. The metabolic profile of S-equol was determined in vivo in Sprague-Dawley rats and cynomolgus monkeys, and in vitro using hepatocytes from rat, monkey, and human. High resolution MS fragmentation patterns indicated that the major metabolite of S-equol in rat plasma and urine was the 4'-glucuronide conjugate, with lesser amounts of unconjugated S-equol, the 7-sulfate conjugate, and the 4'-glucuronide-7-sulfate diconjugate. Monkeys also showed extensive metabolism, with the major species in plasma being the 4'-glucuronide and the 7-sulfate-4'-glucuronide diconjugate; urine contained primarily the 4'-glucuronide, as seen in the rat. In vitro metabolism by hepatocytes was extensive and similar in all species, with fragmentation patterns also indicating that the 4'-glucuronide was the major metabolite. No oxidative metabolites of [(14)C] S-equol were detected in either in vivo or in vitro studies. These findings show that glucuronidation is the primary pathway for the metabolism of S-equol in rat, monkey and man, and that all metabolic routes of S-equol observed in vitro were also observed in vivo.

Toxicokinetics and lack of uterotropic effect of orally administered S-equol.

S-equol is a natural product that is produced by the microbial biotransformation of daidzein, an isoflavone found in soy. Evidence suggests that the health benefits of soy may be related to one's ability to produce S-equol, thus S-equol is being developed for the treatment of vasomotor symptoms in postmenopausal women. The toxicokinetics of S-equol were evaluated in Sprague-Dawley rats and cynomolgus monkeys; S-equol was rapidly absorbed with C(max) occurring between 0.5 h and 1.0 h in the rat and 3h in the monkey. AUC was linear over the doses tested with no differences between male and female animals. Conjugated S-equol was the major metabolite in plasma with less than 1% present as the unconjugated form. S-equol showed a weak induction of liver cytochrome P450s in vivo, and did not significantly inhibit the major human cytochrome P450s in vitro. S-equol was highly protein bound (>95%) in rat, monkey and man in a concentration-independent manner. Orally administered S-equol did not significantly change uterine weight or morphology in either the rat or monkey even at the highest doses tested. These studies show that S-equol has pharmacokinetic parameters suitable for drug development with a low potential for uterotropic effects.

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows.

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. ß-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in ß-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.

[Advances on the study of equol metabolism in human].

Equol, the metabolic end product of soybean isoflavones, is a highly stable molecule, only can be changed to beta-glucuronide (mainly)/sulfate and not be metabolized further. Equol can be quickly absorbed and reached the peak value in blood after 1-2 h. The biological half-life of equol is 7-8 h. It was found that majority of equol was discharged through urine and the discharge ratio was not affected by the person who can produce equol or not. Equol plays an important role in health. The ability of using Equol is different among individuals and the differences are not affected by the person who can produce equol or not.