Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower K_m value and a higher k_cat/K_m value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.
Cyclodextrins ; Genistein ; Glucosyltransferases/metabolism* ; Glycosylation ; Kinetics

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.
Cloning, Molecular ; Genistein ; Glucosyltransferases/metabolism* ; Isoflavones/pharmacology* ; Pueraria/chemistry*

The interaction between genistein and beta-glucosidase was studied using fluorescence quenching method and synchronous fluorimetry. The binding reaction was simultaneously studied by the AutoDock 4.2 molecular docking model. Data from fluorescence spectroscopy indicated that these interactions resulted in the endogenous fluorescence quenching of beta-glucosidase, which belongs to a static quenching mechanism. The calculated binding constants were 3.69 x 10(4), 3.06 x 10(4) and 2.36 x 10(4) L x mol(-1) at 17, 27 and 37 degrees C, respectively. The evidences from synchronous fluorescence showed the effect of genistein on the microenvironment around beta-glucosidase in aqueous solution. The inhibition test showed that the activity of beta-glucosidase could be inhibited by genistein. The determined bimolecular rate constant (k(i)) was 1.2 x 10(3) (mol x L(-1)(-1) x min(-1). Molecular docking was performed to reveal the possible binding mode or mechanism and suggested that genistein could bind strongly to beta-glucosidase. The results revealed that genistein tended to bind with beta-glucosidase mainly by hydrogen bond and hydrophobic interaction as well as electrostatic forces.
Genistein ; metabolism ; Molecular Docking Simulation ; Protein Binding ; Protein Kinase Inhibitors ; metabolism ; Spectrometry, Fluorescence ; beta-Glucosidase ; metabolism

The medium for isoflavone production in Maackia amurensis suspension cells has been optiwised through the artificial neural networks (ANNs) and the real coding based accelerating genetic algorithm (RAGA). Among the ingredients of the medium, nitrogen sources and plant growth regulators were found to be the main factors affecting the production of isoflavone genistein. (NH4)2SO4, KNO3, 2,4-D and 6-BA, 100 approximately 800 mg/L, 1500 approximately 3000 mg/L, 0 approximately 3 mg/L and 0 approximately 1 mg/L respectively, significantly increased genistein yield, in the ranges of effective concentrations. The random ten combinations of these four components generated by RAGA as input data and the genistein yields of ten combinations as output data were used for ANNs-RAGA (the artificial neural networks associated with the accelerating genetic algorithm) modeling. The resultant model showed a high fit between the experimental data and calculating values by ANNs-RAGA. Based on the prediction of the model, the optimum combination of four factors for genistein production was determined on 149.68 mg/L for (NH4)2SO4, 2936.10 mg/L KNO3, 0.01 mg/L 2,4-D and 0.19 mg/L 6-BA. When cells were cultured in the optimized medium, their capability of genistein production was remarkably enhanced to 14.13 mg/L, which was about 19 times higher than that in the original medium. The relative discrepancy between the experimental value and the predictive value of genistein yield from the optimized medium was 7.38%.
Algorithms ; Cells, Cultured ; Culture Media ; Genistein ; metabolism ; Maackia ; metabolism ; Models, Biological ; Neural Networks (Computer) ; Suspensions

Genistein is a high specific and noncompetitive inhibitor of epidermal growth factor receptor tyramine kinase domain (EGFR-TK). In the paper, a molecular docking between genistein and EGFR-TK was studied to explore the mechanism of their interaction and antitumor mechanism of genistein by AUTODOCK 3.05 program. The results indicated that genistein located in the active cavity of EGFR-TK by high affinity (deltaG = -31.2 kJ/mol), and genistein inhibited EGFR-TK by interfering with forming of Lys721/Glu738 ion pair. The inhibition belonged to noncompetitive interaction, in which hydrophobic force and hydrogen bond played key roles.
Catalytic Domain ; Genistein ; metabolism ; pharmacology ; Models, Molecular ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; metabolism

PURPOSE: It has been postulated that soybean isoflavones act as inhibitory factors in several cancers. Recently, various in vitro and in vivo experimental studies have demonstrated that these isoflavones inhibit prostate cancer. Therefore, we investigated whether soybean isoflavones influenced the development of prostate cancer by comparing the levels of circulating isoflavones between prostate cancer patients and controls. MATERIALS AND METHODS: The serum levels of genistein, daidzein and equol were determined using reverse-phase, high-performance liquid chromatography-multiple reaction ion monitoring mass spectrometry(HPLC-MS) and compared in 122 experimental subjects(61 prostate cancer patients and 61 cancer-free controls) from 6 hospitals. RESULTS: The serum concentrations of genistein, daidzein and equol in the patients were 130.7+/-181.4ng/ml, 53.6+/-69.3ng/ml and 11.37+/-43.4ng/ml, with control values of 95.6+/-95.2ng/ml, 55.2+/-121.8ng/ml and 23.2+/-34.5ng/ml, respectively. There was no statistical difference between the 2 groups. Daidzein non-metabolizers who were unable to degrade daidzein into equol were compared between the patients and the controls, and were found to be significantly more common in the patient group (p=0.001, OR=3.44, 95% CI=1.6243-7.2855). However, the equol/daidzein ratio was significantly lower in the patients than in the controls(p=0.0072). No association between age, stage, Gleason score or isoflavone concentrations was found. CONCLUSIONS: These results suggest that the capability to produce equol (i.e., the mechanism for the metabolism of daidzein into equol) is closely involved in the lower incidence of prostate cancer, and that a diet based on soybean isoflavones would be useful in preventing prostate cancer.
Diet ; Equol ; Genistein ; Humans ; Incidence ; Isoflavones ; Metabolism ; Neoplasm Grading ; Prostate* ; Prostatic Neoplasms* ; Soybeans*

OBJECTIVETo observe the effects of Genistein on TGF-beta1 expression and the intracellular free Ca2+ concentration in human hypertrophic scar fibroblasts, and to discuss the mechanism of the anti-fibrosis effect.

METHODSFibroblasts were derived from human hypertrophic scar tissue and cultured in vitro. Genistein in different concentrations (25, 50, 100 micromol/L) was administrated to the fibroblasts, respectively. After 48 hours of co-culture, the expression of TGF-beta1 mRNA and protein were examined by RT-PCR and Western-Blot assay respectively. The intracellular free Ca2+ concentration in hypertrophic scar fibroblasts pretreated by Genistein was determined by laser confocal scanning microscopy with or without the stimulation of bFGF.

RESULTSGenistein inhibited the expression of TGF-beta1 in hypertrophic scar fibroblasts on a concentration-dependent manner. bFGF significantly elevated the intracellular free Ca2+ concentration, however its stimulating effect was remarkably alleviated when the fibroblasts were pre-treated by Genistein.

CONCLUSIONSGenistein can reduce the expression of TGF-beta1 and block the accumulation of intracellular free calcium induced by growth factors. It maybe one of the possible mechanisms of Genistein's antifibrosis effect.

Calcium ; analysis ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Genistein ; pharmacology ; Humans ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVEGenistein, a major soy isoflavone metabolite (SIF), inactivates oxidation activity of bovine lactoperoxidase (LPO). Modification of the heme moiety of LPO by nitrogen-containing compounds has been shown to inactivate LPO. In contrast, SIF mediated inactivation of LPO does not involve a heme modification and the mechanism of SIF inhibition is poorly understood.

METHODSAfter inactivation of LPO by genistein in the presence of H(2)O(2), trypsin-digested LPO peptide fragments were collected and analyzed by MALDI-TOF-MS to characterize the chemical binding of genistein(s) to LPO.

RESULTSThe heme moiety of LPO was not modified by genistein. A covalent binding study showed that (3)H-genistein bound to LPO with a ratio of ∼12 to 1. After HPLC analysis and peak collection, trypsin-digested peptide fragments were analyzed by MALDI-TOF-MS. The 3H-genistein co-eluted peptide fragments (RT=24 min) were putatively identified as 199IVGYLDEEGVLDQNR214 with two bound genistein molecules or a genistein dimer (2 259 Da), 486TPDNIDIWIGGNAEPMVER504 with two bound genistein molecules or a genistein dimer (2 663 Da), and 161ARWLPAEYEDGLALPFGWTQR182 with three bound genistein molecules or a genistein trimer (3 060 Da). The fragment with a mass of 1 792 Da (RT=36 min) was identified as 132CDENSPYR139 with three genistein molecules or a genistein trimer.

CONCLUSIONSThe results suggest that LPO was inactivated by irreversible covalent binding of genistein or genistein polymers to particular peptide fragments constituting regions of the outward domain. No genistein interaction with the prosthetic heme moiety of LPO was observed.

Animals ; Cattle ; Enzyme Activation ; drug effects ; Genistein ; metabolism ; Hydrogen Peroxide ; pharmacology ; Isoflavones ; pharmacology ; Lactoperoxidase ; metabolism ; Placental Lactogen ; Protein Binding

The purpose of the present overview of meta-analysis is to summarize and critically assess the effect of isoflavones and genistein on glucose metabolism among the peri- and post-menopausal women. Two independent authors searched the databases of MEDLINE, Scopus and Cochrane Library for meta-analysis. Three databases were searched from inception to January 2018. Methodological quality of each meta-analysis of randomized controlled trials was evaluated using the AMSTAR (a measurement tool used to assess systematic reviews). Four meta-analyses were included to the current overview. Fasting insulin levels and homeostatic model assessment of insulin resistance (HOMA-IR) values were significantly lower in peri-menopausal and postmenopausal. Two meta-analyses showed that treatment with isoflavones could not alter fasting blood glucose. However, one meta-analysis depicted that isoflavones significantly improved blood glucose levels in non-Asian postmenopausal women. Treatment with genistein could have significant beneficial effects on fasting insulin, blood glucose and HOMA-IR in comparison to the control group. Regardless of the population, the treatment with genistein is effective in improving fasting insulin, HOMA-IR and glucose levels. Nevertheless, the high heterogeneity among studies and poor methodology of reviews made it difficult to draw a definite conclusion on the positive impacts of soy on glucose metabolism.
Blood Glucose ; Fasting ; Female ; Genistein ; Glucose Metabolism Disorders ; Glucose ; Humans ; Insulin ; Insulin Resistance ; Insulins ; Isoflavones ; Menopause ; Metabolism ; Population Characteristics

Actins ; metabolism ; Animals ; Calcium ; metabolism ; Cells, Cultured ; Cytoskeleton ; metabolism ; Enzyme Inhibitors ; pharmacology ; Genistein ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Male ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Rats ; Rats, Wistar