HPLC-UV/HRMS methods for the unambiguous detection of adulterations of Ginkgo biloba leaves with Sophora japonica fruits on an extract level.

CONTEXT: Ginkgo biloba L. (Ginkgoaceae) leaf extract is one of the most frequently sold herbal extracts. There have been reports on poor quality and adulteration of ginkgo leaf extracts or the powdered plant material with extracts or powder of Styphnolobium japonicum (L.) Schott (Fabaceae) (syn. Sophora japonica L.) fruits, which is rich in flavone glycosides. OBJECTIVE: The study investigates whether ginkgo leaves genuinely contain genistein and sophoricoside and whether these two substances could be used as markers to detect adulterations with sophora fruits. MATERIALS AND METHODS: A total of 33 samples of dried ginkgo leaves were sourced from controlled plantations in China, the USA, and France. After extraction, the samples were analyzed using two high-performance liquid chromatography (HPLC) coupled with UV/HRMS methods for the detection of genistein and sophoricoside, respectively. Chromatograms were compared to standard reference materials. RESULTS: In none of the tested ginkgo samples, neither genistein nor sophoricoside could be detected. The applied method was designed to separate genistein from apigenin. The latter is a genuine compound of ginkgo leaves, and its peak may have been previously misidentified as genistein because of the same molecular mass. The method for the detection of sophoricoside allows identification of the adulteration with sophora fruit without prior hydrolysis. By both HPLC methods, it was possible to detect adulterations of ≥2% sophora fruits in the investigated ginkgo extract. CONCLUSION: The methods allow unambiguous detection of adulterations of ginkgo leaves with sophora fruits, using genistein and sophoricoside as marker compounds.

Enhancement of developmentally regulated daidzein secretion from soybean roots in field conditions as compared with hydroponic culture.

Analyses of metabolite secretions by field-grown plants remain scarce. We analyzed daidzein secretion by field-grown soybean. Daidzein secretion was higher during early vegetative stages than reproductive stages, a trend that was also seen for hydroponically grown soybean. Daidzein secretion was up to 10 000-fold higher under field conditions than hydroponic conditions, leading to a more accurate simulation of rhizosphere daidzein content.

Soy Isoflavone Genistein Inhibits an Axillary Osmidrosis Risk Factor ABCC11: In Vitro Screening and Fractional Approach for ABCC11-Inhibitory Activities in Plant Extracts and Dietary Flavonoids.

Axillary osmidrosis (AO) is a common chronic skin condition characterized by unpleasant body odors emanating from the armpits, and its aetiology is not fully understood. AO can seriously impair the psychosocial well-being of the affected individuals; however, no causal therapy has been established for it other than surgical treatment. Recent studies have revealed that human ATP-binding cassette transporter C11 (ABCC11) is an AO risk factor when it is expressed in the axillary apocrine glands-the sources of the offensive odors. Hence, identifying safe ways to inhibit ABCC11 may offer a breakthrough in treating AO. We herein screened for ABCC11-inhibitory activities in 34 natural products derived from plants cultivated for human consumption using an in vitro assay system to measure the ABCC11-mediated transport of radiolabeled dehydroepiandrosterone sulfate (DHEA-S-an ABCC11 substrate). The water extract of soybean (Glycine max) was found to exhibit the strongest transport inhibition. From this extract, via a fractionation approach, we successfully isolated and identified genistein, a soy isoflavone, as a novel ABCC11 inhibitor with a half-maximal inhibitory concentration value of 61.5 µM. Furthermore, we examined the effects of other dietary flavonoids on the ABCC11-mediated DHEA-S transport to uncover the effects of these phytochemicals on ABCC11 function. While further human studies are needed, our findings here about the natural compounds will help develop a non-surgical therapy for AO.

An anti-inflammatory isoflavone from soybean inoculated with a marine fungus Aspergillus terreus C23-3.

A new isoflavone derivative compound 1 (psoralenone) was isolated from soybean inoculated with a marine fungus Aspergillus terreus C23-3, together with seven known compounds including isoflavones 2-6, butyrolactone I (7) and blumenol A (8). Their structures were elucidated by MS, NMR, and ECD. Psoralenone displayed moderate in vitro anti-inflammatory activity in the LPS-induced RAW264.7 cell model. Compound 2 (genistein) showed moderate acetylcholinesterase (AChE) inhibitory activity whereas compounds 2, 5 (biochanin A), 6 (psoralenol), and 7 exhibited potent larvicidal activity against brine shrimp. Compounds 3 (daidzein), 4 (4'-hydroxy-6,7-dimethoxyisoflavone), and 5-7 showed broad-spectrum anti-microbial activity, and compound 7 also showed moderate 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity.

Simultaneous determination of formononetin, biochanin A and their active metabolites in human breast milk, saliva and urine using salting-out assisted liquid-liquid extraction and ultra high performance liquid chromatography-electrospray ionization tandem mass spectrum.

Isoflavonoid phytoestrogens, referred as "dietary estrogens" are widely distributed in the plant kingdom. Formononetin, biochanin A and their active metabolites daidzein and genistein are known to be the most potent among other isoflavonoid phytoestrogens. Thus there is a growing need to determine accurately their concentration in different biological fluids. In the present work, a sensitive analytical method was developed for the quantitative determination of these compounds in human breast milk, saliva and urine. The glycoside conjugates of these compounds were enzymatically hydrolysis prior to salting-out assisted liquid-liquid extraction. Quantitative analysis was done by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The obtained results showed high correlation coefficients (r2 > 0.998) for the linear range established for formononetine, biochanin A, daidzein and genistein. The limits of detection (LODs) and low limits of quantitation (LLOQs) were in the ranges of 0.05-1.0 ng/mL and 1.0-4.0 ng/mL for all analytes in human biological fluids, respectively. The average recoveries ranged from 83.29% to 115.24% for the analytes with relative standard deviation (n = 5) values from 1.84% to 9.75% in samples. Both intra-day and inter-day precisions and accuracy were found to be within 12.53% and ± 12.92% respectively. Under different conditions of stability, the concentrations for four isoflavonoid phytoestrogens deviated within ±12.87% of norminal values. The developed method was successfully validated and applied to human breast milk, saliva and urine. The average concentrations of daidzein and genistein found in breast milk, saliva and urine samples ranged from 0 to 104.2 µg/kg, 18.17 to 786.0 µg/kg, 0 to 10974 µg/kg, respectively. Their presence in breast milk samples shows exposure of breast-fed baby to isoflavones. It also allows for the rapid screening of human biological fluids when testing for formononetin, biochanin A, daidzein and genistein production status in human.

Preparation of DESIGNER extracts of red clover (Trifolium pratense L.) by centrifugal partition chromatography.

Starting with an isoflavone-rich red clover extract (RCE), this study expands on the DESIGNER approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources using countercurrent separation (CCS) methodology. A hydrostatic CCS (also known as centrifugal partition chromatography, CPC) technique was used to enrich and deplete selected bioactive isoflavones of RCE extracts. In order to efficiently prepare large enough DESIGNER extracts from RCE for biological testing including in vivo assays, it was necessary to choose a balance between resolution and a loading capacity of at least 1 g per separation for the selected solvent system (SS). Adding 3 mL of DMSO to the sample containing equal amounts of upper and lower phases of hexanes-ethyl acetate-methanol-water (HEMWat 5.5/4.5/5/5, v/v) allowed 1 g of RCE to be dissolved in the sample without disrupting the chromatographic resolution of the target isoflavones. CPC experiments using other solubility modifiers, acetone and acetonitrile indicated that these modifiers increase solubility significantly, even better than DMSO, but the separation of target compounds was sufficiently disturbed to be unacceptable for producing the desired DESIGNER extracts. The preparation of DESIGNER extracts was achieved with two sequential CPC separations. The first produced a biochanin A enriched fraction (93.60% w/w) with only small amounts of other isoflavones: 2.30% w/w prunetin, 1.17% w/w formononetin, and 0.12% w/w irilone. Gravimetric investigations of this step demonstrated the high efficiency of CCS technology for full and unbiased sample recovery, confirmed experimentally to be 99.80%. A formononetin enriched fraction from this first separation was re-chromatographed on a more polar HEMWat (4/6/4/6, v/v) SS to produce a formononetin enriched DESIGNER fraction of 94.70% w/w purity. The presence of the minor (iso)flavonoids: 3.16% w/w pseudobaptigenin, 0.39% w/w kaempferol, and 0.31% w/w genistein was also monitored in these fractions. Chromatographic fractions, combined fractions, and DESIGNER extracts were analyzed with quantitative 1H NMR (qHNMR) spectroscopy which provided purity information, quantitation, and structural identification of the components.

The application of the supported liquid membrane and molecularly imprinted polymers as solid acceptor phase for selective extraction of biochanin A from urine.

An efficient sample clean-up and preconcentration procedure for phytoestrogens analysis in urine has been developed. It was based on a combination of solid phase extraction with hollow-fiber supported liquid membrane and molecularly imprinted beads (MIPs-HF-SLM-SPE). The molecularly imprinted polymers (MIPs) were synthesized by precipitation polymerization technique with biochanin A (BCA) as a template, giving narrowly dispersed microspheres with a regular shape. As the functional monomer, (dimethylamino)ethyl methacrylate (-DEM) turned out to be better than methacrylic acid (MAA) to get the best-imprinted effects. The MIPs used as sorbents in the MIPs-HF-SLM-SPE extraction process exhibited excellent binding selectivity for BCA, in comparison to non-imprinted polymers as well as its structural analogs (genistein and daidzein). Finally, the developed method was used to detect BCA in urine. Under optimal extraction conditions, the recovery of BCA in urine samples (using 4.5 mL sample spiked with 10 µg L-1) was over 41%, with a coefficient of variation (CV) < 6.6% (n = 5). The detection limit (LOD) and quantification limit (LOQ) for BCA analysis in urine were 0.41 and 1.36 µg L-1, respectively.

Trace analysis of estrogens in milk samples by molecularly imprinted solid phase extraction with genistein as a dummy template molecule and high-performance liquid chromatography-tandem mass spectrometry.

Dummy molecularly imprinted polymer microspheres (DMIPMS) towards estrogens were synthesized by Pickering emulsion polymerization employing genistein (GEN) as a dummy template molecule. The FTIR analysis indicated the successful preparation of the imprinted polymers, and the characterization results of scanning electron microscopy and nitrogen adsorption desorption measurement indicated that the obtained DMIPMS are in possess of regular spherical shapes, porous structures and narrow diameter distribution, a BET surface area of 402.74 m2 g-1, a total pore volume of 0.568 cm3 g-1 and a pore diameter of 3.62 nm. The binding capacity and selectivity of DMIPMS were investigated in equilibrium binding experiments and chromatographic evaluation experiments through scatchard analysis and molecular imprinting factor (IF) analysis, respectively. The MIPs showed high binding capacity and excellent selectivity towards seven selected natural and synthetic estrogens, which are Estrone (E1), 17ß-estradiol (ßE2), estriol (E3), ethinylestradiol (EE2), dienestrol(DS), diethylstilbestrol (DES), and hexestrol (HEX). A method for selective determination of seven estrogens in milk samples via dummy molecularly imprinted solid phase extraction coupled with HPLC-MS/MS was developed, which showed good linearity from 2 to 500 µg L-1 with a correlation coefficient (R2) of more than 0.999. The detection limits were within the range of 0.10-0.35 µg L-1 and the recoveries of the seven estrogens at three spiking levels (10,100,250 µg L-1) ranged from 88.9% to 102.3% with relative standard deviation (RSD, n = 5) for intra-day and inter-day assays varied from 0.8% to 4.5%. The developed method is thus proven to be efficient and reliable for regular monitoring of trace estrogens in complex matrices such as milk samples.

Screening and isolation of cyclooxygenase-2 inhibitors from Trifolium pratense L. via ultrafiltration, enzyme-immobilized magnetic beads, semi-preparative high-performance liquid chromatography and high-speed counter-current chromatography.

Nonsteroidal anti-inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase-2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase-2 inhibitors can also act through cyclooxygenase-independent mechanisms. In this study, using ultrafiltration, enzyme-immobilized magnetic beads, high-performance liquid chromatography, and electrospray-ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi-preparative high-performance liquid chromatography and high-speed counter-current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase-2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase-2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme-immobilized magnetic beads, semi-preparative high-performance liquid chromatography, and high-speed counter-current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.

Solid-liquid extraction of daidzein and genistein from soybean: Kinetic modeling of influential factors.

The extraction of daidzein and genistein from soybean has been studied and the kinetic modeling was established using four modeling equations. The goodness of fit was evaluated by statistical errors including the standard error of means (SEM), the adjusted correlation coefficient (R2), and chi-square (χ2). The best model was considered to be the So and Macdonald model and it could give the most adequate description of solid-liquid extraction of daidzein and genistein from soybean sample. The effect of process parameters on extraction yields of daidzein and genistein also has been investigated. The optimized extraction condition was at 333.2 K using 70% ethanol solvent at a solvent-to-solid ratio of 20 mL g-1 with an agitation speed of 300 rpm. The highest extraction yields of daidzein and genistein from soybean were 0.126 ± 0.006 and 0.184 ± 0.013 mg g-1, respectively. The activation energies for extraction kinetics of soybean were found to be 11.10 kJ mol-1 (washing step) and 13.96 kJ mol-1 (diffusion step) for daidzein, 10.47 kJ mol-1 (washing step) and 19.70 kJ mol-1 (diffusion step) for genistein, respectively.

Forage Legumes as Sources of Bioactive Phytoestrogens for Use in Pharmaceutics: A Review.

BACKGROUND: Alfalfa and red clover are the most widespread and most important perennial legumes, primarily used as a high-quality feed for livestock. Both alfalfa and red clover, as well as some other plant species from Fabaceae family, are a rich natural source of phytoestrogens, nonsteroidal compounds with an estrogenic activity whose beneficial effects in the prevention and treatment of many diseases are demonstrated in numerous scientific studies. OBJECTIVES: Nowadays, various herbal preparations are present on the world market and their use is constantly increasing, as well as the growing demands of consumers for environmentally sound and health-safe production of plant species used as sources of active substances. Because of their widespread distribution, the possibility of organic breeding, and also significant increases in surface area under genetically modified crops in most EU countries, alfalfa and red clover have become more interesting alternative sources of phytoestrogens. The most common phytoestrogens in these forage legumes are genistein, daidzein, glycitein, formononetin, biochanin, coumestrol, kaempferol and apigenin. The content of these substances is significantly influenced by a number of factors including genotype, environment, production technology, plant maturity stage, and individual plant parts. CONCLUSION: Available evidence suggests that forage legumes represent high promising sources of health-promoting phytoestrogens. Due to numerous favorable features, they can find commercial application in different industries such as pharmaceutical, nutraceutical, cosmetic, and agriculture.

The Extracts and Major Compounds Derived from Astragali Radix Alter Mitochondrial Bioenergetics in Cultured Cardiomyocytes: Comparison of Various Polar Solvents and Compounds.

Astragali Radix (AR) is a widely used "Qi-invigorating" herb in China for its tonic effects in strengthening biological tissues. The extract of AR contains abundant antioxidants, including astragalosides and isoflavonoids. However, very few reports have systematically measured the effects of the major components of AR on cell mitochondrial bioenergetics. Here, a systemic approach employing an extracellular flux analyzer was developed to evaluate mitochondrial respiration in cultured cardiomyocyte cells H9C2. The effects of different polar extractives, as well as of the major compounds of AR, were compared. The contents of astragaloside IV, calycosin, formononetin, and genistein in the AR extracts obtained by using water, 50% ethanol, and 90% ethanol were measured by liquid chromatograph-mass spectrometer (LC⁻MS). The antioxidant activities of the AR extracts, as well as of their major compounds, were determined by measuring the free radical scavenging activity and protective effects in tert-butyl hydroperoxide (tBHP)-treated H9C2 cells. By monitoring the real-time oxygen consumption rate (OCR) in tBHP-treated cardiomyocytes with a Seahorse extracellular flux analyzer, the tonic effects of the AR extracts and of their main compounds on mitochondrial bioenergetics were evaluated. AR water extracts possessed the strongest antioxidant activity and protective effects in cardiomyocytes exposed to oxidative stress. The protection was proposed to be mediated via increasing the spare respiratory capacity and mitochondrial ATP production in the stressed cells. The major compounds of AR, astragaloside IV and genistein, showed opposite effects in regulating mitochondrial bioenergetics. These results demonstrate that highly polar extracts of AR, especially astragaloside-enriched extracts, possess better tonic effects on mitochondrial bioenergetics of cultured cardiomyocytes than extracts with a lower polarity.

In silico estrogen-like activity and in vivo osteoclastogenesis inhibitory effect of Cicer arietinum extract.

Postmenopausal osteoporosis is a common disorder accompanied with estrogen deficiency in women. Plants containing phytoestrogens and amino acids have been used in the osteoporosis treatment. The present study aims to evaluate the estrogen-like activity of the Cicer arietinum extract (CAE) and its ability to inhibit osteoclastogenesis process. These achieved by investigating the binding of its active phytoestrogens (genistein, daidzein, formononetin and biochanin A) to the estrogen receptors (ER) α and ß of rats and human in silico. In addition, in vivo study on ovariectomized (OVX) rats is performed. For in vivo study, twenty four rats were divided into four groups (n= 6). Group I is the sham control rats which administered distilled water. Groups II, III, and IV are OVX groups which administered distilled water, CAE (500 mg/kg), and alendronate; respectively. The docking study revealed that the phytoestrogens docked into the protein active site with binding energies comparable with that of estrogens (estriol and ß-estradiol) which means the similarity between the estrogenic contents of CAE and the ensogenous ones. Additionally, in vivo study revealed that CAE reverse TRAP5b and RANKL levels that drastically increased in the untreated OVX group. But, it trigger upregulation of OPG, enhance the OPG/RANKL ratio and modulate the bone and uterus alterations of OVX group. Phytoestrogens and the bone-protective amino acids contents of CAE could be responsible for their estrogen-like effect and antiosteoporotic activity. These results concluded that CAE is an attractive candidate for developing a potential therapeutic cheap agent used as an alternative to the synthetic estrogen replacement therapy. Further, in vivo validation is required for its clinical application.

Isolation and Purification of Two Isoflavones from Hericium erinaceum Mycelium by High-Speed Counter-Current Chromatography.

High-speed counter-current chromatography (HSCCC) was used to separate and purify two isoflavones for the first time from Hericium erinaceum (H. erinaceum) mycelium using a two-phase solvent system composed of chloroform-dichloromethane-methanol-water (4:2:3:2, v/v/v/v). These two isoflavones were identified as genistein (4',5,7-trihydroxyisoflavone, C15H10O5) and daidzein (4',7-dihydroxyisoflavone, C15H10O4), using infrared spectroscopy (IR), electro-spary ionisation mass (ESI-MS), ¹H-nuclear magnetic resonance (NMR) and 13C-NMR spectra. About 23 mg genistein with 95.7% purity and 18 mg daidzein with 97.3% purity were isolated from 150 mg ethanolic extract of H. erinaceum mycelium. The results demonstrated that HSCCC was a feasible method to separate and purify genistein and daidzein from H. erinaceum mycelium.

Soybean soluble polysaccharide enhances absorption of soybean genistein in mice.

This study was designed to probe the promoting effects of soybean soluble polysaccharide (SSPS) on bioavailability of genistein in mice and the underlying molecular mechanism. Male Kunming mice (n=8) were administered intragastrically with either saline, SSPS (5mg/kgbw), genistein (100mg/kgbw), or SSPS (5 or 50mg/kgbw) together with genistein (100mg/kgbw) for consecutive 28days. UPLC-qTOF/MS analysis showed that co-administration of SSPS and genistein in mice caused significant elevation in the urinary levels of genistein and its metabolites (p<0.05). Furthermore, the fecal excretion of genistein was also enhanced by co-administration of SSPS. However, the feces level of dihydrogenistein, a characteristic metabolite of genistein degraded by gut microorganism, was dose-dependently decreased by the combined treatment of SSPS. Additionally, co-treatment of SSPS with genistein also decreased the small intestinal levels of uridinediphosphate-glucuronosyltransferase (UGT), sulfotransferase (SULT), P-glycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP1), and multidrug resistance-associated protein-2 (MRP2) in mice. These findings suggest that the inhibition of SSPS against small intestinal first-pass metabolism of genistein is involved in the promoting effect of genistein bioavailability in mice.

Hypotensive effects of genistein: From chemistry to medicine.

Genistein (4', 5, 7-trihydroxyisoflavone), a naturally occurring flavonoid characteristic of Leguminoseae plants, is a phyto-oestrogen exerting oestrogenic activity as both an agonist and an antagonist substance. A large body of evidence suggests that genistein possesses many physiological and pharmacological properties that make this molecule a potential agent for the prevention and treatment of a number of chronic diseases. Growing evidence suggests that genistein could act as a vasodilating, anti-thrombotic, and anti-atherosclerotic agent, exerting these effects through different mechanisms of action. This paper aims to review data from the literature assessing the beneficial effects of genistein on hypertension, one of the most important cardiovascular disease risk factors along with hyperglycemia and hyperlidipemia. In addition, we discuss the chemistry, main sources and bioavailability of genistein. Scientific findings support genistein's potential as a promising anti-hypertensive agent in different experimental models. However, clinical trials are very limited and more research will be required before genistein intake can be recommended as part of therapies targeting raised blood pressure.

Optimization of Microwave-Assisted Extraction Conditions for Five Major Bioactive Compounds from Flos Sophorae Immaturus (Cultivars of Sophora japonica L.) Using Response Surface Methodology.

Microwave-assisted extraction was applied to extract rutin; quercetin; genistein; kaempferol; and isorhamnetin from Flos Sophorae Immaturus. Six independent variables; namely; solvent type; particle size; extraction frequency; liquid-to-solid ratio; microwave power; and extraction time were examined. Response surface methodology using a central composite design was employed to optimize experimental conditions (liquid-to-solid ratio; microwave power; and extraction time) based on the results of single factor tests to extract the five major components in Flos Sophorae Immaturus. Experimental data were fitted to a second-order polynomial equation using multiple regression analysis. Data were also analyzed using appropriate statistical methods. Optimal extraction conditions were as follows: extraction solvent; 100% methanol; particle size; 100 mesh; extraction frequency; 1; liquid-to-solid ratio; 50:1; microwave power; 287 W; and extraction time; 80 s. A rapid and sensitive ultra-high performance liquid chromatography method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (EIS-Q-TOF MS/MS) was developed and validated for the simultaneous determination of rutin; quercetin; genistein; kaempferol; and isorhamnetin in Flos Sophorae Immaturus. Chromatographic separation was accomplished on a Kinetex C18 column (100 mm × 2.1 mm; 2.6 µm) at 40 °C within 5 min. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile (71:29; v/v). Isocratic elution was carried out at a flow rate of 0.35 mL/min. The constituents of Flos Sophorae Immaturus were simultaneously identified by EIS-Q-TOF MS/MS in multiple reaction monitoring mode. During quantitative analysis; all of the calibration curves showed good linear relationships (R² > 0.999) within the tested ranges; and mean recoveries ranged from 96.0216% to 101.0601%. The precision determined through intra- and inter-day studies showed an RSD% of <2.833%. These results demonstrate that the developed method is accurate and effective and could be readily utilized for the comprehensive quality control of Flos Sophorae Immaturus.

Thonningiiflavanonol A and thonningiiflavanonol B, two novel flavonoids, and other constituents of Ficus thonningii Blume (Moraceae).

A phytochemical study of Ficus thonningii has led to the isolation of two previously unreported compounds, thonningiiflavanonol A and thonningiiflavanonol B together with 16 known compounds: shuterin, naringenin, syringic acid, p-hydroxybenzoic acid, genistein, 5,7,3',4',5'-pentahydroxyflavanone, luteolin, methylparaben, aromadendrin, garbanzol, dihydroquercetin, 5,7,3'-trihydroxyflavanone, ß-sitosterol, sitosterolglucoside, lupeol acetate, and taraxerol. Their structures were elucidated on the basis of spectroscopic data. The new compounds and extracts displayed potent antioxidant activity.

The Stimulatory Effect of Strontium Ions on Phytoestrogens Content in Glycine max (L.) Merr.

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

Compositional changes in (iso)flavonoids and estrogenic activity of three edible Lupinus species by germination and Rhizopus-elicitation.

The effects of germination and elicitation on (iso)flavonoid composition of extracts from three edible lupine species (Lupinus luteus, Lupinus albus, Lupinus angustifolius) were determined by RP-UHPLC-MS(n). The total (iso)flavonoid content of lupine increased over 10-fold upon germination, with the total content and composition of isoflavonoids more affected than those of flavonoids. Glycosylated isoflavones were the most predominant compounds found in lupine seedlings. Lesser amounts of isoflavone aglycones, including prenylated ones, were also accumulated. Elicitation with Rhizopus oryzae, in addition to germination, raised the content of isoflavonoids further: the total content of 2'-hydroxygenistein derivatives was increased considerably, without increasing that of genistein derivatives. Elicitation by fungus triggered prenylation of isoflavonoids, especially of the 2'-hydroxygenistein derivatives. The preferred positions of prenylation differed among the three lupine species. The change in isoflavone composition increased the agonistic activity of the extracts towards the human estrogen receptors, whereas no antagonistic activity was observed.