Homoisoflavonoids from the bulbs of Bellevalia longipes and an assessment of their potential cytotoxic activity.

Seven undescribed homoisoflavonoids were identified from the bulbs of Bellevalia longipes Post (Asparagaceae) as well as thirteen known and one natural homoisoflavonoid that had been reported as a synthetic product previously. A general approach for recognizing homoisoflavonoids via NMR spectroscopy data were presented. The undescribed compounds were: 8-dehydroxy-5-O-demethyl-6-hydroxyscillapersicone, 6-methoxyscillapersicone, 5-O-demethyl-6-methoxyscillapersicone, 8-O-methylscillapersicone, 4'-O-methylscillapersicone, 4',8-O,O-dimethylscillapersicone, 3'-O-methylscillapersicone, and 3-hydroxy-desmethylophiopogonanone A. Structures were determined based on analysis of HRMS and NMR data, while absolute configurations were assigned using ECD spectroscopy. Human cancer cell lines were used to assess the cytotoxic activities of the isolated compounds, where 3-dehydroxy-3'-hydroxyeucomol showed IC50 values of 0.62 µM, 5.36 µM, and 2.52 µM, when tested against MDA-MB-435 (melanoma), MDA-MB-231 (breast), and OVCAR3 (ovarian) cells, respectively.

Safety and Efficacy Assessment of Isoflavones from Pueraria (Kudzu) Flower Extract in Ovariectomised Mice: A Comparison with Soy Isoflavones.

Numerous Foods with Function Claims that contain the extract of Pueraria flower (kudzu) isoflavones (PFI) are available in the Japanese market. These are labelled with function claims of reducing visceral fat. However, these foods have not undergone proper safety assessment such as the evaluation of their oestrogenic activity and effects on drug-metabolising enzymes (cytochrome P-450: CYP) in the liver. This study evaluated the estrogenic effect and the hepatic CYP activity and mRNA expression in normal female mice as a safety assessment of PFI (Experiment 1). In addition, the bone mineral density and visceral fat weight in ovariectomised mice (OVX) compared to soy isoflavones (SI) was evaluated to assess the efficacy of PFI (Experiment 2). OVX control fed a control diet, OVX fed a PFI diet (the recommended human intake of PFI), OVX fed a PFI20 diet (20- times the recommended PFI), OVX fed an SI diet (the recommended human intake of SI), and OVX fed an SI20 diet (20 -times the recommended intake of SI) for 28 days in Experiment 2. Body, liver, and visceral fat weights were not affected by the PFI, PFI20, SI, or SI20 diets. The hepatic CYP1A and CYP3A activities were elevated by the SI20 treatment. Ovariectomy-induced bone loss was inhibited by the SI20 treatment, but not by the PFI20 treatment. These results suggest that (1) PFI intake in human doses had no oestrogenic properties and did not affect CYP activity in the liver; (2) there was no evidence that PFI affects the amount of visceral fat in OVX mice.

Isoflavones in food supplements: chemical profile, label accordance and permeability study in Caco-2 cells.

Consumers nowadays are playing an active role in their health-care. A special case is the increasing number of women, who are reluctant to use exogenous hormone therapy for the treatment of menopausal symptoms and are looking for complementary therapies. However, food supplements are not clearly regulated in Europe. The EFSA has only recently begun to address the issues of botanical safety and purity regulation, leading to a variability of content, standardization, dosage, and purity of available products. In this study, isoflavones (puerarin, daidzin, genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A) from food supplements (n = 15) for menopausal symptoms relief are evaluated and compared with the labelled information. Only four supplements complied with the recommendations made by the EC on the tolerable thresholds. The intestinal bioavailability of these compounds was investigated using Caco-2 cells. The apparent permeability coefficients of the selected isoflavonoids across the Caco-2 cells were affected by the isoflavone concentration and product matrix.

Bitter taste receptor activation by flavonoids and isoflavonoids: modeled structural requirements for activation of hTAS2R14 and hTAS2R39.

Many flavonoids and isoflavonoids have an undesirable bitter taste, which hampers their use as food bioactives. The aim of this study was to investigate the effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 and to predict their structural requirements to activate these receptors. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, among which 58 ligands were overlapping. Their activation threshold values varied over a range of 3 log units between 0.12 and 500 µM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure-activity relationships. The 2D models demonstrated excellent predictive power in identifying bitter (iso)flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 (or hTAS2R39) were determined by 3D-pharmacophore models to be composed of two (or three) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. The additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behaviors on the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)flavonoids and provided a powerful tool in the prediction of their potential bitterness. By understanding the "bitter motif", introduction of bitter taste in functional foods enriched in (iso)flavonoid bioactives might be avoided.

Comparative assessment of dermal wound healing potentials of various Trifolium L. extracts and determination of their isoflavone contents as potential active ingredients.

ETHNOPHARMACOLOGICAL RELEVANCE: Trifolium species are used in Turkish folk medicine as a wound healing agent, expectorant, antiseptic, sedative and to alleviate pain in rheumatism. In the present study, the aqueous methanolic extracts (80%) of 13 Trifolium species (Trifolium ambigum, Trifolium arvense var. arvense, Trifolium campestre, Trifolium canescens, Trifolium hybridum var. anatolicum, Trifolium hybridum var. hybridum, Trifolium pannonicum, Trifolium pratense var. pratense, Trifolium purpureum var. purpureum, Trifolium repens var. repens, Trifolium resupinatum var. microcephalum, Trifolium spadiceum and Trifolium trichocephalum) collected from different regions of Anatolia were evaluated for their in vivo wound healing effects. MATERIALS AND METHODS: In vivo wound healing activities of the plant aqueous methanolic extracts were evaluated by linear incision and circular excision wound models subsequent to histopathological analysis. Active constituents were determined by a validated high performance liquid chromatographic method. Precision of the method was performed by the evaluation of intra-day and inter-day variations of the each standard at limits of quantification (LOQ) levels. RESULTS: The aqueous methanolic extracts of Trifolium canescens and Trifolium pretense var. pratense possessed better wound healing activity compared to the other extracts and control groups. The animal groups treated with the Trifolium canescens extract demonstrated increased contraction (48.96%) on excision and a significant increase in wound tensile strength (35.6%) on incision models. The main compounds were detected as genistein and biochanin A for Trifolium canescens. CONCLUSION: The results of the present study revealed the wound healing potential of Trifolium canescens. This might be due to the combined effect of the isoflavones genistein, formononetin, daidzein, and biochanin A present in the extract.

A facile route to isoflavone quinones via the direct cross-coupling of chromones and quinones.

A straightforward and efficient method for the palladium-catalyzed direct cross-coupling of chromones with various quinones has been developed to rapidly construct isoflavone quinone structural motifs.

Development of bioluminescent enzyme immunoassay for s-equol using firefly luciferase and its application to the assessment of equol-producer status.

In this study, we developed a specific bioluminescent enzyme immunoassay (BLEIA) for S-equol, employing firefly luciferase as a labeling enzyme, as an alternative to HPLC methods. Satisfactory correlation (r=0.992) was shown when this S-equol BLEIA was compared with HPLC. The cross-reactivity with R-equol as its diastereoisomer is <5%, and that with daidzein, which is the substrate of equol, is 0.02%. Frequencies of Japanese equol producers determined using two distinct approaches were compared: a threshold value for urinary S-equol concentration of 232 ng/ml gave frequencies of 32% of men and 19% of women. These values correspond to the results for log(10)-transformed urinary S-equol to daidzein ratio threshold of -1.75, namely, 34% of men and 19% of women. When the changes in concentration of urinary equol and daidzein were measured after ingestion of isoflavone, the maximum concentration (C(max)) of urinary equol appeared after 9.6 h of isoflavone consumption; this C(max) was 2 h later than that for daidzein. The S-equol BLEIA documented in this study is expected to be an important tool for the assessment of equol producer status and demonstration of the bioavailability of isoflavone.

Bioactivity of isoflavones: assessment through a theoretical model as a way to obtain a "Theoretical Efficacy Related to Estradiol (TERE)".

The increase of human life span will have profound implications in Public Health in decades to come. By 2030, there will be an estimated 1.2 billion women in post-menopause. Hormone Replacement Therapy with synthetic hormones is still full of risks and according to the latest developments, should be used for the shortest time possible. Searching for alternative drugs is inevitable in this scenario and science must provide physicians with other substances that can be used to treat the same symptoms with less side effects. Systematic research carried out on this field of study is focusing now on isoflavones but the randomised controlled trials and reviews of meta-analysis concerning post-menopause therapy, that could have an important impact on human health, are very controversial. The aim of the present work was to establish a theoretical calculation suitable for use as a way to estimate the "Theoretical Efficacy (TE)" of a mixture with different bioactive compounds as a way to obtain a "Theoretical Efficacy Related to Estradiol (TERE)". The theoretical calculation that we propose in this paper integrates different knowledge about this subject and sets methodological boundaries that can be used to analyse already published data. The outcome should set some consensus for new clinical trials using isoflavones (isolated or included in mixtures) that will be evaluated to assess their therapeutically activity. This theoretical method for evaluation of a possible efficacy could probably also be applied to other herbal drug extracts when a synergistic or contradictory bio-effect is not verified. In this way, it we may contribute to enlighten and to the development of new therapeutic approaches.

[Assessment of Pueraria lobata isoflavone with self-microemulsifying drug delivery systems in vitro and in vivo].

OBJECTIVE: To evaluate the self-microemulsifying ability and dissolution behavior of pueraria lobata isoflavone in vitro and the pharmacokinetic behavior in rats. METHODS: The self-microemulsifying rate was evaluated by the self-microemulsifying time and the self-microemulsifying efficiency was evaluated by the particle size of resultant microemulsions. The plasma concentrations were evaluated by HPLC and dissolution and pharmacokinetic behavior of self-microemulsifying drug delivery systems were evaluated by comparison with commercial tablets. RESULTS: The system was self-microemulsified in 2 min and the particle size was less than 50 nm. The dis- solution of SMESC in distilled water was more than 90% at 10 min, while those of the commercial tablet were less than 50% at 120 min. 82% increase in the relative bioavailability was observed for the self microemulsifying drug delivery systems compared with Yufengningxin tablets. Tmax was smaller in the self-microemulsifying drug delivery systems compared with Yufengningxin tablets. CONCLUSION: The self-microemulsifying drug delivery systems can increase drug dissolution in vitro and absorption in vivo significantly.

Assessment of the reactivity of selected isoflavones against proteins in comparison to quercetin.

Selected isoflavones (genistein, daidzein, formononetin, prunetin, biochanin A, and two synthetic isoflavones) were allowed to interact with soy and whey proteins. The reaction products were analyzed in terms of covalent binding at the nucleophilic side chains of proteins. Changes in molecular properties of the proteins derivatives were documented by SDS-PAGE, IEF, and SELDI-TOF-MS. The structural changes induced were studied using circular dichroism. The in vitro digestibility was assessed with trypsin. The results show that the occurrence of the catechol moiety, that is, the two adjacent (ortho) aromatic hydroxyl groups on ring B of the flavonoid structural skeleton appears to be prerequisite condition for covalent binding to proteins. The catechol moiety on ring A was less reactive. Its absence lead to a slight or no significant reaction, although noncovalent interactions may still be possible, even when lacking this structural element. A comparison of the data is also made with quercetin representing the flavonols.

Assessment of the estrogenicity of isoflavonoids, using MCF-7-ERE-Luc cells.

In the current study, our research focused on the estrogenic activity of isoflavonoids, mainly genistein, biochanin A and daidzein. Genistein enhanced the reporter gene expression of MCF-7-ERE-Luc cells, at a concentration as low as 10 nM, with a concentration of 100 nM the achieved gene expression effects were similar to those of 10 pM 17beta-estradiol, Based on the estrogenic activities of biochanin A and daidzein, hydroxyl groups at the 4' and 5 positions are needed for the maximal effect of the genistein. The estrogenic effects of these isoflavonoids were inhibited by the concomitant treatment with tamoxifen. The data showed that the estrogenic effects of isoflavonoids were mediated through estrogen receptors. When the isoflavonoids were tested as mixtures, the estrogenic effects were lower than the arithmetic sum of those induced by each individual isoflavonoid. The estrogenic potency of each isoflavonoid was presented at EC50 levels with a 17beta-estradiol equivalent concentration (EEQ) based on the dose response of each chemical. The EC50s and EEQs of genistein, biochanin A and daidzein were 4.15, 0.89 and 0.18 microM, and 15.0, 5.12 and 1.83 microM/M, respectively. Our data clearly demonstrated that the pERE-luciferase reporter gene assay was suited for the sensitive and quantitative measurement, and large scale screening, of the estrogenicity of chemicals in vitro.