Phytoestrogens: Chemistry, potential health benefits, and their medicinal importance.

Phytoestrogens, also known as xenoestrogens, are secondary metabolites derived from plants that have similar structures and biological effects as human estrogens. These compounds do not directly affect biological functions but can act as agonists or antagonists depending on the level of endogenous estrogen in the body. Phytoestrogens may have an epigenetic mechanism of action independent of estrogen receptors. These compounds are found in more than 300 plant species and are synthesized through the phenylpropanoid pathway, with specific enzymes leading to various chemical structures. Phytoestrogens, primarily phenolic compounds, include isoflavonoids, flavonoids, stilbenes, and lignans. Extensive research in animals and humans has demonstrated the protective effects of phytoestrogens on estrogen-dependent diseases. Clinical trials have also shown their potential benefits in conditions such as osteoporosis, Parkinson's disease, and certain types of cancer. This review provides a concise overview of phytoestrogen classification, chemical diversity, and biosynthesis and discusses the potential therapeutic effects of phytoestrogens, as well as their preclinical and clinical development.

Dual Self-Assembly of Puerarin and Silk Fibroin into Supramolecular Nanofibrillar Hydrogel for Infected Wound Treatment.

The treatment of infected wounds remains a challenging biomedical problem. Some bioactive small-molecule hydrogelators with unique rigid structures can self-assemble into supramolecular hydrogels for wound healing. However, they are still suffered from low structural stability and bio-functionality. Herein, a supramolecular hydrogel antibacterial dressing with a dual nanofibrillar network structure is proposed. A nanofibrillar network created by a small-molecule hydrogelator, puerarin extracted from the traditional Chinese medicine Pueraria, is interconnected with a secondary macromolecular silk fibroin nanofibrillar network induced by Ga ions via charge-induced supramolecular self-assembly. The resulting hydrogel features adequate mechanical strength for sustainable retention at wounds. Good biocompatibility and efficient bacterial inhibition are obtained when the Ga ion concentration is 0.05%. Otherwise, the substantial release of Ga ions and puerarin endows the hydrogel with excellent hemostatic and antioxidative properties. In vivo, evaluation of a mouse-infected wound model demonstrates that its healing effect outperformed that of a commercially available silver-containing wound dressing. The experimental group successfully achieves a 100% wound closure rate on day 10. This study sheds new light on the design of nanofibrillar hydrogels based on supramolecular self-assembly of naturally derived bioactive molecules as well as their clinical use for treating chronic infected wounds.

ROS Scavenging Effect of Selected Isoflavones in Provoked Oxidative Stress Conditions in Human Skin Fibroblasts and Keratinocytes.

Isoflavones, belonging to polyphenolic compounds, show structural similarity to natural estrogens, and in this context, they have been extensively studied. Some of them are also applied as cosmetic additives; however, little is known regarding their effects on skin cells. In this investigation, common isoflavones, including genistein, daidzein, glycitein, formononetin, and biochanin A, as well as coumestrol, were evaluated for antioxidant activity and their impact on human skin fibroblasts and keratinocytes. Antioxidant effects were assessed using DPPH, ABTS, and FRAP tests, and the ability to scavenge reactive oxygen species (ROS) was tested in cells with H2O2-provoked oxidative stress. The impact on the activity of antioxidant enzymes (SOD, CAT, GSH) and lipid peroxidation (MDA) was also explored. As shown by Alamar Blue and neutral red uptake assays, the compounds were not toxic within the tested concentration range, and formononetin and coumestrol even demonstrated a stimulatory effect on cells. Coumestrol and biochanin A demonstrated significant antioxidative potential, leading to a significant decrease in ROS in the cells stimulated by H2O2. Furthermore, they influenced enzyme activity, preventing depletion during induced oxidative stress, and also reduced MDA levels, demonstrating protection against lipid peroxidation. In turn, genistein, daidzein, and glycitein exhibited low antioxidant capacity.

An Injectable Puerarin Depot Can Potentiate Chimeric Antigen Receptor Natural Killer Cell Immunotherapy Against Targeted Solid Tumors by Reversing Tumor Immunosuppression.

Chimeric antigen receptor natural killer (CAR-NK) cell therapy represents a potent approach to suppressing tumor growth because it has simultaneously inherited the specificity of CAR and the intrinsic generality of NK cells in recognizing cancer cells. However, its therapeutic potency against solid tumors is still restricted by insufficient tumor infiltration, immunosuppressive tumor microenvironments, and many other biological barriers. Motivated by the high potency of puerarin, a traditional Chinese medicine extract, in dilating tumor blood vessels, an injectable puerarin depot based on a hydrogen peroxide-responsive hydrogel comprising poly(ethylene glycol) dimethacrylate and ferrous chloride is concisely developed. Upon intratumoral fixation, the as-prepared puerarin depot (abbreviated as puerarin@PEGel) can activate nitrogen oxide production inside endothelial cells and thus dilate tumor blood vessels to relieve tumor hypoxia and reverse tumor immunosuppression. Such treatment can thus promote tumor infiltration, survival, and effector functions of customized epidermal growth factor receptor (HER1)-targeted HER1-CAR-NK cells after intravenous administration. Consequently, such puerarin@PEGel-assisted HER1-CAR-NK cell treatment exhibits superior tumor suppression efficacy toward both HER1-overexpressing MDA-MB-468 and NCI-H23 human tumor xenografts in mice without inducing obvious side effects. This study highlights a potent strategy to activate CAR-NK cells for augmented treatment of targeted solid tumors through reprogramming tumor immunosuppression.

Identification of the Metabolites of Both Formononetin in Rat Hepatic S9 and Ononin in Rat Urine Samples and Preliminary Network Pharmacology Evaluation of Their Main Metabolites.

Astragalus membranaceus is a traditional Chinese medicine derived from the roots of Astragalus membranaceus (Fisch.) Bge., which has the same medicinal and edible uses in China. It is also widely used in daily food, and its pharmacological effects mainly include antioxidant effects, vascular softening effects, etc. Currently, it is increasingly widely used in the prevention of hypertension, cerebral ischemia, and stroke in China. Formononetin and its glucopyranoside (ononin) are both important components of Astragalus membranaceuss and may play important roles in the treatment of cardiovascular diseases (CVDs). This study conducted metabolic studies using formononectin and its glucopyranoside (ononin), including a combination of the in vitro metabolism of Formonetin using rat liver S9 and the in vivo metabolism of ononin administered orally to rats. Five metabolites (Sm2, 7, 9, 10, and 12) were obtained from the solution incubated with formononetin and rat hepatic S9 fraction using chromatographic methods. The structures of the five metabolites were elucidated as (Sm2)6,7,4'-trihydroxy-isoflavonoid; (Sm7)7,4'-dihydroxy-isoflavonoid; (Sm9)7,8,4'-trihydroxy-isoflavonoid; (Sm10)7,8,-dihydroxy-4'-methoxy-isoflavonoid; and (Sm12)6,7-dihydroxy-4'-methoxy- isoflavonoid on the basis of UV, NMR, and MS data. Totally, 14 metabolites were identified via HPLC-DAD-ESI-IT-TOF-MSn analysis, from which the formononetin was incubated with rat hepatic S9 fraction, and the main metabolic pathways were hydroxylation, demethylation, and glycosylation. Then, 21 metabolites were identified via HPLC-DAD-ESI-IT-TOF-MSn analysis from the urine samples from SD rats to which ononin was orally administered, and the main metabolic pathways were glucuronidation, hydroxylation, demethylation, and sulfonation. The main difference between the in vitro metabolism of formononetin and the in vivo metabolism of ononin is that ononin undergoes deglycemic transformation into Formonetin in the rat intestine, while Formonetin is absorbed into the bloodstream for metabolism, and the metabolic products also produce combined metabolites during in vivo metabolism. The six metabolites obtained from the aforementioned separation indicate the primary forms of formononetin metabolism, and due to their higher contents of similar isoflavone metabolites, they are considered the main active compounds that are responsible for pharmacological effects. To investigate the metabolites of the active ingredients of formononetin in the rat liver S9 system, network pharmacology was used to evaluate the cardiovascular disease (CVD) activities of the six primary metabolites that were structurally identified. Additionally, the macromolecular docking results of six main components and two core targets (HSP90AA1 and SRC) related to CVD showed that formononetin and its main metabolites, Sm10 and Sm12, may have roles in CVD treatment due to their strong binding activities with the HSP90AA1 receptor, while the Sm7 metabolite may have a role in CVD treatment due to its strong binding activity with the SRC receptor.

Synthesis of proposed structure of ledebourin A.

Naturally occurring homoisoflavonoids have attracted significant attention in the field of medicinal chemistry due to their potential health benefits and diverse range of biological properties. Recently, C-prenylated homoisoflavonoids, namely ledebourin A, B, and C, were isolated from the bulbs of Ledebouria floribunda and have exhibited potent antioxidant activity. In this study, we successfully synthesized ledebourin A and its regioisomer, compounds 1 and 9. By comparing the NMR spectra of the synthesized compounds with those of reported ledebourin A, we observed discrepancies. Nonetheless, our synthesis and subsequent findings offer valuable insights into the structural revision and biological activities of these unique prenylated homoisoflavonoids. Both synthesized compounds 1 and 9 exhibited no toxicity towards Hep-G2 cells and displayed the ability to recover glyceraldehyde-induced cell death, suggesting their potential as protective agents against liver damage.

Phytoestrogens as Biomarkers of Plant Raw Materials Used for Fish Feed Production.

The intensive use of plant materials as a sustainable alternative for fish feed production, combined with their phytochemical content, which affects the growth and production characteristics of farmed fishes, necessitates their monitoring for the presence of raw materials of plant origin. This study reported herein concerns the development, validation and application of a workflow using high-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) for the quantification of 67 natural phytoestrogens in plant-derived raw materials that were used to produce fish feeds. Specifically, we verified the presence of 8 phytoestrogens in rapeseed meal samples, 20 in soybean meal samples, 12 in sunflower meal samples and only 1 in wheat meal samples in quantities enabling their efficient incorporation into clusters. Among the various constituents, the soybean phytoestrogens daidzein, genistein, daidzin, glycitin, apigenin, calycosin and coumestrol, as well as the sunflower neochlorogenic, caffeic and chlorogenic phenolic acids, displayed the highest correlations with their origin descriptions. A hierarchical cluster analysis of the studied samples, based on their phytoestrogen contents, led to the efficient clustering of raw materials. The accuracy and efficiency of this clustering were tested through the incorporation of additional samples of soybean meal, wheat meal and maize meal, which verified the utilization of the phytoestrogen content as a valuable biomarker for the discrimination of raw materials used for fish feed production.

Spatially resolved metabolomics combined with bioactivity analyses to evaluate the pharmacological properties of two Radix Puerariae species.

ETHNOPHARMACOLOGICAL RELEVANCE: P. lobata and P. thomsonii are medicinal plants with similar pharmacological functions but different therapeutic effects. A novel method is presented herein to investigate metabolites in terms of their distribution and qualification, quantification is necessary to elucidate the different therapeutic effects of the two Puerariae species. AIM OF THE STUDY: The aim of the present study was to perform spatially resolved metabolomics combined with bioactivity analyses to systematically compare the metabolite differences in P. lobata and P. thomsonii by distribution, qualification, quantification, and biological activity to evaluate their pharmacological properties. MATERIALS AND METHODS: Air flow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI) was performed to characterize the differences in the metabolite distributions of P. lobata and P. thomsonii. Further qualitative and quantitative analyses of the differential metabolites were performed using liquid chromatography-mass spectrometry (LC-MS). Biological activities correlated with the differences in the metabolites were validated by MTT assays. RESULTS: Some metabolites showed complementary distributions of the phloem and xylem in the two species, saccharide, vitamin, and inosine levels were higher in the phloem of P. thomsonii but higher in the xylem of P. lobata. The 3'-hydroxyl puerarin level was higher in the xylem of P. thomsonii but higher in the phloem of P. lobata. Qualitative and quantitative analyses of the metabolites revealed a total of 52 key differential metabolites. MTT assays showed that daidzein, daidzin, puerarin, ononin, genistin, formononetin, 3'-hydroxy puerarin, 3'-methoxy puerarin, mirificin, and 3'-methoxy daidzin exerted protective effects on H9c2 cells against hypoxia/reoxygenation injury. P. lobata extracts exhibited a significantly better protective efficacy than P. thomsonii extracts. CONCLUSIONS: In this study, AFADESI-MSI combined with LC-MS and biological activities comprehensively elucidated metabolite differences in the distribution, qualification, quantification, and pharmacological properties of P. lobata and P. thomsonii. The results of this study could provide a novel strategy for species identification and quality assessment of similar Chinese herbal medicines.

Scavenging of Superoxide in Aprotic Solvents of Four Isoflavones That Mimic Superoxide Dismutase.

Isoflavones are plant-derived natural products commonly found in legumes that show a large spectrum of biomedical activities. A common antidiabetic remedy in traditional Chinese medicine, Astragalus trimestris L. contains the isoflavone formononetin (FMNT). Literature reports show that FMNT can increase insulin sensitivity and potentially target the peroxisome proliferator-activated receptor gamma, PPARγ, as a partial agonist. PPARγ is highly relevant for diabetes control and plays a major role in Type 2 diabetes mellitus development. In this study, we evaluate the biological role of FMNT, and three related isoflavones, genistein, daidzein and biochanin A, using several computational and experimental procedures. Our results reveal the FMNT X-ray crystal structure has strong intermolecular hydrogen bonding and stacking interactions which are useful for antioxidant action. Cyclovoltammetry rotating ring disk electrode (RRDE) measurements show that all four isoflavones behave in a similar manner when scavenging the superoxide radical. DFT calculations conclude that antioxidant activity is based on the familiar superoxide σ-scavenging mode involving hydrogen capture of ring-A H7(hydroxyl) as well as the π-π (polyphenol-superoxide) scavenging activity. These results suggest the possibility of their mimicking superoxide dismutase (SOD) action and help explain the ability of natural polyphenols to assist in lowering superoxide concentrations. The SOD metalloenzymes all dismutate O2•- to H2O2 plus O2 through metal ion redox chemistry whereas these polyphenolic compounds do so through suitable hydrogen bonding and stacking intermolecular interactions. Additionally, docking calculations suggest FMNT can be a partial agonist of the PPARγ domain. Overall, our work confirms the efficacy in combining multidisciplinary approaches to provide insight into the mechanism of action of small molecule polyphenol antioxidants. Our findings promote the further exploration of other natural products, including those known to be effective in traditional Chinese medicine for potential drug design in diabetes research.

Crystal structure of tubulin-barbigerone complex enables rational design of potent anticancer agents with isoflavone skeleton.

BACKGROUND: Isoflavones possess many biological activities, including anti-inflammatory and anticancer effects. Microtubules (composed of αß-tubulin heterodimers) are described as one possible cellular target of some of these isoflavones. However, the binding of tubulin to isoflavones has not been extensively studied, and until now, no crystal structure of the tubulin-isoflavone complex has been solved, and details of the isoflavone-tubulin interaction remain elusive. PURPOSE: Barbigerone is an isoflavone mainly found in the genus Milletti, such as the edible leguminous plant Millettia ferruginea, with anticancer activity. This study aims to confirm the cellular target of barbigerone and to study its anticancer mechanism. METHOD: Surface plasmon resonance assays and X-ray crystallography were used to study the interaction of barbigerone with tubulin protein. Immunofluorescence, in-cell and in vitro tubulin polymerization assays were employed to investigate the mechanism. MTT assays, cell clonal formation assays, wound healing assays, tube formation assays and H460 xenograft models were conducted to evaluate the in vitro and in vivo anticancer activities of barbigerone and one of its derivatives, 0412. RESULTS: Here, we found that barbigerone binds to tubulin to inhibit tubulin polymerization. Moreover, we solved the X-ray crystal structure of the tubulin-barbigerone complex at 2.33 Å resolution, which unambiguously determined the orientation and position of barbigerone in the colchicine-binding site. Illuminated by the X-ray data, we synthetized and obtained a more active isoflavone, 0412. Both barbigerone and 0412 inhibit cancer cell proliferation, tubulin polymerization, migration of HeLa cells and capillary-like tube formation of HUVECs, induce G2/M phase cell cycle arrest and apoptosis, and exhibit anticancer activity in an H460 xenograft model. CONCLUSION: In all, through biochemical and X-ray crystal structure results, we identified tubulin as the cellular target of one isoflavone, barbigerone, and proved that the tubulin-barbigerone complex plays a guiding role in obtaining a more active compound, 0412. These studies provide a crucial research basis for the development of isoflavones as anticancer candidate compounds.

Two new isoflavones from the barks of Dalbergia hancei Benth.

Two new isoflavone compounds, Dalhancei A (1) and Dalhancei B (2), along with a known compound epicatechin (3) were isolated from 80% methanol extract of the barks of Dalbergia hancei Benth. The structures of compounds 1-3 were elucidated by comparison with the literature and physical data analysis, including optical rotation, MS, 1D and 2D NMR spectra. Compounds 1 and 2 showed weak inhibitory activity against tyrosinase at 16.22 mmol/L, with inhibition rates of 42.23 ± 0.18% and 45.68 ± 0.17%, respectively; compound 1 exhibited weak inhibitory activity against α-glucosidase with the inhibition rate of 43.72 ± 0.22% at 5.41 mmol/L, compounds 2 and 3 had better α-glucosidase inhibitory activity than compound 1 with IC50 values of 0.90 ± 0.18 and 0.41 ± 0.17 mmol/L, respectively.

Acidic natural deep eutectic solvents as dual solvents and catalysts for the solubilization and deglycosylation of soybean isoflavone extracts: Genistin as a model compound.

This study investigated the possibility of using green solvent natural deep eutectic solvents (NADESs) as dual solvent-catalysts for the solubilization and deglycosylation of soybean isoflavones. The deglycosylation behavior of genistin as a model compound in NADESs was compared. Acidic NADESs showed moderate solubility for genistin and could hydrolyze it to form genistein. The onset temperature of deglycosylation in the choline chloride/malic acid (Ch-Ma) was 60 °C. The solubilities of genistin in the Ch-Ma system were modeled. The dissolution process was endothermic and mainly enthalpy-driven. The deglycosylation followed first-order kinetics with a half-life (t1/2) of 40 min at 90 °C. The method was validated using soybean isoflavone extracts as a substrate and the ratio of glycoside to aglycone in the extracts could be adjusted by changing the conditions. The methods have great potential in the extraction and preparation of ready-to-use isoflavone extracts from soybean and other legumes.

Chromosome-Level Genome Assembly and Multi-Omics Dataset Provide Insights into Isoflavone and Puerarin Biosynthesis in Pueraria lobata (Wild.) Ohwi.

Pueraria lobata (wild.) Ohwi is a leguminous plant and one of the traditional Chinese herbal medicines. Its puerarin extract is widely used in the pharmaceutical industry. This study reported a chromosome-level genome assembly for P. lobata and its characteristics. The genome size was ~939.2 Mb, with a contig N50 of 29.51 Mbp. Approximately 97.82% of the assembled sequences were represented by 11 pseudochromosomes. We identified that the repetitive sequences accounted for 63.50% of the P. lobata genome. A total of 33,171 coding genes were predicted, of which 97.34% could predict the function. Compared with other species, P. lobata had 757 species-specific gene families, including 1874 genes. The genome evolution analysis revealed that P. lobata was most closely related to Glycine max and underwent two whole-genome duplication (WGD) events. One was in a gamma event shared by the core dicotyledons at around 65 million years ago, and another was in the common ancestor shared by legume species at around 25 million years ago. The collinearity analysis showed that 61.45% of the genes (54,579 gene pairs) in G. max and P. lobata had collinearity. In this study, six unique PlUGT43 homologous genes were retrieved from the genome of P. lobata, and no 2-hydroxyisoflavanone 8-C-glucoside was found in the metabolites. This also revealed that the puerarin synthesis was mainly from the glycation of daidzein. The combined transcriptome and metabolome analysis suggested that two bHLHs, six MYBs and four WRKYs were involved in the expression regulation of puerarin synthesis structural genes. The genetic information obtained in this study provided novel insights into the biological evolution of P. lobata and leguminous species, and it laid the foundation for further exploring the regulatory mechanism of puerarin synthesis.

Isolation of Mirificin and Other Bioactive Isoflavone Glycosides from the Kudzu Root Lyophilisate Using Centrifugal Partition and Flash Chromatographic Techniques.

Pueraria lobata (Willd.) Ohwi is a legume taxon native to Southeast Asia and widely used in traditional medicine systems of that region. The therapeutic applications of the underground parts of this species (known as kudzu root) are related to its high content of isoflavones, mainly the characteristic C-glycoside derivatives. Within this group, the most explored compound both phytochemically and pharmacologically is puerarin. However, current scientific findings document important anti-biodegenerative effects for some of the minor isoflavones from kudzu roots. Therefore, the main objective of the study was to develop an original preparative method that allowed the efficient isolation of closely related hydrophilic daidzein C-glycosides, including mirificin, from vacuum-dried aqueous-ethanolic extracts of kudzu roots. For this purpose, the combined centrifugal partition (CPC) and flash chromatographic (FC) techniques were elaborated and used. The optimized biphasic solvent system in CPC, with ethyl acetate, ethanol, water, and 0.5% (V/V) acetic acid as a mobile phase modifier, enabled the purification and separation of the polar fraction containing bioactive isoflavones and ultimately the isolation of mirificin, 3'-hydroxy- and 3'-methoxypuerarin, puerarin, and daidzin using FC. The identity of isoflavones was confirmed using spectroscopic (UV absorption and nuclear magnetic resonance) and mass spectrometric methods. The determined purity of isolated mirificin was 63%.

[A new isoflavone from Dalbergia odorifera and inhibitory activity of its tyrosinase].

Twelve flavonoids were isolated and purified from the ethyl acetate fraction of 95% ethanol extract of Dalbergia odorifera by heat reflux extraction, solvent extraction, recrystallization, normal phase silica gel, Sephadex LH-20, MCI gel and HPLC methods. The structures were identified with multiple spectroscopic methods, including 1 D-NMR, 2 D-NMR and MS. The compounds were identified as 6,7,8-trimethoxy-5,4'-dihydroxy isoflavone(1), medicarpin(2), 7,2'-dihydroxy-4'-methoxy-isoflavanol(3), biochanin A(4), prunetin(5), genistein(6), pratensein(7), 3-(4-hydroxyphenyl)-6-isopentenyl-7-methoxy-4H-chromen-4-one(8), tectorigenin(9), irisolidone(10), vestitol(11), and formononetin(12). Compound 1 was a new isoflavone, and compound 8 was isolated from D. odorifera for the first time. The results showed that compounds 1-3 had inhibitory effects on tyrosinase, with inhibition rates of 35.58%, 38.63% and 51.34% at the concentration of 1.0 mmol·L~(-1), respectively.

Derrisrobustones A-D, isoflavones from the twig extract of Derris robusta (DC.) Benth. and their α-glucosidase inhibitory activity.

Three previously undescribed isoflavones, derrisrobustones A-C, and a previously undescribed natural isoflavone, derrisrobustone D, along with eight known isoflavones, were isolated from the twig extract of Derris robusta (DC.) Benth. All structures were identified by extensive spectroscopic analysis. Derrisrobustones A-C were obtained as scalemic mixtures and were resolved by chiral HPLC. The (1″R, 2″R) absolute configuration of (+)-derrisrobustone B was established by single-crystal X-ray crystallography using Cu Kα radiation. The absolute configurations of derrisrobustones A and C were determined by analysis of experimental and calculated ECD data. All compounds were evaluated for their α-glucosidase inhibitory activity. Of these, derrubone displayed the best α-glucosidase inhibitory activity with an IC50 value of 64.2 µM.

Antimicrobial Prenylated Isoflavones from the Leaves of the Amazonian Medicinal Plant Vatairea guianensis Aubl.

Vatairea guianenis Aubl. (Fabaceae) is an Amazonian medicinal plant species traditionally used for treating skin diseases. In an initial screening, a V. guianensis leaf extract and its subextracts showed antibacterial and antifungal activities. The EtOAc subextract was selected for chemical workup and afforded five known (1-4 and 8) and six undescribed isoflavones, vatairenones C-H (5-7 and 9-11). All isoflavones are prenylated in position C-8, displaying either chain-prenylated (1-7) or ring-closed forms (8-11). The most bioactive compound (3) exhibited in vitro activity against clinically relevant bacteria and fungi with IC50 values ranging from 6.8 to 26.9 µM. Due to its broad antimicrobial activity and low general toxicity, compound 3 is a potential lead compound for structural modifications. The results of the present study support the ethnomedicinal use of V. guianensis in the treatment of dermatological disorders. 1H NMR spectra of some of the isolated compounds showed intricate signal patterns, which might explain repeated errors in assigning the correct structure of the isoflavonoid B-ring in the literature and which we resolved by higher order spectra simulations.

Oxidation of methylophiopogonanone A on the surface of TLC plate.

Methylophiopogonanone A (MOPNA, 1) is a characteristic homoisoflavonoid, having two methyl groups on the A ring, isolated from Ophiopogon Root (enlarged part of the root of Ophiopogon japonicus Ker-Gawler, Liliaceae). Although MOPNA is chemically stable in various organic solvents, such as acetone, chloroform, methanol and dimethyl sulfoxide, it gave a spot of higher polarity in addition to the spot of MOPNA on the surface of TLC plate. The spot was isolated and the structure of the major compound was revealed to be a compound with an oxygen atom at C-6 of the A ring. This oxidation on the surface of TLC plate was observed for synthetic intermediates of MOPNA having two methyl groups on a phloroacetophenone-type ring, suggesting that the methyl groups enhanced susceptibility to air oxidation. The structure of the major oxidation product was similar to those of humulone and sufflomin A. Humulone has two prenyl units and sufflomin A has two C-glucosyl moieties instead of the two methyl groups, respectively. As dialkylated phloroacetophenone derivatives seem susceptible to oxidation, air oxidation might be involved in the formation of these compounds.

A new isoflavone from the fruits of Ficus auriculata and its antibacterial activity.

A new isoflavone (Z)-5,7,4'-trihydroxy-3'-[3-hydroxy-3-methyl-1-butenyl] isoflavone (1) together with seven known isoflavones (2-8) were isolated from the fruits of the Ficus auriculata. Their structures were established on the basis of 1 D, 2 D-NMR spectroscopic data and HR-ESI-MS analysis. All compounds were evaluated for their antibacterial activities against five pathogenic bacteria in vitro. Compounds 3 and 4 exhibited significant antibacterial activities against five pathogenic bacteria with the MIC values ranging from 1.25 to 20 µg/mL.[Formula: see text].

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization.

ETHNOPHARMACOLOGICAL RELEVANCE: Glabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. AIM OF THE STUDY: The study identified the toxic impurities of glabridin and their toxicological mechanism. MATERIALS AND METHODS: In total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. RESULTS: Although glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 µM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. CONCLUSION: Glabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.