Effects of Isoflavonoid and Vitamin D Synergism on Bone Mineral Density-A Systematic and Critical Review.

Phytoestrogens are non-steroidal plant compounds, which bind to α and ß estrogen receptors, thereby causing specific effects. The best-known group of phytoestrogens are flavonoids, including isoflavonoids-genistein and daidzein. They play a role in the metabolism of bone tissue, improving its density and preventing bone loss, which contributes to reducing the risk of fractures. Vitamin D is found in the form of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) and is traditionally recognized as a regulator of bone metabolism. The aim of this review was to evaluate the synergistic effect of isoflavonoids and vitamin D on bone mineral density (BMD). The MEDLINE (PubMed), Scopus and Cochrane databases were searched independently by two authors. The search strategy included controlled vocabulary and keywords. Reference publications did not provide consistent data regarding the synergistic effect of isoflavonoids on BMD. Some studies demonstrated a positive synergistic effect of these compounds, whereas in others, the authors did not observe any significant differences. Therefore, further research on the synergism of isoflavonoids and vitamin D may contribute to a significant progress in the prevention and treatment of osteoporosis.

Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.

Radical Scavenging Capability and Mechanism of Three Isoflavonoids Extracted from Radix Astragali: A Theoretical Study.

As a valuable traditional Chinese herbal medicine, Radix Astragali has attracted much attention due to its extensive pharmacological activities. In this study, density functional theory (DFT) was used thermodynamically and kinetically in detail to predict the antioxidant activity and reaction mechanisms involved in the free radical scavenging reactions of three representative isoflavonoids (formononetin, calycosin, and calycosin-7-glucoside) extracted from Radix Astragali. Three main mechanisms, including hydrogen atom transfer (HAT), proton transfer after electron transfer (SET-PT), and sequential proton loss electron transfer (SPLET) were examined by calculating the thermodynamic parameters. It was found that HAT is the predominant mechanism in the gas phase, while SPLET is supported in the solvent environment. The isoflavonoids' order of antioxidant activity was estimated as: calycosin > calycosin-7-glucoside > formononetin. For the calycosin compound, the result revealed the feasibility of double HAT mechanisms, which involve the formation of stable benzodioxazole with significantly reduced energy in the second H+/e- reaction. In addition, the potential energy profiles and kinetic calculations show that the reaction of •OH into the 3'-OH site of calycosin has a lower energy barrier (7.2 kcal/mol) and higher rate constant (4.55 × 109 M-1 s-1) compared with other reactions in the gas phase.

Biochemometry identifies suppressors of pro-inflammatory gene expression in Pterocarpus santalinus heartwood.

The heartwood extract of the Ayurvedic medicinal plant Pterocarpus santalinus L. f. has previously been shown to significantly suppress the expression of CX3CL1 and other pro-inflammatory molecules in IL-1-stimulated human endothelial cells. Here, we identify the pigment-depleted extract PSD as the most promising yet still complex source of metabolites acting as an inhibitor of CX3CL1 gene expression. For the target-oriented identification of the constituents contributing to the observed in vitro anti-inflammatory effect of PSD, the biochemometric approach ELINA (Eliciting Nature's Activities) was applied. ELINA relies on the deconvolution of complex mixtures by generating microfractions with quantitative variances of constituents over several consecutive fractions. Therefore, PSD was separated into 35 microfractions by means of flash chromatography. Their 1H NMR data and bioactivity data were correlated by heterocovariance analysis. Complemented by LC-MS-ELSD data, ELINA differentiated between constituents with positive and detrimental effects towards activity and allowed for the prioritization of compounds to be isolated in the early steps of phytochemical investigation. A hyphenated high-performance counter-current chromatographic device (HPCCC+) was employed for efficient and targeted isolation of bioactive constituents. A total of 15 metabolites were isolated, including four previously unreported constituents and nine that have never been described before from red sandalwood. Nine isolates were probed for their inhibitory effects on CX3CL1 gene expression, of which four isoflavonoids, namely pterosonin A (1), santal (6), 7,3'-dimethylorobol (12) and the previously unreported compound pterosantalin A (2), were identified as pronounced inhibitors of CX3CL1 gene expression in vitro.

Millettia isoflavonoids: a comprehensive review of structural diversity, extraction, isolation, and pharmacological properties.

There are approximately 260 known species in the genus Millettia, many of which are used in traditional medicine to treat human and other animal ailments in various parts of the world. Being in the Leguminosae (Fabaceae) family, Millettia species are rich sources of isoflavonoids. In the past three decades alone, several isoflavonoids originating from Millettia have been isolated, and their pharmacological activities have been evaluated against major diseases, such as cancer, inflammation, and diabetes. Despite such extensive research, no recent and comprehensive review of the phytochemistry and pharmacology of Millettia isoflavonoids is available. Furthermore, the structural diversity of isoflavonoids in Millettia species has rarely been reported. In this review, we comprehensively summarized the structural diversity of Millettia isoflavonoids, the methods used for their extraction and isolation protocols, and their pharmacological properties. According to the literature, 154 structurally diverse isoflavonoids were isolated and reported from the various tissues of nine well-known Millettia species. Prenylated isoflavonoids and rotenoids were the most dominant subclasses of isoflavonoids reported. Other subclasses of reported isoflavonoids include isoflavans, aglycone isoflavones, glycosylated isoflavones, geranylated isoflavonoids, phenylcoumarins, pterocarpans and coumaronochromenes. Although some isolated molecules showed promising pharmacological properties, such as anticancer, anti-inflammatory, estrogenic, and antibacterial activities, others remained untested. In general, this review highlights the potential of Millettia isoflavonoids and could improve their utilization in drug discovery and medicinal use processes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11101-022-09845-w.

Genus Lupinus (Fabaceae): a review of ethnobotanical, phytochemical and biological studies.

OBJECTIVES: Lupinus is a large and diverse genus comprising approximately 200 species, belonging to the family Fabaceae. Lupinus plants have been used for heart stimulants, nerves, urinary tract infections, skin disorders, and psoriasis in folk medicine. This review aims to recap the traditional medicinal uses, nutritional value, phytochemical profile, and biological activities of Lupinus species. KEY FINDINGS: From the literature survey, Lupinus is considered as a factory of various phytochemicals like flavonoids, iso-flavonoids, alkaloids, triterpenoids. The presence of proteins, essential fatty acids, and amino acids, as well as alkaloids, minerals, and dietary fibers, indicated that the plants in this genus had a high nutritional value. The Lupinus extracts displayed promising antidiabetic, anticancer, antimicrobial, antidiabetic, antihypertensive, antioxidant, anti-inflammatory, and antimicrobial activities. CONCLUSIONS: The current review provides updated information that could drive the researchers for further studies. The in vitro and in vivo experiments have demonstrated various pharmacological properties. Some pharmacokinetic and toxicological investigations are warranted to ensure its safety and validity for human use.

RRLC-QTOF/MS-Based Metabolomics Reveal the Mechanism of Chemical Variations and Transformations of Astragali Radix as a Result of the Roasting Process.

Astragali Radix (AR), which is extensively used as a healthy food supplement and medicinal herb, contains two forms of products corresponding to raw Astragalus Radix (RAR) and processed Astragali Radix (PAR), which was obtained by roasting. In this study, a non-targeted rapid resolution liquid chromatography coupled with quadruple time-of-flight mass spectrometry (RRLC-Q/TOF-MS) based metabolomics approach was developed to investigate the chemical changes of AR due to roasting. A total of 63 compounds were identified or tentatively identified. Among them, 23 isoflavonoids (composed of 12 isoflavones, eight pterocarpans, and three isoflavans) and six cycloastragenols were characterized as differential metabolites. Heatmap visualization and high-performance liquid chromatography coupled with photodiode array and evaporative light scattering detector (HPLC-PDA-ELSD) quantitative analysis revealed that malonyl isoflavonoids or cycloastragenols were at higher levels in RAR. These might be converted to corresponding acetyl isoflavonoids and cycloastragenols and related isoflavonoid glycosides during roasting. To prove this prediction, chemical conversion experiments on malonyl isoflavonoids and cycloastragenols were performed to confirm and clarify the chemical transformation mechanism.

UPLC-ESI-MS/MS profiling of the underground parts of common Iris species in relation to their anti-virulence activities against Staphylococcusaureus.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of plant extracts and their phytochemicals as candidates for targeting the microbial resistance inhibition is increasingly focused in last decades. In Mongolian traditional medicine, Irises were long used for the treatment of bacterial infections. Irises have been used since the Ancient Egyptians. AIM OF THE STUDY: Chemical composition and virulence inhibition potential of both polar (PF) and non-polar fractions (NPF) of three common Iris species (I. confusa, I. pseudacorus and I. germanica) were explored. MATERIAL AND METHODS: Secondary metabolites profiling was characterized by the UPLC-HRMS/MS technique. Multi-variate data analysis was performed using Metaboanalyst 3.0. Anti-virulence inhibitory activity was evaluated via anti-haemolytic assay and Quantitative biofilm inhibition assay. RESULTS: I. pseudacorus PF exhibited the most potent effect against S. aureus haemolytic activity. All the tested fractions from all species, except I. pseudacorus NPF, have no significant inhibition on the biofilm formation of methicillin resistant and sensitive (MRSA and MSSA) S. aureus. I. pseudacorus NPF showed potent biofilm inhibitory potential of 71.4 and 85.8% against biofilm formation of MRSA and MSSA, respectively. Metabolite profiling of the investigated species revealed ninety and forty-five metabolites detected in the PFs and NPFs, respectively. Nigricin-type, tectorigenin-type isoflavonids and xanthones allowed the discrimination of I. pseudacorus PF from the other species, highlighting the importance of those metabolites in exerting its promising activity. On the other hand, triterpene acids, iridals, triacylglycerols and ceramides represented the metabolites detected in highest abundance in I. pseudacorus NPF. CONCLUSIONS: This is the sole map represents the secondary metabolites profiling of the PFs and NPFs of common Iris species correlating them with the potent explored Staphylococcus aureus anti-virulence activity.

Natural isoflavonoids in invasive cancer therapy: From bench to bedside.

Cancer is a public health problem worldwide, and one of the crucial steps within tumor progression is the invasion and metastasis of cancer cells, which are directly related to cancer-associated deaths in patients. Recognizing the molecular markers involved in invasion and metastasis is essential to find targeted therapies in cancer. Interestingly, about 50% of the discovered drugs used in chemotherapy have been obtained from natural sources such as plants, including isoflavonoids. Until now, most drugs are used in chemotherapy targeting proliferation and apoptosis-related molecules. Here, we review recent studies about the effect of isoflavonoids on molecular targets and signaling pathways related to invasion and metastasis in cancer cell cultures, in vivo assays, and clinical trials. This review also reports that glycitein, daidzein, and genistein are the isoflavonoids most studied in preclinical and clinical trials and displayed the most anticancer activity targeting invasion-related proteins such as MMP-2 and MMP-9 and also EMT-associated proteins. Therefore, the diversity of isoflavonoids is promising molecules to be used as chemotherapeutic in invasive cancer. In the future, more clinical trials are needed to validate the effectiveness of the various natural isoflavonoids in the treatment of invasive cancer.

Medicinal Potential of Isoflavonoids: Polyphenols That May Cure Diabetes.

In recent years, there is emerging evidence that isoflavonoids, either dietary or obtained from traditional medicinal plants, could play an important role as a supplementary drug in the management of type 2 diabetes mellitus (T2DM) due to their reported pronounced biological effects in relation to multiple metabolic factors associated with diabetes. Hence, in this regard, we have comprehensively reviewed the potential biological effects of isoflavonoids, particularly biochanin A, genistein, daidzein, glycitein, and formononetin on metabolic disorders and long-term complications induced by T2DM in order to understand whether they can be future candidates as a safe antidiabetic agent. Based on in-depth in vitro and in vivo studies evaluations, isoflavonoids have been found to activate gene expression through the stimulation of peroxisome proliferator-activated receptors (PPARs) (α, γ), modulate carbohydrate metabolism, regulate hyperglycemia, induce dyslipidemia, lessen insulin resistance, and modify adipocyte differentiation and tissue metabolism. Moreover, these natural compounds have also been found to attenuate oxidative stress through the oxidative signaling process and inflammatory mechanism. Hence, isoflavonoids have been envisioned to be able to prevent and slow down the progression of long-term diabetes complications including cardiovascular disease, nephropathy, neuropathy, and retinopathy. Further thoroughgoing investigations in human clinical studies are strongly recommended to obtain the optimum and specific dose and regimen required for supplementation with isoflavonoids and derivatives in diabetic patients.

Cicer arietinum L. Sprouts' Influence on Mineralization of Saos-2 and Migration of MCF-7 Cells.

In the present study, we investigated the biological activity of four extracts obtained from Cicer arietinum L. sprouts. The fermentation of the sprouts with Lactobacillus casei and their incubation with ß-glucosidase elevated the concentrations of isoflavonoids, especially coumestrol, formononetin and biochanin A. To study the biological activity of C. arietinum, the human osteosarcoma Saos-2 and human breast cancer MCF-7 cell lines were used. The extracts obtained from fermented sprouts exhibited the strongest ability to decrease intracellular oxidative stress in both types of cells. They augmented mineralization and alkaline phosphatase activity in Saos-2 cells, as well as diminished the secretion of interleukin-6 and tumor necrosis factor α. Simultaneously, the extracts, at the same doses, inhibited the migration of MCF-7 cells. On the other hand, elevated concentrations of C. arietinum induced apoptosis in estrogen-dependent MCF-7 cells, while lower doses stimulated cell proliferation. These results are important for carefully considering the use of fermented C. arietinum sprouts as a dietary supplement component for the prevention of osteoporosis.

Transcriptome and metabolome profiling unveiled mechanisms of tea (Camellia sinensis) quality improvement by moderate drought on pre-harvest shoots.

Conventional wisdom holds that tea (Camellia sinensis) quality can be improved by drought. To clarify the underlying mechanism, a conjoint analysis of transcriptome and metabolome profiling was carried out in tea shoots harvested under different soil water contents (SWCs). Drought had little impact on theanine, catechins and caffeine in field conditions. Besides the flavor contributions of amino acid and their derivatives, organic acids, and nucleotides and their derivatives, the obviously increased isoflavonoids and glycosylflavonoids and the sharply decreased lipids are suggested to play key roles, which is mainly due to substantial increases of type III polyketide synthase B (PKSB), flavonol synthase/flavanone 3-hydroxylase (FLS), and UDP-glycosyltransferases (UGTs), as well as the significant repression of anthocyanidin synthase (ANS) and R2R3MYBs, and downregulated lipid metabolisms. Genes of GDSL esterase/lipase (GDSL), abscisic acid (ABA) and jasmonate (JA) signaling were found to play important roles in both flavonoid accumulation and lipid reduction. These findings increased our understanding of how moderate drought improves taste and aroma of tea by interfering in the metabolism of fresh leaves, which provides new insight into balancing compounds in pre-harvest tea shoots.

SAR Study on Estrogen Receptor α/ß Activity of (Iso)flavonoids: Importance of Prenylation, C-Ring (Un)Saturation, and Hydroxyl Substituents.

Many botanicals used for women's health contain estrogenic (iso)flavonoids. The literature suggests that estrogen receptor beta (ERß) activity can counterbalance estrogen receptor alpha (ERα)-mediated proliferation, thus providing a better safety profile. A structure-activity relationship study of (iso)flavonoids was conducted to identify ERß-preferential structures, overall estrogenic activity, and ER subtype estrogenic activity of botanicals containing these (iso)flavonoids. Results showed that flavonoids with prenylation on C8 position increased estrogenic activity. C8-prenylated flavonoids with C2-C3 unsaturation resulted in increased ERß potency and selectivity [e.g., 8-prenylapigenin (8-PA), EC50 (ERß): 0.0035 ± 0.00040 µM], whereas 4'-methoxy or C3 hydroxy groups reduced activity [e.g., icaritin, EC50 (ERß): 1.7 ± 0.70 µM]. However, nonprenylated and C2-C3 unsaturated isoflavonoids showed increased ERß estrogenic activity [e.g., genistein, EC50 (ERß): 0.0022 ± 0.0004 µM]. Licorice (Glycyrrhiza inflata, [EC50 (ERα): 1.1 ± 0.20; (ERß): 0.60 ± 0.20 µg/mL], containing 8-PA, and red clover [EC50 (ERα): 1.8 ± 0.20; (ERß): 0.45 ± 0.10 µg/mL], with genistein, showed ERß-preferential activity as opposed to hops [EC50 (ERα): 0.030 ± 0.010; (ERß): 0.50 ± 0.050 µg/mL] and Epimedium sagittatum [EC50 (ERα): 3.2 ± 0.20; (ERß): 2.5 ± 0.090 µg/mL], containing 8-prenylnaringenin and icaritin, respectively. Botanicals with ERß-preferential flavonoids could plausibly contribute to ERß-protective benefits in menopausal women.

Dalbergia ecastaphyllum (L.) Taub. and Symphonia globulifera L.f.: The Botanical Sources of Isoflavonoids and Benzophenones in Brazilian Red Propolis.

The Brazilian red propolis (BRP) constitutes an important commercial asset for northeast Brazilian beekeepers. The role of Dalbergia ecastaphyllum (L.) Taub. (Fabaceae) as the main botanical source of this propolis has been previously confirmed. However, in addition to isoflavonoids and other phenolics, which are present in the resin of D. ecastaphyllum, samples of BRP are reported to contain substantial amounts of polyprenylated benzophenones, whose botanical source was unknown. Therefore, field surveys, phytochemical and chromatographic analyses were undertaken to confirm the botanical sources of the red propolis produced in apiaries located in Canavieiras, Bahia, Brazil. The results confirmed D. ecastaphyllum as the botanical source of liquiritigenin (1), isoliquiritigenin (2), formononetin (3), vestitol (4), neovestitol (5), medicarpin (6), and 7-O-neovestitol (7), while Symphonia globulifera L.f. (Clusiaceae) is herein reported for the first time as the botanical source of polyprenylated benzophenones, mainly guttiferone E (8) and oblongifolin B (9), as well as the triterpenoids ß-amyrin (10) and glutinol (11). The chemotaxonomic and economic significance of the occurrence of polyprenylated benzophenones in red propolis is discussed.

Modulation of LPS-induced inflammation in RAW264.7 murine cells by novel isoflavonoids from Millettia pulchra.

Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1ß while pulvones C showed better suppression effect on IL-1ß production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-κB pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-κB(p65) and interrupted IκB phosphorylation. The ADP-Glo™ kinase assay showed pulvones A and C could directedly inhibit the IKKß kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-κB activation by inhibiting IKKß kinase.

Trifolium Repens Blocks Proliferation in Chronic Myelogenous Leukemia via the BCR-ABL/STAT5 Pathway.

Some species of clover are reported to have beneficial effects in human diseases. However, little is known about the activity of the forage plant Trifolium repens, or white clover, which has been recently found to exert a hepatoprotective action. Scientific interest is increasingly focused on identifying new drugs, especially natural products and their derivatives, to treat human diseases including cancer. We analyzed the anticancer effects of T. repens in several cancer cell lines. The phytochemical components of T. repens were first extracted in a methanol solution and then separated into four fractions by ultra-high-performance liquid chromatography. The effects of the total extract and each fraction on cancer cell proliferation were analyzed by MTT assay and Western blotting. T. repens and, more robustly, its isoflavonoid-rich fraction showed high cytotoxic effects in chronic myelogenous leukemia (CML) K562 cells, with IC50 values of 1.67 and 0.092 mg/mL, respectively. The block of cell growth was associated with a total inhibition of BCR-ABL/STAT5 and activation of the p38 signaling pathways. In contrast, these strongly cytotoxic effects did not occur in normal cells. Our findings suggest that the development of novel compounds derived from phytochemical molecules contained in Trifolium might lead to the identification of new therapeutic agents active against CML.

[Determination of 9 isoflavonoids in Puerariae Lobatae Radix with quantitative analysis of multi-components by single marker].

A method for determination of 9 isoflavones in Puerariae Lobatae Radix was established and the accuracy and feasibility of the method were verified. The relative correction factors of eight isoflavonoids,3'-hydroxy puerarin,puerarinapioside,3'-methoxy puerarin,puerarin 6″-O-xyloside,daidzin,genistin,formononetin and daidzein were determined by HPLC method with puerarin as the internal standard. The contents of 9 isoflavonoids in 11 batches of samples were determined by external standard method and QAMS.The accuracy and feasibility of the methods were evaluated by comparison of the quantitative results between external standard method and QAMS. The reproducibility of the relative correction factors was good under different experimental conditions,and there was no significant difference between the external standard method of the 9 compounds and the content of QAMS method. The results showed that using puerarin as an internal standard to simultaneously determine the 8 isoflavonoids mentioned above is accurate and feasible. Thus,it can be used as quality control of Puerariae Lobatae Radix.

[Simultaneous determination of 8 bioactive isoflavonoids in Astragali Radix by UHPLC equipped with core-shell column].

This research aims to develop an UHPLC method, based on core-shell column(i.e. superficially porous particles), for simultaneous determination of eight isoflavonoids including formononetin,(6αR,11αR)-3-hydroxy-9,10-dimethoxypterocarpan, calycosin-7-O-ß-D-glucopyranoside,(3R)-7,2-dihydroxy-3,4-dimethoxyisoflavone, calycosin, ononin,(6αR,11αR)-9,10-dimethoxypterocarpan-3-O-ß-D-glucopyranoside, and(3R)-7,2-dihydroxy-3,4-dimethoxyisoflavan-7-O-ß-D-glucopyranoside in Astragali Radix. The analysis was performed on an Agilent Poroshell EC-C_(18 )column(2.1 mm×100 mm, 2.7 µm) with 0.2% formic acid solution(A)-acetonitrile(B) as mobile phase for gradient elution. The flow rate was 0.5 mL·min~(-1), with column temperature of 40 ℃ and the wavelengths were set at 260 and 280 nm. According to the results, all calibration curves showed good linearity(R~2>0.999 8) within the tested concentration ranges. Both the intra-and inter-day precisions for 8 isoflavonoids were less than 0.80%, with the mean recovery at the range of 94.71%-104.6%. Thus, the newly developed UHPLC method using core-shell column owned the advantages in terms of rapid analysis, low column pressure and less solvent consumption, thus enabling the usage of conventional HPLC systems. Meanwhile, quantitative evaluation was carried out for 22 batches of commercial Astragali Radix. It has been found that great variations occurred for the content of the individual isoflavonoids among different batches; in contrast, the total content of total 8 isoflavonoids(>0.1%) was stable in most samples, indicating that it was reasonable to involve all isoflavonoids as the chemical markers for the quality control of Astragali Radix.

Identification of Three (Iso)flavonoid Glucosyltransferases From Pueraria lobata.

(Iso)flavonoids are one of the largest groups of natural phenolic products conferring great value to the health of plants and humans. Pueraria lobata, a legume, has long been used in Chinese traditional medicine. (Iso)flavonoids mainly present as glycosyl-conjugates and accumulate in P. lobata roots. However, the molecular mechanism underlying the glycosylation processes in (iso)flavonoid biosynthesis are not fully understood. In the current study, three novel UDP-glycosyltransferases (PlUGT4, PlUGT15, and PlUGT57) were identified in P. lobata from RNA-seq data. Biochemical assays of these three recombinant PlUGTs showed all of them were able to glycosylate isoflavones (genistein and daidzein) at the 7-hydroxyl position in vitro. In comparison with the strict substrate specificity for PlUGT15 and PlUGT57, PlUGT4 displayed utilization of a broad range of sugar acceptors. Particularly, PlUGT15 exhibited a much higher catalytic efficiency toward isoflavones (genistein and daidzein) than any other identified 7-O-UGT from P. lobata. Moreover, the transcriptional expression patterns of these PlUGTs correlated with the accumulation of isoflavone glucosides in MeJA-treated P. lobata, suggesting their possible in vivo roles in the glycosylation process.

Simultaneous determination of 8 bioactive isoflavonoids in Astragali Radix by UHPLC equipped with core-shell column / 中国中药杂志

This research aims to develop an UHPLC method, based on core-shell column(i.e. superficially porous particles), for simultaneous determination of eight isoflavonoids including formononetin,(6αR,11αR)-3-hydroxy-9,10-dimethoxypterocarpan, calycosin-7-O-β-D-glucopyranoside,(3R)-7,2-dihydroxy-3,4-dimethoxyisoflavone, calycosin, ononin,(6αR,11αR)-9,10-dimethoxypterocarpan-3-O-β-D-glucopyranoside, and(3R)-7,2-dihydroxy-3,4-dimethoxyisoflavan-7-O-β-D-glucopyranoside in Astragali Radix. The analysis was performed on an Agilent Poroshell EC-C_(18 )column(2.1 mm×100 mm, 2.7 μm) with 0.2% formic acid solution(A)-acetonitrile(B) as mobile phase for gradient elution. The flow rate was 0.5 mL·min~(-1), with column temperature of 40 ℃ and the wavelengths were set at 260 and 280 nm. According to the results, all calibration curves showed good linearity(R~2>0.999 8) within the tested concentration ranges. Both the intra-and inter-day precisions for 8 isoflavonoids were less than 0.80%, with the mean recovery at the range of 94.71%-104.6%. Thus, the newly developed UHPLC method using core-shell column owned the advantages in terms of rapid analysis, low column pressure and less solvent consumption, thus enabling the usage of conventional HPLC systems. Meanwhile, quantitative evaluation was carried out for 22 batches of commercial Astragali Radix. It has been found that great variations occurred for the content of the individual isoflavonoids among different batches; in contrast, the total content of total 8 isoflavonoids(>0.1%) was stable in most samples, indicating that it was reasonable to involve all isoflavonoids as the chemical markers for the quality control of Astragali Radix.