Bioactive Isoflavonoids Research Articles

Formononetin, a bioactive isoflavonoid constituent from Astragalus membranaceus (Fisch.) Bunge, ameliorates type 1 diabetes mellitus via activation of Keap1/Nrf2 signaling pathway: An integrated study supported by network pharmacology and experimental validation

Ethnopharmacological relevanceType 1 diabetes mellitus (T1DM) results from insulin deficiency due to the destruction of pancreatic β-cells. Previously, our studies showed that inhibition of Keap1/Nrf2 signaling pathway promoted the onset of T1DM, which suggests that finding drugs that can activate the Keap1/Nrf2 signaling may be a promising therapeutic strategy for the T1DM treatment. Astragalus membranaceus (Fisch.) Bunge is a common traditional Chinese medicine that has been frequently applied in Chinese clinics for the treatment of diabetes and other diseases. Formononetin (FMNT), one of the major isoflavonoid constituents isolated from this herbal medicine, possesses diverse pharmacological benefits and T1DM therapeutic potential. However, the exact molecular mechanisms underlying the action of FMNT in ameliorating T1DM have yet to be fully elucidated. Aims of the studyThis study is to investigate the regulation of FMNT on the Keap1/Nrf2 signaling pathway to ameliorate T1DM based on network pharmacology approach combined with experimental validation. Materials and methodsA mouse-derived pancreatic islet β-cell line (MIN6) was used for the in vitro studies. An alloxan (ALX)-induced T1DM model in wild-type and Nrf2 knockout (Nrf2−/−) C57BL/6J mice were established for the in vivo experiments. The protective effects of FMNT against ALX-stimulated MIN6 cell injury were evaluated using MTT, EdU, apoptosis and comet assays. The levels of blood glucose in mice were measured by using a blood monitor and test strips. The protein expression was detected by Western blot analysis. Furthermore, the binding affinity of FMNT to Keap1 was evaluated using cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and solvent-induced protein precipitation (SIP) assay. The interaction pattern between FMNT and Keap1 was assessed by molecular docking and molecular dynamics simulation techniques. ResultsNetwork pharmacology analysis revealed that FMNT exerted its therapeutic effect against T1DM by mainly regulating oxidative stress response-associated signaling molecules and pathways, such as Nrf2 regulating anti-oxidant/detoxification enzymes and Keap1-Nrf2 signaling pathway. The in vivo results showed that FMNT significantly deceased the ALX-induced high blood glucose levels and conversely increased the ALX-induced low insulin contents. In vitro, FMNT markedly protected MIN6 cells from ALX-induced cytotoxicity, proliferation inhibition and DNA damage and reduced the ALX-stimulated cell apoptosis. FMNT also inhibited ALX-induced overproduction of intracellular ROS to alleviate oxidative stress. In addition, FMNT could bind to Keap1 to notably activate the Keap1/Nrf2 signaling to upregulate Nrf2 expression and promote the Nrf2 translocation from the cytoplasm to the nucleus, resulting in enhancing the expression of antioxidant proteins HO-1 and NQO1. Inhibition of Keap1/Nrf2 signaling by ALX was also markedly abolished in the cells and mice exposed to FMNT. Moreover, these effects of FMNT in ameliorating T1DM were not observed in Nrf2−/− mice. ConclusionsThis study demonstrates that FMNT could bind to Keap1 to activate the Keap1/Nrf2 signaling to prevent intracellular ROS overproduction, thereby attenuating ALX-induced MIN6 cell injury and ameliorating ALX-stimulated T1DM. Results from this study might provide evidence and new insight into the therapeutic effect of FMNT and indicate that FMNT is a promising candidate agent for the treatment of T1DM in clinics.

The protein conformational basis of isoflavone biosynthesis

Isoflavonoids play important roles in plant defense and also exhibit a range of mammalian health-promoting activities. Their biosynthesis is initiated by two enzymes with unusual catalytic activities; 2-hydroxyisoflavanone synthase (2-HIS), a membrane-bound cytochrome P450 catalyzing a coupled aryl-ring migration and hydroxylation, and 2-hydroxyisoflavanone dehydratase (2-HID), a member of a large carboxylesterase family that paradoxically catalyzes dehydration of 2-hydroxyisoflavanones to isoflavone. Here we report the crystal structures of 2-HIS from Medicago truncatula and 2-HID from Pueraria lobata. The 2-HIS structure reveals a unique cytochrome P450 conformation and heme and substrate binding mode that facilitate the coupled aryl-ring migration and hydroxylation reactions. The 2-HID structure reveals the active site architecture and putative catalytic residues for the dual dehydratase and carboxylesterase activities. Mutagenesis studies revealed key residues involved in substrate binding and specificity. Understanding the structural basis of isoflavone biosynthesis will facilitate the engineering of new bioactive isoflavonoids.

Investigation of facile spectroscopic approaches for rapid calycosin determination in invitro biological samples and pharmaceuticals; application to the content uniformity of capsules

Calycosin, the major bioactive isoflavonoid inAstragali Radix and an important anti-viral drug with a variety of pharmacological actions, is being determined by five different spectroscopic methods. Two spectrophotometric methods have been investigated including measuring the absorption spectra at λmax = 270 nm and the first derivative spectra at λ = 288 nm for methods I and II, respectively. For the first time; the native fluorescence of calycosin is measured without adding any reagents. The fluorescence intensity was measured at 340 nm after excitation at 282 nm in method III. The fourth method involves the direct measuring of a first derivative spectrofluorimetric emission peak at 292 nm. In method V synchronous fluorescence spectra were recorded in methanol at Δλ = 70 nm. The linear range for the fluorescence-based methods was 0.05–1.0 µg/mL and for the UV-based methods was 0.5–10.0 µg/mL. The methods were validated per International Council of Harmonization (ICHQ2R1) guidelines. The limits of detection were found to be down to 0.11 and 0.12 µg/mL for the spectrophotometric methods, and 15.0, 18.0,16.0 ng/ mL, for the spectrofluorimetric approaches respectively, representing the high sensitivity. Accordingly, this permitted the quantitation of calycosin in spiked human plasma samples with satisfactory percentage recoveries (94.50.–102.50 %). The methods were utilized for calycosin analysis in different matrices including plasma and capsules with high precision and accuracy.

In Vitro Anti-Aging Potential Evaluation of Maclura pomifera (Rafin.) Schneider 80% Methanol Extract with Quantitative HPTLC Analysis.

Maclura pomifera (Rafin.) Schneider is a widespread species all around the world, which is also frequently cultured for ornamental purposes. Previous studies revealed that M. pomifera fruits are rich in prenylated isoflavonoids, exhibit noteworthy biological activities, and have probable benefits, particularly, when applied topically. Considering that phenolic compounds are important sources in the development of anti-aging cosmetic products, this study investigated the anti-aging potential of M. pomifera 80% methanolic extract (MPM) by evaluating antioxidant and extracellular matrix degrading enzymes inhibiting activity. For this study, the inhibitory potential of 80% MPM against different enzymes associated with the aging process was evaluated. Given the unequivocal role of oxidative stress in aging, in vitro antioxidant tests were employed as well. Moreover, osajin was determined as the major bioactive isoflavonoid of the sample by high performance thin layer chromatography analysis. Results of the mechanistically different antioxidant assays exhibited notable antioxidant potential of the extract. Inhibition potential of MPM against hyaluronidase, collagenase, and elastase enzymes, which are directly linked to acceleration of the aging process, was investigated and results revealed that MPM inhibited the aforementioned enzymes explicitly. MPM had notable phenolic and flavonoid content; 113.92 ± 2.26 mg gallic acid equivalent/g and 66.41 ± 0.74 mg QE/g, respectively. When total antioxidant capacity assays were considered, it is possible to suggest that MPM is a promising anti-aging agent. As a result, this study discloses that extracts of fruits of M. pomifera have significant anti-aging potential and may be used for this purpose.

Alpinumisoflavone against cancer pro-angiogenic targets: In silico, In vitro, and In ovo evaluation.

Breast cancer is currently the world's most predominant malignancy. In cancer progression, angiogenesis is a requirement for tumor growth and metastasis.Alpinumisoflavone (AIF), a bioactive isoflavonoid, exhibited good binding affinity with the angiogenesis pathway's druggable target through molecular docking. To confirm AIF's angiogenesis inhibitory activity, cytotoxic potential toward breast cancer cells, and druggability. Antiangiogenicactivity was evaluated in six pro-angiogenic proteinsin vitro, duck chorioallantoic membrane (CAM)in ovo, molecular docking and druggabilityin silico. Findings showed that AIF significantly inhibited (p = < 0.001) the HER2(IC50 = 2.96µM), VEGFR-2(IC50 = 4.80µM), MMP-9(IC50 = 23.00µM), FGFR4(IC50 = 57.65µM), EGFR(IC50 = 92.06µM) and RET(IC50 = > 200µM) activity in vitro.AIF at 25µM-200µM significantly inhibited (p = < 0.001) the total number of branch points (IC50 = 14.25μM) and mean length of tubule complexes (IC50 = 3.52μM) of duck CAM comparable (p = > 0.001) with the positive control 200µM celecoxib on both parameters.AIF inhibited the growth of the estrogen-receptor-positive (ER +) human breast cancer cells (MCF-7) by 44.92 ± 1.79% at 100µM while presenting less toxicity to human dermal fibroblast neonatal (HDFn) normal cells.The positive control 100µM doxorubicin showed 86.66 ± 0.93% and 92.97 ± 1.27% inhibition with MCF-7 (IC50 = 3.62μM) and HDFn, (IC50 = 27.16μM) respectively.In docking, AIF has the greatestin silicobinding affinity on HER2 (-10.9kcal/mol) among the key angiogenic molecules tested. In silico rat oral LD50 calculation indicates that AIF is moderate to slightly toxic at 146.4mg/kg with 1.1g/kg and 20.1mg/kg upper and lower 95% confidence limits. Lastly, it sufficientlycomplies with Lipinski's, Veber's, Egan's, Ghose's, and Muegge's Rule, supporting its oral drug-like property. This study revealed that AIF possesses characteristics of a phytoestrogen compound with significant binding affinity, inhibitory activity against pro-angiogenic proteins, and cytotoxic potential against ER + breast cancer cells.The acceptable and considerable safety and drug-likeness profiles of AIF are worthy of further confirmation in vivo and advanced pre-clinical studies so that AIF can be elevated as a promising molecule for breast cancer therapy.

Elicitation of isoflavonoids in Colombian edible legume plants with jasmonates and structurally related compounds

Common bean (Phaseolus vulgaris L.), soybean (Glycine max L.) and mung bean (Vigna radiata L. Wilczek) seedlings were treated with methyl jasmonate (MeJA); then, dose-response and time-course experiments were carried out. Isoflavonoid composition was evaluated by high performance liquid chromatography. As a result of MeJA induction, all leguminous plants increase the amount of isoflavonoids, at levels that depend on the concentration of the elicitor and the time after induction. However, the application of MeJA in concentrations higher than 2.22 mM showed deleterious effects on seedlings and strong decreases in the concentration of isoflavonoids. In addition, a series of compounds structurally related to MeJA, such as jasmonic acid, cis-jasmone, coronatine, and indanoyl derivatives, were evaluated as elicitors. The results show that coronatine and the indanoyl-amino acids conjugates displayed a significant elicitor effect of isoflavonoids in common bean (cvs. Cargamanto Mocho and Corpoica LAS 106) and soybean (cv. Soyica P-34) seedlings, even higher than that found with the recognized elicitors, benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (acibenzolar S-methyl) and benzo-(1,2,3) thiadiazole-7-carbothioic acid (acibenzolar acid). Leguminous plants can be treated with jasmonates and indanoyl derivatives to increase levels of bioactive isoflavonoids and consequently improve biological and functional properties and resistance against pests.

Calycosin, a Common Dietary Isoflavonoid, Suppresses Melanogenesis through the Downregulation of PKA/CREB and p38 MAPK Signaling Pathways.

Calycosin, a bioactive isoflavonoid isolated from root extracts of Astragalus membranaceus, has been reported to inhibit melanogenesis, the mechanism of which remains undefined. In this study, we interrogated the mechanistic basis by which calycosin inhibits melanin production in two model systems, i.e., B16F10 melanoma cells and zebrafish embryos. Calycosin was effective in protecting B16F10 cells from α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis and tyrosinase activity. This anti-melanogenic effect was accompanied by decreased expression levels of microphthalmia-associated transcription factor (MITF), a key protein controlling melanin synthesis, and its target genes tyrosinase and tyrosinase-related protein-2 (TRP-2) in calycosin-treated cells. Mechanistically, we obtained the first evidence that calycosin-mediated MITF downregulation was attributable to its ability to block signaling pathways mediated by cAMP response element-binding protein (CREB) and p38 MAP kinase. The protein kinase A (PKA) inhibitor H-89 and p38 inhibitor SB203580 validated the premise that calycosin inhibits melanin synthesis and tyrosinase activity by regulating the PKA/CREB and p38 MAPK signaling pathways. Moreover, the in vivo anti-melanogenic efficacy of calycosin was manifested by its ability to suppress body pigmentation and tyrosinase activity in zebrafish embryos. Together, these data suggested the translational potential of calycosin to be developed as skin-lightening cosmeceuticals.

De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories

Isoflavonoids comprise a class of plant natural products with great nutraceutical, pharmaceutical and agricultural significance. Their low abundance in nature and structural complexity however hampers access to these phytochemicals through traditional crop-based manufacturing or chemical synthesis. Microbial bioproduction therefore represents an attractive alternative. Here, we engineer the metabolism of Saccharomyces cerevisiae to become a platform for efficient production of daidzein, a core chemical scaffold for isoflavonoid biosynthesis, and demonstrate its application towards producing bioactive glucosides from glucose, following the screening-reconstruction-application engineering framework. First, we rebuild daidzein biosynthesis in yeast and its production is then improved by 94-fold through screening biosynthetic enzymes, identifying rate-limiting steps, implementing dynamic control, engineering substrate trafficking and fine-tuning competing metabolic processes. The optimized strain produces up to 85.4 mg L−1 of daidzein and introducing plant glycosyltransferases in this strain results in production of bioactive puerarin (72.8 mg L−1) and daidzin (73.2 mg L−1). Our work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added isoflavonoids and the multi-phased framework may be extended to engineer pathways of complex natural products in other microbial hosts.

Identification of Transcription Factor Genes and Functional Characterization of PlMYB1 From Pueraria lobata.

Kudzu, Pueraria lobata, is a traditional Chinese food and medicinal herb that has been commonly used since ancient times. Kudzu roots are rich sources of isoflavonoids, e.g., puerarin, with beneficial effects on human health. To gain global information on the isoflavonoid biosynthetic regulation network in kudzu, de novo transcriptome sequencings were performed using two genotypes of kudzu with and without puerarin accumulation in roots. RNAseq data showed that the genes of the isoflavonoid biosynthetic pathway were significantly represented in the upregulated genes in the kudzu with puerarin. To discover regulatory genes, 105, 112, and 143 genes encoding MYB, bHLH, and WD40 transcription regulators were identified and classified, respectively. Among them, three MYB, four bHLHs, and one WD40 gene were found to be highly identical to their orthologs involved in flavonoid biosynthesis in other plants. Notably, the expression profiles of PlMYB1, PlHLH3-4, and PlWD40-1 genes were closely correlated with isoflavonoid accumulation profiles in different tissues and cell cultures of kudzu. Over-expression of PlMYB1 in Arabidopsis thaliana significantly increased the accumulation of anthocyanins in leaves and proanthocyanidins in seeds, by activating AtDFR, AtANR, and AtANS genes. Our study provided valuable comparative transcriptome information for further identification of regulatory or structural genes involved in the isoflavonoid pathway in P. lobata, as well as for bioengineering of bioactive isoflavonoid compounds.

A metabolomics approach to evaluate post‐fermentation enhancement of daidzein and genistein in a green okara extract

Okara is a major agri-industrial by-product of the tofu and soymilk industries. Employing food-wastes as substrates for the green production of natural functional compounds is a recent trend that addresses the dual concepts of sustainable production and a zero-waste ecosystem. Extracts of unfermented okara and okara fermented with Rhizopus oligosporus were obtained using ethanol as extraction solvent, coupled with ultrasound sonication for enhanced extraction. Fermented extracts yielded significantly better results for total phenolic content (TPC) and total flavonoid content (TFC) than unfermented extracts. A qualitative liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis revealed a shift from glucoside forms to respective aglycone forms of the detected isoflavones, post-fermentation. Since the aglycone forms have been associated with numerous health benefits, a quantitative high-performance liquid chromatography (HPLC) analysis was performed. Fermented okara extracts had daidzein and genistein concentrations of 11.782 ± 0.325 μg mL-1 and 10.125 ± 1.028 μg mL-1 , as opposed to that of 6.7 ± 2.42 μg mL-1 and 4.55 ± 0.316 μg mL-1 in raw okara extracts, respectively. Lastly, the detected isoflavones were mapped to their metabolic pathways, to understand the biochemical reactions triggered during the fermentation process. Fermented okara may be implemented as a sustainable solution for production of natural bioactive isoflavonoids genistein and daidzein. © 2021 Society of Chemical Industry.

Discriminating astragali radix from its adulterants using HPLC coupled with chemometric clustering techniques

A simple high-performance liquid chromatography (HPLC) method coupled with principal component analysis (PCA) and hierarchical cluster analysis (HCA) was developed for distinguishing Astragali Radix from its adulterants. Five species, including A. membranaceus var. mongolicus, A. membranaceus, H. polybotrys, A. chrysopterus and A. ernestii, were analyzed by this method. At the same time, four major bioactive isoflavonoids, namely calycosin-7-O-D-glucopyranoside, ononin, calycosin and formononetin, were simultaneously determined in Astragali Radix. This method was proven to have good precision, repeatability, stability and recovery. It was successfully applied to distinguish Astragali Radix from its adulterants species and can be used for the quality control of Astragali Radix.

The dual roles of calycosin in growth inhibition and metastatic progression during pancreatic cancer development: A “TGF-β paradox”

BackgroundCalycosin is a bioactive isoflavonoid of the medicinal plant Astragalus membranaceus that exhibits a wide range of pharmacological properties. In the present study, we have attempted to explore the anti-tumorigenic potential of calycosin in pancreatic cancer. MethodsMTT assay was used to determine cancer cell viability. Cell cycle analysis and detection of apoptosis were performed using flow cytometry. A wound healing assay was employed to study the migratory activity of cancer cells. Western blotting and RT-PCR were used to explore the mechanism by assessing the target proteins and genes. An orthotopic tumor xenograft mouse model was also used to study the drug effects in vivo. ResultsCalycosin inhibited the growth of pancreatic cancer cells by inducing p21Waf1/Cip1-induced cell cycle arrest and caspase-dependent apoptosis. Alternatively, it also promoted MIA PaCa-2 cell migration by eliciting epithelial-mesenchymal transition (EMT) and matrix metalloproteinase activation. In vivo study has confirmed that calycosin would provoke the pro-invasive and angiogenic drive and subsequent EMT in pancreatic tumors. Further mechanistic study suggests that induction of the Raf/MEK/ERK pathway and facilitated polarization of M2 tumor-associated macrophage in the tumor microenvironment both contribute to the pro-metastatic potential of calycosin. These events appear to be associated with increased expression of TGF-β1 at both transcriptional and post-translational levels, which may explain the paradoxical drug actions since TGF-β has been implicated to play dual roles as both tumor suppressor and tumor promoter in pancreatic cancer development. ConclusionFindings of this study provide innovative insights about the impact of calycosin in pancreatic cancer progression through induction of cell cycle arrest and apoptosis while possessing certain tumor-promoting property by modulation of the tumor microenvironment.

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-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.

Simple and efficient approach for enrichment of major isoflavonoids from Astragalus membranaceus with macroporous resins and their nephroprotective activities

Isoflavonoids are abundant in Astragalus membranaceus and exhibit multiple pharmacological activities, including nephroprotective activity. In the present study, a simple, green and effective method for the simultaneous enrichment of four major bioactive isoflavonoids from A. membranaceus with macroporous resin was established. Among ten widely used macroporous resins, AB-8 resin exhibited the highest adsorption capacity and desorption ratio in static tests. The static equilibrium adsorption data fit to the Langmuir isotherm, and the adsorption kinetics followed the pseudo-second order model. Dynamic adsorption and desorption experiments were performed to optimize the separation parameters. After only one cycle of treatment with a stepwise elution on AB-8 resin, the contents of calycosin-7-O-β-d-glucoside (CAG), ononin (ONO), calycosin (CAL) and formononetin (FOR) in the final product were 7.71-, 10.64-, 10.50- and 9.49-fold increased with recovery yields of 70.89, 83.14, 85.01 and 74.62%, respectively. Furthermore, the target isoflavonoid-enriched product exhibited better nephroprotective activity than the crude extract, and both the purity of the isoflavonoids and their bioactivities were significantly enhanced after enrichment with macroporous resin. The developed method is a promising foundation for the scaled-up preparation of bioactive isoflavonoids for their wide use in the pharmaceutical industry.

Biochanin A Reduces Inflammatory Injury and Neuronal Apoptosis following Subarachnoid Hemorrhage via Suppression of the TLRs/TIRAP/MyD88/NF-κB Pathway.

Inflammatory injury and neuronal apoptosis participate in the period of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Suppression of inflammation has recently been shown to reduce neuronal death and neurobehavioral dysfunction post SAH. Biochanin A (BCA), a natural bioactive isoflavonoid, has been confirmed to emerge the anti-inflammatory pharmacological function. This original study was aimed at evaluating and identifying the neuroprotective role of BCA and the underlying molecular mechanism in an experimental Sprague-Dawley rat SAH model. Neurobehavioral function was evaluated via the modified water maze test and modified Garcia neurologic score system. Thus, we confirmed that BCA markedly decreased the activated level of TLRs/TIRAP/MyD88/NF-κB pathway and the production of cytokines. BCA also significantly ameliorated neuronal apoptosis which correlated with the improvement of neurobehavioral dysfunction post SAH. These results indicated that BCA may provide neuroprotection against EBI through the inhibition of inflammatory injury and neuronal apoptosis partially via the TLRs/TIRAP/MyD88/NF-κB signal pathway.

Isoflavonoids from Brazilian red propolis down-regulate the expression of cancer-related target proteins: A pharmacogenomic analysis.

Vestitol and neovestitol are bioactive isoflavonoids isolated from Brazilian red propolis, a unique Apis melifera type of propolis botanically originated from Dalbergia ecastophyllum. Although these molecules have relevant biological effects, including anticancer and immunomodulatory activities, their mechanism(s) of action and the affected pathways remain largely unknown. Here, we carried out a pharmacogenomic analysis to investigate the effects of vestitol and neovestitol on the whole-genome expression in human tumor cells, particularly cancer-related target proteins. HeLa cells were exposed to the compounds at IC20 and genomic information of treated cells was analyzed using the Illumina transcriptome system and GeneGo MetaCore software. Our results showed that vestitol (IC20 =214.7μM) reduced the expression of genes enrolled with the alpha tubulin (fold -3.7), tubulin in microtubules (fold -3.7), and histone h3 (fold=-3.03), and that treatment with neovestitol (IC20 =102.91μM) downregulated prostaglandin E synthase gene (fold=-3.12), which are considered ideal targets for anticancer therapy. These data open avenues for the study of vestitol and neovestitol as potential promising candidates for anticancer therapy. Toxicological, non-clinical, and clinical validation of the findings presented herein is needed.

Proteome analysis of Pueraria mirifica tubers collected in different seasons.

Pueraria mirifica-derived tuberous powder has been long-term consumed in Thailand as female hormone-replacement traditional remedies. The protein profiles of tubers collected in different seasons were evaluated. Phenol extraction, 2D-PAGE, and mass spectrometry were employed for tuberous proteome analysis. Out of the 322 proteins detected, over 59% were functionally classified as being involved in metabolism. The rest proteins were involved in defense, protein synthesis, cell structure, transportation, stress, storage, and also unidentified function. The proteins were found to be differentially expressed with respect to harvest season. Importantly, chalcone isomerase, isoflavone synthase, cytochrome p450, UDP-glycosyltransferase, and isoflavone reductase, which are all involved in the biosynthesis pathway of bioactive isoflavonoids, were most abundantly expressed in the summer-collected tubers. This is the first report on the proteomic patterns in P. mirifica tubers in relevant with seasonal variation. The study enlights the understanding of variance isoflavonoid production in P. mirifica tubers.

ChemInform Abstract: Palladium‐Catalyzed Oxyarylation, Azaarylation and α‐Arylation Reactions in the Synthesis of Bioactive Isoflavonoid Analogues

AbstractReview: 132 refs.

Palladium-Catalyzed Oxyarylation, Azaarylation and &amp;#945;-Arylation Reactions in the Synthesis of Bioactive Isoflavonoid Analogues

Isoflavonoids constitute an important class of natural products, comprising great structural diversity. Known as phytoalexins and phytoestrogens, these compounds exhibit interesting biological activities. The corresponding carba-analogues, in which the oxygen atom at the B-ring is replaced by a methylene, are emerging as an attractive alternative for the preparation of new bioactive compounds. In this review, we report our results on the synthesis of isoflavonoid analogues (5-carba-pterocarpans, 1-carba-isoflavanones, 5-carba-11-aza-pterocarpan and pterocarpanquinones) with anti-cancer, antiparasitic and anti-viral properties. Palladium-catalyzed oxyarylation and azaarylation of dihydronaphthalenes and α-arylation of α- tetralones were the key steps in the developed synthetic approaches. A historical background, as well as a brief mechanistic discussion of these reactions, will be presented. Keywords: Isoflavonoids, Carba-isoflavonoids, Oxyarylation, Azaarylation, α-Arylation, Palladium-catalyzed.

Study of pharmacokinetic profiles and characteristics of active components and their metabolites in rat plasma following oral administration of the water extract of Astragali radix using UPLC–MS/MS

Ethnopharmacological relevanceAstragali radix is one of the well-known traditional Chinese herbal medicine, and possesses various biological functions, such as hepatoprotective and anticancer. In present study, to investigate the metabolism and pharmacokinetics of the major constituents of A. radix, a sensitive ultra-performance liquid chromatography–electrospray ionization–mass spectrometry (UPLC–MS/MS) method with shorter chromatographic running time was developed and validated for simultaneous quantification of formononetin, ononin, calycosin, calycosin-7-β-glucoside, astragaloside IV and their glucuronide metabolites in rat plasma after oral administration of water extract of A. radix at two different doses. Materials and methodsThe chromatographic separation was achieved on a C18 column with gradient elution by using a mixture of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase at a flow rate of 0.3mL/min. A tandem mass spectrometric detection was conducted using multiple-reaction monitoring (MRM) via electrospray ionization (ESI) source in positive ionization mode. Samples were pre-treated by a single-step protein precipitation with methanol, and erlotinib was used as internal standard (IS). ResultsThe current UPLC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery, matrix effects and stability. The lowest limit of quantifications (LLOQ) were 1ng/mL for all analytes. After oral administration, the plasma concentrations of the glucuronides, especially calycosin-3′-glucuronide, were much higher than the parent compounds. The mean half-life (t1/2) was between 1 and 5h, and the metabolites were eliminated faster than the parent constituents. The median (range) time to reach maximum plasma concentration (Tmax) was between 0.5 and 1h. ConclusionsThis is the first study of the pharmacokinetic study of bioactive compounds and their glucuronides in male rat plasma after oral administration of water extract of A. radix. The results demonstrated the biotransformation between the bioactive isoflavonoids and their glucuronides was extensive in rats and provided a significant basis for better understanding the absorption and metabolism mechanism of A. radix. Furthermore, this study could suggest that future studies should focus on the metabolites and biotransformation between the bioactive constituents when conducting a drug efficacy study.