Formononetin,J1 and J2 have different effects on endothelial cells via EWSAT1‐TRAF6 and its downstream pathway

Formononetin is a natural isoflavone compound found mainly in Chinese herbal medicines such as astragalus and red clover. It is considered to be a typical phytooestrogen. In our previous experiments, it was found that formononetin has a two‐way regulatory effect on endothelial cells (ECs): low concentrations promote the proliferation of ECs and high concentrations have an inhibitory effect. To find a specific mechanism of action and provide a better clinical effect, we performed a structural transformation of formononetin and selected better medicinal properties for formononetin modifier J1 and J2 from a variety of modified constructs. The MTT assay measured the effects of drugs on human umbilical vein endothelial cell (HUVEC) activity. Scratch and transwell experiments validated the effects of the drugs on HUVEC migration and invasion. An in vivo assessment effect of the drugs on ovariectomized rats. Long‐chain non‐coding RNA for EWSAT1, which is abnormally highly expressed in HUVEC, was screened by gene chip, and the effect of the drug on its expression was detected by PCR after the drug was applied. The downstream factors and their pathways were analysed, and the changes in the protein levels after drug treatment were evaluated by Western blot. In conclusion, the mechanism of action of formononetin, J1 and J2 on ECs may be through EWSAT1‐TRAF6 and its downstream pathways.     

Differential turnover of isoflavone 7-O-glucoside-6″-O-malonates in Cicer arietinum roots

Pulse labelling experiments and studies with a molecular inhibitor of phenylalanine ammonia lyase showed that in roots of Cicer arietinum formononetin 7-O-glucoside-6″-O-malonate is rapidly metabolized whereas biochanin A 7-O-glucoside-6″-O-malonate appears to be metabolically rather inert.     

Methods of phytochemical standardisation of rhizoma Cimicifugae racemosae

The methanolic extract of roots of Cimicifuga racemosa and its methanolysis products have been analysed by GC-MS. 2-Hexylcyclopropaneoctanoic acid (9,10-methylenehexadecanoic acid) was found to be the marker most specific for the identification for this herb. For the phytochemical standardisation of Rhizoma Cimicifugae racemosae, validated methods for the quantitative analysis of formononetin (by TLC-fluorometry), of isoferulic acid (by GC-MS of the methyl ester), and of total triterpene glycosides (transformed to coloured complexes and measured photometrically) in roots of C. racemosa have been developed. The contents of formononetin, isoferulic acid and total triterpene glycosides (measured as actein) in the herb ranged from 0.0031 to 0.0035, from 1.22 to 1.35 and from 20.09 to 22.06 mg/g dry weight, respectively.     

Formononetin ameliorates muscle atrophy by regulating myostatin-mediated PI3K/Akt/FoxO3a pathway and satellite cell function in chronic kidney disease

Muscle atrophy is a common complication in chronic kidney disease (CKD). Inflammation and myostatin play important roles in CKD muscle atrophy. Formononetin (FMN), which is a major bioactive isoflavone compound in Astragalus membranaceus, exerts anti-inflammatory effects and the promotion of myogenic differentiation. Our study is based on myostatin to explore the effects and mechanisms of FMN in relation to CKD muscle atrophy. In this study, CKD rats and tumour necrosis factor α (TNF-α)-induced C2C12 myotubes were used for in vivo and in vitro models of muscle atrophy. The results showed that FMN significantly improved the renal function, nutritional status and inflammatory markers in CKD rats. Values for bodyweight, weight of tibialis anterior and gastrocnemius muscles, and cross-sectional area (CSA) of skeletal muscles were significantly larger in the FMN treatment rats. Furthermore, FMN significantly suppressed the expressions of MuRF-1, MAFbx and myostatin in the muscles of CKD rats and the TNF-α-induced C2C12 myotubes. Importantly, FMN significantly increased the phosphorylation of PI3K, Akt, and FoxO3a and the expressions of the myogenic proliferation and differentiation markers, myogenic differentiation factor D (MyoD) and myogenin in muscles of CKD rats and the C2C12 myotubes. Similar results were observed in TNF-α-induced C2C12 myotubes transfected with myostatin-small interfering RNA (si-myostatin). Notably, myostatin overexpression plasmid (myostatin OE) abolished the effect of FMN on the phosphorylation of the PI3K/Akt/FoxO3a pathway and the expressions of MyoD and myogenin. Our findings suggest that FMN ameliorates muscle atrophy related to myostatin-mediated PI3K/Akt/FoxO3a pathway and satellite cell function.     

In vitro and in vivo studies of antiosteosarcoma activities of formononetin

Osteogenic sarcoma (OGS) is a primary bone cancer, characterized by aggressive neoplasm from mesenchymal oncogenesis. However, the clinical therapeutic regimen against OGS is limited. Therefore, potential medication warrants to be further developed. Our previous study indicates that formononetin (FN) exerts effective pharmacological activity against OGS. This study aimed to further decipher the molecular mechanism behind this benefit. Patients with OGS were recruited for clinical data assay and immunoassay. Human OGS cell line (U2OS) and tumor-bearing nude mice were subjected to a battery of biochemical analyses and immunoassays for integrative evaluation of FN-exerted anti-OGS effects. In human data, OGS samples showed increased expressions of ERα, p-PI3KCA^Tyr317, and p-AKT ^Ser473 proteins, followed by notably upregulated miR-375 content in comparison with that in OGS-free. In addition, FN-treated U2OS cells showed inhibited cell proliferation, elevated lactic dehydrogenase production and lowered endogenous miR-375 level in cells. Further, reduced immunopositive cells of Ki-67, p-PI3KCA ^Tyr317, and p-AKT ^Ser473 were observed by the treatments of FN, while the intracellular Bax- and Apaf-1-positive cells were increased dose-dependently. Beneficially, FN-treated tumor-bearing mice exhibited reduced tumor mass and intercellular miR-375 expression. Meanwhile, immuno-labeled cells and proteins of Bax, Caspase-3, and Apaf-1 in FN-treated mice were increased dose-dependently, whereas ERα, p-PI3KCA ^Tyr317, and p-AKT ^Ser473 positive cells and proteins were downregulated, respectively. Collectively, our current results elucidate that FN exerts effective therapeutic benefits against OGS, and the pharmacological mechanism may be related to promoting cell apoptosis by inactivating intracellular miR-375/ERα−PI3K/AKT cascaded pathway.     

Influence of phosphorus and formononetin on isozyme expression in the Zea mays‐Glomus intraradices symbiosis

Maize (Zea mays L. cv. Great Lakes 586) plants were either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith, or grown in the presence of the isoflavone formononetin or were provided with both G. intraradices and formononetin. All plants were grown in soil containing one of five levels of inorganic P (between 8 and 110 µg g^?1 soil). By 3 weeks there were significant differences in a number of enzyme activities and in the pattern of isoenzymes in roots colonized by the VAM fungus or treated with formononetin. One NAD-malate dehydrogenase (MDH) isozyme was expressed only in mycorrhizal roots, whether treated or not with formononetin. Despite differences in the soil P level, the expression of this isozyme was not observed in non-mycorrhizal roots, indicating specific expression in the mycorrhizae. We suggest that MDH isozyme could serve as a specific, early indicator of the Zea-Glomus symbiosis. Differences in the esterase (EST) isozyme pattern were not detectable between VAM and non-VAM roots, suggesting that this enzyme system is not a good parameter for the evaluation of mycorrhizal colonization. As available P in the soil increased, total EST activity appeared to increase as well. Interestingly, total peroxidase (POX) activity increased along with P suggesting that as plant P nutrition improved, both cell wall ramification and the quantity of defense peroxidases increased as well. Total POX activity from mycorrhizal roots was inversely correlated with root colonization, indicating that there was suppression of POX activity by the host under low soil P. Most interestingly, formononetin further decreased POX activity regardless of the level of P or mycorrhizal status. This may suggest one mechanism by which formononetin enhances root VAM colonization. The presence of this isoflavone suppressed POX activity in mycorrhizal roots allowing a rapid penetration and spread of the fungus in the root cortex. The interplay between host root, soil P levels, secondary metabolites and endogenous host enzyme activities and a particular VAM fungus has a profound effect on the efficiency, duration and functioning of an endomycorrhizal symbiosis.     

Isoflavone biosynthesis in Onobrychis viciifolia: Formononetin and texasin as precursors of afrormosin

4,2′,4′-Trihydroxychalcone- [carbonyl-¹⁴C], formononetin- [Me-¹⁴C] and texasin- [Me-¹⁴C] were all good precursors of afrormosin (7-hydroxy-6,4′-dimethoxyisoflavone) in Onobrychis viciifolia seedlings, and a biosynthetic pathway involving these intermediates is proposed. 2′,4′-Dihydroxy-4-methoxychalcone- [carbonyl-¹⁴C] and daidzein-[carbonyl-¹⁴C] were poor precursors. Incorporations into formononetin were also recorded.     

芒柄花黄素在体外对内皮细胞细胞周期的影响

目的观察芒柄花黄素在体外对人脐静脉内皮细胞（HUVEC）增殖及周期的影响。方法采用四甲基偶氮唑蓝（MTT）法检测不同芒柄花黄素对HUVEC增殖的影响,流式细胞术检测细胞周期,Western blot检测cyclin D1蛋白表达水平。结果芒柄花黄素呈剂量依赖性促进HUVEC增殖。且药物作用后,S期细胞比例增加,cyclin D1蛋白表达升高。结论芒柄花黄素对人脐静脉内皮细胞有明显的促进增殖作用,可通过上调cyclin D1蛋白表达增加S期细胞比率。     

Fertility and growth of nulliparous ewes after feeding red clover silage with high phyto-oestrogen concentrations

The study aimed to determine the effects of red clover (Trifolium pratense) silage with high phyto-oestrogen content on ewe performance during their first breeding season. Red clover silage containing formononetin, biochanin A, genistein, and daidzein was fed to 10 nulliparous ewes of the prolific Finnish Landrace breed before, during and after the breeding season, for a total of 5 months. A control group of 10 ewes was fed with grass silage. The mean numbers of foetuses per pregnancy were 2.1±0.7 and 2.2±0.8 for the red clover and control groups, respectively. The total mass of the uterus with its contents was significantly greater in ewes of the red clover group compared with those of the control group. This difference was mainly explained by the greater volume of foetal fluids. Serum progesterone concentration in the red clover group was significantly lower over the entire period analysed than in the control group. In conclusion, the fecundity of the ewes was not reduced by red clover feed with high phyto-oestrogen concentrations. The volume of foetal fluids increased that could increase the risk for vaginal prolapse before the term.