Concentrations Of Formononetin Research Articles

Water Stress Influences Phytoestrogen Levels in Red Clover (Trifolium pratense) but Not Kura Clover (T. ambiguum).

Some forage legumes synthesize phytoestrogens. We conducted a glasshouse study to investigate how water stress (drought and waterlogging) influences phytoestrogen accumulation in red clover and kura clover. Compared to the red clover control, the 20 day drought resulted in an over 100% increase in the phytoestrogens formononetin and biochanin A, which together accounted for 91-96% of the total phytoestrogens measured. Waterlogging resulted in elevated concentrations of daidzein, genistein, and prunetin but not formononetin or biochanin A. Concentrations of phytoestrogens in kura clover were low or undetectable, regardless of water stress treatment. Leaf water potential was the most explanatory single-predictor of the variation in concentrations of formononetin, biochanin A, and total phytoestrogens in red clover. These results suggest that drought-stressed red clover may have higher potential to lead to estrogenic effects in ruminant livestock and that kura clover is a promising alternative low- or no-phytoestrogen perennial forage legume.

Formononetin Restrains Tumorigenesis of Breast Tumor by Restraining STING-NF-κB and Interfering with the Activation of PD-L1.

Breast cancer (BC), a common tumor in women, has high morbidity and mortality. Formononetin, an active ingredient in red clover and Astragalus membranaceus, has a wide range of pharmacological applications, including as an anticancer agent. Since immunotherapy is a hot topic in the treatment strategy of BC, it was dedicated to appraising the specific mechanism of formononetin in BC immunotherapy in this research. Different formononetin concentrations (0, 20, 40, 60, 80, 100 μM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot. Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone. Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.

Phytochemical Composition of Different Red Clover Genotypes Based on Plant Part and Genetic Traits.

Red clover (Trifolium pratense L.) is an important legume that is also known as a rich source of isoflavones, which are compounds with mild estrogenic activity. Therefore, this plant is often used as a raw material in the production of dietary supplements recommended in menopause. Many factors can influence isoflavone content, but those genetically related are considered to be the most important. Therefore, the aim of this study was to evaluate the phytochemical profile of different plant parts of 30 red clover genotypes grouped according to ploidy and country of seed origin by analyzing the content of dominant isoflavones, total phenolic content (TPC) and antioxidant activity. It was found that there are significant differences in the examined traits among plant parts. Red clover leaves had the highest total isoflavone content, with biochanin A as the dominant compound, while flower extracts had the highest TPC and antioxidant activity. Diploid and tetraploid genotypes were significantly different concerning the content of daidzein, genistein, formononetin and TPC with higher quantities in tetraploid samples. On the other hand, seed origin was not a useful separating factor for the analyzed samples. The results of this research indicate that ploidy, as a previously poorly studied factor, could influence isoflavone content in red clover.

235 Effects of Curing Extent on the Phytoestrogen Concentrations of Red Clover Hay and Silage Across Storage Phases

Abstract Our objective was to assess the effects of insufficient (WET) or ample (CUR) curing on the phytoestrogen concentrations of red clover silage (29.4 and 45.3% DM) and hay (65.1 and 89.1, respectively). Measurements were taken at the start of storage (STRT), after 14 d (MicA), and once storage processes had stabilized for hay and silage (50 and 78 d, respectively; LATE). Hay was packed into mini-bales (~500 fresh g; ~232 kg DM/m3) and silage into mini-silos (19.5 L; ~214 kg DM/m3). Data were analyzed as a randomized complete block design (5 blocks) with a 2 (curing extents) x 2 (storage methods) x 3 (storage phases) factorial. Differences were declared at P ≤ 0.05. The concentration of formononetin was greater in silage vs. hay at STRT (4,624 vs. 2,893 ± 175 mg/kg of DM, respectively), MicA (5,383 vs. 2,110), and LATE (5,666 vs. 2,569). Also, more formononetin was found in WET vs. CUR silage (5,841 vs. 4,608.1± 154 mg/kg of DM, respectively). However, no differences were observed between WET vs. CUR hay (x̅ = 2,524). At STRT, biochanin A was greater in silage vs. hay (3,105 vs. 2,012 ± 139 mg/kg of DM, respectively). A similar trend was observed at MicA (3,233 vs. 1,057), and LATE (3,295 vs. 1,228). In addition, more biochanin A was observed in WET vs. CUR silage (3,793 vs. 2,628.9 ± 127 mg/kg of DM, respectively). The opposite was observed in WET vs. CUR hay (1,219.5 vs. 1,644.9). The genistein concentration was greater in silage vs. hay at STRT (149 vs. 120 ± 8.36 mg/kg of DM, respectively), MicA (246 vs. 62.6), and LATE (264 vs. 72.9). Also, the concentration of genistein was greater in WET vs. CUR silage (236 vs. 202.3 ± 7.21 mg/kg of DM, respectively) but the opposite was observed in WET vs. CUR hay (72.8 vs. 97.3). In the case of daidzein, hay had a greater concentration of this phytoestrogen than silage (12.8 vs. 6.67 ± 0.43 mg/kg of DM, respectively). In conclusion, red clover silage maintains greater concentrations of formononetin, biochanin A, and genistein than hay during curing period and across the storage phase. Also, adequate curing is more beneficial for silage than hay in terms of decreasing the phytoestrogen concentrations.

Anti-cancer targets and molecular mechanisms of formononetin in treating osteosarcoma based on network pharmacology.

Osteosarcoma (OS) is a multifactorial bone malignancy that accounts for most cancers in children and adolescents. Formononetin has been proven to exhibit various pharmacological effects including anti-tumor, anti-obesity, anti-inflammation, and neuroprotective effects. Few studies have examined the pharmacological activities of formononetin in OS treatment, but the mechanism has not yet been completely elucidated. Network pharmacology is a new method based on the theory of system biology for analyzing the network of biological systems and selecting specific signal nodes for multi-target drug molecular design. Here, we used network pharmacology to explore the possible mechanism of formononetin in OS treatment. Human OS cell line MG63 was processed with four concentrations (0, 2, 5, 8 μg/mL) of formononetin. Subsequently, an MTT assay was performed to test cell proliferation and a scratch test was used to evaluate the migration ability of cancer cells. Caspase-3, p53, p21, and bcl-2 expression levels incubated with different concentrations of formononetin in MG63 cells were determined using Western blotting. After treated with formononetin for 48 h, MG63 cells exhibited marked apoptosis. The results revealed that certain concentrations of formononetin significantly exerted inhibitory effects on MG63 cell proliferation. Furthermore, formononetin decreased the bcl-2 level in MG63 cells but increased caspase-3, p21, and p53 levels in a concentration-dependent manner. Additionally, formononetin suppressed the expression of SATB2. Therefore, formononetin could dose-dependently inhibit MG63 cell proliferation and induce apparent cell apoptosis, providing a candidate treatment for OS, whereas SATB2 could be a potential prognostic biomarker for screening OS and therapeutic target of formononetin.

Formononetin ameliorates the LPS-induced inflammatory response and apoptosis of neuronal cells via NF-κB/NLRP3 signaling pathway.

The objective of this study was to investigate the impact of formononetin on cellular apoptosis and inflammatory responses following spinal cord injury (SCI), as well as the underlying mechanisms involved. In this study, PC12 cells were treated with lipopolysaccharide (LPS) and different concentrations of Formononetin (FT) (50μM, 100μM, 200μM). To confirm the effect of nuclear factor-κB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) signaling pathways, the cells in the phorbol-12-myristate-13-acetate (PMA) group were treated with 0.1μmol/L PMA (NF-κB/NLRP3 signaling pathway activators). The lactate dehydrogenase (LDH) concentration and cell viability, proliferating cell nuclear antigen (PCNA) fluorescence intensity, and cell apoptosis were determined using an LDH kit, Cell Counting Kit-8 (CCK-8), immunofluorescence, and flow cytometry assays, respectively. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-16 (IL-6) expression levels were detected by quantitative ELISA assay. The expression of proteins related to the NF-κB/NLRP3 signaling pathway was detected by western blotting. Our results showed that LPS increased LDH levels in PC12 cells, suggesting that inflammation caused PC12 cell damage. However, the PC12 cell damage was decreased by methylprednisolone. Formononetin promotes cell survival and proliferation, and prevents apoptosis in a concentration-dependent manner. Formononetin reduced the TNF-α, IL-1β, and IL-6 levels in the LPS-treated model. Moreover, formononetin decreased the levels of p-p65 NF-κB and NLRP3 in PC12 cells. We conclude that formononetin ameliorated the inflammatory response and apoptosis in LPS-induced inflammatory injury in neuronal cells via the NF-κB/NLRP3 signaling pathway.

Formononetin Inhibits Progression of Endometriosis via Regulation of p27, pSTAT3, and Progesterone Receptor: In Vitro and In Vivo Studies.

Formononetin is one of the phytoestrogens that functions like a selective estrogen receptor modulator (SERM). In this study, we evaluated the effects of formononetin on endometriosis progression in vitro and in vivo. After pathological confirmation, 10 eutopic and ectopic endometria were collected from patients with endometriosis. Ten eutopic endometria samples were collected from patients who did not have endometriosis. To determine the cytotoxic dose and therapeutic dose of formononetin, the concentration of 70% of the cells that survived after formononetin administration was estimated using a Cell counting kit-8 (CCK 8) assay. Western blot analysis was used to determine the relative expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 in the eutopic endometria without endometriosis, eutopic endometria with endometriosis, and ectopic endometria with endometriosis as the formononetin concentration was increased. We confirmed the effect of formononetin on apoptosis and migration in endometriosis using fluorescence-activated cell sorting (FACS) and wound healing assays, respectively. A mouse model of endometriosis was prepared using a non-surgical method, as previously described. The mice were intraperitoneally administered formononetin for four weeks after dividing them into control, low-dose formononetin (40 mg/kg/day) treatment, and high-dose (80 mg/kg/day) formononetin treatment groups. All the mice were euthanized after formononetin treatment. Endometriotic lesions were retrieved and confirmed using hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) staining of p27 was performed. We set the maximum concentration of formononetin administration to 80 μM through the CCK8 assay. Based on formononetin concentration, the expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 proteins were measured using Western blot analysis (N = 4 per group). The expression level of pERK, p27, and pSTAT3 in eutopic endometrium with endometriosis tended to decrease with increasing formononetin concentration, and a significant decrease was noted at 80 μM. The expression of p27 in ectopic endometrium with endometriosis was also significantly decreased at 80 μM of formononetin. FACS analysis revealed that formononetin did not significantly affect apoptosis. In the wound healing assay, formononetin treatment revealed a more significant decrease in the proliferation of the eutopic endometrium in patients with endometriosis than in the eutopic endometrium without endometriosis. Relative expression of sex hormone receptors decreased with increasing formononetin doses. Although no significant differences were observed in the ER, PR-A, ERβ/ERα, and PR-B/PR-A, significant down-regulation of PR-B expression was noted after formononetin treatment at 80 μM. In the in vivo study, endometriotic lesions in the formononetin-treated group significantly decreased compared to those in the control group. The relative expression of p27 using IHC was highest in the control group and lowest in the high-dose formononetin treatment group. Formononetin treatment was shown to inhibit the proliferation of eutopic and ectopic endometria in patients with endometriosis through the regulation of p27, pSTAT3, and PR-B. In an endometriosis mouse model, formononetin treatment significantly reduced the number of endometriotic lesions with decreased p27 expression. The results of this study suggest that formononetin may be used as a non-hormonal treatment option for endometriosis.

Leaf formononetin content of Trifolium subterraneum increases in response to waterlogging but its proportion of total isoflavones is little changed.

The isoflavone formononetin (F) impacts livestock fertility and cultivars of the pasture legume Trifolium subterraneum L. (subclover) have been selected for F levels ≤0.2% of leaf dry weight. However, the impact of waterlogging (WL) on isoflavones is little studied. We investigated the response of isoflavones, biochanin A (BA), genistein (G) and F, to WL for: (1) Yarloop (high F) and eight low F cultivars each from subspecies subterraneum , brachycalycinum and yanninicum (Experiment 1); and (2) four cultivars and 12 ecotypes of ssp. yanninicum (Experiment 2). WL impacted F: estimated means increased from 0.19% (control) to 0.31% (WL) in Experiment 1 and from 0.61% to 0.97% in Experiment 2. Isoflavones under WL were highly heritable, particularly F (H 2 =95%). The proportions of BA, G and F were little changed by WL, with strong positive correlations between free-drained and waterlogged treatments. Isoflavone contents were not related to WL tolerance, as assessed by shoot relative growth rate. In conclusion, isoflavones varied among genotypes and increased with WL, but the proportion of individual isoflavones in each genotype was stable. High F under WL was unrelated with genotype tolerance to WL. Instead, it was a consequence of inherently high F for that particular genotype.

Formononetin ameliorates ferroptosis-associated fibrosis in renal tubular epithelial cells and in mice with chronic kidney disease by suppressing the Smad3/ATF3/SLC7A11 signaling

AimsChronic kidney disease (CKD) is characterized by interstitial fibrosis, while limited treatment drugs are available. Ferroptosis is a newly identified process that can trigger tubular atrophy and fibrosis. The aim of this study is to investigate the effects of formononetin (FN), a bioflavonoid, on ferroptosis and renal fibrosis. Main methodsIn vivo experiments, unilateral ureteral obstruction (UUO)- and folic acid (FA, 250 mg/kg)-induced CKD models were constructed in C57BL/6 mice of 6–8 weeks old, followed by the administration with 40 mg/kg/day FN by gavage. For in vitro experiments, ferroptosis was induced with RSL3 or erastin in primary mouse renal tubular epithelial cells (TECs), followed by the addition of indicated concentrations of FN. Then, the levels of ferroptosis and fibrosis were analyzed. The translocation of Smad3, ATF3, and Nrf2 from the cytoplasm to the nucleus was checked by western blotting. The interaction of Smad3 and ATF3 was detected by Co-immunoprecipitation. Key findingsFN dramatically ameliorated tubular injury along with reduced expression of the profibrotic genes including α-SMA, Col1a1, and fibronectin in both two CKD mouse models and RSL3/erastin-treated TECs. Furthermore, FN administration also significantly suppressed ferroptosis, as evidenced by increased expression of SLC7A11 and GPX4, and decreased levels of 4-HNE. In mechanism, FN disrupted the interaction between Smad3 and ATF3, resulting in the blocking of their translocation from the cytoplasm to the nucleus. In addition, FN also promoted the separation of the Nrf2/Keap1 complex and enhanced Nrf2 nuclear accumulation. SignificanceFN alleviates CKD by impeding ferroptosis-associated fibrosis by suppressing the Smad3/ATF3/SLC7A11 signaling and could serve as a candidate therapeutic drug for CKD.

Parent-Metabolite Pharmacokinetic Modeling of Formononetin and Its Active Metabolites in Rats after Oral Administration of Formononetin Formulations

Formononetin is a major isoflavone contained in propolis and is reported to exhibit various pharmacological effects. However, the use of formononetin in pharmaceutical industry is limited due to its low bioavailability and solubility. There had been several efforts on formononetin formulation development, but further study is required to acquire optimal formulation. The aim of this study is to conduct pharmacokinetic (PK) evaluations after the oral administration of three formononetin formulations (20 mg/kg) in male Sprague Dawley rats. Then, a parent-metabolite PK model for formononetin was developed and evaluated for the first time. To do this, a simultaneous analysis method for formononetin and its active metabolites, daidzein, dihydrodaidzein and equol in rat plasma was developed using ultra-performance liquid chromatography tandem mass spectrometry. The separation was performed using a gradient elution of water and acetonitrile and a Kinetex C18 column (2.1 mm × 100 mm, 1.7 µm particle size) at a temperature of 30 ± 5 °C. The simultaneous analytical method developed in this study was validated according to international guidance and was successfully applied for the pharmacokinetic study. The time-plasma concentrations of formononetin and daidzein were well described by a two-compartment model combined with a metabolite compartment. Additionally, plasma protein binding assay was conducted in male rat plasma. The findings from the study could be used as a fundamental for the future development of formononetin as a pharmaceutical product.

Differences in Chemical Constituents between Dalbergia oliveri Heartwood and Sapwood and Their Effect on Wood Color.

The purpose of this study was to characterize and quantify the chemical constituents of heartwood and sapwood of Dalbergia oliveri extract in order to investigate the chemical components that determine the formation of heartwood's color. In this work, the types of pigments in heartwood and sapwood extract were analyzed using UV-Visible (UV) Spectrophotometer, and the main pigment components of heartwood and sapwood extract were identified and quantified using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). The results showed that the difference in content of the main components between heartwood and sapwood of Dalbergia oliveri was slight, and the lignin structure between heartwood and sapwood is basically identical; flavonoid pigments were found to be the primary chromophoric components of heartwood and sapwood extract. However, a total of 21 flavonoids were identified in heartwood and sapwood, of which the unique substances to heartwood were vitexin, isorhamnetin, and pelargonidin, and the content of isoliquiritigenin, formononetin, and biochanin A were 253, 37, and 583 times higher in the heartwood than in the sapwood, respectively, which could be the main pigment components affecting the significant color difference between heartwood and sapwood of Dalbergia oliveri. These results will provide a foundation for revealing the underlying mechanism of color difference between heartwood and sapwood and provide a theoretical basis for wood coloring.

Development of an indirect competitive enzyme-linked immunosorbent assay for formononetin and its application in a cell-based assay using MC3T3-E1 cells

Formononetin (FMN) is a methoxy isoflavone found abundantly in leguminous plants and associated foods. Several analytical methods have been developed to detect FMN. However, they are costly, complicated, and time-consuming. This study describes an indirect competitive enzyme-linked immunosorbent assay (icELISA) to determine FMN content in food samples using a monoclonal antibody (mAb) against FMN produced by a newly established hybridoma cell line. Validation studies were conducted, and this assay was found to be sufficiently reliable, with an analytical measurement range of 19.53–1250 ng/mL and a detection limit of 17.42 ng/mL. Furthermore, icELISA was successfully applied for a cell-based assay in which the amount of FMN and ononin uptake was quantified in MC3T3-E1 cells. Hence, icELISA is a simple and reliable method for the detection and quantification of FMN, as well as elucidation of its functions and underlying mechanisms of action.

Natural borneol enhances the anti-cerebral ischaemia efficacy of formononetin in MCAO/R rats by promoting its delivery in the brain.

Objectives Due to its high morbidity, high mortality, and high disability, stroke has been the first cause of death and the major cause of adult disability in China. Natural borneol has been widely utilized in Traditional Chinese Medicine to promote drug absorption. Formononetin is a natural isoflavonoid with potent neuroprotective activity but poor brain delivery. Methods This study aimed to screen the optimum proportion that natural borneol promotes formononetin entry into the brain, evaluate the anti-cerebral ischaemia efficacy of formononetin/natural borneol combination in middle cerebral artery occlusion/reperfusion model rats, and clarify the possible mechanism for natural borneol's promoting formononetin delivery in the brain. Key findings Our studies exhibited that natural borneol remarkably promoted formononetin entry into the brain when combined with formononetin in a 1 : 1 molar ratio and notably improved neuro-behavioural scores and reduced the infarct of middle cerebral artery occlusion/reperfusion model rats. This study further discovered that the enhanced anti-cerebral ischaemia effect resulted from natural borneol increasing the permeability of the blood-brain barrier to elevate formononetin concentration in the brain rather than the pharmacodynamic synergy or addition between formononetin and natural borneol. Conclusions The study provides a good strategy to screen drug combinations for the treatment of brain disease by combining natural borneol with other drugs.

Complementary Effects of Dark Septate Endophytes and Trichoderma Strains on Growth and Active Ingredient Accumulation of Astragalus mongholicus under Drought Stress.

Drought is a major abiotic stress factor affecting plant growth and production, while utilizing beneficial endophytic fungi is one of the most promising strategies for enhancing plant growth and drought tolerance. In the current study, a pot experiment was conducted to investigate the beneficial effects of dark septate endophyte (DSE) (Macrophomina pseudophaseolina, Paraphoma radicina) and Trichoderma (Trichoderma afroharzianum, Trichoderma longibrachiatum) inoculum on Astragalus mongholicus grown in sterile soil under drought stress, alone, or in combination. The addition of Trichoderma enhanced the DSE colonization in roots regardless of the water condition. Under well-watered conditions, M. pseudophaseolina inoculation significantly enhanced the biomass and root length of A. mongholicus. The two DSE and Trichoderma inoculum significantly improved calycosin-7-O-β-D-glucoside content. However, M. pseudophaseolina + T. afroharzianum inoculation better promoted root growth, whereas co-inoculation had higher active ingredient contents compared with single inoculation, except for P. radicina + T. afroharzianum. Under drought stress, DSE and Trichoderma inoculum significantly improved root biomass, root length, calycosin-7-O-β-D-glucoside content, and activities of nitrate reductase and soil urease. P. radicina + T. afroharzianum and P. radicina + T. longibrachiatum better increased root length, and all combinations of DSE and Trichoderma had a greater impact on the increase in formononetin content compared with the single treatments. Additionally, Trichoderma relies on antioxidant enzymes, growth hormones, and the redox system (ascorbic acid–glutathione) to resist drought, while DSE strains have an additional osmotic regulation system in addition to the drought resistance function possessed by Trichoderma, and the effect of co-inoculation (especially M. pseudophaseolina + T. longibrachiatum and P. radicina + T. afroharzianum) on plant physiological parameters was greater than that of single inoculation. This study provides a new research direction for the effects of DSE and Trichoderma on medicinal plant cultivated in dryland.

Hepatoprotective Effects of Astragaloside IV and Formononetin Against Oxidative Injury

The mitochondrial SIRT3 is a major deacetylase protein that regulates important mitochondrial biology, including metabolic pathways, reactive oxygen species detoxification, mitochondrial dynamics, and the mitochondrial unfolded protein response. We found that astragalus aqueous extract increased the activity of SIRT3 in vitro. We further explore the potential antioxidative effects of Astragaloside IV (AST‐IV) and Formononetin (FMR) on induced oxidative stressed AML12 (alpha mouse liver 12) cells. The AML12 cell line was established from healthy hepatocytes from a mouse (CD1 Strain, Line MT42) transgenic for human TFG alpha.We first tested whether AST‐IV or formononetin increases SIRT3 expression in cell model. We cultured AML12 and treated with various concentrations of AST‐IV or formononetin in parallel with untreated control, negative control, and positive control. We found that AST‐IV and formononetin induced SIRT3 expression through Western Blotting (WB) analysis.In addition, we assessed rescue effects of AST‐IV and formononetin on t‐BHP‐induced oxidative injury in AML12 hepatocytes under three separate conditions: pre‐treatment with AST‐IV or formononetin, post‐treatment with AST‐IV or formononetin, and both pre‐treatment and post‐treatment with AST‐IV and formononetin. Through CCK‐8 analysis, we identified that both pre‐treatment and post‐treatment significantly increased cell proliferation on induced‐oxidative stress AML12 cells compared to no treatment and control. Pre‐treatment of AML12 cells before induction of oxidative stress with AST‐IV and formononetin also showed cell proliferation but was less significant compared to AML12 cells that received both pre‐treatment and post‐treatment with AST‐IV or formononetin. Post‐treatment AST‐IV or formononetin after induction of oxidative stress on AML12 cells did not significantly show any difference in cell proliferation compared to non‐treatment and control. Significantly AST‐IV and formononetin showed induction of the SIRT3 protein on tert‐butyl hydroperoxide (t‐BHP) induced oxidative stressed AML12 hepatocytes by WB.Taken together, the increase in cell proliferation is indicative of the antioxidative effects of AST‐IV and formononetin through induction of SIRT3 protein. Further investigation is currently conducted to provide evidence of AST‐IV and formononetin's mechanistic effect on inducing the expression of the SIRT3 protein‐linked downstream pathways for their antioxidative benefit to hepatocytes. The ultimate goal is to utilize these SIRT3 modulators as pharmacologic agents in treating oxidative injury hepatocytes.

Differential Expression of Calycosin-7-O-β-D-glucoside Biosynthesis Genes and Accumulation of Related Metabolites in Different Organs of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao Under Drought Stress.

Calycosin-7-O-β-D-glycoside (CG), as a flavonoid, plays an important role in the abiotic stress response of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). CG is also an active ingredient in A. mongholicus with high medicinal value. However, the response mechanism of the CG biosynthetic pathway of drought stress is not clear. In this research, drought stress was inflicted upon A. mongholicus, and the variations in flavonoid metabolites and the correlating gene expression in CG biosynthesis were studied in roots, stems, and leaves of A. mongholicus by UHPLC-MRM-MS/MS and qRT-PCR. Drought stress reduced the dry weight and increased the content of malondialdehyde (MDA) and proline. Drought was beneficial to the accumulation of L-phenylalanine and 4-coumaric acid in leaves and promoted the accumulation of all target compounds in the roots, except calycosin. Overexpression of AmIOMT was observed in the leaves, but the content of formononetin which is the product of isoflavone O-methyltransferase (IOMT) catalysis was higher in stems than in leaves. This research aims to further understand the acclimation of abiotic stress and the regulation mechanism of flavonoid accumulation in A. mongholicus.

Effects of Physical Properties and Processing Methods on Astragaloside IV and Flavonoids Content in Astragali radix.

The aim of this study was to investigate the effects of the physical properties (diameter size, powder particle size, composition of bark- and wood-tissue, and turnover rate) and processing methods on the content of active ingredients in Astragali radix (AR), a popular Chinese herbal medicine. The astragaloside IV and flavonoid contents increased with decreasing diameter size. Bark-tissue had significantly higher astragaloside IV and formononetin content than that in the wood-tissue. As a higher proportion of bark-tissue is associated with decreasing diameter, a strong correlation was also shown between bark- to wood-tissue ratio and active ingredients’ content. Furthermore, an increase in astragaloside IV content was observed in thin powder as compared to coarse powder ground from the whole root. However, this association between active ingredients’ content and powder particle size was abolished when isolating bark- and wood-tissue individually. Moreover, AR stir-frying with refined honey, a typical processing method of AR, increased formononetin content. The turnover rate of active constituents upon decoction ranged from 61.9–81.4%. Assessing the active constituent contents using its physical properties and processing methods allows for a more comprehensive understanding of optimizing and strengthening the therapeutic potentials of AR used in food and herbal supplements.

The Effects of the Trifolium Pratense L. Extract on the Leukemia Inhibitory Factor and its Receptor in The Endometrial Epithelial Cell Line

Isoflavones have attracted much notice due to their health advantages; however, a comprehensive understanding of the effects of isoflavones on endometrium biology remains undiscovered. The expression and deficiency of leukemia inhibitory factor (LIF) and LIF receptor (LIFR) has been shown to be involved in multiple implantations failures in female infertility. Mechanisms implicated in the failure of implantations require further researches, thus our aim is to investigate the effect of the Trifolium pratense L. isoflavone extract with abundant formononetin content on implantation through assessing LIF and LIFR expressions. The Ishikawa cells were cultured with 20, 30, and 40 µg/mL concentrations of Trifolium pratense L. isoflavone extracts for 24 h and detected staining intensity of LIF and LIFR by immunocytochemistry and immunofluorescence staining using image analysis software. As compared with the control and 20 µg/mL Trifolium pratense L. groups, the staining intensity of LIF and LIFR in 30 and 40 µg/mL Trifolium pratense L. groups were significantly increased ( P < .0001). Our findings suggest that Trifolium pratense L. isoflavone extract may alter the endometrium expression of LIF and LIFR in the human endometrial adenocarcinoma cell line.

FORMONONETIN INHIBITS PROLIFERATION OF ENDOMETRIOSIS VIA DOWN-REGULATION OF p27-PERK-pSTAT3-Twist1 PATHWAY

Formononetin is a phytoestrogen known to function as a selective estrogen receptor modulator. We aimed to evaluate the effect of formononetin on proliferation of endometriosis in this study We obtained eutopic endometrium from patients diagnosed endometriosis after surgery. To determine therapeutic dose of formononetin, the concentration in 70% of cells that survived when formononetin was administered was calculated through the CCK8 assay. While increasing the formononetin concentration up to the corresponding concentration, the target protein expression level of the endometriotic endometrium was measured by western blot. Statistical analysis was calculated using SPSS 25, IBM. Significant differences were assessed using Mann-Whitney tests. A p-value <0.05 was considered statistically significant We set the maximum concentration of formononetin administration to 80μM through the CCK8 assay. The expression levels of pAKT, pERK, p27, p53, and BAX proteins were evaluated by western blot by increasing formononetin in steps of 20μM. (N=4) In this experiment, the expression of pERK, twist1 decreased after 20μM, and p27, pSTAT3 decreased depending on the concentration increase. (p<0.05) On the other hand, pAKT, p53, and BAX did not show any significant difference. Formononetin could inhibit proliferation of endometriosis with dose dependent manner in vitro, with downregulation of p27, pERK, pSTAT3, and Twist1.

Isoflavones of the red and Hungarian clover and possible impact on animal diet

The content of daidzein, genistein, formononetin, and biochanin A isoflavones was studied in natural populations of red and Hungarian clover, to estimate their impact on fodder quality and to determine directions in possible breeding programs. The study included 6 red clover (Trifolium pratense) and 6 Hungarian clover (Trifolium pannonicum) populations, collected in the central Balkans. The differences between the species and among the populations were analysed. The average content of total isoflavones was 1.393 mg g–1 and 0.487 mg g–1 of air dry matter in Hungarian clover, respectively. While the most prevailed isoflavone in red clover was biochanin A (46%), the Hungarian clover populations were rich in genistein (43%). The red clover leaves accumulated the highest content of isoflavones. The Hungarian clover flowers and leaves had an equal amount of isoflavones. The obtained values of the total isoflavones could not affect the overall nutrient quality and therefore, researched natural populations of two clover species could be considered for further breeding programs.