Tetramethoxyflavone Research Articles

Proteomics-based network pharmacology and molecular docking reveal the potential mechanisms of 5,6,7,4′-tetramethoxyflavone against HeLa cancer cells

Recent research has highlighted the therapeutic potential of citrus-derived dietary 5,6,7,4′-tetramethoxyflavone (TMF) against HeLa cancer. Our study aims to elucidate its mechanisms of action through proteomics analysis, network pharmacology, and molecular docking. The results suggested that TMF demonstrated efficacy by upregulating CD40, CD40L, Fas, Fas-L, HSP27, HSP60, IGFBP-1, IGFBP-2, IGF-1sR, Livin, p21, p27, sTNFR2, TRAILR2, TRAILAR3, TRAILR4, XIAP, p-Sre, p-Stat1, p-Stat2 p-c-Fos, p-SMAD1, p-SMAD2, p-SMAD4, p-SMAD5, p-IκBα, p-MSK1, p-NFκB, p-TAK1, p-TBK1, p-ZAP70, and p-MSK2, while downregulating p-EGFR, p-ATF2, p-cJUN, p-HSP27, p-JNK, and p-GSK3A. These targets are primarily involved in MAPK, apoptosis, and TNF signaling pathways. Notably, p21, p27, EGFR, SMAD4, JNK, ATF2, and c-JUN merged as pivotal targets contributing to TMF's anti-cancer efficacy against HeLa cells. This study is first to delineate the potential signaling pathways and core targets of TMF in treating of HeLa cancer, paving the way for further exploration of TMF's medical potential.

Exploration of the mechanism of tetramethoxyflavone in treating osteoarthritis based on network pharmacology and molecular docking

ABSTRACT Objectives: This study aimed to explore the potential mechanisms of TMF (5,7,3’,4’-tetramethoxyflavone) in treating osteoarthritis (OA) using network pharmacology and molecular docking. Materials and Methods: Databases including SwissTargetPrediction, BATMAN-TCM, PharmMapper, TargetNet, SuperPred, and SEA were utilized to screen the targets of TMF. “OA” was used as the disease keyword to predict OA-related genes through GeneCards, Therapeutic Target Database, PharmGKB, Online Mendelian Inheritance in Man, and Comparative Toxicogenomics Database. The Venn diagram was employed to identify the intersection of predicted targets between TMF and OA as potential targets for TMF in treating OA. The intersection targets were input into the STRING 12.0 online database to construct a protein–protein interaction (PPI) network and identify core targets. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Metascape V3.5 online database platform. Finally, molecular docking between TMF and core targets was conducted using AutoDockTools 1.5.6. Results: A total of 228 intersection targets for TMF treating OA were obtained, and PPI network analysis identified 5 core targets: STAT3, SRC, CTNNB1, EGFR, and AKT1. GO enrichment analysis yielded 2736 results, while KEGG analysis identified 203 pathways. Most elated GO and KEGG items of TMF in treating OA may include hormonal responses, antiviral and anticancer effects, anti-inflammation, phosphorus metabolism, phosphate metabolism, nitrogen compound responses, cancer-related pathways, PI3K-Akt signaling pathway, and MAPK signaling pathway. Molecular docking revealed good binding affinities between TMF and all core targets except STAT3. Conclusion: TMF might act on multiple targets and activate diverse pathways to intervene in OA, revealing the molecular processes involved in TMF treatment of OA.

Chemical Composition and Therapeutic Potential of Syngonium podophyllum L. Leaves against Hypercholesterolemia in Rats: Liver, Kidney, and Heart Crosstalk

Background: One of the main risk factors for atherosclerosis is hypercholesterolemia Objective: This study aimed to assess hypercholesterolemia's effect on the liver, heart, and kidney and the impact of Syngonium podophyllum L. leaves methanolic extract as a treating agent in a rat model. Methods: Flavonoid components were isolated and identified from the methanolic extract of Syngonium podophyllum L. leaves. Total serum leptin, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), aspartate and alanine aminotransferases (AST and ALT), urea, and creatinine levels were all measured as part of the biochemical evaluation. The liver tissue was tested for levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and DNA fragmentation. Results: Thirty-nine compounds were identified by GC/MS profiling of the n-hexane fraction of Syngonium podophyllum L leaves. The major volatile constituents were decane, 4-methyl, decane, N-acetyl 3-pentenyl, 1-amine, 2-methyl, 1-hexene, and 3-hydroxy, propanenitrile, while the major phenolic compounds isolated from methanolic extract were luteolin-7- α-L rhamnoside-4'- O-β-glucopyranoside (1), apigenin 6, 8-di-C-β-glucopyranoside (vicenin 2) (2), quercetin-3-Oα-L-rhamnoside (3), quercetin-7-O-β-glucoside compound (4), luteolin-7-O-β-glucoside (5), 5- hydroxy-6,7,8,4'-tetramethoxy flavone (6), gallic acid (7) and quercetin (8). Hypercholesterolemic rats revealed significant alterations (p ≤ 0.05) in the lipid profile, liver and kidney function, DNA fragmentation pattern and antioxidant indices. With oral cholesterol administration of 30 mg/0.3 mL, 0.7% tween/rats fed a high-fat diet for nine weeks, treatment with leaves extract (250 mg/kg body weight) was able to restore all biochemical parameters as well as the architectures of the liver and heart. Conclusion: Due to its abundance in physiologically active phenolic and flavonoid components, the methanolic extract of Syngonium podophyllum L. leaves successfully served as a hypolipidemic, anti-atherosclerotic, and antioxidant therapeutic agent.

5,7,3′,4′-Tetramethoxyflavone suppresses TGF-β1-induced activation of murine fibroblasts in vitro and ameliorates bleomycin-induced pulmonary fibrosis in mice

Objective This study aimed to investigate the use of 5,7,3′,4′-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. In vitro and in vivo models were used to examine the impact of TMF on PF. Methods NIH-3T3 (Mouse Embryonic Fibroblast) were exposed to transforming growth factor‑β1 (TGF-β1) and treated with or without TMF. Cell growth was assessed using the MTT method, and cell migration was evaluated with the scratch wound assay. Protein and messenger ribonucleic acid (mRNA) levels of extracellular matrix (ECM) genes were analyzed by western blotting and quantitative reverse transcription-polymerase chain reaction (RT–PCR), respectively. Downstream molecules affected by TGF-β1 were examined by western blotting. In vivo, mice with bleomycin-induced PF were treated with TMF, and lung tissues were analyzed with staining techniques. Results The in vitro results showed that TMF had no significant impact on cell growth or migration. However, it effectively inhibited myofibroblast activation and ECM production induced by TGF-β1 in NIH-3T3 cells. This inhibition was achieved by suppressing various signaling pathways, including Smad, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT (PI3K/AKT), and WNT/β-catenin. The in vivo experiments demonstrated the therapeutic potential of TMF in reducing PF induced by bleomycin in mice, and there was no significant liver or kidney toxicity observed. Conclusion These findings suggest that TMF has the potential to effectively inhibit myofibroblast activation and could be a promising treatment for PF. TMF achieves this inhibitory effect by targeting TGF-β1/Smad and non-Smad pathways.

TMF suppresses chondrocyte hypertrophy in osteoarthritic cartilage by mediating the FOXO3a/BMPER pathway.

Osteoarthritis (OA) is a disease of the joints, characterized by chronic inflammation, cartilage destruction and extracellular matrix (ECM) remodeling. Aberrant chondrocyte hypertrophy promotes cartilage destruction and OA development. Collagen X, the biomarker of chondrocyte hypertrophy, is upregulated by runt-related transcription factor 2 (Runx2), which is mediated by the bone morphogenetic protein 4 (BMP4)/Smad1 signaling pathway. BMP binding endothelial regulator (BMPER), a secreted glycoprotein, acts as an agonist of BMP4. 5,7,3',4'-tetramethoxyflavone (TMF) is a natural flavonoid derived from Murraya exotica L. Results of our previous study demonstrated that TMF exhibits chondroprotective effects against OA development through the activation of Forkhead box protein O3a (FOXO3a) expression. However, whether TMF suppresses chondrocyte hypertrophy through activation of FOXO3a expression and inhibition of BMPER/BMP4/Smad1 signaling remains unknown. Results of the present study revealed that TMF inhibited collagen X and Runx2 expression, inhibited BMPER/BMP4/Smad1 signaling, and activated FOXO3a expression; thus, protecting against chondrocyte hypertrophy and OA development. However, BMPER overexpression and FOXO3a knockdown impacted the protective effects of TMF. Thus, TMF inhibited chondrocyte hypertrophy in OA cartilage through mediating the FOXO3a/BMPER signaling pathway.

TMF inhibits extracellular matrix degradation by regulating the C/EBPβ/ADAMTS5 signaling pathway in osteoarthritis

Osteoarthritis (OA) is a chronic joint disease, characterized by degenerative destruction of articular cartilage. Chondrocytes, the unique cell type in cartilage, mediate the metabolism of extracellular matrix (ECM), which is mainly constituted by aggrecan and type II collagen. A disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5) is an aggrecanase responsible for the degradation of aggrecan in OA cartilage. CCAAT/enhancer binding protein β (C/EBPβ), a transcription factor in the C/EBP family, has been reported to mediate the expression of ADAMTS5. Our previous study showed that 5,7,3’,4’-tetramethoxyflavone (TMF) could activate the Sirt1/FOXO3a signaling in OA chondrocytes. However, whether TMF protected against ECM degradation by down-regulating C/EBPβ expression was unknown. In this study, we found that aggrecan expression was down-regulated, and ADAMTS5 expression was up-regulated. Knockdown of C/EBPβ could up-regulate aggrecan expression and down-regulate ADAMTS5 expression in IL-1β-treated C28/I2 cells. TMF could compromise the effects of C/EBPβ on OA chondrocytes by activating the Sirt1/FOXO3a signaling. Conclusively, TMF exhibited protective activity against ECM degradation by mediating the Sirt1/FOXO3a/C/EBPβ pathway in OA chondrocytes.

Suppression of HCT116 Human Colon Cancer Cell Motility by Polymethoxyflavones is Associated with Inhibition of Wnt/β-Catenin Signaling

Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). In this study, we examined the effect of polymethoxyflavones present in citrus peels on Wnt/β-catenin signaling in the HCT116 CRC cell line. We found that 5,7,3′,4′-tetra-methoxyflavone (TMF) and 7,8,3′,4′-TMF inhibited the expression of target genes of Wnt/β-catenin signaling and the transcriptional activities of β-catenin/Tcf and suppressed the motility of HCT116 cells. Because the binding of β-catenin to Tcf-4 was disrupted by 5,7,3′,4′-TMF and 7,8,3′,4′- TMF, we suggest that they are inhibitors of the Wnt/β-catenin signaling and may have potential applications in CRC prevention.

Flavonoid glycosides and other bioactive compounds in Citrus reticulate ‘Chachi’ peel analysed by tandem mass spectrometry and their changes during storage

The peel of Citrus reticulate ‘Chachiennsis’ (Chachi) is a well-known functional food with multiple health benefits in Asia. There is an old saying “the longer time Chachi is stored, the better health benefits it has”. Is it convincible? What are the critical bioactive compounds in Chachi? To answer these questions, gas chromatography-mass spectrometry (GC-MS) and ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were used to qualify and quantify the flavonoid glycosides and other bioactive compounds of Chachi with storage time of 5–20 years. Limonene was the representative volatile compound. The level of most volatile compounds decreased along with storage. Sixteen flavonoids glycosides and twenty flavonoids were identified. Nobiletin, hesperitin, tetramethoxy flavone and pentamethoxy flavone were characteristic bioactive compounds for Chachi. Most of them accumulated during 10-year storage, thereafter decreased. Ten years could be the optimal storage time. These results indicated that the old saying should be corrected.

5,7,3',4'-tetramethoxyflavone amelioratescholesterol dysregulation by mediating SIRT1/FOXO3a/ABCA1 signaling in osteoarthritis chondrocytes.

Dyslipidemia has been associated with the development ofosteoarthritis. Our previous study found that 5,7,3',4'-tetramethoxyflavone (TMF) exhibited protective activities against thepathological changes of osteoarthritis. Aim: To investigate the roles of TMF in regulating ABCA1-mediated cholesterol metabolism. Methods: Knockdown and overexpression were employed to study gene functions. Protein-protein interaction was investigated by co-immunoprecipitation, and the subcellular locations of proteins were studied by immunofluorescence. Results: IL-1β decreased ABCA1 expression and induced apoptosis. Therapeutically, TMF ameliorated the effects of IL-1β. FOXO3a knockdown expression abrogated the effects of TMF, and FOXO3a overexpression increased ABCA1 expression by interacting with LXRα. TMF promoted FOXO3a nuclear translocation by activating SIRT1 expression. Conclusions: TMF amelioratescholesterol dysregulation by increasing the expression of FOXO3a/LXRα/ABCA1 signaling through SIRT1 in C28/I2 cells.

Development of Orthosiphon stamineus ethanolic solid dispersions for solubility improvements of lipophilic flavones

This study was conducted to develop an ethanolic solid dispersion of Orthosiphon stamineus (ESD) using polymers as carriers, namely polyvinylpyrrolidone (PVP), poloxamer 188 (P188), and poloxamer 407 (P407) via the solvent evaporation method. The purpose of preparing the formulation is to improve the solubility of the lipophilic flavones, namely sinensetin (SIN), eupatorine (EUP), and 3′-hydroxy-5, 6, 7, 4′-tetramethoxyflavone (TMF) and caffeic acid derivatives, namely rosmarinic acid (RA). The optimized ESD was characterized using High-Performance Liquid Chromatography (HPLC), Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) fingerprints, and physicochemical methods (particle size, zeta potential, TEM, and SEM). The effect of pH on stability and solubility in buffer and water, in-vitro release, and antioxidant properties (DPPH assay) indicated that the nano-formulation ESD using polymers (PVP/P407) with a ratio of extract to polymers (1.0:1.1:0.3 w/w) enhanced the lipophilic flavones (TMF=3.56 ± 0.01% w/w, SIN=2.46 ± 0.01% w/w and EUP=7.87 ± 0.01% w/w) and RA (20.66 ± 0.01% w/w) compared to the same compounds in ethanolic extract (P< 0.0001) with particles size less than 200 nm. In conclusion, the successful development of ESD using water-soluble copolymers (PVP/P407) has enhanced the solubility of lipophilic flavones and other compounds (RA), thereby further improving its pharmacological properties.

Accelerated stability study of Orthosiphon stamineus standardised ethanolic extract and its solid dispersion

The objective of the present study is to develop accelerated stability of Orthosiphon stamineus standardised ethanolic extract (SEE) and its solid dispersion (ESD). The stability study of SEE and ESD has been performed using high-performance liquid chromatography (HPLC) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analyses. The spectroscopic datasets of ESD were applied to the principal component analysis (PCA) to extract the maximum information of the ATR-FTIR spectra. SEE and ESD were stored at three different temperatures with two different humidity conditions (30 °C/75% RH, 40 °C/75% RH and 60 °C/85% RH) for six months. Overall, the degradation of marker compounds; rosmarinic acid (RA), 3’-hydroxy-5, 6, 7, 4’-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) at high temperature (60 °C/85% RH) was higher compared to low temperature (30 °C/75% RH) for both samples. Moreover, the degradation of RA, TMF, SIN and EUP in ESD was slower compared to SEE. The deterioration of marker compounds for both samples followed the first-order reaction kinetics. The shelf life of SEE and ESD is based on the estimated shelf life RA, TMF, SIN, and EUP present in the samples. The shelf life of RA, TMF, SIN, and EUP in ESD were significantly enhanced (p < 0.001) compared to the same markers in SEE with EUP was showing the highest shelf life (15 months), while RA showed the lowest shelf life (7 months) when stored at the temperature below 30 °C. The shelf life of all marker compounds in SEE was less than two months when stored at the same temperature (below 30 °C). Based on ATR-FTIR fingerprinting datasets analysed with PCA, ESD kept at 30 °C/75% RH were still preserved of its chemical properties, which indicates that low temperature is better to keep the formulation.

Complexity of active medicinal ingredients in radix scutellariae with sodium hydrosulfite exposure.

Both plants and animals are living things made up of similar cells as well as organelles, and their essence of life is the same. However, plants face more environmental stress than animals and generate excessive reactive oxygen species (ROS), a group of small molecules that can harm proteins, necessitating distinctive metabolic processes. Secondary metabolites in plants are a group of chemical components that can eliminate ROS and can also exhibit medicinal properties; therefore, herbal medicines are often closely linked to the ecological significance of secondary metabolites. Why plants contain so many, not few, active medicinal ingredients is unknown. The root of Scutellaria baicalensis, a popular herbal medicine, is rich in various flavonoids with diverse structural features. Sodium hydrosulfite (Na2S2O4) can produce O˙-2 radicals and induce physical conditions under environmental stress. Using UHPLC-ESI-Q-TOF-MS/MS analysis, a total of 25 different compounds were identified in the roots of S. baicalensis between the Na2S2O4 groups and suitable conditions. Based on the results of the t-test (P<0.05) performed for the groups and ions with values of VIP ≥ 2, the most significantly different chemical markers with Na2S2O4 treatment were shikimic acid, citric acid, baicalin, wogonoside, baicalein, wogonin, 3,5,7,2',6'-pentahydroxyflavanone, 5,2',6'-trihydroxy-7,8-dimethoxy flavone, chrysin, eriodictyol, 5,8-dihydroxy-6,7 -dimethoxy flavone, skullcapflavone Ⅱ, and 5,7-dihydroxy-6,8,2',3'-tetrame thoxyflavone, and most of them were free flavonoids with many phenolic hydroxyl or methoxyl groups and characteristically high antioxidant activities. S. baicalensis roots modified their ability to eliminate ROS and maintained the equilibrium of ROS through the multitudinous biosynthesis and conversion of flavonoids, which is similar to the equilibrium established by an intricate buffer solution and perfectly explains the diversity and complexity of medicinal plant ingredients.

FORMULATION OF AN ANTI-BACTERIAL CREAM FROM PLANTOXALIS CORNICULATA ANDITS EVALUATION

Objective: Even in areas where modern medicine is available, the interest on herbal medicines and their utilization have been increasing rapidly in recent years. Plant-derived substances and herbal medicines have recently attracted the great interest towards their versatile application as medical plants are the rich source of bioactive compounds used in traditional and modern medicine. The present work is to formulate and evaluate the antibacterial cream of oxalis corniculata extract.&#x0D; Methods: The ethanolic extracts were prepared by using the maceration method.&#x0D; Results: The agrochemical potential of methanolic extract, n-hexane, chloroform, ethyl acetate,and n-butanol soluble sfractions showed excellent activites against Escherichia coli, Shigella dysenteriae, Salmonella typhi, and Bacillus subtilis. Similarly the crude n-hexane and chloroform fractions were also found to have significant activity against fungal strains including Fusarium solani, Aspergillus flexneri, and Aspergillus flavus.&#x0D; Conclusion: Oxalis corniculata is a common medicinal plant widely used against numerous infectious diseases. The two isolated compounds 5-hydroxy-6,7,8,4’-tetra methoxy flavone and 5,7,4’-trihydroxy-6,8-dimethoxyflavone were evaluated for antibacterial and antifungal activities. The results showed that latter compound was more active than that of the former.

Flavonoid Calycopterin Induces Apoptosis in Human Prostate Cancer Cells In-vitro.

Prostate cancer is enumerated as one of the most prevalent cancers in men, with a mortality rate of 18%. Chemotherapy is considered as a common strategy for cancer treatment; however, toxic side effects and drug resistance associated with chemotherapy are major drawbacks with this approach. It is well known that a diet rich in flavonoids can reduce the incidence of many types of cancer in a significant manner, and it was proved that methoxy flavones have greater bioavailability compared to the nonmethylated ones. Calycopterin is a tetramethoxy flavone which was demonstrated to have anti-proliferative effects on colon, gastric, and osteosarcoma cancer cells. Therefore, in the current study, we have evaluated the apoptotic effects of this flavonoid on two prostate cancer cell lines in-vitro. The MTT assay revealed that after 48 h treatment with this flavonoid, cell viability reduced to 50% compared to the control group. However, calycopterin treatment of healthy HUVEC did not cause any significant reduction in cell viability. Moreover, the clonogenic assay demonstrated that after 14 days, colony size and numbers reduced significantly in calycopterin treated cells. In addition, the percentage of the sub-G1 population in calycopterin-treated cells augmented significantly compared to untreated group. Also, calycopterin-treated cells demonstrated shiny condensed nuclei with fragmented DNA indicative of apoptosis. Finally, a significant reduction in the migration ability was observed in both lines subjected to calycopterin after 48 h. To conclude, our results demonstrated the apoptotic and anti-metastatic effects of calycopterin in both hormone-dependent and independent prostate cancer cell lines.

Pharmacognostical, Phytochemical and Pharmacological profile of Colebrookea oppositifolia Smith

Colebrookea oppositifolia commonly known as ‘Bhaman’ is distributed throughout India from the Himalayas down to Deccan. The plant is used traditionally as such as dermatitis, dysentery, fever, headache, peptic ulcer, haemostatic, wounds, as anti-fertility agent, fungicide, and the roots of the plant has been most widely used for the treatment of epilepsy.. Medicinally, it has been proven to possess various pharmacological activities like treating corneal opacity or conjunctivitis, sore eyes due to its anti-inflammatory properties, cardioprotective, hepatoprotective, anti-inflammatory, antihelmintic, antifungal, antioxidant, antimicrobial, antinociceptive, cytotoxic activity, anticonvulsant, antiulcer, antimicrobial, anti-fertility, antipyretic and insecticide. Further, studies reveal the presence of various phytochemical constituents mainly flavone glycosides viz. chrysin, negletein, landenein; leaves contain 5,6,7- tri-methoxyflavone, 5,6,7,4'-tetramethoxyflavone, acteoside, and quercetin in the bark; root contains stearic, palmitic, oleic acids, triacontanol, flavone glycoside echioidin, 5,6,7-trimethoxyflavone and 4',5,6,7- tetra methoxy flavone; sugars and vitamins have also been isolated from this plant. These studies reveal that Colebrookea oppositifolia is a source of medicinally active compounds and have various pharmacological effects; hence, this drug encourage finding its new therapeutic uses.&#x0D; Keywords: Colebrookea oppositifolia, wound healing, anticonvulsant, Lamiaceae

TMF inhibits miR-29a/Wnt/β-catenin signaling through upregulating Foxo3a activity in osteoarthritis chondrocytes.

Background: miR-29a, a downstream factor of Wnt/β-catenin signaling, promotes the activity of the Wnt/β-catenin signaling in a positive feedback loop. Our previous work showed that 5,7,3ʹ,4ʹ-tetramethoxyflavone (TMF), a major constituent from Murraya exotica L., exhibited chondroprotective activity by inhibiting the activity of Wnt/β-catenin signaling.Purpose: To investigate whether TMF showed the inhibitory effects on miR-29a/β-catenin signaling by up regulation of Foxo3a expression.Methods: Rat knee OA models were duplicated by using Hulth’s method. TMF (5 μg/mL and 20 μg/mL) was used for administration to cultured cells, which were isolated from the rat cartilages. Analysis of chondrocytes apoptosis, gene expression, and protein expression were conducted. In addition, miR-29a mimics and pcDNA3.1(+)-Foxo3a vector were used for transfection, luciferase reporter assay for detecting the activity of Wnt/β-catenin signaling, and co-immunoprecipitation for determining proteins interaction.Results: TMF down regulated miR-29a/β-catenin signaling activity and cleaved caspase-3 expression and up regulated Foxo3a expression in OA rat cartilages. In vitro, miR-29a mimics down regulated the expression of Foxo3a and up regulated the activity of Wnt/β-catenin signaling and cleaved caspase-3 expression. TMF ameliorated miR-29a/β-catenin-induced chondrocytes apoptosis by up regulation of Foxo3a expression.Conclusion: TMF exhibited chondroprotective activity by up regulating Foxo3a expression and subsequently inhibiting miR-29a/Wnt/β-catenin signaling activity.

PHYTOCHEMICAL STUDY OF BIOACTIVE CONSTITUENTS FROM SATUREJA MONTANA L. GROWING IN EGYPT AND THEIR ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES

Objective: This work aimed to investigate the lipid constituents and flavonoidal compounds of Satureja montana, in addition to evaluation of different extracts and/or isolated compounds as antimicrobials and antioxidants.Methods: The volatile and lipid constituents were extracted with n-hexane by partition from hydroalcoholic extract of S. montana L. aerial parts, after then were fractionated to unsaponifiable matters and fatty acid methyl esters which were identified by gas–liquid chromatography and/or gas chromatography–mass spectrometry. The phenolic constituents were isolated from the ethyl acetate fraction of the aqueous methanolic extract of the aerial parts of the plant. The antimicrobial activity of different extracts and the isolated compounds was evaluated against Gram-positive, Gram-negative bacteria, yeast, and fungus using a modified Kirby-Bauer disc diffusion method.Results: The identified compounds are luteolin-7-rhamnoside-4’-O-β-glucopyranoside (1), quercetin-3-O-α-L-rhamnopyranoside (2), quercetin- 7-O-glucopyranoside (3), luteolin-7-O-glucopyranoside (4), 5-hydroxy-6,7,8,4’-tetramethoxy flavone (5), gallic acid (6), 2,3-hexahydroxydiphenoyl 1-galloyl glucopyranoside (7), and quercetin (8). The structure of all isolated compounds was established using different chromatographic and spectroscopic measurements (PC, thin-layer chromatography, ultraviolet [UV], 1D, 2D-nuclear magnetic resonance, and MS). Compound-2 showed the highest antibacterial activity against all the tested microorganisms. Hydroalcoholic extract exhibited high antioxidant activity (87.7%). On the other hand, hexane fraction showed a low antioxidant activity (46.4%), in addition to the compound-8 showed the highest antioxidant activity (96.27%) in 2,2-diphenyl-1-picrylhydrazyl assay.Conclusion: It can be concluded that the hydroalcoholic extract of S. montana showed significant antimicrobial and antioxidant activity.

Mechanism of vasorelaxation induced by 3'-hydroxy-5,6,7,4'-tetramethoxyflavone in the rats aortic ring assay.

Previous studies have demonstrated that 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF) content in Orthosiphon stamineus fractions correlate with its vasorelaxation activity. Even with the availability of previous studies, there is still very little information on the vasorelaxation effect of TMF, and few scientific studies have been carried out. Therefore, the present study was designed to investigate the vasorelaxation activity and mechanism of action of the TMF. The vasorelaxation activity and the underlying mechanisms of TMF were evaluated on thoracic aortic rings isolated from Sprague Dawley rats. TMF caused the relaxation of aortic rings with endothelium pre-contracted with phenylephrine. However, the vasorelaxant effect of TMF was significantly decreased in PE-primed endothelium-denuded and potassium chloride-primed endothelium-intact aortic rings. In the presence of Nω-nitro-L-arginine methyl ester, methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, indomethacin, tetraethylammonium, 4-aminopyridine, barium chloride, atropine and propranolol, the relaxation stimulated by TMF was significantly reduced. TMF was also found to reduce Ca2+ release from sarcoplasmic reticulum (via IP3R) and block calcium channels (VOCC). The present study demonstrates the vasorelaxant effect of TMF involves NO/sGC/cGMP and prostacyclin pathways, calcium and potassium channels and muscarinic and beta-adrenergic receptors.

Effects of Polymethoxyflavonoids on Bone Loss Induced by Estrogen Deficiency and by LPS-Dependent Inflammation in Mice.

Polymethoxyflavonoids (PMFs) are a family of the natural compounds that mainly compise nobiletin, tangeretin, heptamethoxyflavone (HMF), and tetramethoxyflavone (TMF) in citrus fruits. PMFs have shown various biological functions, including anti-oxidative effects. We previously showed that nobiletin, tangeretin, and HMF all inhibited interleukin (IL)-1-mediated osteoclast differentiation via the inhibition of prostaglandin E2 synthesis. In this study, we created an original mixture of PMFs (nobiletin, tangeretin, HMF, and TMF) and examined whether or not PMFs exhibit co-operative inhibitory effects on osteoclastogenesis and bone resorption. In a coculture of bone marrow cells and osteoblasts, PMFs dose-dependently inhibited IL-1-induced osteoclast differentiation and bone resorption. The optimum concentration of PMFs was lower than that of nobiletin alone in the suppression of osteoclast differentiation, suggesting that the potency of PMFs was stronger than that of nobiletin in vitro. The oral administration of PMFs recovered the femoral bone loss induced by estrogen deficiency in ovariectomized mice. We further tested the effects of PMFs on lipopolysaccharide-induced bone resorption in mouse alveolar bone. In an ex vivo experimental model for periodontitis, PMFs significantly suppressed the bone-resorbing activity in organ cultures of mouse alveolar bone. These results indicate that a mixture of purified nobiletin, tangeretin, HMF, and TMF exhibits a co-operative inhibitory effect for the protection against bone loss in a mouse model of bone disease, suggesting that PMFs may be potential candidates for the prevention of bone resorption diseases, such as osteoporosis and periodontitis.

5,7,3′,4′-Tetramethoxyflavone protects chondrocytes from ER stress-induced apoptosis through regulation of the IRE1α pathway

ABSTRACTAim of the study: To investigate the roles of endoplasmic reticulum (ER) transmembrane sensor inositol-requiring enzyme-1 (IRE1)α signaling in ER stress-induced chondrocyte apoptosis, and to determine the molecular mechanisms underlying chondroprotective activity of 5,7,3′,4′-tetramethoxyflavone (TMF) from Murraya exotica. Materials and methods: IRE1α was knocked down by siRNA transfection in chondrocytes, which were harvested from rats’ knee cartilages. Chondrocytes with IRE1α deficiency were administrated with tunicamycin (TM) and TMF. Chondrocyte apoptosis was quantified by flow cytometry and DAPI/TUNEL staining. Expression of mRNA and proteins was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western-blot, respectively. Results: IRE1α deficiency significantly increased the rate of TM-induced chondrocyte apoptosis, down-regulated the expression of pro-survival factors XBP1S and Bcl-2, and up-regulated pro-apoptotic factors CHOP, p-JNK, and caspase-3. TMF suppressed TM-induced chondrocyte apoptosis by activating the expression of IRE1α, which reversed the expression patterns of downstream pro-survival and pro-apoptotic factors due to IRE1α deficiency. Conclusion: The mechanism of TMF in protecting chondrocytes against ER stress-induced apoptosis might be associated with regulating the activity of ER sensor IRE1α and its downstream pathway.