5,7-Dimethoxyflavone induces apoptotic cell death in human endometriosis cell lines by activating the endoplasmic reticulum stress pathway.

Endometriosis is a reproductive disorder characterized by the dislocation of endometrial tissues. Approximately 5-20% of women at their reproductive age are diagnosed with endometriosis, which causes chronic pain and infertility. Here, we demonstrated that the bioactive flavonoid, 5,7-dimethoxyflavone (DMF), exhibited antiproliferative and apoptotic effects in VK2/E6E7 and End1/E6E7 cells which were established from vaginal and endocervical tissue taken from a premenopausal woman undergoing hysterectomy for endometriosis. DMF treatment significantly elevated DNA fragmentation resulting in apoptotic cell death in both cell lines. Furthermore, DMF induced loss of mitochondrial membrane potential, dysregulation of intracellular calcium level, and ROS production, which accelerate apoptosis. Additionally, DMF modulated the expression of the signaling molecules related to cell survival and endoplasmic reticulum stress in VK2/E6E7 and End1/E6E7 cells. Overall, DMF may ameliorate endometriosis and can be a potential alternative to hormonal and surgical therapy for endometriosis treatment.

The anti-cancer effect of epigallocatechin-3-O-gallate against multiple myeloma cells is potentiated by 5,7-dimethoxyflavone.

(-)-Epigallocatechin-3-O-gallate (EGCG) is one of the major components of green tea polyphenol. Previous studies have shown that EGCG induces cancer-specific cell death in vitro and in vivo without causing severe side effects. However, the anti-cancer effect of EGCG alone is limited. 5,7-dimethoxyflavone (5,7-DMF), one of the principal functional components of black ginger (Kaempferia parviflora), also exerts anti-cancer effects. Here, we show that 5,7-DMF synergistically enhances the anti-cancer effect of EGCG in multiple myeloma cells by potentiating EGCG-induced intracellular cyclic guanosine monophosphate (cGMP) production. Moreover, the combination of EGCG and 5,7-DMF induces apoptotic cell death in multiple myeloma cells, and this is accompanied by activation of the cGMP/acid sphingomyelinase (ASM)/cleaved caspase-3 pathway. In conclusion, we have shown that 5,7-DMF enhances the anti-cancer effect of EGCG by upregulating cGMP in multiple myeloma cells.

Biotransformation of 5,7-Methoxyflavones by Selected Entomopathogenic Filamentous Fungi.

5,7-Dimethoxyflavone, a chrysin derivative, occurs in many plants and shows very low toxicity, even at high doses. On the basis of this phenomenon, we biotransformed a series of methoxy-derivatives of chrysin, apigenin, and tricetin obtained by chemical synthesis. We used entomopathogenic fungal strains with the confirmed ability of simultaneous hydroxylation/demethylation and glycosylation of flavonoid compounds. Both the amount and the place of attachment of the methoxy group influenced the biotransformation rate and the product's amount nascent. Based on product and semi-product structures, it can be concluded that they are the result of cascading transformations. Only in the case of 5,7,3',4',5'-pentamethoxyflavone, the strains were able to attach a sugar molecule in place of the methoxy substituent to give 3'-O-ß-d-(4″-O-methylglucopyranosyl)-5,7,4',5'-tetramethoxyflavone. However, we observed the tested strains' ability to selectively demethylate/hydroxylate the carbon C-3' and C-4' of ring B of the substrates used. The structures of four hydroxyl-derivatives were determined: 4'-hydroxy-5,7-dimethoxyflavone, 3'-hydroxy-5,7-dimethoxyflavone, 3'-hydroxy-5,7,4',5'-tetramethoxyflavone, and 5,7-dimethoxy-3',4'-dihydroxyflavone (5,7-dimethoxy-luteolin).

The 5,7-Dimethoxyflavone Suppresses Sarcopenia by Regulating Protein Turnover and Mitochondria Biogenesis-Related Pathways.

Sarcopenia is a muscle disease featured by the loss of muscle mass and dysfunction with advancing age. The 5,7-dimethoxyflavone (DMF), a major flavone found in Kaempferia parviflora, has biological activities, including anti-diabetes, anti-obesity, and anti-inflammation. However, its anti-sarcopenic effect remains to be elucidated. This current study investigated the inhibitory activity of DMF on sarcopenia. Eighteen-month-old mice were orally administered DMF at the dose of 25 mg·kg-1·day-1 or 50 mg·kg-1·day-1 for 8 weeks. DMF not only stimulated grip strength and exercise endurance but also increased muscle mass and volume. Besides, DMF stimulated the phosphatidylinositol 3-kinase-Akt pathway, consequently activating the mammalian target of rapamycin-eukaryotic initiation factor 4E-binding protein 1-70-kDa ribosomal protein S6 kinase pathway for protein synthesis. DMF reduced the mRNA expression of E3 ubiquitin ligase- and autophagy-lysosomal-related genes involved in proteolysis via the phosphorylation of Forkhead box O3. DMF upregulated peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, nuclear respiratory factor 1, and mitochondrial transcription factor A along with the increase of relative mitochondrial DNA content. DMF alleviated inflammatory responses by reducing the tumor necrosis factor-alpha and interleukin-6 serum and mRNA levels. Collectively, DMF can be used as a natural agent to inhibit sarcopenia via improving protein turnover and mitochondria function.

Effect of the active ingredient of Kaempferia parviflora, 5,7-dimethoxyflavone, on the pharmacokinetics of midazolam.

5,7-Dimethoxyflavone (5,7-DMF), one of the major components of Kaempferia parviflora, has anti-obesity, anti-inflammatory, and antineoplastic effects. On the other hand, in vitro studies have reported that it directly inhibits the drug metabolizing enzyme family cytochrome P450 (CYP) 3As. In this study, its safety was evaluated from a pharmacokinetic point of view, based on daily ingestion of 5,7-DMF. Midazolam, a substrate of CYP3As, was orally administered to mice treated with 5,7-DMF for 10 days, and its pharmacokinetic properties were investigated. In the group administered 5,7-DMF, the area under the curve (AUC) of midazolam increased by 130% and its biological half-life was extended by approximately 100 min compared to the control group. Compared to the control group, 5,7-DMF markedly decreased the expression of CYP3A11 and CYP3A25 in the liver. These results suggest that continued ingestion of 5,7-DMF decreases the expression of CYP3As in the liver, consequently increasing the blood concentrations of drugs metabolized by CYP3As.

Effect of 5,7-dimethoxyflavone on Bcrp1-mediated transport of sorafenib in vitro and in vivo in mice.

Tyrosine kinase inhibitors (TKI) are a novel and target-specific class of anticancer drugs. One drawback of TKI therapy is cancer resistance to TKI. An important TKI resistance mechanism is enhanced efflux of TKI by efflux transporters, such as Breast Cancer Resistance Protein (BCRP), in cancer cells. 5,7-Dimethoxyflavone (5,7-DMF) is a natural flavonoid which was recently reported to be a potent BCRP inhibitor. In the current study, the effect of 5,7-DMF on the disposition of sorafenib, a TKI which is a good substrate of BCRP, was investigated both in vitro in efflux transporter expressing cells and in vivo in mice. 5,7-DMF significantly inhibited Bcrp1-mediated sorafenib efflux in a concentration dependent manner in MDCK/Bcrp1 cells, with EC50 value of 8.78 µM. The pharmacokinetics and tissue distribution of sorafenib (10 mg/kg) with and without co-administration of 75 mg/kg 5,7-DMF were determined. With 5,7-DMF, the AUC of sorafenib in plasma was 47,400 ±â€¯4790 ng·h/mL, which was significantly higher than 27,300 ±â€¯2650 ng·h/mL in sorafenib alone group. In addition, compared to sorafenib alone group, great increase in sorafenib AUC was observed in most tissues collected when sorafenib was given with 5,7-DMF. Our results indicated that 5,7-DMF may represent a novel and very promising chemosensitizing agent for BCRP-mediated anticancer drug resistance due to its low toxicity and potent BCRP inhibition.

Quality evaluation of Kaempferia parviflora rhizome with reference to 5,7-dimethoxyflavone.

Kaempferia parviflora Wall. ex Baker is a medicinal plant found in the upper Northeastern regions of Thailand, which belongs to Zingiberaceae family. The present study aims to investigate the standardization parameters, to analyze chemical constituents of volatile oil by gas chromatography-mass spectrometry, and to determine the content of 5,7-dimethoxyflavone in K. parviflora rhizomes by thin-layer chromatography (TLC)-densitometry compared to TLC image analysis. K. parviflora rhizomes from 15 different sources throughout Thailand were investigated for morphological and pharmacognostic parameters. 5,7-Dimethoxyflavone contents were determined by TLC-densitometry with winCATS software and TLC image analysis with ImageJ software. The mobile phase for TLC development consisted of toluene: chloroform: Acetone: formic acid (5: 4: 1: 0.2). For the Results, the pharmacognostic parameters of K. parviflora rhizome were demonstrated. The loss on drying, total ash, acid-insoluble ash, water content, volatile oil content, ethanol, and water-soluble extractive values were found to be 8.979 ± 0.041, 5.127 ± 0.060, 2.174 ± 0.092, 9.291 ± 0.458, 0.028 ± 0.003, 5.138 ± 0.092, and 8.254 ± 0.191 g/100 g of dry weight, respectively. K. parviflora volatile oil showed the major components of α-copaene, dauca-5, 8-diene, camphene, ß-pinene, borneol, and linalool. The 5,7-dimethoxyflavone content of K. parviflora rhizomes determined by TLC-densitometry and TLC image analysis were found to be 2.15 ± 0.64 and 1.96 ± 0.51 g/100 g of dry rhizomes, respectively. The 5,7-dimethoxyflavone contents of both methods were not significantly different (P > 0.05) using paired t-test.

6-Hydroxy-5,7-dimethoxy-flavone suppresses the neutrophil respiratory burst via selective PDE4 inhibition to ameliorate acute lung injury.

Over-activated neutrophils produce enormous oxidative stress and play a key role in the development of acute and chronic inflammatory diseases. 6-Hydroxy-5,7-dimethoxy-flavone (UFM24), a flavone isolated from the Annonaceae Uvaria flexuosa, showed inhibitory effects on human neutrophil activation and salutary effects on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. UFM24 potently inhibited superoxide anion (O2•-) generation, reactive oxidants, and CD11b expression, but not elastase release, in N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils. However, UFM24 failed to scavenge O2•- and inhibit the activity of subcellular NADPH oxidase. fMLF-induced phosphorylation of protein kinase B (Akt) was inhibited by UFM24. Noticeably, UFM24 increased cyclic adenosine monophosphate (cAMP) concentration and protein kinase (PK) A activity in activated human neutrophils. PKA inhibitors significantly reversed the inhibitory effects of UFM24, suggesting that the effects of UFM24 were through cAMP/PKA-dependent inhibition of Akt activation. Additionally, activity of cAMP-related phosphodiesterase (PDE)4, but not PDE3 or PDE7, was significantly reduced by UFM24. Furthermore, UFM24 attenuated neutrophil infiltration, myeloperoxidase activity, and pulmonary edema in LPS-induced ALI in mice. In conclusion, our data demonstrated that UFM24 inhibits oxidative burst in human neutrophils through inhibition of PDE4 activity. UFM24 also exhibited significant protection against endotoxin-induced ALI in mice. UFM24 has potential as an anti-inflammatory agent for treating neutrophilic lung damage.

Pharmacokinetics and tissue distribution of 5,7-dimethoxyflavone in mice following single dose oral administration.

5,7-Dimethoxyflavone (5,7-DMF) is a major active constituent of many herbal plants, such as Kaempferia paviflora, Piper caninum, and Leptospermum scoparium. 5,7-DMF has demonstrated many beneficial pharmacological effects in vitro, including anti-infammatory, anti-oxidant, cardioprotection effects, as well as chemopreventive and chemosensitizing properties. In contrast to the extensive in vitro investigations, the information of the pharmacokinetic (PK) profile of 5,7-DMF in vivo is very limited. In this study we investigated the PK and tissue distribution of 5,7-DMF in mice following single oral dose of 10mg/kg 5,7-DMF. Mouse plasma, heart, lung, liver, kidney, intestine, brain, spleen, muscle and fat tissues were collected and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Maximal 5,7-DMF concentrations in plasma and tissues were reached within 30min. The peak plasma concentration (Cmax) was 1870±1190ng/mL, and area under the curve (AUCt) was 532±165hng/mL and terminal half-life was 3.40±2.80h. The volume of distribution was 90.1±62.0L/kg. Clearance was 20.2±7.5L/h/kg. Except for muscle and adipose, other tissues had higher Cmax than plasma, ranging from 1.75- to 9.96-fold. After oral administration, 5,7-DMF was most abundant in gut, followed by liver, kidney, brain, spleen, heart, lung, adipose and muscle. The partition coefficient (Kp) of these tissues were 0.65 to 12.9. In conclusion, we reported for the first time the PK and tissue distribution of 5,7-DMF in mice. These results will be critical in evaluating if those beneficial in vitro effects can be translated in vivo.

5,7-Dimethoxyflavone Attenuates Obesity by Inhibiting Adipogenesis in 3T3-L1 Adipocytes and High-Fat Diet-Induced Obese C57BL/6J Mice.

The antiobesity effect of 5,7-dimethoxyflavone (DMF) was evaluated in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese C57BL/6J mice. The accumulation of lipid droplets and triglycerides in adipocytes was dose dependently suppressed by DMF through inhibition of adipogenesis. DMF downregulated the adipogenic transcription factors (peroxisome proliferator-activated receptor [PPAR]γ, CCAAT/enhancer binding protein [C/EBP]α, and sterol regulatory element-binding protein-1c [SREBP-1c]) and lipid synthesis enzymes (fatty acid synthase [FAS], acetyl-CoA carboxylase [ACC], lipoprotein lipase [LPL], and HMG-CoA reductase [HMGR]). AMP-activated protein kinase (AMPK) and AMPK related lipolytic proteins in differentiated adipocytes were activated by DMF. In the animal model, oral administration of DMF (50 mg/kg/day for 6 weeks) significantly decreased body weight gain without affecting food intake. Elevated serum levels of total cholesterol and low-density lipoprotein cholesterol were suppressed by DMF. Fat pad masses were reduced in DMF-treated obese mice, as evidenced by reduced adipocyte size. DMF altered the expression of adipogenic transcription factors in epididymal fat tissue. In addition, DMF attenuated HFD-induced nonalcoholic fatty liver disease by decreasing hepatic triglyceride accumulation. Overall, these results suggest that DMF is a potential natural agent for attenuating obesity and other obesity-related metabolic syndromes.

Quantification of 5,7-dimethoxyflavone in mouse plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application to a pharmacokinetic study.

5,7-Dimethoxyflavone (5,7-DMF), a natural flavonoid abundant in many plants, has been reported to have many beneficial pharmacological effects, including strong chemopreventive and chemosensitizing properties, anti-oxidant, cardiovascular protectant, and anti-inflammatory activities. However, to date 5,7-DMF was evaluated mainly in vitro and its pharmacokinetics (PK) in vivo remains largely unknown. In addition, current available quantification methods of 5,7-DMF all lack sufficient sensitivity (lower limit of quantification >800 ng/mL). The purposes of our study are to establish a sensitive quantification method of 5,7-DMF using LC-MS/MS and evaluate the PK profile of 5,7-DMF in mouse. Our method was fully validated and all of the fundamental parameters in method validation, including accuracy, precision, sensitivity, selectivity, recovery and stability were evaluated thoroughly in mouse plasma. The calibration curve covered 2-1000 ng/mL with the lower limit of quantification (LLOQ) of 2 ng/mL. The inter-run and intra-run precision and accuracy were less than 15% of nominal concentrations. The matrix effect and recovery yield were within ±15% of nominal concentrations. In the PK study, 5,7-DMF was detectable in mouse plasma up to 21 h, with a terminal half-life of 11.5h. The clearance of 5,7-DMF (CL/F) is 22.3 L/h/kg and area under the curve (AUCinf) is 449 h ng/mL. In conclusion, a fast, accurate, sensitive and selective quantification method of 5,7-DMF was established and the developed method was successfully applied to a PK study of 5,7-DMF following oral administration of 5,7-DMF in mice.

The O-methylation of chrysin markedly improves its intestinal anti-inflammatory properties: Structure-activity relationships of flavones.

The aim of this study was to investigate whether methoxylated flavones versus their unmethylated analogs can modulate the intestinal inflammatory response. Flavone effects were assessed on soluble pro-inflammatory mediator (IL-8, IL-6, macrophage chemoattractant protein-1 (MCP-1), and cyclooxygenase-2 (COX-2)-derived PGE2) production and on nuclear factor (NF)-κB activation in 3d-confluent and 21d-differentiated Caco-2 cells stimulated with interleukin (IL)-1ß. Chrysin (CHRY) showed anti-inflammatory properties by decreasing COX-2-derived PGE2 and reducing NF-κB activation. Compared to CHRY, the dimethoxylated form (CHRY-DM) significantly reduced the secretion of all pro-inflammatory mediators, except IL-8, at both cellular stages (P<0.05); these effects being dose-dependent in 3d-cells. The reduction of NF-κB activation was significantly more pronounced with CHRY-DM. By evaluating other flavones, it was established that several structural dispositions of flavones seemed to be determinant in order to attenuate the intestinal inflammatory response, such as methoxylation of the 5- and 7-hydroxyl groups on the A-ring, non-methoxylation of the 3'-hydroxyl groups on the B-ring, and methoxylation of the 3-hydroxyl group on the C-ring. Of all flavones examined, CHRY-DM exhibited the strongest anti-inflammatory activity. These data indicate that, in the Caco-2 cell model, methoxylation of CHRY greatly improves its anti-inflammatory properties, probably through a more pronounced inhibition of the NF-κB signaling pathway. Nevertheless, methoxylation of other flavones was not systematically beneficial.