5-Hydroxy-6,7,3',4'-tetramethoxyflavone

In vitro human intestinal microbiota biotransformation of nobiletin using liquid chromatography-mass spectrometry analysis and background subtraction strategy.[Pubmed:33682313]

J Sep Sci. 2021 May;44(10):2046-2053.

In this study, the in vitro biotransformation of nobiletin by human intestinal microbiota, which is a bioactive polymethoxyflavone widely presented in Citrus plants, has been investigated via utilizing an anaerobic incubation protocol. The incubation samples were detected using high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. A background subtraction strategy incorporated in Microsoft Office was employed to eliminate the interferences in medium and feces. The parent and three metabolites sinensetin, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, and 5-demethylnobiletin were detected and identified based on the characteristics of their protonated molecules. The proposed metabolic pathway revealed that nobiletin went through phase I metabolism including demethylation and demethoxylation in human intestinal microbiota. The characterization of nobiletin metabolic profile transformed by human intestinal bacteria would be helpful for understanding its efficacy and action mechanism.

Phytochemical Investigation and Biological Activities of Lantana rhodesiensis.[Pubmed:33562771]

Molecules. 2021 Feb 5;26(4). pii: molecules26040846.

Lantana rhodesiensis Moldenke is a plant widely used to treat diseases, such as rheumatism, diabetes, and malaria in traditional medicine. To better understand the traditional uses of this plant, a phytochemical study was undertaken, revealing a higher proportion of polyphenols, including flavonoids in L. rhodesiensis leaf extract and moderate proportion in stem and root extracts. The antioxidant activity of the extracts was also determined using three different assays: the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, the FRAP method (Ferric-reducing antioxidant power) and the beta-carotene bleaching test. The anti-malarial activity of each extract was also evaluated using asexual erythrocyte stages of Plasmodium falciparum, chloroquine-sensitive strain 3D7. The results showed that the leaf extract exhibited higher antioxidant and anti-malarial activities in comparison with the stem and root extracts, probably due to the presence of higher quantities of polyphenols including flavonoids in the leaves. A positive linear correlation was established between the phenolic compound content (total polyphenols including flavonoids and tannins; and total flavonoids) and the antioxidant activity of all extracts. Furthermore, four flavones were isolated from leaf dichloromethane and ethyl acetate fractions: a new flavone named rhodescine (5,6,3',5'-tetrahydroxy-7,4'-dimethoxyflavone) (1), 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone (2), 5-hydroxy-6,7,3',4'-tetramethoxyflavone (3), and 5,6,3'-trihydroxy-7,4'-dimethoxyflavone (4). Their structures were elucidated by (1)H, (13)CNMR, COSY, HSQC, HMBC, and MS-EI spectral methods. Aside from compound 2, all other molecules were described for the first time in this plant species.

Polymethoxylated flavones from Orthosiphon stamineus leaves as antiadhesive compounds against uropathogenic E. coli.[Pubmed:31678632]

Fitoterapia. 2019 Nov;139:104387.

Aqueous and acetone extracts of O. stamineus leaves reduce the adhesion of uropathogenic E. coli (UPEC, strain UTI89) to T24 bladder cells significantly (IC25 ~ 524mg/mL, resp. 40mug/mL). The acteonic extract had no cytotoxic effects against UPEC in concentrations that inhibited the bacterial adhesion. The extract significantly reduced the gene expression of fimH, fimC, fimD, csgA and focG, which are strongly involved in the formation of bacterial adhesins. The antiadhesive effect was due to the presence of polymethoxylated flavones, enriched in the acetonic extract. Five flavones have been isolated by fast centrifugal partition chromatography, followed by preparative HPLC. Eupatorin, ladanein, salvigenin, sinensetin, 5,6,7,4'-tetramethoxyflavone and 5-hydroxy-6,7,3',4'-tetramethoxyflavone were identified as the main polymethoxylated flavones. With the exception of eupatorin, all of these flavones reduced the bacterial adhesion in a concentration depending manner, indicating that B-ring hydroxylation and methoxylation seems to have a major impact on the antiadhesive activity. In addition, this was confirmed by investigation of the flavones chrysoeriol and diosmetin, which had only very weak antiadhesive activity. From these data, Orthosiphon extracts can be assessed to have a pronounced antiadhesive activity against UPEC, based on a variety of polymethoxylated flavones.

Drug Metabolite Cluster-Based Data-Mining Method for Comprehensive Metabolism Study of 5-hydroxy-6,7,3',4'-tetramethoxyflavone in Rats.[Pubmed:31505804]

Molecules. 2019 Sep 9;24(18). pii: molecules24183278.

The screening of drug metabolites in biological matrixes and structural characterization based on product ion spectra is among the most important, but also the most challenging due to the significant interferences from endogenous species. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of prototype drug metabolites using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Although classical techniques are well-suited for achieving the partial characterization of prototype drug metabolites, there is a pressing need for a strategy to enable comprehensive drug metabolism depiction. Therefore, we present drug metabolite clusters (DMCs), different from, but complementary to, traditional approaches for mining the information regarding drugs and their metabolites on the basis of raw, processed, or identified tandem mass spectrometry (MS/MS) data. In this paper, we describe a DMC-based data-mining method for the metabolite identification of 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF), a typical hydroxylated-polymethoxyflavonoid (OH-PMF), which addressed the challenge of creating a thorough metabolic profile. Consequently, eight primary metabolism clusters, sixteen secondary metabolism clusters, and five tertiary metabolism clusters were proposed and 106 metabolites (19 potential metabolites included) were detected and identified positively and tentatively. These metabolites were presumed to generate through oxidation (mono-oxidation, di-oxidation), methylation, demethylation, methoxylation, glucuronidation, sulfation, ring cleavage, and their composite reactions. In conclusion, our study expounded drug metabolites in rats and provided a reference for further research on therapeutic material basis and the mechanism of drugs.

Flavonoids from Perovskia atriplicifolia and Their in Vitro Displacement of the Respective Radioligands for Human Opioid and Cannabinoid Receptors.[Pubmed:26035635]

J Nat Prod. 2015 Jun 26;78(6):1461-5.

Bioassay-guided fractionation of the leaves of Perovskia atriplicifolia (Russian sage) resulted in the isolation of four previously known flavonoid derivatives, 5-hydroxy-6,7,3',4'-tetramethoxyflavone (1), 5,7-dihydroxy-6,3',4'-trimethoxyflavone (2), 5-hydroxy-6,7,4'-trimethoxyflavone (3), and 5,7-dihydroxy-6,4'-dimethoxyflavone (4). Compounds 1, 3, and 4 showed displacement of the radioligand for the cloned human delta opioid receptor with Ki values ranging from 3.1 to 26.0 muM. In addition, the binding mode of the compounds in the active site of the delta opioid receptor was investigated through molecular modeling algorithms. This study may have implications in better understanding non-nitrogenous delta opioid receptor ligands.

[Polymethoxylated flavonoids activate cystic fibrosis transmembrane conductance regulator chloride channel].[Pubmed:25896054]

Sheng Li Xue Bao. 2015 Apr 25;67(2):225-34.

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 mumol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation.

Inhibition of xanthine oxidase activity by gnaphalium affine extract.[Pubmed:25429747]

Chin Med Sci J. 2014 Dec;29(4):225-30.

OBJECTIVE: To evaluate the inhibitory effect of Gnaphalium affine extracts on xanthine oxidase (XO) activity in vitro and to analyze the mechanism of this effect. METHODS: In this in vitro study, Kinetic measurements were performed in 4 different inhibitor concentrations and 5 different xanthine concentrations (60, 100, 200, 300, 400 Mumol/L). Dixon and Lineweaver-Burk plot analysis were used to determine Ki values and the inhibition mode for the compounds isolated from Gnaphalium affine extract. RESULTS: Four potent xanthine oxidase inhibitors were found in 95% ethanolic (v/v) Gnaphalium affine extract. Among them, the flavone Eupatilin exhibited the strongest inhibitory effect on XO with a inhibition constant (Ki) of 0.37 Mumol/L, lower than the Ki of allopurinol (4.56 mol/L), a known synthetic XO inhibitor. Apigenin (Ki of 0.56 Mumol/L, a proportion of 0.0053 per thousand in Gnaphalium affine), luteolin (Ki of 2.63 Mumol/L, 0.0032 per thousand in Gnaphalium affine) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (Ki of 3.15 Mumol/L, 0.0043 per thousand in Gnaphalium affine) also contributed to the inhibitory effect of Gnaphalium affine extract on XO activity. CONCLUSIONS: These results suggest that the use of Gnaphalium affine in the treatment of gout could be attributed to its inhibitory effect on XO. This study provides a rational basis for the traditional use of Gnaphalium affine against gout.

Isolation, cytotoxicity evaluation and HPLC-quantification of the chemical constituents from Artemisia amygdalina Decne.[Pubmed:24148842]

J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Dec 1;940:135-41.

The hexane extracts of both shoot and root parts of Artemisia amygdalina Decne displayed potent cytotoxic effects. Phytochemical analysis of these active extracts led to the isolation of six cytotoxic constituents, viz., Ergostadien-3beta-ol (1), ludartin (2), 5-hydroxy-6,7,3',4'-tetramethoxyflavone (3) (from shoot) and trans-matricaria ester (4), diacetylenic spiroenol ether (5) and cis-matricaria ester (6) (from root) for the first time from this plant. The constituents were identified using spectral techniques in the light of literature. Sulphorhodamine B cytotoxicity screening of the isolated constituents was carried out against four human cancer cell lines including Lung (A-549), Leukaemia (THP-1), Prostate (PC-3) and Colon (HCT-116) cell lines. Ludartin (2) exhibited the highest cytotoxicity with IC50 values of 7.4muM, 3.1muM, 7.5muM and 6.9muM against Lung (A-549), Leukaemia (THP-1), Prostate (PC-3), Colon (HCT-116) cancer cell lines respectively. To test against in vitro skin cancer models [human dermal fibroblasts (CRL-1635)] all the isolates were further subjected to 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) cytotoxicity screening. Ludartin (2) being highly cytotoxic was again evaluated against mouse melanoma (B16F10) and human epidermoid carcinoma (A-431) cells by MTT assay displaying IC50 values of 6.6muM and 19.0muM respectively. Finally a simple and reliable HPLC method was developed (RP-HPLC-DAD) and validated for the simultaneous quantification of these cytotoxic constituents in A. amygdalina Decne. Excellent specificity and high linearity for all the standard calibration curves having regression coefficients of the respective linear equations in the range of 0.9962-0.9999 was observed. Relative recovery rates varied between 98.37+/-0.90 and 105.15+/-1.74 with relative standard deviation less than 4%. Based on our results, the developed method features good quantification parameters, accuracy, precision and can serve as effective quality control method for standardisation of A. amygdalina Decne.

Protective effect of compounds from the flowers of Citrus aurantium L. var. amara Engl against carbon tetrachloride-induced hepatocyte injury.[Pubmed:23985451]

Food Chem Toxicol. 2013 Dec;62:432-5.

5-Hydroxy-6,7,3',4'-tetramethoxyflavone (HTF), limonexic acid (LA) are two compounds isolated from the flowers of Citrus aurantium L. var. amara Engl with various biological activities. This study was designed to investigate their protective effects against carbon tetrachloride (CCl4)-induced hepatocyte injury, using human hepatic cell line HL-7702 to determine the cell cytotoxicity, cell viability, levels of hepatic marker enzymes, malondialdehyde (MDA). Results showed that pretreatment with HTF, LA could significantly reverse CCl4-induced HL-7702 cell viability decrease, LA displayed a higher activity. HTF, LA also showed their capability of decreasing the CCl4-induced leakage of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), inhibiting the lipid peroxidation, HTF showed more significant activity. Given that HTF, LA were not toxic, it is concluded that HTF, LA could effectively protect hepatocyte against CCl4-induced injury.

Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry.[Pubmed:23647150]

J Agric Food Chem. 2013 May 29;61(21):5016-21.

Sinensetin (SIN), one of the major polymethoxyflavones (PMFs) contained mainly in the citrus peels, has been reported to possess various bioactivities, including antifungal, antimutagenic, anticancer, and anti-inflammatory activities. Although the biotransformation of SIN in fungi and insects has been reported, the information about the metabolism of SIN in mammals is still unclear. In this study, formation of SIN metabolites in rats was investigated. Four isotope-labeled SINs ([4'-D3]SIN, [3'-D3]SIN, [5-D3]SIN, and [6-D3]SIN) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. The administered compound and four SIN metabolites were detected in rat urine. These metabolites were identified as 4'-hydroxy-5,6,7,3'-tetramethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 6-hydroxy-5,7,3',4'-tetramethoxyflavone, and 7-hydroxy-5,6,3',4'-tetramethoxyflavone sulfate.

Bioactivity evaluations of ingredients extracted from the flowers of Citrus aurantium L. var. amara Engl.[Pubmed:22980787]

Food Chem. 2012 Dec 15;135(4):2175-81.

Citrus aurantium L. var. amara Engl, a member of genus Citrus (Rutaceae), widespread in China, is used as folk medicine for the treatment of helping digest, phlegm, enteritidis, stomachic and other deceases. In the present research, silica gel column, Sephadex LH-20, mass spectrometer (MS) and nuclear magnetic resonance (NMR) were used to separate and identify the chemical compounds from the flowers of C. aurantium var. amara, and several bioactivity assays were used to evaluate their antioxidant, anti-inflammatory, anti-virus and antitumour activities. Two major compounds, 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) and limonexic acid (LA), were isolated and identified from C. aurantium var. amara for the first time. The results of the bioactivity assays showed that HTF and LA displayed significant antioxidant activities and showed significant inhibition effects on the B16 cell lines at a concentration range from 6.25 to 50 mug/ml, and on the SMCC-7721 cell lines from 12.5 to 200 mug/ml. The antitumour effect, anti-inflammatory activity and the inhibiting expression of HBsAg and HBeAg of 2.2.15 cells displayed the tendency in a concentration-dependent manner. These two compounds from C. aurantium var. amara could potentially be used as a promising natural agent in the pharmaceutical industries.

[Isolation and purification of flavones from Murraya exotica L. by high-speed counter-current chromatography].[Pubmed:20712121]

Se Pu. 2010 Apr;28(4):383-7.

High-speed counter-current chromatography (HSCCC) was used to isolate and purify flavones from Murraya exotica L. The optimum separation conditions were as follows: A two-phase solvent system was petroleum ether-ethyl acetate-methanol-water (5:5:4.8:5, v/v/ v/v). The lower phase as the mobile phase was operated at a flow rate of 2.0 mL/min, while the apparatus rotated at 800 r/min. Each time 200 mg of the sample was loaded. Under these conditions, 54.31 mg of recrystallized 5,7,3',4',5'-pentamethoxyflavone, 107.68 mg of 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 215.54 mg of 5-hydroxy-6,7, 8,3', 4'-pentamethoxyflavone, and 84.36 mg of 5-hydroxy-6,7,8,3',4',5 '-hexamethoxyflavone with their purities over 95% were successfully obtained from 4.0 g of the crude extract of Murraya exotica L. The four compounds were analyzed by high performance liquid chromatography (HPLC), and identified by mass spectrometry (MS), ' H-nuclear magnetic resonance (NMR) and 13C-NMR. The compound 5-hydroxy-6,7,3',4'-tetramethoxyflavone was for the first time isolated and purified from Murraya exotica L.

New Bisabolane-Type Sesquiterpenes from the Aerial Parts of Lippia dulcis.[Pubmed:16141591]

Chem Pharm Bull (Tokyo). 2005 Sep;53(9):1175-7.

Two new bisabolane-type sesquiterpenes, lippidulcine A (3) and epilippidulcine A (4), have been isolated from the aerial parts of Lippia dulcis TREV. along with five known flavonoids, cirsimaritin (5), salvigenin (6), eupatorin (7), 5-hydroxy-6,7,3',4'-tetramethoxyflavone (8) and 5,3'-dihydroxy-6,7,4',5'-tetramethoxyflavone (9), three known phenylethanoid glycosides, decaffeoylverbascoside (10), acteoside (11) and isoacteoside (12), and two known iridoid glucosides, 8-epiloganin (13) and lamiide (14). Their chemical structures have been determined on the basis of spectroscopic data. Among them, 5, 7, and 9 exhibited almost the same activity as that of alpha-tocopherol, and 10-12 were identified as stronger antioxidants than alpha-tocopherol using the ferric thiocyanate method.