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

The purpose of this study was to purify the phytoconstituents and to explore the antibacterial, antifungal, phytotoxic and cytotoxic potential of dichloromethane and methanol extracts of aerial and root parts of Trigonella polycerata. The phytochemical study on methanol extract of aerial parts of the plant led to the isolation and purification of seven compounds that were identified as 3,4-dimethoxycinnamaldehyde, Trigocoumarin, 6,7,8-trimethoxycoumarin, Penduletin, 5-hydroxy-3,6,7,4'-tetramethoxyflavone, 3,5,7-trihydroxy-6,4-dimethoxyflavone and 5-hydroxy-4,7-dimethoxyflavone. These structures were elucidated by interpretation of EI-MS and NMR spectral data. The plant aerial parts methanol extract (TPAM) demonstrated higher antibacterial (78.99%), phytotoxic (85% growth regulation at 1000μg/mL) and cytotoxic activities (LD50: 45.643μg/mL). While the methanol root extract (TPRM) was highly active against bacteria's; Salmonella typhi (71.56%), Staphylococcus aureus (70.15%), Escherichia coli (69%), fungi like Candida albicans (70.21%) and moderately active against Brine shrimp larvae (LD50: 125.663μg/mL). The dichloromethane aerial (TPAD) and root (TPRD) extracts exhibited significant antibacterial (78.03% and 50.21% inhibitions respectively) and phytotoxic (55% growth regulation at 1000μg/mL) potential. Only TPAD indicated the best inhibition against fungi; Aspergillus flavus (75.31%) and moderate inhibition against Microsporum canis (42.21%). This phytochemical and biological work is the first time reported in Trigonella polycerata.

Scope: Previously, we reported that 5-hydroxy polymethoxyflavones (5OH-PMFs) isolated from orange, namely 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5HHMF) and 5-hydroxy-6,7,8,4'-tetramethoxyflavone (5HTMF), potently induced apoptosis and cell-cycle arrest in multiple human colon cancer cells. Herein, using isogenic variants of HCT116 human colon cancer cells, we investigated the effects of p53, Bax and p21 on the apoptosis and cell-cycle arrest induced by different 5OH-PMFs. Methods and results: Annexin V/PI co-staining assay demonstrated that 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (p53(+/+) ) cells but not in HCT116 (p53(-/-) ) cells. Furthermore, 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (Bax(+/-) ) cells, whereas their pro-apoptotic effects on HCT116 (Bax(-/-) ) cells were marginal. All three 5OH-PMFs increased G0/G1 cell population of HCT116 (p53(+/+) ) cells, and these effects were abolished in HCT116 (p53(-/-) ) and HCT116 (p21(-/-) ) cells. Immunoblotting analysis showed that 5HHMF and 5HTMF increased the levels of cleaved caspase-3, cleaved PARP in both HCT116 (p53(+/+) ) and HCT116 (Bax(+/-) ) cells and these effects were much weaker in HCT116 (p53(-/-) ) and HCT116 (Bax(-/-) ) cells. Conclusion: Our results demonstrated that 5OH-PMFs, especially 5HHMF and 5HTMF, induce apoptosis and cell-cycle arrest by p53-, Bax- and p21-dependent mechanism.

Hydroxylated polymethoxyflavones (PMFs) are a class of novel flavonoid compounds mainly found in citrus plants. We studied the effects of three major 5-hydroxy PMFs, namely: 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone, and 5-hydroxy-6,7,8,4'-tetramethoxyflavone, on human colon cancer HCT116 and HT29 cells. Their effects were compared with those produced by their permethoxylated counterparts, namely: nobiletin, 3,5,6,7,8,3',4'-heptamethoxylflavone, and tangeretin. 5-Hydroxy PMFs showed much stronger inhibitory effects on the growth of the colon cancer cells in comparison with their permethoxylated counterparts, suggesting the pivotal role of hydroxyl group at 5-position in the enhanced inhibitory activity by 5-hydroxy PMFs. Flow cytometry analysis demonstrated that three 5-hydroxy PMFs produced different effects on the cell cycle and apoptosis, which may suggest that three 5-hydroxy PMFs act through different mechanisms. For example, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone caused cell cycle arrest at G2/M phase in HT29 cells, while 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone led to significant G0/G1 phase arrest. In contrast, 5-hydroxy-6,7,8,4'-tetramethoxyflavone increased sub-G0/G1 cell population, which has been confirmed to be due to enhanced apoptosis. Our results further demonstrated that the inhibitory effects of 5-hydroxy PMFs were associated with their ability in modulating key signaling proteins related to cell proliferation and apoptosis, such as p21(Cip1/Waf1), CDK-2, CDK-4, phosphor-Rb, Mcl-1, caspases 3 and 8, and poly ADP ribose polymerase (PARP).