Ethyl acetate fraction from methanol extraction of Vitis thunbergii var. taiwaniana induced G0 /G1 phase arrest via inhibition of cyclins D and E and induction of apoptosis through caspase-dependent and -independent pathways in human prostate carcinoma DU145 cells.

Vitis thunbergii var. taiwaniana (VTT) is a wild grape native to Taiwan, belonging to the Vitaceae family and Vitis genus, and widely used as folk herbal medicine. It is traditionally used for the treatment of diarrhea, hypertension, neuroprotection, jaundice, and arthritis. We used the wild-collected VTT and sterilized them to establish the plant tissue culture, and then took the leaves for DNA sequencing to determine its original base. We use methanol to extract VTT in four different solvents: 1-butanol, n-hexane, ethyl acetate, and water. These four preliminary extracts were used to treat human prostate cancer DU145 cells in vitro. We use the flow cytometry to check the cell survival situation. Finally, we found the ethyl acetate layer roughing product (referred VTEA) in human prostate cancer apoptotic effects of cell line DU-145. In the present studies, we use the crude extract of VTT to examine whether or not it can induce apoptosis of DU145 cells in vitro. Viability assays for extracts of VTT treatment showed that it had dose-dependent effect on human prostate cancer DU145 cells. We also found that the extract of VTT induces time-dependent mitochondrial and intrinsic-dependent apoptosis pathways. The in vitro cytotoxic effects were investigated by cell cycle analysis and the determination of apoptotic DNA fragmentation in DU145 cells. The cell cycle analysis showed that extracts of VTT induced a significant increase in the number of cells in G0 /G1 phase. The extract of VTT induced chromatin changes and apoptosis of DU145 cells also were confirmed by DAPI and PI staining that were measured by fluorescence microscopy and flow cytometry, respectively. Finally, the expression of relevant proteins was analyzed by Western blot analysis. These results promoted us to further evaluate apoptosis associated proteins and elucidate the possible signal pathway in DU-145 cells after treated with the extract of VTT.

Establishment of hairy root lines and analysis of iridoids and secoiridoids in the medicinal plant Gentiana scabra.

BACKGROUND: Gentiana scabra is commonly known as 'Longdan' is an important herb in traditional Chinese medicines, commonly used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are main bioactive compounds which attributed to the pharmacological properties of this plant. The use of hairy root cultures as an excellent alternative for the production of pharmaceutically important metabolites in less time period with ensured quality of raw materials. RESULTS: An efficient hairy root culture system of Gentiana scabra and influence of different plant growth regulators (PGRs) on the production of gentiopicroside, swertiamarin and loganic acid constituents were described. Leaf explants were infected with Agrobacterium rhizogenes, which induced hairy roots up to 21%. The transformed hairy root lines were confirmed by PCR using rolB and rolC gene-specific primers. Among various solid and liquid media, B5 liquid medium resulted maximum root biomass (36- fold higher) in 4-weeks. Quantitative analysis showed loganic acid was 6.6- fold higher in the presence of zeatin (1 mg/l) and gentiopicroside accumulation was 1.8- fold higher in the presence of naphthaleneacetic acid (NAA, 1 mg/l), as compared to the roots of plants grown in greenhouse. On the other hand, 1.4- and 2.5- fold higher gentiopicroside and swertiamarin were observed in the presence of 1.0 mg/l NAA as compared to commercial Gentiana herb No. 2. The result also showed iridoid and secoiridoid contents affected greatly by age, physiology and growing environment of the plant. CONCLUSIONS: The use of hairy root cultures is an excellent alternative to harvesting natural or in vitro grown plants to produce pharmaceutically important metabolites in less time with ensured quality.