EXPERIMENTAL PROCEDURE: The microbial limit test (MLT) studies indicated the suitable dosage of minimum and maximum gamma irradiation for leaf extracts as well as dried leaves of all the tested medicinal plants. Quantitative analysis of total phenolic content (TPC) analysis is based on calorimetric measurements determined using the Folin-Ciocalteu reagent with gallic acid (GA) used as the reference. In vitro cytotoxicity assay by using fibroblast (L929) cell lines was performed on each plant to determine the toxicity effect which sodium dodecyl sulfate (SDS) as the positive control. DPPH (2,2-diphenyl-1-picryl-hydrazyl) assay was conducted by using vitamin C and GA as the positive controls to determine the antioxidant property of each plant.
RESULTS AND CONCLUSION: The MLT analysis indicated that the suitable dosage gamma irradiation for leaf extracts was 6-12 kGy and dried leaves were 9-13 kGy. The amount of GA concentration in each plant increased significantly from 30-51 mg GAE g-1 before treatment to 57-103 mg GAE g-1 after treatment with gamma radiation. This showed no significant effect of in vitro cytotoxicity activity before and after treatment with gamma irradiation in this study. Effective concentration (EC50) values of Khaya senegalensis plant reduced significantly (P ≤ 0.005) from 44.510 μg/ml before treatment to 24.691 μg/ml after treatment with gamma radiation, which indicate an increase of free radical scavenging activity.
METHODS: The cytotoxicity activity was measured using MTS assay. The mode of cell death was analysed by early (phosphatidylserine externalization) and late apoptosis (DNA fragmentation). The caspases 8, 9, 3/7 and apoptotic proteins bax, bcl-2 study were done by western blot and ELISA method.
RESULTS: The methanol extract was found to inhibit 50% growth of T-47D cells at the concentration of 79.43µg/ml respectively after 72hr. From seven fractions, fraction F1, F2 and F3 produced cytotoxicity effects in T-47D cell line with IC50 (72hr) < 30µg/ml. The results obtained by Annexin V/PI apoptosis detection assay and TUNEL assay suggest that active fractions of Vitex rotundifolia induced early and late apoptosis (DNA fragmentation) in T-47D cell line. Moreover, western blot analysis and Caspase GloTM luminescent assay demonstrated that fractions F2 and F3 triggered apoptotic cell death via activation of caspases -8, -9 and -3/7 and up-regulation of Bax and down-regulation of Bcl-2 protein. Furthermore, chemical profiling confirms the presence of potential metabolites (vitexicarpin) in fractions of Vitex rotundifolia.
CONCLUSION: Thus, the present study suggests the remarkable potential of active metabolites in fractions of Vitex rotundifolia as future cancer therapeutic agent for the treatment of breast cancer.
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