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The current study was carried out to appraise the Anti-proliferative activity of Herbal Medicine Formulation (HMF) comprising different components of ethno-medicinal plant drugs which are practiced by the local healers in the Tribal Medicine System (TMS). The medicine formulation was subjected for validation with the authorized Ayurvedic Medical Practitioner in order to ascertain the active resources for novel lead constituents present in the drug. In addition, the validated HMF will also facilitate the movement of indigenous practices of traditional medicines to the public domain. The analysis was made on preliminary phyto-chemicals using fractions of HMF drug which reveals the active presence and were known to demonstrate both biomedical and physiological activities. Subsequently, the analysis of Thin Layer Chromatographic confirms the presence of diversified bioactive ingredients in the different components of HMF drug which indicates the efficacy of the HMF drug. The active fractions were prepared by serial extraction with ethanol, methanol, petroleum ether and ethyl acetate respectively based on the standard protocols and tested against MCF-7 (Mammarain Cancer cells) followed by HeLa cancer cells lines in vitro and their probable mechanism of action was critically analyzed. Further, the anti-proliferative activity was evaluated with these diversified HMF drug fractions against cancer cell lines by MTT assay, Trypan Blue assay and Hoechst’s staining methods respectively. The apoptotic effects was determined and correlated for its anti-cancer activity in the extracts of HMF drug. The TLC of extracts showed that, chloroform extract moved to the maximum distance of 15cm followed by ethyl acetate extract with a 14.7 cm as retention factor. Whereas, the ethanolic extract of HMF drug moved the least distance with 6.5cm retention factor and aqueous extract did not show any movement on the stationary phase. The analysis for MTT assay demonstrated that, the cell viability was diminished with the increased concentration of the HMF drug. The inhibition concentration value (IC50) for the MTT assay at 24hrs was found to be 5.1μg/ml and for 48hrs at 5.4μg/ml. Later, the attentiveness of HMF drug against both MCF-7 and HeLa cancer cells was instituted to appraise the anti-proliferative activity. The arresting of cell proliferation and growth of these cancerous cells was observed even in very low concentration of the HMF drug, which may be due to the presence of bio-active constituents in the medicine formulation. Subsequently, the apoptotic assays showed that, the increased fractions of HMF drug (60, 75, 80 and 95%) induced mitochondrial depolarization in MCF cells correspondingly, at those fractions which could have triggered the apoptosis in mitochondrial pathway. Eventually, the ability of curbing and inhibition capability of the free radicals was assessed in the different fractions of HMF drug through biochemical method like, ABTS scavenging assays respectively. The result reveals; significant ABTS free radical scavenging activity of 7± 0.74 was evident in the extract. Hence, the HMF drug was found to be most effective in deactivating these Cancer Cells and justifies the tested herbal formulation, HMF being practiced by the tribal healers in their traditional medicine system. Further, the formulation needs to be evaluated for its clinical trials through the absolute purification process in order to propose the medicine as most potential anticancer herbal drug.

Evaluation of Validated Herbal Medicine Formulation for AntiProliferative and Apoptotic Activities Used In the Tribal Medicine System