Anti-proliferative potentiality of purified anthocyanin from in vitro culture of Clerodendron infortunatum L. against human cervical cancer cells (HeLa)

Abstract

Anthocyanins are polyphenolic compounds possessing anticarcinogenic effects through multiple ways. The proper mechanism for the effect of the anthocyanins againt proliferation of cancer cells is unknown. The present study aims to analyze the pro-apoptotic potential of purified anthocyanin from the in vitro cultures of Clerodendron infortunatum L. The cytotoxic activity was evaluated using MTT cell viability assay. Fluorescence microscopic, DNA fragmentation analysis, cell cycle phase distribution and Annexin V binding assay/Quantification of apoptotic cell death were also carried. Cellular morphological changes by fluorescence microscopy and flow cytometry were used to analyze the effect of anthocyanin on cell cycle and apoptosis. Anthocyanin extracted from the in vitro cultures of C.infortunatum was purified by column chromatography. The mean purity values obtained by HPLC were 90.9% ± 1.9, 80.60% ±2.3 for Oasis MCX, Amberlite XAD-7 +Sephadex LH-20 column respectively. However, the purity by molar absorptivity was found to be less. HPLC chromatogram revealed 12 fractions of anthocyanin. MTT assay revealed that anthocyanin inhibited in vitro cancer cell growth of human cervical cancer cells (HeLa) in a dose- and time-dependent manner as compared to other cell lines. The IC₅₀ values of anthocyanin were found to be 47.2 and 32.5 µM at 24 and 48 h time intervals respectively. Anthocyanin treated cells exhibited apoptotic bodies, which inferred an early apoptotic event. DNA ladder was more apparent and increased with the anthocyanin dose as compared to the control group. Anthocyanin also induced sub-G1 cell cycle arrest and increased fraction of HeLa apoptotic cells. Conclusion: Purified anthocyanin from in vitro culture of Clerodendron infortunatum showed anti proliferative activity against HeLa cells.