Purified Anthocyanin from in vitro Culture of Bridelia retusa (L.) Spreng. Capable of Inhibiting the Growth of Human Oral Squamous Cell Carcinoma Cells

The present study aims in vitro cell suspension culture of Bridelia retusa, isolation of anthocyanin, purification, fractionation and its anti-metastatic potential against oral squamous carcinoma cells. Experimental results reveal that 2, 4-D either alone or in combination with kinetin supplemented in MS medium showed significant initiation of callus from leaf explants than stem. Growth hormones, pH, light, and carbon source influence anthocyanin synthesis. Maximum callus induction was noticed with 2.5 mg/L N6-benzyladenine (BA) + 2 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) (98.9%). Fresh and dry weight of the calli were i.e., 1.9 ± 0.04 and 0.45 ± 0. 03 g respectively. Optimal response was seen with light on MS medium contain 4% glucose + 2.5 mg/L BA and 2 mg/L 2, 4-D at pH 3.5 yielded 2.8 mg /g of anthocyanins. Suspension culture medium fortified with 2, 4-D (2.5 mg/L) + BA (2 mg/L) at pH 5.0 induced anthocyanin production at pH 4.4 – 4.6. HCl-ethanol extraction for 90 min yielded the maximum anthocyanin content. Fractionation of anthocyanin using HPLC coupled with mass spectrometry revealed 07 fractions such as acylated cyanidins, two peonidins, cyanidin 3-p-coumaroyl and feruloyl diglucoside-5-glucosides. In the search of novel therapeutic drugs against cancer, cytotoxicity effect of B.retusa anthocyanin extracts on human oral squamous cell carcinoma (SCC4, SCC9 and SCC25) cells using cell adhesion and cell viability assay was carried. The morphological alterations in SCCs cells after treatment with B.retusa anthocyanin includes nuclear condensation, fragmentation and apoptotic cells as revealed by Hoechst stain. Flow cytometry showed arresting of SCC25 cells mostly in the G0/G1 and S-G2/M stages with a concomitant up regulation of sub-G1 fraction, indicating cell death by apoptosis. Apoptosis was further substantiated by the activation of caspase-3 expression in the SCC25 cells treated with B.retusa anthocyanin. Thus, it is possible to suggest that B.retusa anthocyanin cause apoptosis of SCCs and warrant further investigation using animal models.