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. Key words: Bridelia retusa, Anthocyanin, in vitro culture, Cell suspension, Purification, apoptosis, Anti-metastatic potential. significant role in tumor promotion by direct chemical reaction or alteration of cellular metabolic processes, and their scavengers (SOD, CAT, etc) represent inhibitors at different stages of carcinogenesis. The continuing severity and magnitude of the cancer problems make it imperative to develop chemopreventive strategies utilizing natural antioxidants to block the initiation, or arrest, or reverse the progression of pre-malignant cells. Antioxidants protect the cells against the toxicity of ROS by preventing their formation and neutralization of oxygen-free radicals. Aswathy 5Environmental factors of either biologic or chemical origin, may act as initiators or promoters, of carcinogenesis. Oral squamous cell carcinoma (OSCC) was the most common of the head and neck cancers with a poor prognosis. This may be further explained...
Background: Caesalpinia pulcherrima, belongs to Caesapiniaceae, is a known medicinal plant widely distributed in India and is used in traditional medicine for the treatment of various ailments. Many phytochemicals are reported from the plant as potential source of crude drug. Materials and Methods: An efficient and simple reproducible protocol was developed for callus production using leaf explants of C. pulcherrima. The combination of 2, 4-D, kin and BA, was used for the callus induction. Subsequently, cell suspension culture and quercetin synthesis from in vitro callus was attempted. Role of effect of elicitors (Sucrose, ABA and salicylic acid) in cell suspension culture was carried in MS medium containing 2,4-D + BA + kinetin. Flavonoids was purified, fractionated by HPLC-DAD and NMR. Results: 2, 4-D (2.5 mg/L), BA (2.5 mg/L) + kin (1 mg/mL) was effective for maximum callus induction from leaf explants. Significant cell suspension culture was noticed with liquid MS medium containing 2,4-D (2 mg/L)+ BA (1mg/L)+ kinetin (1.5 mg/L). Sucrose, ABA and salicylic acid (SA) at different concentrations influenced cell biomass and quercetin accumulation. The addition of ABA/SA along with sucrose was found to have no remarkable effect on cell biomass and also quercetin synthesis. However, cells cultured in the medium fortified with 45 g/L sucrose without ABA/ SA showed the highest quercetin content (16.5 mg/g). Flavonoids was purified, fractionated by HPLC-DAD and NMR revealed the presence of 9 components such as quercetin, isoquercetin, quercetrin, rutin, quercetin 3-O-β-D-xyloside, quercetin 3-Oarabinopyranoside, quercetin 3-O-α-arabinopyranosyl (1→2) β-galactopyranoside, isorhamnetin 3-O-rutinoside and an unknown compound. Conclusion: C. pulcherima reveals significant synthesis of quercetin. Quercetin content recorded in cell suspension culture was significantly higher compared with in vivo plants grown in fields and the compounds were identified by NMR.
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