Extraction, Quantification and Anti-Cancer Activity of Important Secondary Metabolite “Shatavarin-IV” from in vitro Callus Culture of Asparagus racemosus Willd.

Objectives: This study investigates the in vitro efficient pathway for the callus induction from nodal explant and isolation of the Shatavarin-IV from the callus extract from fresh callus and Dry callus. The effect of Shatavarin IV was examined on the Human lung carcinoma cell line. Gene expression study was carried out using a specific primer to conform weather it is anticancer compound or not. Materials and Methods: In the present study, 2,4-D and Kn were used for the direct callus induction. Callus culture, organogenesis and cell culture are used to harvest secondary metabolites. Quantification of Shatavarin-IV was performed using a TLC scanner at a wavelength of 425 nm. HPTLC analysis was performed to quantitatively determine the presence of Shatavarin-IV in Fresh Callus (FC), Dry Callus (DC) and Root Extract (RE). The mobile phase used for HPTLC analysis was ethyl acetate: methanol: water: formic acid (7.5:1.5:1:0.2 v/v). Extraction was carried out using 80% methanol and the extract was filtered. The extract was then analyzed using HPTLC methods. Further, the effect of Shatavarin-IV was studied on the human lung carcinoma cell line NCI-H23. The end result of cell cytotoxicity was analyzed using MTT dye. To, confirm whether the cell expressed an apoptotic gene or not RTPCR studies were been carried out. Results: The best hormonal combination for callus induction from nodal explant was found 11.31 μM 2,4-D combined with 4.64 μM Kn on MS as basal media. Shatavarin-IV accumulation in dry callus was 0.0305% w/w, wild-grown plant root extract showed 0.01732% w/w and in fresh callus presence of Shatavarin-IV was 0.0012% w/w. IC50 of the Shatavarin-IV was 0.8 Micromolar dissolved in DMSO. Gene expression study reflects the multifold increase of the BAX and decrease of BCL2, concluded interpreted as Shatavarin-IV causes apoptotic gene expression. Conclusion: It was concluded that, Shatavarin IV reduced the expression of BCL2 and increase BAX expression which positive sign that Shatavarin IV act as an anticancer compound. Shatavarin IV can be isolated from the in vitro callus culture, which can be alternative of whole plant that was exploited for its medicinal properties. (Schematic Abstract).