Optimization of callus induction and cell suspension system in Hemidesmus indicus (L.) R. Br. for improved production of lupeol

Hemidesmus indicus (L.) R.Br. is an ethnopharmacologically important medicinal plant with valuable bioactive compounds and industrially important secondary metabolites. This plant has a promising role in treating a wide spectrum of ailments, such as cancer, respiratory difficulties, liver disorders, diabetes, and neurological diseases. However, the rising demand for this plant and its metabolites has classified it as an endangered species. Thus, to meet the demand for secondary metabolites, in vitro culture systems, especially cell suspension systems are considered as an alternative strategy. Hence, the present study is an attempt to develop a cell suspension culture system in Hemidesmus indicus using leaf explants. In solid culture system, auxins (NAA, IAA, and 2,4-D), photoperiod (16 h light/8 hrs dark or complete darkness), media strengths (1x, 3/4x, 1/2x, and 1/4x), carbon sources (glucose, sucrose, and fructose) and in liquid media auxins, (NAA, IAA, and 2,4-D), media strength (1x, 3/4x, 1/2x, and 1/4x), and carbon sources (glucose, sucrose and fructose) were optimized. In solid media, leaf explants showed maximum results when cultured in 1/2 MS media augmented with 32.2 mu M NAA and 5% sucrose and incubated under dark conditions. In addition, leaf explants showed better regeneration potential compared to internodes. Whereas in the liquid system, an enhanced biomass (19.2 g flask-1) was thrived when friable callus was suspended in 1/2 strength MS medium containing 32.2 mu M NAA and 5% sucrose and incubated under complete dark condition. Assessment of growth kinetics revealed maximum biomass on the 25th day. Additionally, 5% sucrose exhibited higher levels of phenolic, flavonoid, and terpenoid content, which correlated with increased antioxidant activities in both DPPH and FRAP assays. HPLC analysis revealed that 5% sucrose resulted in an enhanced level of lupeol content of 5.68 mg g-1 DW with a 5.2-fold improvement in cell suspension cultures of Hemidesmus indicus compared to the control (1.1mg g-1 DW). To our knowledge, this is the first study that have explored the production of lupeol from the cell suspension culture of Hemidesmus indicus, marking a significant advance towards commercial-scale lupeol enhancement through cell culture systems.