Production of squalene with promising antioxidant properties in callus cultures of Nilgirianthus ciliatus

There is a lot of market for squalene (C₃₀H₅₀) for industrial and therapeutic applications such as oil, biofuel, antioxidant, antimicrobial and anticancer agents. Squalene was used to be derived from liver oil of sharks and whales, but a sustainable alternative source such as plants is needed. An ayurvedic plant, Nilgirianthus ciliatus, was found to contain a limited amount of squalene. This study hypothesizes that squalene content in callus culture of N. ciliatus could produce an increased level of squalene. For efficient callus induction, various parameters such as explants, plant growth regulators, light and dark conditions were optimized. Leaf produced the highest frequency of callus induction (89.5%) with 2.3 fold enhanced squalene production on MS medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 0.4 mg l⁻¹ benzyladenine. The wild and in vitro tissues were evaluated for metabolic profiles using GC–MS and antioxidant properties. The leaf callus exhibited increased level of total phenolic, flavonoid and squalene contents which confers the highest level of antioxidant (IC₅₀ = 88.8 μg ml⁻¹) activity compared to wild explants. The GC–MS profiles of wild and callus tissues showed twelve compounds, in which squalene was 9.3% in wild and 21.4% in callus. Yield of squalene was found to be 70.9 μg g⁻¹ (dry weight) DW and 146.3 μg g^-1 DW for wild and callus tissues, respectively. Furthermore, FT-IR analysis also confirmed the presence and increased level of squalene in callus compared to that of wild. This callus could be an ideal source for mass squalene production without depleting natural sources and simultaneously fulfilling the pharmacological demands.