In vitro elicitation, isolation, and characterization of conessine biomolecule from Holarrhena antidysenterica (L.) Wall. callus and its larvicidal activity against malaria vector, Anopheles stephensi Liston

Abstract

In vitro elicitation of an important compound conessine has been done in the bark-derived callus culture of Holarrhena antidysenterica (L.) Wall. employing different elicitors. For induction of callus, green bark explants excised from field-grown plants were cultured on MS medium augmented with different concentrations (0, 1, 2.5, 5, and 10 μM) of various growth regulators such as BA, IBA, NAA, and 2,4-D either alone or in combinations. The maximum amount of conessine (458.18 ± 0.89^d μg/g dry wt.) was achieved in callus developed on MS medium supplemented with 5 μM BA and 5 μM 2,4-D through HPLC analysis. Elicitation in conessine content in the above callus was achieved employing a variety of organic (phenylalanine, tyrosine, chitosan, tryptophan, casein hydrolysate, proline, sucrose, and yeast extract) as well as inorganic elicitors (Pb(NO₃)₂, As₂O₃, CuSO₄, NaCl, and CdCl₂) in different concentrations. The optimum enhancement in conessine content (3518.58 ± 0.28^g μg/g dry wt.) was seen at the highest concentration (200 mg/L) of phenylalanine. The enhancement was elicitor specific and dose dependent. The overall increment of the conessine content was seen in the order of phenylalanine > tryptophan > Pb(NO₃)₂ > sucrose > NaCl > As₂O₃ > casein hydrolysate > CdCl₂ > chitosan > proline > yeast extract > CuSO₄ > tyrosine. The isolation and purification of conessine was done using methanol as a solvent system through column chromatography (CC) and TLC. The isolated compound was characterized by FT-IR, ¹H-NMR, and HRMS which confirmed with the structure of conessine. The bioassays conducted with the isolated compound revealed a strong larvicidal activity against Anopheles stephensi Liston with LC₅₀ and LC₉₀ values being 1.93 and 5.67 ppm, respectively, without harming the nontarget organism, Mesocyclops thermocyclopoides Harada, after 48 h of treatment. This is our first report for the isolation and elicitation of conessine in the callus culture of H. antidysenterica.