In vitro elicitation of Camptothecin by challenging with biotic elicitors in Nothapodytes nimmoniana (J.Graham) Mabb

Abstract

Camptothecin (CPT) and its analogs are gaining prominence for its promising anti-cancer activity and are used throughout the world for the treatment of colorectal, breast, and lung cancers in particular. Nothapodytes nimmoniana (J. Graham) Mabb. (Olacaceae), a plant native to India's Western Ghats, has been harnessed for the production of CPT. The endangered nature of its wild population has urged plant tissue culture and engineering techniques to increase CPT production in multifold levels by adapting elicitation methods using chemical stress-inducing substances called elicitors. The main objective of this study is to increase the CPT production through in vitro cell suspension culture of N. nimmoniana by treating it with 5 different biotic elicitors such as chitin, chitosan, pullulan, glutathione, and jasmonic acid. The preliminary quantification of CPT in methanolic extracts of wild N. nimmoniana revealed that the bark extracts showed higher level of CPT than the leaf extracts. Further, column chromatography technique was employed to obtain CPT with 99.25 % purity. The elemental composition and structure of the purified compound was confirmed through HR-MS and Proton NMR spectroscopy. Shake flask in vitro elicitation of CPT was observed at a maximum level in 10 % chitin treated elicited culture with a yield of 15.06 μg/150 ml culture followed by 5 % jasmonic acid amounting to 6.025 μg/150 ml of culture as compared with other elicitors and control. LC-MS/MS fragmentation analysis was utilized for the structural confirmation of CPT. In conclusion, the biotic elicitor chitin 10 % treated suspension cells of N. nimmoniana showed maximum level (11.48-fold) of CPT elicitation followed by 4.65-fold in 5 % jasmonic acid treatment as compared with other elicitors and control suspension cells.