IN VITRO PROPAGATION AND IN SILICO STUDIES OF IMPORTANT MEDICINAL PLANTS

Abstract

Blooming and acceptability of traditional system of medicine has drastically increased the demand of medicinal plants. Demand of therapeutically important medicinal species is continuously increasing in national and international markets. Exploring elite accessions among several accessions, collected from different agro-climatic zones, can be a suitable approach to meet the increasing demand of medicinal plants. Biotechnological approaches such as plant tissue culture and molecular marker-based techniques are reliable for producing elite accessions and offer long-term utilization of the herbs. Two pharmaceutically important medicinal plants, Bacopa monnieri(L.) Wettst and Chlorophytum borivilianum Sant and Fern. have been explored in the present study. In vitro conservation protocol was established for 13 accessions of B. monnieri, a medicinal plant with legendry reputation as brain energizer. Sixteen media combinations (MS with varying concentrations of auxins and cytokines) were tested for 13 accessions of B. monnieri to find the optimum media based on growth parameters for in vitro propagation. The accession with maximum number of shoots, shoot length and number of propagules was selected for further study. Furthermore, the concentration of therapeutically important secondary metabolite such as total bacosides, total phenol content and antioxidant potential of 13 accessions of B. monnieri were evaluated. The concentration of total bacosides in 13 accession ranged from 216.93 - 515.47 µg/g dry weight bases. Amount of total bacosides and total phenol content in wild grown plants is generally low. Use of elicitors in cell culture technology not only increased the yield of total bacosides (2.7-3.9 fold) but the production of total phenols and antioxidant potential has also been increased to 5-18 fold and 7.9 fold respectively. Principal Component Analysis (PCA) using correlation matrix was carried out to evaluate the relative contribution of moisture content, antioxidant potential, total bacoside and total phenols to the total variability in the B. monnieri accessions. Moreover, accessions of different agro-climatic zones were assessed for genetic diversity analysis with gene targeted molecular markers SCoT (Start Codon Targeted Polymorphism) and CBDP (CAAT box Derived Polymorphism). Genetically diverse

accessions can be utilized by plant breeders to produce elite accessions having high amount of therapeutically important secondary metabolites. Accession-based study of in vitro plant cultures based on growth parameters, presence of therapeutically important secondary metabolites and antioxidant potential has identified accessions IC 554588, IC 344312 and IC 554585 of B. monnieri as elite accession. The present study also explored the effect of bacoside on age-related neurodegenerative diseases through in silico studies as well as through in vitro assay. AD is associated with depletion in amount of acetylcholine, a neurotransmitter. The results generated by molecular docking and molecular dynamics simulation programs revealed that the AChE complex with bound Bacoside is very stable throughout the simulations period of 30 ns. The second important medicinal plant is C. borivilianum, known to possess stigmasterol, a phytosterol. Ten media combinations have been used for in vitro propagation and root induction. The optimum selected media based on growth parameters such as maximum number of shoots, shoot length, number of roots and root length can be used for rapid proliferation and conservation. Biochemical analyses of pharmaceutically active metabolite, stigmasterol and total phenols along with antioxidant potential have been assessed for the three accessions. Variable amount of stigmasterol (1.18-1.58 mg/g DW), phenol content (89.0 - 140.0 µg/g) and antioxidant potential has been observed in three accessions. The accession with maximum biochemical activity was selected for elicitation study. Elicitation has increased stigmaterol 3.2 fold, 4.3 fold and 17.7 fold. Principal Component Analysis showed antioxidant potential as major contributor for variability. Accession IC 558379 has been identified as promising accession with higher stigmasterol production, phenol content and antioxidant potential. The present study also explored the anticancer activity of stigmasterol through computational approach. The ligand stigmasterol was docked with the target tumor necrosis factor alpha converting enzyme (TACE) to explore its effect on colon cancer. It can be suggested that stigmasterol, a green material may be considered as a possible therapeutic agent in the treatment of colon cancer and the same can certainly be confirmed through in vivo studies and clinical trials to confirm their effectiveness in patients.