ENHANCED PRODUCTION OF BIOACTIVE WITHANOLIDES FROM WITHANIA SOMNIFERA: A METABOLOMICS APPROACH

Abstract

W. somnifera (L.) Dunal also known as Ashwagandha, is a well-known valued medicinal plant belongs to the Solanaceae family. W. somnifera (L.) Dunal, is an important medicinal plant with tremendous use in the herbal drug industry. It is a commended genus described in the Indian Ayurvedic system of medicine and also enlisted as an important herb in Unani and Chinese traditional medicinal systems. This plant is geographically distributed around the world, mainly in tropical and subtropical regions bearing dry weather. The entire plant has important medicinal applications, but the root and leaf extracts in particular are known to have a broad spectrum of biological activities, which include anti-inflammatory, anti-cancer, cardio-protective, neuroprotective, and immunomodulatory. The plant root is used as an anti-inflammatory, anti-aging, anti-tumor, memory boosting, anti-stress, anti-oxidant, immunostimulant, and as diuretic. This plant exhibited several important phytoconstituents such as phenolics, flavonoids alkaloids, and withanolides but Withanolides are the major phytoconstituents responsible for biological activities. Most of the beneficial pharmacological effects have been attributed to phytochemicals present in the plant known as withanolides of which withaferin-A is the most well-studied. There are several techniques like HPTLC, HPLC, GC-MS, LC-MS, etc to identify and quantify these metabolites. Present research work was focused to establish a new method for enhanced extraction efficiency for these metabolites from various plant parts. To achieve this goal, separation, and quantification of these important phytochemicals were done, then robust methodologies of metabolite profiling and metabolomics using HPTLC, HPLC, and Gas Chromatography-Mass Spectrometry (GC-MS) data analysis were established. This allowed a comprehensive analysis of withanolides, fatty acids, sugars, organic acids, amino acids, and secondary metabolites. Develop an easy-to-use analytical method for the separation and identification of withanolides and bioactive phenolics. Three withanolides- withaferin A, withanone, and withanolide A and three phenolic acids- caffeic acid, ferulic acid, and benzoic acid- from different plant parts of Withania somnifera and its two commercially available polyherbal formulations have been simultaneously determined by using an easy, rapid, and selective high-performance thin-layer chromatographic (HPTLC) approach. HPTLC separation was carried out on silica-coated aluminium plates Si 60F254; using toluene, ethyl acetate, and acetic acid (60:40:4). When compared to other phytoconstituents present in W. somnifera extracts, this simple and accurate HPTLC method provided enhanced resolution of the studied analytes. Also, it has been used successfully to analyze and quantify two polyherbal formulations that comprise W. somnifera plant components.However, due to the low abundance of withanolides, it is difficult to fulfill the market demand. There is a serious gap between the demand and supply of these phytoconstituents. So, after that focus, the major problem is the low accumulation of withanolides in this plant. So, there is an urgent need to develop a new method for the enhancement of withanolides. The purpose of this study was to develop a simple method for enhanced withanolide production in W. somnifera field-grown plants. An efficient method to enhance the production of several plant secondary metabolites is to apply exogenous elicitor treatment to field-grown plants. In this study, a high-performance liquid chromatography method was developed for the easy separation of withanolides, phenolic acid, and flavonoids from the leaves. Here we applied new technology to enhance the production of these metabolites. We introduce a non-destructive elicitation strategy using field-grown/green-house-grown plants of W. somnifera for rapid in-planta enhancement of withanolide accumulation. A novel closed polybag approach was used to treat W. somnifera plants that were cultivated in the field by using the methyl jasmonate elicitor. This method involved covering branches in specialized polybags before injecting MeJa into them using a vial that was specifically made for the purpose. Withaferin A, withanolide A, withanolide D, and withanone production increased, respectively, 2.2-fold, 1.9-fold, 2.0-fold, and 2.2-fold, over the course of a 4-hour treatment with methyl-jasmonate. Treatment with methyl-jasmonate also raised the transcript levels of four essential withanolide biosynthesis genes, including 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, and cycloartenol synthase. A substantial cytotoxic impact against a selected human breast cancer cell line was also shown by methyl-jasmonate-treated leaf extracts and extracted pure withanolides. These findings suggest that MeJa-treatment using a closed polybag system is a successful and simple elicitation method for the increased production of bioactive withanolides in field-grown W. somnifera plants. This technology is easily adaptable by industry and farmers to address the rising demand for bioactive withanolides. Also, develop a cisgenically cell suspension culture of W. somnifera overexpressing squalene synthase (SQS) gene from a similar W. somnifera plant for enhanced production of withanolides using in vitro platform. In this work, first, we have cloned the squalene synthase (WsSQS) gene from W. somnifera plants. Thereafter, young leaves of W. somnifera were transformed using a cisgenic strategy to express an extra copy of the native WsSQS 1 gene to increase the expression of WsSQS and subsequent higher SQS enzyme activity. WsSQS overexpressing cell lines exhibited 2.4-fold higher ‘withaferin A’ yields than wild‐type cell suspension cultures. In conclusion, we can say that an increase in WsSQS activity obtained through cisgenic overexpression of WsSQS can improve withaferin production metabolically engineered cell suspension culture of W. somnifera. And also we checked anticancer efficacy and the mechanism of actions of selected withanolides toward selected human cancer cell lines.The major outcome of the thesis is:- • W. somnifera a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids known as Withanolides • Withanolides are present in this plant at very low concentrations up to 0.5% of plant dry weight • So, the enhancement of withanolides was done by using different techniques such as Meja treatment and cis-genic cell culture