THE ENHANCEMENT OF CONTENT OF BIOACTIVE METABOLITES IN ECONOMICALLY IMPORTANT MEDICINAL PLANTS BOERHAVIA DIFFUSA AND RUTA CHALEPENSIS

Abstract

Due to the high cost of modern healthcare, the global population is steadily turning towards herbal medicinal products and other plant-derived medicinal compounds. This has created a huge gap between the demand and supply of plants harboring important natural products. To cater to this need, plant secondary metabolites are either attempted to be synthesized chemically or the plants are harvested from the wild in an unsustainable manner. Even when harvested from nature, these plants are not reliable sources for consistent yield and quality of secondary metabolites. It is critical to investigate the phytochemical profile of plant extracts in order to create sufficient quantities of a plant-based medicine. In order to design viable strategies for increased bioproduction, it is critical to understand the effect of external and internal factors that have an effect on the biosynthesis of these phytochemicals. In current times, biotechnology has become a major contributor in medicinal product research, with the field of plant cell and tissue culture holding the centre stage of research. For the objectives of this thesis, two medicinal plants were chosen based on their commercial importance in the field of drug development: Boerhavia diffusa and Ruta chalepensis. Both B. diffusa and R. chalepensis plant extracts exhibit a wide range of pharmacological properties. Because of their reputed ayurvedic and therapeutic uses, both of these plants are included in the pharmacopoeias of several countries. In order to meet the growing demands for these plants, some alternative strategies were explored. Till date, experimentally validated strategies for the enhanced production of targeted bioactive metabolites identified from these plants using in vitro culture system as well as intact plants remains poorly studied. Initially, plants and saplings of B. diffusa and R. chalepensis were collected from different locations. Three chemotypes of B. diffusa and two of R. chalepensis were screened for elite phytochemical profiles. To assess the chemotypes for best antioxidant activity, various phytochemical methods were employed. The constituent phenolics and flavonoids were compared since they have potential antioxidant activity. Thereafter, free radical scavenging potential was analysed along with the ability to reduce ferric ions. B. diffusa chemotype B and R. chalepensis chemotype A were determined to be elite cultivars. Subsequent experiments were carried out on elite chemotypes only. GC-MS technique was used to obtain comprehensive metabolite profiles. In contrast to chloroform extract, the methanolic extract of B. diffusa leaves, rich in D-pinitol, was effective at accelerating healing of in vitro scratch on keratinocytes and in vivo excision wounds in Albino Wistar rats. Wound-healing activity of D-pinitol was determined in vitro. GC-MS-based metabolic profiles of leaves and roots of R. chalepensis were compared. The coumarin family consisting of umbelliferone, psoralen, xanthotoxin and bergapten were found to be stored in leaves, even though umbelliferone and xanthotoxin were higher in roots, and psoralen and bergapten were absent in roots. Since rutin, an important constituent of R. chalepensis, was not detectable by GC-MS, a simple, rapid and specific HPLC method was developed and validated for the simultaneous detection and quantification of rutin and the 4 coumarins. These five metabolites were tested for anticancer activity in human colon cancer cell line HCT-15. With the objective of enhancing the production of these important metabolites, a variety of biotechnological strategies were explored using in vitro and in vivo methods. Invasive as well as non-invasive approaches were employed to achieve overproduction. For B. diffusa, a controlled application of water deficit resulting in drought stress was successful in eliciting the overproduction of D-pinitol in intact plants. D-pinitol levels increased multifold to 486% by day 21 of imposed water-deficit. For R. chalepensis, two strategies were explored for enhancing metabolite production both in vitro in callus and cell suspension cultures and in vivo in intact plants. First, methyl jasmonate was used to elicit cell suspension cultures, which resulted in enhanced production of coumarin and furanocoumarins. Accumulation of umbelliferone peakes at 24 hpe (hours post elicitation), followed by psoralen at 36 hpe, and xanthotoxin and bergapten at 48 hpe. Foliar application of methyl jasmonate (20 μmol) was carried out for intact healthy R. chalepensis plants, which elicited the overproduction of umbelliferone, psoralen, xanthotoxin and bergapten by 2- to 5- folds. Next, monochromatic LED lights (red, green or blue) were used instead of regular greenhouse lighting over R. chalepensis plants. Blue LED treatment of greenhouse grown plants resulted in maximum 2.8-, 2.3-, and 3.8-fold increase in the contents of umbelliferone, psoralen, and xanthotoxin, respectively. Blue LED was also successful in enhancing the accumulation of umbelliferone, psoralen and xanthotoxin in callus cultures od R. chalepensis.