METABOLOMICS-BASED BIOACTIVITY ASSESSMENT OF PLUMBAGO ZEYLANICA

Abstract

The use of medicinal plants dates back to ancient times, and many of their secondary metabolites are used today both as drugs directly and as raw materials for semi-synthesis. It is costly to chemically synthesize natural compounds due to their structural complexity, and their typically low content necessitates processing large amounts of field-cultivated raw materials. Plant raw materials are limited in quantity and require costly and destructive harvesting processes to obtain on a large scale. It is possible to manufacture such compounds using biotechnological methods with a number of advantages, including predictable, scalable, and year-round sustainability. The in vitro culture of plants could provide an alternative to the extraction of secondary metabolites from plants. In this study, gas-chromatography mass-spectrometry was used to perform metabolite profiling and comparative metabolomics on Plumbago zeylanica L. leaves, stems, and roots. Elicitation of the bioactive compound plumbagin by α-ionone treatment (as a novel elicitor) of the generated hairy root cultures (HRCs) and adventitious root cultures (ARCs) from P. zeylanica L. leaf explants were tested. A series of in vitro assays were performed to characterize the mechanism of action, efficacy, and selectivity of the lead bioactive molecule against selected human cancer cell lines (Hela and HCT-15 cancer cell lines). The entire study thesis is divided into six chapters. Chapter 1 reviewed and discussed P. zeylanica, its medicinal value, bioactive molecules of relevance, and the need for metabolite profiling and improved production of plumbagin, a targeted bioactive compound. Chapter 2 focuses on the biochemical analysis of total phenolics, flavonoids, and antioxidant activity as well as comparative metabolomics of P. zeylanica plant parts (leaf, stem, and root). From the entire plumbago germplasm, 23 metabolites were identified. Benzoic acid, salicylic acid, 4-hydroxybenzoic acid, vanillic acid, plumbagin, syringic acid, coumaric acid, caffeic acid, and catechin were detected as secondary metabolites. Plumbagin was detected only in the roots and was the most abundant metabolite. Chapter 3 described the induction of adventitious roots from leaf explants and the elicitation of these roots by α-ionone. The α-ionone (10 μM) treatment significantly increased plumbagin content (~3 times) in adventitious roots. Chapter 4 described the induction of hairy roots from plumbago leaf explants using Agrobacterium rhizogenes. The plumbagin content in hairy root cultures was 2.3-fold higher than that in control roots. The elicitor treatment with α-ionone significantly increases plumbagin content (3.6-fold) in hairy roots as compared to the control roots. In Chapter 5, the lead metabolites identified from P. zeylanica were examined for their bioactivity potential. Using Hela (cervical cancer) and HCT-15 (colorectal cancer) cell lines, methanolic extracts of leaf, stem, and root were tested for anti-cancer activity. The root extract showed the highest anti-cancer activity, followed by the leaf and stem extracts. As a result of the treatment, the cancer cells appeared to be in the late apoptosis phase upon AO/EB dual staining. After plumbagin treatment, most Hela cells were in the early apoptosis phase and most HCT-15 cells were in the dead phase. PCR analysis of cell cycle regulator genes showed higher expressions of caspases and p53. In addition, plumbagin's bioavailability was studied using Caco-2 cells (intestinal epithelial cell line). It was observed that plumbagin was most readily absorbed between two and four hours after incubation. In Chapter 6, the major findings/conclusions were discussed, as well as the scope of future research.