Dr. Preethy JohnSHANKAR R2024-09-282024-09-282023-03-23https://krishikosh.egranth.ac.in/handle/1/5810215147Cancer is ranked as the primary cause of mortality and a significant roadblock to raising life expectancy in every part of the world. Though being targeted by multimodal therapies, cancer is still the most common aetiology for loss of life worldwide. The adverse effects associated with conventional therapies and the development of resistance to them warrant investigation of alternative therapeutic agents. Thus, the present study was undertaken to evaluate the antineoplastic potential of the methanolic extracts of Magnolia champaca (MMC) and Epipremnum aureum (MEA) leaves in Michigan Cancer Foundation-7 (MCF-7) cell line and to compare the effect with doxorubicin. The leaves of M. champaca and E. aureum were collected, cleaned, shade dried, extracted using methanol with Soxhlet apparatus and stored until use. The phytochemical constituents were analysed qualitatively and with gas chromatography-tandem mass spectrometry (GC-MS/MS) and fourier transform infrared (FTIR) spectroscopy. The phytochemicals identified were screened for its activity using Prediction of Activity Spectra for Substances (PASS). Those compounds with antineoplastic activity were docked with Bcl-2 protein using Autodock4. The cytotoxic potential of MMC and MEA were assessed using MTT assay at the doses of 10,20,40, 80,160 and 320 μg/mL from which per cent cell viability, per cent inhibition and half-maximal inhibitory concentration (IC50) were calculated. Doxorubicin was used as a positive control. With the IC50concentration, the apoptotic changes were studied using dual acridine orange AO)-ethidium bromide (EB) staining. The ability of the extract to generate intracellular ROS in the cells were assessed using 2’,7’ dichlorofluoresceindiacetate (DCF-DA) assay. The effect of MMC and MEA on the expression of anti-apoptotic gene Bcl-2 was studied using real time - quantitative polymerase chain reaction (RT-qPCR) with glyceraldehyde-3-phosphate dehydrogenase(GAPDH) as house-keeping gene.The extraction yields of MMC and MEA were found to be 21.26 and 17.56 per cent respectively. The qualitative phytochemical screening revealed the presence of steroids, alkaloids, flavonoids and terpenes in both the extracts, in addition tannins, phenols and glycosides in MMC. The GC-MS/MS analysis revealed presence of 99 and 93 compounds in MMC and MEA respectively. The FTIR spectroscopic analysis revealed many peaks that depicted the presence of structurally related compounds and confirmed the presence of phytochemicals that were previously screened qualitatively. The in silico docking analysis revealed good interaction of many compounds with the Bcl-2 protein. Among the various compounds, the binding of ethyl iso-allocholate, β-copaene, ascaridole epoxide and caryophyllene oxide with the Bcl-2 protein were significant with a binding energy of -5.71, -5.7, -5.00 and -4.91 kcal/mol, respectively.The MTT assay revealed a dose-dependent inhibition of cell viability in both the extracts. Based on the per cent inhibition, the IC50 values of doxorubicin, MMC and MEA were calculated to be 15.27, 66.61 and 104.4 µg/ mL respectively. On AO/EB staining, the untreated control cells were viable that emitted uniform green fluorescence whereas cells treated with MMC and MEA revealed early apoptotic changes like nuclear condensation and marginalisation. Nuclear fragmentation and late apoptotic changes could be seen in doxorubicin cells. The DCF-DA assay revealed significant generation of ROS in doxorubicin treated cells whereas cells treated with MMC and MEA scavenged the basal ROS, decreasing their intensities. The RT-qPCR revealed, critically up regulation of Bcl-2 in untreated control cells, whereas a significant down regulation of the gene in cells treated with MMC (0.15-fold) and MEA (0.34-fold)was found which was comparable with the doxorubicin (0.23-fold). From the present study, it can be concluded that both MMC and MEA possessed antineoplastic activity that produced apoptosis-induced ROS independent cytotoxicity and MMC found to be comparatively more potent than MEA. However, further studies are essential for developing novel therapeutic candidates against cancer with M. champaca and E. aureum.EnglishThesis