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The endorsement of ethno-botanical data from the under explored folk plant remedies represent an illimitable reservoir of novel compounds for drug discovery. Nature has always been the most imperative source of novel drugs against a number of unremitting and challenging health anomalies since antiquity. The present work was aimed to discover natural compounds from untapped natural source. After an extensive survey and information gathered by local vicinity, Rhus punjabensis Stewart (Anacardiaceae) plant was selected from Pakistan for investigation. For initial screening, a total of 22 extracts from leaf and stem parts of R. punjabensis were prepared by using total ‗11‘ different extraction solvent of escalating polarity. All these extracts were subjected to standard phytochemical and in-vitro biological evaluation. The phytochemical assays included standard chromogenic assays for the determination of total phenolic (TP) and total flavonoid contents (FC) and specific polyphenol quantification by RP-HPLC. Multimode antioxidant, antimicrobial, cytotoxic, antileishmanial, nuclear factor kappa-B (NF-κB) and mitochondrial transmembrane potential (MTP) detection assays were employed to assess the in-vitro biological prospective of the subject plants. All-inclusive, the results of initial screening were promising and led to the selection of R. punjabensis leaves as the most bioactive plant material for further appraisal. Briefly, maximum total phenolic and flavonoid contents were determined in the polar solvents extracts whereas, substantial amounts of gallic acid, rutin, apigenin and catechin were quantified in the moderately polar solvents extracts. A proficient antioxidant stature in terms of DPPH free radical scavenging (FRSA), total antioxidant capacity (TAC) and reducing power potential (RP) was also manifested by the moderately polar extracts of R. punjabensis leaves. A noteworthy antibacterial activity (MIC 1.11 µg/mL) against S. typhimurium. Sulphorhodamine B (SRB) cytotoxicity against human cancer cell lines (DU-145, IC50 value of 11.11 µg/mL), (HL-60, IC50 value of 10.82 µg/mL), and NF-κB based chemo-preventive proficiency was demonstrated by the ethyl acetate leaf extract.. All the extracts/fractions were tested for their anticancer and cancer chemopreventive activity by employing different independent assays. Overall the results of the initial screening were very encouraging and led to the selection of the most potent plant material for further investigation. Keeping in view the results of aforementioned assays, ethyl acetate was selected as the extraction solvent to proceed for preparative scale extraction. A total of 6 fractions were prepared from the crude ethyl acetate extract of leaves (863 g) by employing solid phase extraction as the fractionation technique. Bioassay directed isolation from active fractions using column chromatography led to the isolation of 5 bioactive compounds, n-hexane (n-hex), chloroform+n-hexane (n-hex+CHCl3) and ethyl acetate (EtOAc) fractions showed significant activities against tested bioassays. Structural elucidation of these compounds was conceded by infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (1D and 2D NMR) and mass spectrometry (MS). Compound 1 (RPPEC1) showed substantial antibacterial potential against S. typhimurium and B. bronchiseptica (MIC: 11.11 µg/mL) as well as antileishmanial activity (IC50 11.6 µg/mL). Compound 2 (PEEA-C2) demonstrated noteworthy cytotoxic activities against DU-145 and HL-60 human cancer cell lines with an IC50 value of 11.2 and 15.2 μg/mL, respectively. It also exhibited chemopreventive prospective in terms of inhibition of NF-κB (IC50 value of 19.4 μg/mL) and MTP (IC50 value of 28.6 μg/mL). Compound 3 (PEEA-C3) manifested noteworthy cytotoxicity against DU-145 cell line (IC50 value of 21.72 μg/mL). Compound 5 (RPEA-C5) demonstrated significant antibacterial spectrum against all the tested gram positive and gram negative strains while compound 4 manifested significant antibacterial activity against S. typhimurium (MIC 11.1 μg/mL) and cytotoxicity against DU-145 cell line (IC50 value of 29.5 μg/mL). The present study have been reported for the first time from R. punjabensis total five bioactive compounds were isolated, among them compound 4 (RPEA-C4) is a new compound.
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