Pheophorbide a Extracted from Fresh and Senescent Strobilanthes Crispus Leaves and Its photocytotoxic Effects against HepG2 Cell Line

Lim, King Hoo (2018) Pheophorbide a Extracted from Fresh and Senescent Strobilanthes Crispus Leaves and Its photocytotoxic Effects against HepG2 Cell Line. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Photodynamic treatment (PDT) using pheophorbide a as photosensitizer with the means of improving current understandings of its cytotoxic effects upon cancer cells has attained greater attention recently. Further knowledge on the efficacy of pheophorbide a in PDT upon cancer cells in-vitro strives to assist in the implementation and involvement of the photosensitizer in clinical trials. HepG2 cell line was used as the targeted cancer for investigation. The chlorophyll degradative intermediate, pheophorbide a was extracted and purified from Strobilanthes Crispus leaves in which fresh and senescent leaves were used to compare and analyze their efficacy in PDT. The efficacy study showed that the senescence leaves exhibited higher amount of pheophorbide a than fresh leaves in milligrams per gram by approximately 11%. The extraction from the leaves was performed using maceration with 80% methanol and further isolated into organic phytocompounds with solvent extraction using hexane and water. The resulting extract was separated using preparative thin layer chromatography and pheophorbide a was obtained from the process. HPLC analysis showed a purity of 72% and ±90% for the pheophorbide a obtained from fresh and senescent leaves respectively. MTT assay was performed on HepG2 cells using the purified compound to determine the cytotoxic effects. Cytotoxic effects against HepG2 was evidenced when the dosage level of the isolated pheophorbide a increases. HepG2 growth was inhibited with an IC50 value of 0.9 μg/ml and 0.8 μg/ml for the fresh and senescent isolated compound respectively. Western blot analysis of the proteins obtained from the cells by treatments with and without PDT depicted that the expression for the anti-apoptotic protein, BcL-2 was generally lowered after PDT. Hence, a higher occurrence of apoptosis for HepG2 cell line was believed to have occurred in PDT.