Soheil Zorofchian, Moghadamtousi (2016) In Vitro and In Vivo evaluation of the chemopreventive, gastroprotective and wound healing potential of Annona Muricata / Soheil Zorofchian Moghadamtousi. PhD thesis, University of Malaya.
Abstract
Annona muricata Linn. is a popular fruit tree growing in tropical countries. Its leaves have been extensively employed in folk medicine to treat a variety of ailments and diseases. In this study, anticancer, gastroprotective and wound healing properties of A. muricata leaves and their possible mechanisms of action were determined using in vitro and in vivo models. Solvent extraction yielded crude ethyl acetate extract (AMEAE), which demonstrated remarkable cytotoxicity against different cancer cell lines including A549, HT-29 and HCT-116. Hence, AMEAE anticancer property was investigated against the respective cell lines. In addition, in vivo chemopreventive potential of AMEAE was determined against azoxymethane-induced colonic aberrant crypt foci (ACF) in rats, and AMEAE was subjected to a bioassay-guided approach to isolate the cytotoxic compound and evaluate its apoptosis-inducing effect. AMEAE was found to induce mitochondrial-initiated events in cancer cells, as the treated cells shown disruption of mitochondrial membrane potential, cytochrome c leakage and elevation of Bax expression. Inversely, Bcl-2 expression was lowered in the treated cells. The following experiments suggested apoptosis induction in cancer cells, as was reflected by increase in total nuclear intensity, augmentation in sub-G1 cells, externalization of phosphatidylserine and activation of initiator (-9) and executioner (-3/7) caspases. These findings strongly implied that exposure of AMEAE to cancer cells have resulted in apoptosis induction through the intrinsic pathway. A bioassay-guided investigation on AMEAE led to the isolation of annonaceous acetogenin, annomuricin E which induced significant apoptosis-inducing effects in HT-29 cancer cells through mitochondrialmediated mechanism. The in vivo chemopreventive potential of AMEAE was examined in five groups of rats, namely negative control, cancer control and AMEAE (250, 500 mg/kg) and positive control (5-fluorouracil). Oral treatment of AMEAE at both doses iv decreased the formation of colonic ACF. The expression of PCNA protein, a marker of cell proliferation, was downregulated in treated cells and associated with upregulation of Bax and downregulation of Bcl-2. These results substantiated the traditional use of A. muricata leaves against cancer and tumors. The gastroprotective activity of AMEAE at two doses of 1 g/kg and 2 g/kg was examined against ethanol-induced gastric injury in rats. Gross and histological characterizations suggested the antiulcerogenic property of AMEAE. Immunostaining revealed upregulation of Hsp70 protein and downregulation of Bax protein. This activity was associated with attenuation in oxidative stress evidenced by an increase in the level of enzymatic antioxidants and nitric oxide and decrease in the level of malondialdehyde. These findings revealed promising gastroprotective potential for AMEAE, which was mediated through antioxidant and anti-inflammatory mechanisms. Wound healing potential of AMEAE (5% w/w and 10% w/w) was evaluated against excisional wound models in rats. Significant wound healing activity was observed after topical treatment with AMEAE, assessed by macroscopic and microscopic analyses. This was associated with a decrease in the number of inflammatory cells, supported by upregulation in the expression of Hsp70 protein. In addition, level of enzymatic antioxidants showed augmentation which led to the attenuation in the malondialdehyde formation.
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