Induction of Mitochondrial-Mediated Apoptosis by Solvent Fractions of Methanol Extract of Heliotropium indicum in Rat Liver Cells

Introduction: The biochemical process of apoptosis may occur by a number of mechanism including; extrinsic or death-receptor pathway, intrinsic or mitochondrial-mediated pathway which involves the opening of Mitochondrial Membrane Permeability Transition (MMPT) pore in situations of intracellular calcium overload or oxidative stress or cellular insult and by the perforin/granzyme pathway. There is considerable evidence that bioactive agents in some plants (sulforaphane in cruciferous vegetables, genistein in soybeans and epigallocatechin gallate in green tea etc.) were able to induce MMPT pore opening. Quite a number of these agents have been identified and they currently under preclinical and clinical trials.

Aim: Mitochondrial Membrane Permeability Transition (MMPT) pore has emerged as a promising target for drug development because the release of cytochrome c upon the opening of the pore is a point of no return for mitochondrial-mediated apoptosis to occur. Heliotropium indicum (HI) is as an anti-tumor and wound healing agent in traditional medicine. It is not known whether its mode of action involves the induction of apoptosis via the opening of the MMPT pore.

Methodology: Mitochondria, isolated from male albino rat liver (about 100 g), were exposed to varying concentrations (10, 30, 50, 70, and 90 µg/ml) of  solvent fractions of  methanol extract of HI  i.e  Chloroform (CFHI), Ethylacetate (EFHI), Methanol (MFHI)  and crude  Methanol Extract (MEHI) of HI. Opening of the pore, cytochrome c release, mitochondrial ATPase activity and extent of mitochondrial lipid peroxidation were assessed spectrophotometrically in vitro. Activation of caspases 9 and 3 were also assessed using ELISA kits.

Results: In the absence of Ca2+, CFHI, EFHI, MFHI and MEHI induced the opening of the pore in a concentration-dependent manner with CFHI having the highest induction fold of 26 and MFHI as the lowest having 6.6. All the fractions inhibited lipid peroxidation in a concentration-dependent manner. Also, these fractions induced the release of cytochrome c with CFHI having the highest effect and the least by MFHI. Mitochondrial ATPase activity was enhanced by all the fractions with CFHI having the highest stimulatory effect. Interestingly, intra-peritoneal administration of CFHI and MEHI at 2, 5, 10 and 20 mg/kg body weight for 21 days resulted in significant opening of the pore, the release of cytochrome c and activation of caspases 9 and 3. All these effects were highest with 20 mg/kg body weight.

Conclusion: These findings therefore suggest that Chloroform Fraction of Heliotropium indicum is the most potent of all these fractions and therefore contains the bioactive agent that induces mitochondrial-mediated apoptosis in normal liver cells. The fraction will therefore be useful for further studies for drug development in diseases requesting up-regulation of apoptosis.

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