Moringa oleifera Lam. has received much praise over its potential benefits to health and nutrition, however, not much literature is available to support claims of hepatic safety. The liver, as primary xenobiotic metaboliser, is subject to high levels of cytotoxicity. As such, hepatotoxicity has been a consistent hurdle during the drug discovery and development process. The aim of this study was to assess the in vitro hepatotoxic properties of both hot water and methanol extracts of M. oleifera leaves by assessing common mechanisms of cytotoxicity in the HepG2 cell line. Also, the ability of these extracts to protect against in vitro oleic acid-induced steatosis was investigated.
Tentative phytochemical profiling was done by ultra-performance liquid chromatography coupled to a photodiode array detector and high-definition mass spectrometer. In vitro cytotoxicity was assessed by sulforhodamine B staining (cell density), dihydro-dichlorofluorescein diacetate cleavage (reactive oxygen species), monochlorobimane adduct formation (reduced glutathione), JC-1 ratiometry (mitochondrial membrane potential), nile red staining (fatty acids), Ac-DEVD-AMC cleavage (caspase-3/7 activity), thiobarbituric acid reactive species production (lipid peroxidation), chemiluminescence (adenosine triphosphate; ATP), propidium iodide distribution (cell cycle) and Annexin V-FITC dual staining (mode of cell death). The protective action of the extracts was determined using an oleic acid-induced model of steatosis.
Phytochemical profiling tentatively identified several chemicals within the extracts. Viability assessment indicated slight cytotoxicity at 100 μg/mL after both the hot water and methanol extract exposure (approximately 8–14%), however, this was not reflected by cell density results. The cell cycle remained unperturbed, apart from a slight increase in G0/G1-phase. Although mitochondrial depolarisation was evident, especially after exposure to the methanol extract, it did not translate to cytotoxicity in the form of oxidative stress. Reactive oxygen species generation was only observed at 100 μg/mL hot water extract exposure (1.99-fold), with the rest of the values being below baseline. Slight lipid peroxidation (1.48-fold at 100 μg/mL) was observed after methanol extract exposure. The GSH was unaltered. Fatty acid levels reduced, especially after methanol extract exposure (0.43-fold at 100 μg/mL), which may be indicative of increased mitochondrial respiration. Caspase-3/7 activity and ATP were decreased slightly. Methanol extracts were particularly active in protecting cells from oleic acid-induced cytotoxicity and fatty acid accumulation, with a decrease of up to 27% and 4.1-fold at 3.2 μg/mL, respectively.
Although the in vitro results suggest hepatic safety, further investigation is needed to ascertain the underlying mechanisms of cytotoxicity at concentrations above 100 μg/mL, particularly the effects on mitochondrial membrane stability. Although mitochondrial membranes were depolarised, this may assist with the reduction of fatty acid levels and offer protection against oleic acid-induced steatosis.