The Effects of Moringa oleifera, Quercetin and Myricetin on Cholesterol Levels in HepG2 Cells in vitro as Well as on Enzymes Regulating Cholesterol Homeostasis in silico

Abstract

Hypercholesterolemia, a major risk factor for cardiovascular diseases and atherosclerosis, is primarily managed with statins, which inhibit HMG-CoA reductase (HMGCR) and upregulate LDL receptor expression. However, prolonged statin use is associated with adverse effects, necessitating safer alternatives. Moringa oleifera (MO) has garnered attention for its diverse biological activities, including anti-atherosclerotic, anticancer, antidiabetic, antimicrobial, anti-inflammatory, and hypocholesterolemic effects. Given its widespread consumption as a tea beverage, we explored the effects of aqueous MO leaf extract on cholesterol levels and its potential to enhance statin sensitivity, thereby reducing the required dosage and mitigating side effects. To evaluate this, we employed both in vitro and in silico approaches. We assessed the effects of MO extract, simvastatin, and their combination on cell viability, reactive oxygen species (ROS) levels, and cholesterol homeostasis in HepG2 cells. Cells were treated for 24 and 48 hours with MO extract (0.015% and 0.03%) or simvastatin (10 μM). Both treatments led to a dose-dependent but time-independent decrease in cell viability. Consistently, MO extract (0.03%) significantly increased ROS levels, which were restored to baseline with N-acetylcysteine (NAC) pretreatment, whereas simvastatin had no significant ROS impact. The combination treatment partially restored cell viability, but ROS levels remained comparable to MO treatment alone. Cholesterol levels, normalized to protein content, showed the following reductions: a) 20% with simvastatin alone; b) 58% with MO extract alone; c) 70% with the combined treatment These results suggest an additive rather than synergistic effect, likely due to multiple bioactive compounds in MO extract targeting different pathways. Among these, polyphenols such as quercetin and myricetin are abundant in plant extracts. Notably, quercetin and myricetin were non-toxic at most concentrations, except for myricetin, which caused ~30% cell death at 60 μM, in alignment with ROS levels. Preliminary data showed that both flavonoids significantly reduced cholesterol levels within 24 hours, surpassing simvastatin treatment. Computational tools were employed to assess the binding affinities of simvastatin, quercetin, and myricetin with key proteins involved in cholesterol homeostasis. Docking studies revealed: a) Simvastatin competitively binds to the HMGCR active site, extending into the CoA and NADPH binding pockets; b) Quercetin and myricetin primarily occupy the NADPH binding site, suggesting an alternative mechanism of HMGCR modulation. Additionally, binding affinities of these compounds with cholesterol-regulating proteins (HMGCR, SREBP2, LXR, LCAT, ACAT, and CYP8B1) were strong, indicating their potential as cholesterol-modulating agents. ADME and toxicity profiling further supported their pharmacological viability, as both flavonoids complied with Lipinski's rule of five. This study highlights Moringa oleifera and its polyphenolic components as promising, cost-effective adjuncts to statins for cholesterol management. Their ability to enhance statin efficacy while potentially reducing dosage and side effects warrants further investigation into their therapeutic applications in hypercholesterolemia treatment