POTENTIAL HYPOCHOLESTEROLEMIC EFFECT OF MORINGA OLEIFERA LEAVES’ EXTRACT

Abstract

Cholesterol is a lipid component of membrane structure and a precursor of many biomolecules. Disturbance in serum cholesterol homeostasis leads to an increase in cholesterol level, that underlie cardiovascular diseases and atherosclerosis. While cholesterol biosynthesis occurs in all nucleated cells, the liver plays a key role in distributing and eliminating cholesterol. Biosynthesis of Cholesterol occurs via a complex pathway known as mevalonate pathway with the rate determining step catalyzed by HMG-CoA reductase, an important target for drug development. Statins are competitive inhibitors of HMG-CoA reductase enzyme. Their therapeutic value is not limited to their effect on cholesterol pathway, but to the increase in plasma membrane LDL receptors, consequently hypocholesterolemia. However, the chronic administration of statins, have been reported to cause side effects including myopathies, and liver dysfunction, which necessitated the need to find safer and more affordable medications. Interest in alternative medicine, such as herbal medicine, has increased in recent years, providing a non-costly source for treatment of ailments. Moringa Oleifera (MO) has been recognized for its nutritional, environmental, and medicinal value. All parts of the tree are edible and have been used in curing diseases. The MO leaves are commonly consumed as hot tea beverage. Many beneficial effects have been attributed to the tea drink, including hypoglycemia, antioxidant, and hypocholesterolemic effects. In vitro and in vivo studies, using organic solvents extracts, of MO leaves demonstrated its effect on regulating cholesterol level, by controlling contributors to cholesterol pool. We hereby hypothesize that chronic administration of MO leaves water extract (MOE) will maintain cholesterol homeostasis. We investigate, in this study, the effect of chronic treatment of MOE on key enzymes controlling cholesterol level specifically: HMG-CoA reductase and LDL receptors. The viability of HepG2 cells treated (24hrs) with varying concentration (0.01% -0.1%) of MOE, decreased in a concentration dependent manner; with EC50 ranging between 0.02-0.03%. In all subsequent experiments, we opted to treat cells, for extended periods (24, 48, 72hrs), with MOE, at sub EC50. The metabolic activity was confirmed by assaying: viability (MTT and trypan blue assays); ROS level (NBT assay), and ATP (luciferase assay) level. In addition, HMG-CoA reductase and LDL receptors, transcription (RT-PCR) and translational levels (western blotting, immunostaining) were assessed in MOE treated cells. The effect on intracellular cholesterol level, as well as the direct effect of MOE on pure HMGCOA reductase were also determined. We hereby report in MOE-treated HepG2 cells a concentration dependent, but time independent: a) decrease in the viability; b) increase in ROS and c) decrease in ATP level. Importantly, these findings suggest a strict mechanism controlling ROS and ATP levels maintaining thus metabolic activity of HepG2 cells. Moreover, MOE treated cells caused a significant decrease in HMG-CoA reductase with a concomitant increase in LDL receptors, gene expression levels. No change in the house keeping gene (GAPDH) level was detected. Unexpectedly however, the translated protein level of GAPDH, decreased in MOE treated cells, hence actin was used instead to assess the relative variation in the tested proteins. While a non-significant decrease in the translated HMG-CoA reductase protein was obtained, both pre-mature and mature LDL receptors levels increased significantly. The increase in the level of the LDL receptors was also confirmed using immunostaining. Extracted intracellular cholesterol level showed a small, but not significant, decreased 48-hours following MOE treatment. To sum up, our results demonstrate a hypocholesterolemic potential of MOE. It increases the LDL receptors favoring cholesterol uptake into the cells and decreases HMG-CoA reductase gene expression impacting on cholesterol biosynthetic pathway. In addition, we identified another target of MOE, namely GAPDH enzyme that links glycolysis to electron transport chain. The decrease in the activity of this enzyme will favor flux of glucose into HMP stimulating production of NADPH controlling ROS level in MOE treated cells favoring proliferation and viability. Finally, our findings provide a link between dietary practices and disease prevention. A recommended daily intake of MOE (common tea drink) may have a beneficial effect regulating cholesterol level, hence protecting subjects with low risk from developing heart disease.