The Activity of Ten Natural Extracts Combined in a Unique Blend to Maintain Cholesterol Homeostasis-In Vitro Model

Abstract

Background: Hypercholesterolemia is a major cause of cardiovascular disease and statins, the HMGCoA inhibitors, are the most prescribed drugs. Statins reduce the production of hepatic cholesterol, leading to greater expression of the LDL receptor and greater absorption of circulating LDL, reducing peripheral LDL levels. Unfortunately, statins are believed to induce myopathy and other severe diseases. To overcome this problem, safe nutraceuticals with the same activity as statins could hold great promise in the prevention and treatment of hypercholesterolemia. In this study, the anti-cholesterol efficacy of a new nutraceutical, called Esterol10^®, was evaluated.

Methods: HepG2 cells were used to study the biological mechanisms exerted by Esterol10^® analyzing different processes involved in cholesterol metabolism, also comparing data with Atorvastatin.

Results: Our results indicate that Esterol10^® leads to a reduction in total hepatocyte cholesterol and an improvement in the biosynthesis of free cholesterol and bile acids. Furthermore, the anti-cholesterol activity of Esterol10^® was also confirmed by the modulation of the LDL receptor and by the accumulation of lipids, as well as by the main intracellular pathways involved in the metabolism of cholesterol.

Conclusions: Esterol10^® is safe and effective with anti-cholesterol activity, potentially providing an alternative therapy to those based on statins for hypercholesterolemia disease.

Keywords: cardiovascular disease; cholesterol homeostasis; food supplement; low-density lipoprotein; monacolin K; nutraceuticals; statin.

Figure 1

Cell viability of HepG2 cells incubated with the agents alone and combined, for 24 h. RYR = Red Yeast Rice. From A to E the dose-response study on cell viability was measured by MTT test of each single extract such as vitamin K2 and vitamin D3 (A), quercetin and resveratrol (B), CoQ10 and folic acid (C), astaxanthin and sodium selenite (D), sage extract and RYR (E). In (F) the effects of the better concentration of single agents alone and combined (Esterlo10^®) were compared to RYR and atorvastatin alone. Viability was calculated as the reduction percentage of cells in the culture medium without the addition of test substances. Data are mean ± SD of five independent experiments performed in triplicates. * p < 0.05 vs. control.

Figure 2

HMGCoA reductase activity (A) and HMGCR expression (B) on HepG2 cells. The abbreviations are the same reported in Figure 1. Data are mean ± SD of five independent experiments performed in triplicates. In panel B, the images reported are an example of a Western blot obtained with technical replicates. * p < 0.05 vs. control; ^# p < 0.05 vs. RYR; ^y p < 0.05 vs. Atorvastatin.

Figure 3

Total Cholesterol (A), Free Cholesterol (B), LDLr (C), and LDL uptake quantification (D) on HepG2 cells. The abbreviations are the same reported in Figure 1. Data are mean ± SD of five independent experiments performed in triplicate. In (C), the images reported are an example of a Western blot obtained with technical replicates. * p < 0.05 vs. control; ^# p < 0.05 vs. RYR; ^y p < 0.05 vs. Atorvastatin.

Figure 4

Bile acid production following treatment of hepatic in vitro model. The abbreviations are the same as reported in Figure 1. Data are mean ± SD of five independent experiments performed in triplicate. * p < 0.05 vs. control; ^# p <0.05 vs. RYR and ^y p < 0.05 vs. Atorvastatin.

Figure 5

Intracellular pathways activated in HepG2 cells. In (A,B,E) the protein activity of PCSK9, SRC, and ERK was measured by an ELISA test; in (C,D) the analysis of AMPK and SRC performed by Western Blot and densitometric analysis. The abbreviations are the same reported in Figure 1. Data are mean ± SD of five independent experiments performed in triplicates. The images reported are an example of a Western blot obtained with technical replicates. * p < 0.05 vs. control; ^# p < 0.05 vs. RYR; ^y p < 0.05 vs. Atorvastatin.

Figure 6

Tissue integrity analysis. In (A), ALT activity was measured by an ELISA test. Images captured under the microscope at an original magnification of ×20 and reported in (B) showed the intercellular oil droplets stained by the Oil Red stain. These oil droplets are solubilized for quantification by spectrophotometric analysis reported in (C). The abbreviations are the same as shown in Figure 1. Data are mean ± SD of five independent experiments performed in triplicate. * p < 0.05 compared to the control; ^# p <0.05 vs. RYR; ^y p <0.05 vs. Atorvastatin.