ISOLATION AND EVALUATION OF PHYTOCHEMICALS FROM MUSHROOMS FOR THE INHIBITION OF HMG-CoA REDUCTASE

Abstract

Mushrooms have long been used as food and as a cure for many diseases since they form a low-caloric diet with great amount of proteins, vitamins and minerals. In particular, mushrooms are known to contain phytochemicals that could help in mitigating the development of many diseases such as cancer, diabetes and cardiovascular diseases. 3-hydroxy-3-methylglutaryl-CoA reductase is a first rate limiting enzyme in cholesterol biosynthesis. Inhibition of this enzyme is the prime target of drugs used in the treatment of high serum cholesterol levels in humans. Various synthetic drugs being used in controlling hypercholesterolemia are mostly ineffective in vivo and have high cost of production. Further cholesterol-induced oxidative stress has been considered as a major contributor in the development of atherosclerosis, thus the antioxidant activity of the selected mushrooms (Phellinus baumii, Trametes versicolor, Pleurotus osteratus and Agaricus bisporus) was determined by using DPPH (2, 2-diphenyl-1-picrylhydrazy), H2O2 (Hydrogen peroxide) radical scavenging assays and the total phenolic content was determined by using Folin-Ciocalteu method. The results revealed that the P. baumii exhibited significantly higher scavenging activity against free radicals as compared to other mushrooms under study. Moreover, only a few works have investigated the isolation of compounds from mushrooms that are responsible for lowering blood cholesterol levels. In this regard, this work tested the extracts of the selected mushrooms for their ability to inhibit recombinant HMG-CoA reductase isolated from Drosophila melanogaster. To this end, the cDNA-fragment encoding the 54- kDa catalytic domain of Drosophila HMG-CoA reductase was cloned into a modified pET30b (+) vector, named pET30f using the EcoRI and XhoI sites in the 1 2 multiple cloning site. Expression in this modified vector was under the control of a T7 promoter. The expressed recombinant HMG-CoA reductase was purified by using Ni-NTA chromatography and the molecular weight of the purified enzyme was determined to be 115 kDa by gel filtration chromatography. As a result, it was concluded that this catalytically active HMG-CoA reductase enzyme is a dimer. Moreover, kinetic analysis of this recombinant enzyme revealed the KM values of 0.2 ± 0.02 mM for HMG-CoA and 0.14 ± 0.01 mM for the cofactor NADPH. Extracts from the four evaluated mushroom species showed considerable HMGR inhibitory activity, with P. baumii extract being the most potent (Ki = 0.89 ± 0.18 μg/mL), so further investigations were done on this mushroom for the purification of the compounds responsible for inhibiting HMG-CoA reductase by using silica column chromatography and reverse phase semi-preparative-high performance liquid chromatography (HPLC). The masses and molecular formulas of the purified compounds were determined through Liquid chromatography mass spectrometry (LC-MS), Tandem mass spectrometry (MS-MS), Fourier transform ion cyclotron resonance (FT-ICR) and Gas Chromatography mass spectrometry (GC-MS). The isolated compounds in the most active peak from semi-preparative HPLC were Benzamine, 2,3,5,6-tetrachloro-4 methoxy (259 m/z) identified by using GC-MS while the other two compounds with m/z 399.2 and 457.26294 were detected by using LC-MS and FT-ICR respectively. Thus this study successfully demonstrated the use of catalytic domain of the Drosophila HMGR as a potential tool to identify the inhibitors of enzymatic activity. These preliminary studies on P. baumii have indicated the presence of potential compounds in this mushroom that may be helpful against hypercholesterolemia. So, further studies should be done on the structure elucidation of these compounds as well as isolation of other compounds 3 from P. baumii that may contribute to the development of novel therapeutic agents for lowering blood cholesterol levels.