Preliminary Study Of HMG-CoA Reductase Inhibitors Isolated From Marine Fungus FG319

For a long time, the natural product from microbial source plays an important role in the treatment of human diseases. But in the past decades, the structure of the novel active compounds from the traditional soil microorganisms is in a clear downward trend. Furthermore, in the process of exploring new sources of biologically active natural products, marine microorganisms have attracted wide attention. Marine microorganisms include the microorganisms which living in seawater, marine sediments and symbiotic with marine animals and plants. Marine microorganisms possess unique physiological characteristics, such as salt tolerance, alkali tolerance, and anti-starvation; and they possess special metabolic mechanisms for their defense system, because of their adverse living environments, like oligotrophy, weak light, and low temperature. In this paper, we studied the fermentation of marine fungus FG319, isolation and structure determination of the secondary metabolites MFS from marine fungus FG319. Then the inhibitory effect and mechanism of HMG-Co A reductase in vitro had been studied. Finally, molecular biology means were used to identify fungus FG319.In the first chapter, we summarized the mechanism of statins to therapy for cardiovascular and cerebrovascular diseases based on inhibition of 3-hydroxy-3-methyl-coenzyme A reductase activity. In addition, the treatment of statins on cardiovascular disease, particularly atherosclerosis, showed the endothelial function improvement, vascular smooth muscle cell proliferation inhibition and cell migration as well as it maintained the stability of the plaque, and preventing the formation of foam cells. From this results, it was clearly understood that the pleiotropic drug, stain could also be used as an anti-atherosclerotic agent for the treatment of cardiovascular disease. The present research indicated that more and more active compounds could be discovered from marine microorganism, and the drugs currently used for the treatment of atherosclerosis and other cardiovascular diseases are either expensive or have side effects. With the aim of looking for new drugs to inhibit HMG-Co A reductase, the secondary metabolites of marine fungus FG319 were studied in order to obtain compounds with good activity, and to study its structural features and active effect.In the second chapter, we have studied the isolation condition of HMG-Co A reductase inhibitory compound from the broth of FG319 by the extraction of methanol and ethyl acetate, and semi-preparative HPLC. The result revealed that we could isolate the target compound using the following chromatographic conditions, the column was developed at 40℃ and the detection wavelength was 271 nm and the flow rate was 10 m L/min, through mobile phase 0.1% TFA and within 30 min of acetonitrile from 40% to 85%, the HMG-Co A reductase inhibitory compound MFS was isolated. By the detection of purity, the compound MFS can be used in the structural identification. In addition, the morphological characteristics and molecular biology methods were used to identify FG319. Phylogenetic tree based on ITS r DNA sequences showed that marine fungi FG319(KJ680136) has a high homology with Fusarium solani S-0900(EF152426) and Fusarium solani GFI 91(AJ608989), but was not yet identifiable on species level of taxonomic classification.In the third chapter, On the basis of discovery of HMG-Co A reductase inhibitory compound and single factor tests, Box-Behnken RSM was employed with three factors such as addition of L-ornithine hydrochloride at different concentrations, culture time, and culture temperatures on the yield of MFS. The optimum culture conditions were as follows: the L-ornithine hydrochloride addition was 0.6%(W/V), culture time was 7 d, and culture temperature was 22 °C. Under these conditions the yield of MFS was 20.11 mg/L, which is 4 times increased than without any optimization and the relative error with the theoretical value was 0.64%.In the fourth chapter, we have studied the structure characterization of compound MFS by the spectrum of MS, UV, IR, 1H-HMR, 13C-NMR, 1H-1H COSY, HSQC and HMBC to determine its molecular weight, molecular formula and chemical structure.In the fifth chapter, lovastatin and pravastatin were used as positive controls, we studied and explored inhibition effect of marine fungus FG319 secondary metabolite MFS on HMG-Co A reductase using HMG-Co A and NADPH as substrates. UV spectrophotometry-rate method was used in order to detect the activity of HMG-Co A reductase, and determine inhibition mechanisms of MFS on HMG-Co A reductase in vitro, and investigate mechanisms effect on the cholesterol-lowering. The results show that an in-vitro inhibiting effect of MFS on HMG-Co A reductase was higher than lovastatin and pravastatin with concentration ranging from 0 to 150 μg/m L. And according to the research of inhibition kinetics, it was concluded that MFS occupied a portion of the binding site of HMG-Co A, thus blocking access of this substrate to the active site, whereas the nicotinamide binding pocket of NADPH was unoccupied by the inhibitor molecule.