Preliminary Investigation Of HMG-CoA Reductase Inhibiting Activities Of Metabolites In Marine Microorganisms

Active compounds from microbial secondary metabolism play a vital role in the prevention and treatment on human diseases. Recent years, the widespread abuse of traditional antibiotics have triggered the increasing of bacteria’s resistance. However, the discovery of novel active compounds has become more difficult. Therefore, researchers pay more and more attentions to marine microorganisms. But the knowledges of nutritional needs and metabolic activity of these microorganisms are limited, in which case, the selection and design of appropriate media and culture conditions are critical points of the separation of marine microorganisms. Designing the optimized media and culture conditions make marine organisms fully exploited in outputting active natural products with novel structures. In this research, preliminary findings on ocean Fusarium solani FG319 was discussed.one of the secondary metabolites of FG319,which is named MFS has a significant effect on the inhibition of HMG-Co A reductase. And the the culture, extraction and separation conditions of Fusarium solani FG319 was optimized. Moreover, the activities and structure of MFS was investigated.The first chapter summarized the development of the 3-hydroxy-3-methyl-glutaryl-Co A, HMG-Co A reductase inhibitors, known as Statins, from the first generation of cutting US statins to the latest rosuvastatin; the inhibition of HMG-Co A reductase, stabilize and reversing of the atherosclerotic plaque, inhibition of the migration and proliferation of smooth muscle cells, endothelial cells and regulate vasomotor function and other important pharmacological effects. This article aims to production increasing of HMG-Co A reductase inhibitors and culture, extraction and separation conditions of marine Fusarium solani FG319 were also optimized in order to obtain a pure substance which has potent activity. Furthermore, its structural characteristics was also elucidated.In the second chapter, the maintaining of p H was observed in the broth in which marine Fusarium solani FG319 was cultured and for 5 days in small scale shaker. The crude extract was with maximum absorption at 271 nm was obtained from the end product of the fermentation. With the concentration increasing, the inhibition rate of the HMG-Co A reductase was elevated. When concentration of the crude extract was 150 μg /m L, the inhibition rate reacheed 80%. The crude extract of MFS(Metabolite of Fusarium solani FG319) form the preliminary separation and purification shows the most significant inhibitory activity to HMG-Co A reductase, 9.08 ± 1.38 mg / m L presented in IC50.In the third chapter,the goal was to improve the production of HMG-Co A reductase inhibitors, MFS(Metabolite of Fusarium solani FG319), with the optimizing of the fermentation mediums and conditions of marine microorganism, Fusarium solani FG319. In the present study, following mediums were used to optimize the fermentation conditions by Shake flask fermentation and fermenter: GSB medium, CYM medium, ATCC medium, BPY medium, TYG medium, Oatmeal medium, Czapek-Dox medium and potato dextrose broth culture medium. The result of optimization revealed that Czapek-Dox mediumis was the best medium for Marine microorganism Fusarium solani FG319; maximum production of HMG-Co A reductase inhibitor MFS was achieved on 7th day; inducer L-ornithine hydrochloride had increased the production of MFS(4.07 times);in all sugar type, the marine microorganism Fusarium solani FG319 metabolic MFS quantity was up to 23.47 mg / L without Na Cl(p H = 6.9);The medium selection and optimization of fermentation conditions significantly improved the production of HMG-Co A reductase inhibitor in marine microorganism Fusarium solani FG319.In Chapter IV, strain FG319 fermentation was extracted by ultrasonic with methanol, methylene chloride, ethyl acetate and n-butanol according to the polarity of successive ascending order of extraction. The product extracted with only dichloromethane performed maximum absorption at 271 nm(ε=7.6×104) the maximum absorption peak. The fraction presented by retention time of 36.65 min in HPLC chromatogram as well as tailing of Rf=0.58 in Tl C of dichloromethane organic phase was the target active product MFS. The purities of fractions from column chromatography which dichloroform as eluant were tested by comparison of TLC Chromatogram. After elution TLC plate combined entry point trailing point 3 containing the desired active product MFS. Further purification of dichloromethane crude extract was achieved HPLC which methanol: 50 mmol / L ammonium acetate = 70: 30 as the mobile phase with Shimadzu SCL-10 Avp HPLC separation system.Strains FG 319 was isolated with secondary metabolites inhibiting HMG-Co A reductase. 23.47 mgpurified active compound was obtained from 1L fermentation broth of FG319.In Chapter V: The production of MFS was analyzed using a HPLC by chromogenic substrate. Also, an inhibitory effect of MFS on HMG-Co A reductase was determined in-vitro.in-vitro evaluation system shows inhibition activity of MFS on HMG- Co A reductase is higher than the activity of Lovastatin(1.38 times)and Pravastatin(1.74 times).The inhibition type and inhibition constant of MFS was determined by Lineweaver-Burk plots and slope replots. The results showed that MFS competed with HMG-Co A, whereas it was uncompetitive towards NADPH.In Chapter VI the structure characteristic parameters of the active compound MFS were analyzed based on its ESI-MS mass spectrum, ultraviolet spectrum, nuclear magnetic resonance spectrum(1H-NMR, 13C-NMR), etc., the molecular weight of the compoundwas 719, maximum absorption wavelength was 271 nm, typically NMR signal δH is 0.866(13H, m), 1.138(7H, m), 1.257(10H, m), 1.302(4H, m), 1.345(5H, m), 1.163(6H, m), 2.029(6H, m), 2.342(6H, t, J = 7.33Hz), 2.770(1H, t, J = 6.41Hz), 4.130(1H, m), 5.344(4H, m). Typical compounds of signal carbonδC are14.06,14.11,22.59,22.70,24.69,25.65,27.18,27.20,27.23,29.05,29.08,29.16,29.25,29.34,29.38,29.45,29.54,29.61,29.66,29.69,29.70,29.7229.79,31.55,31.95,34.10,127.93,128.10,129.74,130.03,130.22,180.29,180.33. The compound is Initially speculated to be macrolide compounds.In this research, an optimized culture condition of marine Fusarium solani FG319 fermenter was obtained, followed by separation and purification of crude extract metabolites of MFS. Inhibition of HMG-Co A reductase in-vitro activity of MFS and the structure characteristic parameters attributed to macrolide were investigated. The in-vitro studies revealed that the secondary metabolites, MFS, from marine Fusarium solani FG319 showed inhibition on activities on HMG-Co A reductase and deceleration on the generation of the endogenous cholesterol precursor mevalonate, which can reduce the synthesis of cholesterol and help to prevent heart brain vascular diseases. MFS is expected to become a novel leading compound for the prevention and treatment of atherosclerosis as well as other cardiovascular and cerebrovascular diseases.