시로미와 Streptomyces sp. JR1으로부터 기능성 식품 및 의약선도 소재 개발

Abstract

Natural products have attracted considerable attention due to their potential nutritional and therapeutic effects. This study investigated bioactivity in Empetrum nigrum var. japonicum K. Koch(crowberry; Shiromi called in Korea) and useful biosynthesis gene clusters in metabolite of Streptomyces sp. JR1.

The ethanol extracts of E. nigrum from the Jeju Island of Korea were used to assess polyphenolics and flavonol levels, SOD-like enzyme activity, free radical scavenging activity, protective effects against H2O2-induced CPAE cell cytotoxicity and inhibition of LDL peroxidation. The total content of the flavonols and phenolic compounds in the leaves was higher than the fruit. In the free radical scavenging and SOD-like enzyme assay, all extracts showed strong antioxidative effects. The CPAE viability was greatly reduced and the survival rate was near 37% when the cells were treated with 1.0 mM H2O2 for 24h. The extracts also inhibited lipid peroxidation in human LDL, measured as reduction in MDA production. The results showed that the leaf extracts of E. nigrum significantly protected human LDL from Cu2+-induced oxidation. In-vivo bioactivities of E. nigrum were investigated on the following points, such as GSH concentration, Catalase activity, SOD activity in oxidative stress induced rat and on platelet aggregation, plasma lipids and bone density, in ovariectomized rat model. Thirty two male SD rats were divided into four groups at age of 8 weeks(275g). They were named as ConA for AIN-76 Vit.E free, ConB for AIN-76, EN-L for 0.05% stem and leaf extracts, and EN-F for 0.05% fruit extracts. E. nigrum had positive activities on GSH concentration, Catalase activity, SOD activity in alcoholic oxidative stress induced rat. Twenty two female SD rats were ovariectomized(OVX) and 10 female SD rats were sham-operated at age of 7 weeks(140～150g) and divided into three groups at age of 9 weeks(170～180g) such as control diet for Sham-Control and OVX-Control, 5% Shiromi powder for OVX-EN. They were fed the experimental diets for another four weeks. Platelet aggregation by ovariectomy was inhibited by E. nigrum. The degree of platelet aggregation in the presence of Shiromi was surprisingly decreased for Sham-Control group. Concentration of glucose, total cholesterol, HDL-cholesterol and triglyceride in blood samples were measured by using Enzyme assay kit(ASAN Pharmaceutical Co., Ltd, Korea). OVX-EN had less glucose in plasma than OVX-Control, but higher than Sham-Control. E. nigrum(Shiromi) increased the level of HDL-cholesterol, while it decreased the level of total cholesterol and triglyceride in ovariectomized rats. The results implied that E. nigrum was an excellent potential source of natural antioxidants used as a functional food or an alternative medicine. Ethanol extracts of E. nigrum protected injury of liver from oxidative stress and prevented cardiovascular disorders after menopause.

Streptomyces has been known as the largest antibiotic-producing genus. The history of antibiotics derived from the genus Streptomyces begins with the discovery of the first antibiotic, streptothricin, in 1942. Between 1955 and 1962, about 80% of the antibiotics were originated from actinomycetes, among which the major contributor was Streptomyces. Streptomyces sp. JR1 was isolated from the intestine of Omphalius rusticus(Bomal in Korean) captured at the Jeju coast. Culture medium of S. JR1 showed antibacterial activity against Bacillus subtilis. It also showed week antibacterial activities against Streptococcus pyogenes and Bacillus cereus. Solvent extracts of cell culture medium showed cytotoxicity against breast cancer cell(MCF-7), human promyelocytic leukemia cell(HL-60), melanoma cell(B16F10) and antinflammatory effects against LPS stimulation in RAW 264.7 cell. This study also tried to find the Industrially useful gene cluster of S. JR1. Shotgun and Fosmid DNA library were constructed from genomic DNA of Streptomyces sp. JR1. Then 3360 Shotgun clones and 1152 fosmid clones obtained. Analysis of comparative genome sequence in shotgun DNA clones showed useful gene clusters such as pksE and Daptomycin biosynthesis gene cluster etc. Other genes sequenced can be further analysed to find useful functions. The results suggested that metabolite of S. JR1 producing anticancer and antibacterial materials against MRS(multi-drug resistant strains). The gene cluster found will be used to construct the microbial medicine factory.