Studies On The Screening Of Active Fractions And Chemical Constituents Of Agricultural Antibiotic Wuyiencin

Wuyiencin is a kind of agricultural antibiotic which is produced by Streptomyces ahygroscopicus var. wuyiensis with the characteristic of broad-spectrum, high efficiency, low toxicity. The producing strain of Wuyiencin was isolated from Wuyi Mountain, Fujian Province of China. Antimicrobial assays in greenhouse and field indicated that Wuyiencin is effective to control fungus diseases of vegetables, fruit trees and crops. Great progress has been made in Wuyiencin research in early research, and it was successful to be applied in Wuyiencin production. The titer of the Wuyiencin producing strain was greatly improved by optimization of fermentation broth and protoplast fusion techniques. Wuyiencin has been widely used in the production of agriculture. However, the chemical constituents and structures of Wuyiencin have not been identified, so this limited the research of Wuyiencin in molecular biology and chemical structure modification. In this study, several modern separation and purification methods and determination technology were used to study the chemical constituents of Wuyiencin, antimicrobial activities were routinely monitored during the separation process.1. Preisolation of Wuyiencin fermentation broth. Fermentation broth of Wuyiencin was submitted to AB-8 macroporous adsorptive resin column chromatography, eluted with H2O-MeOH (1: 0, 1: 9, 3: 7, 5: 5, 7: 3, 0: 1), and gave 6 fractions with different polarities. One-column volume was used for each solution. Each fraction was collected, concentrated under vacuum. The dry-weights of 6 fractions were 300 g, 250 g, 245 g, 325 g, 190 g and 60 g, respectively.2. Determination of minimum inhibitory concentrations (MICs). Antimicrobial activities were evaluated for 6 fractions with different polarities by agar diffusion method against 7 indicator microorganisms, Rhodotorula rubra, Bacillus subtilis, Bacillus megaterium, Escherichia coli, Pseudomonas solanacearum, Staphylococus aureus and Cladosporium fulvum. Minimum inhibitory concentrations (MICs) were also determined. Antimicrobial tests showed that all fractions exhibited antimicrobial effects against most microorganisms tested. The fractions eluted with water and methanol showed great antimicrobial activities, with MIC values of 0.0625 mg/ml and 0.5 mg/ml, respectively.3. Separation and purification of Wuyiencin. The fraction eluted with methanol was concentrated under vacuum and then submitted to Diaion HP-20SS macroporous adsorptive resin column chromatography, eluting with H2O-MeOH (1: 0-0: 1), resulting in 10 fractions. Then these fractions were separated by silica gel, Diaion HP-20SS, MCI-gel CHP-20, Sephadex LH-20, Chromatorex ODS column chromatography to give 7 compounds. The dry-weights of 7 compounds were 300 mg, 5 mg, 6 mg, 10 mg, 3 mg, 4mg and 5 mg, respectively.4. Structral identification of compounds. Several spectroscopic technologies (such as EI-MS, ESI-MS, dept, 1H-NMR, 13C-NMR) were used to study the structures of 7 compounds. 3 compounds were identified which were firstly isolated from Wuyiencin. Compound 1 was benzoic acid; molecular formula was C7H6O2 and molecular weight was 122. Compound 2 was flavonoid; molecular formula was C30H18O11 and molecular weight was 554. Compound 7 was organic amine; molecular formula was C8H16N2O2 and molecular weight was 172. The structures of other compounds were analysing.