Ephedra Active Ingredient To Produce The Optimization Of Strains And Their Production Conditions

Five endophytic fungi were isolated from the stem of a medicinal plant Ephedra. Sinicastapf.By the deposition reaction and the special color reaction, two strains— Mh3 and Mh5—were screened out. Then Mh5 was selected out because of its higher yield of alkaloid. Two extracting methods were compared and some conditions in process extraction were optimized. And the optimum extraction solvent was CHCl₃, the temperature was about 40℃ and others were same with the industrial method. The dry mycelia and the culture filtrate of Mh5 were extracted and tested and it was found that the yield of alkaloid in the culture filtrate were more than those in the mycelia.The extract of the culture filtrate of Mh5 was compared with the standards of ephedrine and pseudoephedrine by TLC scan in UV and visible spectrum. It was found that the absorption curves of the extract were basically same as those of standards. The results of IR and HNMR was analyzed and it was supposed that the extract was belong to ephedra alkaloid and might be a mixture of ephedrine and pseudoephedrine.The culture media were selected according to the growth rates of the colonies, the mycelia dry weight and the yields of alkaloid. It was found that the bean-sprout extract medium or PDA were good as solid media for mycelial growth and the synthetic medium was appropriate to for the production of alkaloid. Then the components of the synthetic medium were optimized, including carbon source and nitrogen source. It was found that sucrose and (NH4) 2SO4 were suitable for the mycelial growth and the alkaloid yield.The fermentation conditions of Mh5 were further optimized by the orthogonal experiment. The results showed that sucrose 20g/L, ( NH₄ ) 2SO₄1.5g/L, pH about 7.0 and the temperature about 30℃ were the optimum conditions for its mycelial growth; sucrose 40g/L, (NH4)₂SO₄4.5g/L, pH about 5.5 and the temperature about 25℃ were suitable for alkaloid production.The strain—Mh5— was classified as Alternaria Nees ex Wallr. based on the method of morphological taxonomy.