【作者】 常军；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2008， 博士

【摘要】 藏茵陈主要来源于龙胆科（Gentianaceac）獐牙菜属（Swertia）植物,该属植物多种中药、民族药及民间药均有长期治疗肝炎的临床背景。藏茵陈在《晶珠本草》、《四部医典》及《甘露本草明镜》等藏药经典著作中均有记载,主要功能为清热消炎、利胆退黄。主治各种热病,尤以治疗肝炎后高胆红素血症、急性黄胆肝炎、慢性肝炎而著称。獐牙菜苦苷是藏茵陈的主要活性成分之一。采用三株真菌:黑曲霉、米曲霉和康氏木霉及二株肠内菌:短乳杆菌和大肠杆菌对獐牙菜苦苷转化后发现,短乳杆菌和大肠杆菌对獐牙菜苦苷没有可见的转化作用;三株真菌均对獐牙菜苦苷起到了一定程度的转化作用,生成了三个产物。黑曲霉对獐牙菜苦苷的转化率最高,达到36%,米曲霉和康氏木霉次之,分别为15%和8%。獐牙菜苦苷转化液经分离、纯化得到两个转化产物,经过波谱解析鉴定为:（5Z）-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c]pyridin-1-one（M1）和红百金花内酯（M3）。红百金花内酯是一个已知的活性化合物,具有保护皮肤细胞的作用;M1为一个新化合物。体外抗菌活性研究表明,M1对金黄色葡萄球菌和大肠杆菌具有一定程度的抑制活性。黑曲霉培养液经硫酸铵沉淀、透析及DEAE-Sepharose凝胶柱分离、纯化,得到了一个与獐牙菜苦苷转化相关的β-葡萄糖苷酶。经测定此酶分子量约为88 KDa,最适pH 5.0-6.0,最适温度为50-60℃。在60℃以下有良好的热稳定性。Mg2+和Mn2+对此酶具有一定的激活作用,而Ca2+、Zn2+、Fe3+和Cu2+则能抑制该酶;ρ-NPG与纤维二糖是其合适的底物,獐牙菜苦苷次之;葡萄糖对该酶的抑制常数为0.25 mM,表明葡萄糖对该酶具有较强的抑制能力。转化条件优化结果表明,在变温培养条件下,产M1的最优转化培养基为MgSO₄·7H₂O 5.14 mg/mL,MnSO₄·4H₂O 3.42 mg/mL,葡萄糖9.95 mg/mL,蛋白胨9.24 mg/mL,pH 5.80,M1的最大转化产率为17.64 %;产M3的最优化转化培养基为:MgSO₄·7H₂O 4.09 mg/mL,MnSO₄·4H₂O 6.07 mg/mL,葡萄糖7.37 mg/mL,酵母膏7.37 mg/mL,pH 5.73,M₃的最大转化产率为8.81 %。更多还原

【Abstract】 Three fungi (Aspergillus niger, Aspergillus oryzae and Trichoderma koningii) and two intestinal bacteria (Lactobacillus brevis CICC 6004 and Escherichia coli CICC 10032) were applied to the biotransformation of swertiamarin. The result showed that only the three fungi are able to transform swertiamarin into three metabolites. Of the three fungi, Aspergillus niger was found to has the highest ability of metabolizing swertiamarin (approximately 36%).The purification of the metabolites was carried out by liquid-liquid extraction followed by semi-preparative HPLC separation. As a result, 11 mg of M1 (96% in purification) and 6 mg of M3 (95%in purification) were obtained. The structures of two metabolites were identified by NMR, high resolution MS, UV and IR spectra as (5Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c]pyridin-1-one and erythrocentaurin. Erythrocentaurin is a known active compound and the former was a novel compound. The following study of the in vitro anti-bacteria action showed that the novel compound has an activity of inhibiting the growth of Staphylococcus aureus and Escherichia coli.Theβ-glucosidase participating in the transformation of swertiamarin was isolated and purified from the broth of Aspergillus niger. The molecular weight of this enzyme was determined as approximately 88 KDa. The optimal pH and temperature were tested as 5.0-6.0 and 50-60℃, respectively. The enzyme was stable under the temperature lower than 60℃for 12 h. Mg2+ and Mn2+ activate, but Ca2+, Zn2+, Fe3+ and Cu2+ inhibit the enzyme. The enzyme has a high affinity atρ-NPG and cellobiose, but swertiamarin was proved to be a poor substrate of the enzyme. The inhibition constant of glucose on the enzyme was 0.25mM, which suggested that theβ-glucosidase could be completely inhibited by high concentration of glucose.The optimization of medium showed that the optimal concentrations of components of medium for producing M1 were MgSO4·7H2O 5.14mg/mL, MnSO4·4H2O 3.42 mg/mL, glucose 9.95mg/mL, peptone 9.24mg/mL and pH 5.80, and the biotransformation ratio for M1 was 17.64 %. The optimal concentrations of components of medium for producing M3 were MgSO4·7H2O 4.09mg/mL, MnSO4·4H2O 6.07mg/mL, glucose 7.37mg/mL, yeast extracts 7.37mg/mL and pH 5.73, and biotransformation ratio for M3 was 8.81 %.更多还原