人参皂甙Rb_3的生物转化以及拟青霉菌sp.229中代谢产物的分离纯化鉴定
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摘要
本论文的研究内容包括人参皂甙Rb_3微生物转化和拟青霉菌sp.229代谢产物的研究两部分。
经过长期的研究,本实验室从人参种植区土壤里分离筛选得到含有大量糖苷酶的拟青霉菌,经过诱变、筛选得到拟青霉菌sp.229,能够高效地转化人参皂苷Rb_1为稀有人参皂甙Compound-K(C-K),克分子转化率达到75%以上。人参皂甙Rb_3同属于二醇类人参皂甙,与Rb_1结构很相似,而且在自然界,尤其是三七茎叶中含量较高,但是目前还未见有对人参皂甙Rb_3进行微生物转化的研究报道。
本研究首先探索了人参皂甙Rb_3的HPLC的分离方法,并建立了其标准曲线,对三七茎叶总皂甙中Rb_3的含量进行测定,测定三七茎叶总皂甙中Rb_3的含量。同时对三七茎叶总皂甙进行柱层析分离,经过正相硅胶和反相硅胶反复层析后,制备得到纯度95%以上的人参皂甙Rb_3作为下一步微生物转化的底物。
在积累足够量的人参皂甙Rb_3底物后,进行人参皂甙Rb_3可制备量的发酵转化,并对发酵产物进行分离鉴定,探索人参皂甙Rb_3的代谢途径。已鉴定的代谢产物有人参皂甙Mx以及人参皂甙C-K,其中首次对人参皂甙Mx进行了X-ray衍射分析,并建立人参皂甙Mx的HPLC分离条件和标准曲线。
此后,进一步对人参皂甙Rb_3的代谢途径展开研究,分别对不同时间发酵产物、不同时间粗酶直接转化产物、不同时间固定化酶转化产物、不同投入量底物固定化酶转化产物进行TLC以及HPLC的分析,分析人参皂甙Rb_3的代谢特点,探索其转化途径。推测出人参皂甙Rb_3在拟青霉菌sp.229的作用下可能的转化途径为:Rb_3→Gypenoside IX→Mx→C-K。
本研究继续对人参皂甙Rb_3的发酵条件进行优化,分别进行碳源、氮源筛选,探索了生长因子、表面活性剂对发酵的影响,并通过正交法对人参皂甙Rb_3的发酵条件的六个因素进行了优化,确定最佳的转化条件为:3%蔗糖、3%酵母粉、0.1%麸皮粉、0.1%MgSO_4、0.15%CaCl_2及0.2%(NH_4)_2SO_4,克分子转化效率提高至28.2%。
微生物由于其丰富的代谢产物,越来越受到研究者的重视,除了抗生素外,微生物还产生许多其它活性化合物,包括黄酮、生物碱、皂甙等结构的化合物都有被发现。微生物代谢产物已经成为治疗恶性肿瘤、艾滋病、心脑血管等病症药物的重要来源之一。本研究对拟青霉菌sp.229代谢产物也进行了初步分离,经过反复柱层析分离得到两个甾体类化合物,并制备得到结晶,进行了X-ray衍射分析,鉴定为过氧麦角甾和麦角甾醇。这是首次从该菌株中分离得到这两个化合物,为它们的制备提供了一条新的途径。
Paecilomyces Bainier sp.229 is a fungal strain isolated from the soil of ginseng plantation localities.It shows strong glycosidase activity that can transform ginsenoside Rb_1 to CK efficiently,and the Mol.conversion quotient reaches to 80%.
Ginsenoside Rb_3 is a protopanaxadiol ginsenoside,similar to ginsenoside Rb_1 in chemical structure.Compared to others ginsenosides,it is abundant in natural products,especially in leaves and stems of Panax notoginseng.So far,none study on the microbial transformation of ginsenoside Rb_3 has been reported.
A convenient High performance liquid chromatography standard method was established for determining Rb_3 content in Panax notoginseng saponins.And silica gel chromatography and reverse-phase silica gel chromatography were employed to separate the Panax notoginseng saponins,affording ginsenoside Rb_3 as the substrate for the advanced fermentation.
After preparing enough ginsenoside Rb_3,Microbial-transformation was carried out,and the metabolic products were separated and identified by TLC, HPLC,as well as X-ray diffraction analysis.The metabolizing pathway of ginsenoside Rb_3 was also studied.The metabolites have been identified as ginsenoside Mx and ginsenosidesC-K.X-ray diffraction technology was firstly employed in studying ginsenoside Mx,and a High performance liquid chromatography standard method was established for determining ginsenoside Mx.
To exploring the metabolizing pathway of ginsenoside Rb_3,Samples of microbial-transformation in different time,samples of products transformed by crude enzymes for different hours,samples of products transformed by immobilized crude enzymes for different hours,and samples of different quantities substrate transformed by immobilized crude enzymes,were analyzed by TLC and HPLC.And the metabolizing pathway of ginsenoside Rb_3 was speculated to be:Rb_3→GypenosideⅨ→Mx→C-K.
In this study,the influences of carbon source,nitrogen source,growth factor, and surface acting agent were studied,and then orthogonal experiment was carried out to optimize the conditions of fermentation.The optimal conditions were determined as:3%Sucrose,3%yeast powder,0.1%wheat bran powder, 0.1%MgSO_4、0.15%CaCl_2,and 0.2%(NH_4)_2SO_4.
Because of its abundant metabolites,microorganism gets more and more attention.Apart from antibiotics,lots of compounds with kinds of activities were found in the metabolites,including flavones,alkaloids and saponins etc.microbic metabolites have been one of the important sources of drugs.In this study,the metabolites of Paecilomyces Bainier sp.229 were partly separated,affording two steroid compounds,the crystals of which were prepared,and were identified as Ergosterol Peroxide(EP) and Ergcoterol.This is the first time that these two compounds were separated from Paecilomyces Bainier sp.229,which is a new way to prepare these two compounds.
经过长期的研究,本实验室从人参种植区土壤里分离筛选得到含有大量糖苷酶的拟青霉菌,经过诱变、筛选得到拟青霉菌sp.229,能够高效地转化人参皂苷Rb_1为稀有人参皂甙Compound-K(C-K),克分子转化率达到75%以上。人参皂甙Rb_3同属于二醇类人参皂甙,与Rb_1结构很相似,而且在自然界,尤其是三七茎叶中含量较高,但是目前还未见有对人参皂甙Rb_3进行微生物转化的研究报道。
本研究首先探索了人参皂甙Rb_3的HPLC的分离方法,并建立了其标准曲线,对三七茎叶总皂甙中Rb_3的含量进行测定,测定三七茎叶总皂甙中Rb_3的含量。同时对三七茎叶总皂甙进行柱层析分离,经过正相硅胶和反相硅胶反复层析后,制备得到纯度95%以上的人参皂甙Rb_3作为下一步微生物转化的底物。
在积累足够量的人参皂甙Rb_3底物后,进行人参皂甙Rb_3可制备量的发酵转化,并对发酵产物进行分离鉴定,探索人参皂甙Rb_3的代谢途径。已鉴定的代谢产物有人参皂甙Mx以及人参皂甙C-K,其中首次对人参皂甙Mx进行了X-ray衍射分析,并建立人参皂甙Mx的HPLC分离条件和标准曲线。
此后,进一步对人参皂甙Rb_3的代谢途径展开研究,分别对不同时间发酵产物、不同时间粗酶直接转化产物、不同时间固定化酶转化产物、不同投入量底物固定化酶转化产物进行TLC以及HPLC的分析,分析人参皂甙Rb_3的代谢特点,探索其转化途径。推测出人参皂甙Rb_3在拟青霉菌sp.229的作用下可能的转化途径为:Rb_3→Gypenoside IX→Mx→C-K。
本研究继续对人参皂甙Rb_3的发酵条件进行优化,分别进行碳源、氮源筛选,探索了生长因子、表面活性剂对发酵的影响,并通过正交法对人参皂甙Rb_3的发酵条件的六个因素进行了优化,确定最佳的转化条件为:3%蔗糖、3%酵母粉、0.1%麸皮粉、0.1%MgSO_4、0.15%CaCl_2及0.2%(NH_4)_2SO_4,克分子转化效率提高至28.2%。
微生物由于其丰富的代谢产物,越来越受到研究者的重视,除了抗生素外,微生物还产生许多其它活性化合物,包括黄酮、生物碱、皂甙等结构的化合物都有被发现。微生物代谢产物已经成为治疗恶性肿瘤、艾滋病、心脑血管等病症药物的重要来源之一。本研究对拟青霉菌sp.229代谢产物也进行了初步分离,经过反复柱层析分离得到两个甾体类化合物,并制备得到结晶,进行了X-ray衍射分析,鉴定为过氧麦角甾和麦角甾醇。这是首次从该菌株中分离得到这两个化合物,为它们的制备提供了一条新的途径。
Paecilomyces Bainier sp.229 is a fungal strain isolated from the soil of ginseng plantation localities.It shows strong glycosidase activity that can transform ginsenoside Rb_1 to CK efficiently,and the Mol.conversion quotient reaches to 80%.
Ginsenoside Rb_3 is a protopanaxadiol ginsenoside,similar to ginsenoside Rb_1 in chemical structure.Compared to others ginsenosides,it is abundant in natural products,especially in leaves and stems of Panax notoginseng.So far,none study on the microbial transformation of ginsenoside Rb_3 has been reported.
A convenient High performance liquid chromatography standard method was established for determining Rb_3 content in Panax notoginseng saponins.And silica gel chromatography and reverse-phase silica gel chromatography were employed to separate the Panax notoginseng saponins,affording ginsenoside Rb_3 as the substrate for the advanced fermentation.
After preparing enough ginsenoside Rb_3,Microbial-transformation was carried out,and the metabolic products were separated and identified by TLC, HPLC,as well as X-ray diffraction analysis.The metabolizing pathway of ginsenoside Rb_3 was also studied.The metabolites have been identified as ginsenoside Mx and ginsenosidesC-K.X-ray diffraction technology was firstly employed in studying ginsenoside Mx,and a High performance liquid chromatography standard method was established for determining ginsenoside Mx.
To exploring the metabolizing pathway of ginsenoside Rb_3,Samples of microbial-transformation in different time,samples of products transformed by crude enzymes for different hours,samples of products transformed by immobilized crude enzymes for different hours,and samples of different quantities substrate transformed by immobilized crude enzymes,were analyzed by TLC and HPLC.And the metabolizing pathway of ginsenoside Rb_3 was speculated to be:Rb_3→GypenosideⅨ→Mx→C-K.
In this study,the influences of carbon source,nitrogen source,growth factor, and surface acting agent were studied,and then orthogonal experiment was carried out to optimize the conditions of fermentation.The optimal conditions were determined as:3%Sucrose,3%yeast powder,0.1%wheat bran powder, 0.1%MgSO_4、0.15%CaCl_2,and 0.2%(NH_4)_2SO_4.
Because of its abundant metabolites,microorganism gets more and more attention.Apart from antibiotics,lots of compounds with kinds of activities were found in the metabolites,including flavones,alkaloids and saponins etc.microbic metabolites have been one of the important sources of drugs.In this study,the metabolites of Paecilomyces Bainier sp.229 were partly separated,affording two steroid compounds,the crystals of which were prepared,and were identified as Ergosterol Peroxide(EP) and Ergcoterol.This is the first time that these two compounds were separated from Paecilomyces Bainier sp.229,which is a new way to prepare these two compounds.
引文
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