1、格木内生真菌次生代谢产物的研究2、中药川芎和山楂的药效物质基础研究
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摘要
第一部分
格木(Erthrophleum fordii Oliver),为豆科格木属植物,是我国传统草药,主要生长于广西、广东、福建、台湾、浙江等省区。其树皮和种子可入药,性味辛、平,有毒。主要功效为益气活血,可治疗心气不足所致的气虚血瘀之症。
本课题组前期曾对格木活性部位的化学成分进行过研究,分离得到了一些针对A2780、Bel-7402、BGC-823、MCF-7、HCT-8、KB、Hela、PC-3M、A549、Ketr3这十种人肿瘤细胞株具有选择性细胞毒活性的咖萨因型二萜酰胺类单体化合物。在此基础上,为了进一步寻找格木中具有抗肿瘤活性的化合物,本课题组继续对从该植物中分离的85株内生真菌进行了次生代谢产物的研究。
本论文作者选择了LYB-027菌株进行具体研究,经鉴定该菌株为烟曲霉Aspergillus fumigatu。首先采用CZ-Met培养基对其进行发酵培养,所得的菌丝和菌液分别进行初步的分离和提取,然后针对“菌液的大孔树脂95%乙醇洗脱部位的乙酸乙酯萃取物”进行进一步分离,共得到17个单体化合物。
通过波谱学的方法确定了这17个化合物的结构,它们分别是：8R9R-Dihydroxy-12R,18R-fumitremorgin C (1),8R-Methoxyl-9R-hydroxy-12SS,18R-fumitremorgin C (2),8R-Methoxyl-9R,20-dihydroxy-12R,18R-Fumitremorgin C (3),8R-Methoxyl-9R,19S,20-trihydroxy-12R,18S,19S-fumitremorgin C (4),8S,9S-Dihydroxy-spirotryprostatin A (5),6-Methoxyl-3-(3-methyl-2-butylene)-2-oxobrevianamide F (6), Brevianamide F (7), Fumitremorgin C (8),18-OxotryprostatinA(9), N-Prenyl-cyclo-L-tryptophyl-L-Proline (10), Tryprostatin B (11),8S,9R-Dihydroxy-12SS,18S-fumitremorgin C(12), Cyclo-L-isoleucine-L-proline (13), Cyclo-L-leucine-L-proline (14), PseurotinA (15),4-Hydroxy-phenylacetamide (16), N-acetyltyramine (17)。其中,化合物1-6为新化合物,化合物7-17为已知化合物。
这些化合物包括14个二酮哌嗪类生物碱(化合物1-14),1个氧杂螺内酰胺类化合物(化合物15),2个苯乙酰胺类化合物(化合物16、17)。
第二部分
川芎(Ligusticum chuanxiong Hort),为伞形科藁本属植物川芎的干燥根茎,山楂,为蔷薇科植物山里红(Crataegus pinnatifida Bge.var.major N.E.Br)或山楂(Crataegus pinnatifida Bge.)的干燥成熟果实,均为我国的传统中药。
本课题组应用现代高效、快速、简便的检测手段(如：HPLC-UV、HPLC-MS),对这两味中药中发挥药效的化学物质基础进行了初步的研究。
“川芎嗪”(Tetramethylpyrazine, Ligustrazine, Chuanxiongzine),作为川芎中最具代表性的主要有效成分,本课题组选择以它作为主要的研究对象,决定先对“川芎嗪”在川芎中的含量进行测定,但是令人不解的是,我们采用了“比较用不同溶剂提取川芎的方法”、“研究盐酸川芎嗪和川芎嗪在高效液相上的色谱行为”、“从药材中有针对性地提取川芎嗪”、“重复首次分得川芎嗪的文献方法”等多种方法进行尝试,均未在川芎中发现“川芎嗪”的踪迹。基于这些实验结果,我们无法继续将“川芎嗪”作为川芎发挥药效的重要化学物质来进行研究。于是,我们又选择了川芎中另一个主要化学成分——“阿魏酸”(Ferulic acid),它的化学性质稳定,含量较多,我们对其进行了含量测定及其方法学考察。山楂中的主要成分是黄酮类化合物,我们选择“金丝桃苷”(Hyperoside)作为对照品进行含量测定及其方法学考察。
Part One
Erythrophloeum fordii Oliv, Leguminosae, is one of the Chinese traditional herbal medicine.It widely distributed in some provinces in China, such as Guangxi, Guangdong, Fujian etc., and its bark and seeds are clinically used to facilitate blood circulation and dispersing blood stasis.
Basing on our previous results of the studies on the cytotoxic compounds seperated from barks of Erythrophloeum fordii Oliv, we were going on the studies on secondary metabolites of endophytic fungi LYB-027from roots of Erythrophloeum fordii Oliv.. It has been identified that LYB-027is Aspergillus fumigatu.
Chromatographic methods were applied to purify and separate its secondary metabolites.17compounds were isolated and their structures were identified by spectroscopic methods. They includes14diketopiperazine alkaloids,1oxaspiro lactam and2phenylacetamides.
Compound1-6are new, and7-17are known. They are8R,9R-Dihydroxy-12R,18R-fumitremorgin C (1),8R-Methoxyl-9R-hydroxy-12S,18R-fumitremorgin C (2),8R-Methoxyl-9R,20-dihydroxy-12R18R-Fumitremorgin C (3),8R-Methoxyl-9R,19S,20-trihydroxy-12R,18S,19S-fumitremorgin C (4),8S,9S-Dihydroxy-spirotryprostatin A (5),6-Methoxyl-3-(3-methyl-2-butylene)-2-oxobrevianamide F (6), Brevianamide F (7), Fumitremorgin C (8),18-Oxotryprostatin A (9), N-Prenyl-cyclo-L-tryptophyl-L-proline (10), Tryprostatin B (11),8S,9R-Dihydroxy-12S,18S-fumitremorgin C (12), Cyclo-L-isoleucine-L-proline (13), Cyclo-L-leucine-L-proline (14), Pseurotin A (15),4-Hydroxy-phenylacetamide (16), N-acetyltyramine (17).
Part Two
Ligusticum chuanxiong Hort, the dried rhizome of Ligusticum chuanxiong (Umbelliferae) and Crataegus pinnatifida Bge., the dried ripe fruit of Crataegus pinnatifida are two major traditional Chinese medicines in China.
Some rapid, convenient and efficient analytical methods, including HPLC-UV and HPLC-MS, has been used in the studies on the effective chemical substances in Ligusticum chuanxiong Hort. and Crataegus pinnatifida Bge.
Tetramethylpyrazine(TMP), which is one of the main effective chemical ingredients contributing to the therapeutic effects of Ligusticum chuanxiong Hort., has always been employed as a chemical marker for quality control of it. Four methods were applied. First, abstracting TMP from processed chuanxiong respectively by using95%ethanol and water. Second, studying the same and different properties between TMP and TMP hydrochlorid by means of the preparation of TMP from TMP hydrochlorid. Third, separating TMP directly from processed chuanxiong by cold-soaking extraction and abstracting volatile oils using volatile oil extractor. Forth, repeating the experiment of the original literature in1977about the first isolation and identification of TMP from Ligusticum chuanxiong Hort. By comparing those experimental datas obtained from HPLC-UV and HPLC-MS, any evidence of the existence of TMP was found out. Therefore, it is doubtable that TMP is one of the main ingredients of Ligusticum chuanxiong Hort.. And then, the other major chemical ingredient ferulic acid was chosen as the chemical marker, and fully validated on precision, reproducibility and stability. The results demonstrated that it is available that ferulic acid was the bioactive chemical marker of quality control of chuanxiong.
Crataegus pinnatifida Bge. mainly contains flavonoid compounds, which are the major bioactive ingredients. Hyperoside, one of flavonoid compounds, its precison, reproducibility and stability were fully validated. The results demonstrated that it is available that hyperosidewas the bioactive chemical marker of quality control of Crataegus pinnatifida Bge..
格木(Erthrophleum fordii Oliver),为豆科格木属植物,是我国传统草药,主要生长于广西、广东、福建、台湾、浙江等省区。其树皮和种子可入药,性味辛、平,有毒。主要功效为益气活血,可治疗心气不足所致的气虚血瘀之症。
本课题组前期曾对格木活性部位的化学成分进行过研究,分离得到了一些针对A2780、Bel-7402、BGC-823、MCF-7、HCT-8、KB、Hela、PC-3M、A549、Ketr3这十种人肿瘤细胞株具有选择性细胞毒活性的咖萨因型二萜酰胺类单体化合物。在此基础上,为了进一步寻找格木中具有抗肿瘤活性的化合物,本课题组继续对从该植物中分离的85株内生真菌进行了次生代谢产物的研究。
本论文作者选择了LYB-027菌株进行具体研究,经鉴定该菌株为烟曲霉Aspergillus fumigatu。首先采用CZ-Met培养基对其进行发酵培养,所得的菌丝和菌液分别进行初步的分离和提取,然后针对“菌液的大孔树脂95%乙醇洗脱部位的乙酸乙酯萃取物”进行进一步分离,共得到17个单体化合物。
通过波谱学的方法确定了这17个化合物的结构,它们分别是：8R9R-Dihydroxy-12R,18R-fumitremorgin C (1),8R-Methoxyl-9R-hydroxy-12SS,18R-fumitremorgin C (2),8R-Methoxyl-9R,20-dihydroxy-12R,18R-Fumitremorgin C (3),8R-Methoxyl-9R,19S,20-trihydroxy-12R,18S,19S-fumitremorgin C (4),8S,9S-Dihydroxy-spirotryprostatin A (5),6-Methoxyl-3-(3-methyl-2-butylene)-2-oxobrevianamide F (6), Brevianamide F (7), Fumitremorgin C (8),18-OxotryprostatinA(9), N-Prenyl-cyclo-L-tryptophyl-L-Proline (10), Tryprostatin B (11),8S,9R-Dihydroxy-12SS,18S-fumitremorgin C(12), Cyclo-L-isoleucine-L-proline (13), Cyclo-L-leucine-L-proline (14), PseurotinA (15),4-Hydroxy-phenylacetamide (16), N-acetyltyramine (17)。其中,化合物1-6为新化合物,化合物7-17为已知化合物。
这些化合物包括14个二酮哌嗪类生物碱(化合物1-14),1个氧杂螺内酰胺类化合物(化合物15),2个苯乙酰胺类化合物(化合物16、17)。
第二部分
川芎(Ligusticum chuanxiong Hort),为伞形科藁本属植物川芎的干燥根茎,山楂,为蔷薇科植物山里红(Crataegus pinnatifida Bge.var.major N.E.Br)或山楂(Crataegus pinnatifida Bge.)的干燥成熟果实,均为我国的传统中药。
本课题组应用现代高效、快速、简便的检测手段(如：HPLC-UV、HPLC-MS),对这两味中药中发挥药效的化学物质基础进行了初步的研究。
“川芎嗪”(Tetramethylpyrazine, Ligustrazine, Chuanxiongzine),作为川芎中最具代表性的主要有效成分,本课题组选择以它作为主要的研究对象,决定先对“川芎嗪”在川芎中的含量进行测定,但是令人不解的是,我们采用了“比较用不同溶剂提取川芎的方法”、“研究盐酸川芎嗪和川芎嗪在高效液相上的色谱行为”、“从药材中有针对性地提取川芎嗪”、“重复首次分得川芎嗪的文献方法”等多种方法进行尝试,均未在川芎中发现“川芎嗪”的踪迹。基于这些实验结果,我们无法继续将“川芎嗪”作为川芎发挥药效的重要化学物质来进行研究。于是,我们又选择了川芎中另一个主要化学成分——“阿魏酸”(Ferulic acid),它的化学性质稳定,含量较多,我们对其进行了含量测定及其方法学考察。山楂中的主要成分是黄酮类化合物,我们选择“金丝桃苷”(Hyperoside)作为对照品进行含量测定及其方法学考察。
Part One
Erythrophloeum fordii Oliv, Leguminosae, is one of the Chinese traditional herbal medicine.It widely distributed in some provinces in China, such as Guangxi, Guangdong, Fujian etc., and its bark and seeds are clinically used to facilitate blood circulation and dispersing blood stasis.
Basing on our previous results of the studies on the cytotoxic compounds seperated from barks of Erythrophloeum fordii Oliv, we were going on the studies on secondary metabolites of endophytic fungi LYB-027from roots of Erythrophloeum fordii Oliv.. It has been identified that LYB-027is Aspergillus fumigatu.
Chromatographic methods were applied to purify and separate its secondary metabolites.17compounds were isolated and their structures were identified by spectroscopic methods. They includes14diketopiperazine alkaloids,1oxaspiro lactam and2phenylacetamides.
Compound1-6are new, and7-17are known. They are8R,9R-Dihydroxy-12R,18R-fumitremorgin C (1),8R-Methoxyl-9R-hydroxy-12S,18R-fumitremorgin C (2),8R-Methoxyl-9R,20-dihydroxy-12R18R-Fumitremorgin C (3),8R-Methoxyl-9R,19S,20-trihydroxy-12R,18S,19S-fumitremorgin C (4),8S,9S-Dihydroxy-spirotryprostatin A (5),6-Methoxyl-3-(3-methyl-2-butylene)-2-oxobrevianamide F (6), Brevianamide F (7), Fumitremorgin C (8),18-Oxotryprostatin A (9), N-Prenyl-cyclo-L-tryptophyl-L-proline (10), Tryprostatin B (11),8S,9R-Dihydroxy-12S,18S-fumitremorgin C (12), Cyclo-L-isoleucine-L-proline (13), Cyclo-L-leucine-L-proline (14), Pseurotin A (15),4-Hydroxy-phenylacetamide (16), N-acetyltyramine (17).
Part Two
Ligusticum chuanxiong Hort, the dried rhizome of Ligusticum chuanxiong (Umbelliferae) and Crataegus pinnatifida Bge., the dried ripe fruit of Crataegus pinnatifida are two major traditional Chinese medicines in China.
Some rapid, convenient and efficient analytical methods, including HPLC-UV and HPLC-MS, has been used in the studies on the effective chemical substances in Ligusticum chuanxiong Hort. and Crataegus pinnatifida Bge.
Tetramethylpyrazine(TMP), which is one of the main effective chemical ingredients contributing to the therapeutic effects of Ligusticum chuanxiong Hort., has always been employed as a chemical marker for quality control of it. Four methods were applied. First, abstracting TMP from processed chuanxiong respectively by using95%ethanol and water. Second, studying the same and different properties between TMP and TMP hydrochlorid by means of the preparation of TMP from TMP hydrochlorid. Third, separating TMP directly from processed chuanxiong by cold-soaking extraction and abstracting volatile oils using volatile oil extractor. Forth, repeating the experiment of the original literature in1977about the first isolation and identification of TMP from Ligusticum chuanxiong Hort. By comparing those experimental datas obtained from HPLC-UV and HPLC-MS, any evidence of the existence of TMP was found out. Therefore, it is doubtable that TMP is one of the main ingredients of Ligusticum chuanxiong Hort.. And then, the other major chemical ingredient ferulic acid was chosen as the chemical marker, and fully validated on precision, reproducibility and stability. The results demonstrated that it is available that ferulic acid was the bioactive chemical marker of quality control of chuanxiong.
Crataegus pinnatifida Bge. mainly contains flavonoid compounds, which are the major bioactive ingredients. Hyperoside, one of flavonoid compounds, its precison, reproducibility and stability were fully validated. The results demonstrated that it is available that hyperosidewas the bioactive chemical marker of quality control of Crataegus pinnatifida Bge..
引文
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