几种地衣和地衣内生菌发酵液的活性成分研究
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摘要
地衣(Iichens)是植物界一个特殊的类群,是真菌(fungi)和藻类(algae)合的共生复合体。目前,全球地衣植物约有525属,共计26000多种。其组成真菌绝大多数属于子囊菌,约占地衣型真菌总数的98%。地衣分布极为广泛,从南北两极到赤道,从高山到平原,从森林到荒漠,都有地衣的存在。
地衣次生代谢产物主要包括缩酚酸及酯类、缩酚酸环醚类、多取代单苯环化合物、蒽醌及卤代蒽醌化合物、二苯并呋喃类化合物、萜类、甾体类化合物等及地衣多糖类成分,到目前为止,已经确定出来结构的地衣化学物质700余种,大多数具有广泛的生物学活性。
国内外对地衣内生菌的分离及次生代谢产物的研究现在相对较少,自2007年Priyani A. Paranagama等人首次报道地衣内生菌化学成分至今,主要是车永胜等人对地衣内生菌化学成分报道较多,且报道的化学结构都较新颖,并有一定的抗肿瘤、抗真菌等活性。
本文对四种采自中国青海省祁连县的地衣植物柔扁枝衣(Evernia divaricata (L.) Ach.),金丝带(Lethariella zahlbruckneri)的化学成分进行了系统研究,共分离获得化合物11个,利用波谱学以及X-单晶衍射的方法鉴定其结构,主要包括缩酚酸类(Depsides),二苯并呋喃类(Dibenzofuranes)和缩酚酸、缩酚酸环醚裂解产物。对分离得到的化合物进行了生物活性研究,化合物Usnic acid和Divaric acid显示较强抗菌活性。
采用刮皮层方法对采自云南丽江老君山的21个属的地衣内生菌进行分离培养,获得263株内生菌,通过ITS序列分析,确定出51株地衣内生菌的分类地位,还对其中9株内生菌进行了HPLC结合活性的粗筛,确定出4株较好的内生菌(20102559, 20102550B, 20081189, 20102595A)。
利用广泛的柱层析结合半制备HPLC,从菌株20081189的乙酸乙酯部分分离鉴定19个化合物,包括6个新化合物: 1,8a-dihydro-7,8-dihydro-7,8-dihydroxy- 3,7-dimethyl-2-benzopyran-6-one (7)、5-chloro-7,8-dihydro-7,8-dihydroxy-3,7-dimet hyl-2-benzopyran-6-one (8)、2,3-dihydro-2-n-butyloxyanhydrofulvic acid (13)、2,3- dihydro-2-methoxyanhydrofulvic acid (14)、13-decarboxylanhydrofulvic acid (15)、12-dehydroxylcitromycetin (19)。对分离自菌株20081189的化合物8-19活性实验表明:化合物19和13对人白血病细胞K562表现出中等的生长抑制活性,IC50分别为20.0和32.2μM。化合物9, 10, 11对白色念珠菌(sc5314)具有中等抑制活性,其MIC均为128μg/ml。
对地衣内生菌株20102595A利用柱层析结合半制备HPLC多种分离手段,从乙酸乙酯提取物中分离鉴定3个化合物:采用微量稀释法测定其MIC值,结果显示3个化合物均具有中等抗白色念珠菌活性。
Lichen is a special symbiotic of cyanobacteria and filamentous fungi. at present, there are 26000 species of belongs to 525 families. Most of the fungi are ascomycete, account for 98% of the amount of fungi. The lichens are widespread from the north and south poles to equator, from the mountains to the plain, from the forest to desert.
The metabolisms of lichens include polyols and carbohydrates、xanthones and bis- xanthones、aromatic、dibenzofuranes、chromanes and chromones、depsides、depsidones、cleavage products of depsides and depsidones. To the present, more than 700 lichen compounds have been identified, most of them show good bio-activity.
There are a few reports about the isolation and the metabolism of endolichenic fungi. Most of the research about the chemical constitutes of endolichenic fungi is from Yongsheng Che, from the first report about endolichenic in 2007 by Priyani A. Paranagama. The structure of constitutes is very novel and they show modest antitumor and antifungal activities.
This thesis studied the metabolisms of two species of lichens Evernia divaricata (L.) Ach. and Lethariella zahlbruckneri (Dr.) Krog., from which we afford 11 compounds , and we identified their structure by spectrum and Single crystal X-ray, most of which belongs to depsides、dibenzofuranes and cleavage products of depsides and depsidones. Two of the isolates (usnic acid and divaric acid) showed potent antibacterial activity against four bacterial.
The method of scraping cortex was adapted to isolate endolichenic fungi from Laojun Mountain, Yunnan Province, and 263 strains of fungi was afford from 21 families. 51 strains of fungi’s classic position were determined by ITS analysis, nine of which were selected for antibiotic activity and HPLC analysis, 4 strains were selected for further chemical study.
We isolated and identified 19 compounds from the EtOAc extract of strain of 20081189 by semi-prepared HPLC and silica gel column chromatograph, including 6 new compounds:1,8a-dihydro-7,8-dihydro-7,8-dihydroxy-3,7-dimethyl-2-benzopyra n-6-one (7)、5-chloro-7,8-dihydro-7,8-dihydroxy-3,7-dimethyl-2-benzopyran-6-one (8)、2,3-dihydro-2-n-butyloxyanhydrofulvic acid (13)、2,3-dihydro-2-methoxyanhydroful vic acid (14)、13-decarboxylanhydrofulvic acid (15)、12-dehydroxylcitromycetin (19). The bio-activity assay of compounds 8-19 show: compounds 19 and 13 exhibit modest cytotoxicity against human leukemia cell line K562, with IC50 is 20.0 and 32.2μM respectively. Compounds 9、10、11 show modest inhibitory on Candida albicans (sc5314), with the MIC value is 128μg/ml respectively.
We isolated and identified 3 compounds from the strain of 20102595A with the methods of semi-prepared HPLC and silica gel column chromatograph. All of the isolates showed modest antifungal activity against C. albicans.
地衣次生代谢产物主要包括缩酚酸及酯类、缩酚酸环醚类、多取代单苯环化合物、蒽醌及卤代蒽醌化合物、二苯并呋喃类化合物、萜类、甾体类化合物等及地衣多糖类成分,到目前为止,已经确定出来结构的地衣化学物质700余种,大多数具有广泛的生物学活性。
国内外对地衣内生菌的分离及次生代谢产物的研究现在相对较少,自2007年Priyani A. Paranagama等人首次报道地衣内生菌化学成分至今,主要是车永胜等人对地衣内生菌化学成分报道较多,且报道的化学结构都较新颖,并有一定的抗肿瘤、抗真菌等活性。
本文对四种采自中国青海省祁连县的地衣植物柔扁枝衣(Evernia divaricata (L.) Ach.),金丝带(Lethariella zahlbruckneri)的化学成分进行了系统研究,共分离获得化合物11个,利用波谱学以及X-单晶衍射的方法鉴定其结构,主要包括缩酚酸类(Depsides),二苯并呋喃类(Dibenzofuranes)和缩酚酸、缩酚酸环醚裂解产物。对分离得到的化合物进行了生物活性研究,化合物Usnic acid和Divaric acid显示较强抗菌活性。
采用刮皮层方法对采自云南丽江老君山的21个属的地衣内生菌进行分离培养,获得263株内生菌,通过ITS序列分析,确定出51株地衣内生菌的分类地位,还对其中9株内生菌进行了HPLC结合活性的粗筛,确定出4株较好的内生菌(20102559, 20102550B, 20081189, 20102595A)。
利用广泛的柱层析结合半制备HPLC,从菌株20081189的乙酸乙酯部分分离鉴定19个化合物,包括6个新化合物: 1,8a-dihydro-7,8-dihydro-7,8-dihydroxy- 3,7-dimethyl-2-benzopyran-6-one (7)、5-chloro-7,8-dihydro-7,8-dihydroxy-3,7-dimet hyl-2-benzopyran-6-one (8)、2,3-dihydro-2-n-butyloxyanhydrofulvic acid (13)、2,3- dihydro-2-methoxyanhydrofulvic acid (14)、13-decarboxylanhydrofulvic acid (15)、12-dehydroxylcitromycetin (19)。对分离自菌株20081189的化合物8-19活性实验表明:化合物19和13对人白血病细胞K562表现出中等的生长抑制活性,IC50分别为20.0和32.2μM。化合物9, 10, 11对白色念珠菌(sc5314)具有中等抑制活性,其MIC均为128μg/ml。
对地衣内生菌株20102595A利用柱层析结合半制备HPLC多种分离手段,从乙酸乙酯提取物中分离鉴定3个化合物:采用微量稀释法测定其MIC值,结果显示3个化合物均具有中等抗白色念珠菌活性。
Lichen is a special symbiotic of cyanobacteria and filamentous fungi. at present, there are 26000 species of belongs to 525 families. Most of the fungi are ascomycete, account for 98% of the amount of fungi. The lichens are widespread from the north and south poles to equator, from the mountains to the plain, from the forest to desert.
The metabolisms of lichens include polyols and carbohydrates、xanthones and bis- xanthones、aromatic、dibenzofuranes、chromanes and chromones、depsides、depsidones、cleavage products of depsides and depsidones. To the present, more than 700 lichen compounds have been identified, most of them show good bio-activity.
There are a few reports about the isolation and the metabolism of endolichenic fungi. Most of the research about the chemical constitutes of endolichenic fungi is from Yongsheng Che, from the first report about endolichenic in 2007 by Priyani A. Paranagama. The structure of constitutes is very novel and they show modest antitumor and antifungal activities.
This thesis studied the metabolisms of two species of lichens Evernia divaricata (L.) Ach. and Lethariella zahlbruckneri (Dr.) Krog., from which we afford 11 compounds , and we identified their structure by spectrum and Single crystal X-ray, most of which belongs to depsides、dibenzofuranes and cleavage products of depsides and depsidones. Two of the isolates (usnic acid and divaric acid) showed potent antibacterial activity against four bacterial.
The method of scraping cortex was adapted to isolate endolichenic fungi from Laojun Mountain, Yunnan Province, and 263 strains of fungi was afford from 21 families. 51 strains of fungi’s classic position were determined by ITS analysis, nine of which were selected for antibiotic activity and HPLC analysis, 4 strains were selected for further chemical study.
We isolated and identified 19 compounds from the EtOAc extract of strain of 20081189 by semi-prepared HPLC and silica gel column chromatograph, including 6 new compounds:1,8a-dihydro-7,8-dihydro-7,8-dihydroxy-3,7-dimethyl-2-benzopyra n-6-one (7)、5-chloro-7,8-dihydro-7,8-dihydroxy-3,7-dimethyl-2-benzopyran-6-one (8)、2,3-dihydro-2-n-butyloxyanhydrofulvic acid (13)、2,3-dihydro-2-methoxyanhydroful vic acid (14)、13-decarboxylanhydrofulvic acid (15)、12-dehydroxylcitromycetin (19). The bio-activity assay of compounds 8-19 show: compounds 19 and 13 exhibit modest cytotoxicity against human leukemia cell line K562, with IC50 is 20.0 and 32.2μM respectively. Compounds 9、10、11 show modest inhibitory on Candida albicans (sc5314), with the MIC value is 128μg/ml respectively.
We isolated and identified 3 compounds from the strain of 20102595A with the methods of semi-prepared HPLC and silica gel column chromatograph. All of the isolates showed modest antifungal activity against C. albicans.
引文
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