川芎内生菌与品质相关性研究
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摘要
川芎为伞形科(Umbelliferae)植物川芎(Ligusticum chuanxiong Hort.)的干燥根茎,是著名的川产道地药材,主产于四川,具有活血行气、祛风止痛功效。已有的研究表明:川芎挥发性类成分具有较强的生理活性,是川芎中的主要活性成分;不同产地和品种川芎的挥发油含量和组分存在差异,并认为川芎道地药材的成因主要与生态环境中特定的土壤与气候有关。鉴于宏观生态环境的尺度较大,难以解释不同微环境川芎存在品质差异。为此,我们开展了川芎内环境与川芎品质的相关性研究。
首先,从不同产地和不同品种的川芎根茎共分离得到内生真菌87株,经形态观察鉴定为1纲、3目、4科、17属。在目的分类水平上以丛梗孢目(Moniliales)为优势种群,包含菌株占总菌株数的94.3%;在属的分类水平上以镰孢属(Fusarium)、曲霉属(Aspergillus)、头孢霉属(Cephalosporium)、拟小卵孢属(Ovulariopsis)为优势种群,包含菌株分别占总菌株数的30.0%、19.5%、14.9%、14.9%。首次揭示不同产地及不同品种川芎内生真菌在数量、分布、种群及其组成存在差异;道地产区的内生真菌种群多样性较非道地产区更丰富;不同产地的内生真菌种群组成中有特有种群,聚类分析表明,四川道地产区都江堰石羊的内生真菌种群结构基本稳定。
从川芎根茎中直接提取内生真菌DNA后,以特异性引物扩增真菌的18S rDNA区段,采用扩增核糖体DNA限制性酶切基因分析(ARDRA):不同样品的ARDRA图谱有一定的差异,其中也存在共有的保守条带,体现出不同样品中有效扩增的内生真菌间的亲缘关系与遗传差异。DGGE分离内生真菌只能得到1～2个条带,证实DGGE对500 bp以上的片段检测有局限。
其次,对川芎内生细菌的研究。对细菌16S rDNA区段扩增后,经DGGE分析,共检测37个条带,不同样品均得到了数量不等的条带,彼此并且存在特异条带和共同条带,说明不同产地和不同样品间既存在相似的微生物类群又具有其特异的微生物种群;显示出微生物种群的多样性。
再其次,采用GC-MS对道地产区和非道地产区的川芎油挥发性成分进行了分析,共分离鉴定48个成分。6个样品共有成分7个,其中均以藁本内酯为主要成分,道地产区比非道地产区的含量,与文献报道的相符。主成分分析法(PAC)表明前5个主成分所包含21个化学成分的累积贡献为100%,足以概括挥发性成分的信息,聚类结果表明川芎油挥发性成分与产地有一定相关性。同时不同产地川芎油中含13.69%～38.38%不等的脂肪酸类成分。
最后,用SPSS对内生真菌、内生细菌与挥发油中挥发性成分相关性分析表明:拟小孢卵属的存在与进藁本内酯的呈正相关,而青霉属、沃德属霉属的存在与藁本内酯呈负相关。2006年、2007年的石羊和敖平的内生真菌和川芎油的数据,表明拟小孢卵属的存在对藁本内酯的含量起促进作用。内生细菌与挥发性成分没有发现相关性,其种群结构仅与脂肪酸类成分具有相关性,表明内生细菌仅与植物初生代谢物的形成有关。通过这次研究为川芎道地性的形成提供了一条新解释。
Rhizoma Chuanxiong is dry rhizomes of Umbelliferae plant Ligusticum chuanxiong Hort..It is a famous chinese traditional medicinal crop and its main producing area is Sichuan.It can promote Qi and blood to circulate,dispel wind and restrain pain.Research results in near years indicated that the volatile oil was the main active component,which had strong physiological activity.There were some differences at chemical components and content between different habitations and varieties of Ligusticum chuanxiong Hort.The formation of famous-region of Ligusticum chuanxiong attribute to the specific soil and climate in ecological environment.It is hard to explain quality difference in different micro-environment using method of macroscopical ecology.Therefor,we study the corretation between micro-environment and quality.
Firstly,87 strains endophytic fungi were isolated from the rhizomes of Ligusticum chuanxiong Hort.and they were morphologically identified belonging to 17 genera,4 families,3 orders and 1 classe.Moniliales,which includes 94.3%of the whole strains,is believed the dominant order.While Fusarium,Aspergillus, Cephalosporium、Ovulariopsis are believed the dominant genera,which may include 30.0%、19.5%、14.9%、14.9%of the whole strains.Results firstly revealed the differences of number,distribution,and composition from vary endophytic fungi of Ligusticum chuanxiong Hort..At first time,the diversity of endophytic fungi in the main areas is better than others;.endophytic fungi from different areas have their special population,for esample,the endophytic fungi of Shiyan is stable.
After DNA was extracted directly from Radix Ligusticum chuanxiong,sepefic primers were used to amplify the object gene 18S rDNA,then the object gene was partially restricted.The ARDRA figure showed that difference exist in different samples,which suggested kinship and genetic diversity of entophytic fungi. Entophytic fungi can only get 1 or 2 bands by DGGE,which maybe comfirm DGGE is not good at test product over 500bp.
Secondly,We used DGGE to study endogenetic bacteria.DGGE showed 38 bands with 16S rDNA product separated.The different samples have different type and quantity bands.There are common and specific bands,which illuminats that different habitations and varieties possess similar and specific microorganism population.
Thirdly,48 chemical components were isolated and identified from the volatile components.Chemical components and content of 6 samples were different and Ligustilide is the main component.PAC indicated that the 5 principal components contained 21 chemical composition,and the accumulated dedication rate is up to 100%,which includes all volatile components in principle.Cluster analysis showed that the volatile components is corretate with habitations.Meanwhile,the oil of Ligusticum chuanxiong in different habitats contain different fatty acids range from 13.69%to 38.38%.
Finally,we did correlation analysis among endogenetic fungi,endogenetic bacteria and volatile components in oil of Ligusticum chuanxiong,by SPSS.And results concluded that the existence of Ovulariopsis had positive correlation with content of Ligustilide,in contrast the existence of Penicillium and Wardonmyces had negative correlation with content of Ligustilide.Datas from endogenetic fungi and chuanxiong oil of Ligusticum chuanxiong in Aoping and Shiyang from 2006 to 2007 indicated the existence of Ovulariopsis could promote production of Ligustilide.There was no correlation between endogenetic bacteria and volatile components,but its population constitution had correlation with fatty acids,which suggested endogenetic bacteria had relationship with synthesis of plant primary metabolite.Our research can give some references from a new degree measure to explain the formation of famous-region of Ligusticum chuanxiong.
首先,从不同产地和不同品种的川芎根茎共分离得到内生真菌87株,经形态观察鉴定为1纲、3目、4科、17属。在目的分类水平上以丛梗孢目(Moniliales)为优势种群,包含菌株占总菌株数的94.3%;在属的分类水平上以镰孢属(Fusarium)、曲霉属(Aspergillus)、头孢霉属(Cephalosporium)、拟小卵孢属(Ovulariopsis)为优势种群,包含菌株分别占总菌株数的30.0%、19.5%、14.9%、14.9%。首次揭示不同产地及不同品种川芎内生真菌在数量、分布、种群及其组成存在差异;道地产区的内生真菌种群多样性较非道地产区更丰富;不同产地的内生真菌种群组成中有特有种群,聚类分析表明,四川道地产区都江堰石羊的内生真菌种群结构基本稳定。
从川芎根茎中直接提取内生真菌DNA后,以特异性引物扩增真菌的18S rDNA区段,采用扩增核糖体DNA限制性酶切基因分析(ARDRA):不同样品的ARDRA图谱有一定的差异,其中也存在共有的保守条带,体现出不同样品中有效扩增的内生真菌间的亲缘关系与遗传差异。DGGE分离内生真菌只能得到1～2个条带,证实DGGE对500 bp以上的片段检测有局限。
其次,对川芎内生细菌的研究。对细菌16S rDNA区段扩增后,经DGGE分析,共检测37个条带,不同样品均得到了数量不等的条带,彼此并且存在特异条带和共同条带,说明不同产地和不同样品间既存在相似的微生物类群又具有其特异的微生物种群;显示出微生物种群的多样性。
再其次,采用GC-MS对道地产区和非道地产区的川芎油挥发性成分进行了分析,共分离鉴定48个成分。6个样品共有成分7个,其中均以藁本内酯为主要成分,道地产区比非道地产区的含量,与文献报道的相符。主成分分析法(PAC)表明前5个主成分所包含21个化学成分的累积贡献为100%,足以概括挥发性成分的信息,聚类结果表明川芎油挥发性成分与产地有一定相关性。同时不同产地川芎油中含13.69%～38.38%不等的脂肪酸类成分。
最后,用SPSS对内生真菌、内生细菌与挥发油中挥发性成分相关性分析表明:拟小孢卵属的存在与进藁本内酯的呈正相关,而青霉属、沃德属霉属的存在与藁本内酯呈负相关。2006年、2007年的石羊和敖平的内生真菌和川芎油的数据,表明拟小孢卵属的存在对藁本内酯的含量起促进作用。内生细菌与挥发性成分没有发现相关性,其种群结构仅与脂肪酸类成分具有相关性,表明内生细菌仅与植物初生代谢物的形成有关。通过这次研究为川芎道地性的形成提供了一条新解释。
Rhizoma Chuanxiong is dry rhizomes of Umbelliferae plant Ligusticum chuanxiong Hort..It is a famous chinese traditional medicinal crop and its main producing area is Sichuan.It can promote Qi and blood to circulate,dispel wind and restrain pain.Research results in near years indicated that the volatile oil was the main active component,which had strong physiological activity.There were some differences at chemical components and content between different habitations and varieties of Ligusticum chuanxiong Hort.The formation of famous-region of Ligusticum chuanxiong attribute to the specific soil and climate in ecological environment.It is hard to explain quality difference in different micro-environment using method of macroscopical ecology.Therefor,we study the corretation between micro-environment and quality.
Firstly,87 strains endophytic fungi were isolated from the rhizomes of Ligusticum chuanxiong Hort.and they were morphologically identified belonging to 17 genera,4 families,3 orders and 1 classe.Moniliales,which includes 94.3%of the whole strains,is believed the dominant order.While Fusarium,Aspergillus, Cephalosporium、Ovulariopsis are believed the dominant genera,which may include 30.0%、19.5%、14.9%、14.9%of the whole strains.Results firstly revealed the differences of number,distribution,and composition from vary endophytic fungi of Ligusticum chuanxiong Hort..At first time,the diversity of endophytic fungi in the main areas is better than others;.endophytic fungi from different areas have their special population,for esample,the endophytic fungi of Shiyan is stable.
After DNA was extracted directly from Radix Ligusticum chuanxiong,sepefic primers were used to amplify the object gene 18S rDNA,then the object gene was partially restricted.The ARDRA figure showed that difference exist in different samples,which suggested kinship and genetic diversity of entophytic fungi. Entophytic fungi can only get 1 or 2 bands by DGGE,which maybe comfirm DGGE is not good at test product over 500bp.
Secondly,We used DGGE to study endogenetic bacteria.DGGE showed 38 bands with 16S rDNA product separated.The different samples have different type and quantity bands.There are common and specific bands,which illuminats that different habitations and varieties possess similar and specific microorganism population.
Thirdly,48 chemical components were isolated and identified from the volatile components.Chemical components and content of 6 samples were different and Ligustilide is the main component.PAC indicated that the 5 principal components contained 21 chemical composition,and the accumulated dedication rate is up to 100%,which includes all volatile components in principle.Cluster analysis showed that the volatile components is corretate with habitations.Meanwhile,the oil of Ligusticum chuanxiong in different habitats contain different fatty acids range from 13.69%to 38.38%.
Finally,we did correlation analysis among endogenetic fungi,endogenetic bacteria and volatile components in oil of Ligusticum chuanxiong,by SPSS.And results concluded that the existence of Ovulariopsis had positive correlation with content of Ligustilide,in contrast the existence of Penicillium and Wardonmyces had negative correlation with content of Ligustilide.Datas from endogenetic fungi and chuanxiong oil of Ligusticum chuanxiong in Aoping and Shiyang from 2006 to 2007 indicated the existence of Ovulariopsis could promote production of Ligustilide.There was no correlation between endogenetic bacteria and volatile components,but its population constitution had correlation with fatty acids,which suggested endogenetic bacteria had relationship with synthesis of plant primary metabolite.Our research can give some references from a new degree measure to explain the formation of famous-region of Ligusticum chuanxiong.
引文
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