马勃培养特性、总生物碱含量及体外抑菌活性的研究
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摘要
马勃是较常用的传统药用真菌,广泛分布于世界各地。中国药典(2005年版)收载的马勃主要为脱皮马勃(Lasiosphaera fenzlii Reich),大马勃[Calvatia gigantean(Batsch ex Pers.)Lloyd]或紫色马勃[C.lilacina(Mont.et Berk.)Lloyd]的干燥子实体。脱皮马勃具有消肿、解毒和止血的功用,临床主要用于治疗呼吸道感染所引起的咽喉肿痛、扁桃体炎、咳嗽等症状,也可外用于治疗疮痈、褥疮、冻疮及创伤出血等。褐孢大秃马勃[C.saccata(Vahl:Fr.)Morg]、黑铅色灰球菌(Bovista nigrescens Pers.)、大口静灰球(Bovistella sinensis Lloyd)和灰包菇[Secotium agaricoides(Czern.)Hollos]这四种腹菌纲真菌在我国部分地区也被作为马勃入药,但尚未被中国药典所收载。据相关文献记载,褐孢大秃马勃幼时可食用;黑铅色灰球菌的孢子粉可作为伤口药使用;大口静灰球和灰包菇与脱皮马勃具有相同的药用功效,特别是大口静灰球在吉林、江苏、湖北、江西、福建等多个省市的众多中医门诊中被广泛地作为马勃入药,用于治疗咳血、创伤出血和扁桃体炎等。
我国马勃资源丰富,药用历史悠久,民间和临床使用较广泛,开发利用的潜力巨大,值得对其进一步研究与探讨。本论文对鉴定的灰包菇[Secotium agaricoides(Czern.)Hollos],脱皮马勃(L.fenzlii),褐孢大秃马勃[C.saccata(Vahl:Fr.)Morg],大口静灰球(Bovistella sinensis Lloyd)和黑铅色灰球菌(Bovista nigrescens Pers.)5种马勃的菌丝体培养特性及体外抑菌活性进行研究,并对大口静灰球子实体的总生物碱进行提取和含量测定,以期为马勃的优质培育与资源开发提供实验依据和理论基础。
本实验采用固体培养方法分别研究单因子碳源、氮源、维生素、pH值、温度和光照对以上5种马勃菌丝体固体培养特性的影响,实验结果分别筛选出适合以上5种马勃生长的最佳营养成分和最佳培养条件,根据单因子实验结果设计正交实验L_(16)(4~5),筛选出适合灰包菇和大口静灰球菌丝体生长的最佳培养基配方;另外采用液体培养方法对5种马勃液体培养基中碳源、氮源、初始pH值,液体接种量和培养条件进行研究,筛选出适合5种马勃液体发酵的最佳营养成分和最佳培养条件,从而为5种马勃菌种的优质培育和实现其人工栽培提供理论依据。
对大口静灰球总生物碱的提取方法进行比较研究,采用酸水提取法提取大口静灰球子实体并收集酸水提取液,经732型阳离子交换树脂吸附总生物碱后,使用732型阳离子交换树脂柱洗脱纯化法、静置提取纯化法、热回流提取纯化法和索氏提取纯化法该树脂进行洗脱纯化,获得较纯的总生物碱,通过称重法和酸性染料比色法对不同提取方法获得的总生物碱含量进行测定。实验结果显示,使用732型阳离子交换树脂柱洗脱纯化法获得的总生物碱最多,总生物碱含量达2.68%,其次依次是热回流提取纯化法获得1.13%、索氏提取纯化法获得0.52%和静置提取纯化法获得0.47%。此外,采用二苯碳酰二肼分光光度法对脱皮马勃孢子粉水提液中Cr~(6+)的含量进行测定,结果显示其中Cr~(6+)含量为0.1206～0.2956mg/L。
采用菌饼法(活菌活性测定)和纸片扩散法研究了5种马勃的发酵液、菌丝体水提液、活菌菌饼以及大口静灰球子实体6种溶剂提取液对金黄色葡萄球菌、大肠埃希氏菌、鼠伤寒沙门氏菌、黑曲霉和绿色木霉5种测试菌的抑菌活性。实验结果表明这5种马勃的发酵液中都含有与原菌丝体相同或相似的抑菌活性物质。
Puffball is a kind of traditional medicinal fungus,distributing extensively around the world.Puffballs which recorded by the Pharmacopoeia of the People's Republic of China(Edition of 2005) mainly contain the dried fruiting bodies of Lasiosphaera fenzlii Reichand,Calvatia gigantean(Batsch ex Pers.) Lloyd and C. lilacina(Mont.et Berk.) Lloyd.L.Fenzlii has the functions of detumescence,deintoxication and hematischesis. It mainly used for treating swelling and aching of throat causing by respiratory infection,tonsillitis,cough etc. It also used in clinically for treating sore and anthracia,decubital ulcer,frostbite,traumatic hemorrhage and so on.C.saccata(Vah1:Fr.) Morg,Bovista nigrescens Pers,Bovistella sinensis Lloyd and Secotium agaricoides (Czern.) Hollos,which belong to Gasteromycetes,are also used as puffballs in Chinese civilian,but they haven't been recorded by the Pharmacopoeia of the People's Republic of China yet.According to the records of pertinent literature,the immature C.saccata is edible;the spore of B.Nigrescens can be used as vulneraria; B.sinensis and S.agaricoide have the same medicinal effectiveness as L.Fenzlii.Especially,B.sinensis is extensively used as puffball in numerous traditional Chinese medicine clinics in Jilin,Jiangsu,Hubei,Jiangxi and Fujian province for coughing up blood,traumatic hemorrhage and tonsillitis etc.
The puffballs' resources are abundance in China,and puffballs are using widespread for a long time in folk and clinic,so their exploitation are potential and they are worthy to further investigate.This thesis researched the cultural character and the bacteriostatic activity of 5 kinds of puffballs(S.agaricoide,L. Fenzlii,C.saccata,B.sinensis and B.nigrescens) and also determined the total alkaloids' content of B. sinensis,expected to provide the theoretical foundation for the high-quality cultivation and exploitation resources of puffballs.
Used the method of solid culture to study the single factors of the carbon source,the nitrogen source,the Vitamin,the pH value,the temperature and the illumination respectively influencing on the mycelia growth of five kinds of above-mention puffballs,and then according to the results of the optimal nutritional ingredients and culture conditions,designed the orthogonal test L_(16)(4~5) and screened the optimal prescription of the culture medium.In addition,used the method of liquid culture to study the single factors of the carbon source, the nitrogen source,the pH value and the capacity of inoculation respectively influencing on the mycelia growth of five kinds of above-mention puffballs,and screened out the optimal nutritional ingredients and culture conditions,expecting to provide the theoretical foundation of high-quality cultivation for the five kinds of puffballs and achieving the artificial cultivation.
This experiment researched the different extractioes of total alkaloids of B.sinensis,using the method of the acidic water extractio to extract the crude total alkaloids of B.sinensis and then using 4 different kinds of extractioes such as type-732 cation exchange resin extractio,Soxhlet's extractio,standing extractio and heating circumfluence extractio to purify the initial extraction of total alkaloids and obtain the purified total alkaloids of B.sinensis,then assaying the contents of the total alkaloids using weighting method and acid dye colorimetry.The results showed that the total alkaloids were obtained 2.68%to be most when used the type-732 cation exchange resin extraction,and next in turn were heating circumfluence extraction which obtained 1.13%,standing extraction which obtained 0.52%and Soxhlet's extraction which obtained 0.47%.In addition,the content of Cr~(6+) in L.fenzlii was measured by the method of sym-diphenylcarbazide spectrophotometry.The result showed that the content of Cr~(6+) was from 0.1206mg/L to 0.2956mg/L.
Using the mycelia,the broth and the fungi cakes of the above-mention 5 kinds of puffballs and the six kinds of solvent extractions of B.sinensis as the materials,and using the method of Kirby-Bauer and fungi cakes to study the antimicrobial activities on S.aureus,E.coli,S.typhimurium,A.niger and T.viride.The experimental results indicated that there were some same or similar bacteriostatic active materials between the broth and the promycelium of 5 kinds of puffballs.
我国马勃资源丰富,药用历史悠久,民间和临床使用较广泛,开发利用的潜力巨大,值得对其进一步研究与探讨。本论文对鉴定的灰包菇[Secotium agaricoides(Czern.)Hollos],脱皮马勃(L.fenzlii),褐孢大秃马勃[C.saccata(Vahl:Fr.)Morg],大口静灰球(Bovistella sinensis Lloyd)和黑铅色灰球菌(Bovista nigrescens Pers.)5种马勃的菌丝体培养特性及体外抑菌活性进行研究,并对大口静灰球子实体的总生物碱进行提取和含量测定,以期为马勃的优质培育与资源开发提供实验依据和理论基础。
本实验采用固体培养方法分别研究单因子碳源、氮源、维生素、pH值、温度和光照对以上5种马勃菌丝体固体培养特性的影响,实验结果分别筛选出适合以上5种马勃生长的最佳营养成分和最佳培养条件,根据单因子实验结果设计正交实验L_(16)(4~5),筛选出适合灰包菇和大口静灰球菌丝体生长的最佳培养基配方;另外采用液体培养方法对5种马勃液体培养基中碳源、氮源、初始pH值,液体接种量和培养条件进行研究,筛选出适合5种马勃液体发酵的最佳营养成分和最佳培养条件,从而为5种马勃菌种的优质培育和实现其人工栽培提供理论依据。
对大口静灰球总生物碱的提取方法进行比较研究,采用酸水提取法提取大口静灰球子实体并收集酸水提取液,经732型阳离子交换树脂吸附总生物碱后,使用732型阳离子交换树脂柱洗脱纯化法、静置提取纯化法、热回流提取纯化法和索氏提取纯化法该树脂进行洗脱纯化,获得较纯的总生物碱,通过称重法和酸性染料比色法对不同提取方法获得的总生物碱含量进行测定。实验结果显示,使用732型阳离子交换树脂柱洗脱纯化法获得的总生物碱最多,总生物碱含量达2.68%,其次依次是热回流提取纯化法获得1.13%、索氏提取纯化法获得0.52%和静置提取纯化法获得0.47%。此外,采用二苯碳酰二肼分光光度法对脱皮马勃孢子粉水提液中Cr~(6+)的含量进行测定,结果显示其中Cr~(6+)含量为0.1206～0.2956mg/L。
采用菌饼法(活菌活性测定)和纸片扩散法研究了5种马勃的发酵液、菌丝体水提液、活菌菌饼以及大口静灰球子实体6种溶剂提取液对金黄色葡萄球菌、大肠埃希氏菌、鼠伤寒沙门氏菌、黑曲霉和绿色木霉5种测试菌的抑菌活性。实验结果表明这5种马勃的发酵液中都含有与原菌丝体相同或相似的抑菌活性物质。
Puffball is a kind of traditional medicinal fungus,distributing extensively around the world.Puffballs which recorded by the Pharmacopoeia of the People's Republic of China(Edition of 2005) mainly contain the dried fruiting bodies of Lasiosphaera fenzlii Reichand,Calvatia gigantean(Batsch ex Pers.) Lloyd and C. lilacina(Mont.et Berk.) Lloyd.L.Fenzlii has the functions of detumescence,deintoxication and hematischesis. It mainly used for treating swelling and aching of throat causing by respiratory infection,tonsillitis,cough etc. It also used in clinically for treating sore and anthracia,decubital ulcer,frostbite,traumatic hemorrhage and so on.C.saccata(Vah1:Fr.) Morg,Bovista nigrescens Pers,Bovistella sinensis Lloyd and Secotium agaricoides (Czern.) Hollos,which belong to Gasteromycetes,are also used as puffballs in Chinese civilian,but they haven't been recorded by the Pharmacopoeia of the People's Republic of China yet.According to the records of pertinent literature,the immature C.saccata is edible;the spore of B.Nigrescens can be used as vulneraria; B.sinensis and S.agaricoide have the same medicinal effectiveness as L.Fenzlii.Especially,B.sinensis is extensively used as puffball in numerous traditional Chinese medicine clinics in Jilin,Jiangsu,Hubei,Jiangxi and Fujian province for coughing up blood,traumatic hemorrhage and tonsillitis etc.
The puffballs' resources are abundance in China,and puffballs are using widespread for a long time in folk and clinic,so their exploitation are potential and they are worthy to further investigate.This thesis researched the cultural character and the bacteriostatic activity of 5 kinds of puffballs(S.agaricoide,L. Fenzlii,C.saccata,B.sinensis and B.nigrescens) and also determined the total alkaloids' content of B. sinensis,expected to provide the theoretical foundation for the high-quality cultivation and exploitation resources of puffballs.
Used the method of solid culture to study the single factors of the carbon source,the nitrogen source,the Vitamin,the pH value,the temperature and the illumination respectively influencing on the mycelia growth of five kinds of above-mention puffballs,and then according to the results of the optimal nutritional ingredients and culture conditions,designed the orthogonal test L_(16)(4~5) and screened the optimal prescription of the culture medium.In addition,used the method of liquid culture to study the single factors of the carbon source, the nitrogen source,the pH value and the capacity of inoculation respectively influencing on the mycelia growth of five kinds of above-mention puffballs,and screened out the optimal nutritional ingredients and culture conditions,expecting to provide the theoretical foundation of high-quality cultivation for the five kinds of puffballs and achieving the artificial cultivation.
This experiment researched the different extractioes of total alkaloids of B.sinensis,using the method of the acidic water extractio to extract the crude total alkaloids of B.sinensis and then using 4 different kinds of extractioes such as type-732 cation exchange resin extractio,Soxhlet's extractio,standing extractio and heating circumfluence extractio to purify the initial extraction of total alkaloids and obtain the purified total alkaloids of B.sinensis,then assaying the contents of the total alkaloids using weighting method and acid dye colorimetry.The results showed that the total alkaloids were obtained 2.68%to be most when used the type-732 cation exchange resin extraction,and next in turn were heating circumfluence extraction which obtained 1.13%,standing extraction which obtained 0.52%and Soxhlet's extraction which obtained 0.47%.In addition,the content of Cr~(6+) in L.fenzlii was measured by the method of sym-diphenylcarbazide spectrophotometry.The result showed that the content of Cr~(6+) was from 0.1206mg/L to 0.2956mg/L.
Using the mycelia,the broth and the fungi cakes of the above-mention 5 kinds of puffballs and the six kinds of solvent extractions of B.sinensis as the materials,and using the method of Kirby-Bauer and fungi cakes to study the antimicrobial activities on S.aureus,E.coli,S.typhimurium,A.niger and T.viride.The experimental results indicated that there were some same or similar bacteriostatic active materials between the broth and the promycelium of 5 kinds of puffballs.
引文
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