Leptographiumbhutanense提取物抑菌活性的初步分析
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摘要
内生真菌是动植物体内的微生物类群,其多样性、复杂性和特殊性的特点使其能够产生化学结构新颖、具有多种生物活性的次生代谢产物。本研究采用纸片法检测Leptographium bhutanense发酵提取物对13种致病细菌的抑菌活性,并对抗菌化合物的最低抑菌浓度(MIC)、热稳定性、光稳定性进行检测;同时,采用OSMAC策略寻找产抗菌化合物的最佳培养基、最佳培养时间和最佳接种量。分析显示提取物对3种人体致病细菌(蜡样芽孢杆菌,副溶血性弧菌,溶血性葡萄球菌)具有抑菌活性;其MIC值分别为:12.5mg/m L、6.25 mg/m L和6.25 mg/m L;提取物具有较好的热稳定性和光稳定性。产抗菌化合物的最佳培养基为:MFS,最佳培养时间和最佳接种量分别为15 d和1.0 g/100 m L。本研究为昆虫内生真菌抗菌化合物的开发利用提供帮助。
Endophytic fungi are microorganisms in the body of animals and plants, and their characters of diversity, complexity and specificity make it possible to produce secondary metabolites with novel chemical structure and biological activities. In this study, the antibacterial activity of L. bhutanense fermentation extract on 13 pathogenic bacteria was detected by paper method. We tested the minimum inhibitory concentration(MIC), thermal stability and photo-stability of antimicrobial compounds. Simultaneously, OSMAC strategy was utilized to find the optimum culture medium, culture time and inoculum size. The analysis indicated that the extract of L. bhutanense had antibacterial activity on three kinds of human pathogenic bacteria(Bacillus cereus,Vibrio parahaemolyticus, Staphylococcus aureus), the MIC values of which were 12.5 mg/mL, 6.25 mg/mL, and 6.25 mg/mL respectively. The extract also had well thermal stability and light stability. MFS was the optimum culture medium for producing antibacterial compounds. The optimum culture time and inoculum size were 15 d and 1.0 g/100 mL. The study could provide help for the development and utilization in antibacterial compounts of insect endophytic fungi.
Endophytic fungi are microorganisms in the body of animals and plants, and their characters of diversity, complexity and specificity make it possible to produce secondary metabolites with novel chemical structure and biological activities. In this study, the antibacterial activity of L. bhutanense fermentation extract on 13 pathogenic bacteria was detected by paper method. We tested the minimum inhibitory concentration(MIC), thermal stability and photo-stability of antimicrobial compounds. Simultaneously, OSMAC strategy was utilized to find the optimum culture medium, culture time and inoculum size. The analysis indicated that the extract of L. bhutanense had antibacterial activity on three kinds of human pathogenic bacteria(Bacillus cereus,Vibrio parahaemolyticus, Staphylococcus aureus), the MIC values of which were 12.5 mg/mL, 6.25 mg/mL, and 6.25 mg/mL respectively. The extract also had well thermal stability and light stability. MFS was the optimum culture medium for producing antibacterial compounds. The optimum culture time and inoculum size were 15 d and 1.0 g/100 mL. The study could provide help for the development and utilization in antibacterial compounts of insect endophytic fungi.
引文
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Andersson D.I., and Hughes D., 2010, Antibiotic resistance and its cost: is it possible to reverse resistance, Nature Reviews Microbiology, 8(4): 260-271
Barbier F., and Wolff M., 2010, Multi-drug resistant Pseudomonas aeruginosa: towards a therapeutic dead end, Medecine Sciences M/s, 26(11): 960-968
Bode H.B., Bethe B., H?fs R., and Zeeck A., 2002, Big effects from small changes: possible ways to explore nature's chemical diversity, Chembiochem, 3(7): 619-627
Ge H.M., and Tan R.X., 2009, Symbionts, an important source of new bioactive natural products, Huaxue Jinzhan (Progress in Chemistry), 21(1): 30-46 (戈惠明, 谭仁祥, 2009, 共生菌新活性天然产物的重要来源, 化学进展, 21(1): 30-46)
Guo L.Z., Liu C.X., Xu B., Chen J.F., Zou K., and Guo Z.Y.,2014, Secondary metabolites from the insect-derived endophytic fungus Alternaria sp., Huaxue Yanjiu Yu Yingyong (Chemical Research and Application), 26(2): 306-310 (郭玲芝, 刘呈雄, 徐帮, 陈剑锋, 邹坤, 郭志勇, 2014, 一株昆虫内生真菌 Alternaria sp.次级代谢产物研究, 化学研究与应用, 26(2): 306-310)
Hewage R.T., Aree T., Mahidol C., Mahidol C., Ruchirawat S.,and Kittakoop P., 2014, One strain-many compounds(OSMAC) method for production of polyketides, azaphilones, and an isochromanone using the endophytic fungus Dothideomycete sp., Phytochemistry, 108(1): 87-94
Li D., Zhu W.M., Gu Q.Q., Cui C.B., Zhu T.J., Liu H .B., and Fang Y.C., 2007, Structural identification and anti-tumor activity of Diketopi-perazines from secondary metabolites of marine-derived actinomycete H2003, Haiyang Kexue (Marine Science), 31(5): 45-48 (李冬, 朱伟明, 顾谦群, 崔承彬, 朱天骄, 刘红兵, 方玉春, 2007, 海洋放线菌 H2003 代谢产物中环二肽成分及其抗肿瘤活性的初步研究, 海洋科学, 31(5): 45-48)
Li T., Kong X.Q., and Dong H.Z., 2016, Gene silencing and its application in cotton, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 14(4): 918-928 (李婷, 孔祥强, 董合忠, 2016,基因沉默技术及其在棉花中的应用, 分子植物育种, 14(4): 918-928)
Li W.Y., Jiang Y.L., Deng R.X., Liu P., Gao T., and Yin W.P.,2012, Antibacterial activity research from insects extracts,Guowai Yiyao Kangshengsu Fence (World Notes on Antibiotics), 33(1): 30-33 (李文渊, 姜亚玲, 邓瑞雪, 刘普,高婷, 尹卫平, 2012, 昆虫提取物的抗菌活性研究, 国外医药抗生素分册, 33(1): 30-33)
Liu C.X., Wang L., Chen J.F., Guo Z.Y., Tu X., and Deng Z.X.,2015, Paraconfuranones A-H, eight new furanone analogs from the insec-associated fungus Paraconiothyrium brasiliense MZ-1, Magnetic Resonance Chemistry, 53(4): 317-322
Liu J.H., 2015, The study of secondary metabolites from marine microorganism based on the strategy of OSMAC, Thesis for M.S., Dalian University of Technology, Supervisor: Dong Y.S., pp.13 (刘俊慧, 2015, 基于 OSMAC 策略的海洋微生物次级代谢产物研究, 硕士学位论文, 大连理工大学, 导师: 董悦生, pp.13)
Liu R., Zhu T.J., Zhu W.M., Li D., Cui C.B., and Gu Q.Q., 2006,The antitumor active component from marine derived actinomycete S1001, Zhonguo Kangshengsu Zazhi (Chinese Journal of Antibiotics), 31(1): 36-38 (刘睿, 朱天骄, 朱伟明, 李冬, 崔承彬, 顾谦群, 2006, 海洋放线菌 S1001 中抗肿瘤活性成分的研究, 中国抗生素杂志, 31(1): 36-38)
Liu W.C., Yang F., Zhang R., Shi X., Lu X.H., Luan Y.S., Xiu Z.L., and Dong Y.S., 2016, Production of polyketides with anthelmintic activity by the fungus Talaromyces wortmannii,using one strain-many compounds (OSMAC) method, Phytochemistry Letters, 18: 157-161
Livermore D.M., 2004, The need for new antibiotics, Clinical Microbiol Infect, 10(S4): 1-9
Lu X., Chen G., and Pei Y.H., 2011, One strain many compounds (OSMAC) approach in the research of the lead compounds,Shenyang Yaoke Daxue Xuebao (Journal of Shenyang Pharmaceutical University), 28 (8): 640-645 (卢轩, 陈刚, 裴月湖, 2011, 单菌多产物策略及其在药物先导化合物研发中的应用, 沈阳药科大学学报, 28(8): 640-645)
Ma X.L., Wang W.S., Li E.W., Guo L.D., Pei Y.F., and Gao F.H.,2016, Bioactive secondary metabolites of the entomogenous fungus Phomops is amygdale, Qingdao Keji Daxue Xuebao (Journal of Qingdao University of Science & Technology ),37(3): 286-291 (马晓莉, 王文蜀, 李二伟, 郭良栋, 裴云飞,高锋华, 2016, 一株虫生真菌 Phomopsis amygdali 次级代谢产物研究, 青岛科技大学学报, 37(3): 286-291)
Monroe S., and Polk R., 2000, Antimicrobial use and bacterial resistance, Current Opinion in Microbiology, 3(5): 496-501
Nirma C., Eparvier V., and Stien D., 2013, Antifungal agents from Pseudallescheria boydii SNB-CN73 isolated from a Nasutitermes sp. termite, Journal of Natural Products, 76(5):988-991
Putri S.P., Kinoshita H., Ihara F., Igarashi Y., and Nihira T.,2009, Farinomalein, a maleimide-bearing compound from the entomopathogenic fungus Paecilomyces farinosus, Journal of Natural Products, 72(8): 1544-1546
Song M.D., Wachi M., Doi M., Ishino F., and Matsuhashi M.,1987, Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion,Febs Letters, 221(1): 167-171
Spratt B.G., 1994, Resistance to antibiotics mediated by target alterations, Science, 264(5157): 388-393
Stewart P.S., and Costerton J.W., 2001, Antibiotic resistance of bacteria in biofilms, Lancet, 358(9276): 135-138
Wang L., Hu S.Z., Liu C.X., Zou K., and Guo Z.Y., 2014, Cyclic dipeptides metabolites from the symbiotic fungus paraconiothyrium brasiliense, Huaxue Yanjiu Yu Yingyong (Chemical Research and Application), 26(9): 1478-1482 (汪蕾, 胡圣璋,刘呈雄, 邹坤, 郭志勇, 2014, 共生真菌巴西类壳小圆孢中环二肽类代谢产物研究,化学研究与应用,26(9):1478-1482)
Wei H.J., Lin Z.J., Li H.D., Gu Q.Q., and Zhu T.J., 2010, OSMAC (One Strain Many Compounds) approach in the research of microbial metabolites-a review, Weishengwu Xuebao (Acta Microbiologica Sinica), 50(6): 701-709 (韦洪娟, 林贞建, 李德海, 顾谦群, 朱天骄, 2010, 单菌多次级代谢产物方法及其在微生物代谢产物研究中的应用, 微生物学报,50(6): 701-709)
Wen X., Nangong Z.Y., Zhang Y., Song P., Cao K.Q., and Wang Q.Y., 2012, Anti fungai activity of metabolites from Xenorhabdus nematophila and Photorhabdus luminescens against apple pathogenic fungi, Zhongguo Zhibao Daokan (China Plant Protection), 32(1): 13-16 (文旭, 南宫自艳, 张园, 宋萍, 曹克强, 王勤英, 2012, 2 种昆虫病原线虫共生菌的代谢产物对苹果病原菌真菌的抑菌作用, 中国植保导刊, 32(1): 13-16)
Xie M.C., Li X.G., and Zhang D.H., 2015, A mini review of OSMAC approach and its application in Aspergillus fumigatus secondary metabolites, Tianran Chanwu Yanjiu Yu Kaifa (Research and Development of Natural Products),27(9): 1668-1673 (谢绵测, 李先国, 张大海, 2015, OSMAC策略及其在烟曲霉菌次级代谢产物研究中的应用, 天然产物研究与开发, 27(9): 1668-1673)
Xu B., Zou K., Guo L.Z., Liu C.X., and Cheng F., 2014, Dimeric naphthopyrones from Penicillium oxalicum, a fungus residing in Acrida cinerea, Tianran Chanwu Yanjiu Yu Kaifa (Research and Development of Natural Products), 26 (4):517-520 (徐帮, 邹坤, 郭玲芝, 刘呈雄, 程凡, 2014, 中华剑角蝗共生真菌 Penicillium oxalicum 中苯并吡喃酮二聚体类化合物的研究, 天然产物研究与开发, 26(4): 517-520)
Zhao M., Tan G.W., Tang X.Y., Long L.Z., and Zhou Y., 2011,Study on Minimun Bacteria-inhibiting concentrations of ten Chinese drugs extracts and antibiotics combinationf on pseudomonas aeruginosa in vitro, Hunan Shifan Daxue Xuebao (Journal of Hunan Normal University (Medical Science) ), 8(4): 80-83 (赵敏, 谭钢文, 唐小异, 龙灵芝, 周源, 2011, 十种中药提取物与抗生素合用对铜绿假单胞菌最小抑菌浓度影响的体外实验,湖南师范大学学报(医学版),8(4):80-83)
Zheng Y.L., Zou K., Xu B., and Cheng F., 2014, Secondary metabolites from Penicillium oxalicum of an insect endophytic fungus, Huazhong Shifan Daxue Xuebao (Journal of Central China Normal University ), 48(5): 689-692 (郑燕丽,邹坤, 徐帮, 程凡, 2014, 昆虫内生真菌 Penicillium oxalicum 次生代谢产物的研究, 华中师范大学学报, 48(5): 689-692)
Zhuang H.R., Wu W.S., Lu H.S., and Wang M.Z., 2000, Screening test for antimicrobial activities of 25 species of pteridophytes from Fuzhou, Fujian, Yaredai Zhiwu Kexue (Subtropical Plant Research Communications), 29(1): 5-8 (庄惠如, 吴文珊,卢海声, 王明兹, 2000, 福建福州 25 种蕨类植物抑菌活性筛选研究, 亚热带植物科学, 29(1): 5-8)
Andersson D.I., and Hughes D., 2010, Antibiotic resistance and its cost: is it possible to reverse resistance, Nature Reviews Microbiology, 8(4): 260-271
Barbier F., and Wolff M., 2010, Multi-drug resistant Pseudomonas aeruginosa: towards a therapeutic dead end, Medecine Sciences M/s, 26(11): 960-968
Bode H.B., Bethe B., H?fs R., and Zeeck A., 2002, Big effects from small changes: possible ways to explore nature's chemical diversity, Chembiochem, 3(7): 619-627
Ge H.M., and Tan R.X., 2009, Symbionts, an important source of new bioactive natural products, Huaxue Jinzhan (Progress in Chemistry), 21(1): 30-46 (戈惠明, 谭仁祥, 2009, 共生菌新活性天然产物的重要来源, 化学进展, 21(1): 30-46)
Guo L.Z., Liu C.X., Xu B., Chen J.F., Zou K., and Guo Z.Y.,2014, Secondary metabolites from the insect-derived endophytic fungus Alternaria sp., Huaxue Yanjiu Yu Yingyong (Chemical Research and Application), 26(2): 306-310 (郭玲芝, 刘呈雄, 徐帮, 陈剑锋, 邹坤, 郭志勇, 2014, 一株昆虫内生真菌 Alternaria sp.次级代谢产物研究, 化学研究与应用, 26(2): 306-310)
Hewage R.T., Aree T., Mahidol C., Mahidol C., Ruchirawat S.,and Kittakoop P., 2014, One strain-many compounds(OSMAC) method for production of polyketides, azaphilones, and an isochromanone using the endophytic fungus Dothideomycete sp., Phytochemistry, 108(1): 87-94
Li D., Zhu W.M., Gu Q.Q., Cui C.B., Zhu T.J., Liu H .B., and Fang Y.C., 2007, Structural identification and anti-tumor activity of Diketopi-perazines from secondary metabolites of marine-derived actinomycete H2003, Haiyang Kexue (Marine Science), 31(5): 45-48 (李冬, 朱伟明, 顾谦群, 崔承彬, 朱天骄, 刘红兵, 方玉春, 2007, 海洋放线菌 H2003 代谢产物中环二肽成分及其抗肿瘤活性的初步研究, 海洋科学, 31(5): 45-48)
Li T., Kong X.Q., and Dong H.Z., 2016, Gene silencing and its application in cotton, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 14(4): 918-928 (李婷, 孔祥强, 董合忠, 2016,基因沉默技术及其在棉花中的应用, 分子植物育种, 14(4): 918-928)
Li W.Y., Jiang Y.L., Deng R.X., Liu P., Gao T., and Yin W.P.,2012, Antibacterial activity research from insects extracts,Guowai Yiyao Kangshengsu Fence (World Notes on Antibiotics), 33(1): 30-33 (李文渊, 姜亚玲, 邓瑞雪, 刘普,高婷, 尹卫平, 2012, 昆虫提取物的抗菌活性研究, 国外医药抗生素分册, 33(1): 30-33)
Liu C.X., Wang L., Chen J.F., Guo Z.Y., Tu X., and Deng Z.X.,2015, Paraconfuranones A-H, eight new furanone analogs from the insec-associated fungus Paraconiothyrium brasiliense MZ-1, Magnetic Resonance Chemistry, 53(4): 317-322
Liu J.H., 2015, The study of secondary metabolites from marine microorganism based on the strategy of OSMAC, Thesis for M.S., Dalian University of Technology, Supervisor: Dong Y.S., pp.13 (刘俊慧, 2015, 基于 OSMAC 策略的海洋微生物次级代谢产物研究, 硕士学位论文, 大连理工大学, 导师: 董悦生, pp.13)
Liu R., Zhu T.J., Zhu W.M., Li D., Cui C.B., and Gu Q.Q., 2006,The antitumor active component from marine derived actinomycete S1001, Zhonguo Kangshengsu Zazhi (Chinese Journal of Antibiotics), 31(1): 36-38 (刘睿, 朱天骄, 朱伟明, 李冬, 崔承彬, 顾谦群, 2006, 海洋放线菌 S1001 中抗肿瘤活性成分的研究, 中国抗生素杂志, 31(1): 36-38)
Liu W.C., Yang F., Zhang R., Shi X., Lu X.H., Luan Y.S., Xiu Z.L., and Dong Y.S., 2016, Production of polyketides with anthelmintic activity by the fungus Talaromyces wortmannii,using one strain-many compounds (OSMAC) method, Phytochemistry Letters, 18: 157-161
Livermore D.M., 2004, The need for new antibiotics, Clinical Microbiol Infect, 10(S4): 1-9
Lu X., Chen G., and Pei Y.H., 2011, One strain many compounds (OSMAC) approach in the research of the lead compounds,Shenyang Yaoke Daxue Xuebao (Journal of Shenyang Pharmaceutical University), 28 (8): 640-645 (卢轩, 陈刚, 裴月湖, 2011, 单菌多产物策略及其在药物先导化合物研发中的应用, 沈阳药科大学学报, 28(8): 640-645)
Ma X.L., Wang W.S., Li E.W., Guo L.D., Pei Y.F., and Gao F.H.,2016, Bioactive secondary metabolites of the entomogenous fungus Phomops is amygdale, Qingdao Keji Daxue Xuebao (Journal of Qingdao University of Science & Technology ),37(3): 286-291 (马晓莉, 王文蜀, 李二伟, 郭良栋, 裴云飞,高锋华, 2016, 一株虫生真菌 Phomopsis amygdali 次级代谢产物研究, 青岛科技大学学报, 37(3): 286-291)
Monroe S., and Polk R., 2000, Antimicrobial use and bacterial resistance, Current Opinion in Microbiology, 3(5): 496-501
Nirma C., Eparvier V., and Stien D., 2013, Antifungal agents from Pseudallescheria boydii SNB-CN73 isolated from a Nasutitermes sp. termite, Journal of Natural Products, 76(5):988-991
Putri S.P., Kinoshita H., Ihara F., Igarashi Y., and Nihira T.,2009, Farinomalein, a maleimide-bearing compound from the entomopathogenic fungus Paecilomyces farinosus, Journal of Natural Products, 72(8): 1544-1546
Song M.D., Wachi M., Doi M., Ishino F., and Matsuhashi M.,1987, Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion,Febs Letters, 221(1): 167-171
Spratt B.G., 1994, Resistance to antibiotics mediated by target alterations, Science, 264(5157): 388-393
Stewart P.S., and Costerton J.W., 2001, Antibiotic resistance of bacteria in biofilms, Lancet, 358(9276): 135-138
Wang L., Hu S.Z., Liu C.X., Zou K., and Guo Z.Y., 2014, Cyclic dipeptides metabolites from the symbiotic fungus paraconiothyrium brasiliense, Huaxue Yanjiu Yu Yingyong (Chemical Research and Application), 26(9): 1478-1482 (汪蕾, 胡圣璋,刘呈雄, 邹坤, 郭志勇, 2014, 共生真菌巴西类壳小圆孢中环二肽类代谢产物研究,化学研究与应用,26(9):1478-1482)
Wei H.J., Lin Z.J., Li H.D., Gu Q.Q., and Zhu T.J., 2010, OSMAC (One Strain Many Compounds) approach in the research of microbial metabolites-a review, Weishengwu Xuebao (Acta Microbiologica Sinica), 50(6): 701-709 (韦洪娟, 林贞建, 李德海, 顾谦群, 朱天骄, 2010, 单菌多次级代谢产物方法及其在微生物代谢产物研究中的应用, 微生物学报,50(6): 701-709)
Wen X., Nangong Z.Y., Zhang Y., Song P., Cao K.Q., and Wang Q.Y., 2012, Anti fungai activity of metabolites from Xenorhabdus nematophila and Photorhabdus luminescens against apple pathogenic fungi, Zhongguo Zhibao Daokan (China Plant Protection), 32(1): 13-16 (文旭, 南宫自艳, 张园, 宋萍, 曹克强, 王勤英, 2012, 2 种昆虫病原线虫共生菌的代谢产物对苹果病原菌真菌的抑菌作用, 中国植保导刊, 32(1): 13-16)
Xie M.C., Li X.G., and Zhang D.H., 2015, A mini review of OSMAC approach and its application in Aspergillus fumigatus secondary metabolites, Tianran Chanwu Yanjiu Yu Kaifa (Research and Development of Natural Products),27(9): 1668-1673 (谢绵测, 李先国, 张大海, 2015, OSMAC策略及其在烟曲霉菌次级代谢产物研究中的应用, 天然产物研究与开发, 27(9): 1668-1673)
Xu B., Zou K., Guo L.Z., Liu C.X., and Cheng F., 2014, Dimeric naphthopyrones from Penicillium oxalicum, a fungus residing in Acrida cinerea, Tianran Chanwu Yanjiu Yu Kaifa (Research and Development of Natural Products), 26 (4):517-520 (徐帮, 邹坤, 郭玲芝, 刘呈雄, 程凡, 2014, 中华剑角蝗共生真菌 Penicillium oxalicum 中苯并吡喃酮二聚体类化合物的研究, 天然产物研究与开发, 26(4): 517-520)
Zhao M., Tan G.W., Tang X.Y., Long L.Z., and Zhou Y., 2011,Study on Minimun Bacteria-inhibiting concentrations of ten Chinese drugs extracts and antibiotics combinationf on pseudomonas aeruginosa in vitro, Hunan Shifan Daxue Xuebao (Journal of Hunan Normal University (Medical Science) ), 8(4): 80-83 (赵敏, 谭钢文, 唐小异, 龙灵芝, 周源, 2011, 十种中药提取物与抗生素合用对铜绿假单胞菌最小抑菌浓度影响的体外实验,湖南师范大学学报(医学版),8(4):80-83)
Zheng Y.L., Zou K., Xu B., and Cheng F., 2014, Secondary metabolites from Penicillium oxalicum of an insect endophytic fungus, Huazhong Shifan Daxue Xuebao (Journal of Central China Normal University ), 48(5): 689-692 (郑燕丽,邹坤, 徐帮, 程凡, 2014, 昆虫内生真菌 Penicillium oxalicum 次生代谢产物的研究, 华中师范大学学报, 48(5): 689-692)
Zhuang H.R., Wu W.S., Lu H.S., and Wang M.Z., 2000, Screening test for antimicrobial activities of 25 species of pteridophytes from Fuzhou, Fujian, Yaredai Zhiwu Kexue (Subtropical Plant Research Communications), 29(1): 5-8 (庄惠如, 吴文珊,卢海声, 王明兹, 2000, 福建福州 25 种蕨类植物抑菌活性筛选研究, 亚热带植物科学, 29(1): 5-8)