竹红菌甲素对番茄灰霉病菌的关键抑菌因子
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摘要
为探究竹黄菌Shiraia bambuscola代谢产物竹红菌甲素(HA)对番茄灰霉病菌Botrytis cinerea的关键抑菌因子,依据活性氧淬灭原理,测定了HA的光反应类型;采用单线态氧(~1O_2)捕捉法、芬顿试剂法和核黄素光化学反应法,分析了单线态氧(~1O_2)、羟基自由基(·OH)和超氧阴离子自由基(O_2~(-·)) 3种活性氧浓度与HA对番茄灰霉病菌抑制率的相关性。结果表明:HA是通过光动力作用中的2种光反应类型(TypeⅠ和TypeⅡ)产生的活性氧对番茄灰霉病菌起到抑制作用,其中主要以TypeⅡ为主。~1O_2、·OH和O_2~(-·)的浓度与其对番茄灰霉病菌的抑制率均呈极显著的线性正相关,3种活性氧均为关键抑菌因子。经回归分析,获得最优回归模型为:Y=0.055 9+0.814 5X_1+0.002X_2+0.738 4X_3,其中Y为抑制率,X_1、X_2和不分别为~1O_2、·OH和O_2~(-·)的浓度,R2=0.9619,可见三者抑菌能力顺序为~1O_2>O_2~(-·)>·OH。本论文首次提出HA抑制番茄灰霉病菌的关键因子,为HA作用机理的研究奠定了基础,并为其作为光活化农药的开发利用提供了理论依据。
The key factors in the antifungal activity of Hypocrellin A(HA), the metabolites of Shiraia bambuscola, against the Botrytis cinerea were investigated. The type of photoactivity of HA was determined based on the principle of active oxygen quenching. The correlation between the concentration of three reactive oxygen species, including singlet oxygen(~1O_2), hydroxyl radical(·OH),and the superoxide anion radical(O_2~(-·)), and the inhibition rate of HA against B. cinerea was analyzed by the singlet oxygen capture principle, Fenton reaction, and riboflavin photochemical reaction. Results showed that the antifungal activity of HA against B. cinerea resulted from the active oxygen generated by photodynamic action(Type Ⅰ and Type Ⅱ). Both types exhibited controlling effects against the growth of B. cinerea, but Type Ⅱ exhibited is more active. The concentration of ~1O_2, ·OH, and O_2~(-·)are positively correlated with the bacteriostatic rates, and those three active oxygen species were the key factors in the antifungal activity. Stepwise regression analysis revealed that ~1O_2 is the most critical antifungal factor produced by HA, followed by O_2·-and ·OH. The optimal regression model was obtained as follows: Y= 0.055 9 + 0.814 5 X_1 + 0.002 X_2 + 0.738 4 X_3, in which Y was the inhibition rate,X_1,X_2 and X_3 were the concentration of ~1O_2, ·OH and O_2~(-·), respectively, R~2 = 0.961 9. In this paper, the key factors in the antifungal activity of HA against B. cinerea were investigated for the first time, which laid a foundation for the study of the mechanism of HA and provided a theoretical basis for the development and utilization of HA as a photoactivated pesticide.
The key factors in the antifungal activity of Hypocrellin A(HA), the metabolites of Shiraia bambuscola, against the Botrytis cinerea were investigated. The type of photoactivity of HA was determined based on the principle of active oxygen quenching. The correlation between the concentration of three reactive oxygen species, including singlet oxygen(~1O_2), hydroxyl radical(·OH),and the superoxide anion radical(O_2~(-·)), and the inhibition rate of HA against B. cinerea was analyzed by the singlet oxygen capture principle, Fenton reaction, and riboflavin photochemical reaction. Results showed that the antifungal activity of HA against B. cinerea resulted from the active oxygen generated by photodynamic action(Type Ⅰ and Type Ⅱ). Both types exhibited controlling effects against the growth of B. cinerea, but Type Ⅱ exhibited is more active. The concentration of ~1O_2, ·OH, and O_2~(-·)are positively correlated with the bacteriostatic rates, and those three active oxygen species were the key factors in the antifungal activity. Stepwise regression analysis revealed that ~1O_2 is the most critical antifungal factor produced by HA, followed by O_2·-and ·OH. The optimal regression model was obtained as follows: Y= 0.055 9 + 0.814 5 X_1 + 0.002 X_2 + 0.738 4 X_3, in which Y was the inhibition rate,X_1,X_2 and X_3 were the concentration of ~1O_2, ·OH and O_2~(-·), respectively, R~2 = 0.961 9. In this paper, the key factors in the antifungal activity of HA against B. cinerea were investigated for the first time, which laid a foundation for the study of the mechanism of HA and provided a theoretical basis for the development and utilization of HA as a photoactivated pesticide.
引文
[1]肖仔君,陈惠音,杨汝德.竹红菌素[J].中国食品添加剂,2003(4):74-76.XIAO Z J,CHEN H Y,YANG R D.Hypocrellin[J].China Food Additives,2003(4):74-76.
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[5]JIANG Y,LEUNG A W,WANG X N,et al.Inactivation of Staphylococcus aureus by photodynamic action of Hypocrellin B[J].Photodiagnosis Photodyn Ther,2013,10(4):600-606.
[6]崔荔群.探讨竹红菌素软膏治疗外阴白斑病的临床价值[J].中国卫生标准管理,2017,8(20):108-109.CUI L Q.Investigate the clinical value of bamboo mushroom ointment in the treatment of leukoplakia[J].China Health Standard Managent,2017,8(20):108-109.
[7]赵菊芬,李茜,杨柳风,等.聚焦超声联合竹红菌素软膏治疗外阴白色病变164例疗效观察[J].中国计划生育和妇产科,2012,4(1):61-63.ZHAO J F,LI X,YANG L F,et al.Clinical observation of curative effect on focused-ultrasound combined with hypocrellin-ointment to treat leukoplakia vulvae in 164 cases[J].Chin J Family Plann Gynecotokol,2012,4(1):61-63.
[8]JIN S,ZHOU L J,GU Z J,et al.A new near infrared photosensitizing nanoplatform containing blue-emitting up-conversion nanoparticles and Hypocrellin A for photodynamic therapy of cancer cells[J].Nanoscale,2013,5(23):11910-11918.
[9]HUDSON J B,ZHOU J,CHEN J,et al.Hypocrellin,from Hypocrella bambuase,is phototoxic to human immunodeficiency virus[J].Photochem Photobiol,1994,60(3):253-255.
[10]ZHANG Z Y,ZANG L Y,XU G R,et al.Characteristics of the primary reaction of Hypocrellin A photosensitization[J].Sci China B,1989,32(9):1063-1071.
[11]MAISCH T,BAIER J,FRANZ B,et al.The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria[J].Proc Natl Acad Sci USA,2007,104(17):7223-7228.
[12]赵学,李朋举,朱旭伟,等.竹红菌甲素对番茄灰霉病菌的抑制作用[J].农药学学报,2015,17(2):149-155.ZHAO X,LI P J,ZHU X W,et al.Inhibitory effect of Hypocrellin Aagainst Botrytis cinerea[J].Chin J Pestic Sci,2015,17(2):149-155.
[13]朱齐超,林海萍,李朋举,等.竹红菌甲素对18种植物病原菌的抑制作用[J].植物病理学报,2014,44(1):107-109.ZHU Q C,LIN H P,LI P J,et al.Inhibition of Hypocrellin A against18 pathogenic fungi[J].Acta Phytopathologica Sinica,2014,44(1):107-109.
[14]李平兰,贺稚非.食品微生物学实验原理与技术[M].北京:中国农业出版社,2005.LI P L,HE Z F.Experiments theories and techniques in food microbiology[M].Beijing:China Agriculture Press,2005.
[15]赵学.光活化农药竹红菌甲素对番茄灰霉的关键抑菌因子研究[D].杭州:浙江农林大学,2015.ZHAO X.Key factors of photoactivated pesticide Hypocrellin Aagainst Botrytis cinerea[D].Hangzhou:Zhejiang A&F University,2015.
[16]WHEATLEY R,HACKETT C,BRUCE A,et al.Effect of substrate composition on production of volatile organic compounds from Trichoderma spp.inhibitory to wood decay fungi[J].Int Biodeterior Biodegrad,1997,39(2-3):199-205.
[17]张凯,吴曼莉,辜柳霜,等.胶孢炭疽菌生防放线菌gz-8的鉴定及生物活性初步评价[J].农药学学报,2017,19(6):708-715.ZHANG K,WU M L,GU L S,et al.Identification and preliminary evaluation of a biocontrol actinomycete strain gz-8 against Colletotrichum gloeosporioides[J].Chin J Pestic Sci,2017,19(6):708-715.
[18]GUO X X,YANG X Q,YANG R Y,et al.Salicylic acid and methyl jasmonate but not rose bengal enhance artemisinin production through invoking burst of endogenous singlet oxygen[J].Plant Sci,2010,178(4):390-397.
[19]涂欢,徐涛,易永,等.羟基自由基引起神经细胞[Ca2+]i增高的机制和Ebselen的抑制作用[J].生物物理学报,1996,12(3):522-526.TU H,XU T,YI Y,et al.Mechanism of the enhancement effect of hydroxyl rdicals on[Ca2+]i in rat cerebral cortical neuronal cells and the inhibition of Ebselen[J].Acta Biophysica Sinica,1996,12(3):522-526.
[20]李学梅,李用芳,李培睿,等.安络小皮伞液体发酵过程中还原糖与pH值、菌丝干重及产量的关系[J].中国食用菌,2002,21(2):37-39.LI X M,LI Y F,LI P R,et al.The relations among reducing sugar,pH,dry weight of mycelium and production during the liquid fermentation of Marasmius androsaceus[J].Ed Fungi China,2002,21(2):37-39.
[21]周雪莹,熊文,孔令魁.核黄素光化学法对血浆中革兰阳性和阴性指示菌的减除作用和对血小板P选择蛋白表达的影响[J].中国实验血液学杂志,2010,18(04):1059-1062.ZHOU X Y,XIONG W,KONG L K.Effects of riboflavin combined with potosensitization on reduction of Gram-postitive and gramnegative indicating germs in plasma and P-selectin expression of apheresis platelet concentrates[J].Journal of Experimental Hematology,2010,18(04):1059-1062.
[22]李锦全,陈瑞川,蔡克瑕,等.核黄素光化学反应诱导人胃癌细胞凋亡[J].癌症,2003,22(3):253-256.LI J Q,CHEN R C,CAI K X,et al.Apoptosis of human gastric cancer cell induced by photochemical riboflavin[J].Chin J Cancer,2003,22(3):253-256.
[23]周乃康,尚立群,柳曦.竹红菌乙素-PDT与血卟啉衍生物-PDT对人肺癌细胞杀伤效应的比较研究[J].解放军医学杂志,2004,29(12):1076-1078.ZHOU N K,SHANG L Q,LIU X.Comparative study on the killing effect between Hypocrellins B:photodynamic therapy and hematoporphyrin derivative-photodynamic therapy on human lung cancer cells[J].Med J Chin PLA,2004,29(12):1076-1078.
[24]尚立群,周乃康,顾瑛,等.竹红菌乙素-PDT与血卟啉衍生物-PDT对食管癌细胞杀伤效应的比较研究[J].肿瘤防治杂志,2005,12(15):1139-1142.SHANG L Q,ZHOU N K,GU Y,et al.Comparative study on killing effect to esophageal cancer cell line between Hypocrellins B:photodynamic therapy and hematoporphyrin derivative-photodynamic therapy[J].Chin J Cancer Prev Treat,2005,12(15):1139-1142.
[25]殷红福.光敏化合物竹红菌素抑菌杀虫作用研究[D].杭州:浙江农林大学,2011.YIN H F.Research on fungicidal and insecticidal effect of lightactivated chemical hypocrellin[D].Hangzhou:Zhejiang A&FUniversity,2011
[26]HU Y Z,AN J Y,CHIANG L C.Studies on the photoinduced sulfonation of hypocrellins[J].J Photochem Photobiol A,1993,70(3):301-308.
[27]夏万林,张曼华,蒋丽金.竹红菌乙素与乙醇胺的作用[J].有机化学,1992,12(6):618-623.XIA W L,ZHANG M H,JIANG L J.The reaction between Hypocrellin B ethanolamine[J].Chin J Org Chem,1992,12(6):618-623.
[28]李琼瑶,肖绪玲,何慧珠,等.几种光敏剂光敏氧化效率的比较[J].感光科学与光化学,1989(2):59-63.LI Q Y,XIAO X L,HE H Z,et al.The comparison of photosensitized oxygenation efficiency for several kinds of sensitizers[J].Photograph Sci Photochem,1989(2):59-63.
[2]乔瑞.竹红菌素类光动力药物[J].阜阳师范学院学报(自然科学版),2009,26(03):28-33.QIAO R.Hypocrellin-based photodynamic sensitizers[J].Journal of Fuyang Teachers College(Natural Science),2009,26(03):28-33.
[3]万象义,陈远腾.一种新的光化学疗法药物:竹红菌甲素[J].科学通报,1981,26(11):1040-1042.WAN X Y,CHEN Y T.Hypocrellin A:a new drug for photochemotherapy[J].A Mon J Sci,1981,26(11):1040-1042.
[4]CHIO-SRICHAN S,OUDRHIRI N,BENNACEUR-GRISCELLI A,et al.Toxicity and phototoxicity of Hypocrellin A on malignant human cell lines,evidence of a synergistic action of photodynamic therapy with imatinib mesylate[J].J Photochem Photobiol B,2010,99(2):100-104.
[5]JIANG Y,LEUNG A W,WANG X N,et al.Inactivation of Staphylococcus aureus by photodynamic action of Hypocrellin B[J].Photodiagnosis Photodyn Ther,2013,10(4):600-606.
[6]崔荔群.探讨竹红菌素软膏治疗外阴白斑病的临床价值[J].中国卫生标准管理,2017,8(20):108-109.CUI L Q.Investigate the clinical value of bamboo mushroom ointment in the treatment of leukoplakia[J].China Health Standard Managent,2017,8(20):108-109.
[7]赵菊芬,李茜,杨柳风,等.聚焦超声联合竹红菌素软膏治疗外阴白色病变164例疗效观察[J].中国计划生育和妇产科,2012,4(1):61-63.ZHAO J F,LI X,YANG L F,et al.Clinical observation of curative effect on focused-ultrasound combined with hypocrellin-ointment to treat leukoplakia vulvae in 164 cases[J].Chin J Family Plann Gynecotokol,2012,4(1):61-63.
[8]JIN S,ZHOU L J,GU Z J,et al.A new near infrared photosensitizing nanoplatform containing blue-emitting up-conversion nanoparticles and Hypocrellin A for photodynamic therapy of cancer cells[J].Nanoscale,2013,5(23):11910-11918.
[9]HUDSON J B,ZHOU J,CHEN J,et al.Hypocrellin,from Hypocrella bambuase,is phototoxic to human immunodeficiency virus[J].Photochem Photobiol,1994,60(3):253-255.
[10]ZHANG Z Y,ZANG L Y,XU G R,et al.Characteristics of the primary reaction of Hypocrellin A photosensitization[J].Sci China B,1989,32(9):1063-1071.
[11]MAISCH T,BAIER J,FRANZ B,et al.The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria[J].Proc Natl Acad Sci USA,2007,104(17):7223-7228.
[12]赵学,李朋举,朱旭伟,等.竹红菌甲素对番茄灰霉病菌的抑制作用[J].农药学学报,2015,17(2):149-155.ZHAO X,LI P J,ZHU X W,et al.Inhibitory effect of Hypocrellin Aagainst Botrytis cinerea[J].Chin J Pestic Sci,2015,17(2):149-155.
[13]朱齐超,林海萍,李朋举,等.竹红菌甲素对18种植物病原菌的抑制作用[J].植物病理学报,2014,44(1):107-109.ZHU Q C,LIN H P,LI P J,et al.Inhibition of Hypocrellin A against18 pathogenic fungi[J].Acta Phytopathologica Sinica,2014,44(1):107-109.
[14]李平兰,贺稚非.食品微生物学实验原理与技术[M].北京:中国农业出版社,2005.LI P L,HE Z F.Experiments theories and techniques in food microbiology[M].Beijing:China Agriculture Press,2005.
[15]赵学.光活化农药竹红菌甲素对番茄灰霉的关键抑菌因子研究[D].杭州:浙江农林大学,2015.ZHAO X.Key factors of photoactivated pesticide Hypocrellin Aagainst Botrytis cinerea[D].Hangzhou:Zhejiang A&F University,2015.
[16]WHEATLEY R,HACKETT C,BRUCE A,et al.Effect of substrate composition on production of volatile organic compounds from Trichoderma spp.inhibitory to wood decay fungi[J].Int Biodeterior Biodegrad,1997,39(2-3):199-205.
[17]张凯,吴曼莉,辜柳霜,等.胶孢炭疽菌生防放线菌gz-8的鉴定及生物活性初步评价[J].农药学学报,2017,19(6):708-715.ZHANG K,WU M L,GU L S,et al.Identification and preliminary evaluation of a biocontrol actinomycete strain gz-8 against Colletotrichum gloeosporioides[J].Chin J Pestic Sci,2017,19(6):708-715.
[18]GUO X X,YANG X Q,YANG R Y,et al.Salicylic acid and methyl jasmonate but not rose bengal enhance artemisinin production through invoking burst of endogenous singlet oxygen[J].Plant Sci,2010,178(4):390-397.
[19]涂欢,徐涛,易永,等.羟基自由基引起神经细胞[Ca2+]i增高的机制和Ebselen的抑制作用[J].生物物理学报,1996,12(3):522-526.TU H,XU T,YI Y,et al.Mechanism of the enhancement effect of hydroxyl rdicals on[Ca2+]i in rat cerebral cortical neuronal cells and the inhibition of Ebselen[J].Acta Biophysica Sinica,1996,12(3):522-526.
[20]李学梅,李用芳,李培睿,等.安络小皮伞液体发酵过程中还原糖与pH值、菌丝干重及产量的关系[J].中国食用菌,2002,21(2):37-39.LI X M,LI Y F,LI P R,et al.The relations among reducing sugar,pH,dry weight of mycelium and production during the liquid fermentation of Marasmius androsaceus[J].Ed Fungi China,2002,21(2):37-39.
[21]周雪莹,熊文,孔令魁.核黄素光化学法对血浆中革兰阳性和阴性指示菌的减除作用和对血小板P选择蛋白表达的影响[J].中国实验血液学杂志,2010,18(04):1059-1062.ZHOU X Y,XIONG W,KONG L K.Effects of riboflavin combined with potosensitization on reduction of Gram-postitive and gramnegative indicating germs in plasma and P-selectin expression of apheresis platelet concentrates[J].Journal of Experimental Hematology,2010,18(04):1059-1062.
[22]李锦全,陈瑞川,蔡克瑕,等.核黄素光化学反应诱导人胃癌细胞凋亡[J].癌症,2003,22(3):253-256.LI J Q,CHEN R C,CAI K X,et al.Apoptosis of human gastric cancer cell induced by photochemical riboflavin[J].Chin J Cancer,2003,22(3):253-256.
[23]周乃康,尚立群,柳曦.竹红菌乙素-PDT与血卟啉衍生物-PDT对人肺癌细胞杀伤效应的比较研究[J].解放军医学杂志,2004,29(12):1076-1078.ZHOU N K,SHANG L Q,LIU X.Comparative study on the killing effect between Hypocrellins B:photodynamic therapy and hematoporphyrin derivative-photodynamic therapy on human lung cancer cells[J].Med J Chin PLA,2004,29(12):1076-1078.
[24]尚立群,周乃康,顾瑛,等.竹红菌乙素-PDT与血卟啉衍生物-PDT对食管癌细胞杀伤效应的比较研究[J].肿瘤防治杂志,2005,12(15):1139-1142.SHANG L Q,ZHOU N K,GU Y,et al.Comparative study on killing effect to esophageal cancer cell line between Hypocrellins B:photodynamic therapy and hematoporphyrin derivative-photodynamic therapy[J].Chin J Cancer Prev Treat,2005,12(15):1139-1142.
[25]殷红福.光敏化合物竹红菌素抑菌杀虫作用研究[D].杭州:浙江农林大学,2011.YIN H F.Research on fungicidal and insecticidal effect of lightactivated chemical hypocrellin[D].Hangzhou:Zhejiang A&FUniversity,2011
[26]HU Y Z,AN J Y,CHIANG L C.Studies on the photoinduced sulfonation of hypocrellins[J].J Photochem Photobiol A,1993,70(3):301-308.
[27]夏万林,张曼华,蒋丽金.竹红菌乙素与乙醇胺的作用[J].有机化学,1992,12(6):618-623.XIA W L,ZHANG M H,JIANG L J.The reaction between Hypocrellin B ethanolamine[J].Chin J Org Chem,1992,12(6):618-623.
[28]李琼瑶,肖绪玲,何慧珠,等.几种光敏剂光敏氧化效率的比较[J].感光科学与光化学,1989(2):59-63.LI Q Y,XIAO X L,HE H Z,et al.The comparison of photosensitized oxygenation efficiency for several kinds of sensitizers[J].Photograph Sci Photochem,1989(2):59-63.