isopropylstilbene发酵条件优化及其对灰葡萄孢的抑制作用
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摘要
isopropylstilbene (3, 5-二羟基-4-异丙基二苯乙烯)是发光杆菌Photohabdus temperata产生的一种二苯乙烯类化合物,具有多种生物活性。为促进isopropylstilbene在农业抗生素领域应用价值的开发,采用固相萃取、柱层析等技术从发光杆菌P. temperata SN 35的发酵液中分离纯化得到1个化合物,通过核磁共振氢谱对其结构进行了确认,并采用高效液相色谱测定了其纯度;通过Plackett-Burman试验和响应面法对发光杆菌SN 35摇瓶发酵培养条件进行了优化,并初步研究了所分离化合物对灰葡萄孢Botrytis cinerea的离体抑菌活性及对灰霉病的防治效果。结果表明:所分离的化合物为isopropylstilbene,纯度为93%;对发光杆菌SN 35摇瓶发酵培养条件进行优化后,isopropylstilbene的产量是优化前的2.22倍。离体抑菌试验结果表明:isopropylstilbene对灰葡萄孢的菌丝生长具有较好的抑制作用,其EC_(50)值为(9.17±0.17)μg/mL,但低于对照药剂百菌清[EC_(50)值为(0.90±0.08)μg/mL];在10μg/mL下,isopropylstilbene对灰葡萄孢孢子萌发的抑制率为63.01%。活体盆栽试验结果表明,在200μg/mL下,isopropylstilbene对黄瓜灰霉病的防治效果达92.19%,与百菌清的防效(97.63%)相当,对番茄灰霉病的防治效果为72.12%,略低于百菌清(81.61%)。本研究通过对isopropylstilbene发酵条件的优化,为其工业化生产奠定了理论基础。该化合物对灰霉病具有很好的防治效果,可为天然杀菌剂的研究与开发提供参考。
Isopropylstilbene(3, 5-dihydroxy-4-isopropyl stilbene) is a stilbene compound produced by Photohabdus temperata, which has various biological activities. In order to investigate the potential application of isopropylstilbene in the field of agricultural antibiotics, this compound was isolated from the fermentation broth of Photohabdus temperata SN35 by solid phase extraction and and purified by column chromatography. Its structure was confirmed by 1 H NMR and the purity was determined by high performance liquid chromatography. The shake flask fermentation conditions of the Photohabdus temperata SN35 were optimized by the Plackett-Burman test and the response surface method. The in vitro and in vivo antifungal activities of the compound against Botrytis cinerea were studied. The results showed that the isolated compound was isopropylstilbene with the purity of 93%. The yield of isopropylstilbene was 2.22 times higher than that before the optimization. The in vitro test results showed that the EC_(50) value of isopropylstilbene against the mycelial growth of B. cinerea was(9.17 ±0.17) μg/mL, which was lower than that of the control, chlorothalonil [EC_(50) value(0.90 ± 0.08) μg/mL].And at 10 μg/mL, the inhibition rate against spore germination was 63.01%. The results of pot experiments showed that the control effect of isopropylstilbene on cucumber gray mould was 92.19% at200 μg/mL, which was almost the same with the control effect of chlorothalonil(97.63%). The control effect on tomato gray mold was 72.12%, which slightly lower than that of chlorothalonil(81.61%). This study laid a theoretical foundation for the industrial production by optimizing the fermentation conditions of isopropylstilbene. The compound has a good control effect against gray mold which will be useful for the research and development of natural fungicides.
Isopropylstilbene(3, 5-dihydroxy-4-isopropyl stilbene) is a stilbene compound produced by Photohabdus temperata, which has various biological activities. In order to investigate the potential application of isopropylstilbene in the field of agricultural antibiotics, this compound was isolated from the fermentation broth of Photohabdus temperata SN35 by solid phase extraction and and purified by column chromatography. Its structure was confirmed by 1 H NMR and the purity was determined by high performance liquid chromatography. The shake flask fermentation conditions of the Photohabdus temperata SN35 were optimized by the Plackett-Burman test and the response surface method. The in vitro and in vivo antifungal activities of the compound against Botrytis cinerea were studied. The results showed that the isolated compound was isopropylstilbene with the purity of 93%. The yield of isopropylstilbene was 2.22 times higher than that before the optimization. The in vitro test results showed that the EC_(50) value of isopropylstilbene against the mycelial growth of B. cinerea was(9.17 ±0.17) μg/mL, which was lower than that of the control, chlorothalonil [EC_(50) value(0.90 ± 0.08) μg/mL].And at 10 μg/mL, the inhibition rate against spore germination was 63.01%. The results of pot experiments showed that the control effect of isopropylstilbene on cucumber gray mould was 92.19% at200 μg/mL, which was almost the same with the control effect of chlorothalonil(97.63%). The control effect on tomato gray mold was 72.12%, which slightly lower than that of chlorothalonil(81.61%). This study laid a theoretical foundation for the industrial production by optimizing the fermentation conditions of isopropylstilbene. The compound has a good control effect against gray mold which will be useful for the research and development of natural fungicides.
引文
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[8]BUSCATóE,BüTTNER D,BRüGGERHOFF A,et al.From a multipotent stilbene to soluble epoxide hydrolase inhibitors with antiproliferative properties[J].ChemMedChem,2013,8(6):919-923.
[9]BISSONNETTE R,POULIN Y,ZHOU Y,et al.Efficacy and safety of topical WBI-1001 in patients with mild to severe atopic dermatitis:results from a 12-week,multicentre,randomized,placebo-controlled double-blind trial[J].Br J Dermatol,2012,166(4):853-860.
[10]CHEN G,LI J,LIU W,et al.Novel bioactive diphenyl ethene compounds and their therapeutic applications:WO2004031117A1[P/OL].2004-04-15.http://www.google.com/patents/WO2004031117A1?cl=en.
[11]CHEN G,WEBSTER J M,LI J,et al.Anti-inflammatory and psoriasis treatment and protein kinase inhibition by hydroxy stilbenes and novel stilbene derivatives and analogues:US 007868047B2[P/OL].2011-01-11.http://www.google.com/patents/US7868047.
[12]HU K J,LI J X,LI B,et al.A novel antimicrobial epoxide isolated from larval Galleria mellonella infected by the ematode symbiont,Photorhabdus luminescens(Enterobacteriaceae)[J].Bioorg Med Chem,2006,14(13):4677-4681.
[13]PARK H B,CRAWFORD J M.Lumiquinone A,anα-aminomalonate-derived aminobenzoquinone from Photorhabdus luminescens[J].J Nat Prod,2015,78(6):1437-1441.
[14]CHALLINOR V L,BODE H B.Bioactive natural products from novel microbial sources[J].Ann N Y Acad Sci,2015,1354:82-97.
[15]LI J,CHEN G,WU H,et al.Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens[J].Appl Environ Microbiol,1995,61(12):4329-4333.
[16]KUMAR S N,SIJI J V,RAJASEKHARAN K N,et al.Bioactive stilbenes from a Bacillus sp.N strain associated with a novel rhabditid entomopathogenic nematode[J].Lett Appl Microbiol,2012,54(5):410-417.
[17]SHI D S,AN R,ZHANG W B,et al.Stilbene derivatives from Photorhabdus temperata SN259 and their antifungal activities against Phytopathogenic fungi[J].J Agric Food Chem,2017,65(1):60-65.
[18]KUMAR V,BHALLA A,RATHORE A S.Design of experiments applications in bioprocessing:concepts and approach[J].Biotechnol Prog,2014,30(1):86-99.
[19]MEAD R,PIKE D J.A review of response surface methodology from a biometric viewpoint[J].Biometrics,1975,31(4):803-851.
[20]吴文君.植物化学保护实验技术导论[M].西安:陕西科学与技术出版社,1987.WU W J.Introduction to experimental techniques of plant chemical protection[M].Xi'an:Shaanxi Scientific and Technical Press,1987.
[21]方中达.植病研究方法[M].3版.北京:中国农业出版社,1998:146-155.FANG Z D.Methodology on plant pathology research[M].3rd ed.Beijing:China Agriculture Press,1998:146-155.
[22]农药室内生物测定试验准则杀菌剂第9部分:抑制灰霉病菌试验叶片法:NY/T 1156.9-2008[S].北京:中国标准出版社,2008.Guideline for laboratory bioassay of pesticides.Part 9:detached leaf test for fungicide control Botrytis cinerea Pers:NY/T 1156.9-2008[S].Beijing:Standards Press of China,2008.
[23]农药田间药效试验准则(一)杀菌剂防治蔬菜灰霉病:GB/T17980.28-2000[S].北京:中国标准出版社,2000.Pesticide-Guidelines for the field efficacy trials(I):Fungicides against grey mould of vegetables:GB/T 17980.28-2000[S].Beijing:Standards Press of China,2000.
[24]PAUL V J,FRAUTSCHY S,FENICAL W,et al.Antibiotics in microbial ecology:Isolation and structure assignment of several new antibacterial compounds from the insect-symbiotic bacteria Xenorhabdus spp.[J].J Chem Ecol,1981,7(3):589-597.
[25]HIMABINDU M,POTUMARTHI R,JETTY A.Enhancement of gentamicin production by mutagenesis and non-nutritional stress conditions in Micromonospora echinospora[J].Process Biochemistry,2007,42(9):1352-1356.
[26]SHI YM,BODE H B.Chemical language and warfare of bacterial natural products in bacteria-nematode-insect interactions[J].Nat Prod Rep,2018,35(4):309-335.
[2]赵建江,王文桥,马志强,等.两种新杀菌剂对番茄灰霉病的作用方式及田间防效[J].中国蔬菜,2016(5):18-21.ZHAO J J,WANG W Q,MA Z Q,et al.Action mode and field control efficiency of two new fungicides against tomato gray mould[J].China Vegetables,2016(5):18-21.
[3]纪军建,张小风,韩秀英,等.8种杀菌剂对番茄灰霉病菌的毒力及田间番茄灰霉病菌对咯菌腈的敏感性[J].植物保护,2012,38(6):144-146.JI J J,ZHANG X F,HAN X Y,et al.Fungitoxicity of eight fungicides against Botrytis cinerea and the sensitivity of field strains to fludioxonil[J].Plant Prot,2012,38(6):144-146.
[4]张新虎,何静,沈慧敏.苍耳提取物对番茄灰霉病菌的抑制作用及抑菌机理初探[J].草业学报,2008,17(3):99-104.ZHANG X H,HE J,SHEN H M.Primary study on fungistasis and fungistatic mechanisms of Xanthium sibiricum on Botrytis cinerea[J].Acta Prataculturae Sinica,2008,17(3):99-104.
[5]FORST S,NEALSON K.Molecular biology of the symbioticpathogenic bacteria Xenorhabdus spp.[J].Microbiol Rev,1996,60(1):21-43.
[6]KRONENWERTH M,BRACHMANN A O,KAISER M,et al.Bioactive derivatives of isopropylstilbene from mutasynthesis and chemical synthesis[J].Chembiochem,2014,15(18):2689-2691.
[7]KRONENWERTH M,DAUTH C,KAISER M,et al.Facile synthesis of cyclohexanediones and dialkylresorcinols-bioactive natural products from entomopathogenic bacteria[J].Eur J Org Chem,2014,2014(36):8026-8028.
[8]BUSCATóE,BüTTNER D,BRüGGERHOFF A,et al.From a multipotent stilbene to soluble epoxide hydrolase inhibitors with antiproliferative properties[J].ChemMedChem,2013,8(6):919-923.
[9]BISSONNETTE R,POULIN Y,ZHOU Y,et al.Efficacy and safety of topical WBI-1001 in patients with mild to severe atopic dermatitis:results from a 12-week,multicentre,randomized,placebo-controlled double-blind trial[J].Br J Dermatol,2012,166(4):853-860.
[10]CHEN G,LI J,LIU W,et al.Novel bioactive diphenyl ethene compounds and their therapeutic applications:WO2004031117A1[P/OL].2004-04-15.http://www.google.com/patents/WO2004031117A1?cl=en.
[11]CHEN G,WEBSTER J M,LI J,et al.Anti-inflammatory and psoriasis treatment and protein kinase inhibition by hydroxy stilbenes and novel stilbene derivatives and analogues:US 007868047B2[P/OL].2011-01-11.http://www.google.com/patents/US7868047.
[12]HU K J,LI J X,LI B,et al.A novel antimicrobial epoxide isolated from larval Galleria mellonella infected by the ematode symbiont,Photorhabdus luminescens(Enterobacteriaceae)[J].Bioorg Med Chem,2006,14(13):4677-4681.
[13]PARK H B,CRAWFORD J M.Lumiquinone A,anα-aminomalonate-derived aminobenzoquinone from Photorhabdus luminescens[J].J Nat Prod,2015,78(6):1437-1441.
[14]CHALLINOR V L,BODE H B.Bioactive natural products from novel microbial sources[J].Ann N Y Acad Sci,2015,1354:82-97.
[15]LI J,CHEN G,WU H,et al.Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens[J].Appl Environ Microbiol,1995,61(12):4329-4333.
[16]KUMAR S N,SIJI J V,RAJASEKHARAN K N,et al.Bioactive stilbenes from a Bacillus sp.N strain associated with a novel rhabditid entomopathogenic nematode[J].Lett Appl Microbiol,2012,54(5):410-417.
[17]SHI D S,AN R,ZHANG W B,et al.Stilbene derivatives from Photorhabdus temperata SN259 and their antifungal activities against Phytopathogenic fungi[J].J Agric Food Chem,2017,65(1):60-65.
[18]KUMAR V,BHALLA A,RATHORE A S.Design of experiments applications in bioprocessing:concepts and approach[J].Biotechnol Prog,2014,30(1):86-99.
[19]MEAD R,PIKE D J.A review of response surface methodology from a biometric viewpoint[J].Biometrics,1975,31(4):803-851.
[20]吴文君.植物化学保护实验技术导论[M].西安:陕西科学与技术出版社,1987.WU W J.Introduction to experimental techniques of plant chemical protection[M].Xi'an:Shaanxi Scientific and Technical Press,1987.
[21]方中达.植病研究方法[M].3版.北京:中国农业出版社,1998:146-155.FANG Z D.Methodology on plant pathology research[M].3rd ed.Beijing:China Agriculture Press,1998:146-155.
[22]农药室内生物测定试验准则杀菌剂第9部分:抑制灰霉病菌试验叶片法:NY/T 1156.9-2008[S].北京:中国标准出版社,2008.Guideline for laboratory bioassay of pesticides.Part 9:detached leaf test for fungicide control Botrytis cinerea Pers:NY/T 1156.9-2008[S].Beijing:Standards Press of China,2008.
[23]农药田间药效试验准则(一)杀菌剂防治蔬菜灰霉病:GB/T17980.28-2000[S].北京:中国标准出版社,2000.Pesticide-Guidelines for the field efficacy trials(I):Fungicides against grey mould of vegetables:GB/T 17980.28-2000[S].Beijing:Standards Press of China,2000.
[24]PAUL V J,FRAUTSCHY S,FENICAL W,et al.Antibiotics in microbial ecology:Isolation and structure assignment of several new antibacterial compounds from the insect-symbiotic bacteria Xenorhabdus spp.[J].J Chem Ecol,1981,7(3):589-597.
[25]HIMABINDU M,POTUMARTHI R,JETTY A.Enhancement of gentamicin production by mutagenesis and non-nutritional stress conditions in Micromonospora echinospora[J].Process Biochemistry,2007,42(9):1352-1356.
[26]SHI YM,BODE H B.Chemical language and warfare of bacterial natural products in bacteria-nematode-insect interactions[J].Nat Prod Rep,2018,35(4):309-335.