龙脑樟树叶内生真菌的分离及生物活性菌株的筛选鉴定
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摘要
旨在从龙脑樟树叶片中分离内生真菌并对其发酵产物的体外抑菌、抗肿瘤和抗氧化活性进行测定,筛选鉴定具有较强生物活性的菌株。对分离出的内生真菌,用K-b纸片法测定其抑菌活性,MTT法测定抗肿瘤活性,紫外分光光度法测定其抗氧化活性,ITS(Internal transcribed spacer)序列测序分析确定活性菌株的种属。结果显示,共从龙脑樟树叶片中分离得到内生真菌39株,其中具有对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、铜绿假单胞菌较强抑制活性的菌株7株。又从7株真菌中筛选出具有较强抗肿瘤活性和抗氧化活性的菌株ZS14,其对人乳腺癌细胞MCF-7抑制率达到73.00%,对DPPH清除率70.92%,对羟自由基清除率78.83%,过氧化氢清除率81.36%,据ITS测序结果将其命名为Diaporthales sp. ZS14。分离得到的菌株Diaporthales sp. ZS14具有较强的抑菌、抗肿瘤、抗氧化活性,可以作为药物开发的潜在来源进行进一步研究。
This work aims to isolate endophytic fungi from leaves of Cinnamomum camphora,to determine its anti-bacterial activity,anti-tumor activity and anti-oxidation activity in vitro,and to screen and identify strains with strong biological activity. On the isolated endophytic fungi,K-b disk method was used to determine its anti-bacterial activity,MTT assay to determine its anti-tumor activity,ultraviolet spectrophotometry to determine its anti-oxidation activity,and ITS(Internal transcribed spacer)to analyze and determine the species of active strains. Results showed that total 39 endophytic fungi were isolated from the leaves of C. camphora. Among them,there were 7 strains highly inhibiting Escherichia coli,Staphylococcus aureus,Bacillus subtilis and Pseudomonas aeruginosa. Further,the strain ZS14 with anti-tumor and anti-oxidation activities from the 7 strains was screened,its inhibition rate on human breast cancer cell MCF-7 reached 73.00%,clearance rate of DPPH was 70.92%,scavenging rate of hydroxyl radical was 78.83%,and clearance rate of hydrogen peroxide was 81.36%. With ITS sequencing results it was designated as Diaporthales sp. ZS14. In sum,the isolated strain Diaporthales sp. ZS14 had strong anti-bacterial,antitumor and anti-oxidation activities,and it can be used as potential sources for drug development in further study.
This work aims to isolate endophytic fungi from leaves of Cinnamomum camphora,to determine its anti-bacterial activity,anti-tumor activity and anti-oxidation activity in vitro,and to screen and identify strains with strong biological activity. On the isolated endophytic fungi,K-b disk method was used to determine its anti-bacterial activity,MTT assay to determine its anti-tumor activity,ultraviolet spectrophotometry to determine its anti-oxidation activity,and ITS(Internal transcribed spacer)to analyze and determine the species of active strains. Results showed that total 39 endophytic fungi were isolated from the leaves of C. camphora. Among them,there were 7 strains highly inhibiting Escherichia coli,Staphylococcus aureus,Bacillus subtilis and Pseudomonas aeruginosa. Further,the strain ZS14 with anti-tumor and anti-oxidation activities from the 7 strains was screened,its inhibition rate on human breast cancer cell MCF-7 reached 73.00%,clearance rate of DPPH was 70.92%,scavenging rate of hydroxyl radical was 78.83%,and clearance rate of hydrogen peroxide was 81.36%. With ITS sequencing results it was designated as Diaporthales sp. ZS14. In sum,the isolated strain Diaporthales sp. ZS14 had strong anti-bacterial,antitumor and anti-oxidation activities,and it can be used as potential sources for drug development in further study.
引文
[1]丁雄,苏健裕,石磊,等.龙脑樟鲜叶挥发油成分及其抗菌活性的研究[J].食品工业科技, 2012, 33(18):167-170.
[2]许韧.三株药用植物内生真菌次级代谢产物的初步研究[D].厦门:厦门大学, 2008.
[3]Hazalin NA, Ramasamy K, Lim SM, et al. Induction of apoptosis against cancer cell lines by four ascomycetes from Malaysian rainforest[J]. Phytomedicine, 2012, 19(7):609-612.
[4]谢少朵,陈向东,汪辉,等.雷公藤内生真菌的分离鉴定及活性研究[J].微生物学通报, 2018, 45(2):413-419.
[5]Amal HA, Abdessamad D, Julia K, et al. Fungal endophytes from higher plants:a prolific source of phytochemicals and other bioactive natural products[J]. Fungal Diversity, 2010, 41:1-16.
[6]Stierle A, Strobel G, Stierle D. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific Yew[J].Science, 1993, 260(5105):214-216.
[7]Rehman S, Shawl SA, Kour AR, et al. An endophytic Neurospora sp.form Nophatodytes foetida producing camptothecin[J]. Applied Biochemistry and Microbiology, 2008, 44(2):203-209.
[8]Isaka M, Yangchuma A, Rachtawee P, et al. Dihydronaphthalenones form the endophytic fungus Botryosphaeria sp. BBC 8200[J].Phytochem Letter, 2009, 72(9):1712-1715.
[9]KharwarRN,MauryaAL,VermaVC,etal.Diversityand antimicrobial activity of endophytic fungal community isolated from medicinal plant Cinnamomum camphora[J]. Proc Natl Acad Sci USA, 2012, 82:557-565.
[10]游玲,王涛,等. 78株油樟内生真菌发酵产物的挥发性组分分析[J].西北农林科技大学学报, 2009, 37(9):193-198.
[11]Xiong ZQ, Yang YY, Zhao N, et al. Diversity of endophytic fungi and screening of fungal paclitaxel producer from Anglojap yew,taxus x media[J]. BMC Microbiology, 2013, 13:71-75.
[12]StefanakisMK,TouloupakisE,AnastasopoulosE,etal.Antibacterial activity of essential oils from plants of the genus Origanum[J]. Food Control, 2013, 34:539-546.
[13]Vasundhara M, Baranwal M, Kumar A. Fusarium tricinctum, An endophytic fungus exhibits cell growth inhibition and antioxidant activity[J]. Ind J Microbiol, 2016, 56(4):433-438.
[14]Concepcion SM, Jose AL, Fulgencio SC. A procedure to measure the antiradical efficiency of polyphenols[J]. Science of Food and Agriculture, 1998, 76:270-276.
[15]Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes[J]. Phytochemistry, 1989, 28(4):1057-1060.
[16]Du X, Mu H, Zhou S, et al. Chemical analysis and antioxidant activity of polysaccharides extracted from inonotus obliquus sclerotia[J]. Int J Biol Macromol, 2013, 62:691-696.
[17]Benitez AU, Romero YMM, Fernandez RES, et al. Phytotoxic and antimicrobial activity of volatile and semi-volatile organic compounds from the endophyte Hypoxylon anthochroum strain Blaci isolated from Bursera lancifolia(Burseraceae)[J]. J Appl Microbiol, 2016, 121:380-400.
[18]郑有坤,刘凯,等.药用植物内生放线菌多样性及天然活性物质研究进展[J].中草药, 2014, 45(14):2089-2099.
[19]程龙.南方红豆杉中内共生真菌的分离、鉴定及耦合培养初探[D].无锡:江南大学, 2006.
[20]姜建昌,曹雷,姚瑜洁,等.冰片的临床应用研究概况[J].现代中西医结合杂志, 2013, 22(2):221-223.
[21]郑元,王歆,陈海涛,等.樟树研究进展与展望[J].江西科学,2014, 32(5):640-645.
[22]王沫,陈玉婵,孙章华,等.广藿香内生真菌Diaporthe longicolla A616细胞毒活性次级代谢产物研究[J].中国中药杂志, 2016, 46(11):2112-2117.
[23]Sousa JPB, Aguilar MM, Arnold AE, et al. Chemical constituents and their antibacterial activity from the tropical endophytic fungus Diaporthe sp. F2934[J]. J Appl Microbiol, 2016, 120:1501-1508.
[24]Ding XW, Liu KH, Deng BW, et al. Isolation and characterization of endophytic fungi from Camptotheca acumiate[J]. W J Microbiol Biotechnol, 2013, 29:1831-1838.
[2]许韧.三株药用植物内生真菌次级代谢产物的初步研究[D].厦门:厦门大学, 2008.
[3]Hazalin NA, Ramasamy K, Lim SM, et al. Induction of apoptosis against cancer cell lines by four ascomycetes from Malaysian rainforest[J]. Phytomedicine, 2012, 19(7):609-612.
[4]谢少朵,陈向东,汪辉,等.雷公藤内生真菌的分离鉴定及活性研究[J].微生物学通报, 2018, 45(2):413-419.
[5]Amal HA, Abdessamad D, Julia K, et al. Fungal endophytes from higher plants:a prolific source of phytochemicals and other bioactive natural products[J]. Fungal Diversity, 2010, 41:1-16.
[6]Stierle A, Strobel G, Stierle D. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific Yew[J].Science, 1993, 260(5105):214-216.
[7]Rehman S, Shawl SA, Kour AR, et al. An endophytic Neurospora sp.form Nophatodytes foetida producing camptothecin[J]. Applied Biochemistry and Microbiology, 2008, 44(2):203-209.
[8]Isaka M, Yangchuma A, Rachtawee P, et al. Dihydronaphthalenones form the endophytic fungus Botryosphaeria sp. BBC 8200[J].Phytochem Letter, 2009, 72(9):1712-1715.
[9]KharwarRN,MauryaAL,VermaVC,etal.Diversityand antimicrobial activity of endophytic fungal community isolated from medicinal plant Cinnamomum camphora[J]. Proc Natl Acad Sci USA, 2012, 82:557-565.
[10]游玲,王涛,等. 78株油樟内生真菌发酵产物的挥发性组分分析[J].西北农林科技大学学报, 2009, 37(9):193-198.
[11]Xiong ZQ, Yang YY, Zhao N, et al. Diversity of endophytic fungi and screening of fungal paclitaxel producer from Anglojap yew,taxus x media[J]. BMC Microbiology, 2013, 13:71-75.
[12]StefanakisMK,TouloupakisE,AnastasopoulosE,etal.Antibacterial activity of essential oils from plants of the genus Origanum[J]. Food Control, 2013, 34:539-546.
[13]Vasundhara M, Baranwal M, Kumar A. Fusarium tricinctum, An endophytic fungus exhibits cell growth inhibition and antioxidant activity[J]. Ind J Microbiol, 2016, 56(4):433-438.
[14]Concepcion SM, Jose AL, Fulgencio SC. A procedure to measure the antiradical efficiency of polyphenols[J]. Science of Food and Agriculture, 1998, 76:270-276.
[15]Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes[J]. Phytochemistry, 1989, 28(4):1057-1060.
[16]Du X, Mu H, Zhou S, et al. Chemical analysis and antioxidant activity of polysaccharides extracted from inonotus obliquus sclerotia[J]. Int J Biol Macromol, 2013, 62:691-696.
[17]Benitez AU, Romero YMM, Fernandez RES, et al. Phytotoxic and antimicrobial activity of volatile and semi-volatile organic compounds from the endophyte Hypoxylon anthochroum strain Blaci isolated from Bursera lancifolia(Burseraceae)[J]. J Appl Microbiol, 2016, 121:380-400.
[18]郑有坤,刘凯,等.药用植物内生放线菌多样性及天然活性物质研究进展[J].中草药, 2014, 45(14):2089-2099.
[19]程龙.南方红豆杉中内共生真菌的分离、鉴定及耦合培养初探[D].无锡:江南大学, 2006.
[20]姜建昌,曹雷,姚瑜洁,等.冰片的临床应用研究概况[J].现代中西医结合杂志, 2013, 22(2):221-223.
[21]郑元,王歆,陈海涛,等.樟树研究进展与展望[J].江西科学,2014, 32(5):640-645.
[22]王沫,陈玉婵,孙章华,等.广藿香内生真菌Diaporthe longicolla A616细胞毒活性次级代谢产物研究[J].中国中药杂志, 2016, 46(11):2112-2117.
[23]Sousa JPB, Aguilar MM, Arnold AE, et al. Chemical constituents and their antibacterial activity from the tropical endophytic fungus Diaporthe sp. F2934[J]. J Appl Microbiol, 2016, 120:1501-1508.
[24]Ding XW, Liu KH, Deng BW, et al. Isolation and characterization of endophytic fungi from Camptotheca acumiate[J]. W J Microbiol Biotechnol, 2013, 29:1831-1838.