苦蘵内生真菌Trichoderma harzianum的次级代谢产物
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摘要
目的:研究茄科酸浆属植物苦蘵(Physalis angulata)的内生真菌哈茨木霉(Trichoderma harzianum)的次级代谢产物。方法:菌株采用震荡培养箱扩大培养,得到的菌液用乙酸乙酯等体积萃取3次,菌丝体则以丙酮浸泡超声,回收溶剂后将所得提取物合并,再经液相-质谱联用技术,开放ODS反相柱色谱,LH-20羟丙基葡聚糖凝胶柱色谱(sephadex LH-20),高效液相色谱等方法进行成分的分析与分离纯化活性次级代谢产物。利用其理化性质和光谱数据(~1H-NMR,~(13)C-NMR)等鉴定化合物结构。采用噻唑蓝(MTT)法测定化合物的细胞毒性。结果:从苦蘵内生真菌哈茨木霉中分离得到10个次级代谢产物,分别鉴定为destruxin A_2(1),destruxin B_2(2),环(亮-脯)二肽(3),环(苯丙-脯)二肽(4),cyclonerodiol(5),brevianamide F(6),N-乙酰色胺(7),9-羟基-邻苯二甲酸二异丁基酯(8),5-羟基-3-羟甲基-2-甲基-7-甲氧基色酮(9)和苯丙酸(10)。细胞毒性试验结果显示,化合物1～10对肺癌细胞株A549均未表现出显著的细胞毒性(半抑制浓度≥20 mg·L~(-1))。结论:化合物1～10均首次从哈茨木霉中分离得到。
Objective: To study the secondary metabolites of the endophytic fungus Trichoderma harzianum,which was isolated from Physalis angulata. Method: The strains were cultured in a big-scale by oscillating incubator. Then the obtained culture liquid and mycelia were extracted by EtOAC and actone,respectively. The extracts were integrated after recovery of solvents,and then the active secondary metabolites were isolated and purified by comprehensive use of open ODS flash column,sephadex LH-20,HPLC,and LC-MS analysis techniques. Their structures were identified according to their physico-chemical properties and nuclear magnetic resonance( NMR) data. The cytotoxicities of the compounds were determined by methyl thiazolyl tetrazolium( MTT) assay. Result: Ten compounds isolated from T. harzianum were identified as destruxin A2( 1),destruxin B2( 2),3-isobutyl-pyrrolopiperazine-2,5-dione [cyclo( leu-pro dipeptide) ]( 3),cyclo( Phen-pro) dipeptide( 4),cyclonerodiol( 5),brevianamide F( 6),N-acetyltryptamine( 7),9-hydroxyl-( 2-methylpropyl) isobutyl phthalate( 8),5-hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone( 9),and phenylpropionic acid( 10). Compounds 1-10 didn't exhibit significant cytotoxicity to lung cancer cell line A549 in the MTT assay( IC50≥20 mg·L~(-1)). Conclusion: All compounds were isolated from T. harzianum for the first time.
Objective: To study the secondary metabolites of the endophytic fungus Trichoderma harzianum,which was isolated from Physalis angulata. Method: The strains were cultured in a big-scale by oscillating incubator. Then the obtained culture liquid and mycelia were extracted by EtOAC and actone,respectively. The extracts were integrated after recovery of solvents,and then the active secondary metabolites were isolated and purified by comprehensive use of open ODS flash column,sephadex LH-20,HPLC,and LC-MS analysis techniques. Their structures were identified according to their physico-chemical properties and nuclear magnetic resonance( NMR) data. The cytotoxicities of the compounds were determined by methyl thiazolyl tetrazolium( MTT) assay. Result: Ten compounds isolated from T. harzianum were identified as destruxin A2( 1),destruxin B2( 2),3-isobutyl-pyrrolopiperazine-2,5-dione [cyclo( leu-pro dipeptide) ]( 3),cyclo( Phen-pro) dipeptide( 4),cyclonerodiol( 5),brevianamide F( 6),N-acetyltryptamine( 7),9-hydroxyl-( 2-methylpropyl) isobutyl phthalate( 8),5-hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone( 9),and phenylpropionic acid( 10). Compounds 1-10 didn't exhibit significant cytotoxicity to lung cancer cell line A549 in the MTT assay( IC50≥20 mg·L~(-1)). Conclusion: All compounds were isolated from T. harzianum for the first time.
引文
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[13] Asiri I A M,Badr J M,Youssef D T A. Penicillivinacine,antimigratory diketopiperazinealkaloid from the marinederived fungus Penicillium vinaceum[J]. Phytochem Lett,2015,13:53-58.
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[15] LI G,ZHANG Q,WANG Y. Chemical constituents from roots of Peuraria labata[J]. Chin J Chin Mat Med,2010,35(23):3156-3160.
[16] Yukiko Takenaka,Takao Tanahashi,Naotaka Nagakura,et al. 2,3-Dialkylchromones from mycobiont cultures of the Lichen Graphis scripta[J]. Heterocycles,2000,53(7):1589-1593.
[17]刘震,赵静怡,李勇,等.娃儿藤内生真菌Diaporthe sp.次生代谢产物研究[J].中国中药杂志,2018,43(14):2944-2949.
[18]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,2016,41(11):2112-2117.
[19]马宗敏,刘佳段,绪红,等.黄芩苷发酵转化成黄芩素和千层纸素A的黄芩内生真菌的筛选及鉴定[J].中国实验方剂学杂志,2019,25(9):166-171.
[20]佟永春,刘雅茹,何蕾,等.水母雪莲内生真菌Alterraria sp.的次级代谢产物鉴定[J].中国实验方剂学杂志,2017,23(7):64-67.
[2]吕长鑫,王晶晶,杨玲辉,等.药膳两用植物灯笼果开发利用的研究进展[J].食品工业科技,2013,34(10):391-394.
[3] Ismail N,Alam M. A novel cytotoxic flavonoid glycoside from Physalis angulata[J]. Fitoterapia,2001,72(6):676-679.
[4] JIN Z, Mashuta M S, Stolowich N J, et al.Physangulidines A,Band C:three newantiproliferative withanolides from Physalis angulata L.[J]. Org Lett,2012,14(5):1230-1233.
[5] Schulz B,Boyle C,Draeger S,et al. Endophytic fungi:a source of novel biologically active secondary metabolites[J]. Mycol Res,2002,106(9):996-1004.
[6] Strobel G,Daisy B,Castillo U,et al. Natural products from endophytic microorganisms[J]. J Nat Prod,2004,67(2):257-268.
[7]吕黎,许丽媛,罗志威,等.哈茨木霉生物防治研究进展[J].湖南农业科学,2013(17):92-95.
[8] Sandeep G,Donald W R,Renwick J A. Insecticida I cyclodepsipeptides from Metarhizium anisopliae[J]. J Chem Soc Perkin Trans,1989(12):2347-2357.
[9] SUN S,TIAN L,WANG Y N,et al. A novel natural product from the fermentation liquid of marine fungus Trichoderma atroviride G20-12[J]. Asian J Trad Med,2009,4(3):123-127.
[10] Jayatilake G S,Thornton M P,Leonard A C,et al.Metabolites from an antarctic sponge-associated bacterium,Pseudomonas aeruginosa[J]. J Nat Prod,1996,59(3):293-296.
[11] Furtado N A J C,Pupo M T,Carvalho I,et al.Diketopiperazines produced by an Aspergillus fumigatus Brazilian strain[J]. J Braz Chem Soc,2005,16(6B):1448-1453.
[12]赵丹奇,吴泽宏,刘东,等.海绵共附生真菌Hansfordia sp.次级代谢产物的分离与鉴定[J].沈阳药科大学学报,2013,30(5):342-345.
[13] Asiri I A M,Badr J M,Youssef D T A. Penicillivinacine,antimigratory diketopiperazinealkaloid from the marinederived fungus Penicillium vinaceum[J]. Phytochem Lett,2015,13:53-58.
[14]罗雄明,漆淑华,田新朋,等.海洋放线菌Salinispora pacifica发酵液的化学成分研究[J].中草药,2009,40(11):1710-1712.
[15] LI G,ZHANG Q,WANG Y. Chemical constituents from roots of Peuraria labata[J]. Chin J Chin Mat Med,2010,35(23):3156-3160.
[16] Yukiko Takenaka,Takao Tanahashi,Naotaka Nagakura,et al. 2,3-Dialkylchromones from mycobiont cultures of the Lichen Graphis scripta[J]. Heterocycles,2000,53(7):1589-1593.
[17]刘震,赵静怡,李勇,等.娃儿藤内生真菌Diaporthe sp.次生代谢产物研究[J].中国中药杂志,2018,43(14):2944-2949.
[18]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,2016,41(11):2112-2117.
[19]马宗敏,刘佳段,绪红,等.黄芩苷发酵转化成黄芩素和千层纸素A的黄芩内生真菌的筛选及鉴定[J].中国实验方剂学杂志,2019,25(9):166-171.
[20]佟永春,刘雅茹,何蕾,等.水母雪莲内生真菌Alterraria sp.的次级代谢产物鉴定[J].中国实验方剂学杂志,2017,23(7):64-67.
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