银杏内生真菌Aspergillus oryzae YX-5抗肿瘤活性物质的分离与鉴定
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摘要
【背景】植物内生真菌是天然活性物质的重要来源。【目的】对一株具有抗肿瘤活性的银杏内生真菌米曲霉Aspergillus oryzae YX-5进行活性物质的分离与鉴定。【方法】将该菌株发酵培养后,发酵液经乙酸乙酯萃取,采用减压柱层析、葡聚糖凝胶柱层析和高效液相色谱分析,从其代谢产物中分离活性化合物,在分离过程中以MTT法跟踪检测分离到的各组分及纯化合物的抗肿瘤活性。【结果】从菌株YX-5的发酵产物中分离纯化得到4个化合物。经核磁共振和高分辨质谱分析,将其分别鉴定为羟基曲霉酸(1)、环(4-羟脯氨酸-苯丙氨酸)(2)、环(亮氨酸-苯丙氨酸)(3)和环(丙氨酸-苯丙氨酸)(4)。其中羟基曲霉酸对人宫颈癌HeLa细胞具有显著的细胞毒活性,其IC_(50)为1.07μg/mL。【结论】报道羟基曲霉酸在抗肿瘤方面的活性,表明米曲霉及羟基曲霉酸在抗肿瘤天然产物开发中具有一定的应用潜力。
[Background] Plant endophytic fungi are an important source of natural bioactive substances.[Objective] The active substance of an antitumor endophytic fungus Aspergillus oryzae YX-5 isolated from Ginkgo biloba was investigated. [Methods] First, strain YX-5 was fermented. Then, the culture broth was extracted with ethyl acetate. By using vacuum liquid chromatography, Sephadex gel chromatography and high performance liquid chromatography, the active compound was isolated from the crude extract and the antitumor activity was measured by MTT assay. [Results] Four pure compounds were isolated from the crude extract of strain YX-5. Based on the analysis of NMR and HR-ESI-MS, these compounds were identified as hydroxyaspergillic acid(1), cyclo(4-Hydroxypro-Phe)(2), Cyclo(Leu-Phe)(3) and Cyclo(Ala-Phe)(4). Hydroxyaspergillic acid exhibited significant cytotoxic activity against HeLa cell with IC_(50) of 1.07 μg/mL. [Conclusion] Hydroxyaspergillic acid exhibited antitumor activity, indicating that Aspergillus oryzae and hydroxyaspergillic acid might have application potential in antitumor natural products development.
[Background] Plant endophytic fungi are an important source of natural bioactive substances.[Objective] The active substance of an antitumor endophytic fungus Aspergillus oryzae YX-5 isolated from Ginkgo biloba was investigated. [Methods] First, strain YX-5 was fermented. Then, the culture broth was extracted with ethyl acetate. By using vacuum liquid chromatography, Sephadex gel chromatography and high performance liquid chromatography, the active compound was isolated from the crude extract and the antitumor activity was measured by MTT assay. [Results] Four pure compounds were isolated from the crude extract of strain YX-5. Based on the analysis of NMR and HR-ESI-MS, these compounds were identified as hydroxyaspergillic acid(1), cyclo(4-Hydroxypro-Phe)(2), Cyclo(Leu-Phe)(3) and Cyclo(Ala-Phe)(4). Hydroxyaspergillic acid exhibited significant cytotoxic activity against HeLa cell with IC_(50) of 1.07 μg/mL. [Conclusion] Hydroxyaspergillic acid exhibited antitumor activity, indicating that Aspergillus oryzae and hydroxyaspergillic acid might have application potential in antitumor natural products development.
引文
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[18]Qin L,Li BG,Guan JF,et al.Chemical study on Aspergillus sp.136[J].Chinese Journal of Applied&Environmental Biology,2007,13(1):66-68
[19]Si SY,Hong B,Jiang W,et al.Deoxyneohydroxyaspergillic acid and its prepn and use:CN,200410029901.X[P].2009-01-14(in Chinese)司书毅,洪斌,姜威,等.脱氧新羟曲霉酸及制备及其用途:中国,200410029901.X[P].2009-01-14
[20]Chen XW,Li CW,Hua W,et al.Metabolites of Aspergillus sp.16-02-1 isolated from a deep sea sediment and preliminary test of their antitumor and antifungal activities[J].Chinese Journal of Marine Drugs,2013,32(3):1-10(in Chinese)陈修文,李长伟,华威,等.深海来源曲霉16-02-1的代谢产物及其抗肿瘤抗真菌活性初步测评[J].中国海洋药物,2013,32(3):1-10
[2]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
[3]dos Santos Souza B,dos Santos TT.Endophytic fungi in economically important plants:ecological aspects,diversity and potential biotechnological applications[J].Journal of Bioenergy and Food Science,2017,4(2):113-126
[4]Singh B,Kaur P,Singh GRD,et al.Biology and chemistry of Ginkgo biloba[J].Fitoterapia,2008,79(6):401-418
[5]Zhang YQ,Chen AY,Li M,et al.Ginkgo biloba extract kaempferol inhibits cell proliferation and induces apoptosis in pancreatic cancer cells[J].Journal of Surgical Research,2008,148(1):17-23
[6]Li HQ,Gao Y,Li XJ.Advances of endophyte associated with Ginkgo biloba[J].Chinese Agricultural Science Bulletin,2014,30(25):1-6(in Chinese)李虎强,高义,李晓君.银杏内生真菌研究进展[J].中国农学通报,2014,30(25):1-6
[7]Miao L,Wang YY,Zhu L,et al.Endophytic fungi from four plant species:their isolation and antitumor activity[J].Microbiology China,2009,36(6):865-869(in Chinese)缪莉,王元元,朱磊,等.四种植物内生真菌的分离及其抗肿瘤活性的筛选[J].微生物学通报,2009,36(6):865-869
[8]Zhou HR,Luan HB,Wang H,et al.Optimization of the fermentation medium of an antitumor endophytic fungus Aspergillus oryzae YX-5 isolated from Ginkgo biloba[J].Microbiology China,2014,41(7):1358-1367(in Chinese)周惠茹,栾海彬,王慧,等.抗肿瘤银杏内生真菌Aspergillus oryzae YX-5发酵培养基的优化[J].微生物学通报,2014,41(7):1358-1367
[9]Zhang ZR,Kang JC,Lei BX,et al.Anti-tumor activity of extracts from Ginkgo endophytic fungi[J].Journal of Mountain Agriculture and Biology,2015,34(1):52-55,65(in Chinese)张峥嵘,康冀川,雷帮星,等.银杏内生真菌及发酵液粗提物抗肿瘤活性研究[J].山地农业生物学报,2015,34(1):52-55,65
[10]Sano Y,Ishikawa Y,Muramatsu S,et al.Study on the Koji mold producing substrate-possible application as a food supplement[J].Food Science and Technology Research,2007,13(1):13-19
[11]Liu H,Chen J,Deng ZW,et al.Secondary metabolites from marine derived fungus Penicillium terrestre[J].Journal of Chinese Pharmaceutical Sciences,2010,19(6):482-486
[12]Stark T,Hofmann T.Structures,sensory activity,and dose/response functions of 2,5-diketopiperazines in roasted cocoa nibs(Theobroma cacao)[J].Journal of Agricultural and Food Chemistry,2005,53(18):7222-7231
[13]Ma JY,Zhuge B,Fang HY,et al.Purification and characterization of proteases from Aspergillus oryzae[J].Microbiology China,2014,41(1):83-89(in Chinese)马俊阳,诸葛斌,方慧英,等.米曲霉蛋白酶的分离纯化及酶学性质研究[J].微生物学通报,2014,41(1):83-89
[14]Ge L,Xie JC,Wu T,et al.Purification and characterisation of a novelα-L-rhamnosidase exhibiting transglycosylating activity from Aspergillus oryzae[J].International Journal of Food Science&Technology,2017,52(12):2596-2603
[15]Liu L,Bao L,Wang L,et al.Asperorydines A-M:prenylated tryptophan-derived alkaloids with neurotrophic effects from Aspergillus oryzae[J].The Journal of Organic Chemistry,2018,83(2):812-822
[16]Zhu F,Hu GP,Chen X,et al.Studies on the secondary metabolites produced by mixed cultures of two epiphytic fungi isolated from a fruit of mangrove Avicennia marina[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2009,48(6):79-82(in Chinese)朱峰,胡谷平,陈忻,等.榄钱两株附生真菌混合培养次级代谢产物研究[J].中山大学学报:自然科学版,2009,48(6):79-82
[17]Qin L,Guan JF,Li BG,et al.New use of fumigacin type steroids in producing medicine for anti-drug fast bacteria:CN,200510021822.9[P].2007-04-11(in Chinese)秦岭,官家发,李伯刚,等.一种烟曲霉酸型甾体在制造抗耐药菌药物中的新用途:中国,200510021822.9[P].2007-04-11
[18]Qin L,Li BG,Guan JF,et al.Chemical study on Aspergillus sp.136[J].Chinese Journal of Applied&Environmental Biology,2007,13(1):66-68
[19]Si SY,Hong B,Jiang W,et al.Deoxyneohydroxyaspergillic acid and its prepn and use:CN,200410029901.X[P].2009-01-14(in Chinese)司书毅,洪斌,姜威,等.脱氧新羟曲霉酸及制备及其用途:中国,200410029901.X[P].2009-01-14
[20]Chen XW,Li CW,Hua W,et al.Metabolites of Aspergillus sp.16-02-1 isolated from a deep sea sediment and preliminary test of their antitumor and antifungal activities[J].Chinese Journal of Marine Drugs,2013,32(3):1-10(in Chinese)陈修文,李长伟,华威,等.深海来源曲霉16-02-1的代谢产物及其抗肿瘤抗真菌活性初步测评[J].中国海洋药物,2013,32(3):1-10