HPLC法测定Streptomyces sp.BOR-0331发酵液中疏螺旋体素的含量
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摘要
目的:建立放线菌Streptomyces sp. BOR-0331发酵液中疏螺旋体素含量测定的方法。方法:采用高效液相色谱-四极杆飞行时间串联质谱(HPLC-Q-TOF-MS)分析技术进行定性鉴别;高效液相色谱(HPLC)法进行含量测定,采用Agilent ZORBAX SB-C_(18)色谱柱(5μm,4.6 mm×250 mm),流动相为乙腈-0.1%三氟乙酸溶液(50∶50),流速为1.5 mL·min~(-1),检测波长为257 nm。结果:疏螺旋体素浓度范围在12.5～200μg·mL~(-1)线性良好,相关系数为0.9997(n=5);平均回收率为102.8%(RSD为0.86%),精密度RSD为1.3%;3批发酵液中疏螺旋体素的含量分别为61.18、69.92、61.08μg·mL~(-1)。结论:方法准确可靠,可以用于疏螺旋体素发酵、提炼及纯化过程的含量测定。
Objective:To develop an HPLC method for the determination of borrelidinin in fermentation broth of Streptomyces sp.BOR-0331.Methods:HPLC-Q-TOF-MS was used to qualitatively determine borrelidin in fermentation broth,and HPLC was used to quantitatively determine the content of borrelidin.Sample was achieved on a ZORBAX SB-C_(18)(5 μm,4.6 mm×250 mm)column and detected at 257 nm.The mobile phase consisted of acetonitrile and 0.1% trifluoroacetic acid solution(50∶50,v/v) at a flow rate of 1.5 mL·min~(-1).Results:The calibration curve was linear over the range of 12.5-200 μg·m L~(-1) with the correlation of 0.999 7(n=5).The average recovery of borrelidin was 102.8%(RSD=0.86%).The contents of borrelidinin in three batches of fermentation broths were 61.18,69.92 and 61.08 μg·m L~(-1),respectively.Conclusion:The method is accurate,simple with good reproducibility,which is suitable for the determination of borrelidinin in fermentation broth.
Objective:To develop an HPLC method for the determination of borrelidinin in fermentation broth of Streptomyces sp.BOR-0331.Methods:HPLC-Q-TOF-MS was used to qualitatively determine borrelidin in fermentation broth,and HPLC was used to quantitatively determine the content of borrelidin.Sample was achieved on a ZORBAX SB-C_(18)(5 μm,4.6 mm×250 mm)column and detected at 257 nm.The mobile phase consisted of acetonitrile and 0.1% trifluoroacetic acid solution(50∶50,v/v) at a flow rate of 1.5 mL·min~(-1).Results:The calibration curve was linear over the range of 12.5-200 μg·m L~(-1) with the correlation of 0.999 7(n=5).The average recovery of borrelidin was 102.8%(RSD=0.86%).The contents of borrelidinin in three batches of fermentation broths were 61.18,69.92 and 61.08 μg·m L~(-1),respectively.Conclusion:The method is accurate,simple with good reproducibility,which is suitable for the determination of borrelidinin in fermentation broth.
引文
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[14]李周,赵圣印.娄彻氏链霉菌ATCC10739产抗生素Borrelidin发酵条件优化及其分离纯化[J].微生物学通报,2011,38(7):1051LI Z,ZHAO SY.Borrelidin production by Streptomycete rochei ATCC10739:optimization of fermentation,separation and purification conditions[J].Microbiol China,2011,38(7):1051
[15]周剑,陈宏,杨煌建,等.响应面法优化疏螺旋体素发酵培养基[J].中国抗生素杂志,2016,41(5):344ZHOU J,CHEN H,YANG HJ,et al.Optimization of fermentation medium for production of borrelidin by response surface methodology[J].Chin J Antibiot,2016,41(5):344
[2]SAISIVAM S,BHIKSHAPATHI DVRN,KRISHNAVENI J,et al.Isolation of borrelidin from Streptomyces californicus-An Indian soil isolate[J].Indian J Biotechnol,2008,7(3):349
[3]FANG P,YU X,JEONG SJ,et al.Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase[J].Nat Commun,2015,6(1):11
[4]LUMB M,MACEY PE,SPYVEE J,et al.Isolation of vivomycin and borrelidin,two antibiotics with anti-viral activity,from a species of Streptomyces(C2989)[J].Nature,1965,206(981):263
[5]GAO YM,WANG XJ,ZHANG J,et al.Borrelidin,a potent antifungal agent:insight into the antifungal mechanism against Phytophthora sojae[J].J Agric Food Chem,2012,60(39):9874
[6]LIU CX,ZHANG J,WANG XJ,et al.Antifungal activity of borrelidin produced by a Streptomyces strain isolated from soybean[J].J Agric Food Chem,2012,60(5):1251
[7]SUGAWARA A,TANAKA T,HIROSR T,et al.Borrelidin analogues with antimalarial activity:design,synthesis and biological evaluation against Plasmodium falciparum parasites[8].Bioorg Med Chem Lett,2013,23(8):2302
[9]WAKABAYASHI T,KAGEYAMA R,NARUSE N,et al.Borrelidin is an angiogenesis inhibitor;Disruption of angiogenic capillary vessels in a rat aorta matrix culture model[J].J Antibiot,1997,50(8):671
[10]KAWAMURA T,LIU D,TOWLE MJ,et al.Anti-angiogenesis effects of borrelidin are mediated through distinct pathways:threonyl-tRNA synthetase and caspases are independently involved in suppression of proliferation and induction of apoptosis in endothelial cells[J].J Antibiot(Tokyo),2003,56(8):709
[11]BHIKSHAPATHI DVRN,KUMAR YS,RAO YM,et al.Borrelidin:a prospective drug[J].Indian J Biotechnol,2010,9(1):18
[12]NAGAMITSU T,TAKANO D,MARUMOTO K,et al.Total synthesis of borrelidin[J].J Org Chem,2007,72(8):2744
[13]萨拉斯J A,门德斯C,奥拉诺C,等.产生疏螺旋体素的聚酮化合物合酶及其用途:CN1732264A[P].2003-12-24SALAS JA,MéNDEZ C,OLANO C,et al.Borrelidin-producing polyketide synthase and its use:CN1732264A[P].2003-12-24
[14]李周,赵圣印.娄彻氏链霉菌ATCC10739产抗生素Borrelidin发酵条件优化及其分离纯化[J].微生物学通报,2011,38(7):1051LI Z,ZHAO SY.Borrelidin production by Streptomycete rochei ATCC10739:optimization of fermentation,separation and purification conditions[J].Microbiol China,2011,38(7):1051
[15]周剑,陈宏,杨煌建,等.响应面法优化疏螺旋体素发酵培养基[J].中国抗生素杂志,2016,41(5):344ZHOU J,CHEN H,YANG HJ,et al.Optimization of fermentation medium for production of borrelidin by response surface methodology[J].Chin J Antibiot,2016,41(5):344