HPLC法测定Streptomyces sp.发酵液中bafilomycin A1的含量
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摘要
目的:建立放线菌Streptomyces sp.发酵液中bafilomycin A1的鉴别及含量测定的方法。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;采用高效液相色谱法(HPLC法)进行含量测定,使用Ultimate LP-C_(18)色谱柱(4.6 mm×250 mm,5μm),流动相为甲醇-水(85∶15),流速1.0 mL·min~(-1),检测波长247 nm,柱温40℃。结果:bafilomycin A1质量浓度在18.75～600μg·mL~(-1)范围内线性良好,相关系数为1.0(n=6);平均回收率为101.4%,RSD为1.3%(<2.0%);方法的精密度RSD为1.2%。3批发酵样品中bafilomycin A1含量分别为183.69、196.29、198.63μg·mL~(-1),均在有效测定范围内。结论:本方法可以用于bafilomycin A1发酵、提炼及纯化过程的含量测定。
Objective:To develop an HPLC method for the determination of bafilomycin A1 in Streptomyces sp.fermentation broth.Methods:HPLC-Q-TOF-MS was used to qualitatively identify bafilomycin A1 in fermentation broth.HPLC was used to quantitatively determine the content of bafilomycin A1 in Streptomyces sp.fermentation broth.The separation was achieved on a Ultimate LP-C_(18)( 4.6 mm×250 mm,5 μm)column at 40 ℃ and detected at247 nm,the mobile phase consisted of methanol and water(85∶15)at a flow rate of 1.0 mL·min~(-1).Results:The calibration curve was linear over the range of 18.75-600 μg·mL~(-1) with the correlation coefficient of 1.0(n=6).The average recovery of bafilomycin A1 was 101.4%(RSD=1.3%).Bafilomycin A1 contents in three batches of samples were 183.69,196.29,198.63 μg·m L-1 within the effective measurement range,respectively.Conclusion:The method is suitable for the determination of bafilomycin A1 in fermentation broth.
Objective:To develop an HPLC method for the determination of bafilomycin A1 in Streptomyces sp.fermentation broth.Methods:HPLC-Q-TOF-MS was used to qualitatively identify bafilomycin A1 in fermentation broth.HPLC was used to quantitatively determine the content of bafilomycin A1 in Streptomyces sp.fermentation broth.The separation was achieved on a Ultimate LP-C_(18)( 4.6 mm×250 mm,5 μm)column at 40 ℃ and detected at247 nm,the mobile phase consisted of methanol and water(85∶15)at a flow rate of 1.0 mL·min~(-1).Results:The calibration curve was linear over the range of 18.75-600 μg·mL~(-1) with the correlation coefficient of 1.0(n=6).The average recovery of bafilomycin A1 was 101.4%(RSD=1.3%).Bafilomycin A1 contents in three batches of samples were 183.69,196.29,198.63 μg·m L-1 within the effective measurement range,respectively.Conclusion:The method is suitable for the determination of bafilomycin A1 in fermentation broth.
引文
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[12]张欣,杨煌建,周剑,等.链霉菌BAF-0711产生的抗肿瘤活性成分的分离纯化和结构鉴定[J].天然产物研究与开发,2016,28(5):696ZHANG X,YANG HJ,ZHOU J,et al.Isolation,purification and structural identification of component with antitumor activity produced by Streptomyces strain[J].Nat Prod Res Dev,2016,28(5):696
[2]WEERNER G,HAGENMAIER H,DRAUTZ H,et al.Metabolic products of microorganisms.224.Bafilomycins,a new group of macrolide antibiotics.Production,isolation,chemical structure and biological activity[J].J Antibiot(Tokyo),1984,37(2):110
[3]KRETSCHMERE A,DORGERLOH M,DEEG M,et al.The structures of novel insecticidal macrolides bafilomycins D and E,and oxohygrolidin[J].Agric Biol Chem,1985,49(8):2509
[4]UYEDA M,KONDO KI,ITO A,et al.New antiherpetic agent produced by Streptomyces sp.strain No.758[J].J Antibiot,1995,48(11):1234
[5]YOSHIMORI T,YAMAMOTO A,MORIYAMA Y,et al.Bafilomycin A1,a specific inhibitor of vacuolar-type H+-ATPase,inhibits acidification and protein degradation in lysosomes of cultured cells[J].J Biol Chem,1991,266(26):17707
[6]TAKEUCHI M,ASHIHARA E,YAMAZAKI Y,et al.Bafilomycin A1 A effectively induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells[J].Cancer Sci,2011,102(3):591
[7]CARR G,WILLIAMS DE,DIAZ-MARRERO AR,et al.Bafilomycins produced in culture by Streptomyces spp.isolated from marine habitats are potent inhibitors of autophagy[J].J Nat Prod,2010,73(3):422
[8]SUN-WADA GH,WADA Y.Vacuolar-type proton pump ATPases:acidification and pathological relationships[J].Histol Histopathol,2013,28(7):805
[9]YU Z,ZHAO LX,JIANG CL,et al.Bafilomycins produced by an endophytic actinomycete Streptomyces sp.YIM56209[J].JAntibiot,2011,64(1):159
[10]魏刚,苏超,张道敬,等.海洋放线菌Y12-26代谢产物bafilomycins分离纯化及结构鉴定[J].中国抗生素杂志,2011,36(8):571WEI G,SU C,ZHANG DJ,et al.Isolation,purification and structure identification of secondary metabolites produced by marine actinomycete Y12-26[J].Chin J Antibiot,2011,36(8):571
[11]杨巍民,斯聪聪,杨星,等.海洋放线菌Y-0117农用活性代谢产物的研究[J].化学与生物工程,2013,30(1):1672YANG WM,SI CC,YANG X,et al.Study of agro-active metabolites of marine Actinomycete Y-0117[J].Chem Bioeng,2013,30(1):1672
[12]张欣,杨煌建,周剑,等.链霉菌BAF-0711产生的抗肿瘤活性成分的分离纯化和结构鉴定[J].天然产物研究与开发,2016,28(5):696ZHANG X,YANG HJ,ZHOU J,et al.Isolation,purification and structural identification of component with antitumor activity produced by Streptomyces strain[J].Nat Prod Res Dev,2016,28(5):696