剧毒卡罗藻GM5株中标志性毒素4,5-dihydro-KmTx2的色谱制备
PDF下载 (546)陈 蓉,王如坤,李 伟,黄立明,曹嘉懿,周成旭,李 双,徐继林.剧毒卡罗藻GM5株中标志性毒素4,5-dihydro-KmTx2的色谱制备[J].宁波大学学报(理工版),2020,33(2):1-6.DOI:
CHEN Rong,WANG Rukun,LI Wei,HUANG Liming,CAO Jiayi,ZHOU Chengxu,LI Shuang,XU Jilin.Chromatographic preparation of 4,5-dihydro-KmTx2 from Karlodinium veneficum GM5 strain[J].Journal of Ningbo University(Natural Science & Engineering Edition),2020,33(2):1-6.DOI:
| Title: | Chromatographic preparation of 4,5-dihydro-KmTx2 from Karlodinium veneficum GM5 strain |
| 作者: | 陈 蓉, 王如坤, 李 伟, 黄立明, 曹嘉懿, 周成旭, 李 双, 徐继林 |
| Author(s): | CHEN Rong, WANG Rukun, LI Wei, HUANG Liming, CAO Jiayi, ZHOU Chengxu, LI Shuang, XU Jilin |
| 关键词: | 液相色谱; 四极杆/静电场轨道阱高分辨质谱; 剧毒卡罗藻GM5株 |
| Keywords: | 4,5-dihydro-KmTx2; liquid chromatography; quadrupole/electrostatic field orbitrap high-resolution mass spectrometry; Karlodinium veneficum strain GM5 |
| 分类号: | O657 |
| 文献标识码: | A |
| 摘要: | 建立了剧毒卡罗藻GM5株中标志性毒素4,5-dihydro-KmTx2的分离纯化体系, 通过高效液相色谱-四极杆/静电场轨道阱高分辨质谱(UHPLC-Q-Orbitrap HRMS)对其纯化效果进行评价. 过滤后的培养液经大型固相萃取柱(YMC-GEL ODS-A-HG, 80 cm×10 cm, 12 nm, S-50 μm)进行富集, 依次用体积分数为10%、40%和60%的甲醇溶液进行淋洗, 体积分数为80%的甲醇溶液进行洗脱, 得到目标化合物粗提液(纯度为51.3％). 采用半制备型高效液相色谱技术进一步分离纯化, 使得目标化合物纯度高达93.2％. 该方法提取及纯化效率高, 可操作性强, 可以为4,5-dihydro- KmTx2的规模化生产及功能活性研究提供技术支持. |
| Abstract: | A separation and purification system for the toxin 4,5-dihydro-KmTx2 in Karlodinium veneficum GM5 strain at the end of the platform was established. The purification effect of the system was evaluated by high-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectro- metry (UHPLC-Q-Orbitrap HRMS). First, the collected culture solution following the removal of the algae was enriched on a large solid-phase extraction column (YMC-GEL ODS-A-HG, 10 cm×80 cm, 12 nm, S-50 μm). The results showed that leaching was accomplished in sequence by using 10%, 40％ and 60％ methanol solution. The purity of the crude extract obtained by elution with 80％ methanol was 51.3％. The crude extract was separated and purified by semi-preparative HPLC. The purity of the 4,5-dihydro-KmTx2 monomer was 93.2％ with gradient elution using a methanol-water mixture as the mobile phase. The method has high extraction efficiency and operability, which can provide technical support for large-scale production and functional activity research of 4,5-dihydro-KmTx2. |
| 参考文献 /References: | [1] 周成旭, 傅永静, 严小军. 新型赤潮种类微小卡罗藻的溶血毒性检测[J]. 海洋通报, 2007, 26(3):112-116. [2] 许亚如, 何山, 周成旭, 等. 卡罗藻毒素研究进展[J]. 海洋科学, 2014, 38(10):113-118. [3] Waggett R J, Tester P A, Place A R. Anti-grazing properties of the toxic dinoflagellate Karlodinium veneficum during predator-prey interactions with the copepod Acartia tonsa[J]. Marine Ecology Progress Series, 2008, 366:31-42. [4] Sheng J, Malkiel E, Katz J, et al. A dinoflagellate exploits toxins to immobilize prey prior to ingestion[J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(5):2082-2087. [5] 邴晖, 高炳淼, 于海鹏, 等. 海洋生物毒素研究新进展[J]. 海南大学学报(自然科学版), 2011, 29(1):78-85. [6] Houdai T, Matsuoka S, Morsy N, et al. Hairpin conformation of amphidinols possibly accounting for potent membrane permeabilizing activities[J]. Tetrahedron, 2005, 61(11):2795-2802. [7] Place A R, Bowers H A, Bachvaroff T R, et al. Karlodinium veneficum—The little dinoflagellate with a big bite[J]. Harmful Algae, 2012, 14:179-195. [8] Bachvaroff T R, Adolf J E, Place A R. Strain variation in Karlodinium veneficum (dinophyceae): Toxin profiles, pigments, and growth characteristics[J]. Journal of Phycology, 2009, 45(1):137-153. [9] Cai P J, He S, Zhou C X, et al. Two new karlotoxins found in Karlodinium veneficum (strain GM2) from the East China Sea[J]. Harmful Algae, 2016, 58:66-73. [10] 沈斐, 许燕娟, 姜晟, 等. 超高效液相色谱-串联质谱法测定水华样品中微囊藻毒素及其基质效应的消除[J]. 色谱, 2017, 35(7):772-777. [11] 郑立洋, 陈娟娟, 徐继林, 等. 高效液相色谱-三重四极杆质谱同时测定与筛查海洋浮游藻类中的5种色素[J]. 色谱, 2014, 32(9):992-998. [12] 吴海燕, 郭萌萌, 赵春霞, 等. 液相色谱-串联质谱法筛查原多甲藻酸毒素及其代谢产物[J]. 色谱, 2016, 34(4):401-406. [13] 徐娜, 逄少军, 刘峰. 一株桑沟湾赤潮藻的分子鉴定[J]. 海洋科学, 2012, 36(4):13-18. [14] Bachvaroff T R, Adolf J E, Squier A H, et al. Characterization and quantification of karlotoxins by liquid chromatography-mass spectrometry[J]. Harmful Algae, 2008, 7(4):473-484. [15] Deeds J R, Hoesch R E, Place A R, et al. The cytotoxic mechanism of karlotoxin2 (KmTx2) from Karlodinium veneficum (dinophyceae)[J]. Aquatic Toxicology, 2015, 159(1):148-155. [16] 陈永芳. 微囊藻毒素MC-LR纯品的制备工艺研究[D]. 济南: 济南大学, 2011. [17] 卫涛. 微囊藻毒素的分离、分析和制备[D]. 南京: 东南大学, 2005. [18] 丁西飞, 王彦霏, 章群, 等. 珠江口海域3种重要有害藻类的溶血毒性研究[J]. 生态毒理学报, 2018, 13(2): 66-76. [19] Krock B, Busch J A, Tillmann U, et al. LC-MS/MS detection of karlotoxins reveals new variants in strains of the marine dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean)[J]. Marine Drugs, 2017, 15(12):391-411. [20] Guibal R, Lissalde S, Charriau A, et al. Coupling passive sampling and time of flight mass spectrometry for a better estimation of polar pesticide freshwater contamination: Simultaneous target quantification and screening analysis [J]. Journal of Chromatography A, 2015, 1387:75-85. |
| 备注/Memo: | 收稿日期: 2019－09－23. 宁波大学学报（理工版）网址: http://journallg.nbu.edu.cn/ 基金项目: 国家重点研发计划（2019YFD0900400）; 国家现代农业产业技术体系建设专项（CARS-49）; 浙江省分析测试科技计划项目（2018C37072）; 浙江省科技重大专项（2019C02057）; 宁波市“科技创新2025”重大专项（2019B10006）. 第一作者: 陈蓉（1992－）, 女, 山西太原人, 在读硕士研究生, 主要研究方向: 海洋生物化学. E-mail: chenrong321@163.com 宁波大学学报（理工版）网址：http://journallg.nbu.edu.cn/*通信作者: 曹嘉懿（1989－）, 女, 江苏南通人, 讲师, 主要研究方向: 海洋微藻生物. E-mail: caojiayi@nbu.edu.cn |
