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Volume 40 Issue 1
Jan.  2021
Article Contents

Citation:

Extraction and purification of yessotoxin in the dinoflagellate Protoceratium reticulatum

  • Received Date: 2019-06-26
    Accepted Date: 2019-09-06
  • Marine dinoflagellate Protoceratium reticulatum producing yessotoxins was mass cultured. Then obtained algal cells were extracted by ultrasonic crushing with methanol for yessotoxins. Macroporous adsorption resin (HP20) was used to adsorb the extracellular toxin secreted in the culture medium, and the yessotoxins adsorbed on the resin were eluted with 70% ethanol. Nine of yessotoxin and their analogues were detected by high performance liquid chromatography-tandem mass spectrometry in extracts. The toxin of yessotoxin accounted for 95.1% of the total toxin group in the extracts, derivative 41YTX accounted for 3.65%, and other trace analogues accounted for 1.25%. Yessotoxin was purified through high performance preparation liquid chromatography for 5 times. The molecular weight and molecular formula was confirmed as 1142.4747 and C55H82O21S2, respectively, by high resolution mass spectrometry. The purity of yessotoxin detected by quantitative nuclear magnetic method was more than 99%, meeting the requirement for preparation of yessotoxin standard material.
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  • [1] MILES C O, SAMDAL I A, AASEN J A G, et al. Evidence for numerous analogs of yessotoxin in Protoceratium reticulatum[J]. Harmful Algae, 2005, 4(6): 1075-1091. doi: 10.1016/j.hal.2005.03.005
    [2] LIU L, WEI N, GOU Y X, et al. Seasonal variability of Protoceratium reticulatum and yessotoxins in Japanese scallop Patinopecten yessoensis in northern Yellow Sea of China[J]. Toxicon, 2017, 139: 31-40. doi: 10.1016/j.toxicon.2017.09.015
    [3] SUZUKI T, HORIE Y, KOIKE K, et al. Yessotoxin analogues in several strains of Protoceratium reticulatum in Japan determined by liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry[J]. Journal of Chromatography A, 2007, 1142(2): 172-177. doi: 10.1016/j.chroma.2006.12.048
    [4] PAZ B, DARANAS A H, NORTE M, et al. Yessotoxins, a group of marine polyether toxins: an overview[J]. Marine Drugs, 2008, 6(2): 73-102. doi: 10.3390/md6020073
    [5] (EU) No 786/2013, Amending Annex III to Regulation (EC) No 853/2004 of the European Parliament and of the Council as regards the permitted limits of yessotoxins in live bivalve molluscs[S].
    [6] PAZ B, BLANCO J, FRANCO J M. Yessotoxins production during the culture of Protoceratium reticulatum strains isolated from Galician Rias Baixas (NW Spain)[J]. Harmful Algae, 2013, 21-22: 13-19. doi: 10.1016/j.hal.2012.11.002
    [7] KONISHI M, YANG X M, LI B, et al. Highly cytotoxic metabolites from the culture supernatant of the temperate dinoflagellate Protoceratium cf. reticulatum[J]. Journal of Natural Products, 2004, 67(8): 1309-1313. doi: 10.1021/np040008c
    [8] PAZ B, RIOBO P, RAMILO I, et al. Yessotoxins profile in strains of Protoceratium reticulatum from Spain and USA[J]. Toxicon, 2007, 50(1): 1-17. doi: 10.1016/j.toxicon.2007.02.005
    [9] 高春蕾, 孙 萍, 贾智慧, 等. 温度和营养盐限制对网状原角藻生长与产毒的影响[J]. 生态学报, 2017, 37(12): 4217-4226.
    [10] GUERRINI F, CIMINIELLO P, Dell’AVERSANO C, et al. Influence of temperature, salinity and nutrient limitation on yessotoxin production and release by the dinoflagellate Protoceratium reticulatum in batch-cultures[J]. Harmful Algae, 2007, 6(5): 707-717. doi: 10.1016/j.hal.2007.02.006
    [11] MITROVIC S M, HAMILTON B, MCKENZIE L, et al. Persistence of yessotoxin under light and dark conditions[J]. Marine Environmental Research, 2005, 60(3): 397-401. doi: 10.1016/j.marenvres.2005.01.001
    [12] SALA-PEREZ M, ALPERMANN T J, KROCK B, et al. Growth and bioactive secondary metabolites of arctic Protoceratium reticulatum (Dinophyceae)[J]. Harmful Algae, 2016, 55: 85-96. doi: 10.1016/j.hal.2016.02.004
    [13] ÁLVAREZ G, URIBE E, DÍAZ R, et al. Bloom of the Yessotoxin producing dinoflagellate Protoceratium reticulatum (Dinophyceae) in Northern Chile[J]. Journal of Sea Research, 2011, 65(4): 427-434. doi: 10.1016/j.seares.2011.03.008
    [14] 渠佩佩, 杨晶晶, 徐轶肖, 等. 固相吸附毒素跟踪技术(SPATT)在浙江南麂海域的应用[J]. 海洋与湖沼, 2016, 47(4): 795-803. doi: 10.11693/hyhz20160100007
    [15] 宿志伟, 赵 峰, 刘远平, 等. 固相吸附毒素跟踪技术监测牡蛎养殖区中腹泻性贝毒[J]. 渔业科学进展, 2016, 37(6): 144-150. doi: 10.11758/yykxjz.20151012001
    [16] RUNDBERGET T, SANDVIK M, LARSEN K, et al. Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2[J]. Toxicon, 2007, 50(7): 960-970. doi: 10.1016/j.toxicon.2007.07.003
    [17] 刘 丽. 网状原角藻(Protoceratium reticulatum)产毒特性及虾夷扇贝毒素的提取与纯化研究[D]. 大连: 大连海洋大学, 2016.
    [18] 杨 林, 荣明强, 刘少华, 等. 舟山黄海葵兴奋性毒素AX-1的分离纯化及鉴定[J]. 中国海洋药物, 2012, 31(2): 25-33.
    [19] 刘仁沿, 梁玉波, 刘 磊, 等. 液相色谱结合串联质谱方法研究中国沿海贝类中脂溶性藻毒素的种类结构和分布规律[J]. 生态环境学报, 2014, 23(8): 1320-1326. doi: 10.3969/j.issn.1674-5906.2014.08.011
    [20] ORELLANA G, VAN MEULEBROEK L, VAN VOOREN S, et al. Quantification and profiling of lipophilic marine toxins in microalgae by UHPLC coupled to high-resolution orbitrap mass spectrometry[J]. Analytical and Bioanalytical Chemistry, 2015, 407(21): 6345-6356. doi: 10.1007/s00216-015-8637-y
    [21] GERSSEN A, MULDER P P J, DE BOER J. Screening of lipophilic marine toxins in shellfish and algae: development of a library using liquid chromatography coupled to orbitrap mass spectrometry[J]. Analytica Chimica Acta, 2011, 685(2): 176-185. doi: 10.1016/j.aca.2010.11.036
    [22] BURTON I W, QUILLIAM M A, WALTER J A. Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products[J]. Analytical Chemistry, 2005, 77(10): 3123-3131. doi: 10.1021/ac048385h
    [23] LIU Y, YU R C, KONG F Z, et al. Contamination status of lipophilic marine toxins in shellfish samples from the Bohai Sea, China[J]. Environmental Pollution, 2019, 249: 171-180. doi: 10.1016/j.envpol.2019.02.050
    [24] 高春蕾, 刘仁沿, 梁玉波, 等. 虾夷扇贝毒素yessotoxins(YTXs), 中国沿海贝类中首次发现的一组贝类生物毒素[J]. 海洋学报, 2010, 32(3): 129-137.
    [25] 陈建华, 于仁成, 孔凡洲, 等. 北黄海海域虾夷扇贝体内脂溶性藻毒素分析[J]. 海洋与湖沼, 2014, 45(4): 855-863. doi: 10.11693/hyhz20130400016
    [26] MONTECHIARO F, GIORDANO M. Compositional homeostasis of the dinoflagellate Protoceratium reticulatum grown at three different pCO2[J]. Journal of Plant Physiology, 2010, 167(2): 110-113. doi: 10.1016/j.jplph.2009.07.013
    [27] RODER K, HANTZSCHE F M, GEBÜHR C, et al. Effects of salinity, temperature and nutrients on growth, cellular characteristics and yessotoxin production of Protoceratium reticulatum[J]. Harmful Algae, 2012, 15: 59-70. doi: 10.1016/j.hal.2011.11.006
    [28] 高莉媛, 王艳龙, 陈军辉, 等. 高效液相色谱-离子阱质谱法测定海洋微藻藻粉中的8种脂溶性毒素[J]. 海洋科学, 2016, 40(10): 113-119. doi: 10.11759//hykx20151225002
    [29] 郭萌萌, 谭志军, 吴海燕, 等. 液相色谱-串联质谱法同时测定贝类中大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素[J]. 色谱, 2012, 30(3): 256-261.
    [30] MACKENZIE L, BEUZENBERG V, HOLLAND P, et al. Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves[J]. Toxicon, 2004, 44(8): 901-918. doi: 10.1016/j.toxicon.2004.08.020
    [31] RUNDBERGET T, GUSTAD E, SAMDAL I A, et al. A convenient and cost-effective method for monitoring marine algal toxins with passive samplers[J]. Toxicon, 2009, 53(5): 543-550. doi: 10.1016/j.toxicon.2009.01.010
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Extraction and purification of yessotoxin in the dinoflagellate Protoceratium reticulatum

  • 1. National Marine Environmental Monitoring Center, Dalian 116023, China
  • 2. Jinan University, Guangzhou 510632, China
  • 3. Dalian key laboratory of algal toxin, Dalian 116023, China
  • 4. Dalian modern agricultural production development service center, Dalian 116023, China
  • 5. Dalian University of Technology, Dalian 116024, China

Abstract: Marine dinoflagellate Protoceratium reticulatum producing yessotoxins was mass cultured. Then obtained algal cells were extracted by ultrasonic crushing with methanol for yessotoxins. Macroporous adsorption resin (HP20) was used to adsorb the extracellular toxin secreted in the culture medium, and the yessotoxins adsorbed on the resin were eluted with 70% ethanol. Nine of yessotoxin and their analogues were detected by high performance liquid chromatography-tandem mass spectrometry in extracts. The toxin of yessotoxin accounted for 95.1% of the total toxin group in the extracts, derivative 41YTX accounted for 3.65%, and other trace analogues accounted for 1.25%. Yessotoxin was purified through high performance preparation liquid chromatography for 5 times. The molecular weight and molecular formula was confirmed as 1142.4747 and C55H82O21S2, respectively, by high resolution mass spectrometry. The purity of yessotoxin detected by quantitative nuclear magnetic method was more than 99%, meeting the requirement for preparation of yessotoxin standard material.

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  • 虾夷扇贝毒素(yessotoxin,YTX)是一种硫酸酯聚醚类的海洋生物毒素(见图1[1],已报道的YTX及其类似物有百余种,称为虾夷扇贝毒素组(yessotoxins,YTXs)[1-3]。虾夷扇贝毒素对人体的心脏、肝脏、胰脏和神经系统等组织器官可造成损伤,潜在威胁人类健康剂量为80~750 mg/kg[4]。欧盟规定,海产品中虾夷扇贝毒素安全食用限量为3.75 mg/kg[5]。虾夷扇贝毒素主要由网状原角藻(Protoceratium reticulatum)、多边舌甲藻(Lingulodinium polyedrum)和具刺膝沟藻(Gonyaulax spinifera)等海洋甲藻产生。网状原角藻是常见的产YTXs的微藻,分布于新西兰、日本、挪威、意大利、英国、加拿大、西班牙和美国等近岸海域[6]。不同海域的网状原角藻中,YTXs的组成不同,一般YTX为其主要组分,但在日本[7]和西班牙[8]近岸海域网状原角藻以homoYTX组分为主。不同株系的网状原角藻单细胞产毒量变动很大,0~79 pg不等[4],其产毒能力可受到温度、光照强度、生长时期和营养条件等多种因素影响[6, 9-11]。虾夷扇贝毒素属于脂溶性毒素,可存在于藻细胞内及细胞外。胞内毒素可采用甲醇溶液超声提取[2, 3, 8, 12-13];胞外毒素可采用大孔吸附树脂(HP20)富集[1, 14],这类树脂主要为苯乙烯聚合物,依靠范德华力和氢键吸附毒素,可富集水中的大田软海绵酸及鳍藻毒素等脂溶性毒素[15-16]。海洋脂溶性毒素纯化可采用低压柱,填料有三氧化二铝、C18等[17],但填装常压柱费时费力,且重复性差。制备液相色谱问世以来,有效地解决了这一问题,现已广泛应用于海洋生物毒素纯化[18]。虾夷扇贝毒素可用高效液相色谱-串联质谱(high performance liquid chromatography-tandem mass spectrometry,HPLC-MS/MS)法检测[2, 19],其分子量可通过高分辨质谱(High resolution mass spectrometry,HRMS)测定[20-21],其纯度可通过核磁共振波谱(Nuclear magnetic resonance spectroscopy,NMR)确定[1, 22]

    Figure 1.  Chemical structural formula for yessotoxin[1]

    近几年,在我国近海虾夷扇贝、栉孔扇贝、牡蛎、贻贝等双壳贝类体内虾夷扇贝毒素常有检出[2, 19, 23-25],在北黄海虾夷扇贝消化腺中YTX时有超出欧盟限量标准[2]。海水酸性条件下,网状原角藻产毒量明显增多[26]。海水中磷比例降低有利于网状原角藻细胞内的毒素产生[9, 10, 27]。全球气候变暖、海水酸化及营养盐比例失调等因素,使我国近海虾夷扇贝毒素的污染有不断加重的趋势。因此,加强海洋生物(虾夷扇贝)毒素的监测,保障我国海产品食用和生态环境的安全是非常必要的。然而,我国至今尚无虾夷扇贝毒素标准物质(有证参考物质),严重制约了我国虾夷扇贝毒素日常检测工作的开展。标准物质的制备需要毫克级以上的高纯度毒素,本研究通过批量培养网状原角藻,提取纯化其胞内和胞外毒素,可满足标准物质制备的需求。

1.   材料与方法

    1.1.   试剂

  • 无水甲醇(CH3OH)、乙醇(CH3CH2OH)为分析纯,购自天津市科密欧化学试剂公司;大孔吸附树脂(HP20)购自日本三菱化学株式会社;虾夷扇贝毒素标准物质购自加拿大海洋生物科学研究所;乙腈为色谱纯,购自德国Merck公司;氨水为色谱纯,购自天津市科密欧化学试剂公司;色谱仪器用水均为蒸馏水,购自屈臣氏公司;0.22 μm孔径滤膜,购自上海兴亚净化材料厂。

  • 1.2.   网状原角藻培养

  • 网状原角藻分离自獐子岛海域[2],在实验室连续培养。一般20 d左右浓度可达到104 cells/mL,经连续流离心机浓缩,收集藻泥用于胞内毒素提取;收集海水培养液用于胞外毒素富集。

  • 1.3.   网状原角藻胞内虾夷扇贝毒素提取

  • 称取6份网状原角藻藻泥,每份5 g,分别添加0%、20%、40%、60%、80%和100%的甲醇溶液各15 mL,涡旋混合均匀后超声破碎,功率为20 W,脉冲间歇时间为3 s,破碎时间为12、24和36 min时分别取样,10000 r/min离心30 min,收集上清液,通过减压旋转蒸发仪、氮吹仪去除有机溶剂,冷冻干燥去除水分,甲醇重溶,过0.22 μm孔径有机滤膜后,高效液相色谱-串联质谱法分析毒素组成及含量,确定最佳胞内毒素提取方法,用该法提取剩余藻泥中的毒素,去除水分后的毒素干粉于−20 ℃暂时保存,待进一步分离纯化。

  • 1.4.   网状原角藻胞外虾夷扇贝毒素富集

  • 网状原角藻培养液离心后的海水经过大孔吸附树脂(HP20)层析柱(76 mm×500 mm)富集胞外毒素,流速约为6 L/h,过滤后,5倍柱体积纯水冲洗除盐后,将填料混匀,湿法填装成小柱(32 mm×160 mm),分别用10%、20%、30%、40%、50%、60%、70%、80%、90%和100%的500 mL(约四倍柱体积)乙醇水溶液洗脱毒素,由洗脱液中YTX的含量来判断洗脱效果。将含YTX的洗脱液旋蒸浓缩,去除有机溶剂,冷冻干燥去除水分,毒素干粉于−20 ℃暂时保存,待进一步分离纯化。

  • 1.5.   虾夷扇贝毒素分离纯化

  • 将1.3节和1.4节中得到的毒素干粉用初始流动相(20%乙腈)重溶,过0.22 μm孔径有机滤膜后通过制备液相色谱(谱道P2100)对虾夷扇贝毒素进行两次分离纯化。A流动相为乙腈,B流动相为水。最大上样量为10 mL。第一次纯化,每3 min收集一次。合并所有含有YTX的收集液,旋转蒸发去除有机相后冷冻干燥。20%乙腈重新溶解后,过0.22 μm有机滤膜,每次制备的收集液均按此方法处理。第二次纯化,每1 min收集一次。

    采用半制备液相色谱仪(安捷伦1260)再进行3次分离纯化。最大上样量为100 µL。A流动相为乙腈+1‰氨水,B流动相为水+1‰氨水。少量分流由单四级杆质谱检测器监测YTX碎片m/z=570。第三、四、五次分离纯化均手动收集虾夷扇贝毒素洗脱液。

  • 1.6.   虾夷扇贝毒素检测方法

  • YTXs检测方法及数据处理参考Liu等[2]。对获得的高纯度虾夷扇贝毒素通过四级杆飞行时间高分辨质谱法(HRMS)确认分子量及分子式,通过定量核磁法进行纯度鉴定。

2.   结果与讨论

    2.1.   网状原角藻中的虾夷扇贝毒素及其类似物

  • 本研究中的网状原角藻藻泥经甲醇超声提取,高效液相色谱-串联质谱分析检测到9种毒素组分(见图2),各组分相对百分含量从高到低依次为:YTX(95.10%),41YTX(3.65%),homoYTX(0.79%),45、46、47-trinorYTX(0.22%),45、46、47- trinorhomo YTX(0.08%),45-OH YTX(0.06%),45-OH homo YTX(0.04%),38YTX(0.03%),49YTX(0.03%)。与Liu等[2]所用藻种源自同一株系,但发现毒素组分由原来的15种减少至9种。与一株源自日本的网状原角藻中4个主要组分YTX,homoYTX,45、46、47-trinorYTX,45、46、47-trinorhomoYTX相似,但不包含组分noroxoYTX enone[3]

    Figure 2.  Multiple reaction monitoring (MRM) chromatogram showing the presence of numerous yessotoxins in the strain Protoceratium reticulatum cells

    根据毒素量及细胞数可知,本研究中网状原角藻胞内YTX含量为(2.2±0.7)pg/cell,胞外为(1.7±0.6) pg/cell。比Liu等[2]研究中的胞内及胞外YTX毒素含量大幅度降低,这可能是由本藻株经室内条件下长时间培养,产毒能力明显下降所致。意大利东岸的一株网状原角藻培养于f/2营养液中,指数期胞内YTX含量为(5.60±0.36)pg,胞外含量为(1.60±0.13)pg;稳定期胞内含量为(14.1±1.3)pg,胞外为(8.1±0.8)pg[10]。北海德国湾的网状原角藻中的YTX占YTXs总量的94%以上,指数期末的胞内YTX含量为(7.22±0.20)pg[27]。西班牙的3株网状原角藻进入稳定期的时间及YTX含量均不同[6]。在营养盐磷限制条件下培养,胞内毒素含量都有明显增加[9-10, 27]。虽然与其他海域株系相比,本实验室的网状原角藻产毒量略低,但YTX占总毒素的95%以上,且已适应室内高密度培养,因此适用于虾夷扇贝毒素提取。

  • 2.2.   网状原角藻胞内虾夷扇贝毒素的提取方法

  • 5 g藻泥分别用0%、20%、40%、60%、80%和100%的甲醇-水作为提取溶液,超声12 min、24 min和36 min后检测YTX含量(n=3)。结果表明,采用100%甲醇超声破碎24 min得到的毒素含量最高,每克藻泥可获得大约(91±31) μg YTX(见图3)。高莉媛等[28]采用异丙醇、甲醇、乙酸乙酯、二氯甲烷、乙醇和丙酮6种溶剂,超声波辅助提取海洋微藻中的脂溶性毒素时发现甲醇对脂溶性藻毒素的提取效果最好。郭萌萌等[29]提取贝类样品中的YTX时,二氯甲烷提取液含有悬浮物,不利于后续净化;甲醇和80%甲醇水的提取率分别为90%和不足80%。Liu等[2]和Sala-Pe´rez等[12]在提取脂溶性毒素时均选用无水甲醇作为提取溶液。另外,100%甲醇提取液易于浓缩,有利于后续净化。因此,100%甲醇为超声辅助提取胞内脂溶性毒素的最佳溶剂。

    Figure 3.  The content of YTX per gram of algal mud under different extraction conditions

  • 2.3.   网状原角藻胞外虾夷扇贝毒素的富集

  • 海水培养液(离心后,约290 L)经大孔树脂(HP20)吸附后,用纯水冲洗去盐,湿法填装小柱,分别用10%、20%、30%、40%、50%、60%、70%、80%、90%和100%乙醇-水溶液洗脱YTX。结果表明,500 mL 70%乙醇的洗脱液中YTX含量最高,可达(304±113)µg;而50%以下的乙醇洗脱液几乎检测不到YTX(见图4)。HP20广泛用于富集YTX。MacKenzie等[30]首次提出原位监测有毒藻赤潮,并发现DIAION®HP20、DIAION®SP70和DIAION® HP2MG3种吸附树脂对YTX的平均相对回收率分别为100%、36%和62%。Rundberget等[31]使用HP20树脂成功监测到YTX等9种藻毒素。宿志伟等[15]使用HP20在黄海牡蛎养殖区水体中检测到4种脂溶性毒素。渠佩佩等[14]使用HP20在浙江南麂海域监测到7种脂溶性毒素(含YTX)。Miles等[1]用HP20吸附藻滤液中的YTX。Rundberget等[16]亦采用HP20成功吸附大量海水中的多种脂溶性毒素。虽然从HP20上洗脱YTX多用甲醇[1, 16],但乙醇毒性小,本文中70%乙醇对YTX在HP20上的解吸能力最强。

    Figure 4.  Ethanol elution efficiency of yessotoxins on macroporous resin HP20 adsorption column

  • 2.4.   高效制备液相色谱分离纯化虾夷扇贝毒素效果

  • 经过高效制备液相色谱5次分离纯化(见表1),最终获得高纯度的YTX固体。第一次制备杂质最多,有机相初始比例设定较低(20%),洗脱时间最长达40 min;第二次制备有机相初始比例提高到38%;第三次采用粒径(5 μm)更小的制备柱实现分离纯化;第四次通过降低流速实现分离;第五次通过缩短采集时段提高YTX纯度。

    制备次数使用仪器使用制备柱洗脱条件收集t/min
    第一次谱道P2100Welch Ultimate XB-C18,粒径10 μm,20×250 mmA相,20%~80%梯度洗脱0~40 min,流速15 mL/min18~27
    第二次谱道P2100同上A相,38%~58%梯度洗脱0~20 min,流速15 mL/min10~12
    第三次安捷伦1260Welch Xitimate C18,粒径5 μm,10×250 mmA相,20%~90%梯度洗脱0~15 min,流速4 mL/min7~10
    第四次安捷伦1260同上A相,40%~70%梯度洗脱0~15 min,流速2.36 mL/min4~9.6
    第五次安捷伦1260同上同上4.2~9.6

    Table 1.  Purification conditions and collection time of YTX via high performance preparation liquid chromatography

    将高纯度YTX固体溶于甲醇中,高分辨质谱扫描的主要质谱峰m/z=1141.4747为分子离子峰(M-H)(见图5),分子量为1142.4747,分子式为C55H82O21S2,同YTX一致。核磁谱图亦证实了该物质为YTX(见图6),由定量核磁法测得YTX的纯度为99.3%。

    Figure 5.  High resolution mass spectra of YTX

    Figure 6.  Hydrogen spectra of yessotoxin by NMR spectroscopy

3.   结 论
  • (1)本研究中的网状原角藻,适宜批量培养,所含毒素以虾夷扇贝毒素为主,可用作获得高纯毒素的原料。

    (2)100%甲醇-超声波辅助提取是网状原角藻胞内毒素的适宜提取方式。

    (3)大孔吸附树脂HP20适用于胞外虾夷扇贝毒素富集,70%乙醇是富集于树脂上虾夷扇贝毒素的适宜洗脱溶剂。

    (4)经过高效制备液相色谱5次分离纯化,可获得纯度超过99%的虾夷扇贝毒素。

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