赤潮赤潮异弯藻培养液CDOM分析及其潜在的化感作用
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摘要
有色可溶性有机物质(chromophoric dissolvable organic matter, CDOM)是海洋溶解有机物储库中一个重要组分,在影响海洋水色、初级生产力和生态系统结构以及海-气物质交换等方面都起着至关重要的作用,因此已成为近年来相关领域的前沿热点问题。但由于其组成复杂,分离困难,迄今为止国内外的研究仍主要是从宏观上对其光学行为进行探讨,研究对象多为海洋湖泊等自然水域。目前,国内外对CDOM的化学分离及结构鉴定的研究工作尚处于起始阶段。
本文以针胞藻纲赤潮异弯藻Heterosigma akashiwo (Hada)的培养液为研究对象,选取HP-20大孔吸附树脂富集保留水体中的有色可溶性有机物质,对甲醇/水梯度洗脱后得到的六个洗脱组分分别进行了光化学性质分析、液相色谱质谱联用分析及其潜在化感物质溶血活性的研究,结果如下:
通过对各洗脱组分的吸收及荧光光谱、光降解等光化学性质的研究表明:随着洗脱液极性的降低,赤潮异弯藻CDOM洗脱物的平均分子量下降,芳香结构数量减少、富里酸/腐植酸比例升高;在荧光光谱图中共出现了8个峰,包括3个类腐殖质荧光峰、4个类蛋白荧光峰及一个类色素荧光峰,各洗脱组分中类腐殖质的相对含量随着洗脱液极性的降低呈指数下降(R2=0.9686),类蛋白质呈线性上升(R2=0.9664);紫外照射结果证实了赤潮异弯藻CDOM各洗脱组分在接受短期的紫外光照射下均容易发生光化学降解,吸收系数衰减随时间的变化可按一阶动力学方程拟合,一阶动力学常数位于0.2~0.35之间。
其次,运用超高效液相色谱-四级杆飞行时间质谱联用分析系统(UPLC-Q-TOF-MS)对各洗脱组分进行LC-MS分析,建立了有效分离分析各组分的方法,确定了各组分的平均质荷比(m/z)位于300-700之间。另外,总结了各组分中主要离子的精确m/z比值及其碎片离子,为进一步的化学结构研究提供了支持。
最后,对各组分进行的溶血活性测定结果表明:赤潮异弯藻溶血毒素主要出现在低极性洗脱组。运用高效液相色谱对溶血活性最高的a5组分进一步细分,结果显示a5组分中至少含有两组溶血物质,一组为具有183.01(m/z)稳定母核的同系物,结构鉴定为3-烷基苯羟基亚硫酸;另一组溶血物质推测为不饱和脂肪酸C18:3(n-6)、C18:2(n-6)、C18:1(n-9)及C20:1(n-9)。
以上结果表明,赤潮异弯藻CDOM多为低分子量物质,富里酸等较低极性物质的含量低于以腐植酸为主的高极性物质。其中溶血活性物质主要出现在低极性洗脱组,结构鉴定分别为3-烷基苯羟基亚硫酸与不饱和脂肪酸,与紫外吸收物质不存在一致性,两者无确切相关关系。
The chromophoric dissolvable organic matter (CDOM) , one of an important components in the marine dissolvable organic matter storage, play an important role in influencing ocean color, primary productivity and ecosystem structure, as well as the air-sea exchange. Therefore, study on CDOM has became a hot topic in recent years. As its composition is too complex to fractionate, researches at home and abroad are mainly carried on optics behavior of CDOM in natural waters, such as oceans and lakes, while isolation and identification of CDOM are still in an exploratory stage.
In this study, the CDOM excreted by Raphidophyte microalga Heterosigma akashiwo (Hada) was used for detailed characterization. The CDOM was selectively absorbed on HP-20 macroporous resin, and eluted gradiently by methyl alcohol/water to obtain six fractions, which was used for further discussion on photochemistry nature, chemical analysis by UPLC-Q-TOF-MS, and assessment of hematolysis activeness. The results were summarized described as follows:
Firstly, the photochemistry nature of six fractions, such as absorption spectrum, fluorescence spectrum and photodegradation was studied. The results showed that as eluent polarity reduced, the average molecular weight of eluate decreased, aromatic substance lessened. There were eight fluorophores in all: three humic-like fluorophores, four protein-like and one pigment-like fluorophores, besides, comparative content of humic-like fluorescence was down, while protein-like’s was up. The photodegradation processes of absorption coefficient followed the first-order dynamic equation, and the kinetic constant was about 0.2~0.35.
Secondly, six fractions were all analyzed by using Ultra Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-ESI-Q-TOF-MS). Method that can isolate various components effectively was established and their average m/z was calculated at 300~700. Efforts were also focused on identifying the main ions of CDOM from heterosigma akashiwo culture, corresponding fragment ions besides. All of these results had provided support for further chemical constitution research.
Finally, hemolytic activity of different eluates was determined. The experiment demonstrated that the low polar materials are main factors responsible for hemolysis excreted by heterosigma akashiwo. Utilizing Highly Performance Liquid Chromatography to further separate eluate a5, the fraction had the highest hemolytic degree, finally found two groups of hemolytic compounds in eluate a5: one was homolog with a core structure (m/z 183.01), and it had been speculated as 3-dodecylphenyl hydrogen sulfite; the other was unsaturated fatty acid (UFA), containing octadecenoic C18:1(n-9), octadecadienoic C18:2(n-6), octadecatrienoic C18:3(n-6) and dodecenoic C20:1(n-9) acids.
In conclusion, CDOM excreted by Raphidophyte microalga Heterosigma akashiwo (Hada) were mainly low molecular weight materials, and the low polar materials, such as fulvic acids (FA), were lower than the high polar materials of which majority were humic acids (HA). While the hemolytic toxin mainly appeared in the low polar elution group, the structure were respectively speculated as 3-aklyl benzenes hydroxyl sulfurous acid and the unsaturated fatty acid, hemolytic compounds and ultraviolet absorption materials had no correlativity.
本文以针胞藻纲赤潮异弯藻Heterosigma akashiwo (Hada)的培养液为研究对象,选取HP-20大孔吸附树脂富集保留水体中的有色可溶性有机物质,对甲醇/水梯度洗脱后得到的六个洗脱组分分别进行了光化学性质分析、液相色谱质谱联用分析及其潜在化感物质溶血活性的研究,结果如下:
通过对各洗脱组分的吸收及荧光光谱、光降解等光化学性质的研究表明:随着洗脱液极性的降低,赤潮异弯藻CDOM洗脱物的平均分子量下降,芳香结构数量减少、富里酸/腐植酸比例升高;在荧光光谱图中共出现了8个峰,包括3个类腐殖质荧光峰、4个类蛋白荧光峰及一个类色素荧光峰,各洗脱组分中类腐殖质的相对含量随着洗脱液极性的降低呈指数下降(R2=0.9686),类蛋白质呈线性上升(R2=0.9664);紫外照射结果证实了赤潮异弯藻CDOM各洗脱组分在接受短期的紫外光照射下均容易发生光化学降解,吸收系数衰减随时间的变化可按一阶动力学方程拟合,一阶动力学常数位于0.2~0.35之间。
其次,运用超高效液相色谱-四级杆飞行时间质谱联用分析系统(UPLC-Q-TOF-MS)对各洗脱组分进行LC-MS分析,建立了有效分离分析各组分的方法,确定了各组分的平均质荷比(m/z)位于300-700之间。另外,总结了各组分中主要离子的精确m/z比值及其碎片离子,为进一步的化学结构研究提供了支持。
最后,对各组分进行的溶血活性测定结果表明:赤潮异弯藻溶血毒素主要出现在低极性洗脱组。运用高效液相色谱对溶血活性最高的a5组分进一步细分,结果显示a5组分中至少含有两组溶血物质,一组为具有183.01(m/z)稳定母核的同系物,结构鉴定为3-烷基苯羟基亚硫酸;另一组溶血物质推测为不饱和脂肪酸C18:3(n-6)、C18:2(n-6)、C18:1(n-9)及C20:1(n-9)。
以上结果表明,赤潮异弯藻CDOM多为低分子量物质,富里酸等较低极性物质的含量低于以腐植酸为主的高极性物质。其中溶血活性物质主要出现在低极性洗脱组,结构鉴定分别为3-烷基苯羟基亚硫酸与不饱和脂肪酸,与紫外吸收物质不存在一致性,两者无确切相关关系。
The chromophoric dissolvable organic matter (CDOM) , one of an important components in the marine dissolvable organic matter storage, play an important role in influencing ocean color, primary productivity and ecosystem structure, as well as the air-sea exchange. Therefore, study on CDOM has became a hot topic in recent years. As its composition is too complex to fractionate, researches at home and abroad are mainly carried on optics behavior of CDOM in natural waters, such as oceans and lakes, while isolation and identification of CDOM are still in an exploratory stage.
In this study, the CDOM excreted by Raphidophyte microalga Heterosigma akashiwo (Hada) was used for detailed characterization. The CDOM was selectively absorbed on HP-20 macroporous resin, and eluted gradiently by methyl alcohol/water to obtain six fractions, which was used for further discussion on photochemistry nature, chemical analysis by UPLC-Q-TOF-MS, and assessment of hematolysis activeness. The results were summarized described as follows:
Firstly, the photochemistry nature of six fractions, such as absorption spectrum, fluorescence spectrum and photodegradation was studied. The results showed that as eluent polarity reduced, the average molecular weight of eluate decreased, aromatic substance lessened. There were eight fluorophores in all: three humic-like fluorophores, four protein-like and one pigment-like fluorophores, besides, comparative content of humic-like fluorescence was down, while protein-like’s was up. The photodegradation processes of absorption coefficient followed the first-order dynamic equation, and the kinetic constant was about 0.2~0.35.
Secondly, six fractions were all analyzed by using Ultra Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-ESI-Q-TOF-MS). Method that can isolate various components effectively was established and their average m/z was calculated at 300~700. Efforts were also focused on identifying the main ions of CDOM from heterosigma akashiwo culture, corresponding fragment ions besides. All of these results had provided support for further chemical constitution research.
Finally, hemolytic activity of different eluates was determined. The experiment demonstrated that the low polar materials are main factors responsible for hemolysis excreted by heterosigma akashiwo. Utilizing Highly Performance Liquid Chromatography to further separate eluate a5, the fraction had the highest hemolytic degree, finally found two groups of hemolytic compounds in eluate a5: one was homolog with a core structure (m/z 183.01), and it had been speculated as 3-dodecylphenyl hydrogen sulfite; the other was unsaturated fatty acid (UFA), containing octadecenoic C18:1(n-9), octadecadienoic C18:2(n-6), octadecatrienoic C18:3(n-6) and dodecenoic C20:1(n-9) acids.
In conclusion, CDOM excreted by Raphidophyte microalga Heterosigma akashiwo (Hada) were mainly low molecular weight materials, and the low polar materials, such as fulvic acids (FA), were lower than the high polar materials of which majority were humic acids (HA). While the hemolytic toxin mainly appeared in the low polar elution group, the structure were respectively speculated as 3-aklyl benzenes hydroxyl sulfurous acid and the unsaturated fatty acid, hemolytic compounds and ultraviolet absorption materials had no correlativity.
引文
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