海参脑苷脂的分离纯化、结构分析及其生物活性研究
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摘要
海参是一种名贵的海洋滋补品,富含多种生物活性成分,具有较高的营养价值和药用价值。海参脑苷脂及长链碱是存在于海参体壁中结构独特的鞘脂类化合物,具有抗肿瘤、免疫调节、神经保护、抗菌等生理活性。海参脑苷脂包括三种主要系列物,每种系列物中含有几十种单体化合物。本论文对仿刺参、海地瓜和叶瓜参三种海参的脑苷脂及长链碱的分离纯化、分子组成和结构以及生理活性进行了较为系统的研究,对于海洋新原料来源的发现、海参新型营养功效的补充、海参的高值化开发利用具有重要意义。本文研究内容和结果如下:
1.建立海参中脑苷脂含量简便准确测定的新方法。采用高效液相色谱-蒸发光散射检测法(HPLC-ELSD),以TSKgel CN-80Ts柱为分离柱,正己烷-异丙醇-二氯甲烷-甲醇为流动相,在13min内实现了脑苷脂和神经酰胺的同时测定。其中,脑苷脂的检出限为0.01μg,加标平均回收率为94.28%,RSD为5.67%,方法稳定性良好。在所测13种水产样品中,海星的脑苷脂和神经酰胺含量最高,在所测8种海参中叶瓜参脑苷脂含量最高。结果表明该方法可行,样品前处理简单,快速准确。结果提示包括海参在内的海洋食品可以作为功能性鞘脂类的良好资源。
2.建立一套用于各种海参脑苷脂分离纯化的快速高效的完整新体系。首次应用高速逆流色谱(HSCCC)技术成功优化了海参脑苷脂的分离过程。(1)海参总脑苷脂:采用石油醚-甲醇-水(5:4:1, V/V)为溶剂体系的高速逆流色谱法,从海参总脂中一步纯化制备出海参总脑苷脂,以总脂计得率约为7.6~12.4%,经正相HPLC-ELSD法检测其纯度达90%以上。(2)海参脑苷脂系列物:先经一次正相硅胶柱层析,收集氯仿:甲醇(80:20,V/V)洗脱组分,再采用乙酸乙酯-乙醇-水(5:3.5:3.5, V/V)为溶剂体系的高速逆流色谱技术,将上述洗脱组分中严重干扰海参脑苷脂纯化的硫酸酯化甾醇去除,制得海参脑苷脂系列物的总得率约为1.1~6.4%,纯度均在90%以上。并以优化出的正相HPLC制备法进行了验证比较。(3)海参脑苷脂单体化合物:采用反相HPLC法,C18半制备柱,98%甲醇水溶液洗脱,从海地瓜脑苷脂系列物中分离出4个脑苷脂单体化合物,纯度在90%以上。结果表明,所建立的脑苷脂纯化体系具有得率高、速度快、成本低等突出优点,从根本上解决了海参脑苷脂纯化的难题。
3.建立海参脑苷脂分子种组成和结构筛选的高通量分析方法。
先采用气相色谱质谱联用(GC-MS)法,分析比较了三种海参脑苷脂系列物的脂肪酸、长链碱和糖基组成差异。用10%盐酸/甲醇水解,正己烷萃取脂肪酸甲脂组分,甲醇保留长链碱和糖基组分。脂肪酸甲酯直接用INNOWax柱分析,长链碱和甲基糖经硅烷化衍生后用HP-5MS柱分析。测定结果:所有脑苷脂系列物的糖基都为葡萄糖基;脑苷脂系列物Ⅰ的脂肪酸以非羟基脂肪酸为主,系列物Ⅱ和Ⅲ的脂肪酸以羟基脂肪酸为主;系列物Ⅰ和Ⅱ中,少见的d17:1长链碱比例较高,系列物Ⅲ中以t17:0长链碱为主,三种海参相比,仿刺参与叶瓜参的长链碱组成相似,主要为d18:2和d17:1,而海地瓜长链碱组成较简单,主要为d17:1和t17:0。并推测出9种海参脑苷脂系列物的结构通式。
又采用高效液相色谱-电喷雾离子阱-飞行时间质谱联用(LC-ESI-IT-TOF)技术,筛选分析三种海参总脑苷脂的脑苷脂分子种组成。采用TSKgel ODS-100Z柱分离,95%甲醇/水溶液洗脱,正ESI电离模式,根据一级质谱中同时出现的[M+H]~+、[M+Na]~+和[M+H-18]~+峰确定母离子,通过自动MS/MS采集m/z 200 ~ 300子离子数据,并分析它们的结构。分别从海地瓜、叶瓜参和仿刺参总脑苷脂中高通量筛选出12、41、26个脑苷脂化合物,其中包含疑似新化合物28个。最后,应用高效液相色谱-电喷雾四极杆-飞行时间质谱联用(LC-ESI-Q-TOF)技术,成功建立了高通量、准确筛选分析脑苷脂分子种组成及结构的新方法。采用Plus C18柱,95%乙腈/水洗脱,负ESI电离模式,自动MS/MS采集数据,一级质谱扫描范围m/z 500 ~ 1000,利用软件自动提取含m/z 179子离子的所有化合物,再根据二级子离子扫描筛出其中中性丢失为162的化合物。利用此方法,从海地瓜的三种脑苷脂系列物中分别筛选出28、20、12个脑苷脂化合物,其中31个为疑似新化合物。实验结果表明,与GC-MS和LC-ESI-IT-TOF法相比,此法测定灵敏度更高,结构信息全面,推测结果更准确。该方法的建立,为脑苷脂单体化合物的研究提供了捷径,为构效关系的深入研究提供了结构信息。
4.建立海参长链碱的高效制备新方法。海参总脂经皂化酸解后,采用溶剂体系为正己烷-甲醇-水(1:2:1,V/V)的高速逆流色谱法分离,以正ESI-MS法确证了长链碱目标洗脱组分,分离制备出三种海参长链碱,得率约为6.3%~10.9%,纯度均达到92%以上。采用LC-MS法分析了海参长链碱的组成和结构。该法具有制备量大、纯化周期短、得率高等突出优点。说明高速逆流色谱技术在长链碱类的快速纯化制备研究中具有很强的应用潜力。
5.研究海参脑苷脂及长链碱的抗肿瘤和抗炎活性,初步探讨其构效关系。
(1)抗肿瘤作用:主要采用MTT法,分别考察了不同浓度、不同作用时间下海参脑苷脂及长链碱对Caco-2,S180和HL-7702三种细胞系的增殖抑制作用情况。结果表明,海参总脑苷脂对Caco-2癌细胞增殖均有极显著的抑制作用;不同脑苷脂系列物对Caco-2细胞的抑制率存在显著差异,且活性效果优于GalCer,海参长链碱的抗肿瘤细胞增殖活性远强于脑苷脂本身;提示脑苷脂抗肿瘤活性与化学结构之间存在构效关系。(2)抗炎作用:采用Caco-2分化成的小肠上皮细胞,通过添加TNF-α和不同浓度的叶瓜参脑苷脂,荧光定量PCR法检测Caco-2细胞炎症因子IL-8 mRNA的表达量,表明叶瓜参脑苷脂具有较强的抗炎作用。以上结果提示,海参脑苷脂尤其是长链碱是重要生物活性成分,在新型海洋功能食品或海洋药物开发领域具有一定的应用前景。
通过以上研究,本文应用高速逆流色谱技术,解决了海参脑苷脂及长链碱分离纯化中存在的难题,针对海参脑苷脂组成复杂结构鉴定困难,采用液相色谱-串联质谱联用技术,实现了脑苷脂分子种的快速高通量筛选,并筛选出大量疑似新化合物,为海参鞘脂的深入研究及海参的营养和食疗评价提供了可靠的理论依据和研究基础。
Sea cucumber is a valuable tonic sea food, which is rich in a variety of biological active ingredients with high nutritional value and medicinal value. Cerebrosides and long-chain bases are the unique structure sphingolipids present in the body wall of sea cucumbers, which are proved to exhibit various physiological activities, including anti-tumor, immunomodulatory, neuroprotective, anti-bacterial. Sea cucumber cerebrosides are composed of three major series, which contains dozens of cerebroside molecular species. This thesis made a systemic study on the isolation, purification, molecular composition, structure analysis and physiological activities of cerebrosides and long-chain bases in three sea cucumbers (Apostichopus japonicus, Acaudina molpadioides and Cucumaria frondosa). These researches are important to discover new marine sources of raw materials and add new nutritional benefits of sea cucumbers. These results would provide theoretical basis for the development and high value utilization of sea cucumbers, to promote the healthy development of sea cucumber industry. In this paper, the main reserch contents and results are as follows:
1. A new simple and accurate method is established for the determination of the cerebroside content in sea cucumbers. By high performance liquid chromatography- evaporative light scattering detector (HPLC-ELSD), the TSKgel CN-80Ts column for separation, n-hexane-isopropyl alcohol-methylene chloride-methanol as mobile phase at 13 min to achieve the separation of cerebroside and ceramide. Among them, the detection limit of cerebroside is 0.01μg, average recovery rate was 94.28%, relative standard deviation is 5.67%, and the method has a good stability. In 13 species of aquatic products, the contents of cerebroside and ceramide of starfish are the highest, and cerebroside content of Cucumaria frondosa is the highest in eight species of sea cucumber samples. The results show that it is a feasible, simple, fast and accurate method to determine the contents of cerebroside and ceramide. The results suggest that marine foods, including sea cucumbers, can be useful resources of the functional sphingolipids.
2. A quickly, efficient, complete and new system is established for purification of sea cucumber cerebrosides. The first application of high-speed countercurrent chromatography (HSCCC) technique successfully optimized the separation process of sea cucumber cerebrosides. (1) Total cerebrosides from sea cucumber: using petroleum ether-methanol-water (5:4:1, V/V) as the solvent system of high-speed countercurrent chromatography, the total cerebrosides can be purifed in one step from the total lipids of sea cucumber. The yield of total cerebrosides of total lipids is about 7.6 ~ 12.4%, and its purity which detected by normal phase HPLC-ELSD method is more than 90%. (2) Cerebroside series from sea cucumber: firstly, using normal phase silica gel column chromatography, the fractions are collected eluting with chloroform/methanol (80:20, V/V) which contained cerebrosides. Then, ethyl acetate-ethanol-water (5:3.5:3.5, V/V) is used as the solvent system of high-speed countercurrent chromatography, to completely remove sulfated sterol which seriously interferes with the purification of sea cucumber cerebrosides. The yield of cerebroside series is about 1.1 ~ 6.4%, and its purity is more than 90%. The purification effect of HSCCC is validated and compared the optimized positive-phase HPLC method. (3) Sea cucumber cerebroside compounds: with the reversed phase-HPLC method which uses 98% methanol solution as mobile phase, four compounds are isolated from cerebroside series of sea cucumber Acaudina molpadioides, and their purities are more than 90%. The results show that the established system to purify cerebroside is high speed, low cost, with higher collection rate and outstanding merits. It is a fundamental solution to purification of sea cucumber cerebroside problems encountered in the process.
3. The cerebroside series separated from the three sea cucumbers respectively are firstly analyzed by gaschromatographic mass spectrometry (GC-MS) to acquire the differences among the compositions of their long-chain bases (LCBs), fatty acids (FAs) and glycosides. The cerebroside samples are hydrolyzed in 10% HCl-methanol to produce a mixture of fatty acid methyl ester (FAMEs), LCBs and methyl glycosides. After extraction with n-hexane, the upper phase containing FAMEs is directly subject to a GC-MS instrument equipped with an INNOWax column. The LCBs and methyl glycosides retained in the lower phase are react with TMS regent and then inject into the GC-MS instrument equipped with an HP-5MS column. The results show that the glycoside of all cerebroside series is glucoside. There is a significant difference in the composition of LCBs. The major fatty acid is nonhydroxy fatty acid for cerebroside seriesⅠ, however, hydroxy fatty acids are mainly FA for seriesⅡandⅢ. For Cucumaria frondosa and Stichopus japonicus sea cucumbers, the major LCBs of cerebroside seriesⅠ,Ⅱare d17:1 and d18:2, it is only d17:1 LCB in Acaudina molpadioides. For all the three sea cucumbers, the major LCB of cerebroside seriesⅢis t17:0. Finally, 9 series structural formula of cerebrosides in three sea cucumbers are speculated.
Secondly, a LC-ESI-IT-TOF technology is applied to screen the cerebroside compounds from the total cerebrosides of the three kinds of sea cucumbers. The preseperation of cerebroside compounds is performed on an ODS-100Z column, 95% methanol-water as eluent. The mass spectrometer is operated at positive electrospray ionization mode. The ions due to [M+H]+, [M+Na]+, [M-H2O+H]+ in MS scan spectra are used for determination of precusor ions. The product ions from m/z 200 to 300 are recorded automatically for structure determination. According to the methodology above, 12, 41, 26 cerebroside compounds are high throughput screened from Acaudina molpadioides, Cucumaria frondosa and Stichopus japonicus cerebrosides respectively, among which 28 suspected new compounds are found.
Finally, a more sensitive LC-ESI-Q-TOF methodology is successfully applied to the screening and structure determination of cerebroside molecular species. The preseperation is performed on a Plus C18 column, 95% acetonitrile-water as eluent. Mass spectrometer is operated at negative electrospray ionization mode, the data is recorded automatically. For mass scan, the full scan range is seted from 500 to 1000; for precursor ion scan, m/z 179 ion is used as the product ion. For product ion scan, the moleculars produce a fragment ion [M-162]- due to a neutral loss of a glucoside was remarked as cerebroside compounds. By application of the methodology above 28, 20, 12 cerebroside compounds were screened out from cerebroside seriesⅠ,ⅡandⅢrespectively, among which 31 suspected new compounds are found. The methodology is sensitive, and the information reflecting the structure is obtained more comprehensively and accurately compared to GC-MS and LC-ESI-IT-TOF method. The establishment of methodology provides a new approach for analysis of cerebroside monomers, and afforded the structure information in the relationship of structure-activity reasearch.
4. A new efficient method of preparing the sea cucumber long-chain base using high speed countercurrent chromatography (HSCCC) is established. The two-phase solvent systems were composed of hexane - methanol - water (1:2:1, V/V). The total lipids of sea cucumber are hydrolyzed with acid after saponification, then separated by HSCCC and monitored with the ESI-MS, the long-chain base is yield 6.3% ~ 10.9% with purities higher than 92%. The composition and structure of the sea cucumber is analyzed with LC-MS. Compared with the traditional Si-gel chromatographic separation method, the HSCCC separation process is a simple, solvent-saving, efficiency and of high recovery. The technology of HSCCC has a broad application prospect on the research of long-chain base.
5. Studies on the anti-tumor and anti-inflammatory activities of sea cucumber cerebrosides and long-chain bases, does pilot studies on their structure-activity relationship. (1) Anti-tumor effects: all total cerebrosides of three sea cucumbers have anti-proliferation activities on Caco-2 colon cancer cells in different concentrations, different time by MTT method. Compared with galactocerebrosides from mammals, cerebroside series from sea cucumbers suppresses the proliferation of Caco-2 colon cancer cells. The anti-tumor cell proliferation activity of long-chain bases from sea cucumber Cucumaria frondosa is much stronger than cerebrosides. These results suggest that antitumor activity of cerebroside have the relationship with its chemical structure. (2) Anti-inflammatory effect: Caco-2 cells can convert to normal intestinal epithelial cells after 14 days incubation, adds TNF-αand Cucumaria frondosa cerebroside, extracts RNA, and analyzes by semi-quantitative RT-PCR. The results suggest that the participation of different concentrations of cerebroside can significantly reduce the inflammatory cytokines IL-8 mRNA expression, indicating its strong anti-inflammatory effect. All of above elucidates that sea cucumber cerebrosides, especially long-chain bases, are important bioactive components, which suggests their potential applications on the new ocean marine functional food or a certain area of drug development.
In short, the puzzle is solved which exists in separating and purificating sea cucumber cerebrosides and long-chain bases using high-speed counter-current chromatography in this paper. On account of complex structure and difficulties identified of sea cucumber, we use chromatographic-tandem mass spectrometry to realize rapid high-throughput screening of cerebroside molecular species, and lots of suspected new compounds are found by this method. Researches in this thesis would provide reliable theoretical basis and experimental foundation for the further study, evaluation of nutrition and diet sea cucumbers.
1.建立海参中脑苷脂含量简便准确测定的新方法。采用高效液相色谱-蒸发光散射检测法(HPLC-ELSD),以TSKgel CN-80Ts柱为分离柱,正己烷-异丙醇-二氯甲烷-甲醇为流动相,在13min内实现了脑苷脂和神经酰胺的同时测定。其中,脑苷脂的检出限为0.01μg,加标平均回收率为94.28%,RSD为5.67%,方法稳定性良好。在所测13种水产样品中,海星的脑苷脂和神经酰胺含量最高,在所测8种海参中叶瓜参脑苷脂含量最高。结果表明该方法可行,样品前处理简单,快速准确。结果提示包括海参在内的海洋食品可以作为功能性鞘脂类的良好资源。
2.建立一套用于各种海参脑苷脂分离纯化的快速高效的完整新体系。首次应用高速逆流色谱(HSCCC)技术成功优化了海参脑苷脂的分离过程。(1)海参总脑苷脂:采用石油醚-甲醇-水(5:4:1, V/V)为溶剂体系的高速逆流色谱法,从海参总脂中一步纯化制备出海参总脑苷脂,以总脂计得率约为7.6~12.4%,经正相HPLC-ELSD法检测其纯度达90%以上。(2)海参脑苷脂系列物:先经一次正相硅胶柱层析,收集氯仿:甲醇(80:20,V/V)洗脱组分,再采用乙酸乙酯-乙醇-水(5:3.5:3.5, V/V)为溶剂体系的高速逆流色谱技术,将上述洗脱组分中严重干扰海参脑苷脂纯化的硫酸酯化甾醇去除,制得海参脑苷脂系列物的总得率约为1.1~6.4%,纯度均在90%以上。并以优化出的正相HPLC制备法进行了验证比较。(3)海参脑苷脂单体化合物:采用反相HPLC法,C18半制备柱,98%甲醇水溶液洗脱,从海地瓜脑苷脂系列物中分离出4个脑苷脂单体化合物,纯度在90%以上。结果表明,所建立的脑苷脂纯化体系具有得率高、速度快、成本低等突出优点,从根本上解决了海参脑苷脂纯化的难题。
3.建立海参脑苷脂分子种组成和结构筛选的高通量分析方法。
先采用气相色谱质谱联用(GC-MS)法,分析比较了三种海参脑苷脂系列物的脂肪酸、长链碱和糖基组成差异。用10%盐酸/甲醇水解,正己烷萃取脂肪酸甲脂组分,甲醇保留长链碱和糖基组分。脂肪酸甲酯直接用INNOWax柱分析,长链碱和甲基糖经硅烷化衍生后用HP-5MS柱分析。测定结果:所有脑苷脂系列物的糖基都为葡萄糖基;脑苷脂系列物Ⅰ的脂肪酸以非羟基脂肪酸为主,系列物Ⅱ和Ⅲ的脂肪酸以羟基脂肪酸为主;系列物Ⅰ和Ⅱ中,少见的d17:1长链碱比例较高,系列物Ⅲ中以t17:0长链碱为主,三种海参相比,仿刺参与叶瓜参的长链碱组成相似,主要为d18:2和d17:1,而海地瓜长链碱组成较简单,主要为d17:1和t17:0。并推测出9种海参脑苷脂系列物的结构通式。
又采用高效液相色谱-电喷雾离子阱-飞行时间质谱联用(LC-ESI-IT-TOF)技术,筛选分析三种海参总脑苷脂的脑苷脂分子种组成。采用TSKgel ODS-100Z柱分离,95%甲醇/水溶液洗脱,正ESI电离模式,根据一级质谱中同时出现的[M+H]~+、[M+Na]~+和[M+H-18]~+峰确定母离子,通过自动MS/MS采集m/z 200 ~ 300子离子数据,并分析它们的结构。分别从海地瓜、叶瓜参和仿刺参总脑苷脂中高通量筛选出12、41、26个脑苷脂化合物,其中包含疑似新化合物28个。最后,应用高效液相色谱-电喷雾四极杆-飞行时间质谱联用(LC-ESI-Q-TOF)技术,成功建立了高通量、准确筛选分析脑苷脂分子种组成及结构的新方法。采用Plus C18柱,95%乙腈/水洗脱,负ESI电离模式,自动MS/MS采集数据,一级质谱扫描范围m/z 500 ~ 1000,利用软件自动提取含m/z 179子离子的所有化合物,再根据二级子离子扫描筛出其中中性丢失为162的化合物。利用此方法,从海地瓜的三种脑苷脂系列物中分别筛选出28、20、12个脑苷脂化合物,其中31个为疑似新化合物。实验结果表明,与GC-MS和LC-ESI-IT-TOF法相比,此法测定灵敏度更高,结构信息全面,推测结果更准确。该方法的建立,为脑苷脂单体化合物的研究提供了捷径,为构效关系的深入研究提供了结构信息。
4.建立海参长链碱的高效制备新方法。海参总脂经皂化酸解后,采用溶剂体系为正己烷-甲醇-水(1:2:1,V/V)的高速逆流色谱法分离,以正ESI-MS法确证了长链碱目标洗脱组分,分离制备出三种海参长链碱,得率约为6.3%~10.9%,纯度均达到92%以上。采用LC-MS法分析了海参长链碱的组成和结构。该法具有制备量大、纯化周期短、得率高等突出优点。说明高速逆流色谱技术在长链碱类的快速纯化制备研究中具有很强的应用潜力。
5.研究海参脑苷脂及长链碱的抗肿瘤和抗炎活性,初步探讨其构效关系。
(1)抗肿瘤作用:主要采用MTT法,分别考察了不同浓度、不同作用时间下海参脑苷脂及长链碱对Caco-2,S180和HL-7702三种细胞系的增殖抑制作用情况。结果表明,海参总脑苷脂对Caco-2癌细胞增殖均有极显著的抑制作用;不同脑苷脂系列物对Caco-2细胞的抑制率存在显著差异,且活性效果优于GalCer,海参长链碱的抗肿瘤细胞增殖活性远强于脑苷脂本身;提示脑苷脂抗肿瘤活性与化学结构之间存在构效关系。(2)抗炎作用:采用Caco-2分化成的小肠上皮细胞,通过添加TNF-α和不同浓度的叶瓜参脑苷脂,荧光定量PCR法检测Caco-2细胞炎症因子IL-8 mRNA的表达量,表明叶瓜参脑苷脂具有较强的抗炎作用。以上结果提示,海参脑苷脂尤其是长链碱是重要生物活性成分,在新型海洋功能食品或海洋药物开发领域具有一定的应用前景。
通过以上研究,本文应用高速逆流色谱技术,解决了海参脑苷脂及长链碱分离纯化中存在的难题,针对海参脑苷脂组成复杂结构鉴定困难,采用液相色谱-串联质谱联用技术,实现了脑苷脂分子种的快速高通量筛选,并筛选出大量疑似新化合物,为海参鞘脂的深入研究及海参的营养和食疗评价提供了可靠的理论依据和研究基础。
Sea cucumber is a valuable tonic sea food, which is rich in a variety of biological active ingredients with high nutritional value and medicinal value. Cerebrosides and long-chain bases are the unique structure sphingolipids present in the body wall of sea cucumbers, which are proved to exhibit various physiological activities, including anti-tumor, immunomodulatory, neuroprotective, anti-bacterial. Sea cucumber cerebrosides are composed of three major series, which contains dozens of cerebroside molecular species. This thesis made a systemic study on the isolation, purification, molecular composition, structure analysis and physiological activities of cerebrosides and long-chain bases in three sea cucumbers (Apostichopus japonicus, Acaudina molpadioides and Cucumaria frondosa). These researches are important to discover new marine sources of raw materials and add new nutritional benefits of sea cucumbers. These results would provide theoretical basis for the development and high value utilization of sea cucumbers, to promote the healthy development of sea cucumber industry. In this paper, the main reserch contents and results are as follows:
1. A new simple and accurate method is established for the determination of the cerebroside content in sea cucumbers. By high performance liquid chromatography- evaporative light scattering detector (HPLC-ELSD), the TSKgel CN-80Ts column for separation, n-hexane-isopropyl alcohol-methylene chloride-methanol as mobile phase at 13 min to achieve the separation of cerebroside and ceramide. Among them, the detection limit of cerebroside is 0.01μg, average recovery rate was 94.28%, relative standard deviation is 5.67%, and the method has a good stability. In 13 species of aquatic products, the contents of cerebroside and ceramide of starfish are the highest, and cerebroside content of Cucumaria frondosa is the highest in eight species of sea cucumber samples. The results show that it is a feasible, simple, fast and accurate method to determine the contents of cerebroside and ceramide. The results suggest that marine foods, including sea cucumbers, can be useful resources of the functional sphingolipids.
2. A quickly, efficient, complete and new system is established for purification of sea cucumber cerebrosides. The first application of high-speed countercurrent chromatography (HSCCC) technique successfully optimized the separation process of sea cucumber cerebrosides. (1) Total cerebrosides from sea cucumber: using petroleum ether-methanol-water (5:4:1, V/V) as the solvent system of high-speed countercurrent chromatography, the total cerebrosides can be purifed in one step from the total lipids of sea cucumber. The yield of total cerebrosides of total lipids is about 7.6 ~ 12.4%, and its purity which detected by normal phase HPLC-ELSD method is more than 90%. (2) Cerebroside series from sea cucumber: firstly, using normal phase silica gel column chromatography, the fractions are collected eluting with chloroform/methanol (80:20, V/V) which contained cerebrosides. Then, ethyl acetate-ethanol-water (5:3.5:3.5, V/V) is used as the solvent system of high-speed countercurrent chromatography, to completely remove sulfated sterol which seriously interferes with the purification of sea cucumber cerebrosides. The yield of cerebroside series is about 1.1 ~ 6.4%, and its purity is more than 90%. The purification effect of HSCCC is validated and compared the optimized positive-phase HPLC method. (3) Sea cucumber cerebroside compounds: with the reversed phase-HPLC method which uses 98% methanol solution as mobile phase, four compounds are isolated from cerebroside series of sea cucumber Acaudina molpadioides, and their purities are more than 90%. The results show that the established system to purify cerebroside is high speed, low cost, with higher collection rate and outstanding merits. It is a fundamental solution to purification of sea cucumber cerebroside problems encountered in the process.
3. The cerebroside series separated from the three sea cucumbers respectively are firstly analyzed by gaschromatographic mass spectrometry (GC-MS) to acquire the differences among the compositions of their long-chain bases (LCBs), fatty acids (FAs) and glycosides. The cerebroside samples are hydrolyzed in 10% HCl-methanol to produce a mixture of fatty acid methyl ester (FAMEs), LCBs and methyl glycosides. After extraction with n-hexane, the upper phase containing FAMEs is directly subject to a GC-MS instrument equipped with an INNOWax column. The LCBs and methyl glycosides retained in the lower phase are react with TMS regent and then inject into the GC-MS instrument equipped with an HP-5MS column. The results show that the glycoside of all cerebroside series is glucoside. There is a significant difference in the composition of LCBs. The major fatty acid is nonhydroxy fatty acid for cerebroside seriesⅠ, however, hydroxy fatty acids are mainly FA for seriesⅡandⅢ. For Cucumaria frondosa and Stichopus japonicus sea cucumbers, the major LCBs of cerebroside seriesⅠ,Ⅱare d17:1 and d18:2, it is only d17:1 LCB in Acaudina molpadioides. For all the three sea cucumbers, the major LCB of cerebroside seriesⅢis t17:0. Finally, 9 series structural formula of cerebrosides in three sea cucumbers are speculated.
Secondly, a LC-ESI-IT-TOF technology is applied to screen the cerebroside compounds from the total cerebrosides of the three kinds of sea cucumbers. The preseperation of cerebroside compounds is performed on an ODS-100Z column, 95% methanol-water as eluent. The mass spectrometer is operated at positive electrospray ionization mode. The ions due to [M+H]+, [M+Na]+, [M-H2O+H]+ in MS scan spectra are used for determination of precusor ions. The product ions from m/z 200 to 300 are recorded automatically for structure determination. According to the methodology above, 12, 41, 26 cerebroside compounds are high throughput screened from Acaudina molpadioides, Cucumaria frondosa and Stichopus japonicus cerebrosides respectively, among which 28 suspected new compounds are found.
Finally, a more sensitive LC-ESI-Q-TOF methodology is successfully applied to the screening and structure determination of cerebroside molecular species. The preseperation is performed on a Plus C18 column, 95% acetonitrile-water as eluent. Mass spectrometer is operated at negative electrospray ionization mode, the data is recorded automatically. For mass scan, the full scan range is seted from 500 to 1000; for precursor ion scan, m/z 179 ion is used as the product ion. For product ion scan, the moleculars produce a fragment ion [M-162]- due to a neutral loss of a glucoside was remarked as cerebroside compounds. By application of the methodology above 28, 20, 12 cerebroside compounds were screened out from cerebroside seriesⅠ,ⅡandⅢrespectively, among which 31 suspected new compounds are found. The methodology is sensitive, and the information reflecting the structure is obtained more comprehensively and accurately compared to GC-MS and LC-ESI-IT-TOF method. The establishment of methodology provides a new approach for analysis of cerebroside monomers, and afforded the structure information in the relationship of structure-activity reasearch.
4. A new efficient method of preparing the sea cucumber long-chain base using high speed countercurrent chromatography (HSCCC) is established. The two-phase solvent systems were composed of hexane - methanol - water (1:2:1, V/V). The total lipids of sea cucumber are hydrolyzed with acid after saponification, then separated by HSCCC and monitored with the ESI-MS, the long-chain base is yield 6.3% ~ 10.9% with purities higher than 92%. The composition and structure of the sea cucumber is analyzed with LC-MS. Compared with the traditional Si-gel chromatographic separation method, the HSCCC separation process is a simple, solvent-saving, efficiency and of high recovery. The technology of HSCCC has a broad application prospect on the research of long-chain base.
5. Studies on the anti-tumor and anti-inflammatory activities of sea cucumber cerebrosides and long-chain bases, does pilot studies on their structure-activity relationship. (1) Anti-tumor effects: all total cerebrosides of three sea cucumbers have anti-proliferation activities on Caco-2 colon cancer cells in different concentrations, different time by MTT method. Compared with galactocerebrosides from mammals, cerebroside series from sea cucumbers suppresses the proliferation of Caco-2 colon cancer cells. The anti-tumor cell proliferation activity of long-chain bases from sea cucumber Cucumaria frondosa is much stronger than cerebrosides. These results suggest that antitumor activity of cerebroside have the relationship with its chemical structure. (2) Anti-inflammatory effect: Caco-2 cells can convert to normal intestinal epithelial cells after 14 days incubation, adds TNF-αand Cucumaria frondosa cerebroside, extracts RNA, and analyzes by semi-quantitative RT-PCR. The results suggest that the participation of different concentrations of cerebroside can significantly reduce the inflammatory cytokines IL-8 mRNA expression, indicating its strong anti-inflammatory effect. All of above elucidates that sea cucumber cerebrosides, especially long-chain bases, are important bioactive components, which suggests their potential applications on the new ocean marine functional food or a certain area of drug development.
In short, the puzzle is solved which exists in separating and purificating sea cucumber cerebrosides and long-chain bases using high-speed counter-current chromatography in this paper. On account of complex structure and difficulties identified of sea cucumber, we use chromatographic-tandem mass spectrometry to realize rapid high-throughput screening of cerebroside molecular species, and lots of suspected new compounds are found by this method. Researches in this thesis would provide reliable theoretical basis and experimental foundation for the further study, evaluation of nutrition and diet sea cucumbers.
引文
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