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Studies On The Purification, Analysis And Bioactivities Of Cerebrosides From Sea Cucumbers

Posted on:2012-05-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:J XuFull Text:PDF
GTID:1114330338465628Subject:Food Science
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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.
Keywords/Search Tags:Cerebrosides, Long-chain bases, Sea cucumbers, High-speed counter-current chromatography, Liquid chromatography-electrospray ionization tandem mass spectrometry, Biological activities
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