| Sea cucumber is a traditional costful tonic sea food in China, which contains multi-kinds of bioactive components. It is well known that holothurians are of high nutrition and pharmic values. Triterpene glycosides, one of the most important bioactive compositions of sea cucumbers, are proved to exhibit various biological activities, including anti-tumor, antifungal, immunomodulatory, cytotoxic and hemolytic effects. This thesis make a study on the isolation and identification on triterpene glycosides in sea cucumber Apostichopus japonicus, their fragmentation patterns are investigated using electrospray ionization-mass spectrometry (ESI-MS) method, and finally establish the fingerprint chromatograms of triterpene glycosides in S. japonicus by high performance liquid chromatography (HPLC). This method is successfully used for the characteristic spectrum and structure analyze of eight other different sea cucumbers. The researches will provide some academic and scientific groundwork for the identification of different types of sea cucumbers, also for quality control and nutrition evaluation of sea cucumber. The main researches in this thesis are listed below:1. To study the constituents and proportion of triterpene glycosides from Apostichopus japonicus, macroporous resin, Si-gel chromatography and semi-preparative HPLC are used. Six triterpene glycosides are found, chemical structures of three compounds are identified. They are cladoloside B, holotoxin A1 and holotoxin B1 based on the chemical evidence and spectral data.2. The fragment pathways of nine kinds of triterpene glycosides from sea cucumbers has been investigated in negative ion mode by electrospray ionization -time of flight-mass spectrometry (ESI-TOF-MS). The[M-H]-/ [M-Na]-ions are observed by ESI-MS, from which the molecular weights are obtained. The collision induced dissociation (CID) data of the [M - H] - / [M - Na] - ions provide fragmentation pathways of the triterpene glycosides. In the MS/MS spectra, results show that the cleavage of non-sulfated triterpene glycosides is occurred on the sugar chain. Product ions are produced by consecutively losses of monosaccharide residue from sugar chain, and finally yield a signature ion of aglycone. These characteristic fragmentations will be helpful in identifying the structures of triterpene glycosides. The sulfated triterpene glycosides have different fragmentation pattern, the cleavage is occured on the agalycon, generating a more stable structure. After solvolysis, the desufated productions show signature ions by the cleavage of glucosidic bonds again. The fragmentation patterns are used for the structural conjecture of three minor triterpene glycosides from Apostichopus japonicus, and their structures are concluded by the information of MS/MS spectra.3. The fingerprint chromatograms of triterpene glycosides in S. japonicus are established for its quality control. The samples are prepared by solid phase extraction (SPE). The analysis is performed on a Zorbax SB-C18 column (250 mm×4.6 mm, 5μm) with acetonitrile-water (containing 0.1% phosphoric acid) as mobile phase, gradient elution, at a flow rate of 1.0 mL/min, at a column temperature of 30℃, and the detection wavelength is 205nm. Ten different original samples are analyzed, and 6 peaks are identified as common fingerprint peaks using the similarity evaluation system for chromatographic fingerprint of TCM(Version 2004 A)recommended by State Pharmacopoeia Committee of China. The similarity of the fingerprints is greater than 0.97. The method set up is proved to be stable and repeatable and can be utilized as a quality control for S. japonicus.4. A SPE-HPLC methord is established for the species identification by comparing the composition of triterpene glycosides among different sea cucumbers. The analysis is performed on a Zorbax SB-C18 column (250mm×4.6 mm, 5μm), use acetonitrile- water (containing 0.1% trifluoroacetic acid) as mobile phase, gradient elution, at a flow rate of 1.0 mL/min, at a column temperature of 30℃, and the detection wavelength is set at 205nm. The HPLC analysis of triterpene glycosides from different sea cucumbers is established for the first time. Among the obtained characteristic spectrums, all the detected peaks are separated effectively. The results show that triterpene glycosides in different sea cucumbers are significant difference. The method established is simple, stable, and can be utilized as a quality control for sea cucumber.5. A convenient method based on high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) has been developed for analysis and characterization of triterpene saponins in extract of different sea cucumbers. Samples are analyzed on a Eclipse XDB-C18 column ( 150mm×4.6mm, 5μm ) , with acetonitrile-water (containing 0.1% ammonium acid carbonate) as mobile phase, gradient elution. ESI-TOF-MS is used to obtain accurate molecular weight and molecular formula information of triterpene glycosides. Fragmentation behavior is obtained by CID experiment. Several chromatographic peaks are chosen for signature peaks for different sea cucumbers, and their unique compositions are verified. Combined with literature review, some triterpene glycosides are primary identified. This method has the advantages of simple operation, rapid measurement, and it is a powerful tool for identification of new triterpene glycosides. |
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