Quantitative Analysis For HolotoxinA₁in Apostichopus Japonicus And The Study On The Characteristic Chromatograms Of Triterpene Glycosides From Diffetent Sea Cucumbers

Sea cucumber is a kind of marine animals belonging to class Holothurioidea andphylum Echinodermate. It contains significant nutrition and medicinal values. Thetriterpene glycosides which possess various pharmacological activities, includinganti-tumor, antifungal, hemolysis and immunomodulatory effects, are anindispensable index for the quality assessment of sea cucumber. Affected by the seaarea and growing environment, the triterpene glycosides in various sea cucumbers areof great difference in their composition and content. In this article, we described anew method of combination of HPLC-ELSD and HPLC-MS assay for thedetermination and identification of triterpene glycosides. Then the method wasapplied to the quality assessment on triterpene glycosides of Apostichopus japonicuswhich ownes the highest value in the edible and medicinal sea cucumber. Moreover,the characteristic chromatograms of triterpene glycosides from diffetent sea cucumberwere established by this method. The research will make up the blank ofquantification for triterpene glycosides due to the lack of standard substance andprovide reliable means and theoretical foundation on the quality evaluation as well asthe characterization and classification of sea cucumber. Furthermore. It will be a greatsignificance for building the overall and regulatory quality criterion of sea cucumber.The main researches in this paper are listed below:The Apostichopus japonicus was refluxed with60%ethanol（75℃）. TheHolotoxin A₁was separated and purified by organic solvents, macroporous resin andHPLC. The structure of Holotoxin A₁was characterized by the MS and NMR and canbe used as reference substance by the HPLC-ELSD analysis.The HPLC-ELSDmethod was performed on a Hypersil Gold C18（150mm×2.1mm，3μm）, usingacetonitrile! water (containing5mmol L^-1ammonium acetate) as the mobile phase with gradient elution. Then the method was applied to the quality analysis onApostichopus japonicus which was purified by ODS-SPE coulum using the HolotoxinA₁as reference substance. The established method of HPLC-ELSD indicated that thecalibration curve shows good linearity from0.2μg mL^-1to25μg mL^-1（r=0.999） andthe lower limit of detection was0.05μg mL^-1at a signal to noise ratio of3. Due to thevery high sensitivity, precision and accuracy, the method described here is proven tobe a reproducible and reliable tool for fast analysis of triterpene glycosides. Thecontent of HolotoxinA₁is of great difference in Apostichopus japonicus, whichimproves from south to north. The Apostichopus japonicus from east coast of SouthKorea is the highest(588.20μg g^-1)and the Apostichopus japonicus from Fujianprovince is the leas(t21.33μg g^-1)according to the analysis.In this research, the issueon lacking of standard substance to quantify and containing complex components toanalysis was resolved by this accurate and sensitive method. The research willprovide scientific basis for the quality of future sea cucumbers.Two kinds of sea cucumber from Stichopodidae and three kinds of sea cucumberfrom Holothuriidae were analysised by HPLC-MS with the established method. Theprobable triterpene glycosides were speculated based on every molecular ion peak inHPLC-MS and the specific chromatograms of different sea cucumbers wereestablished. Affected by the sea area and growing environment, the triterpeneglycosides in these five sea cucumbers are of great difference in their composition andcontent. However, the triterpene glycosides in the same family also show greatsimilarity and peculiarity, which can be a great evidence for their affinity on thetaxonomy. Theoretical foundation and scientific basis for the quick identification oftriterpene glycosides, species discrimination and quality assessment of sea cucumberare provided by the results of this paper.