Isolation, Purification And Characterization Of Different Kinds Of Sea Cucumber Polysaccharides

Sea cucumber is a traditional costful tonic food in China,which contains multi-kinds of bioactive components. It is well known that holothurians are of have high nutrition and pharmic values.In our country, the demand for sea cucumber and sea cucumber products is increasing with the living standard improving. Polysaccharides is one of the most important bioactive composition in sea cucumber,it has been proved to exhibit various biological activities, including anti-coagulation, anti-thrombosis, reduce blood fat and tumor suppression activity. There were mainly two kinds of sea cucumber polysaccharides,sea cucumber chondroitin sulfate (SC-CHS) and sea cucumber fucoidan (SC-FUC). We made a systemic study on the biochemical analysis of the SC-CHS and SC-FUC from the body wall of 9 kinds of sea cucumbers ,which have different types or in different growth maritime space. The results would provide theoretical basis for the identification of differernt types of sea cucumbers, also for quality assessment of sea cucumber products.The paper mainly includes the following four parts:1. Crude polysaccharides were prepared by mild enzymatic extraction from 9 kinds of sea cucumbers.Two polysaccharides,SC-CHS and SC-FUC,were isolated after DEAE-52 ion-exchange chromatography.The purity was determined by high-performance gel filtration chromatography (HPSEC) and cellulose acetate membrane electrophoresis. SC-FUC have a molecular weight more than 700KD,comparing with SC-CHS of a molecular weight of 124.9KD-215.3KD. The results showed that the temperate sea cucumber is rich in SC-CHS, tropical and subtropical sea cucumber is rich in SC-FUC, and those live in frigid waters was among them.2. Monosaccharides composition of SC-CHS and SC-FUC were analyzed by pre-column derivatization-HPLC and sulfate content was determined by Ion Chromatography respectively.The monosaccharides composition of the SC-CHS were similar to that of chondroitin sulfate,mainly with nearly same molar ratio of glucuronic acid (GlcUA),galactosamine (GlaN),but with large amount of fucose (Fuc).The molar ratio of monosaccharides of the SC-CHS shows differences among the 9 species of sea cucumbers. Fucose is the only main monosaccharide in the SC-FUC.Crude sea cucumber polysaccharides has a sulfate content of 23.61%-31.03%,and the sulfate content of SC-CHS and the SC-FUC were 27.52%-32.66% and 23.42%-29.31% respectively. The SC-CHSs have higher sulfate content than the SC-FUCs with the same sea cucumber species.Monosaccharides composition analysis and the sulfate content determination were of great importance for the establishment of the quality evaluation criteria of different kinds of sea cucumbers, as well as futher research on the bioactivity of sea cucumber polysaccharides.3. IR and 1H NMR analysis of SC-CHS and SC-FUC.Sulfate substitutions on branched fucose of the SC-CHS were preliminary analysised by IR.Certain characteristic differences existed among different SC-CHS on infrared absorption. As a simple, inexpensive method,FT-IR could be used as the initial tool on SC-CHS analysis.1H NMR analysis of anomer hydrogen signals of sulfate fucose in SC-CHSs living in different water conditions shows that,sulfate substitutions on branched fucose of SC-CHS from sea cucumbers could be splitted into 4 types:1) Sea cucumber live in frigid zone:mainly 2,4-di OSO₃ and 3,4-di OSO₃ disubstituted, also with minor 4-OSO₃ substitution; 2) Temperate sea cucumber:mainly 2,4-di OSO₃ ,also with partially 4-OSO₃ substitute; 3)Sea cucumbers live in tropical and subtropical waters have 2 types of substitution:one is only 2,4-di OSO₃ substitution,the other is mainly 2,4-di OSO₃ and 4-OSO₃,with minor non-sulfated; 4) Sand fish (Holothuria scabra Jaeger)from subtropical and tropical waters has a similar sulfate substitution style on branched fucose of SC-CHS to that of the temperate sea cucumbers, but with more 4-OSO₃ substitution.Sulfate substitutions styles on sulfated branched fucose of SC-CHS,which ascertained by 1H NMR,could provided important proves for the study on the structure-activity relationship of SC-CHS.4. The HPLC method to determine sea cucumber polysaccharides content in different commercial sea cucumber products was established. PMP-HPLC was used to determine sea cucumber polysaccharides content in different products ,with fucose conversion coefficient as media.This method was accurate and sensitive,could be applied to determination of the total polysaccharide content of a variety of sea cucumber product,and help to establish the quality standards of sea cucumber products.