| Gastric ulcer refers to mucous layer, sunmucous layer, even serosa which is regulated by gastric juice,gastric acid and pepsin in the alimentary tract harm by several factors as a limitation tissue damage disease. Gastric ulcer was characterized by recurrence and difficulty in prevention. To improve quality of ulcer healing is the point to accure gastric ulcer. Quality of ulcer healing closely related to repair injured mucosa which require not only to restore mucosa surface but also to renovate underlayer. By way of research gastric ulcer pathogenesis it was concluded that antiinflammtory and protect stomach and intestinal mucosa are important principles for ulcer treatment.As one of basic materials of life, mucopolysaccharide participates in overwhelming majority vital activity. The distinctive bioactivity of mucopolysaccharide has become hot topic in the field of biochemistry and medicinal research. Over hundreds of bioactive carbohydrates were found, more and more researches about their structure and function are published. The anti-inflammatory activity is the hottest spot of this realm for the carbohydrate's diverse activity mechanisms. There are also some reports announced that the bioactivity of carbohydrate could be promoted through chemical modification.In view of gastric ulcer pathology, sulglycotide is applied to research in our study. First, glycotide was prepared from swine duodenum, then it was sulfated to obtain sulglycotide. Based on it we designed heparan as raw material to find a new way to prepare sulglycotide and optimize the process of preparation, and make the quality specification of sulglycotide. The activity of sulfonation products were screened by rat ulcer model. And the active sulglycotide was purified and the molecular weight was tested. The carbohydrateof sulglycotide was released by trypsin enzymolysis to research the pharmacology activity relationship between sulglycotide and carbohydrates. In order to investigate the potential anti-inflammatory and anti-ulcer activities of sulglycotide, the effects on macrophage cell secreted TNF-a and IL-4 and effects on experiment gastric ulcer rat were studied.The results and conclusions of our research were as follows:1 The preparation and sulfationprocess optimization of glycotideFirst, swine duodenum was skimmed, enzymed and anion exchange resin chromatography purification to get glycotide, the yield was 0.96%-1.17%, which was almost as twice as reported. Glycotide was confirmed relatively purified by cellulose acetate membrane electrophoresis. The total saccharide content is (22.47±1.41)% tested by phenol-sulfuric method, the protein content is (14.15±1.06)% tested by BCA protein reagent measuring kit. Glycotide was modified by three sulfation menthods, the definite method was cholrosulfonic acid-formamide with the product SG Single factor experiments and orthogonal design (3 factors & 4 levels) experiments were designed to optimize sulphation process showed that the most suitable sulphation condition was as follows:cholrosulfonic acid-formamide, reaction is controlled by 23℃to 8 hour with pH9.5 to oxidize and remove ammonia. Three heparans were carried sulfation modified by optimal sulfationprocess. The products were marked HPS-1, HPS-2, HPS-3. The products purified with ultrafiltration for desalinization and the purity was confirmed by PAGE.2 The anti-ulcer activity screening of sulglycotide and purificationThe four products were screened with gastric ulcer model, and the result which judged by ulcer index and pathology slice showed that SG and HPS-1 possessed anti-ulcer activity. The results showed that the anti-ulcer activity could be related with sulphuric acid content and total saccharide content. SG and HPS-1 were separated with QFF anion exchange resin chromatography to obtain different ionic strength constituent. They are SG-1,SG-2,SG-3,SG-4,SG-5 and HPS-1-1,HPS-1-2.3 glycochains release from sulglycotide and test the average molecular weightThe glycochains was released by enzymolysis from sulglycotide, the products were S-1 and H-1. UV spectra revealed that there were both saccharide and protein in sulglycotide while only saccharide not protein with glycochains. The average molecular weight of compositions SG-1, HPS-1 are respectively:99.54kDa,33.97kDa, detected by the technology of Multi-angle Laser Scattering.4. Effects of each component of sulglycotide and their glycochains on cell growth factors extracted from rat abdominal macrophages cellThe effects of each component of sulglycotide and their glycochains on cultured rat macrophage cell vitality were determined by trypan blue exclusion and MTT method, followed by TNF-αand IL-4 content determination via ELISA kit. The result suggested that sulglycotide and their glycochains with different concentration decreased TNF-αand IL-4 secretion, especially SG-3 and SG-4. Besides inhibition effects could be strengthen by concentration rising. The contents of rat TNF-a were 307.40±25.71 pg/mL and 230.59±24.42 pg/mL which the contents of SG-3 were 250μg/mL and 500μg/mL. The contents of rat TNF-αwere 243.55±4.18 pg/mL and 207.18±35.68 pg/mL which the contents of SG-4 were 250μg/mL and 500μg/mL. The contents of rat IL-4 were 54.98±1.03 pg/mL and 46.43±0.55 pg/mL which the contents of SG-3 were 250μg/mL and 500μg/mL. The contents of rat IL-4 were 49.98±1.68 pg/mL and 43.40±1.20 pg/mL which the contents of SG-4 were 250μg/mL and 500μg/mL. Those were obviously lower than model group. It can be initially concluded that each component of sulglycotide and their glycochains exhibited inhibition on the secretion of pro-inflammation cytokines.5. Therapeutic effects of SG-3 and SG-4 on experimental gastric ulcer ratRat gastric ulcer model was established through improved Okabe method, so as to observe the therapeutic effects of SG-3 and SG-4. The experiment that ulcer tissue was dyed through HE suggested that both SG-3 and SG-4 exhibit therapeutic effects on model rat. Expressions of caspase 3, EGFR, NO, NOS, and ET-1 were investigated. After healing 16 days, higher contents of SG processed special effects on them as the following:the inhibition rate of caspase 3 was (81.22±1.62)%, the rising of EGFR was 2.25±0.11 folds, the content of NO declined to (34.55±2.38)μmol/L, the content of cNOS rised to (8.43±0.84) U/mL, the content of iNOS declined to (6.81±1.99) U/mL, the content of EGF rised to (35.49±2.67) pg/mL, the content of ET-1 declined to (6.31±0.32) pg/mL. Those were obviously lower than model group. The conclusion is that sulglycotide can repair gastric mucosal injury entirely so as to prevent gastric ulcer recurrence by SG improving those index obviously. |
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