Preparation Of Zinc Hyaluronate And Quality Control Method By X-ray Fluorescence

Ulcerative colitis is a kind of inflammatory bowel disease,the etiology is not clear.It is mostly found in people aged 20-30.The lesion is located in the sigmoid colon and rectum,which is characterized by a abdominal pain,blood in the stool,weight loss,tenesmus,and vomiting.The main clinical feature is ulceration in the inner wall of colon.Some studies have shown that zinc,as an essential human trace element,is involved in the synthesis of various enzymes,nucleic acids and proteins in the body.It is closely related to cell division,growth and regeneration in the human body,and it also has some antibacterial effects.In the process of wound healing,the hyaluronic acid has the effect of reducing the level of inflammatory mediators and at the same time,it can form a slightly acidic environment on the surface of the ulcer and inhibit bacteria.The zinc hyaluronate which was formed by zinc and HA can yield a good synergistic effect and enhance therapeutic effects and it can treat diseases such as corneal ulcer and diabetic foot ulcer.In this paper,the preparation process of zinc hyaluronate was studied,and the zinc content was determined by EDXRF method,and the animal model of ulcerative colitis was successfully constructed.The inhibitory effect of zinc hyaluronate on ulcerative colitis was studied by HE staining,immunohistochemistry and other animal experiments.The main research contents included the following three aspects:(1)The effects of sodium hyaluronate molecular weight,zinc salt type,ethanol concentration,zinc salt concentration,reaction temperature and reaction time on the glucuronic acid content in zinc hyaluronate were investigated by single factor,and then the optimal experimental protocol was selected through orthogonal experiments.The optimal preparation conditions for zinc hyaluronate are: sodium hyaluronate molecular weight 1.3 million,zinc acetate,ethanol concentration 80%,zinc salt concentration 2%,reaction time 40℃,reaction time 1 hour.The glucuronic acid content of zinc hyaluronate which was prepared under these conditions was 43.18%.(2)The EDXRF method was established for the determination of zinc in zinc hyaluronate.A 0.3% concentration sodium hyaluronate solution was used as a blank matrix,and various amounts of zinc sulfate solution were precisely added to establish an elemental standard curve.A 0.3% concentration of zinc hyaluronate solution was used as a sample solution.The zinc content was determined under the conditions of a tube pressure of 40 KV,a tube flow of 150 mA,a No.1 collimator,and a No.3 filter.In addition,this method was compared with the EDTA-2Na method which in the pharmacopoeia was for determine of zinc,and the results showed no significant difference in t test(P>0.05).(3)Forty Balb/C mice were randomly divided into normal group,model group,zinc sulphate group,sodium hyaluronate group,positive drug sulfasalazine(SASP)group,low-,moderate-,and high-dose zinc hyaluronate groups,5 in each group.In addition to the normal group,other seven groups were given free drinking of 5% Dextran sodium sulfate(DSS)for 7 days to induce ulcerative colitis.Except the normal and model group,other six groups received medication by gavage.The inhibitory effect of ZnHA on ulcerative colitis was detected by HE staining,immunohistochemistry,inflammatory cytokines and Western blot as well.The HE staining revealed that the ulcer areas in the moderate-dose ZnHA group and the SASP group were smaller in the moderate-dose group ZnHA and the SASP group.Immunohistochemistry also indicated that the positive rate of these two groups was higher than that of the other medication groups.As compared with those of the normal group,there were no significant differences in the levels of inflammatory cytokines and the closely-linked protein(P>0.05).Conclusion:ZnHA had a certain inhibitory effect on ulcerative colitis.In summary,this article identifies the optimal preparation process of zinc hyaluronate,establishes a new method to determine its zinc content which can be used for its quality control and process control,and animal model of ulcerative colitis was constructed successfully.The inhibitory effect of zinc hyaluronate on ulcerative colitis was verified by pharmacological experiments.