Preparation, Characterization And Activity Of Bi³⁺-hyaluronate Complex

Background and purpose Peptic ulcer disease is a chronic disease of high morbidity in digestive system.The pathophysiology of peptic ulcer disease has centered on an imbalance between aggressive and protective factors to the gastrointestinal mucosa.It has been acknowledged that H.pylori infection is a major aggressive factor to the gastrointestinal mucosa.Bismuth compounds have been used in treatment of peptic ulcer widely because of the anti-H,pylori activity and mucosa protective effect.Hyaluronic acid(HA)is a glycosaminoglycan composed of D-glucuronic acid and D-N-acetylglucosamine with versatile biological activities, such as matrix construction,water retention and lubricating action,wound-healing effect,etc.HA and its derivatives have been utilized widely in medicine.Recent years, interests have been focused on the metal complex of HA.To develop new bismuth compounds utilized in the treatment of peptic ulcer,Bi³⁺-hyaluronate complexes were prepared and characterized,and the anti-H,pylori and anti-ulcerogenic activity of the complexes were evaluated.Methods Bi³⁺-hyaluronate complexes(BiHA)using HA with Mr of 1.24×10⁶, 6.75×10⁵ and 2.40×10⁵ and Bi(NO₃)₃·5H₂O as materials were prepared.The complexes were characterized by elemental analysis,¹³C-NMR,FT-IR,CD, DSC-TGA,XRD and XPS.The MICs of BiHA against three H.pylori international type strains were determined by agar dilution method.The effects of BiHA on the ultrastructure of H.pylori were observed by transmission electron microscopy(TEM). The effects of BiHA on cell cycle of H.pylori were evaluated by flow cytometry.The anti-ulcerogenic activities of BiHA were evaluated using chronic gastric ulcer model induced with acetic acid and acute gastric mucosa injury model induced with acidified alcohol and aspirin.The mechanism of ulcer-healing effect of the complexes was also explored.Results BiHA after purification were white amorphous powder.The complexes could readily resolve in water to form alkaline colloidal solution with high viscosity. In BiHA,the molar ratio of Bi³⁺to disaccharide unity(C₁₄H₂₀O₁₁N)was about 0.8∶1. The participation of-OH,-NH₂ and -COO^- in the coordination of HA with Bi³⁺was confirmed.After coordination,the conformation of the glycosaminoglycan chains changed and Bi³⁺was scattered thoroughly on the glycosaminoglycan chains.The thermal chemistry properties of BiHA changed a lot compared with HA.The coordination of Bi³⁺with HA through O and N as coordination atoms was confirmed by XPS.The MICs of the complexes and bismuth potassium citrate(BPC)against three H.pylori type strains were both 5μg/ml,indicating the similar antibacterial activity in vitro of BiHA and BPC.Under the effect of BiHA which was similar with that of BPC,the cytoplasm of H.pylori was not homogeneous and contained cytoplasmic aggregates.Large amounts of H.pylori tumed into coccoid forms.Both BiHA and BPC could arrest the cell cycle of H.pylori in G₁ phase,thus inhibit the proliferation of H.pylori.Among the complexes,BiHA prepared with HA with Mr of 1.24×10⁶ and 6.75×10⁵ could accelerate the healing of chronic ulcer and increase the quality of ulcer healing.The ulcer-healing effect of BiHA was better than that of BPC. However,the prophylatic effect of BiHA on acute gastric mucosa injury was poorer than that of BPC.The ulcer-healing mechanism of BiHA may owing to the increase of EGF expression and HA content in the gastric mucosa.Conclusion and significance Bi³⁺-complexes show favorable anti-H,pylori activity and anti-ulcerogenic activity and may be developed into a new bismuth containing drug utilized in the treatment of peptic ulcer disease.