Preparation And Evaluation Of PNS-BSP Composite Microspheres For Gastric Mucosal Protection

In recent years,based on the obvious advantages in structural novelty,functional diversity and biocompatibility,active ingredients of traditional Chinese medicine have occupied an important position in the development of new drugs and clinical medication.The active constituents of Baiji and Sanqi in Sanqi Baiji San are bletilla striata polysaccharide(BSP)and panax notoginseng saponins(PNS).In this study,they were selected as the main components of the preparations for formulation research and pharmacodynamic evaluation.Because of the adhesion of BSP,it was combined with sodium alginate(SA)as a composite carrier.PNS was used as an encapsulated drug to develop a gastrointestinal bioadhesive drug delivery system,playing the role of gastric mucosa protection.Firstly,the rheological and mucoadhesive properties of BSP were studied.The characteristics of BSP as a polymer material were fully understood,which provided a basis for the preparation.The results showed that the BSP solution had certain viscosity characteristics and belonged to non-Newtonian fluids,showing shear-thinning flow characteristics.The viscosity of BSP solution was increased with the increase of solution concentrations;it was affected by temperature and decreased with the increase of the temperature;it was less affected by acidic pH variations,and the viscosity was increased under high pH condition,with more aggregated state reached;the BSP mixture was less affected by electrolytes,but the effect of divalent cations(Ca2+)on the viscosity of BSP was more pronounced than that of monovalent cations(Na+)at a high salt concentration(0.3 mol/L).BSP had a synergistic effect on the viscosity of common bioadhesive polymers such as sodium alginate(SA),sodium carboxymethyl cellulose(CMC-Na),hydroxymethyl cellulose(HPMC)and chitosan(CS),and an anionic polymer(SA)had a remarkable synergistic effect on rheology at low concentrations..BSP had a certain interaction with gastric mucin and had certain mucoadhesive properties,but it was relatively weak compared with commonly used bioadhesive polymers.Secondly,SA-BSP composite microsphere carrier was constructed on the basis of the rheological and mucoadhesive properties of BSP.The composite microsphere was prepared by W/W ion cross-linking method.The appearance shape,particle size,thermodynamic properties,swelling properties,properties of mucin adsorption,the retention rate of microspheres in rat stomach in vitro and in vivo were used to investigate the prescription factors(different ratios of SA and BSP)and process factors(CaCl2)concentration and crosslinking time)on the properties of microspheres.The results showed that SA-BSP composite microsphere has good roundness;the addition of BSP made the microspheres more flexible;the addition of BSP increased the swelling property of the microspheres,increased the adsorption rate of microspheres to gastric mucin and adhesion to gastric mucosa,and the performance was more obvious when SA and BSP were 1:2 and 1:3;the results of in vivo adhesion test showed that the retention time of SA-BSP(1:2)microspheres in the stomach and duodenum was more than 6 h,and the retention effect was better than SA microspheres.Therefore,BSP plays an important role in enhancing adhesion in the whole microsphere system,and the SA-BSP carrier can be used for encapsulation of drugs to exert the mucoadhesive properties of the preparation in the gastrointestinal tract.In addition,process factors such as CaCl2 concentration and crosslinking time had little effect on the mucoadhesive properties of the microspheres.Thirdly,PNS was dispersed in hydrophobic polymers by a liquid phase dispersion method to prepare PNS dispersions to enhance its hydrophobicityreduce,in order to reduce the leakage of the drug during preparation of the microsphere.The cumulative release was used as an evaluation index to investigate the effects of different types and proportions of carriers on the cumulative release of dispersions.The results showed that the release of the PNS was significantly slowed down after it was prepared into a dispersion.Polyacrylic resin Ⅱ and ethylcellulose N7(EC-N7)scored the lowest.PNS-Polyacrylic resin Ⅱ dispersion and PNS-EC-N7 dispersion were characterized.It was found that PNS itself had low crystallinity.The carrier played the role of coating PNS,so that PNS was uniformly dispersed in the carrer.At the same time,it acted as a binder to cause the drug particles to aggregate,then the particle size became larger and the specific surface area was reduced,resulting in slow release of the drug in water.The drug particles of PNS-EC-N7 dispersion had a relatively lower degree of aggregation than PNS-Polyacrylic resin Ⅱ dispersion.The extent of coating by the carrier was also less than that of PNS-Polyacrylic resin Ⅱ dispersion,but it also had a sustained release effect.PNS dispersion was then encapsulated in the SA-BSP composite microsphere carrer to prepare PNS-BSP composite microsphere.The dispersion factors(carrier type,powder particle size),prescription factors(mass ratio of SA to BSP,mass ratio of SA to PNS dispersion)and process factors(CaCl2 concentration,crosslinking time,stirring speed)were investigated.The evaluation index were the appearance of microsphere,drug loading and encapsulation efficiency.Then the carrier type,powder particle size and mass ratio of SA to BSP were determined,and the remaining factors were used as orthogonal design factors for further optimization.The final formulation process was as follows:SA:BSP:PNS dispersion was 1:2:1.5;the concentration of CaCl2 was 5%;the crosslinking time was 20 min;the stirring speed was 200 rpm.The prescription process was verified,and the prepared three batches of microspheres were stable in process and reproducible.Compared with the microspheres prepared by directly adding PNS,the drug loading,encapsulation efficiency and yield of PNS dispersion microspheres increased significantly,which were 10.34%,51.25%,and 82.21%,respectively.The drug loading,encapsulation efficiency,and yield were 4.04%,12.16%,and 61.35%of PNS microspheres.The characterization results of BSP-PNS composite microspheres were as follows:BSP-PNS composite microspheres had good roundness,rough surface and wrinkles;PNS was uniformly dispersed in microspheres in an amorphous state;it had strong swelling performance and high retention rate in rat stomach.The release of five saponins in microspheres were slow in water,and the cumulative release of notoginsenoside R1,ginsenoside Rg1 and ginsenoside Re for 12 h was high,all of which were above 90%.They were released faster in phosphate buffer solution of pH 6.8 than in water,and the cumulative release of five saponins for 12 h was about 90%.The release model fitting curves in different media all satisfied the Weibull equation,and the release of the drug was dominated by diffusion and dissolution.The results of stability influencing factors test indicated that the microspheres were unstable under high temperature,high humidity and strong light irradiation conditions,and should be kept at low temperature,protected from light and sealed.Finally,in order to study whether the composite carrier based on BSP could enhance the efficacy of PNS,pharmacodynamic evaluation of PNS-BSP composite microspheres was performed.The results showed that gastric mucosal damage of rats were lighten after administration.PNS and PNS-BSP microspheres had protective effects on acute gastric ulcer induced by absolute ethanol.Compared with the PNS group,the microsphere group significantly reduced the ulcer index,increased the SOD activity in the serum,decreased the MDA content,and decreased the IL-1β content,but there was no significant difference between the TNF-α and IL-6 levels.SOD activity and PGE2 content in gastric tissue were also increased,but there was no difference in VEGF-A content.It was indicated that encapsulation of PNS in mucoadhesive microspheres constructed by BSP could improve the pharmacodynamic effect of PNS wth better curative effect.There was no significant difference in all indicators except MDA compared with the positive drug group.The protective mechanism of microspheres may be anti-oxidation,gastric mucosal protection and promotion of ulcer healing,followed by anti-inflammatory effects.