| Objective:Notoginsenoside R1is derived from the dried roots and rhizomes of panax notoginseng,and is one of the main components of Notoginsenoside.Previous studies have found that Notoginsenoside R1can significantly improve the symptoms of colon shortening in dss-induced ulcerative colitis model mice,as well as reduce the damage of inflammatory infiltration tissue,inhibit weight loss,diarrhea and blood stool.As a class III pharmaceutical classification system,Notoginsenoside R1has good solubility but poor absorbability,is unstable under moderate gastric acidic conditions,and has low oral bioavailability,which limits its clinical application.In order to improve its intestinal absorption characteristics and bioavailability,two oral colon-targeted release systems were prepared:phospholipid complex enteric capsules?R1-PC?and ph-dependent colon-soluble solid dispersions?R1-SD?,and the stability,efficacy,intestinal absorption characteristics and in vivo and in vitro release of these two preparations were evaluated.Methods:The detection method,solubility,oil-water distribution coefficient,stability and other basic physical and chemical properties of Notoginsenoside R1were investigated by high performance liquid chromatography.Preparation of R1-PC:The compound rate of Notoginsenoside R1and lecithin was taken as the evaluation index.And the preparation process was determined by optimizing the influencing factors such as reaction solvent,drug lipid ratio?molar ratio?,drug concentration,reaction temperature and reaction time.Finally,X-ray diffraction?XRD?and Differential scanning calorimetry?DSC?were performed for R1-PC.Preparation of R1-SD:Eudrigit S100,a Colon-soluble material,was used as the basic carrier,and the dissolution degree was used as the evaluation index.Single factor experiment was used to investigate the influencing factors such as the type of promoter,the proportion of drugs and the carrier,and determine the preparation process.Scanning electron micrographs?SEM?,DSC and XRD were performed on the prepared R1-SD.R1-PC and R1-SD formulations were tested for influencing factors and long-term stability.Methods for evaluating the in vivo efficacy of two preparations:UC mice induced by DSS were used as the model to observe the effects of R1and two preparations on UC mice by taking the morphological length of colon,incidence of blood stool,body weight,spleen weight,pathological tissue sections and other indicators.In vitro intestinal absorption and in vivo and in vitro release of the two preparations:The intestinal absorption characteristics of the two preparations were investigated by in vitro intestinal absorption test.Dissolution apparatus was used to investigate the release of the two preparations in vitro.Taking advantage of the fact that phenol red is not absorbed in the body,Phenol-red ph-dependent solid dispersion was prepared by replacing the R1with phenol-red,and the in vivo localization and release experiment was conducted.HPLC method was used to determine the release of phenolic red in various tissues to detect the localization and release of R1-SD.Results:The peak time of R1established by HPLC method was 12.5min with no interference peak.The linear equation was Y=4197.6X+57.678,R2=0.9999.The linear relationship between R1and R1was good between 11.75?3000?g·m L-1.Its specificity,sensitivity and recovery are good.The solubility of R1in water is 6.12mg·ml-1,and R1is slightly soluble in water.R1is soluble in methanol,anhydrous ethanol,ethyl acetate and tetrahydrofuran,slightly soluble in ethyl acetate and very slightly soluble in trichloromethane.The oil-water balance coefficient of R1was-0.203,The results showed that the water solubility was better and the fat solubility was worse.The experimental results of influencing factors show that R1has certain hygroscopicity and is unstable in strong light environment.Preparation and characterization of R1-PC:By single factor experiment,the preparation process of R1-PC was optimized as follows:Respectively weighing Notoginsenoside R1and soybean lecithin into the triangular flask,,adding certain tetrahydrofuran,then stiring for 2 hour with the magnetic stirrer at 40?,the solvent being evaporated,and dried in a vacuum oven at room temperature for 12 h in last.,The recombination rate of R1-PC can reach 99.00%;DSC and XRD indicated that both the endothermic and crystallization peaks of R1in R1-PC disappeared,which verified the formation of Notoginsenoside R1phospholipid complex.Preparation and characterization of R1-SD:By single factor experiment,the preparation process of R1-SD was optimized as follows:According to the formulation ratio,Weighing R1,Eudragit S100and PEG4000in a small beaker.And adding certain of anhydrous ethanol(drug concentration of 2 mg·m L-1)in 45?on the magnetic stirrer800 r.min-12 h,make a clear solution,and then transferred to pear-shaped flask,50?on the rotary evaporation apparatus spin dry solvents,into vacuum oven is at room temperature,24 h for a quick drying,and finally,through the fourth sieve.In vitro dissolution results showed that R1-SD was insoluble in hydrochloride at p H1.2.And the phosphate buffer at p H6.8 was dissolved for about 22%in 4 hours,and the phosphate buffer at p H7.6 was balanced for 6 hours,and the final dissolution reached more than90%.DSC,XRD and other methods proved that both the endothermic peak and the crystallization peak of R1disappeared.Under SEM scanning electron microscope,it was observed that the surface of R1-SD was smooth and the strip crystal structure of R1disappeared.Stability results:Under the condition of high temperature and high humidity,the appearance,R1content and moisture absorption and weight gain of R1-PC preparation did not change significantly.Under strong light,the color of the preparation gradually changed from pale yellow to white,and the content decreased by 4%.In the long-term stability test,there was no significant change in appearance and content of R1,indicating that R1-PC was relatively stable in long-term storage at dark and room temperature.R1-SD preparation showed no significant change in appearance,R1content and release degree in influence factors and long-term stability test,indicating that R1-SD preparation was relatively stable in long-term storage at room temperature.Effects on DSS induced UC model mice:?1?Symptoms of colon shortening were significantly improved:The degree of congestion and swelling was improved and the area of ulcer necrosis was reduced in the R1group and R1-PC and R1-SD groups.However,the R1-PC and R1-SD groups significantly inhibited colon shortening and formed feces were found in the colon,which was statistically significant compared with the DSS group?P<0.05,P<0.01?.?2?Control diarrhea and bloody stools:Compared with the R1group,R1-PC and R1-SD groups presented only mild diarrhea.Compared with R1-PC and R1-SD 2 preparations,R1-SD significantly inhibited blood stool of UC mice from day 7,and R1-PC significantly inhibited blood stool of UC mice from day 9,so R1-SD was superior to R1-PC.?3?Inhibiting weight loss:R1-PC and R1-SD groups significantly inhibited weight loss after 5 days of DSS compared with DSS group?P<0.05,P<0.001?,and R1-SD group was superior to R1-PC group..?4?Inhibits splenic weight gain:The R1group,R1-PC group and R1-SD group all significantly inhibited the increase of spleen weight?P<0.001?,which was close to the normal group,moreover,R1-SD tends to be superior to R1-PC.?5?Reduces inflammatory infiltration of tissue damage:However,R1-SD was closer to the normal group than R1-PC in gland structure and cell arrangement,so R1-SD was superior to R1-PC.In vitro dissolution results:R1-SD ordinary capsules were not dissolved in p H1.2,and accumulated dissolution was less than 30%within 4 h in p H6.8 dissolution medium,and reached 85.63%within 3 h in p H7.6.R1-PC colon capsule was not dissolved in p H1.2 and p H6.8 for 4 h,and the dissolution rate reached 87.12%at 9 h in p H7.6..All of them met the requirements of colonic preparation dissolution.Phenol red test results?colon-specific drug release results in R1-SD?:A very small amount of phenolic red can be detected in the stomach within 2?24 h.It is possible that phenolic red on the surface of the solid dispersed body is released in the stomach after intragastric administration and always adheres to the gastric mucosa,so a small amount of phenolic red can be detected within 24 h.Phenolic red can be detected in the small intestine and colon for 4?12h,but the content of phenolic red in the colon is significantly higher than that in the small intestine,and the content of phenolic red in the colon reaches the maximum within 8?10h,indicating that the preparation is hardly released in the stomach and small intestine,but is released in large quantities in the colon.The in vivo release result is similar to that in vitro,indicating that the preparation has colon-targeted function.Conclusion:In this paper,two preparations of R1-PC and R1-SD were successfully prepared,which can meet the requirements of Colon-targeted drug release and increase the intestinal absorption of R1.UC mice induced by DSS all showed significant improvement in colon shortening symptoms,as well as reduced inflammatory infiltration tissue damage,weight loss,diarrhea,blood stool and other effects,and the preparation R1-SD is superior to R1-PC. |
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