| ObjectiveAccording to our previous research of the cell model and animal model,to validate the improvement of ulcerative colitis in rats by the SFE and phenolic acid extract of Angelica sinensis,we use the dextran sulfate sodium(DSS)to induce the ulcerative colitis in rats.According to its biological characteristics,we use extrusion spheronization and coating method to prepare the colon specific pellets to make it play a better role.The performance of colon specific pellets was evaluated by in vitro dissolution and in vivo imaging,and ulcerative colitis in mice model induced by DSS to further verify the effect of the effective components of Angelica sinensis colon specific pellets.1.Dextran sulfate sodium was used to induce ulcerative colitis in rats,and validate the effects of the improvement of ulcerative colitis in rats of SFE and phenolic acid extract of Angelica sinensis;2.Prepared colon pellets of the effective component of Angelica sinensis by extrusion spheronization and coated,and make the drug released in the colon after oral administration to improve the concentration of the drug in the colon to play a role of local and systemic treatment;3.The performance of colon targeting pellets was evaluated by in vivo and in vitro evaluation,and to further verify the effect of the effective component of Angelica sinensis colon specific pellets by the mice model with ulcerative colitis induced by DSS.The aim was to provide theoretical basis for further development and utilization of Angelica sinensis,and do some useful research for the development of the new oral colon specific preparation and the establishment of the system of evaluation.Method1.Protective effects of different ratios of effective components of Angelica sinensis on ulcerative colitis in rats:The rat model of ulcerative colitis was induced by 3%dextran sulfate sodium,and administration from second days,once a day.Daily enema at the same time,and before the enema,stimulate the rat anus to evacuation as far as possible and weighing.And connect the stomach filling needle with the syringe after extracting medicine for 1 mL.Then the rats were inserted and in situ 30s after push.Blank group and model group given the same amount of saline retention enema,continue feeding for 6 days.And observe the effects of the combination use of SFE and phenolic acid extract of Angelica angelica on rat body weight and stool situation.After the last administration,fasting for 12 hours and make the rat euthanasia by excessive injection of 10%chloral hydrate.Then abdominal anatomy to measure the entire segment of the intestine from the anus to the cecum.And gross morphology of colon was observed and graded after longitudinal dissection and rinse with ice saline.The pathological damage and the degree of inflammatory cell infiltration were analyzed by HE staining.To verify the anti-inflammatory effect of the effective components of Angelica sinensis on rat colon,and discuss the protective effect of colon in the course of the development of colitis in rats.2.Study on the preparation technology of effective components of Angelica sinensis colon targeting pellets:Microcrystalline cellulose(MCC)as excipient,CMC-Na as binder,with water as the wetting agent,and use extrusion spheronization to prepare the effective component pellets of Angelica sinensis.With 18-24 mesh yield and plane critical angle as index,the single factor was used to investigate the amount of binder,binder concentration,wetting agent,drug dosage,time and speed of spheronization.And determine the main factors affecting the forming,18-24 mesh yield and plane critical angle of pellets.Then use the Box-Behnken experimental design to optimize the prescription of pill core.And performe the process validation according to the optimal prescription.Coating the optimized pill with a coating pot,and the release was investigated based on in vitro to investigate the effects of different coating materials,different prescription and different coating thickness on the colon targeting.Then determine the best coating process and carry out three batch process as validation test,and f2 similarity factor method was used to evaluate the consistency of the release process.3.Evaluation the quality and pharmacodynamics of the effective components of Angelica sinensis colon targeting pellets:The quality of coated pellets was investigated with the surface morphology,roundness,bulk density and brittleness of coated pellets.And the pellets and the coated pellets are sealed in a clean container place at a temperature of 60? and relative humidity of(75±5)%respectively for 10 days,detection the stability of color,character and content at 0,5,10 days.Using the combination of small animal imaging and X-ray to determine the location of pellets at different times in animals to evaluate the colon targeting of pellets.Finally,3%DSS was used to prepare the mice model of ulcerative colitis to observe the body weight,stool,stool,colon length,pathological damage and inflammatory cell infiltration of mice after administration,and study the protective effect of the effective components of Angelica sinensis on mice colon.Result1.Protective effects of effective components of Angelica sinensis on ulcerative colitis in rats:In the experimental,the inflammatory cell infiltration was observed in every group,but the drug administration groups showed delayed and improved effects on the occurrence and development of ulcerative colitis when given drug intervention,whether it is from the observation of the animal's daily behavior or the gross score of the colon or pathological analysis.Among them the pharmacological effect of SFE:PAE(enrichment)=1:2 enema group showed a certain anti-inflammatory effect similar to that of cell model.The effects of SFE:PAE =1:2 enema group were better than those of other groups in rats.It is suggested that the other components of the extracts,which are not enriched in addition to ferulic acid,have better anti-inflammatory activities.2.Study on the preparation technology of effective components of Angelica sinensis colon targeting pellets:The effective component of Angelica sinensis pellets prepared by extrusion spheronization,and establish a method for determination of content of ligustilide and ferulic acid which are index components in pellets by HPLC.Single factor experiments were carried out to investigate the influence of formulation on the formation of pellets,the yield of 18-24 pellets and plane critical angle.Then the Box-Behnken experiment design was used to optimize the prescription,and the best prescription was determined by model fitting.The best prescription is as follows:MCC 20 g,drug 9 g,wetting agent is water 12 g,adhesive is 0.8%CMC-Na 7.4 g,extrusion speed 40 Hz,spheronization speed 30 Hz,rounding time 120 s.Carry out the 3 batch verification experiment and the predicted values are close to the measured values,indicating the predictive ability of the established prediction model is good.By using the method,the effective components of the Angelica sinensis pellets were obtained,which the yield is high and the plane critical angle is small.Study in vitro release find the cumulative release reached 80%at 20 minutes of the uncoated pellets,and reached about 95%at 2 hours.We can see that ligustilide and ferulic acid are able to maintain simultaneous release.In the coating process,coating weight which the inner layer coating acid soluble and the outer coating enteric gain more than 30%were dissolved a lot in simulated gastric fluid.And when internal coating delay type RS PO,outer coating enteric can not meet the requirements of the release.Later,no longer consider the prescription.In accordance with the original coating prescription weight gain of 15%can achieve the purpose of colon localization.But the cumulative release more than 20%in simulated gastric fluid,it need to optimize.During optimizing prescription,when internal coating delay type RS PO can delay the dissolution.Especially in the colon it is difficult to reach 80%in 6 hours,which can not achieve the purpose of rapid delivery in colon.In accordance with the original coating prescription weight gain of 16%can achieve the purpose of colon localization.Therefore,the final selection of coating material is Eudragit FS 30D,the weight gain of pellets was 16%,which could achieve the colon targeting.3.Evaluation of the quality and pharmacodynamics of the effective components of Angelica sinensis colon targeting pellets:According to the results of roundness,bulk density and brittleness of the coated pellets,it showed the colon targeting pellets were homogeneous.By scanning electron microscope,the surface of coated pellets was smooth and uniform.Under the condition of 60? and relative humidity of(75±5)%,the morphology and content of pellets did not change significantly after 10 days;but vnder high temperature conditions(60?),there was partial adhesion of coated pellets,which affected its release at 10 days.Therefore,it is better to keep the pellets at room temperature.Through the fluorescence imaging system and X-ray imaging results,it can be seen that the pill core has a rapid release and metabolism in mice.With the passage of time,coated pellets appeared in different parts of mice,and the expression behavior is basically the same in normal mice and inflammatory mice,which indicate it can achieve the purpose of colon localization and the release is good.In the pharmacodynamics experiment,the overall condition of the mice in the model group was poor and there are mice died during modeling.And there was a large area of deep inflammatory cell infiltration in the colon from the pathological section.The drug group can improve the mice daily state and blood in the stool,and delay the occurrence and development of colitis.It can be seen from the results that the effect of the pellet group is better than that of the positive group,which may be due to the rapid release of the drug when the pellets arrive at the lesion and play a role in improving local symptoms.ConclusionThe combined use of SFE and phenolic acid extract of Angelica sinensis can delay and inhibit the effects on the occurrence and development of ulcerative colitis in rats.At the same time,we compared the phenolic acid extract group,the results showed that the two groups have good anti-inflammatory effect.And drug loaded pellets containing SFE and phenolic acid extracts were successfully prepared,and the preparation process is reasonable,stable,simple and feasible and the pellets have good release.The results of in vitro release and in vivo animal imaging showed that the colon targeting of pellets is good.Pharmacodynamic studies showed that pellets can improve the overall state of mice and have the inhibition of the occurrence and development of ulcerative colitis in mice.The results of the study reached the expected goal. |
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