| Background:Rhizoma Coptidis is exacted from the thizome of CoPtis chinensis Franeh,which contains many types of alkaloids,such as Betherine,Palmatine,Coptisine,Jatrorrhizine,Magnoflorineete,Worenine,etc.Rhizoma Coptidis,a traditional Chinese medicine used commonly,has the effects of clearing away heatness.The researchs show that its broad-spectrum anti-microbial role in the inhibition of Gram-positive and-negative bacteria,protozoa and other influenza viruses,fungi,etc.The total alkaloids from Coptis can alleviate colonic inflammation in mice by antioxidant free radical,inhibition of inflammatory cell activation?migration and NF-?B activation.Rhizoma Coptidis has a protective effect on the gastric mucosa,the effect may be related to increasing the gastric mucosal barrier function,improvement of gastric mucosal blood supply,etc.Ulcerative colitis is a common chronic intestinal diseases.The morbidity of this disease is rising in recent years while the precise etiology remains unclear.Now,many factors are thought to be associated with it.Which immune abnormalities is considered to be an important factor in disease incidence,including autoantibodies,cellular immunity,cytokines,cyclooxygenase,and matrix metalloproteinases,oxygen radicals and nitric oxide,etc.And others also play a role in its occurrence and development,such as genetic factors,infectious factors,psychological factors,dietary factors,etc.The colon specific targeting drug delivery system is a drug delivery system that the drug does not release in alimentary tract but in colon,then it play partial or systematic effect of treatment in human body.Targeted drug delivery can significantly increase the concentration of the drug in treatment site,and decrease the dosage of drugs,cost of treatment and the drug's adverse effects on the body.Colon targeted drug delivery system can be divided into three types in accordance with its design principles:pH-dependent,time-dependent and flora-dependent.Colon targeted drug delivery agents,is particularly suitable for the treatment of intestinal diseases.The total alkaloids from Coptis was prepared as colon targeted drug delivery agents and the following objectives can be to achieve:? The drug almost does not release from Colon targeted drug delivery system in the gastrointestinal tract,this eases stimulation of the gastrointestinal tract.? Colon targeting agents release little drug in the gastrointestinal tract,so it avoids the first pass effect of hepar.? It can achieves a higher drug concentration in the colon area after the preparations were taken.Objectives:1?To establish the method of determination of of Coptis total alkaloids and berberine in Coptis extract,and measure their contents.2?To compare the analgesic and antiinflammatory of the total alkaloids of Coptis and berberine.3?To compare Ulcerative Colitis Treatment of the total alkaloids of Coptis and berberine.4?To determine the preparation of the micropills of the total alkaloids from Coptis.5?To optimize the prescription of colon targeted micropills,and to evaluate release from micropills in vitro and in vivo.Methords:1?Measure the contents of the total alkaloids from Coptis by UV spectrophotometry.The standard curve of berberine hydrochloride and the contents of the total alkaloids from Coptis were measured at 350nm.2?Measure the contents of berberine hydrochloride by HPLC.The condition of HPLC method:a Agilent Agilent ZORBAX SB—C18 column;mobile phase:acetonitrile:water(57:43),which added to the water 0.015ml/L sodium dodecyl sulfate,phosphoric acid pH to 1.5.Column temperature:30 ?;flow rate:1mL/min;detection wavelength:345nm.3?Pharmacodynamics Comparison of the total alkaloids of Coptis and berberine.The indicators were analgesic and anti-inflammatory.Analgesic experiments using 0.6%acetic acid-induced writhing in mice,writhing were counted to calculate the inhibition of writhing.The anti-inflammatory experiments using xylene induced ear edema in mice to observe the impact of drugs on mouse ear swelling.4?Comparison of total alkaloids from Coptis root and berberine for ulcerative colitis treatment.The ulcerative colitis model was established by 5%dextran sulfate sodium(DSS)solution(ig)with intra colonic administration of 5%acetic acid solution.Mice were divided randomly into normal group,model group,sulfasalazine group,large,medium,low dosage(360mg·kg-1?180mg·kg-1?90mg·kg-1)TAC treated groups and large,medium,low dosage(170mg·kg-1?85mg·kg-1?42.5mg·kg-1)BER treated groups.The changes of disease activity index(DAI),contents of MDA in serum and histopathology of colon were obtained.5?The process of total alkaloids micropills.The total alkaloids micropills are made through the pan method.Study the impact of different concentrations of ethanol and Dextrin dosage on micropills roundness and hardness influence.Determine the process of micropills,and calculate the yield of micropills,and the micropills Micromeritic indicators bulk density,angle of repose were evaluated.6?Preparation and release evaluation in vitro of the coated pellets.The release in vitro method is:take a certain amount of micropills,put them in hydrochloric acid solution 2h at first,and then the solution is replaced by the pH6.8 phosphate buffer in 4h,and last place micropills on the pH7.8-8.0 phosphate buffer in 3h.Draw 5ml solution every 1h,filtered by 0.8?m membrane to determine its absorbance at 350nm,and then supplement with the corresponding solution.Release media are 500ml,ultrasound to remove air bubbles before used.Use this method to study the effect of the release of coated micropills of the L100-55 and S100 in different proportions,and to evaluate the impact of the different amount of ethylcellulose on the release effect,determine the best coating process prescription.7?The evaluation of coated pellets release in vivo.Using polyethylene tube intubation gavage coated pellets administered,and measure release contents of coated pellets in rats in vivo at 2h,3h,5h,7h,9h,11h.Results:1?Content determination of the total alkaloids of Coptis.The content of total alkaloids is 64.3%of the Coptis extract;berberine hydrochloride accounted for 30.5%of the Coptis extract by HPLC.2?Comparison of the total alkaloids of Coptis and berberine on analgesic effect.Rotundine and total alkaloids 2 compaired with model group,P<0.05,inhibition of writhing in more than 50%.There is an analgesic effect when total alkaloids were given at 340mg/kg-1 dose.Total alkaloids 1 group compaired with model group,P<0.05,but the inhibition of writhing below 50%,its analgesic effect is not good;berberine group compaired with model group,P>0.05,and the inhibition of writhing below 50%,indicating that berberine has no analgesic effect in the dose.3?Comparison of the total alkaloids of Coptis and berberine on anti-inflammatory effects.Swelling of dexamethasone group,berberine group and total alkaloids group 2 compared with the model group(P<0.05),indicating that three groups can inhibit xylene-induced ear edema in mice,total alkaloids group 1 compared with the model group,P>0.05,has no anti-inflammatory effects may be due to the lower dose.4?Comparison of total alkaloids from Coptis root and berberine for ulcerative colitis treatment.Addition to the normal group,varying degrees of bloody stools,diarrhea and weight loss and other symptoms in each group after the start of the modeling.Compared with model group,In addition to the BER in the dose,the rest of the treatment group can reduce the degree of weight loss(P<0.05).Amount of weight loss and weight loss percentage of the TAC middle dose group were lower than the BER high dose group(P<0.05)and BER middle dose group(P<0.01);The DAI of TAC high doses group is less than the BER high doses(P<0.01),the DAI of TAC middle doses is less than BER high and low-dose group,the DAI of TAC low dose group is less than the BER low dose group.Colon weight of mice increased after modeling,TAC high-dose and TAC middle dose can be antagonistic colon weight increase,compared to the model group were significantly different(P<0.05);after modeling,the length of the mouse colon become shorter.Compared with modle group,TAC high-dose model has a significant difference(P<0.05);colon index of TAC high-dose group is greater than the BER high dose group(P<0.05);the colon index of TAC middle dose group is greater than BER in dose group(P<0.01).Compared with model group,the positive control group,TAC high dose group,BER high dose group,the BER middle dose group MDA content decreased,the difference was statistically significant(P<0.05).Model mice colon apparent significantly ulcers,colon become thicker,shorter and edema.The mice colon of sulfasalazine drug group,TAC high dose group,TAC middle dose group,TAC low dose group,BER high-dose group appearance of a certain improvement,but the effect of BER middle dose and the BER low-dose is not obvious.Colonic mucosa of normal mice complete injury,arranged in order of glands,crypt significantly;Colonic mucosa of model mice deficit serious,obvious swelling,glandular crypt distortion,goblet cell damage,inflammatory cell infiltration and bleeding;Colonic mucosa of Sulfasalazine drug group,the TAC group and BER high-dose group damage is mild,crypt significantly less breakage,goblet cells and have less inflammatory cells;The mouse colonic mucosa of BER middle and low dose group is serious injured,goblet cells appear damaged,and more inflammatory cells.The results showed that the treatment group compared with the model group were significantly different(P<0.01),in addition to BER middle and low dose group.5?The process of total alkaloids micropills.Prescription of the drug-containing pellets identified as:70%microcrystalline cellulose,20%dextrin,10%powder,the binder of 95%ethanol;Preparation method:in accordance with the prescription drug are made into softness and hardness of the soft material,16-mesh sieve granulation.Promptly put particles into a coating pan,coating pan speed controlled in the 40-60rpm,rounded.Dry forming micropills in a 60 ? oven,16-24 mesh,collected between micropills.The yield of micropills is about 53%.Bulk density of the micropills is 0.4726g·ml-1,and the angle of repose is 29.12 °.6?Preparation and release evaluation in vitro of the coated pellets.When coat micropills with S100 alone,30%drug release within 1-2 hours,2-6 hours,the release is slow and less emission.When the ratio of L100-55 and S100 is 1:4,the coated mcropills release about 10%drug in two hours,in the artificial small intestine to release more than 70%.The micropills coated with the L100-55 and S100 ratio of 1:9 release very little in artificial gastric juice,only about 1%,but rapid release in the artificial intestinal fluid.Three micropills can release fully in the artificial colonic fluid,cumulative release are more than 95%.The final prescription:coat micropills with S100 first,the weight gain to 20 percent,and then use the L100-55 and the S100(1:9)coating liquid coating,with a total weight gain of 30%.The obtained micropills release below 10%in the six hours and quickly release in the artificial colon fluid,the final release is more than 90%.7?The release of coated pellets in vivo results showed that the coated pellets can make drugs not release in the stomach and the preceding of small intestine,and start release in the last paragraph of small intestine,a large number of enrichment in the cecum,and the advancing to the colon.Conclusion:1?The total alkaloid content in the extract and berberine hydrochloride content was determined by UV spectrophotometry and HPLC,and the method is accurate,reliable,simple,can be used as the detection method of the effective parts of the Alkaloids of Coptis chinensis.2?The analgesic effect of total alkaloid is better than berberine,and this may be due to other components of total alkaloids in Coptis play this efficacy;both of them have anti-inflammatory effect.3?In total,the treatment effect of TAC on UC is better than BER.This reflects the advantages of traditional Chinese medicine ingredients in a certain extent.4?The micropills yield is not high.Powder indexes show that the mobility of the micropills is ideal,and this in favor of coating.5?In vitro and vivo release results showed that with the double-layer coating,the micropills can achieve the purpose of colon targeted drug delivery. |
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