Study On The Preparation Of Aloperine Enteric Capsules

Ulcerative colitis (UC) is a chronic nonspecific inflammatory disease with unknown etiology, the main diseased region is at colonic mucosa layer, contact with immunization,heredity,infection and circumstance etc.,the major clinical symptoms are diarrhea, bellyache, mucus bloody purulent stool and tenesmus, colonic mucosa chronic inflammation and ulceration are the main pathological features. The incidence of UC is approximately2-20per105per year and the prevalence is50-100per105,main people in15-30years old. The number of UC patients has increased significantly with upturn living standards,dietary structure and mode of life changes in recent years.Due to the long course,differences in patient’s condition and high rate of relapse of UC which has a serious impact on the living quality of patients,and about5-10%get canceration,but to data there is no effective treatment measures in clinic.Therefore,UC is thought as one of the morden knotty disease by the WHO.To date,chemical drugs have been main used in the treatment of UC,such as salazosulfapyridine (SASP), glucocorticoid and immunosuppressor etc.,but the effect is not well,and have much side effects. SASP is suitable for mild, moderate and chronic UC patients,and commonly used in clinic at present.The efficiency of SASP is less than50%,but side effects up to37.5%,and the recurrence rate is33%when under the dose of no more than4g per day.The curative effect and side effects have direct proportion with the dosage,about10-15%of the patient stop using this drug due to rash and gastrointestinal discomfort.In traditional Chinese medicine,the treatment based on syndrome differentiation,the most way is taking decoction of Chinese medicine and assisted by clysis.But this have much disadvantages,such as bad compliance of the patient,big workload of the medical worker and curative effect can not get guaranteed.Aloperine is a effective monomer composition extracted from Sophora alopecuroides L.(a traditional Chinese medicine), the chemical structure shown in Figl.It is a kind of colorless and prismatic crystals, melting point is between71℃and73℃,the optical rotation is [alpha] D+86.59º, which has obvious anti-inflammatory, anti allergy, antiviral, antibiosis, anticancer and antiarrhythmic effect. Studies have shown that aloperine can inhibit acute inflammation induced by various inflammatory agent,â…¢, â…£ type allergic reactions and adjuvant arthritis obviously.The anti-inflammation and anti allergic effect does not depend on pituitary adrenocortical system,but contact with inhibition migration of WBC, stabilization lysosomal membrane,promotion radical scavenging,inhibition of lymphocyte proliferation and synthesis,release and activity of PG, histamine and lymphokine.My research group has studied the therapeutical effect and mechanism in acute stage mice UC model induced by dextran sulfate sodium(DSS),the result show that aloperine can reduce diarrhea,hemafecia and itestinal mucosal inflammatory state significantly,both the disease activity index(DAI)and histology grading are below the model group (P<0.01),This show aloperine has a good curative effect on UC mice. The mechanism may through decreasing NF-κBp65expression and regulating balance of inflammatory cytok ines.Now, my research group is applying for a new national patent named the application of aloperine in preparation drugs treatment on UC(Accept number:200910192776.7). At present,there is no effective drug that treatment on UC, the most commonly used in clinical is SASP,its efficiency is less than50%,but side effects up to37.5%,and the recurrence rate is33%.As a new drug treatment on UC,aloperine has a broad market application prospect with curative effect,low side effects, and the very rich resources of sophora alopecuroides L. in our country.Enteric preparations have many advantages,such as to avoid drugs destroyed by gastric acid and enzymes in the stomach;avert stimulative effect to the stomach which can cause nausea and vomit;deliver the drug to intestinal tract to play local treatment; increase the local drug concentration and delay the drug absorption,etc.At the formal study basis of pharmacological effects of aloperine treatment on ulcerative colitis, my research group propose to develop aloperine to enteric capsules treatment on UC which can cover the amargoso of aloperine; avert stimulative effect to the gastric mucosa and boost the absorption.First to determine aloperine in different parts(leaf,stem,flower,legume and seed) of Sophora alopecuroides L.,so that can provide a reference for choosing the optimal medicinal part to extract aloperine. Second, study on the preparation of aloperine enteric capsule,including preparation technology, middle scale test, establishing quality control standard and stress testing,accelerated testing,long-term testing of the preparation,aim to provide relevant data for later industrial production.l.ObjectiveThrough studying on the extracting and detecting conditions of aloperine,then determined aloperine in different parts(leaf,stem,seed,legume,and flower) of sophora alopcuroides L.,hope to provide a reference for choosing the optimal medicinal part of extracting aloperine.Using humidity absorption,angle of repose, tap density and in vitro release as index,the optimal composition and preparation procedure of aloperine enteric capsules were confirmed.Through determining the critical relative humidity(CRH) of the prescription,as a reference for the control of humidity in the process of production.Through pilot scale test,make sure the preparation process is feasibility.Establishing the quality standard of aloperine enteric capsules. To discuss the impacting factors of drug inherent stability,controlling the environmental conditions in production, transport, storage,choosing of packaging materials and setting the period of validity,influential factor test,accelerating test and long-term test were studied.2.Methods2.1Determination of aloperine in different parts of sophora alopcuroides L.Applying comparison aloperine HPLC chromatograms method, studied the impact on aloperine extracting efficiency by solvent types, infiltration, ultrasonic extraction, heating reflux extraction, extracting time and extracting times,choosing the optimal extraction method.Using HPLC method to determine aloperine in sophora alopcuroides L., the separation was carried out on a Kromasil NH2column(250mm×4.6mm,5μm),the mobile phase was acetonitrile-absolute alcohol-3%phosphonic acid water solution (70:15:15),the detective wavelength was set at205nm,the column temperature was at25℃,the flow rate was1.0mL-min-1.Meanwhile,the methodological validation was investigated, including linear relation, precision, iterancy, stability and sample recovery rate test.2.2The preparation technology research of aloperine enteric capsulesApplying fixed funnel method to determine angle of repose, applying measuring cylinder method to determine tap density, stirring paddle method was carried out to inspect in vitro release,determined humidity absorption of different prescriptions, according to the results of the above tests to choose the optimal supplementary materials and proportion.To provide a reference for the humidity control in production,the CRH of confirmed prescription has been determined. With the equipment and experimental conditions requirment of GMP,using10times inventory of the prescription for pilot scale test,and then investigating the rate of finished products, load difference and content uniformity.2.3Establishing the quality standard of aloperine enteric capsulesIdentifying aloperine in aloperine enteric capsules by TLC. Using HPLC to determine aloperine in aloperine enteric capsules.Applying HPLC to inspect related substance, the specificity was investigated by destructive test.Using stirring paddle method to study in vitro release. The quality standard of aloperine enteric capsules was established.2.4The stability test of aloperine enteric capsulesInfluential factor test:Using one batch preparation with the outer packing removed,put it under the condition of high temperature(60℃), high humidity(RH90±5%,25℃)and hard light(4500±5001x)for10days,detected at0,5th and10th day respectively,focus on changes in external properties, related substance, content and dissolution rate.Accelerating test:Using three batches preparation with commercially available package,put it under the condition of40±2℃and RH75±5%for6months,detected at0,1st,2nd,3rd,and6th month respectively,focus on changes in external properties, related substance, content and dissolution rate.Long-term test:Using three batches preparation with commercially available package,put it under the condition of25±2℃and RH60±10%for12months,detected at0,3rd,6th,9th and12th month respectively,focus on changes in external properties, related substance, content and dissolution rate.After12months, continue to inspect,detect at18th,24th and36th month.Compare with the0month,to make the period of validity.Due to the dispersibility of the experimental data,usually use95%confidence limit statistical analysis to obtain a rational period of validity. 3.Results3.1Determination of aloperine in different parts of sophora alopcuroides L.The optimum extraction conditions of aloperine is Using methanol as extraction solvent, ultrasonic extraction for one time, the extraction time is30minutes,using a moderate amount of ammonia to infiltrate medicinal materials powder for30minutes before extraction.The content of aloperine in sophora alopcuroides L. leaf,stem were0.38%and0.02%, but aloperine was not detected in legume,seed and flower.3.2The preparation technology research of aloperine enteric capsulesThe mixture of starch, microcrystalline cellulose and aloperine has the lowest hydroscopicity.When starch, microcrystalline cellulose and aloperine at the ration of10:15:5,there was no release in acid liquor(pH=1.2) within2hours,but released99.24%in buffer solution(pH=6.8) within45minutes,the average angle of repose was29.5°,the average tap density was0.3640g/ml.The final prescription of aloperine enteric capsules is aloperine(67g),starch(100g) and microcrystalline cellulose(33g),mixing well,put into the enteric hollow capsules,up to1000granules. The rate of finished products of three batch of pilot samples are94.6%,93.1%and94.9%,both the load difference and content uniformity meet the requirements.3.3Establishing the quality standard of aloperine enteric capsulesThe TLC identification of aloperine in aloperine enteric capsules, the speckle is distinct and has good specificity,Rf=0.69.Using HPLC to determine aloperine in aloperine enteric capsules,according to the result of methodological validation,the method is stable, accurate with good repeatability.Applying HPLC to determine related substance in destroyed samples,the impurities and principal component has a good separating effect,so that the method has specificity.The release behavior can fit the relevant provisions.3.4The influential factor test,accelerating test and long-term test of aloperine enteric capsulesThe influential factor test suggests that aloperine enteric capsules under the condition of high temperature, high humidity and hard light for10days,compared with the0day, the external properties, related substance, content and dissolution rate do not have significant changes.The accelerating test suggests that aloperine enteric capsules under the condition of40±2℃and RH75±5%for6months,compared with the0day, the external properties, related substance, content and dissolution rate do not have significant changes.The long-term test suggests that aloperine enteric capsules under the condition of25±2℃and RH60±10%for6months,compared with the0day, the external properties, related substance, content and dissolution rate do not have significant changes.4.ConclusionThe established HPLC method is accurate, stable with good repeatability,and can be used as a method for determination of aloperine in sophora alopcuroides L.The leaves have the highest content of aloperine,so it can be used as the optimal medicinal part for extrcting aloperine.The aloperine enteric capsules has a stable preparation technology,the quality is easy to control,have good enteric performance,and is suitable for industrial production.