| Objective According to the characteristics of Bioadhesive Drug Delivery System,improve the conditions of extraction process of Rhizoma Coptidis,increase the extraction rate of total alkaloids from Coptidis,firewood of total alkaloids from Coptidis medicine prepared bioadhesive suppository.In order to provide a new route of Administration for the treatment of total alkaloids from Coptidis yeast vaginitis and its recurrence.Methods 1.With berberine hydrochloride as the standard,the determination method of the content of total alkaloid of Coptis established by UV.With Berberine,Palmatine,Coptisine,Epiberberine and Jatrorrhizine 5 monomeric alkaloids as standard,using HPLC to establish a method for determination of 5 kinds of main alkaloids in Coptis chinensis to total alkaloid monomer.With the total alkaloids extraction rate as the evaluation index,based on single factor test,orthogonal design is used to optimize the extracting process of water decoction.2.Methods the static adsorption and dynamic adsorption inspection unifies,as total Coptis alkaloids the index to screening 6 different properties of macroporous resin.Through the study of the concentration of sample,sample volume,flow rate,ratio of diameter to height,impurity removing solvent,elution solvent and other factors,the final preliminary establishment of purification process of total alkaloids from Coptidis by D101 macroporous resin.3.Through extract content determination results of the total alkaloid of Coptidis in different batches,initially formulated the content limit of total alkaloid extract and 5 main alkaloid monomer of Coptidis.Then conduct stability influence factor test,the determination of equilibrium solubility,apparent oil-water distribution determination,determination of critical relative humidity of total alkaloids of Coptidis.4.Coptis decoction,Alcohol extract,Total alkaloid in Rhizoma Coptidis,Clotrimazole,Berberine hydrochloride by MIC value compared with antibacterial assay in vitro,to preliminary evaluation of the effectiveness of total alkaloids in Rhizoma Coptidis.The safety of total alkaloids in Rhizoma Coptidis was primarily evaluated by mouse acute toxicity test.We evaluate the irritation of total alkaloids from Rhizoma coptidis by the rabbit vaginal stimulation test.5.Manual irrigation model by melting method,with the suppository appearance,hardness,melting time as indexes,used for preliminary screening matrix of blank suppositories.Further by the central composite design,with the cumulative release amount of 2h,6h,8h as index optimized formulation preparation of total alkaloids in Rhizoma Coptidis vaginal adhesion suppository.And through the shape of the appearance,weight difference,melting time,drug content and viscosity evaluate the quality of total alkaloids in Rhizoma Coptidis vaginal adhesion bolt.And preliminary investigate stability by high temperature,high humidity,strong illumination tests on total alkaloids in Rhizoma Coptidis vaginal adhesion suppository.Results 1.To establish the UV quantitative analysis method of total alkaloids in Rhizoma Coptidis,the detection wavelength at 348nm,within the range of 2.52?15.12 mg·mL-1 exhibited good linearity.Precision,stability,repeatability,recovery rate are in line with the requirements.To establish HPLC content analysis method of Berberine,Palmatine,Coptisine,Epiberberine and Jatrorrhizine.There was a good linear relationship between the five kinds of alkaloid monomer respectively in range of 0.161?0.966?g,0.0525?0.525?g,0.0695?0.695?g,0.0515?0.515?g,0.0200?0.400?g.And precision,stability,repeatability,recovery rate test all meet the requirements.The extraction technology of total alkaloids in Rhizoma Coptidis conditions determined as:with 12 times amount of water,direct fire reflux extraction 3 times,each time 2h.The preparation technology of Coptis extract,three verification test total alkaloids extraction rate was 89.84%,the rate of total alkaloid extract 25.32%,purity of 38.81%,purity of Berberine,Palmatine,Coptisine,Epiberberine and Jatrorrhizine were 22.05%,5.76%,7.59%,3.92%,1.31%.2.Technical conditions for purification of total alkaloids in Rhizoma Coptidis,namely preparation concentration was 0.25 g/mL of sample solution(the content of alkaloid 26.7mg·mL-1)join D101 macroporous resin column size:high=1:9,at a speed of 1-2 BV/h sample is about 1.5 BV,first with about 2BV water to remove impurities,and then with a velocity 1-2 BV/h about 2BV 60%ethanol elution,collecting the eluted liquid.Then with the temperature of 60? vacuum concentration to the content of alkaloid 150mg·mL-1 and spray drying to obtain the total alkaloid of Coptis chinensis extract with 180? and 25%speed.The content of total alkaloids in Rhizoma Coptidis total purified curing at more than 60%,and the transfer rate of about 65%.3.To establish the content determination of alkaloids in Rhizoma Coptidis,which the product is brown yellow powder,fine powder,gas micro,taste extremely bitter,the moisture check is below 5%,the heavy metal content not exceed limited standard,the content of alkaloid?54%,Berberine?27%,Palmatine?5.9%,Coptisine?9.4%,Epiber-berine?3.4%and Jatrorrhizine?1%,by UV and HPLC.The linearity,precision,stability,repeatability,recovery rate are in line with the requirements.The properties of total alkaloids in Rhizoma Coptidis is stability at high temperature of 60?,75%high humidity and high light conditions.Solubility of alkaloids in Rhizoma Coptidis in methanol,ethanol,ultra pure water,saline water,artificial gastric juice(pH 1.2)and artificial intestinal fluid(pH 6.8)for each solvent respectively 317.15±8.60mg·mL-1,273.27±1.57mg·mL-1,157.18±0.78mg·mL-1,20.55±0.79mg·mL-1,322.72±7.10mg·mL-1,283.65±3.71mg·mL-1;in pH 7.8,pH 7.4,pH 6.8,pH 5.8,pH 5.0,pH 4.5,pH 4.0,pH 2.0 PBS are 340.06±10.97mg·mL-1,359.69±9.68mg·mL-1,384.33±5.16mg·mL-1,346.88±7.42mg·mL-1,339.35±9.04mg·mL-1,374.61±4.68mg·mL-1,372.10±3.71mg·mL-1,265.88±2.20mg·mL-1,254.77±3.69mg·mL-1.Apparent oil-water distribution coefficient in pH1.2 HCl,pH1.5,pH 2.0,pH 4.0,pH4.5,pH 5.0,pH 6.8,pH7.4,pH8.0 PBS are 0.146,0.179,0.104,2.929,0.557,0.438,0.259,0.328,0.107.4.Coptis decoction,Alcohol extract,Total alkaloid in Rhizoma Coptidis,clotrimazole,berberine hydrochloride by MIC which are 4.68mg·mL-1,2.34mg·mL-1,1.17mg·mL-1,0.25mg·mL-1,0.25mg·mL-1,value compared with antibacterial assay in vitro,the5.antibacterial effect in vitro of total alkaloids in Rhizoma Coptidis fairly and berberine hydrochloride.The safety of total alkaloids in Rhizoma Coptidis was primarily evaluated by mouse acute toxicity test.The results show that the oral nontoxic LD50 is 1153.99mg·kg-1 and the 95%confidence interval is(940.80?1478.26)mg·kg-1,the vaginal mucosa irritation of small.6.To establish the content determination of alkaloids in Rhizoma Coptidis by UV and HPLC.The linearity,precision,stability,repeatability,recovery rate are in line with the requirements.The total alkaloids from Rhizoma Coptidis vaginal adhesion to total alkaloids in Rhizoma Coptidis:12.00%,PEG6000:60.63%,PEG 1500:13.80%,HPMC:13.57%in the proportion of mixed matrix prepared from average weight is 2.74g,the weight difference between 0.00%?3.28%,melting time within 45min,viscosity 20?23g·cm2.The release results of the release in vitro were 39.497%,72.814%and 86.382%in 2h,6h and 12h,respectively,slow release in 12h.Conclusion Through single factor and orthogonal experiment to optimize the extraction and purification process of total alkaloids extract conditions,obtained the content is above 60%.And use UV and HPLC to control the quality of the component index of total alkaloids in Rhizoma Coptidis.Secondly,the stability influence factors test and long-term stability test to determine the extract has good stability,physical study found that the chemical properties of soluble in water,easily absorb moisture;by bacteriostatic test in vitro,acute toxicity and irritation test preliminary determine the inhibitory effect of total alkaloids in Rhizoma Coptidis on Candida albicans and yeast are smooth,and good safety,small irritation.Total alkaloids from Coptis vaginal adhesion suppository was prepared for the final result,are in line with the "Pharmacopoeia of the people's Republic of China"(2010 Edition)regulations,laid a solid foundation for the preparation of traditional Chinese medicine research and development of high quality. |
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