| The study of Chinese Traditional Medicine’s effective fraction is the research focus for that it’s clarification and enrichment of the bioactive fractions,which are beneficial to the application of modern extraction,purification technologies and new agents.In this study,the best extraction,separation,purification methods of the effective fractions of Yuhuanglian were established and quality control method of total alkaloid of Yuhuanglian was studied.The results will be helpful to the developments of new effective fraction agents,and may bring out convient,effective, and safe medicines for patients with digestive system diseases.The main content followed:1.Studies on the separation technique of effective fractions of Yuhuanglian with ethanolMethods:(1) The indexes were investigated including total alkaloid content and extract yield.(2) Through L9(34) orthogonal experimental,the impacts of ethanol concentration,solvent amount,extraction frequencies and extraction time on the Yuhuanglian total alkaloid extract rate were studied.(3) The results were statisticed by analysis of variance to determine the optimal conditions.(4) The optimal technique was verified and single-factor studied.Results:(1) Ethnal was determined as the extracting solvent.(2) The conditons were as follows:80%ethanol extracting for 2 times and 1.5 hours during each extraction,solvent amount was 12 times of the weight of Yuhuanglian.(3) The results of verification test were:solide yield:31.40%,total alkaloid transfer rate:91.65%. 2.Studies on the separation and purification techniques of the effective fractions of YuhuanglianMethods:(1) With the total alkaloid content and the yield of purified material as the indicators,the method of acid-precipitation and salting-out were developed.(2) The filtrate and washing liquid after treatment with acid-precipitation and salting-out were put on macroporous resin,and the adsorption and desorption abilities of several different types of macroporous resin were studied.Results:(1) After the recovery of ethanol,extract was adjusted to pH=2.0 with hydrochloric acid,then added 10%of the volume of solution of sodium chloride, placed for 24h,filtrated,and washed,dried at 80℃to constant weight,then effective partⅠwas acquired.(2) HPD-100 macroporous adsorption resin possessed the best adsorption,adsorption capacity of total alkaloid of Yuhuanglian.The volume of capacity was 6.686mg/mL(wet resin).The optimum conditions were as follows: The total alkaloids with the concentration of 1.0～1.5mg/mL in scope,pH=5.0, sample flow rate 1.5mL/min,the sample volume 5BV,adsorption resin column after the elution with the first 7.5 BV of distilled water,and then 4BV 70%ethanol.After the recovery of ethnol,the elute was concentrated,dried in vacuum at 80℃to constant weight,then effective partⅡof total alkaloids was acquired.(3) The effective part of the total alkaloids was acquired after the mixing of effevtive parts of acid-precipitation and salting-out and purification with macroporous adsorption resin. Effective part of yield was 12.72%,with a total alkaloid content of 83.41%,of total alkaloids in the transfer rate of 84.47%.3.Studies on the quality control method of effective fractions of YuhuanglianMethods:(1) UV spectrophotometry and high-performance liquid chromatography were used respectively to establish an analysis method of total alkaloids,Berberine Hydrochloride and Palmatine Hydrochloride in the effective part. (2) Evodiamine and rutaecarpine in effective part were qualitatively identificated by the thin-layer chromatography.(3) The fingerprints of effective parts were studied by high-performance liquid chromatography.Results:(1) When determinating total alkaloids,the berberine hydrochloride in the range of 2.804～14.02μg/mL performanced good linear relationship,γ=0.9997, average recovery was 97.43%,RSD 2.01%,effective total alkaloids part of the total alkaloid was up to 83.41%.(3) During the analysis of berberine hydrochloride and palmatine hydrochloric acid,they had good linear relationship in 0.1526μg 1.2208μg and 0.115μg～0.920μg respectively;γ=0.9992,γ=0.9991;The average recoveries were 99.54%and 98.54%;RSD were 1.5%and 1.89%;berberine hydrochloride and hydrochloric acid content average were 41.27%and 13.26%g.(2) TLC results showed that the effective prt of Yuhuanglian contained evodiamine and rutaecarpine illustrating the preparation of the total alkaloids remained the main active ingredient evodiamine and rutaecarpine.(3) A total of nine peaks were acquired through the analysis of 7 batchs of effective fractions.With Berberine Hydrochloride(peak 9) as a reference peak,the total alkaloid of Yuhuanglian was studied by chromatographic fingerprint similarity calculating.The results showed that fingerprint of different batches of Yuhuanglian intermediates have good similarity, Established fingerprint method is feasible and can be used to monitor the preparation process of effective parts of Yuhuanglian. |
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