Study On Preparation Technology And Process Engineering Of The Flavonoid Component With Less Glycoside From Herba Epimedii

The preparation technology and process engineering of traditional Chinese medicine is an important step forward to modernization and industrialization of traditional Chinese medicine production.In the process of process engineering of TCM component preparation technology,reasonable adjustment of process,multi-level,whole process and dynamic quality control should be carried out under the guidance of GMP and other requirements,combining existing production technology and automated advanced production equipment,which is conducive to improving the controllability of dosage,safety and effectiveness of future component preparations of traditional Chinese medicine.Only in this way can we have a firm foothold in today’s pharmaceutical market,better promote the development of internationalization of traditional Chinese medicine,and improve the recognition of traditional Chinese medicine in the international pharmaceutical marketEpimedium is a perennial herb of the genus Epimedium(Berberidaceae).Our research group has applied for and carried out a number of projects of the National Natural Fund around it Previous studies have shown that Epimedium has a definite anti-osteoporosis effect.The main active ingredient of Epimedium is flavonoid,which mostly exist in the form of flavonoid glycosides.With the increase of the number of glycosyl carriers,the solubility increased,but the absorption of less glycosides was worse.The absorption of less glycosides was better than that of polysaccharide glycosides,and the effective substance was less glycosides from Herba Epimedii However,heat processing and intestinal bacterial metabolism contribute to the conversion of Epimedium polysaccharide glycoside flavonoids to less glycoside flavonoids,but not to a full extent.The aim of this paper is to establish a set of technological methods to enhance the content of less glycoside flavonoids and to prepare high purity of the flavonoid component with less glycoside from Herba Epimedii.The research is divided into several parts:1.Based on the evaluation criteria of single component index or rough determination of total flavonoids absorbed by ultraviolet light in the previous study of extraction technology,Four polysaccharide glycoside flavonoids representing the whole total flavonoids of Herba Epimedii,,namely Epimedin A,Epimedin B,Epimedin C,Icariin,whose contents is relatively high in decoction pieces of Epimedium,are selected as the index ingredients to evaluate the quality of the process.Through single factor experiments,the factors and levels of the ethanol extraction conditions of the total flavonoids from Herba Epimedii were preliminarily screened:ethanol concentration(40%,50%,60%),extraction times(1,2,3 times),ethanol volume(18,20,22 times)and extraction duration(1.5,2.0,2.5 h);on this basis,the best extraction process conditions were further optimized by orthogonal test:20 times of 50%ethanol was added to the decoction pieces of Herba Epimedii,which was heated and refluxed for 3 times,2.0 h each time.In order to maximize economic benefits and minimize manpower and material resources,the best extraction process was adjusted by combining with actual production conditions.The final extraction conditions of total flavonoids from Herba Epimedii were determined as follows:adding 20 times 50%ethanol into decoction pieces of Herba Epimedii,heating reflux twice,2.0 h each time.2、A set of equipment which could recover ethanol and concentrate extraction in large quantities at room temperature was built:ceramic membrane + reverse osmosis,ceramic membrane device could filter and remove impurities further.The concentration of recovered ethanol solution obtained while concentrating the extraction in reverse osmosis device is closer to that of ethanol in ethanol extraction process,which is conducive to direct secondary use.Furthermore,the pilot scale technology of the two devices is relatively mature,and it would be put into industrialized production of traditional Chinese medicine in the future with high feasibility、high application prospect and value.3、The effects of cellulase on the transformation of Epimedium polysaccharide monomers were studied.The results showed that only C-7 glucosyl groups were removed from Epimedin A,Epimedin B,Epimedin C,Icariin under cellulase,which were completely converted into corresponding less glycosides Sagittatoside A,Sagittatoside B,2"-0-rhamnosyl-icariside Ⅱ,Baohuoside I.It was further suggested that introducing the process of enzymatic hydrolysis in vitro was helpful for the transformation of polysaccharide glycosides into less glycosides in the total flavonoids and to increase the content of less glycoside flavonoids.The optimum conditions of enzymatic hydrolysis were obtained by single factor experiments.Under 65 ℃ conditions,cellulase(material-liquid ratio 20:0.40)reacted with total flavonoids from Herba Epimedii for 3 hours,and then all the polysaccharide glycoside flavonoids could be converted into les glycoside flavonoids.4、On the basis of simple and rough separation and purification technology,gradient elution process was further refined,and recycling purification and enrichment operation w as adopted for local links in order to improve the purity of the flavonoid component wit h less glycoside from Herba Epimedi and reduce the loss of target components in the pr ocess of separation,purification and enrichment.The separation and purification process fo r preparing the flavonoid component with less glycoside from Herba Epimedii was establ ished.Gradient elution was carried out with water,40%,60%and 80%ethanol in turn.The 60%elution part was collected for secondary separation and purification.80%ethan of eluent in two times separation and purification was collected and combined.the flavon oid component with less glycoside from Herba Epimedii was obtained by crystallization with purity over 80%.Six main components in the flavonoid component with less glycos ide from Herba Epimedii were identified by HPLC-ESI-QTOF-MS,whose names,relative molecular weights and chemical formulas were Baohuoside Ⅱ(500、C26H28O10),Sagittatos ide A(676、C33H40⒚15),Sagittatoside B(646、C32H38⒚14),2"-0-rhamnosyl-icariside Ⅱ(660、C33H40O14),Anhydroicaritin-3-O-rha-dideoxyfuranose(658、C33H38O14),Baohuoside 1(514、C 27H30O10),respectively.5、Based on the preparation technology of the flavonoid component with less glycoside from Herba Epimedi,this paper continued the process engineering research.And a whole process flow chart,detailed steps and quality control points of the preparation technology were planned,which aimed at clearly displaying the operation and requirements of the whole preparation process,so as to ensure the standardization,systematization and rationality of the process engineering.