Preparation Process And Quality Control Standard Of Two Different Specifications Of Scutellaria Baicalensis Georgi Extracts

BACKGROUND:Radix Scutellariae,a member of Labiatae,is the dried root of Scutellaria baicalensis Georgi(SBG).It's cold-natured and bitter in taste.It has the effects of heat-clearing,dampness,purging fire,detoxification,stanching blood and tocolysis efficacy.It's one of the popular herbal medicines in Shanxi.The production of SBG in Shanxi accounted for more than 40%of the national production.However,the raw materials is mainly used with simple initial processing;the proportion of extracting and deep processing herbs is less than 1%,so its added value is very low.SBG extracts are obtained by extraction and purification from SBG and the pharm-acopoeia provide that the content of baicalin in it can not be less than 85%.There are a variety of different specifications of the extracts on the market at present,widely used in pharmaceutical,cosmetics,veterinary drugs and other fields.In the pharmaceutical field,SBG extracts with baicalin greater than 85%and 90%in content is widely used as raw material.Among them,the SBG extract with baicalin greater than 85%in content,namely 85%SBG extract is used as raw materials a total of 12 kinds of preparation such as the Yinhuang granules,Yinzhihuang injection,Shuanghuanglian and Qingkailing preparation and so on;90%SBG extract is used as raw materials of 3 kinds of preparation,namely Yinhuang Oral liquid,Qingkailing oral liquid and Qingkailing Injection.All of these formulations are widely used traditional Chinese medicine and their market demands are very large.Therefore,it is significant to study the 85%and 90%SBG extract.The studies of SBG extracts are very much,but there are still problems as follows:(1)The harvest season of SBG is not sure and the evaluation index is monotonous,so its quality is difficult to control.(2)The existing preparation process is cumber-some,low recovery rate and heavy environmental pollution,so there's a lack of a fast,efficient and environmental friendly preparation method.(3)The standard of extract is monotonous,Chinese Pharmacopoeia only included the quality standard of 85%SBG extract,there is a lack of differentiated quality control methods.We will study the above problems in this paper.OBJECTIVE:1.To compare the quality of SBG from different harvest periods and determine the optimum harvest season of SBG,provide a refer-ence for the source of raw materials of SBG extract.2.To establish the preparation process of 85%and 90%SBG extracts respectively and then verify them.3.To establish the quality control standards of 85%and 90%SBG extracts respectively.METHODS:1.With the one-year and two-year SBG samples from different harvest periods as experimental object,the dynamic changes of the chemical composition in kinds and contents were detected by UPLC-MS/MS metabolomics means,and then search for and identify biomarker metabolites,and determine the optimum harvest period of SBG.2.With the content of baicalin and extraction rate in extracts as assessing indexes,different extraction methods and solventions were investigated to evaluate their influences on the extraction process and the the optimal level of each factor was selected.Further the optimal level of each factor were selected by single factor test and the SBG extraction process were determin-ed;and then with the content of baicalin and yield rate in refined products as assessing indexes,the effects of acid precipitation,macroporous resin purify-cation and high speed counter-current chromatography(HSCCC)were tested respectively.Different methods of extraction and purification were combined to establish the preparation process of 85%and 90%SBG extracts respectiv-ely and then verify the processes.3.To establish the quality standards of 85%and 90%SBG extracts respectively,and determine the standard limit from the determined data.Quality indicators including the contents of water and residue,TLC identi-fication and HPLC.RESULTS:1.A SBG plant metabolomics methodology based on UPLC-MS/MS technology was established.The dynamic changes of the chemical composition in kinds and contents of the one-year and two-year SBG samples from different harvest periods were studied;the results showed that the chemical composition of different harvest seasons had obvious difference,and showed regular changes:the contents of flavonoid glycosides from spring to summer and autumn showed a trend of "decrease-increase-decrease-increase again";the variation of the content of flavonoid glycosides were on the contrary,they showed a trend of "increase-decrease-increase-decrease again"from spring to summer and autumn.Although the changes of the two kinds of chemical composition are obviously different,the content in the early winter(October)return to the near level of the end in spring(April).In addition,the content of baicalin,as the the main component of the SBG extracts,reached the maximum value in April and August,so the best harvest time is April and August.2.The preparation process of 85%SBG extract was established.The process route was as follows:Scutellaria baicalensis Georgi herbs was reflux extracted twice with 10 folds 70%ethanol at 80 ? for 1.5 hours each time.After filtrated,the extracts were combined together,and then the pH of the filtrate was adjusted to 1.0 with hydrochloric acid at the concentration of 6 mol/L.The solution was kept in a water bath with the temperature of 80? for 60 min,then laid aside 12 hours in order to deposit completely.The upper liquid was centrifuged to spill out.The deposit was washed with water,50%ethanol and 95%ethanol for once,respectively,till the pH value to neutral.After the solution was volatiled completely,the 85%SBG extract can be obtained.3.Two different preparation processes of 90%SBG extract was established.The first process route was as follows:Scutellaria baicalensis Georgi herbs was reflux extracted twice with 10 folds 70%ethanol at 80? for 1.5 hours each time.After filtrated,the extracts were combined together,then dried and concentrated with a rotary evaporator to obtain a crude extract.The crude extract was then dissolved in water and loaded onto HPD100 macro-porous resin,first eluted to remove impurities with 4 column volumes(BV)water,then followed by 3BV 50%ethanol to elute,collect the 50%ethanol eluent,dried and concentrated the solvent with a rotary evaporator to obtain a more refined extract.And next the resulting refined extract was injected into the HSCCC system with ethyl acetat-n-butanol-methanol-water(4:1:0.5:6,v/v/v/v)as its two-phase solution system for further purification,collect the components eluent according to the chromatographic peak,combined baicalin component together,the 90%SBG extract can be obtained.This preparation process route is simple;its production cycle is short and the recovery rate and purity of the final product are both high.Compared to the traditional preparation method,it had avoided to produce large amount of acid water;it was more environmental friendly and safe.Because HSCCC technology has not yet been applied to the industrial production,this route can be used as a method to prepare the SBG extract.The second preparation process route was as follows:Scutellaria baicalensis Georgi herbs was reflux extracted twice with 10 folds pH 4-5 70%ethanol at 80? for 1.5 hours each time.After filtrated,the extracts were combined together,and then the pH of the filtrate was adjusted to 1.0 with hydrochloric acid at the concentration of 6mol/L.The solution was kept in a water bath with the temperature of 80? for 60 min,then laid aside 12 hours in order to deposit completely.The upper liquid was centrifuged to spill out.The deposit was washed with water,50%ethanol and 95%ethanol for once respectively,till the pH value to neutral.After the solvention was volat-iled completely,dissolved the resulting powder in water,and loaded onto the HPD100 macroporous resin,first eluted to remove impurities with 4 BV water,then followed by 3BV 50%ethanol to elute,collect the 50%ethanol eluent,dried and concentrated the solvent with a rotary evaporator to obtain the target extract.The route is simple in operation;it can avoid the environ-mental pollution brought by the commonly used two-step acid precipitation method and have the advantages of high repeatability,strong operability,and is suitable for industrial production.4.Established a new content determination method of baicalin and two kinds of TLC identification methods of two different specifications of SBG extracts.Provide that according to the dry matter,the moisture content of 85%SBG extract should not be more than 4.04%;the flaming residue of 85%SBG should not be more than 0.48%;the moisture content of 90%SBG extract should not be more than 3.92%;the flaming residue of 90%SBG should not be more than 0.53%.CONCLUSIONS:1.This study established a SBG plant metabolomics methodology based on UPLC-MS/MS technology.The quality of SBG from different harvest time were compared to determine the best harvest time of SBG.It can provide a reference for the harvest of SBG plant.2.This study established the preparation process of 85%and 90%SBG extracts respectively and the process verification were carried out respective-ly.These processes were stable,feasible and more efficient and less environ-mental pollution compared to the traditional preparation processes.3.This study established the quality standards of the 85%and 90%SBG extracts respectively,which were higher than the standards of Pharmacopoeia standards.They can provide the basis for quality control of different specifications of SBG extracts.