| The active ingredients are the main medicinal components in traditional Chinese medicine(TCM).They are usually a group of compounds with similar structure and sometimes without ultraviolet absorption,such as saponins and polysaccharides.So the separation and purification of the active ingredients in TCM are of great significance for the study of their pharmacological effects and quality control.Evaporative light scattering detector(ELSD)is a kind of universal instrument,as its signal strength is related to the compound quality.It has the advantages of high sensitivity and stable baseline.So it has great advantages in detecting components with no/low UV absorption.Preparative high performance liquid chromatography combined evaporative light scattering detector(Prep-HPLC-ESLD)is an advanced preparation technology.Compared with column chromatography,Prep-HPLC-ESLD has many advantages,such as high resolution,high automation,direct detection of components in eluent,and convenient control of mobile phase gradient.Prep-HPLCESLD has a great advantage in the separation and purification of Chinese traditional medicine with no/weak UV absorption of active components.This thesis is divided into five chapters:In chapter 1,the structure,advantages and application of ELSD and Prep-HPLC in the study of TCM were reviewed.The classification,pharmacological action and preparation methods of ginkgolides and Ginkgo biloba flavone in Ginkgo biloba L.and ziyuglycosides in Sanguisorba officinalis L.were described in detail.In chapter 2,a method for the rapid,convenient preparation of 5 kinds of ginkgolides from ginkgo biloba leaves was established,including extraction,purification and components’ separation.During the extraction process,the extraction efficiency of ginkgolides and ginkgolide B was investigated by solvent,solid-liquid ratio,extraction duration and extraction times,and the optimal reflux extraction conditions were obtained: extracted by 50% ethanol of 12 times(g/m L),extracting 1.5 h for3 times;and as a result,the yield of ginkgolides was 4.54‰,and the yield of ginkgolide B was 0.63‰.During the purification process,the dosage and extraction efficiency of ethyl acetate were investigated.The crude extract of ginkgo biloba was adsorbed by acid alumina,extracted by petroleum ether,extracted by ethyl acetate and recrystallized by methanol,and then the purity of ginkgo biloba was up to 60%.After optimization of separation conditions and sample size,5 ginkgolide monomers were prepared using C18 semi-preparative column within 65 min,with purity up to 97%.In chapter 3,a method was established to prepare 5 kinds of ginkgolide and 3 kinds of ginkgo flavone aglycones from ginkgo biloba leaves at the same time.This method can make full use of ginkgo biloba leaves,and its operation was easy and convenient.The crude extracts in Chapter 2 were raw material.In the polyamide column,ginkgolides was eluted by 10% ethanol elution first and then gingko flavonoid glycosides were eluted by 80% ethanol elution,their purities was about 80% and 35%respectively.Then the gingko flavonoid glycosides were hydrolyzed,in which 4 conditions were investigated and the result showed when the methanol proportion was 70%,hydrochloric acid concentration was 24%,hydrolysis time was 3 hours,hydrolysis temperature is 70℃,solid-liquid ratio was 10 times or more,hydrolysis efficiency could be more than95%.The mass fraction of three ginkgo flavone aglycones was about 40%after extraction with ethyl ether-ethyl acetate mixed solvent.After optimization of separation conditions and sample size,three ginkgo flavone aglycones were prepared using C18 semi-prepared column within40 min,with purity up to 98%.5 kinds of ginkgolides with purity up to 98%were prepared by the method described in chapter 2.In chapter 4,the preparation method of ziyuglycoside I and ziyuglycoside II from Sanguisorba officinalis L.was established,which was mainly divided into three steps: extraction,purification and separation.During the extraction process,the extraction efficiency of ziyuglycosides was investigated,result was shown that when the proportion of ethanol solution was 80%,solid-liquid ratio was 1:10,each extracting time was 20 min,extracting frequency was three times,the extraction rate of ziyuglycosides was about 0.49%.In the purification part,the optimal p H value of precipitation was 13,at that time,the content of ziyuglycosides was the highest.After optimization of separation conditions and sample size,gradient elution was carried out with methanol-0.1% formic acid as mobile phase,and the ziyuglycoside I and II were prepared by C18 semipreparative column(10 mm I.D.× 25 cm,5 μm)within 45 min.The purity was up to 97%.This simple and fast preparative method is the first simultaneous preparation of ziyuglycoside I and ziyuglycoside II reported.In chapter 5,we summarized the whole thesis and looked forward to possible rirections in the future. |
PDF Full Text Request