Study On The High Performance Liquid Chromatography Applied To The Analysis Of Some Medicinal Plants

As a precious heritage of our China,Chinese medicine has been accepted widely by our people during the last five thousand years.As time goes on,Chinese medicine also had a larger impact internationally.In many Asian countries Chinese medicine is used as an important tool to improve the health of people.The importances of natural medicine and traditional medicine are paid more and more attention by some developed countries like Europe and America.Separation and analysis of active components in traditional Chinese medicine is playing the vital role in the modernization of traditional Chinese herb medicine.But Chinese medicine is a typically complicated unknown sample,to illustrate its chemical constitution is one of the key scientific subjects of Traditional Chinese Medicine(TCM) modernization. Liquid chromatography(LC) is the most common technology to be used in the analysis of TCM,as well as the essential separating method combined with MS or NMR. Therefore,based on the chromatography theory,how to construct a systematic and fast development platform for the liquid chromatography analysis method of Chinese medicine gets more and more attention,and this plays a very important role in the basic chemical substances research of Chinese medicine.On the basis of the previous literatures,the following major innovative works were carried out in this dissertation:(1) A novel HPLC method was developed for simultaneous determination of six organic acids in propolis liquid.The developed method may provide the basis for the determination of organic acids in propolis liquid and identification for quality of the related propolis products.(2) The separation and determination of four secondary metabolites(Chlorogenic acid,P-hydroxybenzoic,Phenol and Coumarins) in both mulberry leaves and cudrania tricuspidata leaves.It was first to research the type and content differences of four secondary metabolites in both mulberry leaves and cudrania tricuspidata leaves.(3) A novel RP-HPLC with fluorescence detection was developed for the determination of Toosendanin(TSN) in the fruit of Melia azedarach,which was obtained by using a variety of separation and purification technology.(4) We established HPLC chromatographic fingerprint of semen cassia before and after processing to provide a scientific basement for the quality evaluation of it.This dissertation consists of five chapters.Chapter 1:In this chapter,the current status of HPLC technique is given by complete description at characteristic and development of HPLC and the application of HPLC in study of Chinese herbal preparations.Chapter 2:A reversed-phase high performance liquid chromatographic method was employed to the determination of six organic acids in propolis liquid.The six compounds were analyzed by an Eclipse XDB-C₁₈ column(4.6 mm×150 mm;5μm), the mobile phase was acetonitrile- 0.4%acetic acids(30:70,V/V,pH 2.9),the flow rate was 0.g mL/min,column temperature was 28℃.The detection wavelength was performed according to time program:0-2.6 min,325 nm;2.6-3.0 min,260 nm;3.0-4.0 min,325 nm;4.0-9.0 min,280 nm.The recoveries of sample added standard ranged from 89.35%to 104.5%,the relative standard deviation was below 3%.The method was simple,rapid and has shown good sensitivity according with the results obtained by HPLC-UV detection,the method can be successfully applied to the separation and determination of the six compounds in propolis liquid.Chpter 3:A reversed-phase high performance liquid chromatography method for the separation and determination of four secondary metabolites(Chlorogenic acid, P-hydroxybenzoic,Phenol and Coumarins) was developed.The column was Eclipse XDB-C₁₈(150 mm×4.6 mm,5μm).The mobile phase was a mixture of methanolwater (adjust to pH 3.5 or so with 0.1%H₃PO₄)(30:70,V/V),column temperature was 20℃,DAD detector was performed atλ₁=325 nm,λ₂=254 nm,λ₃=218 nm.External standard was used and the calibration curves showed good linearity over the range of 440～1.35 mg/L(r=0.9995),400～1.25 mg/L(r=0.9990),960～3.00 mg/L(r = 0.9990),400～1.25 mg/L(r = 0.9990) for chlorogenic acid,p-hydroxybenzoic,phenol and coumarins,respectively.Chapter 4:A rapid and selective reversed-phase high performance liquid chromatography with fluorescence detection was developed and validated for the determination of Toosendanin in the fruit of Melia azedarach.The raw material was subjected to a series of steps,viz.,extraction,column chromatography and solvent recrystallization.The purified material was analysed on a high performance liquid chromatography with fluorescence detection system,using Eclipse XDB-C₁₈ column (4.6 mm×150 mm;5μm) and a mobile phase consisting of methanol-water at the flow rater of 1.0 mL/min.Column temperature was 25℃.Detection wavelength was set atλ_Ex = 230 nm,λ_Em = 320 nm.The recoveries of sample added standard ranged from 98.4%to 101.5%,and the relative standard deviation was below 3.0%.A detection limit(LOD) 4.68μg/mL was reached and a linear range was found between peak area and concentration in the range of 7.625-610.0μg/mL for TSN.Meanwhile, the effluent composition of TSN collected was also successfully characterized by IR and MS analysis.Based on the above researches,the method can be successfully used to investigate the level of TSN component in herb samples with satisfactory results.Chapter 5:HPLC chromatographic fingerprints of semen cassia before and after processing were established.The samples were analysed by a Eclipse XDB-C₁₈(150 mm×4.6 mm,5μm),and the mobile phase was acetonitrile-water(pH was adjusted 3.0 with 0.1%aqueous phosphoric acid),gradient elution,the flow rate was 1.0 mL/min,column temperature was 30℃,DAD-detection was performed at 278 nm. The proposed method is accurate,reliable and could provide a scientific basement for the quality control of semen cassia.