Study On The Chemistry Of Fructus Jujubae Resources

This work was supported by the National Natural Science Foundation of China (No.30672678) and 2006 "Qinglan Project" scientific and technological innovation team training program of Jiangsu College and University.This dissertation is divided into two major parts.The first part was a detailed review about the latest research progress of Ziziphus jujuba,including germplasm resources, chemical constituents,pharmacological effects,nutritive value and quality standard.The second part was divided into five chapters,where the resource distribution of fructus jujubae, chemical constutuents,quality evaluation,quality standard and bioactivities about Ziziphus jujuba were studied as following.In the first chapter,the present condition of fructus jujubae resource distribution was investigated by literature investigation and field survey.Based on the variance of topography and geomorphology,hydrology and the major cultivation cultuvars,the cultivation regions of fructus jujubae in north of China were preliminarily divided into thirteen main areas. Furthermore,the present situation of fructus jujubae resource using for TCM was also investigated.In the second chapter,the chemical constituents of Z jujuba were studied.The compounds were isolated by silica gel and polyamide column chromatography,and their structures were identified on the basis of spectral analysis and physicochemical data.As a result,twenty three compounds were obtained from the water extract and ethanol extract, and their structures were elucidated as zizyberanal acid(1),zizyberanone(2),zizyberenalic acid(3),ceanothenic acid(4),ursonic acid(5),betulinic acid(6),zizyberanalic acid(7), alphitolic acid(8),ursolic acid(9),ceanothic acid(10),epiceanothic acid(11),oleanolic acid (12),maslinic acid(13),2α-hydroxyursolic acid(14),(2S,3S,4R,8E)-2-[(2'R)-2'-hydroxytetra-cosanamino]-8-octadecene-1,3,4-triol(15),1-O-β-D-glucopyranosyl-(2S,3S,4R, 8E)-2-[(2'R)-2'-hydroxytetra-cosanamino]-8-octadecene-1,3,4-triol(16),3β,6β-stigmast-4-en-3,6-diol(17),β-sitosterol(18),daucosterol(19),heptadecanoic acid(20),tetracosanoic acid(21),rutin(22) and D-glucose(23).Among these compounds,1 and 2 were new compounds.Compounds 15 and 16 were the ceramide-type constituents which obtained from the genus Ziziphus for the first time,and compounds 5,11,14,17,20 and 21 were obtained from this plant for the first time.In the third chapter,an HPLC-DAD method conjugated with chemometrics approach was established and used to evaluate the quality of fructus jujubae.Firstly,a simple and sensitive HPLC-DAD method has been developed for the first time to simultaneously determine ten triterpenoid acids(ceanothic acid,alphitolic acid,zizyberanal acid, zizyberanalic acid,epiceanothic acid,ceanothenic acid,betulinic acid,oleanolic acid, ursonic acid and zizyberenalic acid) in fructus jujubae.This HPLC assay was performed on a reversed-phase C₁₈ column(250 mm×4.6 mm,5μm) with the column temperature at 35℃. The mobile phase was composed of A(acetonitrile) and B(0.05%aqueous phosphoric acid, v/v).The flow rate was 1.0 mL/min and the detection wavelength was set at 205 nm for reference compounds 1-9 and 238 nm for reference compound 10.All calibration curves showed good linear regression(r²ï¼ž0.9999) within the test range.The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.43-1.72%and 0.53-2.45%,respectively,and overall recoveries of 94.98-104.09%for the ten compounds analyzed.The validated method was successfully applied for the simultaneous determination of the 10 triterpenoid acids in 42 batches of fructus jujubae which contained 36 cultivars from 22 cultivation regions,and were investigated and authenticated as Z.jujuba.Secondly,hierarchical clustering analysis(HCA) and principal components analysis(PCA) were performed to differentiate and classify the samples based on the contents of the ten triterpenoid acids.The results showed that the total content of the ten triterpenoid acids in the cultivar of Dongzao(Cultivared in Zhanhua,Shandong Province) was triple of other samples,which suggested that Dongzao is a potential cultivar of Z.jujuba considering the content of triterpenoid acids.Besides,the results of HCA and PCA showed that the diversity of Dongzao was most obvious to the other cultivars of Z.jujuba in the 36 tested cultivars.The presented HPLC-DAD method conjugated with chemometrics approach was demonstrated to be very helpful in using fructus jujubae resources,and was possibly useful in chemotaxonomic characterization.In the fourth chapter,the quality standard of fructus jujubae was studied.In order to obtain TLC chromatograms with better resolution for betulinic acid and oleanolic acid as reference substances,different extracting solvents,chromatographic conditions and colorimetric methods were compared.Then,the TLC method used for fructus jujubae identification has been established.Furthermore,an HPLC-ELSD method has been developed for the first time to determine the quantity of betulinic acid in fructus jujubae. This HPLC assay was performed on a reversed-phase C₁₈ column(250 mm×4.6 mm,5μm) with the column temperature at 25℃.The mobile phase was composed of acetonitrile-0.1% aqueous acetic acid(81:19).The flow rate was 0.4 mL/min.The parameters of ELSD were as follows:gas pressure:35 psi(2.28 slpm);drift tube temperature:65℃;nebulizer temperature:36℃.Based on the analysis results of 14 batches of fructus jujubae,the least quantity standard of betulinic acid in fructus jujubae was set as 0.025%(g/g).In the fifth chapter,the cytotoxicities of the 12 compounds,such as zizyberanal acid (1),zizyberanone(2),zizyberenalic acid(3),ceanothenic acid(4),ursonic acid(5),betulinic acid(6),zizyberanalic acid(7),alphitolic acid(8),ursolic acid(9),ceanothic acid(10), epiceanothic acid(11) and oleanolic acid(12),were investigated in vitro against MGC-803, HT-29,NCI-H460 and HepG-2 tumor cell lines by the MTT method.Among these compounds,ursolic acid,zizyberenalic acid,zizyberanalic acid and oleanolic acid showed cytotoxic activities against these tumor cell lines.The results suggested that the above four compounds may be potential as tumor inhibitors or lead compounds of anti-tumor drugs.The main features and innovations in this dissertation are presented:1.Using column chromatographic method and spectral technology,twenty three compounds were isolated from the cultivar of Z.jujuba using for TCM.Among these compounds,zizyberanal acid was new triterpenoid acid,and zizyberanone was new naphthaquinone.Furthermore,the ceramide-type constituents were obtained from the genus Ziziphus for the first time.2.By using the HPLC-DAD method conjugated with chemometrics approach,the qualities of fructus jujubae contained various cultivars from different cultivation regions were evaluated based on their contents of triterpoid acids.This method was demonstrated to be very helpful in utilizing fructus jujubae resources efficiently.3.The quality standard of fructus jujubae in Chinese Pharmacopoeia(2005 year's edition) was revised.The draft of the quality standard of betulinic acid as a reference substance has been established for the first time.And the HPLC-ELSD method has been developed for the first time to determine the content of betulinic acid in fructus jujubae, which further enhanced and standardized the quality standard of fructus jujubae.