Quality And Safety Evaluation Of Diaphragma Juglandis Fructus From Yunnan And Antimalarial Compounds Of Dendrobenthamia Capitata

This dissertation is composed of three parts: chapter one expounded the quality and safety evaluation of Diaphragma Juglandis Fructus(DJF)from Yunnan;chapter two introduced the isolation and identification of antimalarial compounds from Dendrobenthamia capitata;chapter three was a mini-review about the research advance study on the DJF.Objective: To study the common constituents of aqueous extracts of the DJFs from different regions.To establish safety and quality evaluation system of the DJF based on these common constituents and comparison of the acute toxicity of aqueous extracts of the DJFs from Dali and Kashgar.To study antimalarial activity of D.capitata to lay the foundation for the development of novel antimalarial drugs from plants.Method: Chemical fingerprints of aqueous extracts of the DJFs from different regions were established by HPLC analyses.The common chemical constituents were evaluated based on HPLC fingerprint,and the constituents were traced,isolated,purified,and identified from Dali DJF by chromatography and spectroscopy techniques.The common constituents from different regions' DJFs were quantitatively analyzed by HPLC and their stabilities were determined based on Dali DJF which was analyzed at different stored period.Contents of some elements between aqueous extracts and plant powders of Dali DJF were determined and compared by AFS,AAS,and ICP-AES.The LD50 values of the acute toxicity of the DJFs from Dali and Kashgar toward Kunming mice were calculated by Bliss method,and the tissue histological examinations of the mice were performed by HE stain.?-Hematin formation inhibition experiment was used to evaluate antimalarial activity in vitro,and the active constituents from D.capitata were traced,isolated,purified,and identified by chromatography and spectroscopy techniques.Results: HPLC fingerprints of aqueous extracts of the DJFs from different regions were compared and analyzed,and a total of seven common constituents were obtained.The similarity values of the DJFs from different regions were higher than 0.78,except for the DJFs from Dali(0.61)and Hancheng(0.66).According to relative amounts of common constituents,the DJFs from different regions could be divided into three types by hierarchical cluster analysis,while two principal components could be concluded by the Principal Component Analysis.As a result,Lijiang DJF showed the best quality among the testing DJF samples based on multiple index evaluation.Nine compounds were isolated from the fraction of ethyl acetate of 75% ethanol extracts of Dali DJF and identified as protocatechuic acid(1),vanillin(2),methyl gallate(3),vanillic acid(4),syringic acid(5),4'-dihydro-phaseic acid(6),gallic acid(7),juglanoside E(8),and ellagic acid(9).Meanwhile,compounds 1,4-6,and 9,could be assigned to the index components of aqueous extracts from different regions DJF.The contents range values of these five index components for the DJFs from different regions were 27.75 ~ 300.94 mg/kg,20.33 ~ 151.56 mg/kg,13.48 ~ 168.84 mg/kg,13.53 ~ 793.85 mg/kg,and 125.77 ~ 516.13 mg/kg,respectively.The contents range values of these five common constituents contents for the Dali DJF detected at different stored period were 11.54 ~ 163.66 mg/kg,42.05 ~ 112.38 mg/kg,13.48 ~ 41.33 mg/kg,13.53 ~ 261.55 mg/kg,and 108.58 ~ 1468.16 mg/kg,respectively.Based on the analyses of the DJF from Dali,elements Se,Cd,Pb,Hg,As,Cr,Co,and Ni were not detected in both aqueous extracts and plant powders,and the contents of Cu,Zn,Mn,Mg,Ti,and K in aqueous extracts were higher than that in plant powders,but the contents of Fe,La,Li Sr,and Ba in aqueous extracts were lower than which in plant powders.The LD50 values of the acute toxicity toward Kunming mice about the aqueous extracts of the DJFs from Dali and Kashgar were 21.50 g/kg and 12.40 g/kg,respectively.Pathologic tissue examination showed that both aqueous extracts of the DJFs from Dali and Kashgar were toxic against cardiac myolytic necrosis,and could cause extensive dissolving and necrosis of the liver,but exhibited no toxicity for spleen,lung,kidney,and engender.Four compounds were obtained from the ethyl acetate part of ethanol extract of twigs and leaves of D.capitata and identified as catechin(1),quercetin(2),isoquercitrin(3),and 2?-hydroxyursolic acid(4).All compounds were tested for the antimalarial activity by ?-Hematin formation inhibition assay,and compound 2 showed the IC50 value of 155.7 ± 6.5 ?g/m L,while the inhibition ratios of compounds 1 and 3 were 32.2% and 28.4% at the highest concentration,but compound 4 did not show activity.Conclusion: The study on the quality evaluation of the common constituents in aqueous extracts of DJFs from different regions showed that the plant origin of the DJFs from Dali and Hancheng has close relatives.Meanwhile,both the DJFs from Dali and Lijiang have the best quality.The isolation,purification,and identification of common constituents indicated that phenolic compounds were the main common constituents of aqueous extracts of the DJFs from different regions.However,the contents of these common constituents were significantly different(P < 0.01).Moreover,the contents of index components from Dali DJF tested at different stored period were of significant difference(P < 0.01).The relative contents of elements Zn,Mn,and Cu which were thought to be kidney disease-related elements in the DJF were increased after poaching,which is accordance with the traditional folk application of aqueous extracts of the DJF.Aqueous extracts of the DJFs from Dali and Kashgar revealed acute lethal toxicity toward Kunming mice,and showed cardiotoxicity and hepatic toxicity.Flavonoids may be the substance of D.capitata for antimalarial activity.The study not only provided a data reference for the antimalarial actives of D.capitata,but also provided a basis for the development of new antimalarial drugs.