Studies On Quality Control Method Of Cortex Juglandis Mandshuricae And Pharmacodynamics And Pharmacokinetics Of Relative Ingredients

Cortex Juglandis Mandshuricae(CJM),the bark of Juglansmandshurica Maxim.(JMM),belongs to the Juglandaceae family.For hundreds of years,CJM has been used as a folk medicine for the treatment of cancer,diarrhea and dysentery.Modern medical researches reveal that CJM has various pharmacological activities,such as anti-oxidant,anti-tumor,anti-inflammatory and anti-parasitic actions.So CJM is a kind of medicinal plants with high value for development.In this study,the quality control method of Cortex Juglandis Mandshuricae,pharmacodynamics and pharmacokinetics of relative ingredients were investigated systematically as follows:1.Microwave extraction process and quality control of CJMA novel and rapid microwave extraction(ME)and ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method was developed for the simultaneous determination of two quinones,seven flavonoids,and gallic acid in Cortex Juglandis Mandshuricae.The operation of ME optimized through orthogonal array design experiments was performed at 70℃ for 8 min with ethanol-water(70:30,v/v)as the extracting solvent at the ratio of solid to solvent equal to 1:30.The method was fully validated with acceptable linearity(r>0.999),intra-and inter-day precision,reproducibility,and extraction recoveries.And the proposed method was successfully applied to analyzing 15 batches of CJM obtained from different regions of Northern China.The hierarchical cluster analysis(HCA)was used to differentiate and classified the obtained data,indicating that the influence of CJM cultivation regions on the contents of index constituents was very obvious.The developed procedure is a promising analytical tool for the overall quality control of CJM.2.Preparation of total flavonoids from CJM and their protective effect on acute liver injuryA simple and efficient chromatographic separation method was developed for preparative separation and enrichment of total flavonoids(TFs)from Cortex Juglandis Mandshuricae(CJM)extracts.Enrichment and purification of TFs from CJM extracts were studied using six macroporous resins and HPD-750 resin was selected as the best resin according to its adsorption and desorption properties.The operating parameters of resin column chromatography were optimized.Under the optimal conditions,TFs from CJM with purity larger than 50%were produced and their antioxidant activity was further evaluated in vitro(DPPH· free radical).Then,the obtained TFs was used to investigate the protective effect against CCl4-induced acute liver injury in mice.The mice were orally administrated with the purified TFs for seven days and then given CCl4(0.3%,10 mL/kg i.p.).The results showed that TFs of CJM significantly attenuated the activities of serum aspartate transaminase(AST)and alanine transaminase(ALT)compared with model group as well as the relative liver weight.Histopathological observation also revealed that TFs reduced the incidence of liver lesions and improved the hepatocyte abnormality.Moreover,oral administration of TFs significantly enhanced antioxidant enzyme activities(superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px))and decreased the content of malondialdehyde(MDA).All of the histopathological and biochemical results elicited that TFs of CJM have significant hepatoprotective activity comparable to the standard silymarin.The current work is the first time to reveal the protective actions of the TFs from CJM against CCl4-induced liver damage in mice and this natural product should be developed as a new drug for treatment of live injury in the future.3.Study on the interaction between Juglone and human serum albuminJuglone(JL),as one of the major bioactive components present in the bark of Juglans mandshruica Maxim,exhibits versatile bioactivities,especially anti-cancer activity.To better understand the pharmacokinetic properties of juglone,the protein binding rate of juglone was determined by ultrafiltration method,and the binding affinity and mechanism between JL and human serum albumin(HSA)was investigated in vitro through multi-spectroscopic,thermodynamic,and molecular modeling methods.The binding degree of JL was measured more than 95.0%which suggested that JL had high binding ability to serum albumin.Fluorescence data showed that juglone quench the intrinsic fluorescence of HSA upon forming the JL-HSA non fluorescent complex at 1:1 stoichiometric proportion,and the complex formation had a high affinity of 104 L·mol-1.Meanwhile,the site marker competitive experiments and the thermodynamic parameters(ΔG=-26.08kJ·mol-1,ΔH=-16.34kJ·mol-1,ΔS=32.69J·mol-1·K-1)indicated that juglone could spontaneously bound to the site I(subdomain ⅡA)of has through hydrophobic and hydrogen bonding interactions.As further revealed by the synchronous fluorescence,three-dimensional fluorescence,Fourier transform infrared and circular dichroism spectroscopy,JL could cause conformational and structural alterations of HSA.Additionally,molecular docking was employed to further define the specific binding site and the result was in accordance with the conclusion of experimental analysis.The present work provided reasonable models helping us further understand the pharmacokinetics,pharmacological and toxic effects of JL in vivo and supplied an important insight for the applications of JL in the clinical research.4.Study on the pharmacokinetics of JugloneA new method was developed for the quantification of juglone in rat plasma by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)after derivatization with 2-mercaptoethanol via Michael addition reaction.The derivatization conditions,such as amount of the derivatization reagent,reaction temperature and reaction time were optimized and the derivative had been identified by MS,1H-NMR and 13C-NMR spectra.The method showed a good linearity and other validation parameters for specificity,extraction recovery,matrix effect,precision,accuracy and stability were within acceptance range.The validated method was successfully applied to the comparative pharmacokinetic study of juglone in rat plasma after oral administration of CJM extract and single juglone,respectively.The Cmax T1/2 and AUC of juglone were considerably increased(P<0.05)after oral administration of the CJM extract in comparison with the equivalent dose of single component administration.This comparative pharmacokinetic study indicated that existence of other compounds in the extract of CJM might play an cooperation and promotive role on the pharmacokinetics of Juglone.In the thesis,CJM as well as juglone were investigated in the aspects of extraction,quality control,pharmacodynamics and pharmacokinetics by utilizing various professional knowledge and techniques such as phytochemistry,analytical chemistry,pharmacology,pharmacokinetics and computer science.The novel and rapid microwave extraction(ME)method was optimized and the quality control of CJM based on UPLC-MS/MS method was developed.The simple and efficient chromatographic separation method was developed for preparative separation and enrichment of TFs from CJM extracts and the protective effect of TFs against CCl4-induced acute liver injury in mice was investigated.On the other hand,the interaction between julone and human serum albumin was investigated by spectroscopic and molecular modeling methods.The novel derivative method for the pretreatment of plasma sample containing juglone had been developed and analytical method using UPLC-MS/MS was employed for the pharmacokinetics study of juglone.All of the achieved results in the thesis would provide the scientific basis for the exploration and utilization of CJM.