| 1Contents analysis and distribution of flavonoids in Celastrus orbiculartus Thunb. by capillary zone electrophoresisA new capillary zone electrophoresis （CZE） method was developed for the simultaneous quantification of rutin, kaempferol and quercetin in Celastrus orbiculatus Thunb.. Analysedresults differences and discussed the medicinal quality control methods: The electrophoreticanalysis was performed on30mmol L-1sodium borate solution（pH=9.0）,20kV electrophoresis voltage and254nm wavelength.Rutin, kaempferol and quercetin were separated successfully in12minutes by CZE,a good linearity between peak area and concentration was found inrange of2.5-500μg m L-1for three compounds:YR=841.7C+4919.3（r=0.998）;YK=2599.1C+12999（r=0.999）;YQ=5111.8C+13570（r=0.999）.The contents of rutin in bark and leaf were1.41,1.37mg g-1, respectively.The content of kaempferol in bark is2.16mg g-1,thecontent of quercetin in root was0.016mg g-1.Flavonoids were abundant in Celastrus orbiculatus Thunb. and the contents of three flavonoids were distinct difference in different parts.Simultaneous determination rutin, kaempferol and quercetin by CZE method has a good accuracyand precision.It is simple, fast and applicable for clinical and scientific research to determine three flavonoids.This provided a technology method and solid theoretical data for Celastrus orbiculatus Thunb. of medicinal quality control.2Optimization extraction process and contents distribution of ursolic acidand oleanoic acid in Celastrus orbiculatus Thunb. by Ultrasonicextraction and capillary zone electrophoresisA ultrasonic-assisted extraction （UAE） method was optimized using a centralcomposite experimental design by Response Surface Methodology （RSM）, and anefficient Capillary zone electrophoresis （CZE）analysis method was developed for fastextraction and simultaneous determination of ursolic acid and oleanolic acid in Celastrusorbiculatus Thunb.（C.orbiculatus Thunb.）,then determined the distributing of ursolic acidand oleanolic acid in different parts of C.orbiculatus Thunb.: Different concentration solvents, solvent to material ratio and extraction time were all as examing factors to design acentral composite experimental of three factors and three levels for optimizing extractionprocess of ursolic acid and oleanolic acid.Concentration of Sodium borate buffersolution,the value of pH and separation voltage were all as examing factors to filter the bestseparation condition of ursolic acid and oleanolic acid.Results showed that the extractioncondition were80%methanol, solvent to material ratio （m/v）20.0mL g-1and extractiontime50.0min,optimized CZE conditions were80mmol L-1Sodium borate buffer solution（pH=9.80） as mobile phase,20kV separation voltage. The UV detection wavelength was210nm. Under the conditions, ursolic acid and oleanolic acid were separated and detectedwithin22min. Linea ranges of two compounds were0.0348-0.1043μg μL-1,YUA=300801x+1210.5（r=0.9992） and0.0624-0.187μg μL-1,YOA=349265x+4118.3（r=0.9992）,respectively.The recoveries were97.23%and96.45%,RSD<5.00%（n=6）.The contents of ursolic acid in root was0.37mg g-1and it was about five times inthe leaves. The contents of oleanolic acid in stem and bark were0.67,0.52mg g-1they wereabout five times in the leaves. Response surface methodology and capillary zoneelectrophoresis technology optimized the extraction processes and determined the contentsdistributing of ursolic acid and oleanolic acid in Celastrus orbiculatus Thunb. has a goodaccuracy and precision.This rapid,sensitive, accurate and stable method is suitable inresearch practice and clinical settings.Ursolic acid and oleanolic acid are distributing in root,stem, leaf and bark and there are remarkable differences.3Optimization the extraction process and content distribution of Celastrolin Celastrus orbiculatus Thunb. by Ultrasonic extraction and capillary zoneelectrophoresisA ultrasonic-assisted extraction （UAE） method was optimized using a centralcomposite experimental design by Response Surface Methodology（RSM）, and an efficientCapillary zone electrophoresis （CZE）analysis method was developed for fast extractionand simultaneous determination of celastrol in Celastrus orbiculatus Thunb.（C.orbiculatusThunb.）,then determined the distributing of celastrol in different parts of C.orbiculatusThunb.: Different concentration solvents, solvent to material ratio and extraction time were allas examing factors to design a central composite experimental of three factors and three levelsfor optimizing extraction process of celastrol.Concentration of Sodium borate buffersolution,the value of pH and separation voltage were all as examing factors to filter the best separation condition of celastrol.Results showed that the extraction condition were80%methanol, solvent to material ratio （m/v）20.0mL g-1and extraction time50.0min,optimized CZE conditions were10%methanol and70mM Sodium borate buffer solution（pH=9.00） as mobile phase,22kV separation voltage. The UV detection wavelength was210nm. Under the conditions, celastrol was separated and detected within15min. Linearanges of the compound was0.039-0.117μg μL-1, YCL=543282x+1967.2（r=0.9920）. Therecoveries was101.13%，RSD<4.00%（n=6）.The contents of celastrol in root,stem,barkand leaf were5.13,1.51,0.32and0.34mg g-1respectively and the content in root was aboutfive times in the stem. Response surface methodology and capillary zone electrophoresistechnology optimized the extraction process and determined the content distributing ofcelastrol in Celastrus orbiculatus Thunb. has a good accuracy and precision.Thisrapid,sensitive,accurate and stable method is suitable in research practice and clinical settings.Celastrol is distributing in root, stem, leaf and bark and there are remarkable differences.4Study on toxicity and safety of Celastrus orbiculatus Thunb. ethanolextractTo study the drug toxicity and safety of Celastrus orbiculatus Thunb. ethanol extract:The experimental project was designed by Kaerber methods to determine the maximumtolerated dose（MTD） and median lethal dose（LD50） of Celastrus orbiculatus Thunb.ethanol extract given intragastrically in mice. In the range of MTD, different doses ofCelastrus orbiculatus Thunb. ethanol extract（0.0,2.5,5.0,10.0g kg-1） were given to themice and the toxic and side effects were recorded. Meanwhile, the changes of hepaticmorphology and index were analyzed and the level of ALT and AST were detected byenzyme method. Results showed that the MTD of Celastrus orbiculatus Thunb. ethanolextract given intragastrically was10.0g kg-1in mice. Its LD50was38.17g kg-1and theconfidence limit of95%was38.14-38.19g kg-1. After treatment within the range of MTD ofthe herb, there were no significant changes in both hepatic morphology and index. Comparedwith that of control group, the level of ALT increased significantly in5and10.0g kg-1groups（P<0.01or P<0.001）and the level of AST promoted significantly in10g kg-1group（P<0.01）. So the MTD of Celastrus orbiculatus Thunb. ethanol extract givenintragastrically was10.0g kg-1in mice. Within the range of MTD, the herb is safe and thetoxicity is very low. 5Celastrus orbiculatus Thunb. improves blood fat phenotype ofhyperlipidemia guinea pigs and attenuates lipid deposition in aortic wallIt is to characterize the underlying effects and molecular mechanisms of Celastrusorbiculatus Thunb.（c. orbiculatus Thunb.） on the lipid metabolism and lipid deposition inhyperlipidemia guinea pig:48male England short-hair guinea pigs（260-310g,5monthsold） were randomly divided into control diet group （CD, grass, n=8）,high fat diet group（HFD,10%lard+10%yolk power+0.30%cholesterol+79.7%grass, n=8）, three c.orbiculatus Thunb．groups,low-dose group （HFD-L,2.5g kg-1d-1）, middle-dose group（HFD-M,5.0g kg-1d-1）, high-dose group（HFD-L,10.0g kg-1d-1） and simvastatingroup （HFD-S,20mg kg-1d-1）.After consecutive drug intervention for8weeks till the endof the experiment, concentrations of total cholesterol （TC）, total triglyceride（TG）, highdensity cholesterol （HDL-C） and non-high density cholesterol （non-HDL-C） inplasma were determined by enzymatic methods （BioSino）. Lipoprotein profiles wereobtained by fast protein liquid chromatography （FPLC）, lipoproteins（VLDL,LDL andHDL）containing equal amounts of cholesterol were loaded on a4%to15%sodium dodecylsulfate （SDS） polyacrylamide gradient gel （PAGE）.The real-time quantitativepolymerase chain reaction （RT-PCR） analyzed the expression of liver cholesterol reversetransport action related genes scavenger receptor-BI （SR-BI）,low density cholesterolreceptor（LDL-R）,3-hydroxy-3-methyl glutaryl （HMG）-CoA reductase （HMG-CoAreductase） and cholesterol7-alpha hydroxylase （CYP7α-1）. Arterial wall lipiddisposition displayed by oil red O,fat venereal change and morphology of liver displayed byhematoxylin and eosin staining.Compared with HFD group,the levels of TC,TG and non-HDL-C were decreased45%,21%and49%in HFD-H group respectively.The levels ofHDL-C were increased16%and20%in HFD-M and HFD-H. The contents of apolipoproteinB （apoB） and apolipoprotein E （apoE） in LDL and VLDL,while the contents ofapolipoprotein AI （apoAI） in plasma HDL were increased by c. orbiculatusThunb.treatment. SR-BI, LDL-R,HMG）-CoA reductase and CYP7α-1were all increased byc. orbiculatus Thunb. treatment. Arterial wall lipid disposition and fat venereal change werereduced significantly by c. orbiculatus Thunb. treatment. Furthermore,the levels ofTC,TG,FC and CE in guinea pig liver were decreased by c. orbiculatus Thunb. The studyshowed that c. orbiculatus Thunb. can inhibit lipid disposition in hyperlipidemia guinea pig aortic wall, the mechanism is related to improving blood fat phenotype,alleviating lipidoxidation and strengthening the expression of liver cholesterol reverse transport action relatedgenes.6Celastrus orbiculatus Thunb. improves blood fat inflammation and attenu-ates inflammatory response in hyperlipidemia guinea pigs aortic wallIt is to characterize the underlying effects and molecular mechanisms of Celastrusorbiculatus Thunb.（c.orbiculatus Thunb.） on the lipid metabolism and aortic wallinflammation in hyperlipidemia guinea pig:48male England short-hair guinea pigs（260-310g,5months old） were randomly divided into control diet group （CD,grass,n=8）,highfat diet group （HFD,10%lard+10%yolk power+0.30%cholesterol+79.7%grass,n=8）,three c. orbiculatus Thunb．groups,low-dose group（HFD-L,2.5g kg-1d-1）,middle-dose group （HFD-M,5.0g kg-1d-1）,high-dose group（HFD-L,10.0g kg-1d-1） andsimvastatin group （HFD-S,20mgkg-1d-1）.After consecutive drug intervention for8weekstill the end of the experiment. ELISA assay revealed that the production of tumor necrosisfactor-α,C-reaction protein and interleukin-6in plasma. In order to investigate theantioxidation of c. orbiculatus Thunb.,the levels of superoxide dismutase （SOD） andmalondialdehyde （MDA） in plasma and liver were determined by a spectrophotometricmeasurement of thiobarbituric acid-reactive substances （TBARS） and nitrobluetetrazolium-reactive substances （NBTRS） respectiveely. Immunohistochemistry andwestern blot were used to detect the expression of nuclear factor-kappaBp65（NF-κBp65）and cluster of differentiation antigen68（CD68） in aortic wall. In order to know the effectof C. orbiculatus Thunb. on endothelial dysfunction,the levels of nitric oxide（NO） andinducible nitric oxide synthase （iNOS） in liver were detected by nitrate reduetase methods（NRM）and arginine reforming process methods （ARPM） respecti-vely.At last thelevels of alanine transferase（ALT） and aspartate transferase（AST） in liver weredetected by Reitman-Frankel.The results showed that,compared with HFD group, C.orbiculatus Thunb. can inhibit the expression of NF-κBp65and CD68in aortic wall andrecuce significantly（P<0.05or P<0.01） the levels of inflammatory factors such as TNF-α,CRP and IL-6with dose-dependent.The levels of SOD in liver were increased significantly（p<0.05） in HFD-M and HFD-H, the levels of MDA in liver and plasma were reducededsignificantly （p<0.05） by c. orbiculatus Thunb.. C. orbiculatus Thunb. inhibited the high expression of NO,iNOS, ALT and AST in liver which induced by high fat diet significantly（P<0.05or P<0.01）.This study proved that c. orbiculatus Thunb.can inhibit theinflammation in plasma and aortic wall of hyperlipidemia guinea pigs which mechanism isrelated to improving blood fat phenotype, strengthening the expressions of anti-oxidationfactor and attenuating the expressions of correlation factors of inflamattory response in cell. |
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