【作者】 张颖；

【导师】 王建华； 秦树存；

【作者基本信息】 山东农业大学 ， 植物资源学， 2013， 博士

【摘要】 1毛细管电泳法分析南蛇藤中黄酮类物质成分及其含量分布建立同时测定南蛇藤中芦丁、山奈酚及槲皮素含量的毛细管电泳法，分析结果差异，探讨南蛇藤的药用质量控制方法：采用30mmol L-1硼酸钠缓冲液（PH=9.0），20kV分离电压条件下，254nm波长下毛细管电泳法测定芦丁、山萘酚、槲皮素含量。结果黄酮类物质芦丁、山萘酚和槲皮素的对照品在12min内实现完全分离，在2.5-500μg mL-1范围内呈良好的线性关系： YRT=841.7C+4919.3（r=0.998）；YKF=2599.1C+12999（r=0.999）；YQC=5111.8C+13570（r=0.999）。芦丁在皮、叶中含量高达1.41、1.37mg g-1；山奈酚在皮中含量高达2.16mg g-1；槲皮素在根中含量可达0.016mg g-1。研究表明南蛇藤中黄酮类物质含量丰富，不同部位中各黄酮含量有明显差异。毛细管电泳技术同时测定南蛇藤中芦丁、山奈酚与槲皮素具有较好的准确度和精密性，为临床和科研提供了一种简单快速的检测方法，同时为南蛇藤的药用质量控制提供了技术方法和坚实的理论数据。2超声波提取-毛细管电泳技术联用优化南蛇藤中熊果酸与齐墩果酸提取工艺及其含量测定超声波技术-响应曲面法优化南蛇藤中熊果酸与齐墩果酸的提取工艺，建立一种毛细管电泳法同时测定南蛇藤中熊果酸和齐墩果酸含量的方法，并检测熊果酸与齐墩果酸在南蛇藤不同部位中的含量分布：分别以提取溶剂浓度、料液比、超声时间为考察因素，采用三因素三水平设计中心组合实验，优化熊果酸与齐墩果酸的提取工艺。以硼酸盐缓冲液浓度、pH值、分离电压为考察因素，筛选熊果酸与齐墩果酸的最佳分离条件。结果熊果酸和齐墩果酸的最佳提取条件为：以80%甲醇为提取溶剂，料液比（m/v）为1：20，超声时间50min。以80mmol L-1的硼酸盐缓冲液（pH=9.80）作为电离介质，20kV分离电压，210nm波长下，22min内熊果酸和齐墩果酸得到很好的分离。二者线性方程分别为：YUA=300801x+1210.5（r=0.9992）和YOA=349265x+4118.3（r=0.9992），检测范围分别为0.0624-0.187μg μL-1，0.0348-0.1043μg μL-1，回收率分别为97.23%、96.45%，变异系数（RSD）<5.00%（n=6）。熊果酸在根中含量为0.37mg g-1，约为叶中的5.5倍。齐墩果酸在茎、皮中的含量为0.67、0.52mg g-1，约为叶中的3.7倍和2.8倍。超声技术-响应曲面优化熊果酸与齐墩果酸的提取工艺，毛细管电泳同时检测熊果酸与齐墩果酸含量，具有较好的准确度和精密性，为临床和科研提供了一种简单、快速、准确的检测方法。熊果酸与齐墩果酸在南蛇藤根、茎、叶、皮中均有分布，且不同部位中含量存在明显差异。3超声波提取-毛细管电泳技术联用优化南蛇藤中南蛇藤素的提取工艺及其含量测定超声波提取-响应曲面法优化南蛇藤中南蛇藤素的提取工艺，建立毛细管电泳法测定南蛇藤素含量的方法并检测其在南蛇藤不同部位中的分布：分别以提取溶剂浓度、料液比、超声时间为考察因素，采用三因素三水平设计中心组合实验，优化南蛇藤素的提取工艺。以硼酸盐缓冲液浓度、pH值、分离电压为考察因素，筛选南蛇藤素的最佳电泳分离条件。结果南蛇藤素的最佳提取条件为：80%甲醇为提取溶剂，料液比（m/v）为1：20，超声时间50min。以10%甲醇和70mmol L-1硼酸盐缓冲液（pH=9.00）作为电解液，分离电压22kV，检测波长210nm作为检测条件。南蛇藤素在15min内得到分离，YCL=543282x+1967.2（r=0.9920），线性检测范围为0.039-0.117μg μL-1。回收率为101.13%，变异系数（RSD）<4.00%（n=6）。南蛇藤素在根、茎、皮、叶中的含量分别为5.13、1.51、0.32、0.34mg g-1。南蛇藤素主要存在于根中，约为茎中的3.4倍，皮和叶的15倍。南蛇藤素在南蛇藤根、茎、叶、皮中均有分布，且不同部位中含量存在明显差异。超声波提取-响应曲面法优化南蛇藤素的提取工艺，同时毛细管电泳检测其含量，具有较好的准确度和精密性，为临床和科研提供了一种简单、快速、准确的检测方法，同时为临床用药提供了参考数据。4南蛇藤醇提物毒性和安全性实验研究研究中药南蛇藤醇提物的药物毒性和安全性：采用Kaerben法设计实验方案，寻找小鼠灌胃给予南蛇藤醇提物的最大耐受量（MTD）和半数致死量（LD50）；在MTD范围内，灌胃给予小鼠0.0、2.5、5.0、10.0g kg-1南蛇藤醇提物21d，记录药物对小鼠毒副作用情况，分析肝脏形态及肝脏指数变化，酶法检测小鼠血清中谷丙转氨酶（ALT）和谷草转氨酶（AST）的水平。结果表明南蛇藤醇提物的MTD为10.0g kg-1，LD50为38.17g kg-1，95%可信限为38.14-38.19g kg-1。在MTD范围内灌胃给药，与对照组相比，肝脏形态学和肝脏指数无明显变化；5.0、10.0g kg-1组ALT升高显著（P<0.01, P<0.001），10g kg-1组AST明显升高（P<0.01）。因此确定南蛇藤醇提物最大耐受量为10.0g kg-1，在此范围内用药对机体毒性较小，能保证用药安全。5南蛇藤改善高脂血症豚鼠血脂表型和减轻主动脉壁脂质沉积探讨南蛇藤对高脂血症豚鼠脂质代谢、动脉壁脂质沉积的作用及机制：48只英格兰雄性短毛豚鼠（260-310g，5月龄）随机分为普通饮食对照组（CD），高脂饮食模型组（HFD），南蛇藤3个浓度处理组，即低剂量组（HFD-L，2.5g kg-1d-1）、中剂量组（HFD-M，5.0g kg-1d-1）和高剂量组（HFD-H，10.0g kg-1d-1），以及辛伐他汀组（HFD-S，20m g kg-1d-1）。连续药物干预8周后，测定血浆总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）和非高密度脂蛋白胆固醇（Non-HDL-C）含量。快速蛋白分析（FPLC）技术分离血浆组分，聚丙烯酰胺凝胶电泳技术（SDS-PAGE）分析极低密度脂蛋白（VLDL）、低密度脂蛋白（LDL）和HDL-C的蛋白组分。采用实时定量聚合酶链反应（RT-PCR）分析肝脏胆固醇逆向转运相关基因清道夫受体-BI（SR-BI）、低密度脂蛋白受体（LDL-R）、3-羟基-3-甲基戊二酰辅酶A还原酶（HMG-CoA reductase）和胆固醇7α-羟化酶1（CYP7α-1）表达。分别采用油红O染色和HE染色分析主动脉壁脂质沉积与肝脏脂肪性病变。采用Applygen试剂盒测定肝脏中TC、TG、游离胆固醇（FC）、胆固醇酯（CE）。结果显示，与高脂模型组比较，南蛇藤高剂量组TC、TG和Non-HDL-C分别下降45%，21%和49%，南蛇藤中、高剂量组分别升高HDL-C16%和20%。LDL和VLDL中apoB和apoE表达显著降低，而HDL中apoA I表达明显增加；南蛇藤可显著上调SR-BI、LDL-R、CYP7α1和HMG-CoA mRNA水平。南蛇藤显著减少动脉壁中脂质沉积与肝脏脂肪病变，明显降低肝脏中TC、TG、FC和CE水平。研究证明南蛇藤可抑制高脂血症豚鼠主动脉壁脂质沉积，其机制与改善高脂血症豚鼠血脂表型、减轻脂质氧化、增强促胆固醇逆向转运相关基因表达有关。6南蛇藤减轻高脂血症豚鼠血脂和主动脉壁的炎症反应探讨南蛇藤对高脂血症豚鼠血浆动脉壁炎症反应的作用及机制：48只英格兰雄性短毛豚鼠（260-310g，5月龄）随机分为普通饮食对照组（CD），高脂饮食组（HFD），南蛇藤3个浓度处理组，即低剂量组（HFD-L，2.5g kg-1）、中剂量组（HFD-M，5.0g kg-1d-1）和高剂量组（HFD-H，10.0g kg-1d-1），以及辛伐他汀组（HFD-S，20mg kg-1d-1）。连续药物干预8周后，酶联免疫吸附法（ELISA）法检测血浆中肿瘤坏死因子（TNF-α），C-反应蛋白（CRP）和白介素6（IL-6）含量，分别用硫代巴比妥酸钠法（TBARS）和硝基蓝四氮唑法（NBTRS）测定血浆和肝组织中超氧化物歧化酶（SOD）和丙二醛（MDA）表达水平，检测南蛇藤抗氧化作用。免疫组化（IMHC）和免疫蛋白印记法（Western blot）检测核转录因子（NF-κBp65）和白细胞分化抗原68（CD68）在动脉壁的表达。硝酸还原酶法（NRM）和精氨酸法（ARPM）分别测定肝组织中一氧化氮（NO）和诱导型一氧化氮合酶（iNOS）表达水平。赖氏法（Reitman-Frankel）检测了肝组织中谷丙转氨酶（ALT）和谷草转氨酶（AST）的含量。结果显示，与高脂模型组比较，南蛇藤显著抑制NF-κBp65和CD68在主动脉的表达，并呈浓度依赖性地降低血浆TNF-α、CRP和IL-6等炎症因子的水平（P<0.05or P<0.01）。南蛇藤中、高剂量组显著升高肝脏中SOD活性，明显降低血浆与肝脏中MDA水平。南蛇藤显著抑制高脂食物诱导的NO、 iNOS在肝脏中的高表达以及ALT、AST在肝脏中的高表达（P<0.05or P<0.01）。研究证明南蛇藤可显著抑制高脂血症豚鼠血浆和主动脉壁的炎症反应，其机制与改善高脂血症豚鼠血脂表型、增强抗氧化因子表达和抑制细胞炎症反应转录因子表达有关。更多还原

【Abstract】 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.更多还原