【作者】 宋淑珍；

【导师】 田亚平； 王成彬； 汪德清；

【作者基本信息】 中国人民解放军军医进修学院 ， 临床生化， 2002， 硕士

【摘要】 葛根为常用中草药之一，为探讨其不同活性部位的生物学效能及中草药双向调节机制和物质基础，利用现代分离提取技术同时获得两类不同性质的活性部位，经分析鉴定确认分别为黄酮类和多糖类。同时利用化学发光、免疫组化、流式细胞术、蛋白芯片等技术，以体外试验、细胞培养和动物实验等多层次对葛根总黄酮(TFP)和总多糖(TPP)的抗氧化活性、免疫调节活性及动物实验条件下的生物学活性进行系统研究，发现在中草药葛根中所获得的两类活性部位：黄酮和多糖对生物体的抗氧化能力、免疫活性具有相反的调节活性，为中草药防治疾病的机理提供了新的思路。 一．葛根总黄酮和总多糖的提取及鉴定：本研究首次利用化学提取技术从中草药葛根中同时获得葛根总黄酮(TFP)和总多糖(TPP)两个活性部位，根据《现代中药学大辞典》的理化鉴别方法，分别以薄层层析和显色反应进行定性鉴定，确认两个活性部位的的性质。同时以葛根素和葡萄糖分别作为TFP和TPP的参考物定量鉴定，TFP中葛根素含量为31.79％，TPP中相当于葡萄糖含量29％。 二．TFP和TPP对免疫细胞所释放活性介质的调控：分离 ＃MFBd＃＄ryIZMI＃＃＃KkMNWANNMgZ lzzvxH 人外周血多形核白细胞、淋巴细胞及嗜酸细胞，利用佛 波豆惹乙酸脂（PMA）激活此类免疫细胞产生呼吸爆 发，进而介导发光剂鲁米诺产生化学发光（CL）作为 实验模型，研究葛根不同活性部位（TFP和TPP）对 活性氧和活性氮介质的效能。发现TPP具有提高免疫 细胞释放活性介质的作用，且其增强效应与葛根总多糖 浓度呈正相关；TFP则具有浓度依赖性清除活性介质 的功效。 三．TFP和TPP对PC12细胞活性的调控及氧化损伤的防 护：以培养PC12细胞为实验对象，用MTT法检测细胞 活性，用Gness方法测定培养液中亚硝酸盐含量，用 次黄膘吟l黄瞟吟氧化酶体系测定培养液中的抗氧化活 性，研究葛根总黄酮和总多糖对体外培养细胞生长活性 的调控及对外源性HZOZ所致培养细胞氧化损伤的影 响。发现葛根总黄酮和总多糖对培养细胞均有一定生长 调控作用，葛根总黄酮在 11 plml浓度范围可提高 细胞外液中的抗氧化活性，抑制亚硝酸盐形成，对HZOZ 所致PC12氧化损伤具有明显防护作用；而葛根总多糖 在 11 uglml浓度范围未见防护效能，反而可降低 细胞外液中抗氧化活性，并促进亚硝酸盐形成，对PC12 2 grmpPlef＃＄＆MI＃gwffkHMWngkWgi’ ，nvxH 细胞的生长繁殖具有一定抑制作用。 四．过敏性哮喘动物模型的建立及防护：受试动物实验干 预分组：SD雌性大鼠（解放军总医院动物实验中心提 供）6g只，体重200t509，64只大鼠随机分为8组， 每组8只。正常对照组（A），第一天腹腔注射生理盐 水 lml，两周后超声雾化喷雾生理盐水 20分钟，每日 一次，共2周；卵蛋白激发哮喘发作组（B），用卵蛋 白致敏并激发建立大鼠哮喘模型，第一天每只大鼠腹腔 注射抗原液 lml（含 10％卵蛋白 100mg，灭活百日咳疫 苗5X109，氢氧化铝干粉100tog）致敏，两周后超声雾 化喷雾 1％卵蛋白（OVA）20分钟，每日一次，持续 2 周；TFP灌胃组（C）：300mglkg TFP灌胃每日一次， 共 4周；TFP干预组（D）：激发同 B组，但每日在雾 化吸入 1％VOA之前灌胃 300mglkg TFP一次，共 4 周；TPP灌胃组（E人 900mglkg TPP灌胃每日一次， 共 4周：TPP干预组吓人 卵蛋白激发同 B组，每日 在雾化吸入 1％VOA之前灌胃 900mglkg TPP一次， 共 4周；精氨酸灌胃组（G人 100mglkg精氨酸灌胃 每日一次，共4周；精氨酸干预组（H）：激发同B组， 但每日在雾化吸入 1％VOA之前灌胃 100mglkg精氨 3更多还原

【Abstract】 Two different active components have been isolated from Kudzuvine Root(Radix puerariae), one was the ethanol extraction portion- total flavone (TFP) and another was the water extraction portion-total polysaccharide(TPP). For exploring the biological efficacy and the mechanism of Bi-direction modulation effects of different Pueraria lobata active components, a series studies have been carried out by using chemiluminescence> Biochip> Cell culture^ flow cytometry technology and animal experiments.The results showed as following:1. Extraction and indentifcation of total flavone(TFP) and total polysaccharide of Puerariae(TPP): ethanol soluble portion and water soluble portion were saperated simultaneously from Puerariae by using a improved chemical technology. They were proved to be TFP and TPP by using qualitative as well as quantitative analysis according to thestandards of Chinese herbal medicine identification.2. Different concentration of two active componentshad been studied by using PMA stimulated immunol cells initiated chemiluminescent system, to evaluate the inhibitory or stimulation effects of TFP and TPP on respiratory burst emitted ROS. The results indicated that TPP could enhance chemiluminescence in concentration dependently whereas TFP could inhibit the chemiluminescence significantly.3. H202 initiated PCi2 cell cytotoxicity had been usedas experimental model to investigate the biological effects of TFP and TPP on the cultured PCi2 cells. The activity of cultured PCi2 cell was monitored by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays, the antioxidant activity of the culture media was monitored by hypoxanthine/xanthine oxidase initiated chemiluminescent system and the nitritewas measured by Griess reagents. The results showed thatTFP could protect the PC12 cells from the oxidative damages of H2O2 at 1-100 ng/ml while TPP could enhance the oxidative damages on the cells at 1-1000 ng/ml.4. To investigate the preventive effect of TFP and TPP on asthmatic model rats, 64 rats were randomly divided into eight groups. Control group(Group A): the rats was raised in normal condition; asthmatic instant stimulated group(Group B); TFP control group(Group C): 300mg/kg TFP only was administrated 28 for days TFP protection group(Group D): As Group B, 300mg/kg TFP was administrated before the rats were inhaled with 1% OA aerosol. TPP control group(Group E): 900mg/kg TPP was administrated for 28 days. TPP intervention group(Group F): As Group B, 900mg/kg TPP was administrated before the rats were inhaled with 1% OA aerosol. L-arginine control group(Group G): 100mg/kg TPP wasadministrated for 28 days. L-arginine intervention group(Group H): As Group B, 100mg/kg L-arginine was administrated before the rats were inhaled with 1% OA aerosol. T cells subsets in spleen were examined and the changes of lung tissues in rat asthmatic model were observed using histopathologic methods. Control rats did not have eosinophils in their submucosal of airways, whereas eosinophils as well as lymphocytes were apparently increased in the airways of Group B, and the percentages of CD4+ as well as the ratio of CD4+/CD8+ were significantly higher in group B than Control group; The application of TFP in Group D not only inhibited the increase of EOS, IL-4> IL-5> CD4\ CD4*VCD8+ , but also depress the airway inflammation, the differences were statistically significant. Results showed that TFP can prevent allergic asthma by inhibit the increasing of EOSx IL-4, IL-5^ CD4\ CD4+/CD8+; On the contrary,TPP can enhance the immunity of normal rats, increasing CD4+> CD4+/CD8+, but have no effects on hyperactive immune rats;5. Effects of TFP and TPP on blood gas of asthmaticrats: PaO2 . pH of asthmatic rats decreased sharply , while PaCO2 increased (p<0.01), which showed that the asthmatic rats was in serious anoxic situation, In group D, PaO2> pH and PaCO2 have no significant difference compared to Control group; in group F, the value of PaO2 ^ pH and PaCO2 were si更多还原