【作者】 易金娥；

【导师】 袁慧；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2010， 博士

【摘要】 随着高效分离和提取技术的发展,从植物中提取生物活性物质作为药物使用越来越受到重视。桦木酸(betulinic acid,BA)属于五环羽扇豆烷型三萜类物质,广泛分布于多种植物中,如白桦树、鼠李科、桃金娘科和莲类植物等,以白桦树中含量最高。BA具有抗肿瘤,抗艾滋病,抗炎,抗疟疾,抗寄生虫等广泛的药理活性。BA药理作用的发挥可能是调节机体免疫力,不一定直接作用于感染或肿瘤细胞,而且很多从植物中提取的天然产物都具有免疫调控的能力,因此BA很有可能是一个免疫调节剂。本论文以纯化BA为实验材料,系统研究了BA对小鼠机体免疫功能及其对淋巴细胞凋亡的影响。主要研究内容和结果如下：1.以白桦树皮为试验原料,采用两步法合成了BA。从白桦树皮中提取桦木醇,经琼斯试剂氧化制备中间产物桦木酮酸,再用硼氢化钠还原合成BA,采用IR,HPLC-MS,1H-NMR以及13C-NMR对桦木醇和BA的结构进行鉴定,确定最佳提取与合成工艺,并建立了HPLC测定BA含量的方法,色谱柱：Zorbax Eclipse XDB-C8, (4.6mm×150mm,5μm);流动相：甲醇-水(4g／L甲酸铵)=40：60,流速1.0ml/min,波长210nm,柱温为室温。结果表明,最佳提取条件为：以甲醇为溶剂,白桦树皮与甲醇比率为15／200(g／mL),70℃提取时间为3h；最佳合成条件：桦木醇与琼斯试剂的摩尔比为1：6,20-22℃反应3h,桦木酮酸与硼氢化钠的质量比为1：1时,其转化率最高。BA的线性范围为0.03125-0.5mg/mL,回归方程为：Y=379381X-2397(R2=0.9988),平均回收率可达97.97%,BA的含量为96.53%。2.通过体内给药途径,研究了0,0.25,0.5,1mg/kgBA对正常小鼠免疫功能调节和抗氧化作用的影响。采用MTT比色法检测了淋巴细胞的增殖,流式细胞术检测脾和胸腺淋巴细胞亚群,溶血空斑法和血清溶血素法检测体液免疫功能；吞噬中性红能力检测巨噬细胞的吞噬能力,ELISA法检测细胞因子的分泌,并检测了BA对超氧化物歧化酶(Superoxide Dismutase,SOD)、谷胱甘肽过氧化物酶(Glutathione Peroxidase, GSH-Px)、丙二醛(Malondialdehyd, MDA)、溶菌酶(lysozyme, LSZ)和总抗氧化能力(Total Antioxidant capacity, T-AOC)的影响。结果表明,BA能提高小鼠的免疫器官指数,协同促进ConA或LPS诱导的小鼠脾脏T淋巴细胞或B淋巴细胞的增殖活性,提高胸腺淋巴细胞CD4+百分率,脾淋巴细胞的CD19+百分率以及CD4+/CD8+的百分比,表明BA从增强淋巴细胞活性和改变T、B淋巴细胞的数目或亚群,来提高机体的细胞免疫。BA增加SRBC免疫小鼠溶血空斑数,降低血清免疫球蛋白IgG和IgM的抗体滴度,提高血清溶菌酶含量,表明BA作为抗原刺激物,活化的B细胞数量增多,功能增强,从而提高机体的体液免疫功能。BA显著增强腹腔巨噬细胞吞噬中性红的能力,提高腹腔巨噬细胞肿瘤坏死因子(Tumor Necrosis Factor, TNF-α)水平,这表明BA能够刺激巨噬细胞,增强巨噬细胞的吞噬能力。BA降低小鼠血清IL-2和IL-6的分泌量,略提高血清IL-10的水平,说明BA混合调控Th1/Th2类细胞因子。BA提高小鼠胸腺和脾脏的T-AOC, SOD和GSH-Px活性,降低MDA含量。说明BA能消除自由基和活性氧以免引发脂质过氧化,减少活细胞内过氧化物,保护机体免受自由基的损伤,通过提高机体的抗氧化能力,从而提高机体的免疫力。3.采用体外培养方式,研究了BA对巨噬细胞能量代谢水平、吞噬能力、胞外NO释放量、TNF-α分泌量与GSH-Px活性以及胞内SOD与LSZ活性的影响。结果表明,BA在2.5～1μg/mL浓度范围内,能够显著性地促进小鼠腹腔巨噬细胞增殖,5～20μg/mLBA能促进巨噬细胞吞噬中性红的能力；BA在1.25～10μg/mL浓度范围内,能增强胞外NO的释放量与GSH-Px活性以及胞内SOD与LSZ的活性,以1.25μg/mLBA作用最强,BA在1.25～20μg/mL浓度范围内,能显著地提高TNF-α的分泌量。这说明BA能激活小鼠腹腔巨噬细胞,提高其吞噬能力和能量代谢水平,增强抗氧化能力,提高机体的免疫力。4.以地塞米松(Dexamethasone, Dex)诱发淋巴细胞凋亡为模型,利用荧光显微镜、电子透射显微镜和相差倒置显微镜进行细胞形态学及亚细胞结构变化的观察；通过琼脂糖凝胶电泳观察细胞凋亡过程中DNA的裂解以及用流式细胞仪分析并测定细胞凋亡率。在细胞形态学、生物化学以及细胞与分子生物学等水平上从体内给药和体外细胞培养两方面研究了BA对淋巴细胞凋亡的影响。体内研究表明,BA能改善和拮抗地塞米松诱导的小鼠淋巴细胞凋亡,并随着剂量的增加,拮抗作用越强,细胞凋亡减少,呈一定的量效关系。体外研究表明,低剂量的BA对地塞米松诱导的淋巴细胞凋亡具有一定的保护作用,但随着剂量的增加,保护作用越来越弱。这表明BA对Dex诱导的淋巴细胞凋亡具有干预作用,在一定程度上起到了预防性的保护作用,但作用的强弱与剂量有关。BA对机体免疫细胞的保护作用,可能通过抗氧化机制发挥免疫调节作用,其机制有待进一步研究。更多还原

【Abstract】 It has recently attracted considerable scientific interest and attention to promote human health by enhancing natural immune protection via the potential of bioactive compound derived from the plant, with recent advances in the hightech instruments for isolation and characterisation of plant natural products, and the developments in the biological assay systems, there has been a growing attention to the importance of plants as a source of biologically-active substances. Betulinic acid (BA) is a pentacyclic triterpene which was found in the stem bark of the plant white birch, and also in various other plants widespread in tropical regions, such as Tryphyllum peltaum, Ancistrocladus heyneaus, Diospyoros leucomelas, Tetracera boliviana, and Syzygium formosanum. BA and its derivatives have been the subject of intense studies with focuses on their anti-cancer effects, anti-HIV, anti-bacterial, anti-inflammatory, antimalarial, as well as anti-helminthic and other pharmaceutical properties. These effects may be due to their ability to modulate immune function rather than having a direct effect on infections and on cancer cells. In addition, various bioactive materials derived from plants exhibit the immunomodulatory ability. Therefore, we proposed that BA may be another valuable immunomodulator. In the present study, the immunomodulatory effects of BA on mice were demonstrated by using the purified BA.1. BA was synthesized by oxidizing betulin from the bark of the white birth with Jone’s reagent, followed by selective oxidation of the resulting betulonic acid with NaBH4. IR, HPLC-MS,1H-NMR and 13C-NMR were used identified the structure of betulin and betulinic acid. To compare extraction efficiency several solvents were evaluated, also extraction time and extraction temperature were optimized, and a quantitative analysis method of BA by HPLC was formed with ethanol-water (4g/L ammonium formate) as the mobile phase on Zorbax Eclipse XDB-C8 column (4.6 mm×150 mm,5μm). The flow-rate was 1 mL/min and the UV detection was set at 210 nm, with a column temperature of room temperature. The results showed that the optimal experimental conditions were as follows:the white birch bark was extracted 3 h with methanol under reflux at 70℃, and the ratio of the bark to the extraction solvent was 15/200(g/mL), and the ratio of betulin to Jone’s reagent was 1:6 and the ratio of betulonic acid to NaBH4; was 1:1. A good linearity of betulinic acid was in the range of 0.03125 mg/mL to 0.5mg/mL (Y=379381x-2397, R2=0.9988), the recovery rate was 97.97%and the RSD (n=9) was 1.64%, and the content of betulinic acid was 96.53%.2. Female mice were orally administered with BA for 14 days at the dose of 0,0.25, 0.5, and 1 mg/kg body weight. We found that BA significantly enhanced the thymus and spleen indices and stimulated lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide as shown by MTT assay. Flow cytometry revealed that BAincreased the percentage of CD4+in thymus as well as the percentage of CD19+B cells and the ratios of CD4+/CD8+cells in spleens. BA increased the number of plaque forming cell and the macrophage phagocytic activity as indicated by neutral red dye uptake assay, and the peritoneal macrophages levels of TNF-a were also increased by BA. In contrast, the serum level of anti-Sheep red blood cell antibodies (IgG and IgM) and the serum concentration of cytokine (IL-2 and IL-6) were decreased significantly in BA-treated mice compared to the control as assayed by haemagglutination test and ELISA assays, respectively. At the same time, BA significantly increased GSH-Px, SOD, T-AOC, LSZ activity and decreased MDA content. Considered together, these results suggest that BA enhances mouse cellular immunity, humoral immunity, and activity of macrophages and antioxidant. Thus, BA is a potential immune stimulator and may strengthen the immune response of its host.3. To investigate immune function and antioxidant activity effects of BA on mouse peritoneal macrophages. The effects of BA on the phagocytosis capability, energy metabolism level, nitrous oxide (NO) release, the product of tumor necrosis factor-α(TNF-α), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and lysozyme (LSZ) activity were assayed by adding different concentration of BA into cultured mouse peritoneal macrophages in vitro. The results showed that phagocytosis capability was increased at the dose of 2.5～10μg/ml BA and energy metabolism level was significantly enhanced at the dose of 5～20μg/ml BA, and the NO release, GSH-Px, SOD, LSZ activity were significantly higher at the dose of 1.25～10μg/ml BA, and the product of TNF-αwas augmented at the dose of 1.25～20μg/ml betulinic acid, compared to the control group. Thus, BA is a possible immune stimulant because it can activate macrophages, enhance its oxidation resistance.4. The protective effects of BA against the Dexamethasone (Dex) induced apoptosis of murine lymphocytes were investigated in vivo and in vitro. Morphological assessment of apoptosis was performed with fluorescence microscope and transmission electron microscope microscope. DNA fragmentation was visualized by agarose gel electrophoresis. The amount of apoptotic cells was measured by flow cytometry. The results showed that in the test of in vivo, apoptotic cells in spleen and thymus of mice treated with BA and Dex (25mg/kg) were less than those in Dex treatment alone. DNA fragmentation assay showed that BA (0.25,0.5, 1mg/mL) obviously reduced Dex-induced ladder bands. Flow cytometry analysis showed that BA decreased the ratio of apoptosis, Thus, BA reduced Dex-induced apoptosis in a dose dependent manner, In the test of in vitro,5μg/mL BA reduced Dex-induced apoptosis, the protective activity of BA was decreased in a dose dependent manner (5,10,20μg/mL BA). Thus, BA can reduced Dex-induced apoptosis, and protect lymphocytes, but the protective activity of preventive was in relation to dosage of BA.更多还原