油茶蒲抑制良性前列腺增生活性成分的分离纯化及其作用研究

发布时间：2018-03-08 15:08

本文选题：油茶蒲　切入点：5α-还原酶　出处：《浙江大学》2014年硕士论文　论文类型：学位论文

【摘要】：良性前列腺增生症(benign prostatic hyperplasia,BPH)是常见的老年男性病,大约有80%老年男性的生活质量因此受到严重影响,5α-还原酶对BPH的形成起着重要作用。本文以5α-还原酶的抑制率为指标,研究确定了油茶蒲中抑制5α-还原酶活性成分的分离纯化方法,采用细胞模型和动物实验探究了活性成分对BPH的作用,为开发高效低毒的BPH治疗药物提供理论依据。主要的研究内容和结果如下： (1)优化5α-还原酶活性的紫外分光光度检测法,以睾酮(T)替代传统的NADPH作为检测对象,测定方法为：2mL测活体系中T20μmol/L,NADPH40μmol/L,酶提取物260μg,37℃测定4min内248nm处的吸光度值。该法检测结果与高效液相色谱法(HPLC)相当。 (2)建立油茶蒲活性成分的提取、纯化、精制及制备方法： ⅰ)活性成分的提取：选择50%7,醇(v/v)作为提取溶剂,该醇提物对5a-还原酶的抑制率为36.31%4±2.76%,得率11.96%。 ⅱ)活性成分的纯化：选择AB-840%乙醇洗脱相(OCE)作大孔树脂柱层析条件,OCE对5a-还原酶抑制率为57.79%±1.67%,得率26.5%。 ⅲ)活性成分的精制：以正丁醇-甲醇-水-冰醋酸(3：2：2：1)为展开剂,0.5%(w/v)FeCl3乙醇溶液为显色剂,应用聚酰胺色谱法代替硅胶色谱法得到分离度良好的Fr1-Fr7流份(见P37图3.1),Rf为0.85～0.0375,其中Fr6、Fr5、Fr4对5α-还原酶的抑制率分别为85.38%±3.71%、76.28%±1.37%、73.01%±2.93%,较纯化前OCE的抑制率显著增加(p0.01),得率分别为3.79%、2.10%、0.64%。明确了油茶蒲的特征酚类物质没食子酸、鞣花酸及MEAG不是抑制5α-还原酶活性的关键化合物。 ⅳ)关键化合物的制备：采用半制备HPLC分离Fr5得到活性单体F0和F0',对5α-还原酶的抑制率分别为76.47%4±0.02%和92.08%±0.18%,对应得率为5.17%和1.19%,由LC-MS推测出F0和F0'的分子量分别为449和335。 (3)采用MTT法和Annexin V/PI双染色法测定油茶蒲活性成分抑制人良性前列腺增生细胞BPH-1增殖和诱导细胞凋亡作用。在浓度为50μg/mL时,抑制增殖作用的强弱顺序为Fr6非那雄胺Fr5≈F0Fr4≈Fr3OCEFr2,其中Fr6抑制活性最高(抑制率50.0%±0.7%),Fr5和FO次之(抑制率分别为43.7%±2.1%和41.4%±2.1%),且抑制作用呈时间-剂量依赖性。F0诱导细胞凋亡作用呈剂量依赖性,Fr6在浓度为25μg/mL时诱导的细胞凋亡率为12.1%±3.8%,与非那雄胺的效果相近,极具研究和开发价值。 (4)油茶蒲活性成分对BPH-1细胞增殖的抑制作用,与其对5α-还原酶活性的抑制作用结果一致,证实了油茶蒲活性成分作为5α-还原酶抑制剂对改善前列腺增生的有效性。 (5)油茶蒲醇提物能有效改善丙酸睾酮致大鼠前列腺增生症状,显著降低大鼠前列腺和腹叶指数(p0.05),改变增生组织病理学形态,抑制大鼠前列腺内5α-还原酶和酸性磷酸酶的活性(p0.01),并提高大鼠体内的抗氧化水平。综上所述,油茶蒲活性成分可通过降低5a-还原酶活性、抑制前列腺细胞增殖、诱导细胞凋亡、清除自由基等多个靶点的作用,有效预防并治疗BPH。
[Abstract]:Benign prostatic hyperplasia (benign prostatic, hyperplasia, BPH) is a common disease in older men, about 80% of the quality of life of older men so severely affected, 5 alpha reductase plays an important role in the formation of BPH. The inhibition of 5 alpha reductase as index, determine the research method for isolation and purification of reductase activity the inhibition of alpha 5 components in the fruit shell of Camellia oleifera Abel, explores the role of active components on BPH cell model and animal experiment, and provide a theoretical basis for the development of drugs for the treatment of BPH with high efficiency and low toxicity. The main research contents and results are as follows:
(1) the optimization of 5 alpha reductase activity in UV spectrophotometric detection of testosterone (T), to replace the traditional NADPH as detection object, determination method: 2mL measurement of activity of T20 in the system mol/L, NADPH40 mol/L, enzyme extract 260 g, 4min 248nm to determine the absorbance value at 37 DEG C test results of this method with high performance liquid chromatography (HPLC).
(2) to establish the extraction, purification, purification and preparation of the active ingredients of Camellia oleifera:
I) to extract the active ingredient: 50%7 alcohol (v/v) as the extraction solvent, the extraction of 5a- reductase inhibition rate for 36.31%4 + 2.76%, the yield of 11.96%.
II) purification of active ingredients: AB-840% ethanol elution phase (OCE) for macroporous resin column chromatography, OCE 5a- reductase inhibition rate was 57.79% + 1.67%, the yield of 26.5%.
III) refined active ingredients: n-butanol methanol water glacial acetic acid (3:2:2:1) as the agent, 0.5% (w/v) FeCl3 ethanol solution as the chromogenic agent, the application of polyamide chromatography silica gel chromatography separation instead of get good Fr1-Fr7 flow (see Fig. 3.1 P37), Rf was 0.85 ~ 0.0375 among them, Fr6, Fr5, Fr4 of the 5 alpha reductase inhibition rates were 85.38% + 3.71%, 76.28% + 1.37%, 73.01% + 2.93%, compared with the inhibition rate of OCE increased significantly before purification (P0.01), the yield were 3.79%, 2.10%, 0.64%. defined the characteristics of the fruit shell of Camellia oleifera Abel phenols gallic acid, the key ellagic acid and MEAG 5 alpha reductase activity was not inhibited.
IV) key compounds prepared by semi preparative HPLC separation of Fr5 active monomer F0 and F0', inhibition of 5 alpha reductase were 76.47%4 + 0.02% and 92.08% + 0.18%, corresponding to a yield of 5.17% and 1.19%, that by LC-MS molecules F0 and F0' were 449 and 335.
(3) using MTT and Annexin V/PI double staining method for the determination of active components of the fruit shell of Camellia oleifera Abel inhibits the proliferation of human benign prostatic hyperplasia of BPH-1 cells and induce apoptosis. At the concentration of 50 g/mL, inhibit the proliferation of the role of the strength in the order of Fr6 finasteride Fr5 = F0Fr4 = Fr3OCEFr2, the highest Fr6 inhibitory activity (the inhibition rate of 50% + 0.7%), Fr5 and FO (the inhibition rate were 43.7% + 2.1% and 41.4% + 2.1%), and the inhibition was dependent on both dose and time of.F0 induced apoptosis in a dose-dependent manner, Fr6 concentrations in cell apoptosis induced by 25 g/mL at the rate of 12.1% + 3.8%. With finasteride effect, high value in research and development.
(4) the inhibitory effect of active ingredients of Camellia oleifera on BPH-1 cell proliferation is consistent with its inhibitory effect on the activity of 5 alpha reductase, which confirms that the active ingredient of Camellia oleifera as a 5 alpha reductase inhibitor is effective in improving prostate hyperplasia.
(5) the fruit shell of Camellia oleifera Abel alcohol extract can effectively improve the symptoms of prostate hyperplasia induced by testosterone propionate in rats, significantly reduce the rat ventral prostate and index (P0.05), change the morphology of hyperplastic tissue pathology, inhibition of rat prostate in 5 alpha reductase and the activity of acid phosphatase (P0.01), and improve the rats the antioxidant level.
In conclusion, active ingredients of Camellia oleifera can effectively prevent and treat BPH. by reducing 5a- reductase activity, inhibiting proliferation of prostate cells, inducing apoptosis and eliminating free radicals.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R697.32

【参考文献】