本文選題：油茶蒲　切入點：5α-還原酶　出處：《浙江大學》2014年碩士論文　論文類型：學位論文

【摘要】：良性前列腺增生癥(benign prostatic hyperplasia,BPH)是常見的老年男性病,大約有80%老年男性的生活質(zhì)量因此受到嚴重影響,5α-還原酶對BPH的形成起著重要作用。本文以5α-還原酶的抑制率為指標,研究確定了油茶蒲中抑制5α-還原酶活性成分的分離純化方法,采用細胞模型和動物實驗探究了活性成分對BPH的作用,為開發(fā)高效低毒的BPH治療藥物提供理論依據(jù)。主要的研究內(nèi)容和結(jié)果如下： (1)優(yōu)化5α-還原酶活性的紫外分光光度檢測法,以睪酮(T)替代傳統(tǒng)的NADPH作為檢測對象,測定方法為：2mL測活體系中T20μmol/L,NADPH40μmol/L,酶提取物260μg,37℃測定4min內(nèi)248nm處的吸光度值。該法檢測結(jié)果與高效液相色譜法(HPLC)相當。 (2)建立油茶蒲活性成分的提取、純化、精制及制備方法： ⅰ)活性成分的提�。哼x擇50%7,醇(v/v)作為提取溶劑,該醇提物對5a-還原酶的抑制率為36.31%4±2.76%,得率11.96%。 ⅱ)活性成分的純化：選擇AB-840%乙醇洗脫相(OCE)作大孔樹脂柱層析條件,OCE對5a-還原酶抑制率為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)為展開劑,0.5%(w/v)FeCl3乙醇溶液為顯色劑,應用聚酰胺色譜法代替硅膠色譜法得到分離度良好的Fr1-Fr7流份(見P37圖3.1),Rf為0.85～0.0375,其中Fr6、Fr5、Fr4對5α-還原酶的抑制率分別為85.38%±3.71%、76.28%±1.37%、73.01%±2.93%,較純化前OCE的抑制率顯著增加(p0.01),得率分別為3.79%、2.10%、0.64%。明確了油茶蒲的特征酚類物質(zhì)沒食子酸、鞣花酸及MEAG不是抑制5α-還原酶活性的關(guān)鍵化合物。 ⅳ)關(guān)鍵化合物的制備：采用半制備HPLC分離Fr5得到活性單體F0和F0',對5α-還原酶的抑制率分別為76.47%4±0.02%和92.08%±0.18%,對應得率為5.17%和1.19%,由LC-MS推測出F0和F0'的分子量分別為449和335。 (3)采用MTT法和Annexin V/PI雙染色法測定油茶蒲活性成分抑制人良性前列腺增生細胞BPH-1增殖和誘導細胞凋亡作用。在濃度為50μg/mL時,抑制增殖作用的強弱順序為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(抑制率分別為43.7%±2.1%和41.4%±2.1%),且抑制作用呈時間-劑量依賴性。F0誘導細胞凋亡作用呈劑量依賴性,Fr6在濃度為25μg/mL時誘導的細胞凋亡率為12.1%±3.8%,與非那雄胺的效果相近,極具研究和開發(fā)價值。 (4)油茶蒲活性成分對BPH-1細胞增殖的抑制作用,與其對5α-還原酶活性的抑制作用結(jié)果一致,證實了油茶蒲活性成分作為5α-還原酶抑制劑對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睪酮致大鼠前列腺增生癥狀,顯著降低大鼠前列腺和腹葉指數(shù)(p0.05),改變增生組織病理學形態(tài),抑制大鼠前列腺內(nèi)5α-還原酶和酸性磷酸酶的活性(p0.01),并提高大鼠體內(nèi)的抗氧化水平。 綜上所述,油茶蒲活性成分可通過降低5a-還原酶活性、抑制前列腺細胞增殖、誘導細胞凋亡、清除自由基等多個靶點的作用,有效預防并治療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.

【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R697.32

【參考文獻】

相關(guān)期刊論文 前10條

1 程芳;陳光亮;;植物藥治療良性前列腺增生癥的研究進展[J];安徽醫(yī)藥;2009年11期

2 王夢芝;;良性前列腺增生癥的藥物治療進展[J];當代醫(yī)學;2010年14期

3 張石革;5-α還原酶抑制劑進展與前列腺增生治療[J];中國醫(yī)藥導刊;2005年01期

4 歐敏銳,戴小福,薛逢春,張怡,許小平;高效液相色譜法檢測5α-還原酶活性[J];福州大學學報(自然科學版);2004年02期

5 彭永德,陳家倫,許曼音;類固醇5α還原酶的研究進展[J];國外醫(yī)學(內(nèi)分泌學分冊);2000年03期

6 孫華君;劉少明;;5α還原酶抑制劑臨床研究進展[J];世界臨床藥物;2006年02期

7 畢夫景;趙詠桔;;良性前列腺增生癥與部分代謝因素相關(guān)性概述[J];國際老年醫(yī)學雜志;2010年01期

8 黃世杰;目前和未來良性前列腺增生的藥物治療[J];國外醫(yī)學.藥學分冊;2003年04期

9 孫宏斌,夏術(shù)階;前列腺組織中5a-還原酶的活性[J];國外醫(yī)學.泌尿系統(tǒng)分冊;2005年01期

10 歐敏銳,戴小福,周訓勝,陳贊秋 ,許小平;5α-還原酶抑制劑的研究進展[J];海峽藥學;2003年06期

相關(guān)博士學位論文 前1條

1 陳秋平;油茶蒲減肥降脂功能因子研究[D];浙江大學;2011年

，

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