【作者】 陈杰波；

【导师】 许小平；

【作者基本信息】 福州大学 ， 化学工程， 2005， 硕士

【摘要】 良性前列腺增生（BPH）是常见的、多发性的男性老年病之一。由于前列腺增生 使尿道变得狭窄从而引起排尿障碍,因此BPH是老年男性尿急、尿频和排尿困难的 主要原因。随着社会的老龄化日趋明显,对BPH的治疗已引起广泛关注,其治疗方-- 法的研究也就具有十分重要的意义。对此,本文提出了一种前列腺增生的新疗法--5α 还原酶抑制与光动力学疗法,同时提出了5α-还原酶抑制剂的高效液相色谱评价与筛 选模型,利用该模型对天然产物进行了筛选,确定了白芥子乙醇提取物为5α-还原酶 的抑制剂,研究了白芥子的乙醇提取及其药效。然后概述了光敏剂、光动力学疗法, 研究了光动力学疗法在前列腺增生中的应用。本文的研究工作包括以下三个部分:第一部分:天然产物白芥子的乙醇提取及其药效确定本部分以乙醇为溶剂采用溶剂提取法对白芥子的提取工艺做了研究,考察了提取 溶剂浓度、白芥子粒度、提取次数、料液比、提取温度及提取时间对提取率的影响, 确立了乙醇提取白芥子的最佳工艺条件:选用80%乙醇/水作为提取溶剂;白芥子粒 度为40目;提取2次;料液比为1:6;浸提温度80口;浸提时间8h,在此工艺条 件下白芥子的提取率可达9.52%。同时,设立正常对照组、动物模型组以及白芥子乙 醇提取物三种剂量（3 0mg/kg/d、60mg/kg/d和1 20mg/kg/d）治疗组,用丙酸睾酮诱导 的小鼠前列腺增生模型确定了白芥子乙醇提取物的药效。结果表明白芥子乙醇提取物 能明显抑制丙酸睾酮诱发的小鼠前列腺增生（p<0.05）,减轻小鼠前列腺的湿重,降低 血清酸性磷酸酶（ACP）的活性,提示白芥子乙醇提取物对丙酸睾酮诱导的前列腺增 生具有治疗作用。第二部分:5α-还原酶抑制剂的高效液相色谱法体外筛选和评价模型的建立建立了一种体外筛选和评价甾体5α-还原酶抑制剂的高效液相色谱（HPLC）模 型。确定了测定甾体5α-还原酶活性的液相色谱条件:Lichrospher-RpC₁₈,5μm, 250mmx3.9mm;温度:室温;检测波长:254nm;流动相配比:甲醇/水,70:30(VN 1; 流速:0.7mL/min。在此色谱条件下,15min之内实现了对底物睾酮与其它物质的分 离,测得睾酮的精密度为2.5-3.6%,回收率为89.70%-95.52%,证明了高效液相色谱 法是测定甾体50r-还原酶活性的一种简便有效的分析方法。在确定的色谱条件下,测 定了大鼠肝脏甾体50r-还原酶活性,将数据按Lineweaver-Burk倒数法作图,求得非 那甾胺抑制常数K_i=1.14⁰.23^tmol/L,爱普列特抑制常数Ki=4.32±0.38^tmol/L,白芥更多还原

【Abstract】 Benign prostatic hyperplasia (BPH) is common and frequently occurs among elder men. BPH is the main reason of urinary obstruction. Therefore, the treatment for BPH has attracted the wide concerns, and it has an important significance for the investigation of treating BPH. In this paper, the base information of BPH, Photodynamic therapy (PDT), photosencitizers and 5α-reductatse inhibitors are introduced firstly. Then, following three studies have been executed.In first section of the studies, the extraction efficiency of ethanol extract from seeds of Brassica alba has been studied. A number of factors, including extraction temperature and time, the ratio of seeds of Brassica alba and solvent, as well as the concentration of the solvent, have been discussed. The optimal conditions for the extraction of ethanol extract were found: seeds of Brassica alba (50g) was extracted twice with 80% ethanol (300mL) at 80 for 8 hours. Under these conditions, the yield of ethanol extract was 9.52%. Then, the therapeutical efficacy of ethanol extract from seeds of Brassica alba on the benign prostatic hyperplasia rats was investigated. Rats were administered with testosterone propionate 5 mg/(kg·d) subcutaneously for 15 days, and then administered with the ethanol extract from seeds of Brassica alba 30, 60, 120 mg/(kgd) orally respectively for 15 days. The results showed that ethanol extract from seeds of Brassica alba could significantly suppress BPH caused by testosterone propionate in rats, decrease the wet-weight of prostate remarkably, and the serum acid phosphatase (ACP) was observably decreased compared with the model group. It is indicated that ethanol extract from seeds of Brassica alba has positive effect on BPH caused by testosterone propionate.In the second section of the studies, a HPLC model screening and evaluating the steroid 5α-reductatse inhibitors was proposed. Proper HPLC operating conditions were investigated, which could determine the activity of the steroid 5α-reductatse. These conditions include column, temperature, solvent, mobile phase, flow rate, and detectionwavelength. The analysis of testosterone and other compounds investigated required no longer than 15 minutes. Suitable chromatographic condition: the column was RPC 18 column, the column temperature was ambient; the mobile phase was mixture of methanol and water (70/30, v/v) maintained at a flow rate of 0.7mL/min, and the UV detector was used at a wavelength of 254nm. Under these conditions, it can separate and analyze testosterone and other compounds. The precision is 0.9%~1.5% and recoveries are 89.70%~95.52%. Under the proper HPLC operating conditions, the activity of the steroid 5α-reductatse was determined. The activity of the enzymatic is defined as the descending rate of the concentration of testosterone. And inhibition constant (Ki) of the steroid 5α-reductatse inhibitors could be calculated according to the Lineweaver-Burk plots. The inhibition constants (Ki) of ethanol extract from seeds of Brassica alba, Finasteride and Epristeride were 3.68±0.31μmol/L, 1.14±0.23μmol/L and 4.32±0.38μmol/L, respectively. And the inhibit efficacy of ethanol extract from seeds of Brassica alba increased with the increase of concentration of ethanol extract. The results were consistent with those of the studies in first section. It is indicated that this method of HPLC is simple, rapid and reproducible well to screen and evaluate steroid 5α-reductatse inhibitors. In the third section of the studies, the effects of HMME-PDT, ZnPcS2P2-PDT and Chlorin e6-PDT on BPH, using the rat model of benign prostatic hyperplasia as the object, were firstly investigated. Rats were divided into seven groups: the normal control, the rat model of BPH, the photosensitizer alone, laser irradiation alone, and HMME(3mg/kg)-PDT, ZnPcS2P2(3mg/kg)-PDT, Chlorin e6(3mg/kg)-PDT. The results of this study demonstrated that the photosensitizer alone and laser irradiation alone could be not efficacious for treatment of BPH. However, the cooperation between the photosensitizer and PDT could kill the prostate cells remarkably (P<0.05). And the photodynamic activity of HMME is considerably better than those of ZnPcS2P2 and Chlorin e6. This indicated that HMME-PDT can be used to treat BPH of rats more effective. Then, using HMME as the photosensitizer, the rats received HMME-PDT treatment with 1mg/kg、3mg/kg、5mg/kg HMME and application of 45J/cm2、90 J/cm2、135 J/cm2、180J/cm2 light at 670nm, respectively. The results showed that the efficacy of HMME-PDT increased significantly with the increase of concentration of HMME. HMME-PDT was almost no efficacy with low energy density of light (<45J/cm2), and with the increase of the energy density of light the photodynamic activity increased. However, Photodynamic activity decreased when the energy density of light exceeded 135 J/cm2. And the pathology change of prostate also proved the conclusions mentioned above. Then the pathology change prostate of the rats was observed and analyzed to explore the mechanism of photodynamic therapy, affordtheoretic basis for PDT of BPH in the clinic. Finally, the effects of cooperation between ethanol extract from seeds of Brassica alba and PDT on BPH caused by testosterone propionate in rats were investigated. The results indicated that the cooperation between ethanol extract from seeds of Brassica alba and PDT could treat BPH more effective than ethanol extract from seeds of Brassica alba or PDT alone.更多还原