麦芽治疗高泌乳素血症有效部位筛选及其作用机制研究

发布时间：2018-09-17 07:23

【摘要】：目的:本研究在前期试验基础上,继续研究麦芽中总生物碱物质的提取纯化工艺。利用现代化药理学研究方法,复制高泌乳素血症动物疾病模型,对麦芽不同极性部位和不同化学部位,以及纯化后生物碱物质开展药效学试验,筛选出麦芽治疗HPRL有效部位,并阐明其作用机制。方法:(一)称取适量麦芽用70%乙醇回流提取浸膏,加水溶解制成混悬液后依次加入石油醚,乙酸乙酯,正丁醇萃取得到不同极性部位提取物,再对各个部位中的化学成分进行含量测定。根据相关文献,选择水做溶剂,40℃水浴,提取得到麦芽总多糖粗提物;用70%乙醇回流提取,然后通过聚酰胺树脂柱进行简单的纯化,得到麦芽总黄酮粗提物;用70%乙醇超声提取,然后通过HPD-600大孔树脂柱进行简单的纯化,得到麦芽总酚粗提物。麦芽总生物碱粗提物制备和含量测定方法依据本课题组前期的实验方法进行。(二)根据单因素实验选择麦芽中生物碱物质的提取溶剂,再进行正交试验,以总生物碱和大麦芽碱提取量的综合评分为评价指标,优选出麦芽生物碱物质的最佳提取工艺。再以总生物碱的转移率和纯度为评价指标,比较柱层析法和溶剂萃取法对麦芽总生物碱的纯化效果,同时对溶剂萃取法中脱脂溶剂、水浴温度以及氨水碱化溶液至不同PH值进行考察,优选麦芽总生物碱的最佳纯化工艺。(三)高泌乳素血症模型的建立:除正常组大鼠外,其它各组大鼠背部皮下注射盐酸甲氧氯普胺注射液50mg/kg体重,每天上午下午分别定时注射1次,连续5天。分组给药:将120只雌性未孕SD大鼠随机分为12组:正常组、模型组、石油醚和乙酸乙酯部位组、正丁醇部位组、水部位组、原药材水提物组、总生物碱粗提部位组、总多糖粗提部位组、总黄酮粗提部位组、总酚粗提部位组、生物碱低浓度组、生物碱高浓度组,每组10只。模型制备后开始给药,石油醚和乙酸乙酯部位组给药剂量为114.70mg·kg-1·d-1;正丁醇部位组给药剂量为124.17mg·kg-1·d-1;水部位组给药剂量为645.96mg·kg-1·d-1;原药材水提物组给药剂量为4.74g·kg-1·d-1;总生物碱粗提部位组给药剂量为592.43mg·kg-1·d-1;总多糖粗提部位组给药剂量为1.57g·kg-1·d-1;总黄酮粗提部位组给药剂量为57.83mg·kg-1·d-1;总酚粗提部位组给药剂量为103.39mg·kg-1·d-1;生物碱低浓度组给药剂量为0.87mg·kg-1·d-1;生物碱高浓度组给药剂量为1.74 mg·kg-1·d-1。正常组和模型组给予等容量的蒸馏水,连续灌胃30d。对高泌乳素血症大鼠血清激素含量的检测:末次给药24h后,所有大鼠禁食12小时,自由饮水。各组大鼠于次日上午心脏取血2ml,血液静置后以3000转/min的速度离心5min,取血清供检测。采用酶联免疫吸附法测定各组大鼠血清中泌乳素(PRL)、雌激素(E2)、孕酮(P)的含量,严格按照ELISA试剂盒说明书中操作流程进行。对高泌乳素血症大鼠脑垂体泌乳素阳性细胞含量及mRNA表达的影响:正常组、模型组、总生物碱粗提部位组、总多糖粗提部位组、总黄酮粗提部位组、总酚粗提部位组、生物碱低浓度组和生物碱高浓度组大鼠末次给药24h后,于次日上午用20%乌拉坦麻醉后取其脑垂体。取1/2大鼠脑垂体于组织固定液中充分固定24小时,石蜡包埋切片后,进行免疫组化实验,测定PRL细胞阳性的累积光密度值(IOD)。取另外的1/2大鼠脑垂体进行PRL mRNA荧光定量PCR实验,计算扩张倍数,进行统计学分析。对高泌乳素血症大鼠乳腺增生情况的影响实验:正常组、模型组、原药材组水提物组、总生物碱粗提部位组、总多糖粗提部位组、总黄酮粗提部位组、总酚粗提部位组、生物碱低浓度组和生物碱高浓度组大鼠末次给药24h后,于次日上午用20%乌拉坦麻醉后取其第二对乳腺组织,于4%多聚甲醛组织固定液中充分固定24小时后,包埋,切片进行HE染色,在显微镜下观察各组大鼠乳腺组织情况,包括乳腺小叶腺泡数、导管的形状及上皮细胞数、腺泡和导管内有无分泌物。结果:(一)水部位中得到的浸膏量最多,其次是正丁醇部位,总生物碱物质集中存在于正丁醇和水部位中,总黄酮物质主要在石油醚和乙酸乙酯部位中含量最高,三个不同极性部位中总酚物质含量差异不大。提取到的总多糖粗提物中总多糖的含量达到了98.09%,总黄酮粗提物中总黄酮的含量为9.83%,总酚粗提物中总酚的含量为4.84%。(二)优选出总生物碱的提取纯化工艺为:称取麦芽粗粉,加入5倍量80%甲醇,超声提取3次,每次提取45 min,收集滤液浓缩蒸干后用PH 1~2的HCl水溶液溶解,过滤,滤液中加入3倍量石油醚萃取3次,收集水层溶液用浓氨水调溶液PH=11,在冰箱中静置,收集沉淀,沉淀用PH=11的氨水溶液洗涤数次后弃去,合并滤液于分液漏斗中,加入3倍量的氯仿萃取5次,1h/次,合并氯仿层溶液于60℃水浴蒸干溶剂,用0.03%HCl水溶液溶解,滤过,即得总生物碱部位纯度为56.64%。不同产地生麦芽中生物碱物质含量相差较大,安徽亳州产的生麦芽中生物碱物质含量最高,生大麦中不含大麦芽碱,炒麦芽中生物碱物质的含量比生麦芽要低。(三)对血清中PRL含量的影响:与正常组(4.714±5.159 pg·ml-1)比较,模型组大鼠血清中PRL的含量明显升高(30.404±5.516 pg·ml-1,P0.01);与模型组比较,不同极性部位组大鼠血清中PRL的含量均有所降低,仅总生物碱粗提部位组有显著性差异(11.051±5.516pg·ml-1,P0.01),该组大鼠血清中PRL的含量最低接近正常组大鼠血清中PRL的含量。与模型组比较,总生物碱粗提部位组、生物碱低浓度组(11.571±4.263 pg·ml-1)和生物碱高浓度组(7.004±4.044 pg·ml-1)均有极显著性差异(P0.01),其中生物碱高浓度组大鼠血清中PRL的含量最低接近正常值。对血清中P含量的影响:不同极性部位比较可知,与正常组(1.283±0.116 ng·ml-1)相比较,模型组大鼠血清中P的含量明显升高(1.993±0.116 ng·ml-1,P0.01);与模型组比较,石油醚和乙酸乙酯部位组、正丁醇部位组以及水部位组大鼠血清中P的含量均有升高。原药材水提物组和总生物碱粗提部位组大鼠血清中P的含量均有降低,其中总生物碱粗提部位组有极显著性差异(1.46±0.135ng·ml-1,P0.01)。不同化学部位比较可知,与模型组比较,各组大鼠血清中P的含量均有所降低,且相差不大。其中总生物碱粗提部位组、总多糖粗提部位组、总黄酮粗提部位组、总酚粗提部位组和生物碱高浓度组有极显著性差异。对血清中E2含量的影响:不同极性部位比较可知,与正常组(69.919±5.986 pg·ml-1)相比较,模型组大鼠血清中E2的含量明显升高(100.825±5.986 pg·ml-1,P0.01);与模型组比较,原药材水提物组(68.949±6.309 pg·ml-1)和总生物碱粗提部位组(64.550±5.986 pg·ml-1)有显著性降低(P0.01),其余各组均无显著性差异。不同化学部位比较可知,与模型组比较,各组大鼠血清中E2的含量均有所降低。泌乳素阳性染色于胞浆中,呈棕黄色。泌乳素细胞的累积光密度值越大表明阳性反应越大。与正常组(0.0068±0.0016)比较,模型组有显著性差异(0.0553±0.0172,P0.05),且泌乳素细胞的单位面积平均光密度值明显高于正常组。与模型组比较,总生物碱粗提部位组(0.0097±0.0026)、生物碱低浓度组(0.0087±0.0028)和生物碱高浓度组(0.0070±0.0019)均有显著性差异(P0.05),总生物碱粗提部位组泌乳素细胞的阳性反应比其他三个化学部位要小,但比生物碱高浓度组要大。PRL mRNA在各组大鼠脑垂体组织中都有表达,与正常组(6.23±1.12)比较,模型组有显著性差异(22.11±3.86,P0.05)。与模型组比较,总生物碱粗提部位组(5.95±1.07)、生物碱低浓度组(6.14±1.36)和生物碱高浓度组(4.32±1.08)均有显著性差异(P0.05),PRL mRNA的表达水平明显低于模型组。正常组大鼠乳腺组织中小叶不增生,腺泡少,导管不扩张,无分泌物,为0级增生。模型组大鼠乳腺组织小叶腺泡部分增生,导管扩张,无分泌物,为二级增生,表明高泌乳素血症伴随乳腺增生。总生物碱粗提部位组、总黄酮粗提部位组和总酚粗提部位组大鼠乳腺组织中小叶个别腺泡增生,导管不扩张,无分泌物,为一级增生。生物碱高浓度组大鼠乳腺组织中小叶不增生,腺泡少,导管不扩张,无分泌物,为0级增生。结论:(一)结果显示麦芽中生物碱物质含量较低,传统水提方法无法将其大量提取出,为中医临床大剂量使用麦芽回乳的用法找到了物质基础依据。(二)优选后的纯化方法可获得纯度大于50%的麦芽总生物碱部位,为药效学实验及化学成分分析研究提供了符合要求的样品。(三)确证了麦芽治疗高泌乳素血症的药效物质为其所含生物碱,作用机制是通过减少大鼠脑垂体PRL阳性细胞数量,下调脑垂体PRL细胞mRNA的表达,从而降低了大鼠体内PRL含量,达到治疗HPRL的目的。
[Abstract]:OBJECTIVE: On the basis of previous experiments, this study continued to study the extraction and purification process of total alkaloids from malt. Using modern pharmacological research methods, animal models of hyperprolactinemia were established. Pharmacodynamic tests were carried out on different polar parts, chemical parts and purified alkaloids of malt to screen out malt. METHODS: (1) A proper amount of malt was extracted with 70% ethanol reflux extract, and then the suspension was dissolved in water. Petroleum ether, ethyl acetate and n-butanol were added to the suspension in turn to extract the different polar parts, and then the chemical components in each part were determined. Total malt polysaccharides were extracted with 70% ethanol and then purified by polyamide resin column. Total malt flavonoids were extracted with 70% ethanol and then purified by HPD-600 macroporous resin column. The preparation and determination methods of alkaloid crude extracts were based on the previous experimental methods of our group. (2) According to the single factor experiment, the extraction solvent of alkaloid substances in malt was selected, and then the orthogonal test was carried out. Taking the comprehensive score of total alkaloids and malt alkaloids as the evaluation index, the best extraction method of alkaloid substances in malt was selected. The purification efficiency of malt total alkaloids by column chromatography and solvent extraction was compared with that by using the transfer rate and purity of total alkaloids as evaluation indexes. The degreasing solvent, water bath temperature and ammonia alkalinization solution to different PH values were investigated in solvent extraction method to optimize the purification process of malt total alkaloids. (3) High lactation. Establishment of vegetaemia model: In addition to normal group rats, other groups of rats were subcutaneously injected with metoclopramide hydrochloride injection 50mg/kg body weight, once a day in the morning and afternoon, respectively, for five consecutive days. Grouping: 120 female infertile SD rats were randomly divided into 12 groups: normal group, model group, petroleum ether and ethyl acetate fraction group, n-butyl group. The alcohol extract group, water extract group, water extract group, total alkaloid extract group, total polysaccharide extract group, total flavonoid extract group, total phenol extract group, alkaloid low concentration group, alkaloid high concentration group, 10 rats in each group. The dosage of n-butanol fraction group was 124.17 mg kg-1 d-1, water fraction group was 645.96 mg kg-1 d-1, water extract group was 4.74 g kg-1 d-1, crude alkaloid fraction group was 592.43 mg kg-1 d-1, crude polysaccharide fraction group was 1.57 g kg-1 d-1, crude flavonoid fraction group was 4.74 g kg-1 d-1, The dosage was 57.83 mg 65507 Determination of serum prolactin (PRL), estrogen (E2) and progesterone (P) in serum of rats in each group were determined by enzyme linked immunosorbent assay (ELISA). Effects of ELISA kit instructions on the content and mRNA expression of prolactin-positive cells in pituitary gland of rats with hyperprolactinemia: normal group, model group, total alkaloid crude extract group, total polysaccharide crude extract group, total flavone crude extract group, total phenol crude extract group, low alkaloid concentration group and high alkaloid concentration group The pituitary gland of 1/2 rats was immobilized in tissue stationary fluid for 24 hours. Immunohistochemistry was performed after paraffin embedded sections. The accumulative optical density (IOD) of PRL positive cells was measured. The other 1/2 pituitary gland of rats was taken for PRL mRNA fluorescence quantitative PC. R experiment, calculating the expansion multiple, statistical analysis. The effect of hyperplasia of mammary gland in rats with hyperprolactinemia: normal group, model group, water extract group, total alkaloid crude extract group, total polysaccharide crude extract group, total flavone crude extract group, total phenol crude extract group, low alkaloid concentration group and high alkaloid concentration group The second pair of mammary gland tissues were taken from the rats in group A after 24 hours of the last administration. The second pair of mammary gland tissues were immobilized in 4% paraformaldehyde tissue fixative for 24 hours. The sections were stained with HE. The mammary gland tissues of the rats in each group were observed under microscope, including the number of lobular acini, the shape of duct and the number of epithelial cells. Results: (1) The amount of extract was the most in water part, followed by n-butanol part, and the total alkaloids were concentrated in n-butanol and water part. The content of total flavonoids was the highest in petroleum ether and ethyl acetate part, and the content of total phenols in three different polar parts was not significantly different. The content of total polysaccharides in the crude polysaccharides was 98.09%, the content of total flavonoids in the crude flavonoids was 9.83%, and the content of total phenols in the crude phenolics was 4.84%. (2) The extraction and purification process of total alkaloids was optimized as follows: weighing malt powder, adding 5 times of 80% methanol, ultrasonic extraction three times, extracting 45 minutes each time, and collecting filtrate to concentrate. After steaming and drying, the solution is dissolved in the HCl solution of PH 1~2, filtered, and the filtrate is extracted three times by adding 3 times of petroleum ether. The water layer solution is collected and mixed with concentrated ammonia water to adjust the solution PH=11. The solution is left in the refrigerator, collected and precipitated. The precipitation is washed several times with the ammonia solution of PH=11 and discarded. The purity of total alkaloids was 56.64%. The content of alkaloids in malt from different producing areas varied greatly. The content of alkaloids in malt from Bozhou, Anhui Province was the highest, and barley did not contain barley malt alkaloids, and the content ratio of alkaloids in stir-fried malt was 56.64%. (3) Effect of raw malt on PRL content in serum: Compared with the normal group (4.714+5.159 pg.ml-1), the content of PRL in serum of model group increased significantly (30.404+5.516 pg.ml-1, P 0.01); Compared with the model group, the content of PRL in serum of rats in different polar parts decreased, only the total alkaloid crude extract group had significant difference. The content of PRL in the serum of the rats in this group was the lowest, which was close to that of the normal group. Compared with the model group, the content of PRL in the crude extract part of total alkaloids, the low alkaloids concentration group (11.571 4.263 PG ml-1) and the high alkaloids concentration group (7.004.044 PG 65507 The content of PRL in serum of rats with high concentration of alkaloids was the lowest close to the normal value. The effect of different polar parts on the content of P in serum: Compared with the normal group (1.283.116 ng 0.116 ng 65507 The contents of P in serum of rats in the water extract group and the crude extract group of total alkaloids were significantly lower than those in the water extract group and the crude extract group of total alkaloids (1.46.135ng 65 The content of P in serum of rats in each group was decreased, and the difference was not significant. There were significant differences in the content of E2 between the crude extracts of total alkaloids, polysaccharides, flavonoids, phenols and alkaloids. Compared with the model group, the content of E2 in serum of rats in the model group was significantly increased (100.825.986 PG, P 0.01 Compared with the model group, the content of E2 in the serum of rats in each group was decreased. The positive staining of prolactin in the cytoplasm was brown and yellow. The higher the cumulative optical density of prolactin cells, the greater the positive reaction. Compared with the normal group (0.0068.0016), the model group had significant difference (0.0553.0172, P 0.05), and the prolactin cells had significant difference (0.0553.0172, P 0.05). Compared with the model group, there were significant differences in the positive reactions of prolactin cells in the crude extracts of total alkaloids (0.0097.0026), the low concentration of alkaloids (0.0087.0028) and the high concentration of alkaloids (0.0070.0019) (P 0.05). PRL mRNA was expressed in pituitary tissue of rats in each group, and there was significant difference between the model group and the normal group (6.23 + 1.12). Compared with the model group, the total alkaloid crude extract group (5.95 + 1.07), the low alkaloid concentration group (6.14 + 1.36) and the high alkaloid concentration group (6.14 + 1.36), the total alkaloid crude extract group (22.11 + 3.86, P 0.05). The expression of PRL mRNA was significantly lower than that of the model group (P 0.05). The normal group had no hyperplasia of lobules, few acini, no ductal dilatation, NO secretion and grade 0 hyperplasia. The model group had partial hyperplasia of acinar lobules, ductal dilatation, no secretion, and secondary hyperplasia, indicating hyperplasia of hyperprolactin blood. In the high alkaloid concentration group, the lobules were not proliferated, the acini were few, the ducts were not dilated, and there was no secretion. The mammary gland was grade 0 hyperplasia. CONCLUSIONS: (1) The results showed that the content of alkaloids in malt was low, and the traditional water extraction method could not extract them in large quantities, which provided the material basis for the usage of large-dose malt milk in TCM clinic. (2) The optimized purification method could obtain the total alkaloids in malt with purity of more than 50%, which was a pharmacodynamic experiment and chemical composition. (3) It was confirmed that the effective substance of malt in the treatment of hyperprolactinemia was the alkaloid. The mechanism was to reduce the number of PRL-positive cells in pituitary gland and down-regulate the expression of PRL-positive cells mRNA in pituitary gland of rats, thereby reducing the PRL content in vivo and achieving the purpose of treating HPRL.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285.5

【参考文献】