【摘要】：目的:本研究在前期試驗基礎(chǔ)上,繼續(xù)研究麥芽中總生物堿物質(zhì)的提取純化工藝。利用現(xiàn)代化藥理學(xué)研究方法,復(fù)制高泌乳素血癥動物疾病模型,對麥芽不同極性部位和不同化學(xué)部位,以及純化后生物堿物質(zhì)開展藥效學(xué)試驗,篩選出麥芽治療HPRL有效部位,并闡明其作用機(jī)制。方法:(一)稱取適量麥芽用70%乙醇回流提取浸膏,加水溶解制成混懸液后依次加入石油醚,乙酸乙酯,正丁醇萃取得到不同極性部位提取物,再對各個部位中的化學(xué)成分進(jìn)行含量測定。根據(jù)相關(guān)文獻(xiàn),選擇水做溶劑,40℃水浴,提取得到麥芽總多糖粗提物;用70%乙醇回流提取,然后通過聚酰胺樹脂柱進(jìn)行簡單的純化,得到麥芽總黃酮粗提物;用70%乙醇超聲提取,然后通過HPD-600大孔樹脂柱進(jìn)行簡單的純化,得到麥芽總酚粗提物。麥芽總生物堿粗提物制備和含量測定方法依據(jù)本課題組前期的實(shí)驗方法進(jìn)行。(二)根據(jù)單因素實(shí)驗選擇麥芽中生物堿物質(zhì)的提取溶劑,再進(jìn)行正交試驗,以總生物堿和大麥芽堿提取量的綜合評分為評價指標(biāo),優(yōu)選出麥芽生物堿物質(zhì)的最佳提取工藝。再以總生物堿的轉(zhuǎn)移率和純度為評價指標(biāo),比較柱層析法和溶劑萃取法對麥芽總生物堿的純化效果,同時對溶劑萃取法中脫脂溶劑、水浴溫度以及氨水堿化溶液至不同PH值進(jìn)行考察,優(yōu)選麥芽總生物堿的最佳純化工藝。(三)高泌乳素血癥模型的建立:除正常組大鼠外,其它各組大鼠背部皮下注射鹽酸甲氧氯普胺注射液50mg/kg體重,每天上午下午分別定時注射1次,連續(xù)5天。分組給藥:將120只雌性未孕SD大鼠隨機(jī)分為12組:正常組、模型組、石油醚和乙酸乙酯部位組、正丁醇部位組、水部位組、原藥材水提物組、總生物堿粗提部位組、總多糖粗提部位組、總黃酮粗提部位組、總酚粗提部位組、生物堿低濃度組、生物堿高濃度組,每組10只。模型制備后開始給藥,石油醚和乙酸乙酯部位組給藥劑量為114.70mg·kg-1·d-1;正丁醇部位組給藥劑量為124.17mg·kg-1·d-1;水部位組給藥劑量為645.96mg·kg-1·d-1;原藥材水提物組給藥劑量為4.74g·kg-1·d-1;總生物堿粗提部位組給藥劑量為592.43mg·kg-1·d-1;總多糖粗提部位組給藥劑量為1.57g·kg-1·d-1;總黃酮粗提部位組給藥劑量為57.83mg·kg-1·d-1;總酚粗提部位組給藥劑量為103.39mg·kg-1·d-1;生物堿低濃度組給藥劑量為0.87mg·kg-1·d-1;生物堿高濃度組給藥劑量為1.74 mg·kg-1·d-1。正常組和模型組給予等容量的蒸餾水,連續(xù)灌胃30d。對高泌乳素血癥大鼠血清激素含量的檢測:末次給藥24h后,所有大鼠禁食12小時,自由飲水。各組大鼠于次日上午心臟取血2ml,血液靜置后以3000轉(zhuǎn)/min的速度離心5min,取血清供檢測。采用酶聯(lián)免疫吸附法測定各組大鼠血清中泌乳素(PRL)、雌激素(E2)、孕酮(P)的含量,嚴(yán)格按照ELISA試劑盒說明書中操作流程進(jìn)行。對高泌乳素血癥大鼠腦垂體泌乳素陽性細(xì)胞含量及mRNA表達(dá)的影響:正常組、模型組、總生物堿粗提部位組、總多糖粗提部位組、總黃酮粗提部位組、總酚粗提部位組、生物堿低濃度組和生物堿高濃度組大鼠末次給藥24h后,于次日上午用20%烏拉坦麻醉后取其腦垂體。取1/2大鼠腦垂體于組織固定液中充分固定24小時,石蠟包埋切片后,進(jìn)行免疫組化實(shí)驗,測定PRL細(xì)胞陽性的累積光密度值(IOD)。取另外的1/2大鼠腦垂體進(jìn)行PRL mRNA熒光定量PCR實(shí)驗,計算擴(kuò)張倍數(shù),進(jìn)行統(tǒng)計學(xué)分析。對高泌乳素血癥大鼠乳腺增生情況的影響實(shí)驗:正常組、模型組、原藥材組水提物組、總生物堿粗提部位組、總多糖粗提部位組、總黃酮粗提部位組、總酚粗提部位組、生物堿低濃度組和生物堿高濃度組大鼠末次給藥24h后,于次日上午用20%烏拉坦麻醉后取其第二對乳腺組織,于4%多聚甲醛組織固定液中充分固定24小時后,包埋,切片進(jìn)行HE染色,在顯微鏡下觀察各組大鼠乳腺組織情況,包括乳腺小葉腺泡數(shù)、導(dǎo)管的形狀及上皮細(xì)胞數(shù)、腺泡和導(dǎo)管內(nèi)有無分泌物。結(jié)果:(一)水部位中得到的浸膏量最多,其次是正丁醇部位,總生物堿物質(zhì)集中存在于正丁醇和水部位中,總黃酮物質(zhì)主要在石油醚和乙酸乙酯部位中含量最高,三個不同極性部位中總酚物質(zhì)含量差異不大。提取到的總多糖粗提物中總多糖的含量達(dá)到了98.09%,總黃酮粗提物中總黃酮的含量為9.83%,總酚粗提物中總酚的含量為4.84%。(二)優(yōu)選出總生物堿的提取純化工藝為:稱取麥芽粗粉,加入5倍量80%甲醇,超聲提取3次,每次提取45 min,收集濾液濃縮蒸干后用PH 1~2的HCl水溶液溶解,過濾,濾液中加入3倍量石油醚萃取3次,收集水層溶液用濃氨水調(diào)溶液PH=11,在冰箱中靜置,收集沉淀,沉淀用PH=11的氨水溶液洗滌數(shù)次后棄去,合并濾液于分液漏斗中,加入3倍量的氯仿萃取5次,1h/次,合并氯仿層溶液于60℃水浴蒸干溶劑,用0.03%HCl水溶液溶解,濾過,即得總生物堿部位純度為56.64%。不同產(chǎn)地生麥芽中生物堿物質(zhì)含量相差較大,安徽亳州產(chǎn)的生麥芽中生物堿物質(zhì)含量最高,生大麥中不含大麥芽堿,炒麥芽中生物堿物質(zhì)的含量比生麥芽要低。(三)對血清中PRL含量的影響:與正常組(4.714±5.159 pg·ml-1)比較,模型組大鼠血清中PRL的含量明顯升高(30.404±5.516 pg·ml-1,P0.01);與模型組比較,不同極性部位組大鼠血清中PRL的含量均有所降低,僅總生物堿粗提部位組有顯著性差異(11.051±5.516pg·ml-1,P0.01),該組大鼠血清中PRL的含量最低接近正常組大鼠血清中PRL的含量。與模型組比較,總生物堿粗提部位組、生物堿低濃度組(11.571±4.263 pg·ml-1)和生物堿高濃度組(7.004±4.044 pg·ml-1)均有極顯著性差異(P0.01),其中生物堿高濃度組大鼠血清中PRL的含量最低接近正常值。對血清中P含量的影響:不同極性部位比較可知,與正常組(1.283±0.116 ng·ml-1)相比較,模型組大鼠血清中P的含量明顯升高(1.993±0.116 ng·ml-1,P0.01);與模型組比較,石油醚和乙酸乙酯部位組、正丁醇部位組以及水部位組大鼠血清中P的含量均有升高。原藥材水提物組和總生物堿粗提部位組大鼠血清中P的含量均有降低,其中總生物堿粗提部位組有極顯著性差異(1.46±0.135ng·ml-1,P0.01)。不同化學(xué)部位比較可知,與模型組比較,各組大鼠血清中P的含量均有所降低,且相差不大。其中總生物堿粗提部位組、總多糖粗提部位組、總黃酮粗提部位組、總酚粗提部位組和生物堿高濃度組有極顯著性差異。對血清中E2含量的影響:不同極性部位比較可知,與正常組(69.919±5.986 pg·ml-1)相比較,模型組大鼠血清中E2的含量明顯升高(100.825±5.986 pg·ml-1,P0.01);與模型組比較,原藥材水提物組(68.949±6.309 pg·ml-1)和總生物堿粗提部位組(64.550±5.986 pg·ml-1)有顯著性降低(P0.01),其余各組均無顯著性差異。不同化學(xué)部位比較可知,與模型組比較,各組大鼠血清中E2的含量均有所降低。泌乳素陽性染色于胞漿中,呈棕黃色。泌乳素細(xì)胞的累積光密度值越大表明陽性反應(yīng)越大。與正常組(0.0068±0.0016)比較,模型組有顯著性差異(0.0553±0.0172,P0.05),且泌乳素細(xì)胞的單位面積平均光密度值明顯高于正常組。與模型組比較,總生物堿粗提部位組(0.0097±0.0026)、生物堿低濃度組(0.0087±0.0028)和生物堿高濃度組(0.0070±0.0019)均有顯著性差異(P0.05),總生物堿粗提部位組泌乳素細(xì)胞的陽性反應(yīng)比其他三個化學(xué)部位要小,但比生物堿高濃度組要大。PRL mRNA在各組大鼠腦垂體組織中都有表達(dá),與正常組(6.23±1.12)比較,模型組有顯著性差異(22.11±3.86,P0.05)。與模型組比較,總生物堿粗提部位組(5.95±1.07)、生物堿低濃度組(6.14±1.36)和生物堿高濃度組(4.32±1.08)均有顯著性差異(P0.05),PRL mRNA的表達(dá)水平明顯低于模型組。正常組大鼠乳腺組織中小葉不增生,腺泡少,導(dǎo)管不擴(kuò)張,無分泌物,為0級增生。模型組大鼠乳腺組織小葉腺泡部分增生,導(dǎo)管擴(kuò)張,無分泌物,為二級增生,表明高泌乳素血癥伴隨乳腺增生�？偵飰A粗提部位組、總黃酮粗提部位組和總酚粗提部位組大鼠乳腺組織中小葉個別腺泡增生,導(dǎo)管不擴(kuò)張,無分泌物,為一級增生。生物堿高濃度組大鼠乳腺組織中小葉不增生,腺泡少,導(dǎo)管不擴(kuò)張,無分泌物,為0級增生。結(jié)論:(一)結(jié)果顯示麥芽中生物堿物質(zhì)含量較低,傳統(tǒng)水提方法無法將其大量提取出,為中醫(yī)臨床大劑量使用麥芽回乳的用法找到了物質(zhì)基礎(chǔ)依據(jù)。(二)優(yōu)選后的純化方法可獲得純度大于50%的麥芽總生物堿部位,為藥效學(xué)實(shí)驗及化學(xué)成分分析研究提供了符合要求的樣品。(三)確證了麥芽治療高泌乳素血癥的藥效物質(zhì)為其所含生物堿,作用機(jī)制是通過減少大鼠腦垂體PRL陽性細(xì)胞數(shù)量,下調(diào)腦垂體PRL細(xì)胞mRNA的表達(dá),從而降低了大鼠體內(nèi)PRL含量,達(dá)到治療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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284;R285.5

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