披针叶黄华碱的提取纯化及生物活性研究

发布时间：2018-03-29 18:32

本文选题：披针叶黄华碱　切入点：提取　出处：《甘肃农业大学》2016年博士论文

【摘要】：研究显示,披针叶黄华中主要含有生物碱、有机酸、黄酮、酚类和多糖等化学成分,而其所含的生物碱是主要的有效成分与有毒成分。目前国内外关于披针叶黄华碱的提取纯化和药理活性方面的研究报道很少。据此,本试验针对披针叶黄华全草中所含生物碱成分进行了提取、分离和鉴定,获取了纯度较高的单体碱,并对总碱与单体碱进行了药理活性方面的研究。具体内容如下:1.披针叶黄华化学成分的预试验通过对披针叶黄华化学成分的预实验研究,以确定其所含化学成分的种类。采用试管预试验法和薄层色谱分析法,对披针叶黄华的水浸液、酸性乙醇液、乙醚提取液进行研究,通过不同指示剂、显色剂发生的沉淀反应或颜色反应,确定披针叶黄华中可能含有的化学组份。试验结果显示:披针叶黄华中可能含有的化学成分有生物碱、多糖、苷类、黄酮、蛋白质、多肽、氨基酸、鞣质、酚类、挥发油等物质,不含香豆素类物质。薄层色谱分析(TLC)结果表明,披针叶黄华中含有的生物碱是主要的有效成分和有毒成分。2.披针叶黄华碱的提取工艺研究本试验选用甲醇溶液做为提取溶剂,通过热回流提取和超声波辅助热回流提取法,分别从相同质量的披针叶黄华全草中提取总生物碱。结果显示:热回流提取法最终的出碱率为0.66%,超声辅助法的出碱率为0.76%。对总生物碱的TLC分析检测结果显示,氯仿萃取剂的部分含有5种生物碱,正丁醇萃取的部分含有2种生物碱。总生物碱在乙酸乙酯:甲醇(3:2)作展开剂时的层析效果最好,共呈现出5个点。3.披针叶黄华碱的柱层析分离采用硅胶吸附柱色谱法对披针叶黄华碱进行分离,共获得2种结晶体(晶体Ⅰ和晶体Ⅱ)和A、E、G、J四段含有生物碱的油状混合物。其中晶体Ⅰ为淡黄色针状结晶体,TLC检测出现2个橘黄色斑点,无荧光。晶体Ⅱ为淡黄色片状结晶体,TLC检测出现5个红色斑点,无荧光。4.披针叶黄华碱的纯化与鉴定收集由硅胶吸附柱色谱法分离得到的晶体I和晶体II,将其分别与黄花碱、金雀花碱标准品对照,并进行TLC和气相色谱-质谱联用仪(GC-MC)分析。结果显示:淡黄色针状晶体Ⅰ为黄华碱,淡黄色片状晶体Ⅱ推测为金雀花碱。在相同条件下的GC-MC分析检测中,离子流色谱图显示黄华碱标准品的出峰时间为12.2,这与晶体Ⅰ中的出峰时间几乎相同;而金雀花碱标准品的出峰时间为7.5,晶体II的出峰时间为7.8,两者之间略有差异。5.披针叶黄华碱的GC-MS分析披针叶黄华碱浸膏经酸溶解过滤、碱中和处理、氯仿萃取后,用70%的甲醇溶解,再经GC-MS分析,共检测出14种生物碱单体,经质谱数据库检索,最终确定了其中的5种,即d-鹰爪豆碱(Sparteine)、Dodecahydro-7,14-methanodipyrido[1.2-a:1’,2’-E][1,5]Diazocine、阿金亭碱(Argentamin)、金雀花碱(Cytisine)、黄华碱(Thermopsine)。其中Dodecahydro-7,14-methanodipyrido[1.2-a:1,2’-E][1,5]Diazocine含量很少,仅占总量的0.26%。而d-鹰爪豆碱(Sparteine)的含量最大,占总碱比例的55.61%。6.披针叶黄华碱的毒性试验研究通过披针叶黄华碱对小鼠毒性试验的研究,复制出了披针叶黄华碱中毒的病理学模型。结果发现,按照200mg/kg的药量对小鼠进行灌服时,即出现全群急性中毒死亡的现象,由此大致可推断披针叶黄华碱的LD50≤200mg/kg;在发生慢性中毒小鼠的血液生化检查中发现,披针叶黄华碱可使小鼠体内血红蛋白、红细胞、白细胞数量升高,谷丙转氨酶(ALT)、谷草转氨酶(AST)、肌酸激酶(CK)含量升高,尿素氮(BUN)浓度下降;对慢性中毒小鼠的临床病理学检查中发现,以心、肝、肺、肾实质器官的细胞变性坏死为主,尤其是以肝脏、肾脏的损伤最严重。7.披针叶黄华碱对奶牛子宫内膜炎致病菌的研究本研究针对患病奶牛子宫中的病原菌进行分离鉴定。结果显示:所收集的病料中以感染大肠埃希氏菌和溶血性链球菌为主,以混合感染为主要感染方式。在披针叶黄华总碱、黄华碱、阿金亭碱对大肠埃希氏菌、溶血性链球菌、非溶血性链球菌、沙门氏菌的体外抑菌活性试验中发现,3种生物碱均具有一定的抗菌活性,其中非溶血性链球菌最为敏感,其次为沙门氏菌、大肠埃希氏菌和溶血性链球菌。而金雀花碱则对以上4种病原菌无抗菌活性。8.披针叶黄华碱对小鼠免疫功能的研究本试验通过与对照组进行对比试验,观察披针叶黄华碱对实验小鼠体重、脏器质量、脏器指数、巨噬细胞吞噬力和T淋巴细胞数量的影响。结果发现在对实验小鼠灌服了低、中、高3个不同药物剂量的披针叶黄华碱后,小鼠的体重、脏器质量、脏器指数、巨噬细胞吞噬力以及T淋巴细胞的数量均有所下降。其中,低浓度的披针叶黄华碱对小鼠的细胞免疫功能的作用不明显,而中高浓度的披针叶黄华碱对小鼠免疫功能还具有一定的抑制作用。9.披针叶黄华碱对细胞毒性的研究利用MTT比色法依次测定披针叶黄华总碱、金雀花碱、羟喜树碱对乳仓鼠肾细胞(BHK)、人结肠癌细胞(SW480)的体外细胞毒性作用。结果显示:披针叶黄华总碱抑制SW480的生长作用显著,其抑制率随被测药液剂量不断增加而逐渐增强。羟喜树碱对BHK和SW480在各浓度梯度组中均有较强的抑制作用。披针叶黄华碱和金雀花碱对BHK细胞的毒性稍低,对SW480的生长均具有一定的抑制作用。
[Abstract]:Research shows that lanceolatum Huang Huazhong mainly contains alkaloids, organic acids, flavonoids, phenolic constituents and polysaccharides, alkaloids and the content of effective components and toxic components. The main current research reports and pharmacological activities at home and abroad on the extraction and purification of Lanceleaf Huang Huajian little. Accordingly, the test for extraction Phi alkaloids yellow grass in China coniferous composition, isolation and identification, to obtain high purity alkali monomer, and the total monomer of alkali and alkali was studied pharmacological activities. The specific contents are as follows: 1. pre test with chemical composition of coniferous Huang Hua through the study on the chemical composition of pre Phi Huang Hua needles, to determine the type of chemical components. Analysis by test tube pre test method and thin layer chromatography of Lanceleaf Huang Hua water extract, acidic ethanol and ether extract were studied by different. The indicator, significant precipitation reaction or color reaction agent, determine lanceolatum may contain the chemical composition of Huang Huazhong. The experiment results show that the chemical composition of Huang Huazhong lanceolatum may contain alkaloids, polysaccharides, glycosides, flavonoids, proteins, peptides, amino acids, tannins, phenols, volatile oil and other substances, not containing coumarin. Thin layer chromatography (TLC) results show that phi is Huang Huazhong containing alkaloids needle extraction.2. effective ingredients and toxic components of the main lanceolatum Huang Huajian study the methanol solution as extraction solvent, by heat reflux extraction and ultrasonic assisted hot reflux extraction, extraction of total alkaloids from the same respectively. The quality of Huang Huaquan lanceolatum grass. The results showed that heat reflux extraction the final alkali was 0.66%, ultrasonic assisted method the alkali rate of 0.76%. on total alkaloid TLC analysis Showed that the chloroform extraction agent containing 5 alkaloids, n-butanol part contains 2 kinds of alkaloids. The total alkaloids in ethyl acetate: methanol (3:2) as developing agent when the best effect of chromatography, were presented separated by silica gel adsorption column chromatography for separation of Lanceleaf Huang Huajian 5 points with.3. column chromatography Huang Huajian's 2 needles, crystals were obtained (crystal crystal I and II) and A, E, G, J four alkaloid containing oil mixture. The crystal of light yellow needle crystal, 2 orange spots, TLC detected no fluorescence. The crystal of light yellow flake crystal 5 red spots, TLC detection, without purification and identification were collected by silica gel adsorption column chromatography separation of crystal I and crystal II fluorescent.4. lanceolatum Huang Huajian, which respectively with Thermopsine, cytisine standard control, and TLC and gas chromatography-mass spectrometry Spectrometer (GC-MC) analysis. The results showed that: light yellow needle crystal of Huang Huajian, pale yellow crystal flower base for genistein. II that under the same conditions of GC-MC detection, ion chromatogram showed the peak time of Huang Huajian standard is 12.2, and the crystal of peak time almost the same; peak time and cytisine standard 7.5, II crystal, the peak time was 7.8, between the two slightly different.5. lanceolatum Huang Huajian GC-MS analysis lanceolatum Huang Hua alkali extract dissolved by acid alkali neutralization, filtration, chloroform, dissolved in methanol 70%, followed by GC-MS analysis detected a total of 14 kinds of alkaloid monomers by mass spectrometry database retrieval, and ultimately determine the 5 of them, namely d- sparteine (Sparteine), Dodecahydro-7,14-methanodipyrido[1.2-a:1 ', 2' -E][1,5]Diazocine, Kim Ting alkali (Argentamin), genistein (Cytisin E), Huang Huajian (Thermopsine). The Dodecahydro-7,14-methanodipyrido[1.2-a:1,2 '-E][1,5]Diazocine content rarely, only accounted for 0.26%. of the total d- and sparteine (Sparteine) content, toxicity test of total alkali ratio 55.61%.6. lanceolatum Huang Huajian through the study of mouse toxicity test lanceolatum Huang Huajian, established a model with pathological needles Huang Hua alkalosis. The results showed that the dose of 200mg/kg on mice were orally given according to that the whole group of acute poisoning death phenomenon, which can be roughly inferred lanceolatum Huang Hua base LD50 = 200mg/kg; found in the blood biochemical examination of chronic poisoning in mice, Lanceleaf Huang Hua base can make the hemoglobin. Mice red blood cells, elevated white blood cell count, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) content increased, blood urea nitrogen (BUN) concentration decreased; The clinical and pathological examination of chronic poisoning of mice found in heart, liver, lung, cell degeneration and necrosis of renal parenchyma organs, especially in liver, kidney damage and serious study.7. lanceolatum Huang Huajian on pathogenic bacteria of cow's Endometritis based on pathogen prevalence of milk cow uterus were isolated and identified. The results showed: the disease material collected in the infection with Escherichia coli and Streptococcus, with mixed infection was the main infection way. In Lanceleaf Huang Hua total alkali, yellow Thermopsine, A Jin Ting alkali on Escherichia coli, Streptococcus, non hemolytic streptococcus, found in vitro antibacterial activity test of Salmonella in 3 kinds of alkaloids have certain antibacterial activity, non hemolytic streptococcus was the most sensitive, followed by Salmonella, Escherichia coli and Streptococcus. And genistein on the above 4 No pathogenic bacteria antibacterial activity.8. lanceolatum Huang Huajian research on the immune function of the mice in this experiment were compared with the control group, the observation Huang Huajian lanceolatum on mice body weight, organ weight, organ index, and affect the number of macrophage phagocytosis of T lymphocytes. The results found in mice fed a low, high 3 different doses of lanceolatum after Huang Huajian, the weight of mice, organ weight, organ index, phagocytosis of the macrophages and the number of T lymphocytes decreased. Among them, the low concentration of Huang Huajian lanceolatum on mice immune function is not obvious, while the high concentration of Lanceleaf Huang Huajian on immune function in mice also has a certain inhibitory effect of.9. lanceolatum of Huang Huajian on cell toxicity were determined lanceolatum Huang Hua total alkaloids by MTT colorimetry, cytisine, HCPT on Baby hamster kidney cells (BHK), human colon cancer cells (SW480) in vitro cytotoxicity. The results showed that the growth inhibition of SW480 lanceolatum Huang Hua total alkaloids significantly, the inhibition rate of the tested liquid dosage increasing gradually. Hydroxycamptothecin on BHK and SW480 in different concentration gradient group were in a strong inhibitory effect. Lanceolatum Huang Huajian and genistein on BHK cell toxicity was low, the growth of SW480 were inhibited.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S859.87;S853.74

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