本文選題：披針葉黃華堿　切入點(diǎn)：提取　出處：《甘肅農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：研究顯示,披針葉黃華中主要含有生物堿、有機(jī)酸、黃酮、酚類和多糖等化學(xué)成分,而其所含的生物堿是主要的有效成分與有毒成分。目前國內(nèi)外關(guān)于披針葉黃華堿的提取純化和藥理活性方面的研究報(bào)道很少。據(jù)此,本試驗(yàn)針對披針葉黃華全草中所含生物堿成分進(jìn)行了提取、分離和鑒定,獲取了純度較高的單體堿,并對總堿與單體堿進(jìn)行了藥理活性方面的研究。具體內(nèi)容如下:1.披針葉黃華化學(xué)成分的預(yù)試驗(yàn)通過對披針葉黃華化學(xué)成分的預(yù)實(shí)驗(yàn)研究,以確定其所含化學(xué)成分的種類。采用試管預(yù)試驗(yàn)法和薄層色譜分析法,對披針葉黃華的水浸液、酸性乙醇液、乙醚提取液進(jìn)行研究,通過不同指示劑、顯色劑發(fā)生的沉淀反應(yīng)或顏色反應(yīng),確定披針葉黃華中可能含有的化學(xué)組份。試驗(yàn)結(jié)果顯示:披針葉黃華中可能含有的化學(xué)成分有生物堿、多糖、苷類、黃酮、蛋白質(zhì)、多肽、氨基酸、鞣質(zhì)、酚類、揮發(fā)油等物質(zhì),不含香豆素類物質(zhì)。薄層色譜分析(TLC)結(jié)果表明,披針葉黃華中含有的生物堿是主要的有效成分和有毒成分。2.披針葉黃華堿的提取工藝研究本試驗(yàn)選用甲醇溶液做為提取溶劑,通過熱回流提取和超聲波輔助熱回流提取法,分別從相同質(zhì)量的披針葉黃華全草中提取總生物堿。結(jié)果顯示:熱回流提取法最終的出堿率為0.66%,超聲輔助法的出堿率為0.76%。對總生物堿的TLC分析檢測結(jié)果顯示,氯仿萃取劑的部分含有5種生物堿,正丁醇萃取的部分含有2種生物堿�？偵飰A在乙酸乙酯:甲醇(3:2)作展開劑時(shí)的層析效果最好,共呈現(xiàn)出5個(gè)點(diǎn)。3.披針葉黃華堿的柱層析分離采用硅膠吸附柱色譜法對披針葉黃華堿進(jìn)行分離,共獲得2種結(jié)晶體(晶體Ⅰ和晶體Ⅱ)和A、E、G、J四段含有生物堿的油狀混合物。其中晶體Ⅰ為淡黃色針狀結(jié)晶體,TLC檢測出現(xiàn)2個(gè)橘黃色斑點(diǎn),無熒光。晶體Ⅱ?yàn)榈S色片狀結(jié)晶體,TLC檢測出現(xiàn)5個(gè)紅色斑點(diǎn),無熒光。4.披針葉黃華堿的純化與鑒定收集由硅膠吸附柱色譜法分離得到的晶體I和晶體II,將其分別與黃花堿、金雀花堿標(biāo)準(zhǔn)品對照,并進(jìn)行TLC和氣相色譜-質(zhì)譜聯(lián)用儀(GC-MC)分析。結(jié)果顯示:淡黃色針狀晶體Ⅰ為黃華堿,淡黃色片狀晶體Ⅱ推測為金雀花堿。在相同條件下的GC-MC分析檢測中,離子流色譜圖顯示黃華堿標(biāo)準(zhǔn)品的出峰時(shí)間為12.2,這與晶體Ⅰ中的出峰時(shí)間幾乎相同;而金雀花堿標(biāo)準(zhǔn)品的出峰時(shí)間為7.5,晶體II的出峰時(shí)間為7.8,兩者之間略有差異。5.披針葉黃華堿的GC-MS分析披針葉黃華堿浸膏經(jīng)酸溶解過濾、堿中和處理、氯仿萃取后,用70%的甲醇溶解,再經(jīng)GC-MS分析,共檢測出14種生物堿單體,經(jīng)質(zhì)譜數(shù)據(jù)庫檢索,最終確定了其中的5種,即d-鷹爪豆堿(Sparteine)、Dodecahydro-7,14-methanodipyrido[1.2-a:1’,2’-E][1,5]Diazocine、阿金亭堿(Argentamin)、金雀花堿(Cytisine)、黃華堿(Thermopsine)。其中Dodecahydro-7,14-methanodipyrido[1.2-a:1,2’-E][1,5]Diazocine含量很少,僅占總量的0.26%。而d-鷹爪豆堿(Sparteine)的含量最大,占總堿比例的55.61%。6.披針葉黃華堿的毒性試驗(yàn)研究通過披針葉黃華堿對小鼠毒性試驗(yàn)的研究,復(fù)制出了披針葉黃華堿中毒的病理學(xué)模型。結(jié)果發(fā)現(xiàn),按照200mg/kg的藥量對小鼠進(jìn)行灌服時(shí),即出現(xiàn)全群急性中毒死亡的現(xiàn)象,由此大致可推斷披針葉黃華堿的LD50≤200mg/kg;在發(fā)生慢性中毒小鼠的血液生化檢查中發(fā)現(xiàn),披針葉黃華堿可使小鼠體內(nèi)血紅蛋白、紅細(xì)胞、白細(xì)胞數(shù)量升高,谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、肌酸激酶(CK)含量升高,尿素氮(BUN)濃度下降;對慢性中毒小鼠的臨床病理學(xué)檢查中發(fā)現(xiàn),以心、肝、肺、腎實(shí)質(zhì)器官的細(xì)胞變性壞死為主,尤其是以肝臟、腎臟的損傷最嚴(yán)重。7.披針葉黃華堿對奶牛子宮內(nèi)膜炎致病菌的研究本研究針對患病奶牛子宮中的病原菌進(jìn)行分離鑒定。結(jié)果顯示:所收集的病料中以感染大腸埃希氏菌和溶血性鏈球菌為主,以混合感染為主要感染方式。在披針葉黃華總堿、黃華堿、阿金亭堿對大腸埃希氏菌、溶血性鏈球菌、非溶血性鏈球菌、沙門氏菌的體外抑菌活性試驗(yàn)中發(fā)現(xiàn),3種生物堿均具有一定的抗菌活性,其中非溶血性鏈球菌最為敏感,其次為沙門氏菌、大腸埃希氏菌和溶血性鏈球菌。而金雀花堿則對以上4種病原菌無抗菌活性。8.披針葉黃華堿對小鼠免疫功能的研究本試驗(yàn)通過與對照組進(jìn)行對比試驗(yàn),觀察披針葉黃華堿對實(shí)驗(yàn)小鼠體重、臟器質(zhì)量、臟器指數(shù)、巨噬細(xì)胞吞噬力和T淋巴細(xì)胞數(shù)量的影響。結(jié)果發(fā)現(xiàn)在對實(shí)驗(yàn)小鼠灌服了低、中、高3個(gè)不同藥物劑量的披針葉黃華堿后,小鼠的體重、臟器質(zhì)量、臟器指數(shù)、巨噬細(xì)胞吞噬力以及T淋巴細(xì)胞的數(shù)量均有所下降。其中,低濃度的披針葉黃華堿對小鼠的細(xì)胞免疫功能的作用不明顯,而中高濃度的披針葉黃華堿對小鼠免疫功能還具有一定的抑制作用。9.披針葉黃華堿對細(xì)胞毒性的研究利用MTT比色法依次測定披針葉黃華總堿、金雀花堿、羥喜樹堿對乳倉鼠腎細(xì)胞(BHK)、人結(jié)腸癌細(xì)胞(SW480)的體外細(xì)胞毒性作用。結(jié)果顯示:披針葉黃華總堿抑制SW480的生長作用顯著,其抑制率隨被測藥液劑量不斷增加而逐漸增強(qiáng)。羥喜樹堿對BHK和SW480在各濃度梯度組中均有較強(qiáng)的抑制作用。披針葉黃華堿和金雀花堿對BHK細(xì)胞的毒性稍低,對SW480的生長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.

【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S859.87;S853.74

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