【作者】 陈建新；

【导师】 叶文才；

【作者基本信息】 暨南大学 ， 生物医学工程， 2010， 博士

【摘要】 本论文对半边莲全草的化学成分及其体内外抗流感病毒活性进行了研究。采用多种色谱分离手段,从半边莲中共分离得到37个化合物,综合运用NMR、HR-ESI-MS、CD等现代谱学技术和化学方法鉴定了其中32个化合物的结构,7个为新化合物。本论文还系统地评价了半边莲醇提物及其不同极性部位体内外抗流感病毒活性。此外,还对分离得到的哌啶生物碱类和木脂素类化合物的电子轰击电离(EI)质谱裂解规律进行了研究和总结。一、半边莲化学成分研究半边莲(Lobelia chinensis Lour.)为桔梗科(Campanulacea)半边莲属植物,为一年生矮小草本。半边莲全草具利尿消肿、清热解毒之功效,常用于治疗带状疱疹、急性肾炎、蛇咬伤等疾病。本课题在抗流感病毒中药筛选中发现,半边莲醇提物对感染流感小鼠有显著保护作用。为寻找其药效成分,本论文对半边莲的乙醇提取物进行了系统的化学研究。采用各种色谱技术分离得到37个化合物,并通过NMR、MS及CD等现代谱学技术和化学方法鉴定了其中32个化合物的结构。其中8个为哌啶类生物碱,分别为cis-2-(2-butanone)-6-(2-hydroxy butyl)-piperidine (LC-1), (2R,4R,6R,2"S)-N-methyl-4-hydroxyl-2-(2-butanone)-6-(2-hydroxybutyl)-piperidine (LC-2), rel-(2R, 4R,6S)-N-methyl-4-hydroxyl-2-(2-butanone)-6-(2-hydroxybutyl)-piperidine (LC-3), cis-N-methyl-2-(2-hydroxybutyl)-6-[2-[(1,3)-dioxolan-4-yl-methanol]-propyl]-piperidine (LC-4),trans-N-methyl-2,6-bis(2-hydroxybutyl)-△3-piperideine (LC-5),trans-8,10-diethyl-lobelidiol (LC-6), trans-10-ethyl-8-methyl-lobelidiol (LC-7), trans-8,10-diethyl-lobelionol (LC-8);11个为木脂素和新木脂素类化合物,分别为(2R,3S)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxy-methyl-7-methoxy-5-benzofuran propanoic acid ethyl ester ((+) LC-9), (2S,3R)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxy-methyl-7-methoxy-5-benzofuran propanoic acid ethyl ester ((-)LC-9), (2R,3S)-2,3-dihydro-2-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-5-benzofuran propanol acetate ((+)LC-10), (2S,3R)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-5-benzofuran propanol acetate ((-)LC-10), (+)-pinoresinol (LC-11), (+)-epipinoresinol (LC-12), (+)-medioresinol (LC-13), (-)-syringaresinol (LC-14), (-)-episyringaresinol (LC-15); 4个为香豆素类化合物,分别为6,7-二甲氧基香豆素(LC-16),6-羟基-5,7-二甲氧基香豆素(LC-17),5-羟基-7-甲氧基香豆素(LC-18),5-羟基-6,7-甲氧基香豆素(LC-19)；7个为黄酮类化合物,分别为3’-羟基芫花素(LC-20),芹菜素(LC-21),槲皮素(LC-22),木犀草素(LC-23),山柰酚(LC-24), luteolin 3’,4’-dimethylether-7-O-(3-D-glucoside (LC-25),蒙花苷(LC-26)；2个苯丙素类化合物,分别为异阿魏酸(LC-27),迷迭香酸乙酯(LC-28)；及1个长链多羟基酯类2,3,10-三羟基-4,9-二甲基-6(E)-十二烯二酸二乙酯(LC-29)和1个多炔类化合物lobetyolin (LC-30),以上化合物中,LC-1～LC-4, (+) LC-9, (-) LC-9及LC-29为7个新化合物,其它16个化合物为首次从半边莲属植物中分离得到。二、哌啶生物碱类和木脂素类化合物的EI电离质谱裂解规律研究本论文对从半边莲中分离获得的2,6-取代哌啶生物碱类、双四氢呋喃木脂素和二氢苯并呋喃木脂素类化合物的EI电离质谱裂解特征进行了研究,并得出如下规律：1.2,6-取代哌啶生物碱类化合物这类化合物的分子离子很不稳定,质谱中通常不显示分子离子。α断裂同时发生氢重排是其主要裂解方式。侧链取代基β碳原子上是否含羰基对这类分子的裂解有重要影响。如果分子侧链β碳原子上不含羰基,则生成N-甲基脱氢哌啶基离子(m／z 98)；如果分子中侧链β碳原子上含有羰基,则哌啶环上p碳原子上的H原子发生重排转移到羰基氧原子上,生成N-甲基氢化吡啶基特征离子(m／z 96)；对于环上含有双键的去氢哌啶生物碱或环上连接羟基的哌啶生物碱,如果侧链上p碳原子不含羰基,则生成m／z 96特征离子,如侧链p碳原子含有羰基,则生成特征的吡啶基(m／z 94)离子。2.双四氢呋喃木脂素和二氢苯并呋喃木脂素类化合物这类化合物有较稳定的分子离子峰。同时发生α断裂,造成呋喃环的裂开,随即再发生a断裂并伴随H转移,生成特征的m／z 151或m／z 181离子。该离子进一步电离,生成相应的子离子m／z 137或m／z 167。对于呋喃环上含有-CH2OH基团的苯并四氢呋喃类新木脂素,分子离子容易丢失一个H2O或CH2O分子,生成基峰离子[M-H2O]+和较高丰度的[M-CH2O]+离子,同时也发生四氢呋喃环开裂,生成m／z 151和m／z 137离子对。哌啶生物碱和木脂素类化合物EI电离质谱规律研究结果为利用质谱快速推测这两类化合物的结构提供了依据。三、半边莲醇提物及其不同极性部位体内外抗流感病毒作用研究本论文建立了用于评价药物抗流感病毒药效的MDCK细胞感染模型和BALB/c小鼠感染模型,系统地评价了半边莲醇提物及其乙酸乙酯萃取物、氯仿萃取物(总生物碱)、正丁醇萃取物和水溶物体内外抗流感病毒效果。结果表明,半边莲醇提物及其总生物碱对感染H9N2及H1N1亚型流感病毒小鼠有显著的保护作用。灌喂500 mg/kg/d的半边莲醇提物对感染H9N2及H1N1亚型流感病毒小鼠的肺指数抑制率分别39.7%和44.8%(p<0.01),对感染小鼠的生命保护率分别为70%和80%(p<0.01)；灌喂125 mg/kg/d的半边莲总生物碱对感染H9N2亚型流感病毒小鼠的肺指数抑制率为35.8%(p<0.05),生命保护率为60%(p<0.05),灌喂250mg/kg/d的半边莲总生物碱对感染H1N1亚型流感病毒小鼠的肺指数抑制率为39.3%(p<0.05),生命保护率为60%(p<0.05)。在MDCK细胞中,半边莲醇提物、总生物碱及其分离单体LC-8和盐酸洛贝林(Lobeline)对感染H9N2、H1N1和H5N1三种亚型流感病毒均有不同程度的抑制作用。由此推测生物碱是半边莲抗流感的活性成分组。但8种从半边莲中分离得到的哌啶生物碱对流感病毒神经氨酸酶(NA)没有抑制活性,表明NA不是其作用靶点。根据文献报道半边莲中分离的生物碱洛贝林具有显著的兴奋呼吸作用,本论文推测半边莲的抗流感作用可能与其生物碱的兴奋呼吸作用有关。更多还原

【Abstract】 The studies on chemical constituents and inhibitory activities against influenza viruses in vivo and in vitro of Lobelia chinensis (L. chinensis) had been carried out in the dissertation.37 compounds were isolated from the plant through various chromatographic methods. The chemical structures of 32 compounds were elucidated on the basis of NMR, HR-ESI-MS, CD analysis and other chemical techniques. Among them,7 compounds are novel compounds. The EI-MS fragmentation mechanisms of piperidine-type alkaloids, ditetrahydrofuran-type lignans and dihydrobenzofuran-type neolignans isolated from L. chinensis were presumed, and the fragmentation rules of the two type compounds were separately summarized. Furthermore, the inhibitory activities against influenza viruses in vivo and in vitro of ethanol extract and different fractions from L. chinensis were systematically evaluated.1. Studies on the chemical constituents of Lobelia chinensisLobelia chinensis is an annual plant and belongs to the genus Lobelia in the family Campanulacea. The plant has been known as a traditional Chinese medicine with functions of prompting diuresis and detumescence, clearing away heat and toxicity, and has been widely used in the treatment of herpes, acute nephritis, snake bite and other diseases. In our screening for anti-influenza activity of Chinese herbal medicines, the alcohol extract of L. chinensis showed a remarkable anti-influenza viral activity. This result inspired us to initiate a phytochemical study on the plant. A total of 37 compounds were isolated from the plant through various chromatographic methods. The chemical structures of 32 compounds were elucidated on the basis of NMR, HR-ESI-MS, CD analysis and other chemical techniques. Their structure were elucidated as cis-2-(2-butanone)-6-(2-hydroxy butyl)-piperidine (LC-1), (2R, 4R,6R,2"S)-N-methyl-4-hydroxyl-2-(2-butanone)-6-(2-hydroxybutyl)-piperidine (LC-2), rel-(2R, 4R,6S)-N-methyl-4-hydroxyl-2-(2-butanone)-6-(2-hydroxybutyl)-piperidine (LC-3), cis-N-methyl-2-(2-hydroxybutyl)-6-[2-[(1,3)-dioxolan-4-yl-methanol]-propyl]-piperidine (LC-4), trans-N-methyl-2,6-bis(2-hydroxy butyl)-A3-piperideine (LC-5), trans-8,10-diethyl-lobelidiol (LC-6), trans-10-ethyl-8-methyl-lobelidiol (LC-7), trans-8,10-diethyl-lobelionol (LC-8), (2R,3S)-2,3-dihydro-2-(4-hydroxy-3-methoxy phenyl)-3-hydroxy-methyl-7-methoxy-5-benzofuran propanoic acid ethyl ester ((+) LC-9), (2S,3R)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxy-methyl-7-methoxy-5-benzofuran propanoic acid ethyl ester ((-)LC-9), (2R,3S)-2,3-dihydro-2-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-5-benzofuran propanol acetate ((+)LC-10), (2S, 3R)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-5-benzofuran propanol acetate ((-)LC-10), (+)-pinoresinol (LC-11), (+)-epipinoresinol (LC-12), (+)-medioresinol (LC-13), (-)-syringaresinol (LC-14), (-)-episyringaresinol (LC-15),6, 7-dimethoxycoumarin (LC-16), fraxinol (LC-17),7-methoxy-5-hydroxycoumarin (LC-18), tomentin (LC-19),3’-hydroxy-genkwanin (LC-20), apigenin (LC-21), quercetin (LC-22), luteolin (LC-23), kaempferol (LC-24), luteolin 3’,4’-dimethyl ether-7-O-β-D-glucoside (LC-25), linarin (LC-26), isoferulic acid (LC-27), ethyl rosmarinate (LC-28),2,3,10-trihydroxy-4,9-dimethyl-dodec-6-enedioic acid diethyl ester (LC-29), lobetyolin (LC-30). Among them, LC-1～LC-4, (+) LC-9, (-) LC-9 and LC-29 are new compounds, and 16 compounds were isolated from the genus Lobelia for the first time.2. Studies on the c behavoir of piperidine-type alkaloids and lignans & neolignans using electron ionization (EI) mass spectrometry The fragmentation behavoir of 2,6-substitued piperidine-type alkaloids, ditetrahydro furan-type lignans and dihydrobenzofuran-type neolignans neolignans in EI mass spectrometry had been studied in this dissertation. The fragmentation rules were summarized as follows:2.1.2,6-Substitued piperidine-type alkaloidsPiperidine-type alkaloids usually did not show molecular ion peaks in the mass spectrum due to the instability of these compounds in EI-MS. The major fragment ions were produced by a cleavage and hydrogen rearrangement. It was found that the carbonyl group at theβ-C position of the side chain residue played an important effect on the fragmentation behavior of these compounds. For compounds without a carbonyl group atβ-C position of the side chain residue, N-methyl dehydropiperidine ion with m/z 98 was observed as the base peak in the mass spectrum. However, for compounds with a carbonyl group at P-C position of the side chain residue, N-methyl dihydropyridine ion with m/z 96 arised as the base peak in the mass spectrum due to hydrogen transfer from theβcarbon of the piperidine ring to theβcarbonyl group of the side chain residue. For compounds with an unsaturated bond or a hydroxyl at the piperidine ring, N-methyl dihydropyridine ion with m/z 96 was observed as the base peak in the mass spectrum of those compounds withoutβcarbonyl group in the side chain residue, and N-methyl pyridine ion with m/z 94 arised as the base peak in the mass spectrum of those compounds withβcarbonyl group in the side chain residue, respectively.2.2. Ditetrahydrofuran-type lignans and dihydrobenzofuran-type neolignansThe molecular ions could be steadily observed in the mass spectrum of these compounds. The characteristic ions of m/z 151 or m/z 181 were produced from the molecular ions by a cleavage and hydrogen rearrangement, and their daughter ions m/z 137 or m/z 167 could be produced from m/z 151 or m/z 181 by a cleavage, respectively. [M-H20]+ion (base peak) and [M-CH20]+ion (the second abundant ion) were observed in the spectrum of dihydrobenzofuran-type neolignans with a hydroxylmethyl group at the furan ring. The characteristic ions of m/z 151 and m/z 137 were also observed in neolignans due to the cleavage of the furan ring.The results of presumed fragmentation mechanisms of piperidine-type alkaloids and lignans & neolignans by El mass spectrometry could provide information to the structural investigation for these two types of compounds. 3. Studies on the inhibitory activities against influenza viruses of ethanol extract from L. chinensis and it’s fractions with different polarity in vivo and in vitroThe inhibitory activities against influenza viruses of ethanol extract from L. chinensis and it’s fractions extracted by ethyl acetate, chloroform, n-butanol and water soluble fraction had been systematically evaluated using MDCK model (in vitro) and BALB/c mice model (in vivo), respectively. Ethanol extract from L. chinensis and the fraction extracted by chloroform (alkaloids) showed significant protective effect on mice infected by the H9N2 and H1N1 influenza viruses. For ethanol extract from L. chinensis,500 mg/kg/day was the most effective dose with a 39.7% and 44.8%(p<0.01) lung index inhibition rate, and a 70% and 80% survival rate, respectively, after infection with the H9N2 and H1N1 viruses. For the fraction of total alkaloids, a 35.8%(p<0.05) lung index inhibition rate and 60% survival rate was achieved at the dose of 125 mg/kg/d after infection with the H9N2 viruses, and a 39.3%(p<0.05) lung index inhibition rate and 60% survival rate was achieved at the dose of 250 mg/kg/d after infection with the H1N1 viruses. Furthermore, Ethanol extract from L. chinensis, alkaloids fraction, LC-8 and lobeline showed considerable inhibitory activities against H9N2, H1N1, and H5N1 influenza viruses in MDCK cells. These results indicated that alkaloids were bioactive constituents of L. chinensis for inhibiting influenza viruses.8 piperidine-type alkaloids from L. chinensis failed to show inhibitory activities on neuramidinase (NA) of influenza viruese in vitro, and it indicated that NA was not the molecular target of alkaloids inhibiting viruses. It was reasonably presumed that the anti-influenza activity of L. chinensis in mice might be attributed to the respiratory excitation of piperidine-type alkaloids based on reported pharmacological action of lobeline on respiratory.更多还原