| 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.
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