| 英文摘要 |
Objective: Fructus Liquidambaris was first published in supplement to "compendium of materia medica". It is the dried ripe fruit inflorescence of Liquidambar formosana Hance, a member of the hamamelis family. It possesses the effect of removing wind and activating collaterals, benefiting water and clearing collaterals. Modern pharmacological research shows that fructus liquidambaris has the effects of protecting liver, anti-tumor and anti-inflammatory. "Su wen" pointed out: "the role in the context, determine to live or die, cure a hundred diseases, regulate excess or deficiency, can not be impasability". Modern Chinese medicine believes that tumor is the result of the stagnation of qi and blood stasis in the meridians for a long time. The meridian system is closely related to the occurrence, development and treatment of cancer diseases and syndromes. Fructus Liquidambaris has porous body, belongs to liver kidney meridians, with the function of passing twelve sutras. The study of this topic is under the guidance of TCM theory “relieving rigidity of muscle and activating collaterals to cure cancer”. At the early stage, the pharmacological and pharmacodynamic effects of fructus liquidambaris on hepatic tumor were clarified through the pharmacological model of DEN induced hepatocellular carcinoma in rats. On the basis of the above research, the anti-liver tumor pharmacodynamic components or activc monomer components and the mechanism of action were clarified, which provided experimental support for revealing the pharmacodynamic material basis and treatment mechanism of fructus liquidambaris treating liver tumor, and also provided scientific basis for the treatment mechanism of relieving rigidity of muscle and activating collaterals to cure cancer. Method: 1. High performance liquid chromatography (HPLC) was used to establish the fingerprints of different polar parts and different producing areas of fructus liquidambaris. The in vitro pharmacodynamic evaluation method was used to evaluate the in vitro pharmacodynamic effects of different polar parts, different places of origin and different dosages. Two analytical methods, gray correlation analysis and partial least squares regression, were used to analyze the spectral activity relationship, and the fingerprint peak content in the fingerprint was correlated with the in vitro pharmacodynamic inhibition rate index, so as to obtain the contribution rate of the main pharmacodynamic monomer components in fructus liquidambaris against liver tumors. 2.Liquid-mass coupling (LC-MS) technique combined with reference comparison and literature retrieval was used to analyze the pharmacodynamic components of fructus liquidambaris. Fingerprint technique was used to construct the quality control method of fructus liquidambaris from different producing areas. At the same time, cluster analysis and principal component analysis were used to carry out the research on the relationship of relatives and genera of medicinal materials from different areas, and the comparative study between the content index and the pharmacodynamic index. 3.Methods as silica gel column chromatography, ODS column chromatography, Sephadex LH-20 gel column chromatography, analytical and preparative high performance liquid chromatography and preparative thin layer silica gel chromatography and so on were used to study the chemical constituents of the ethyl acetate layer of fructus liquidambaris systematically. 4.Flow cytometry and Annexin v-fitc /PI double staining and PI single staining were used to investigate the effects of pharmacodynamic components and monomer components in fructus liquidambaris on the cell cycle and apoptosis of SMMC-7721 human liver cancer cell. RT-PCR and western Blot technology were used to detect the relative expression levels of related genes and proteins in the apoptosis and migration pathways of hepatocellular carcinoma cells to explore the mechanism of action of fructus liquidambaris against liver tumor. 5.The serum pharmacochemical method and HPLC-QTOF-MS technique were used to investigate the blood components of ethyl acetate extract from fructus liquidambaris after administrated with the equivalent dose of 30 times and 10 times that of clinical humans on rats. DEN induced hepatocellular carcinoma model in rats was used to study the pharmacodynamics of anti-liver tumor in vivo of ethyl acetate extract, fructus liquidambaris oil and fructus liquidambaris acid taking organ indexes and pathological sections as indicators. HPLC-QTOF-MS technology and MPP analysis software were used to investigate the effects of ethyl acetate extract, fructus liquidambaris oil and fructus liquidambaris acid on the plasma differential metabolites and metabolic pathways in DEN indused hepatocellular carcinoma rats, to elucidate the mechanism from metabolomics. Results: 1. This study shows that different polarity substances of fructus liquidambaris all have certain inhibitory effect on tumor cells of 6 different organs, in which the effect on human liver cancer cell line SMMC-7721, human stomach cancer cell line HGC-27, human non-small cell lung cancer cell line A549 are better than the other three. The extracts of dichloromethane and ethyl acetate show more significant antitumor activity, and the extract of ethyl acetate is better. In the study of the spectral activity relationship based on the anti-tumor activity of different polarity components, more active components were 5, 6, 8, 12, 13, 14, 18, 19 and 20, a total of 9 chromatographic peaks. In the study of the spectral activity relationship based on the anti-tumor activity of different origins, more active components were 1, 2, 3, 4, 12, 14, 16, 19, 21, 25, 26 and 27, a total of 12 chromatographic peaks. 2.In this study, a total of 17 chemical components were identified by means of standard substancc comparison, Metlin and other related database query and mass spectrometry analysis, respectively No. 4-Phellopterin, No. 6-Imperatorin, No. 13-Sesquichamaenol, No. 14-2-hydroxy-3-oxo-l , 4(5)-oleanadien-28-oic acid, No. 15-2α, 3β-dihydroxy -23-norestolate -4 (24) -12 (13) -diene-28 - carboxylic acid, No. 16-Lantanoic acid, No. 17-Camarolide, No. 18-Arjunolic acid, No. 19-6β-hydroxyl-3-oxolup-20(29)-en-28-oic acid, No. 20-3, 4-seco-olean-12-ene-3, 28-dioic acid, No. 21-3-oxo-11α, 12α-epoxy-oleanan-28, 13β-olide, No. 22-Betulinic acid, No. 23-3 , 6-dion-20(29)-lupen-28-oic acid, No. 24-3-oxoursoleana-9(11) , 12-dien-28-oic acid, No. 25-Betulonic acid, No. 26-3-oxo-oleana-12-en-28-oic acid, No. 27-3-oxo-ursolic acid. 3.Cluster analysis divided the 11 fructus liquidambaris from different places into four categories, among which baoding and jiangsu zhu wenzhong Chinese medicine for a class, hebei dingchen for a class, jiangsu minle qinhuangdao and jiangsu chengda radius for a class, the others for a class. Principal component analysis (PCA) was used to screen the components with different contents in 11 different producing areas. A total of 12 peaks (3, 4, 7, 11, 12, 14, 15, 17, 19, 20, 21, 22, 24, 27) were screened out by using the content as indicators. A total of 17 peaks (1, 2, 4, 5, 6, 8, 12, 13, 16, 17, 18, 19, 20, 21, 24, 25, 26) were screened out by using the anti-tumor active ingredients of fructus liquidambaris as indicators. Compared with them there are 7 identical peaks. 4.A total of 31 known compounds were isolated from the ethyl acetate extract of fructus liquidambaris, respectively ursolic acid, 3-carbonyl ursolic acid, betulinic acid, 3,6-dion-20 (29) -lupen-28-oic acid, 3-oxoursoleana-9 (11 ) , 12-dien-28-oic acid, sesquichamaenol, 5, 7,4’-trimethoxy flavane-3-ol, 3-oxo-11α, 12α-epoxy-oleanan-28, 13β-olide, 3-oxo-11, 13 (18)-oleanadien-28-oic acid, 4-hydroxy-4, 7-dimethyl-1-tetralone, imperatorin, phellopterin, 2-hydroxy-3-oxo-l,4 (5) -oleanadien-28-oic acid, 3α-hydroxy-llα, 12α-epoxyoleanan-28, 13β-olide, camarolide, 6β-hydroxy-3-oxolup-20 ( 29 ) -en-28-oic acid, melliferone, 3 , 4-seco-ursan-4 (23) , 12-dien-3, 28-dioic acid, oleanolic acid, 3, 4-seco-olean-12-ene-3, 28-dioic acid, 3, 4-seco-ursan-12-ene-3 , 28-dioic acid, 3-oxo-12α-hydroxyoleanan-28 , 13β-olide, 3, 4-seco-olean-4 (23) , 12-dien-3, 28-dioic acid, falcarindiol, betulonic acid, 3-oxo-oleana-12-en-28-oic acid, arjunolic acid, eucalyptanoic acid, 23-trans-p-coumaroyloxy-2α ,3β-dihydroxyolean-12-en-28-oic acid, 3-oxo-28-norolean-12-en and lantanoic acid. 5.By flow cytometry technique findout that ethyl acetate extract and monomer composition of fructus liquidambaris possess certain effect of inducing SMMC-7721 cells in human liver cancer apoptosis. The cell cycle changed after the stimulation of drug administration, and it showed different degrees of S phase and G2/M phase arrest. Therefore, it can delay the division cycle of tumor cells and inhibit the proliferation of liver cancer SMMC-7721 cells. 6.Each monomer component and ethyl acetate extract of fructus liquidambaris can regulate the expression of 10 genes including PTEN, PI3K, AKT, PDCD4, VEGFA, Caspase 9, Caspase 3, Bcl-2, Bax and Bad, and 7 proteins including PTEN, PI3K, p-PI3K, AKT, p-AKT, Caspase 9 and VEGFA in smmc-7721 cells of human liver cancer. The relative expression levels of PTEN, PDCD4, Caspase 9, Caspase 3, Bax and Bad increased, while the relative expression levels of PI3K, p-PI3K, AKT, p-AKT, VEGFA and Bcl-2 decreased. However, the regulatory effects of ethyl acetate extract and different monomer components were significantly different. 7.Eight prototype blood components were obtained from the plasma chemical composition analysis of rats in the high dose group and the blank group, in which 6β-hydroxyl-3-oxolup-20 (29) -en-28-oic acid, Sesquichamaenol and 3-carbonyl-ursolic acid were determined. Imperatorin, Camarolide/3-oxoursoleana-9 ( 11 ) , 12-dien-28-oic acid, Phellopterin, 2-hydroxy-3-oxo-1, 4 (5) -oleanadien-28-oic acid and 2α, 3β-dihydroxy-23-norestolate -4 (24) -12 (13) -diene-28 - carboxylic acid were speculated. Eleven prototype blood components were obtained from the plasma chemical composition analysis of rats in the low dose group and the blank group, in which Lantanoic acid, Arjunolic acid, 6β-hydroxyl-3-oxolup-20(29 )-en-28-oic acid, 3-oxo-11α, 12α-epoxy-oleanan-28, 13β-olide, 3 , 6-dion-20 ( 29 ) -lupen-28-oic acid, Sesquichamaenol and 3-carbonyl-ursolic acid were determined. Imperatorin, Phellopterin, 2-hydroxy-3-oxo-1 , 4 (5) -oleanadien-28-oic acid and 2α, 3β-dihydroxy -23- norestolate -4 (24) -12 (13) -diene-28 - carboxylic acid were speculated. 8.The in vivo efficacy results based on DEN-induced hepatocellular carcinoma model in rats showed that, the liver tissue of blank group was ruddy and smooth with soft texture, the model group were densely covered with nodules, the diameter of some tumor exceeded 1㎜. Compared with the model group, the liver tissues of ethyl acetate extract of fructus liquidambaris group, fructus liquidambaris oil group and fructus liquidambaris acid group were varying degrees of improvement. The spleen index decreased in different degrees and thymus index ascended. 9.HPLC-QTOF-MS technology and database identification or standard substance comparative method were used to explore changes of endogenous metabolites in rat plasma after the intervention of ethyl acetate extract of fructus liquidambaris, fructus liquidambaris oil and fructus liquidambaris acid. A total of 78 potential biomarkers were identified, such as LysoPC ( 14:0/0:0 ) , Eicosapentaenoic acid and sphingosine-1-phosphate etc. It was speculated that the changes of metabolites in DEN-induced hepatocellular carcinoma rats were mainly related to lipid metabolism, unsaturated fatty acid metabolism, phosphosphenol metabolism, arachidonic acid metabolism, acylcamitine metabolism, amino acid metabolism and other pathways. Conclusion: 1. Spectrum efficiency technology combined with multi-dimensional statistical analysis method were used to establish the fingerprints of different polar parts, different producing areas and different doses of fructus liquidambaris, based on which gray correlation analysis and partial least squares regression methods were used to analyze monomer components with strong contribution to the efficacy. A set of new methods for screening, evaluation and verification of medicinal substances in traditional Chinese medicine and compound prescription were formed. This method is reasonable and scientific, can provide new ideas for screening medicinal substances of traditional Chinese medicine and compound prescription. 2.A total of 17 chemical ingredients were identified by means of mass spectrometry analysis and standard substance comparison, in which 10 were major mcdicinal ingredients. Cluster analysis and principal component analysis methods were used to establish the quality control method for the fingerprint of fructus liquidambaris. Meanwhile the comparative study of chemical index components and pharmacodynamic index components was carried out, which provide a basis for the rationality of the research based on the screening of pharmacodynamic index components. 3.By means of silica gel column chromatography and other means of analysis and separation, the chemical constituents of ethyl acetate extract of fructus liquidambaris were systematically studied. A total of 31 known compounds were identified, in which 18 compounds including falcarindiol, eucalyptanoic acid, 23-trans-p-coumaroyloxy-2α,3β-dihydroxyolean-12-en-28-oic acid and so on were first isolated from liquidambar. 4.Flow cytometry combined with related gene and protein detection methods was used to study the mechanism of action of pharmacodynamic components and monomer components on cells. The ethyl acetate extract and 6 monomer components of fructus liquidambaris can induce the early apoptosis of liver cancer cells to different degrees. Its function is achieved by regulating different stages of cell mitosis, and the mechanism of action is to regulate the PI3K-AKT/Caspase 9 and PI3K-AKT/VEGF signaling pathways, increase the expression of PTEN, reduce the expression of PI3K, p-PI3K, AKT, p-AKT, Bcl-2, Bax and Bad to inhibit the growth and division of tumor and promote the apoptosis of tumor 5.Serum pharmacochemistry method was used to investigate the blood transfusion components of high and low dose groups of ethyl acetate extract from fructus liquidambaris. A total of 11 prototype blood transfusion components were found, among which Camarolide/3-oxoursoleana-9 (11) and 12-dien-28-oic acid were the specific components of the high dose group. The 4 components of Lantanoic acid, Arjunolic acid, 3-oxo-11α, 12α-epoxy-oleanan-28, 13β-olide, 3, 6-didion -20 (29) -lupen-28-oic acid were the special components of low dose group. 6.DEN-induced hepatocellular carcinoma model in rats is adopted to carry out the pharmacological efficacy study of ethyl acetate extract, fructus liquidambaris oil, fructus liquidambaris acid resisting liver tumors. The results show that all of the above material have excellent therapeutic effect on liver tumors, and ethyl acetate extract and ethyl acetate extract not only have the effect of killing tumor, but also possess the function of the liver repair. Metabonomics method are adopted to explore the changes of metabolites in rats plasma and possible metabolic pathways after the intervention of ethyl acetate extract, fructus liquidambaris oil and fructus liquidambaris acid in DEN-induced hepatocellular carcinoma rats. The results showed that the metabolites and metabolic pathways in rats regulated by ethyl acetate extract, fructus liquidambaris oil and fructus liquidambaris acid were different, which reflects the characteristics of multi-target and multi-path regulation and provides targets for the study of the active part and the mechanism of the monomer component of fructus liquidambaris, and provides a great deal of experimental basis for the development of new anti-liver cancer drugs of fructus liquidambaris. Key words: Liquidambar formosana fruits; liver tumors; spectrum-effect relationship; pharmacodynamic material basis; mechanism of action
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