Chemical Constituents And Biological Activities Of Liuyueqing

Liuyueqing (LYQ) belongs to the family Acanthaceae, which has been used in Chinese traditional medicine with a long history. The whole herb of this plant can be used as a medicine to treat various diseases including hypertension, acute or chronic hepatitis and jaundice. Early researches showed that the extractives from LYQ had the positive effects on scavenging free radical, anti-hepatic injury, anti-hepatic fibrosis, anti-HBV and anti-tumor. The results show that the main constituents were saponins, lactones, hydroxybenzene, organic acids, tannis, glucides, glycosides etc. Howerver, until now, the specific active component in LYQ was unknown. This study was designed to separate and purify the LYQ chemical constituents and identify their molecular structures. Anti-oxidation, anti-tumor, anti-HBV and anti-hepatic fibrosis of LYQ monomers were investigated. This paper estabilished a experimental basis for investigation of LYQ monomers founction and mechanism. And provided the theoretical basis for the development of new drugs.Part Ⅰ. Chemical constituents from LiuyueqingObjective:To study the the chemical constitutents of Liuyueqing. Method:Compounds were isolated by chromatography method, and their structrues were identified by NMR and MS analyses. Result:Twenty compounds were isolated and identified as taraffinisoside A (1), descaffeoyl crenatoside (2), deacyl martynoslde (3),3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→>)]-β-D-glucopyranoside (4), hydrangeifolin Ⅰ(5),(—)-syringaresinol4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (6), hydrolytichazaleanin A (7),(6R,7S,8S)-7α-[(β-glucopyranosyl)oxy]lyoniresinol (8)(6S,7R,8R)-7a-[(β-glucopyranosyl)oxy] lyoniresinol (9),(-)-4-epi-lyoniresinol3α-O-β-D-glucopyranoside (10), and (—)-lyoniresinol2a-O-β-D-glucopyranoside (11), betulin (12), daucosterol (13), p-hydroxybenzaldehyde (14),4’-hydroxyacetophenone (15), salicylic acid (16), gaidic acid(17), linoleic acid(18),9,12,15-octadecatrienoic acid(19). Compound20can not be identified at this moment and thus need further identification. Conclusion:The present paper, for the first time, describes the isolation and structure elucidation of compound1by detailed analysis of their NMR spectra. Compounds2-19were isolated for the first time from this plant.Part Ⅱ. Antioxidative effect of chemical constituents from LiuyueqingObjective:To study the antioxidant activity of eight chemical constituents from Liuyueqing, including taraffinisoside A (1), descaffeoyl crenatoside (2), deacyl martynoslde (3),3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside (4), hydrangeifolin I (5),(—)-syringaresinol4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (6),(6R,7S,8S)-7a-[(β-glucopyranosyl)oxy]lyoniresinol(8) and betulin(12). Methods: With microtitration assay and UV-Vis spectrophotometry, effect of eight chemical constituents from Liuyueqing on clearance rate of DPPH· hydroxyl radical·OH and superoxide anion O2-·were determined with Vc as positive control. Results: Components1,2,3,4,6,8have scavenging capability against DPPH·, IC50values were10.36μM,19.73μM,43.95μM,15.30μM,46.04μM,35.59μM, and have a good dose-responses with the concentration. Components1,2,3,4,6,8have scavenging capability against OH, IC50values were17.61μM,20.65μM,36.16μM,29.38μM,41.26μM,31.23μM, and have a good dose-responses with the concentration. Components1,2,3,4,6,8have scavenging capability against O2-·, IC50values were19.68μM,20.61μM,24.39μM,19.71μM,47.84μM,31.20μM, and have a good dose-responses with the concentration. Components5and12have no scavenging capability against DPPH·,·OH and O2-·. Conclusion:A series of chemical constituents including compounds1、2、3、4、6and8from Liuyueqing have the stronger antioxidant activities in viro.Part Ⅲ. Anti-tumor effect of chemical constituents from LiuyueqingObjective:To study the anti-tumor activity of eight chemical constituents from Liuyueqing, including taraffinisoside A (1), descaffeoyl crenatoside (2), deacyl martynoslde (3),3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside (4), hydrangeifolin Ⅰ(5),(-)-syringaresinol4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (6),(6R,7S,8S)-7a-[(β-glucopyranosyl)oxy]lyoniresinol (8) and betulin (12) Methods:With DDP as positive control, eight chemical constituents from Liuyueqing were evaluated the antiproliferative activity by MTT assay on five human cancer cell lines, such as nasopharyngeal carcinoma cell (CNE2), hepatoma carcinoma cell,(HepG2), lung carcinoma cell (A549), tongue carcinoma cell (Tca8113), and cervical cell (Hela). Results:Betulin has significant activity against five human cancer cell lines, such as nasopharyngeal carcinoma cell (CNE2), hepatoma carcinoma cell,(HepG2), lung carcinoma cell (A549), tongue carcinoma cell (Tca8113), and cervical cell (Hela). IC50values were15.73μM,15.14μM,21.68μM,13.36μM,28.15μM and have a good dose-responses with the concentration. Their morphological characteristics were changed significantly. Components1,2,3,4,5,6,8have no significant activity against the five human cancer cell lines. Conclusion:Betulin from Liuyueqing has significant activity against five human cancer cell lines.Part Ⅳ. The effect of chemical constituents from Liuyueqing on HSC-T6cellsObjective:To study the anti-hepatic fibrosis effect of eight chemical constituents from Liuyueqing, including taraffinisoside A (1), descaffeoyl crenatoside (2), deacyl martynoslde (3),3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside (4), hydrangeifolin (5)1,(—)-syringaresinol4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (6)(6R,7S,8S)-7α-[(β-glucopyranosyl)oxy]lyoniresinol(8) and betulin(12). Methods: HSC-T6cell line was used as cell model. MTT assay was adopt to evaluate the inhibitory effect of chemical constituents from Liuyueqing. MTT assay was adopt to evaluate the inhibitory effect of active monomers on BRL cell line. HSC-T6cell apoptosis was detected by Hoechst33258staining. HSC-T6cell cycle was detected by flow cytometry with PI staining. Results:Betulin concentration-dependently attenuated HSC-T6cell proliferative activity. However, betulin caused little affections on BRL cell. Betulin induced apoptosis and blocked cell cycle progression at S. We determined if betulin regulates apoptosis. Hoechst33258staining showed that betulin was capable of inducing apoptosis in HSC-T6cells, which was characterized by features of apoptotic nuclei such as nuclear shrinkage and chromatin condensation. Conclusion:Betulin from Liuyueqing have the anti-hepatic fibrosis effect in viro.Part V. Anti-HBV effect of chemical constituents from LiuyueqingObjective:To study the anti-HBV effect of seven chemical constituents from Liuyueqing, including taraffinisoside A (1), descaffeoyl crenatoside (2), deacyl martynoslde (3),3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside (4), hydrangeifolin (5) I,(—)-syringaresinol4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (6) and (6R,7S,8S)-7α-[(β-glucopyranosyl)oxy]lyoniresinol(8). Methods:HepG2.2.15cell line was used as cell model. MTT assay was adopt to evaluate the toxicity of chemical constituents from Liuyueqing. The HBsAg and HBeAg in supernate were detected by ELISA. The HBV DNA in supernate was detected by real-time fluorescent quantitative PCR. Results:Compound1,2and4showed potent HBsAg, HBeAg and HBV DNA inhibitory activity in a dose-dependent manner. The time-dependent manner was also showed by Compound1and2. The IC50values of the treatment on HBsAg for9days were0.50,0.72and0.26mM. The IC5o values of the treatment on HBeAg for9days were0.93,0.42and0.07mM. The inhibitive rates HBV DNA were94.23%,96.75%and99.47%with the maximum concentration. Conclusion:The results suggest that taraffinisoside (1), descaffeoyl crenatoside (2) and3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside (4) isolated form Tarphochlamys affinis have inhibory effect on hepatitis B virus.In summary, a new phenylethanoid glycoside taraffinisoside A (1), together with eighteen known chemical constituents from Liuyueqing were obtained. The results showed a series of LYQ monomers have antioxidant, anti-tumor anti anti-HBV or anti-hepatic fibrosis activites in vitro.