六月青化学成分及生物活性研究
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摘要
六月青是广西壮族的特色药材,系爵床科(Acanthaceae)植物,又叫肖鸡笼或顶头马兰[Tarphochlamys affinis (Griff) Bremekhu]或rStrobilanthes affinis (griff) Y. C. Tang]的干燥地上部分,具有广泛的保肝抗肝炎等药理活性,目前国内外只有本课题组对其进行了长期深入的研究。研究证实,六月青提取物具有较强的抗氧化、抗肝损伤、抗肝纤维化、抗乙肝以及抗肿瘤等作用。由系统预分析实验可知,六月青中含有皂苷、内酯类、酚类、有机酸、鞣质类及糖和苷类等成分。随着课题的深入,六月青具体含有哪些有效的活性单体成分有待于我们进一步研究。本实验在前期研究的基础上,对六月青化学成分进行提取、分离、纯化和结构鉴定,并对六月青化学成分进行了体外清除氧自由基、抗肿瘤、抗乙肝以及对肝星状细胞影响的研究,以筛选六月青有效的活性成分,为进一步研究六月青单体生物活性及作用机制奠定基础,并为开发广西新药提供理论依据。
第一部分六月青化学成分的研究
目的：研究六月青的化学成分。方法：采用色谱技术分离纯化,并通过波谱技术及理化性质鉴定化合物结构。结果：从六月青中分离得到20个化合物,分别为：taraffinisoside A (1), descaffeoyl crenatoside (2), deacyl martynoslde (3),3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3glucopyranoside(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-lyoniresinol3a-O-β-D-glucopyranoside (10),(—)-lyoniresinol2a-O-β-D-glucopyranoside (11),桦木醇(12),β-胡萝卜苷(13),对羟基苯甲醛(14),对羟基苯乙酮(15),水杨酸(16),十六碳烯酸(17),亚油酸(18),9,12,15-十八碳三烯酸(19),化合物20结构待定。结论：化合物1为新化合物,其它化合物均为首次从该植物中分离得到。
第二部分六月青化学成分抗氧化活性研究
目的：研究六月青8种化学成分——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)-7α-[(β-glucopyranosyl)oxy]lyoniresinol (8)和桦木醇(12)的抗氧化活性。方法：采用酶标仪微量法和紫外分光光度法,以维生素C为阳性对照,分别测定六月青8种化学成分对1,1-二苯基-2-三硝基苯肼(DPPH·)自由基、羟基自由基(·OH)和超氧阴离子自由基(02-·)的清除能力。结果：化合物1、2、3、4、6、8对DPPH表现出明显的清除作用,半数清除率IC50分别为10.36pM,19.73μM,43.95μM,15.30μM,46.04μM和35.59μM,且成明显的量效关系。化合物1、2、3、4、6、8对OH自由基表现出明显的清除作用,半数清除率IC50分别为17.61μM,20.65μM,36.16μM,29.38μM,41.26pM和31.23μM,且成明显的量效关系。化合物1、2、3、4、6、8对超氧阴离子自由基表现出明显的清除作用,IC50分别为19.68μM,20.61μM,24.39μM,19.71μM,47.84μM和31.20μM,且成明显的量效关系。化合物5和12对三种自由基均未表现出清除作用。结论：六月青含有一系列体外抗氧化活性成分,包括化合物1、2、3、4、6、8。
第三部分六月青化学成分体外抗肿瘤活性研究
目的：研究六月青8种化学成分———taraffinisoside A (1)、descaffeoyl crenatoside (2)、deacylmartynoslde (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)-7α-[(β-glucopyranosyl)oxy]lyoniresinol(8)和桦木醇(12)的体外抗肿瘤活性。方法：采用体外细胞培养技术,以顺铂DDP为阳性对照药,用MTT法分别测定六月青8种化学成分对人鼻咽癌细胞株CNE2、人肝癌细胞株HepG2、人舌癌细胞株Tca8113人肺癌细胞株A549和人宫颈癌细胞株Hela的增殖抑制作用。结果：化合物桦木醇(12)对人鼻咽癌细胞株CNE2、人肝癌细胞株HepG2、人舌癌细胞株Tca8113、人肺癌细胞株A549和人宫颈癌细胞株Hela的增殖表现出明显抑制作用,半数抑制率IC50分别为15.73μM,15.14μM,21.68μM,13.36μM和28.15μM,且成明显的量效关系。桦木醇作用于肿瘤细胞后,细胞形态发生明显改变。化合物1、2、3、4、5、6、8对5种肿瘤细胞的增殖均未表现出明显的抑制作用。结论：六月青一种三萜类化合物桦木醇对肿瘤细胞有明显的抑制活性。
第四部分六月青化学成分对肝星状细胞的影响
目的：研究六月青8种化学成分——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)-7α-[(β-glucopyranosyl)oxy]lyoniresinol (8)和桦木醇(12)的体外抗肝纤维化活性。方法：建立HSC-T6细胞模型,以秋水仙素为阳性对照药,用MTT法从六月青8种化学成分中筛选出具有抑制HSC-T6细胞作用的单体；采用MTT法检测活性单体对大鼠肝正常细胞(BRL)毒性；采用Hoechst33258荧光染色法检测HSC-T6细胞凋亡；采用流式细胞仪PI单染法检测HSC-T6细胞周期。结果：化合物(12)桦木醇对HSC-T6细胞的增殖表现出明显抑制作用,且成明显的量效关系,但对BRL细胞毒性较低；桦木醇能够诱导HSC-T6细胞凋亡并且造成细胞周期S期阻滞；Hoechst33258染色后可以观察到HSC-T6细胞核体积缩小,染色质浓缩,凋亡细胞数随药物浓度增大而增多。结论：六月青一种三萜类化合物桦木醇具有体外抗肝纤维化活性。
第五部分六月青化学成分体外抗乙肝病毒研究
目的：研究六月青7种化学成分——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)、(—)-syringaresinol4-0-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (6)和(6R,7S,8S)-7α-[(β-glucopyranosyl)oxy]lyoniresinol (8)的体外抗乙肝活性。方法：以HepG2.2.15细胞株为模型,采用MTT法检测六月青化学成分对HepG2.2.15细胞的细胞毒作用；用酶联免疫吸附法(ELISA)检测HepG2.2.15细胞上清液乙肝表面抗原(HBsAg)和乙肝e抗原(HBeAg)表达水平。采用实时荧光定量PCR法检测细胞上清液HBV DNA的含量。结果：化合物taraffinisoside (1), descaffeoyl crenatoside (2)和3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside(4)具有良好的体外抑制HBsAg、HBeAg和HBVDNA分泌的活性。抑制作用随药物浓度的增加而逐渐增强。其中化合物1和2对HBsAg和HBeAg的抑制作用呈明显的时间依赖性。药物干预9天后,化合物1、2和4对细胞分泌HBsAg的IC50分别为0.50、0.72和0.26mM,对细胞分泌HBeAg的IC50分别为0.93、0.42和0.07mM。随着化合物1、2和4浓度的增大,对细胞上清HBV DNA的抑制作用逐渐增强,最大浓度抑制率分别达到94.23%、96.75%和99.47%。结论：通过体外抗乙肝病毒筛选研究证实,六月青化学成分taraffinisoside (1), descaffeoyl crenatoside (2)和3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside (4)具有一定的体外抗HBV作用。
综上所述,我们从六月青中分离得到1个新的苯乙醇苷化合物和18个已知化合物。通过体外实验筛选出一系列具有抗氧化,抗肿瘤,抗肝纤维化和抗乙肝病毒的活性单体成分。
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 Liuyueqing
Objective: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 Liuyueqing
Objective: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 Liuyueqing
Objective: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-T6cells
Objective: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 Liuyueqing
Objective: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.
第一部分六月青化学成分的研究
目的：研究六月青的化学成分。方法：采用色谱技术分离纯化,并通过波谱技术及理化性质鉴定化合物结构。结果：从六月青中分离得到20个化合物,分别为：taraffinisoside A (1), descaffeoyl crenatoside (2), deacyl martynoslde (3),3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3glucopyranoside(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-lyoniresinol3a-O-β-D-glucopyranoside (10),(—)-lyoniresinol2a-O-β-D-glucopyranoside (11),桦木醇(12),β-胡萝卜苷(13),对羟基苯甲醛(14),对羟基苯乙酮(15),水杨酸(16),十六碳烯酸(17),亚油酸(18),9,12,15-十八碳三烯酸(19),化合物20结构待定。结论：化合物1为新化合物,其它化合物均为首次从该植物中分离得到。
第二部分六月青化学成分抗氧化活性研究
目的：研究六月青8种化学成分——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)-7α-[(β-glucopyranosyl)oxy]lyoniresinol (8)和桦木醇(12)的抗氧化活性。方法：采用酶标仪微量法和紫外分光光度法,以维生素C为阳性对照,分别测定六月青8种化学成分对1,1-二苯基-2-三硝基苯肼(DPPH·)自由基、羟基自由基(·OH)和超氧阴离子自由基(02-·)的清除能力。结果：化合物1、2、3、4、6、8对DPPH表现出明显的清除作用,半数清除率IC50分别为10.36pM,19.73μM,43.95μM,15.30μM,46.04μM和35.59μM,且成明显的量效关系。化合物1、2、3、4、6、8对OH自由基表现出明显的清除作用,半数清除率IC50分别为17.61μM,20.65μM,36.16μM,29.38μM,41.26pM和31.23μM,且成明显的量效关系。化合物1、2、3、4、6、8对超氧阴离子自由基表现出明显的清除作用,IC50分别为19.68μM,20.61μM,24.39μM,19.71μM,47.84μM和31.20μM,且成明显的量效关系。化合物5和12对三种自由基均未表现出清除作用。结论：六月青含有一系列体外抗氧化活性成分,包括化合物1、2、3、4、6、8。
第三部分六月青化学成分体外抗肿瘤活性研究
目的：研究六月青8种化学成分———taraffinisoside A (1)、descaffeoyl crenatoside (2)、deacylmartynoslde (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)-7α-[(β-glucopyranosyl)oxy]lyoniresinol(8)和桦木醇(12)的体外抗肿瘤活性。方法：采用体外细胞培养技术,以顺铂DDP为阳性对照药,用MTT法分别测定六月青8种化学成分对人鼻咽癌细胞株CNE2、人肝癌细胞株HepG2、人舌癌细胞株Tca8113人肺癌细胞株A549和人宫颈癌细胞株Hela的增殖抑制作用。结果：化合物桦木醇(12)对人鼻咽癌细胞株CNE2、人肝癌细胞株HepG2、人舌癌细胞株Tca8113、人肺癌细胞株A549和人宫颈癌细胞株Hela的增殖表现出明显抑制作用,半数抑制率IC50分别为15.73μM,15.14μM,21.68μM,13.36μM和28.15μM,且成明显的量效关系。桦木醇作用于肿瘤细胞后,细胞形态发生明显改变。化合物1、2、3、4、5、6、8对5种肿瘤细胞的增殖均未表现出明显的抑制作用。结论：六月青一种三萜类化合物桦木醇对肿瘤细胞有明显的抑制活性。
第四部分六月青化学成分对肝星状细胞的影响
目的：研究六月青8种化学成分——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)-7α-[(β-glucopyranosyl)oxy]lyoniresinol (8)和桦木醇(12)的体外抗肝纤维化活性。方法：建立HSC-T6细胞模型,以秋水仙素为阳性对照药,用MTT法从六月青8种化学成分中筛选出具有抑制HSC-T6细胞作用的单体；采用MTT法检测活性单体对大鼠肝正常细胞(BRL)毒性；采用Hoechst33258荧光染色法检测HSC-T6细胞凋亡；采用流式细胞仪PI单染法检测HSC-T6细胞周期。结果：化合物(12)桦木醇对HSC-T6细胞的增殖表现出明显抑制作用,且成明显的量效关系,但对BRL细胞毒性较低；桦木醇能够诱导HSC-T6细胞凋亡并且造成细胞周期S期阻滞；Hoechst33258染色后可以观察到HSC-T6细胞核体积缩小,染色质浓缩,凋亡细胞数随药物浓度增大而增多。结论：六月青一种三萜类化合物桦木醇具有体外抗肝纤维化活性。
第五部分六月青化学成分体外抗乙肝病毒研究
目的：研究六月青7种化学成分——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)、(—)-syringaresinol4-0-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (6)和(6R,7S,8S)-7α-[(β-glucopyranosyl)oxy]lyoniresinol (8)的体外抗乙肝活性。方法：以HepG2.2.15细胞株为模型,采用MTT法检测六月青化学成分对HepG2.2.15细胞的细胞毒作用；用酶联免疫吸附法(ELISA)检测HepG2.2.15细胞上清液乙肝表面抗原(HBsAg)和乙肝e抗原(HBeAg)表达水平。采用实时荧光定量PCR法检测细胞上清液HBV DNA的含量。结果：化合物taraffinisoside (1), descaffeoyl crenatoside (2)和3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside(4)具有良好的体外抑制HBsAg、HBeAg和HBVDNA分泌的活性。抑制作用随药物浓度的增加而逐渐增强。其中化合物1和2对HBsAg和HBeAg的抑制作用呈明显的时间依赖性。药物干预9天后,化合物1、2和4对细胞分泌HBsAg的IC50分别为0.50、0.72和0.26mM,对细胞分泌HBeAg的IC50分别为0.93、0.42和0.07mM。随着化合物1、2和4浓度的增大,对细胞上清HBV DNA的抑制作用逐渐增强,最大浓度抑制率分别达到94.23%、96.75%和99.47%。结论：通过体外抗乙肝病毒筛选研究证实,六月青化学成分taraffinisoside (1), descaffeoyl crenatoside (2)和3,4-dihydroxyphenylethanol-8-O-[β-D-apiofuranosyl (1→3)]-β-D-glucopyranoside (4)具有一定的体外抗HBV作用。
综上所述,我们从六月青中分离得到1个新的苯乙醇苷化合物和18个已知化合物。通过体外实验筛选出一系列具有抗氧化,抗肿瘤,抗肝纤维化和抗乙肝病毒的活性单体成分。
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 Liuyueqing
Objective: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 Liuyueqing
Objective: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 Liuyueqing
Objective: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-T6cells
Objective: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 Liuyueqing
Objective: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.
引文
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