翠绿针毛蕨化学成分及其抗肿瘤活性的研究
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摘要
翠绿针毛蕨Macrothelypteris viridifrons (Tagawa) Ching是金星蕨科针毛蕨属植物,广泛分布于长江流域以南各省区,具有清热解毒,利尿,止血等功效。目前,国内外有关其化学成分和生物活性的报道很少,为了开发翠绿针毛蕨的药用价值和研究其生物活性,我们对翠绿针毛蕨总黄酮活性部位的纯化工艺、化学成分及抗肿瘤作用进行了研究,同时,对新化合物DHEC抗肿瘤作用机制进行了探讨,并建立了翠绿针毛蕨药材的质量标准。
在对总黄酮纯化工艺的研究中,采用碱溶酸沉法结合大孔树脂纯化法,建立了翠绿针毛蕨总黄酮的纯化工艺,最终得到转移率不低于59.52%,纯度不低于54.85%的翠绿针毛蕨总黄酮。最佳提取工艺为：以70%乙醇为溶剂,料液比1：20,80℃下回流提取2次,每次60min；最佳纯化工艺为：碱溶酸沉法(碱液溶解最佳PH值为7,酸液沉淀最佳PH值为1-2)；大孔树脂法(选用HPD500树脂,上样液PH值为5,上样液浓度为4.0mg/mL,吸附流速为2.0BV/h,洗脱剂为70%乙醇,洗脱剂体积为3.0BV,洗脱流速为2.0BV/h)。
通过体内外抗肿瘤活性的对比研究,结果显示翠绿针毛蕨总黄酮可显著抑制多种肿瘤细胞的增殖以及小鼠S180肉瘤模型肿瘤的生长,其活性明显优于总提取物。另外,以HepG2人肝癌细胞为模型,采用MTT法评价细胞增殖,PI染色法检测细胞周期,Annexin V-FITC/PI双染法和Hoechst33258荧光染色法检测细胞凋亡等手段来进一步研究总黄酮的体外抗肿瘤作用,结果显示翠绿针毛蕨总黄酮可诱导HepG2细胞凋亡,且能使细胞周期停滞于G2/M期。同时,在小鼠H22肝癌模型中,采用抑瘤率,生化指标检测,HE染色,免疫组化等方法来深入分析翠绿针毛蕨总黄酮的抗肿瘤作用,结果显示翠绿针毛蕨总黄酮可以显著抑制肿瘤的生长,且对血管形成具有一定的抑制作用,提示其作用机制可能与抑制肿瘤组织血管生成和诱导肿瘤细胞凋亡有关。
在总黄酮化学成分研究中,采用现代色谱和光谱技术,从翠绿针毛蕨总黄酮中分离得到19个化合物,其中9个是结构新颖罕见的B环非苯环的黄酮类化合物,包括1个新化合物8,分别为5,7-二羟基色原酮(1)；5,7-二羟基-2-(1-羟基-2,6-二甲氧基-4-酮-环己烷)-色原酮(2)；5,7-二羟基-2-(1,2-异丙二氧基-4-酮-环己-5-烯)-色原酮(3)；原芹菜素(4)；芹菜素(5)；柚皮素(6)；山奈酚(7)；2-(顺-1,2-二羟基-4酮-环己-5-烯)-5-羟基-7-乙氧基色原酮(8)；2-(顺-1,2-二羟基-4-酮-环己-5-烯)-5,7-二羟基-色原酮(9)；槲皮素(10)；2-(反-1,4-二二羟基环己烷)-5,7-二羟基-色原酮(11)；5,7-二羟基-2-(1,4-二羟基-环己-2,5-二烯)-色原酮(12)；芹菜素-4'-葡萄糖苷(13)；柚皮素-4'-葡萄糖苷(14)；山奈酚-3-葡萄糖苷(15)；5,7-二羟基-2-(1,4-二二羟基环己烷)-色原酮-4'-O-葡萄糖苷(16)；4'-羟基原芹菜素-4'-葡萄糖苷(17)；山奈酚-3-芦丁糖苷(18)；槲皮素-3-芦丁糖苷(19)。所有化合物均为首次从该植物中分离得到。
同时,采用MTT法、流式细胞术、荧光显微镜、免疫印迹实验等方法对新化合物2-(顺-1,2-二羟基-4酮-环己-5-烯)-5-羟基-7-乙氧基色原酮(DHEC)诱导HT-29人结肠癌细胞凋亡的机制进行了深入探讨,结果显示DHEC能显著抑制HT-29细胞增殖和诱导其凋亡,并能引起线粒体内膜电位(MMP)显著降低,细胞内活性氧(ROS)的异常升高,细胞色素C的大量释放,PARP、procaspase-3、-8、-9的活化裂解,促凋亡蛋白Bax和Bad蛋白表达显著增加,抗凋亡蛋白Bcl-2、 Bcl-xl表达显著降低；同时,引起MAPK信号传导通路的3个成员(ERK、JNK、 P38MAPK)发生磷酸化激活；所有结果表明DHEC可经凋亡途径抑制HT-29人结肠癌细胞生长增殖,其中ROS介导的,Bcl-2家族蛋白调控的,Caspase途径依赖的线粒体功能紊乱是DHEC诱导HT-29细胞凋亡的重要机制,而且MAPK信号传导通路也参与了该过程。
另外,参照2010年版药典,对其性状、鉴别方法、指标成分含量测定、浸出物、灰分及水分进行研究,建立了翠绿针毛蕨药材的质量标准。
Macrothelypteris viridifrons (Tagawa) Ching is widely distributed in south of China, which belongs to the genus Macrothelypteris and has been frequently used as a folk medicine for the effects of heat-cleaning and detoxification, the treatment of diseases such as hydropsy and traumatic bleeding. Up to now, there was little report about the chemical constituents and bioactivity of M. viridifrons. In order to explore this folk medicinal resource and research its bioactivity, we did the investigation on the extraction and purification technology, chemical constituents, anti-tumor activity in vitro and in vivo of the crude flavonoids from M. viridifrons. Moreover, the effect on inducing apoptosis of a novel non-aromatic B-ring flavonoid (DHEC) and its putative molecular mechanism of action were evaluated. Meanwhile, the quality standard of M. viridifrons was established.
We investigated the extraction and purification technology of the crude flavonoids from M. viridifrons and established its preparation method via the merge alkali-soluble and acid-isolation with macroporous resin, so that the final flavonoids yield was not less than56.57%, the content of flavonoids was not less than53.37%. The best extraction process:using70%ethanol,1:20solid-liquid ratio, reflux extraction60min with twice at80℃; The best purification process:alkali-soluble and acid-isolation method (the best alkali-soluble PH=7, the best acid-isolation PH=1-2), macroporous resin method (the best resin: HPD500, the best PH of sample solution:5, the best concentration of sample solution:4.0mg/mL, the best adsorption velocity:2.0BV/h, the best eluent:70%ethanol; the best elution volume:3.0BV, the best elution velocity:2.0BV/h)
We comparatively studied the anti-tumor activities of total extract and crude flavonoids from M. viridifron by MTT colorimetric method in vitro and S180sarcoma mouse model in vivo. Research results showed the better anti-tumor activity of crude flavonoids from M. viridifrons. Furthermore, we deeply analyzed the in vitro anti-tumor activity of crude flavonoids from M. viridifrons with MTT method for detecting cell proliferation, PI method for detecting cell cycle, Annexin V-FITC/PI method and Hoechst33258method for detecting cell apoptosis in human hepatoma HepG2cell, and its in vivo anti-tumor effect with the inhibiting ratio of tumor growth, the detection of biochemical indicators, HE dyeing method, immunohistochemical method in H22hepatoma mouse model. Research results showed that crude flavonoids from M. viridifrons could induce apoptosis and G2/M phase arrest in HepG2cell, and could block the growth of tumor and inhibit the formation of blood vessels in H22hepatoma mouse model. All together, crude flavonoids from M. viridifrons exhibits potential anti-tumor activity in vitro and in vivo, which may highly be associated with the effect on induction of apoptosis and its anti-angiogenic activity.
On the basis of normal and reverse phase silica gel and Sephadex LH-20gel column chromatography, nineteen flavonoids (containing nine non-aromatic B-ring flavonoids and one new compound) were isolated from the rhizomes of M. viridifrons and identified as5,7-dihydroxychromone(1);5,7-dihydroxy-2-(1-hydroxy-2,6-dimethoxy-4-oxo-cyclohex)-chromen-4-one(2);5,7-dihydroxy-2-(1,2-isopropyldioxy-4-oxocyclohex-5-enyl)-chromen-4-one(3); protoapigenone(4); apigenin(5); narigenin (6); kaempferol(7);2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5-hydroxy-7-ethoxy-chromone(8);2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5,7-dihydroxy-chromone (9); quercetin(10);2-(trans-1,4-dihydroxy-cyclohexyl)-5,7-dihydroxy-chromone(11); protoapigenin(12); apigenin-4'-O-glucoside(13); narigenin-4'-O-glucoside(14); kaempferol-3-O-glucoside(15);5,7-dihydroxy-2-(1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-chromone-4'-O-glucoside(16); protoapigenin-4'-O-glucoside(17); kaempferol-3-O-rutinose(18); quercetin-3-O-rutinose(19). All compounds were firstly obtained from this plant.
Moreover, on the basis of MTT assay, flow cytometry, microscope and Western blot methods, the effect of2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5-hydroxy-7-ethoxy-chromone (DHEC) on induction of apoptosis and its putative molecular mechanism of action in human colon HT-29cancer cell were evaluated. After treatment of HT-29cell with DHEC, we observed the inhibition of proliferation, the happen of apoptosis, the loss of MMP, the accumulation of intracellular ROS, the releasing of cytochrome c, the cleavage of PARP, the activation of caspase-3,-8and-9, the increase of expression of Bax and Bad, the decrease of expression of Bcl-2、 Bcl-xl, and the phosphorylation of MAPK members (ERK, JNK, P38MAPK). All results suggest that DHEC exhibits potential anti-tumor activity in HT-29cell through induction of apoptosis, which may highly be associated with ROS-mitochondrial dysfunction and involving of caspases family, as well as activation of MAPK signaling pathway.
Besides, on the basis of2010China Pharmacopoeia, we analyzed the traits and microscopic characteristics, the identification method, the content determination, the extract, total ash and moisture content. Thus, the quality standard of M. viridifrons was established.
在对总黄酮纯化工艺的研究中,采用碱溶酸沉法结合大孔树脂纯化法,建立了翠绿针毛蕨总黄酮的纯化工艺,最终得到转移率不低于59.52%,纯度不低于54.85%的翠绿针毛蕨总黄酮。最佳提取工艺为：以70%乙醇为溶剂,料液比1：20,80℃下回流提取2次,每次60min；最佳纯化工艺为：碱溶酸沉法(碱液溶解最佳PH值为7,酸液沉淀最佳PH值为1-2)；大孔树脂法(选用HPD500树脂,上样液PH值为5,上样液浓度为4.0mg/mL,吸附流速为2.0BV/h,洗脱剂为70%乙醇,洗脱剂体积为3.0BV,洗脱流速为2.0BV/h)。
通过体内外抗肿瘤活性的对比研究,结果显示翠绿针毛蕨总黄酮可显著抑制多种肿瘤细胞的增殖以及小鼠S180肉瘤模型肿瘤的生长,其活性明显优于总提取物。另外,以HepG2人肝癌细胞为模型,采用MTT法评价细胞增殖,PI染色法检测细胞周期,Annexin V-FITC/PI双染法和Hoechst33258荧光染色法检测细胞凋亡等手段来进一步研究总黄酮的体外抗肿瘤作用,结果显示翠绿针毛蕨总黄酮可诱导HepG2细胞凋亡,且能使细胞周期停滞于G2/M期。同时,在小鼠H22肝癌模型中,采用抑瘤率,生化指标检测,HE染色,免疫组化等方法来深入分析翠绿针毛蕨总黄酮的抗肿瘤作用,结果显示翠绿针毛蕨总黄酮可以显著抑制肿瘤的生长,且对血管形成具有一定的抑制作用,提示其作用机制可能与抑制肿瘤组织血管生成和诱导肿瘤细胞凋亡有关。
在总黄酮化学成分研究中,采用现代色谱和光谱技术,从翠绿针毛蕨总黄酮中分离得到19个化合物,其中9个是结构新颖罕见的B环非苯环的黄酮类化合物,包括1个新化合物8,分别为5,7-二羟基色原酮(1)；5,7-二羟基-2-(1-羟基-2,6-二甲氧基-4-酮-环己烷)-色原酮(2)；5,7-二羟基-2-(1,2-异丙二氧基-4-酮-环己-5-烯)-色原酮(3)；原芹菜素(4)；芹菜素(5)；柚皮素(6)；山奈酚(7)；2-(顺-1,2-二羟基-4酮-环己-5-烯)-5-羟基-7-乙氧基色原酮(8)；2-(顺-1,2-二羟基-4-酮-环己-5-烯)-5,7-二羟基-色原酮(9)；槲皮素(10)；2-(反-1,4-二二羟基环己烷)-5,7-二羟基-色原酮(11)；5,7-二羟基-2-(1,4-二羟基-环己-2,5-二烯)-色原酮(12)；芹菜素-4'-葡萄糖苷(13)；柚皮素-4'-葡萄糖苷(14)；山奈酚-3-葡萄糖苷(15)；5,7-二羟基-2-(1,4-二二羟基环己烷)-色原酮-4'-O-葡萄糖苷(16)；4'-羟基原芹菜素-4'-葡萄糖苷(17)；山奈酚-3-芦丁糖苷(18)；槲皮素-3-芦丁糖苷(19)。所有化合物均为首次从该植物中分离得到。
同时,采用MTT法、流式细胞术、荧光显微镜、免疫印迹实验等方法对新化合物2-(顺-1,2-二羟基-4酮-环己-5-烯)-5-羟基-7-乙氧基色原酮(DHEC)诱导HT-29人结肠癌细胞凋亡的机制进行了深入探讨,结果显示DHEC能显著抑制HT-29细胞增殖和诱导其凋亡,并能引起线粒体内膜电位(MMP)显著降低,细胞内活性氧(ROS)的异常升高,细胞色素C的大量释放,PARP、procaspase-3、-8、-9的活化裂解,促凋亡蛋白Bax和Bad蛋白表达显著增加,抗凋亡蛋白Bcl-2、 Bcl-xl表达显著降低；同时,引起MAPK信号传导通路的3个成员(ERK、JNK、 P38MAPK)发生磷酸化激活；所有结果表明DHEC可经凋亡途径抑制HT-29人结肠癌细胞生长增殖,其中ROS介导的,Bcl-2家族蛋白调控的,Caspase途径依赖的线粒体功能紊乱是DHEC诱导HT-29细胞凋亡的重要机制,而且MAPK信号传导通路也参与了该过程。
另外,参照2010年版药典,对其性状、鉴别方法、指标成分含量测定、浸出物、灰分及水分进行研究,建立了翠绿针毛蕨药材的质量标准。
Macrothelypteris viridifrons (Tagawa) Ching is widely distributed in south of China, which belongs to the genus Macrothelypteris and has been frequently used as a folk medicine for the effects of heat-cleaning and detoxification, the treatment of diseases such as hydropsy and traumatic bleeding. Up to now, there was little report about the chemical constituents and bioactivity of M. viridifrons. In order to explore this folk medicinal resource and research its bioactivity, we did the investigation on the extraction and purification technology, chemical constituents, anti-tumor activity in vitro and in vivo of the crude flavonoids from M. viridifrons. Moreover, the effect on inducing apoptosis of a novel non-aromatic B-ring flavonoid (DHEC) and its putative molecular mechanism of action were evaluated. Meanwhile, the quality standard of M. viridifrons was established.
We investigated the extraction and purification technology of the crude flavonoids from M. viridifrons and established its preparation method via the merge alkali-soluble and acid-isolation with macroporous resin, so that the final flavonoids yield was not less than56.57%, the content of flavonoids was not less than53.37%. The best extraction process:using70%ethanol,1:20solid-liquid ratio, reflux extraction60min with twice at80℃; The best purification process:alkali-soluble and acid-isolation method (the best alkali-soluble PH=7, the best acid-isolation PH=1-2), macroporous resin method (the best resin: HPD500, the best PH of sample solution:5, the best concentration of sample solution:4.0mg/mL, the best adsorption velocity:2.0BV/h, the best eluent:70%ethanol; the best elution volume:3.0BV, the best elution velocity:2.0BV/h)
We comparatively studied the anti-tumor activities of total extract and crude flavonoids from M. viridifron by MTT colorimetric method in vitro and S180sarcoma mouse model in vivo. Research results showed the better anti-tumor activity of crude flavonoids from M. viridifrons. Furthermore, we deeply analyzed the in vitro anti-tumor activity of crude flavonoids from M. viridifrons with MTT method for detecting cell proliferation, PI method for detecting cell cycle, Annexin V-FITC/PI method and Hoechst33258method for detecting cell apoptosis in human hepatoma HepG2cell, and its in vivo anti-tumor effect with the inhibiting ratio of tumor growth, the detection of biochemical indicators, HE dyeing method, immunohistochemical method in H22hepatoma mouse model. Research results showed that crude flavonoids from M. viridifrons could induce apoptosis and G2/M phase arrest in HepG2cell, and could block the growth of tumor and inhibit the formation of blood vessels in H22hepatoma mouse model. All together, crude flavonoids from M. viridifrons exhibits potential anti-tumor activity in vitro and in vivo, which may highly be associated with the effect on induction of apoptosis and its anti-angiogenic activity.
On the basis of normal and reverse phase silica gel and Sephadex LH-20gel column chromatography, nineteen flavonoids (containing nine non-aromatic B-ring flavonoids and one new compound) were isolated from the rhizomes of M. viridifrons and identified as5,7-dihydroxychromone(1);5,7-dihydroxy-2-(1-hydroxy-2,6-dimethoxy-4-oxo-cyclohex)-chromen-4-one(2);5,7-dihydroxy-2-(1,2-isopropyldioxy-4-oxocyclohex-5-enyl)-chromen-4-one(3); protoapigenone(4); apigenin(5); narigenin (6); kaempferol(7);2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5-hydroxy-7-ethoxy-chromone(8);2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5,7-dihydroxy-chromone (9); quercetin(10);2-(trans-1,4-dihydroxy-cyclohexyl)-5,7-dihydroxy-chromone(11); protoapigenin(12); apigenin-4'-O-glucoside(13); narigenin-4'-O-glucoside(14); kaempferol-3-O-glucoside(15);5,7-dihydroxy-2-(1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-chromone-4'-O-glucoside(16); protoapigenin-4'-O-glucoside(17); kaempferol-3-O-rutinose(18); quercetin-3-O-rutinose(19). All compounds were firstly obtained from this plant.
Moreover, on the basis of MTT assay, flow cytometry, microscope and Western blot methods, the effect of2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5-hydroxy-7-ethoxy-chromone (DHEC) on induction of apoptosis and its putative molecular mechanism of action in human colon HT-29cancer cell were evaluated. After treatment of HT-29cell with DHEC, we observed the inhibition of proliferation, the happen of apoptosis, the loss of MMP, the accumulation of intracellular ROS, the releasing of cytochrome c, the cleavage of PARP, the activation of caspase-3,-8and-9, the increase of expression of Bax and Bad, the decrease of expression of Bcl-2、 Bcl-xl, and the phosphorylation of MAPK members (ERK, JNK, P38MAPK). All results suggest that DHEC exhibits potential anti-tumor activity in HT-29cell through induction of apoptosis, which may highly be associated with ROS-mitochondrial dysfunction and involving of caspases family, as well as activation of MAPK signaling pathway.
Besides, on the basis of2010China Pharmacopoeia, we analyzed the traits and microscopic characteristics, the identification method, the content determination, the extract, total ash and moisture content. Thus, the quality standard of M. viridifrons was established.
引文
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