北五味子三萜类化合物分离纯化、结构鉴定及抗肝癌活性研究
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摘要
北五味子,木兰科五味子属植物,主要分布于辽宁、吉林、黑龙江等地,是一种十分珍贵的药材。五味子与人参相似,具有“适应原样”作用,能增强机体对非特异性刺激的防御能力,是国家中药现代化科技产业基地的五大品种之一。三萜和木脂素类化合物是五味子中最重要的具有广泛生物活性的物质被大量用于功能食品、药品等领域。目前国内外对北五味子研究多集中在果实和种子部位,化学成分研究集中在木脂素、多糖、挥发油成分上,而对北五味子加工过程中副产物藤茎三萜化学成分及其生物活性的研究报道较少。本文以北五味子藤茎为原料,研究北五味子三萜的提取、分离纯化工艺、并对其抗氧化性质、抗肝癌活性进行深入研究,以期为北五味子的产业化开发奠定基础。主要结论如下：
1.采用超声波辅助浸提法及微波辅助浸提法对北五味子三萜类化合物的提取工艺进行研究,通过Central Composite响应面优化试验及验证试验确定超声波辅助浸提法最佳提取条件为：超声时间48min,乙醇浓度64%,超声波功率68W,料液比1：60,北五味子三萜的得率2.53±0.24%。确定微波辅助浸提法最佳提取条件为：乙醇浓度53%,预处理剂倍数4.6倍,预处理时间2min,北五味子三萜的得率2.02±0.12%。采用Central-Composite设计试验法对北五味子总三萜提取条件进行优化,不仅科学合理,而且快速高效,具有很强的实际生产指导作用。
2.利用大孔吸附树脂法对北五味子三萜纯化工艺进行研究,通过静态吸附试验对5种不同树脂的静态吸附特性和解吸特性的比较,发现AB-8树脂具有较大的吸附总三萜能力,同时解吸率高。选择AB-8为试验树脂,确定其洗脱效果的最佳工艺参数：样品溶液pH为6,上样流速为2BV/h,进样浓度为1.5mg/mL时,洗脱率达90.52%,纯度达34.26%。这种分离纯化方法避开了有毒的有机溶剂,仅用到乙醇,且大孔吸附树脂可以再生,该法不失为一种简便、清洁、有效的分离纯化北五味子三萜的方法。
3.采用硅胶柱对大孔树脂分离后的三萜进一步纯化,并采用薄层色谱法对纯化后样品进行显色定性分析。通过氯仿：甲醇为洗脱剂(20：1-3：1)进行梯度洗脱,用薄层色谱法对纯化后的样品进行了研究,确定最佳洗脱剂为氯仿：甲醇=5：1,定性鉴定了三萜类化合物的存在,为下一步高速逆流色谱制备单体提供参考依据。
4.采用高速逆流色谱(HSCCC)现代分离技术结合传统的超声波辅助萃取、大孔树脂纯化、硅胶柱层析等方法从北五味子藤茎中分离得到4个三萜单体化合物。运用UV、IR、HPLC-MS、NMR、HMBC、HSQC波谱手段及化学反应方法鉴定单体化合物分别为：corosolic acid、lancifodilactone C、nigranoic acid、oleanolic acid。利用SciFinder Scholar数据库联机检索,其中corosolic acid、lancifodilactone C化合物均为首次从该种植物中分离得到。
5.采用总还原力测定法、Fenton法、改良的邻苯三酚自氧化法、DPPH法、猪油体系抗氧化法,分别对北五味子三萜提取物的总还原力、羟自由基、超氧阴离子自由基及DPPH自由基的清除能力、抗油脂氧化能力进行测定。证实了北五味子三萜类化合物是主要抗氧化活性物质。结果表明,北五味子三萜有较好的还原能力；对羟自由基和DPPH自由基有较强的清除作用,且清除率50%所对应的质量浓度ICso分别为0.6mg/mL和0.077mg/mL,但对超氧阴离子自由基清除效果不显著；有一定的抑制油脂氧化的作用但弱于抗坏血酸、TBHQ。抗坏血酸对三萜的抗氧化性有协同增效作用。
6.采用四甲基偶氮哇盐(MTT)比色法,对分离得到的北五味子四种单体化合物抑制人肝癌细胞HepG2、SMMC7721和Bel-7402及肝纤维化星型细胞HSC-T6的抑制增殖作用进行研究,并对corosolic acid诱导HSC-T6细胞凋亡的关键基因及作用机理进行研究。结果表明corosolic acid对于Bel-7402、SMMC7721、HepG2和HSC-T6细胞均具有抑制作用,IC5o在12.5～25μg/mL、12.5μ～25μg/mL,12.5～25μ～g/mL、5～25μg/mL; oleanolic acid仅对HepG2细胞有抑制作用,IC50=52μg/mL,而对于SMMC7721、HSC-T6、Bel-7402细胞的抑制增殖作用较弱；lancifodilactone C对SMMC7721和HSC-T6细胞无抑制作用,对HepG2和Bel-7402细胞具有抑制作用,IC50为30μg/mL、10μg/mL; nigranoic acid对SMMC7721细胞无抑制作用；对HepG2、Bel-7402和HSC-T6细胞的增殖具有较好的抑制作用,IC5o分别为21μg/mL、16μg/mL、25μg/mL。
7.采用流式细胞仪检测corosolic acid对HSC-T6凋亡的影响,光镜观察corosolic acid作用后细胞形态学变化,通过免疫细胞化学术检测HSC-T6中Caspase3和Bcl-2蛋白的表达。结果显示细胞爬片观察到25μg/mL浓度的corosolic acid作用HSC-T6细胞48h后,细胞缩小、变圆,细胞核浓缩,形状不规则,可见细胞碎片。流式细胞仪检测显示：5μg/mL、15μg/mL、25μg/mL、corosolic acid作用HSC-T6细胞48h后,细胞凋亡率分别为(11.86±2.75)%,(24.62±3.42)%,(48.24±4.75)%,比对照组(2.44±1.95)%明显升高(P<0.01)。免疫细胞化学结果显示不同浓度corosolic acid作用于HSC-T6细胞48h后,Caspase3蛋白表达较对照组升高,且呈剂量依赖,但对Bcl-2蛋白无明显影响。推测Corosolic acid诱导HSC-T6细胞凋亡的机理可能是通过降低Bcl-2蛋白表达,促使线粒体通透性增加,再激活Caspase3蛋白诱发细胞凋亡。表明corosolic acid将来可以作为一种抗肝纤维化的组分。
Schisandra chinensis(Turcz.)Baill(Magnoliaceae schisandra plantsis) is a perennial lignifying liana coming from Heilongjiang, Jilin, Liaoning and other places in China.The fruits and seeds of Schisandra chinensis(Turcz.)Baill are similar to ginseng,which are used in traditional Chinese medicine as stimulants and adaptogenic drugs. Schisandra chinensis(Turcz.)Baill is one of five varieties of national modernization of traditional Chinese medicine industry base. The main compositions of Schisandra chinensis(Turcz.)Baill are lignan, triterpenes,that have been widely applied to functional food and medicine field. In recent years, much research for Schisandra chinensis(Turcz,)Baill has been concentrated on it's fruit and seeds. At the same time there are some research to analyze the content of the lignans, polysaccharide, volatile oil etc.However few works was done on the chemical compositionsin caculis of Schisandra chinensis(Turcz.)Baill. The caculis is used as raw materials in this study, the triterpenes extracting technology, purification technology, physical and chemical properties, structure, antioxidant activity and inhibiting liver-carcinoma-cells activity of the triterpenes were studied.It will lay the foundation developping the industrialization of Schisandra chinensis(Turcz.)Baill and the main results are as follow:
1. The triterpenes from caculis of Schisandra Chinensis(Turcz.) baill were extracted by ultrasonic and microwave,the optimual technologys were found through single test and central composite response surface methodology,respectively.In the extraction process by ultrasonic,the optimual result:the extraction time is 48min, the alcohol content was 64%, the frequency of ultrasound was 68W, the ratio of solid and liquid is 1:60, and the extraction percent is 2.53±0.24%. In the extraction process by microwave, the optimual result:the alcohol content was 53%,the multiple of pretreating is 4.6, the pretreating time is 2 min, the extraction percent is 2.02±0.12%. The response surface methodology (RSM) was used to optimize the process parameters of the triterpenes from caculis of Schisandra Chinensis(Turcz.) Baill.This method not only scientific reasonable and efficient, and has a strong guidance to practical production.
2. The purification of triterpenes with macroporous resin from caculis of Schisandra Chinensis(Turcz.) baill were researched.The triterpenes from caculis of Schisandra Chinensis(Turcz.) baill were purification using different types of resin adsorption. AB-8 resin was found to be the best for the purification of triterpenes components.The results showed that AB-8 was the most appropriate resin for the purification total triterpenes,with working solution pH6, flow velocity 2BV/h,Injection concentration 1.5 mg/mL.After purification desorption ratio and product purity reached 90.52% and 34.26%% respectively.This separation and purification methods avoid a toxic organic solvent, only use ethanol, and macroporous adsorption resin can be recycled.
3.The triterpenoids are further purified by silica column chromatography, and qualitative colour-display was analysised by TLC method. the best eluant solvent system is chloroform: methanol=5:1 through with different gradient elution from chloroform:methanol=20:1 to 3:1.The results not only confirm the existence of triterpenoids.but also be used to preparate monomer by high speed countercurrent chromatography.
4.Four compounds were isolated from caculis of Schisandra Chinensis(Turcz.) baill by various chromatographic methods such as macroporous resin chromatography,silicagel column chromatography, high speed countercurrent chromatography(HSCCC), assistant with traditional methods like ultrasonic extraction.The structures of the four compounds had been identified on the basis of UV, IR, HPLC-MS, NMR, HMBC, HSQC spectrum means and chemical evidence.They are corosolic acid,lancifodilactone C,nigranoic acid,oleanolic acid。The compounds were first isolated from the caculis of Schisandra Chinensis(Turcz.) baill, corosolic acid,lancifodilactone C by using SciFinder scholar database online retrieval
5. The anti-oxidative activities of ethanol extracts were evaluated using various established in vitro systems,including total reducing power,fenton method, adjacent pyrogallol since oxidation, diphenyl picryl-hydrazyl, resistance to oxidation and the POV value. The experimental results showed:triterpenes had better reducing power, stronger antioxidation effect on the·OH and DPPH·, and the quality concentration 50%(IC50) was 0.6mg/mL and 0.077mg/mL to inhibit. It had less effect on inhibiting O2-·. Triterpenes could inhibit peroxidation of lard, and have less effect of lard peroxidation than Vc and TBHQ. Triterpenes exhibit remarkable synergistic antioxidation activity with VC in lard.
6. The inhibiting activities of the four monomer compounds isolated from the caculis of Schisandra Chinensis(Turcz.) baill against HepG2,SMMC7721,Bel-7402, HSC-T6 in vitro were studied by cell culture methods.The cell toxicities of the samples were tested by3-(4,5-dimethylthiazol-2-yl)-2-5-diphe-nyltetrazolium bromide (MTT) assaying.To investigate the effect of corosolic acid on proliferation andapoptosis of HSC in vitro and explore the mechanisms of corosolic acid inducing apoptosis of HSC by studying the expressions of apoptosis-regulating proteins Caspase3 and Bcl-2 in HSC were studied. The results showed that the corosolic acid had better inhibitory effects against Bel-7402,SMMC7721,HepG2,HSC-T6 cells with the IC50 value of 12.5～25μg/mL,12.5～25μg/mL,12.5～25μg/mL,5～25μg/mL. The oleanolic acid had certain inhibitory effects against HepG2 cell with the IC50 value of 52μg/mL.The lancifodilactone C had certain inhibitory effects against HepG2 and Bel-7402 cells with the IC50 value of 30μg/mL,10μg/mL. The nigranoic acid had certain inhibitory effects against HepG2,Bel-7402 and HSC-T6 cells with the IC50 value of 21μg/mL、16μg/mL、25μg/mL.
7.The rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed by light microscopy.And then the expression of apoptosis-regulating protein, Caspase3, Bcl-2 in HSC-T6 after apoptosis induced by corosolic acid were examined by immunocytochemical staining assay. HE staining:Treatment with 25μg/mL concentrations of corosolic acid for 48h resulted in morphologic changes of HSC-T6,including karyorrhexis and cytoplasm vacuolization. Flow cytometry quantitation:After treating HSC-T6 with corosolic acid at concentration of 5 u g/mL,15μg/mL,25μg/mL for 48h,the apoptosis ratio of HSC-T6 were(11.86±2.75)%,(24.62±3.42)%,(48.24±4.75)%, respectively,which were significantly higher than that of control group(2.44±1.95)%%(P<0.01). Immunocytochemistry:the expressions of caspase3 protein in HSC-T6 cells were up-regulated in dose-dependent manner,but expressions of bcl-2 protein were not significantly different from that of the control group. The results suggested that HSC-T6 cells apoptosis induced by corosolic acid occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.The study also indicated that corosolic acid might be a potential Chinese medical component for inhibiting liver fibrosis.
1.采用超声波辅助浸提法及微波辅助浸提法对北五味子三萜类化合物的提取工艺进行研究,通过Central Composite响应面优化试验及验证试验确定超声波辅助浸提法最佳提取条件为：超声时间48min,乙醇浓度64%,超声波功率68W,料液比1：60,北五味子三萜的得率2.53±0.24%。确定微波辅助浸提法最佳提取条件为：乙醇浓度53%,预处理剂倍数4.6倍,预处理时间2min,北五味子三萜的得率2.02±0.12%。采用Central-Composite设计试验法对北五味子总三萜提取条件进行优化,不仅科学合理,而且快速高效,具有很强的实际生产指导作用。
2.利用大孔吸附树脂法对北五味子三萜纯化工艺进行研究,通过静态吸附试验对5种不同树脂的静态吸附特性和解吸特性的比较,发现AB-8树脂具有较大的吸附总三萜能力,同时解吸率高。选择AB-8为试验树脂,确定其洗脱效果的最佳工艺参数：样品溶液pH为6,上样流速为2BV/h,进样浓度为1.5mg/mL时,洗脱率达90.52%,纯度达34.26%。这种分离纯化方法避开了有毒的有机溶剂,仅用到乙醇,且大孔吸附树脂可以再生,该法不失为一种简便、清洁、有效的分离纯化北五味子三萜的方法。
3.采用硅胶柱对大孔树脂分离后的三萜进一步纯化,并采用薄层色谱法对纯化后样品进行显色定性分析。通过氯仿：甲醇为洗脱剂(20：1-3：1)进行梯度洗脱,用薄层色谱法对纯化后的样品进行了研究,确定最佳洗脱剂为氯仿：甲醇=5：1,定性鉴定了三萜类化合物的存在,为下一步高速逆流色谱制备单体提供参考依据。
4.采用高速逆流色谱(HSCCC)现代分离技术结合传统的超声波辅助萃取、大孔树脂纯化、硅胶柱层析等方法从北五味子藤茎中分离得到4个三萜单体化合物。运用UV、IR、HPLC-MS、NMR、HMBC、HSQC波谱手段及化学反应方法鉴定单体化合物分别为：corosolic acid、lancifodilactone C、nigranoic acid、oleanolic acid。利用SciFinder Scholar数据库联机检索,其中corosolic acid、lancifodilactone C化合物均为首次从该种植物中分离得到。
5.采用总还原力测定法、Fenton法、改良的邻苯三酚自氧化法、DPPH法、猪油体系抗氧化法,分别对北五味子三萜提取物的总还原力、羟自由基、超氧阴离子自由基及DPPH自由基的清除能力、抗油脂氧化能力进行测定。证实了北五味子三萜类化合物是主要抗氧化活性物质。结果表明,北五味子三萜有较好的还原能力；对羟自由基和DPPH自由基有较强的清除作用,且清除率50%所对应的质量浓度ICso分别为0.6mg/mL和0.077mg/mL,但对超氧阴离子自由基清除效果不显著；有一定的抑制油脂氧化的作用但弱于抗坏血酸、TBHQ。抗坏血酸对三萜的抗氧化性有协同增效作用。
6.采用四甲基偶氮哇盐(MTT)比色法,对分离得到的北五味子四种单体化合物抑制人肝癌细胞HepG2、SMMC7721和Bel-7402及肝纤维化星型细胞HSC-T6的抑制增殖作用进行研究,并对corosolic acid诱导HSC-T6细胞凋亡的关键基因及作用机理进行研究。结果表明corosolic acid对于Bel-7402、SMMC7721、HepG2和HSC-T6细胞均具有抑制作用,IC5o在12.5～25μg/mL、12.5μ～25μg/mL,12.5～25μ～g/mL、5～25μg/mL; oleanolic acid仅对HepG2细胞有抑制作用,IC50=52μg/mL,而对于SMMC7721、HSC-T6、Bel-7402细胞的抑制增殖作用较弱；lancifodilactone C对SMMC7721和HSC-T6细胞无抑制作用,对HepG2和Bel-7402细胞具有抑制作用,IC50为30μg/mL、10μg/mL; nigranoic acid对SMMC7721细胞无抑制作用；对HepG2、Bel-7402和HSC-T6细胞的增殖具有较好的抑制作用,IC5o分别为21μg/mL、16μg/mL、25μg/mL。
7.采用流式细胞仪检测corosolic acid对HSC-T6凋亡的影响,光镜观察corosolic acid作用后细胞形态学变化,通过免疫细胞化学术检测HSC-T6中Caspase3和Bcl-2蛋白的表达。结果显示细胞爬片观察到25μg/mL浓度的corosolic acid作用HSC-T6细胞48h后,细胞缩小、变圆,细胞核浓缩,形状不规则,可见细胞碎片。流式细胞仪检测显示：5μg/mL、15μg/mL、25μg/mL、corosolic acid作用HSC-T6细胞48h后,细胞凋亡率分别为(11.86±2.75)%,(24.62±3.42)%,(48.24±4.75)%,比对照组(2.44±1.95)%明显升高(P<0.01)。免疫细胞化学结果显示不同浓度corosolic acid作用于HSC-T6细胞48h后,Caspase3蛋白表达较对照组升高,且呈剂量依赖,但对Bcl-2蛋白无明显影响。推测Corosolic acid诱导HSC-T6细胞凋亡的机理可能是通过降低Bcl-2蛋白表达,促使线粒体通透性增加,再激活Caspase3蛋白诱发细胞凋亡。表明corosolic acid将来可以作为一种抗肝纤维化的组分。
Schisandra chinensis(Turcz.)Baill(Magnoliaceae schisandra plantsis) is a perennial lignifying liana coming from Heilongjiang, Jilin, Liaoning and other places in China.The fruits and seeds of Schisandra chinensis(Turcz.)Baill are similar to ginseng,which are used in traditional Chinese medicine as stimulants and adaptogenic drugs. Schisandra chinensis(Turcz.)Baill is one of five varieties of national modernization of traditional Chinese medicine industry base. The main compositions of Schisandra chinensis(Turcz.)Baill are lignan, triterpenes,that have been widely applied to functional food and medicine field. In recent years, much research for Schisandra chinensis(Turcz,)Baill has been concentrated on it's fruit and seeds. At the same time there are some research to analyze the content of the lignans, polysaccharide, volatile oil etc.However few works was done on the chemical compositionsin caculis of Schisandra chinensis(Turcz.)Baill. The caculis is used as raw materials in this study, the triterpenes extracting technology, purification technology, physical and chemical properties, structure, antioxidant activity and inhibiting liver-carcinoma-cells activity of the triterpenes were studied.It will lay the foundation developping the industrialization of Schisandra chinensis(Turcz.)Baill and the main results are as follow:
1. The triterpenes from caculis of Schisandra Chinensis(Turcz.) baill were extracted by ultrasonic and microwave,the optimual technologys were found through single test and central composite response surface methodology,respectively.In the extraction process by ultrasonic,the optimual result:the extraction time is 48min, the alcohol content was 64%, the frequency of ultrasound was 68W, the ratio of solid and liquid is 1:60, and the extraction percent is 2.53±0.24%. In the extraction process by microwave, the optimual result:the alcohol content was 53%,the multiple of pretreating is 4.6, the pretreating time is 2 min, the extraction percent is 2.02±0.12%. The response surface methodology (RSM) was used to optimize the process parameters of the triterpenes from caculis of Schisandra Chinensis(Turcz.) Baill.This method not only scientific reasonable and efficient, and has a strong guidance to practical production.
2. The purification of triterpenes with macroporous resin from caculis of Schisandra Chinensis(Turcz.) baill were researched.The triterpenes from caculis of Schisandra Chinensis(Turcz.) baill were purification using different types of resin adsorption. AB-8 resin was found to be the best for the purification of triterpenes components.The results showed that AB-8 was the most appropriate resin for the purification total triterpenes,with working solution pH6, flow velocity 2BV/h,Injection concentration 1.5 mg/mL.After purification desorption ratio and product purity reached 90.52% and 34.26%% respectively.This separation and purification methods avoid a toxic organic solvent, only use ethanol, and macroporous adsorption resin can be recycled.
3.The triterpenoids are further purified by silica column chromatography, and qualitative colour-display was analysised by TLC method. the best eluant solvent system is chloroform: methanol=5:1 through with different gradient elution from chloroform:methanol=20:1 to 3:1.The results not only confirm the existence of triterpenoids.but also be used to preparate monomer by high speed countercurrent chromatography.
4.Four compounds were isolated from caculis of Schisandra Chinensis(Turcz.) baill by various chromatographic methods such as macroporous resin chromatography,silicagel column chromatography, high speed countercurrent chromatography(HSCCC), assistant with traditional methods like ultrasonic extraction.The structures of the four compounds had been identified on the basis of UV, IR, HPLC-MS, NMR, HMBC, HSQC spectrum means and chemical evidence.They are corosolic acid,lancifodilactone C,nigranoic acid,oleanolic acid。The compounds were first isolated from the caculis of Schisandra Chinensis(Turcz.) baill, corosolic acid,lancifodilactone C by using SciFinder scholar database online retrieval
5. The anti-oxidative activities of ethanol extracts were evaluated using various established in vitro systems,including total reducing power,fenton method, adjacent pyrogallol since oxidation, diphenyl picryl-hydrazyl, resistance to oxidation and the POV value. The experimental results showed:triterpenes had better reducing power, stronger antioxidation effect on the·OH and DPPH·, and the quality concentration 50%(IC50) was 0.6mg/mL and 0.077mg/mL to inhibit. It had less effect on inhibiting O2-·. Triterpenes could inhibit peroxidation of lard, and have less effect of lard peroxidation than Vc and TBHQ. Triterpenes exhibit remarkable synergistic antioxidation activity with VC in lard.
6. The inhibiting activities of the four monomer compounds isolated from the caculis of Schisandra Chinensis(Turcz.) baill against HepG2,SMMC7721,Bel-7402, HSC-T6 in vitro were studied by cell culture methods.The cell toxicities of the samples were tested by3-(4,5-dimethylthiazol-2-yl)-2-5-diphe-nyltetrazolium bromide (MTT) assaying.To investigate the effect of corosolic acid on proliferation andapoptosis of HSC in vitro and explore the mechanisms of corosolic acid inducing apoptosis of HSC by studying the expressions of apoptosis-regulating proteins Caspase3 and Bcl-2 in HSC were studied. The results showed that the corosolic acid had better inhibitory effects against Bel-7402,SMMC7721,HepG2,HSC-T6 cells with the IC50 value of 12.5～25μg/mL,12.5～25μg/mL,12.5～25μg/mL,5～25μg/mL. The oleanolic acid had certain inhibitory effects against HepG2 cell with the IC50 value of 52μg/mL.The lancifodilactone C had certain inhibitory effects against HepG2 and Bel-7402 cells with the IC50 value of 30μg/mL,10μg/mL. The nigranoic acid had certain inhibitory effects against HepG2,Bel-7402 and HSC-T6 cells with the IC50 value of 21μg/mL、16μg/mL、25μg/mL.
7.The rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed by light microscopy.And then the expression of apoptosis-regulating protein, Caspase3, Bcl-2 in HSC-T6 after apoptosis induced by corosolic acid were examined by immunocytochemical staining assay. HE staining:Treatment with 25μg/mL concentrations of corosolic acid for 48h resulted in morphologic changes of HSC-T6,including karyorrhexis and cytoplasm vacuolization. Flow cytometry quantitation:After treating HSC-T6 with corosolic acid at concentration of 5 u g/mL,15μg/mL,25μg/mL for 48h,the apoptosis ratio of HSC-T6 were(11.86±2.75)%,(24.62±3.42)%,(48.24±4.75)%, respectively,which were significantly higher than that of control group(2.44±1.95)%%(P<0.01). Immunocytochemistry:the expressions of caspase3 protein in HSC-T6 cells were up-regulated in dose-dependent manner,but expressions of bcl-2 protein were not significantly different from that of the control group. The results suggested that HSC-T6 cells apoptosis induced by corosolic acid occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.The study also indicated that corosolic acid might be a potential Chinese medical component for inhibiting liver fibrosis.
引文
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