活性指导下中药独活与延胡索抗肿瘤有效成分的分离及其抑瘤作用研究
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摘要
第一部分研究背景
恶性肿瘤严重威胁人类健康。在抗肿瘤治疗的手段中,药物治疗占据着十分重要的地位。目前抗肿瘤药物的研发和应用的方向,主要是化疗药物的个体化运用和分子靶向药物的研制,然而这些药物带来的不良反应、药物作用靶点的单一性及其昂贵的费用等,是其发展的瓶颈。中医药在抗肿瘤方面的应用有着悠久的历史和坚实的基础,并有着广袤的药用资源。运用现代科技手段对中医药抗肿瘤的作用机制的研究也取得了斐然的成果,包括中药在内的天然药物中的香豆素、生物碱等化合物均被证实具有显著而独特的抗肿瘤活性。从中医药中寻找和挖掘高效、低毒、广谱、廉价的抗肿瘤药物,具有良好的前景,也是中医药工作者努力的方向。
第二部分中药独活抗肿瘤有效单体的提取、分离及其作用研究
目的:提取分离并鉴定中药独活中具有抗肿瘤活性的单体化合物;考察其活性单体的抗肿瘤作用及其机制。
方法:应用硅胶柱色谱、重结晶及波谱分析等方法从中药独活的乙醇提取物中提取分离并鉴定其抗肿瘤活性单体物质;运用四甲基偶氮唑蓝(MTT)法考察该单体化合物的体外抗肿瘤作用;运用MTT法和细胞克隆形成法考察该单体化合物对人胃癌细胞MKN28、MKN45的放射增敏作用;运用流式细胞术、荧光定量PCR等方法考察该化合物放射增敏的作用机制。
结果:
1.从中药独活乙醇提取物中共分离得到4个单体化合物,分别为甲氧基欧芹素、二十四烷酸、二氢欧山芹素、二氢欧山芹醇乙酸酯。其中甲氧基欧芹素为其抗肿瘤活性的主要物质。
2.甲氧基欧芹素对不同组织来源的10种人源肿瘤细胞均有较强的增殖抑制作用,IC_(50)在17.35～34.40μg/ml。
3.甲氧基欧芹素10μg/ml预处理24h对人胃癌细胞系MKN28有放射增敏作用,其增敏比SER_(D0)=1.137。而对MKN45细胞无增敏作用。
4.甲氧基欧芹素10μg/ml预处理24h后放射治疗人胃癌MKN28细胞,可将细胞阻滞在G_0/G_1期,减少S期细胞比例;甲氧基欧芹素可增加放射诱导的细胞凋亡率,并提高放射引起的细胞内活性氧簇(ROS)的水平;放射处理后的极早期,甲氧基欧芹素可下调细胞BRCA1 mRNA的表达水平。
结论:甲氧基欧芹素是中药独活乙醇提取物抗肿瘤作用的主要活性物质,体外具有较强的抑制肿瘤细胞增殖作用,并对人胃癌细胞MKN28有放射增敏作用,其作用机制可能与诱导细胞凋亡、改变细胞周期时相分布、提高细胞内ROS水平以及下调DNA损伤修复相关基因BRCA1 mRNA表达水平下降有关。
第三部分中药延胡索抗肿瘤有效部位的提取、分离及其作用研究
目的:提取分离中药延胡索中具有抗肿瘤作用的有效部位;考察该有效部位的抗肿瘤作用及其机制。
方法:应用系统溶剂法中药延胡索的乙醇提取物中提取分离其抗肿瘤活性部位;运用四甲基偶氮唑蓝(MTT)法考察该有效部位的体外抗肿瘤作用;运用运用流式细胞术、荧光定量PCR等方法考察该化合物抗肿瘤的作用机制。
结果:
1.从中药延胡索中提取分离出其主要抗肿瘤有效部位——延胡索总生物碱。
2.延胡索总生物碱对不同组织来源的10种人源肿瘤细胞均有较强的增殖抑制作用,IC_(50)在18.39～40.24μg/ml。
3.延胡索总生物碱可诱导人肝癌HepG2细胞凋亡,并将HepG2细胞阻滞在S期,有浓度和时间依赖性;IC50浓度延胡索总碱作用于人肝癌HepG2细胞24或48h后,15种miRNAs均被检测到有不同程度的变化,其中尤以let-7a表达的上调和miR-221、miR-222表达的下调较为突出。
结论:延胡索总生物碱是中药延胡索乙醇提取物抗肿瘤作用的主要活性部位,体外具有较强的抑制肿瘤细胞增殖作用,其机制可能与诱导细胞凋亡、改变细胞周期时相分布、改变HepG2细胞miRNA表达谱有关。
PART I:BACKGROUD
Cancer is a kind of disease that extremely threatens human health. Treatment by drugs is one ofthe main methods on cancer. Nowaday, trends in anticancer drugs are personalized usage of chemotherapeutic agents and discovery on molecular targeted ones. However, those roles are limited in clinic by severe side effects and huge costs. It's an important task for us to find out some drugs that possess higher anticancer activity and lower toxicity and are even cheaper. Traditional Chinese medicine (TCM) may be a useful model for scientific research because of its standardized system of therapies and long-time practices. Chinese medicine provides a rich pool of novel and efficacious agents for treating a variety of cancer. So, isolation and identification of new effective constituents of Chinese medicine and investigation its mechanism using molecular biochemistry methods are ascendant aspects of drug-development in our country.
PART II ISOLATION OF ANTITUMOR-ACTIVE CONSTITUENTS FROM ANGELICAPUBESCENS AND INVESTIGATION ON THEIR EFFECTS
Objective To isolate the antitumor-active constituents from Angelica pubescens guided byactivity and investigate their effects on tumor in vitro.
Methods The effective extract of Angelica pubescens was separated by using several types ofcolumn chromtograpys and recrystallization to obtain the compounds, which structures weredetermined by spectroscopic methods, as well as comparing of their spectral data with those ofknown compounds. MTT assay was applied to detect the inhibitory effect of osthol. MTT andclonogenic assay were used to investigate radiosensitivity of osthol on human gastric cancer celllines MKN28 and MKN45. Apoptosis, cell cycle phases and ROS levels of MKN28 treated byosthol combining with radiotherapy were inspected by flow cytometery (FCM). And Realtime-PCR method was used to detect the mRNA expressions of BRCA1 after exposure to OSTand RT.
Results
1. Four compounds were isolated from the EtOH extract of this plant: osthol, columbianadin,lignoceric acid and columbianetin acetate. Among them, osthol is the main compound of thisherb to display antitumor activity.
2. Osthol has a significant cytotoxic effect on ten kinds of human cancer cells in vitro, and IC50 numerical values of OST are between 17.35 and 34.40μg/ml.
3. The enhanced radiosensitivity of gastric cancer cells MKN28 but not MKN45 has been observed. The sensitivity enhancement ratio (SER) at 37% survival level is 1.137 for 24h pretreatment of OST at dose of 10μg/ml.
4. Pretreatment of OST can increase apoptosis, cell cycle arrest at G_0/G_1 and intracellular ROS production induced by RT. And at the very early time after RT, OST can downregulate the expression of BRCA1 mRNA in MKN28.
Conclusions Osthol which is the main antitumor-active compound of Angelica pubescens has a significant cytotoxic effect in vitro and can sensitize MKN28 to radiotherapy. This sensitization may be associated with increasing apoptosis, cell cycle arrest at G_0/G_1 and intracellular ROS production and downregulating the expression of BRCA1 mRNA by OST.
PART III ISOLATION OF ANTITUMOR-ACTIVE PARTS FROM CORYDALISYANHUSUO AND INVESTIGATION ON THEIR EFFECTS
Objective To isolate the antitumor-active parts from Corydalis yanhusuo guided by activityand investigate their effects on tumor in vitro.
Methods The effective extract of Corydalis yanhusuo was separated by systematic solventextraction to obtain the parts. MTT assay was applied to detect the inhibitory effect of totalalkaloid fraction. Apoptosis and cell cycle phases of HepG2 treated by the total alkaloid wereinspected by flow cytometery (FCM). And the expression profile of microRNA in HepG2 wasdetermined by quantitative real-time PCR.
Results
1. The total alkaloid which was isolated from the EtOH extract of this plant is the main fraction displaying antitumor activity.
2. The total alkaloid fraction of C. yanhusuo has a significant cytotoxic effect on ten kinds of human cancer cells in vitro, and IC50 numerical values of it are between 18.39 and 40.24μg/ml.
3. The result of FCM in the range of 25μg/ml, 50μg/ml and 100μg/ml for 24h or 48h on HepG2 cells showed higher apoptosis rates. The ratio of S phase was significantly increased after exposure to the total alkaloid fraction for 24h or 48h compared with the control group. After exposure to the alkaloid for 24h or 48h, the expression of 15 cancer related microRNAs in HepG2 was altered, among which the up-regulation of let-7a and down-regulation of miR-221, mir-222 was the most significant.
Conclusions The total alkaloid fraction which is the main antitumor-active compound of C. yanhusuo has a significant cytotoxic effect in vitro. The mechanism is probably related with its regulatory effects on cell cycle, apoptosis and expression of miRNA in cancer cells.
恶性肿瘤严重威胁人类健康。在抗肿瘤治疗的手段中,药物治疗占据着十分重要的地位。目前抗肿瘤药物的研发和应用的方向,主要是化疗药物的个体化运用和分子靶向药物的研制,然而这些药物带来的不良反应、药物作用靶点的单一性及其昂贵的费用等,是其发展的瓶颈。中医药在抗肿瘤方面的应用有着悠久的历史和坚实的基础,并有着广袤的药用资源。运用现代科技手段对中医药抗肿瘤的作用机制的研究也取得了斐然的成果,包括中药在内的天然药物中的香豆素、生物碱等化合物均被证实具有显著而独特的抗肿瘤活性。从中医药中寻找和挖掘高效、低毒、广谱、廉价的抗肿瘤药物,具有良好的前景,也是中医药工作者努力的方向。
第二部分中药独活抗肿瘤有效单体的提取、分离及其作用研究
目的:提取分离并鉴定中药独活中具有抗肿瘤活性的单体化合物;考察其活性单体的抗肿瘤作用及其机制。
方法:应用硅胶柱色谱、重结晶及波谱分析等方法从中药独活的乙醇提取物中提取分离并鉴定其抗肿瘤活性单体物质;运用四甲基偶氮唑蓝(MTT)法考察该单体化合物的体外抗肿瘤作用;运用MTT法和细胞克隆形成法考察该单体化合物对人胃癌细胞MKN28、MKN45的放射增敏作用;运用流式细胞术、荧光定量PCR等方法考察该化合物放射增敏的作用机制。
结果:
1.从中药独活乙醇提取物中共分离得到4个单体化合物,分别为甲氧基欧芹素、二十四烷酸、二氢欧山芹素、二氢欧山芹醇乙酸酯。其中甲氧基欧芹素为其抗肿瘤活性的主要物质。
2.甲氧基欧芹素对不同组织来源的10种人源肿瘤细胞均有较强的增殖抑制作用,IC_(50)在17.35～34.40μg/ml。
3.甲氧基欧芹素10μg/ml预处理24h对人胃癌细胞系MKN28有放射增敏作用,其增敏比SER_(D0)=1.137。而对MKN45细胞无增敏作用。
4.甲氧基欧芹素10μg/ml预处理24h后放射治疗人胃癌MKN28细胞,可将细胞阻滞在G_0/G_1期,减少S期细胞比例;甲氧基欧芹素可增加放射诱导的细胞凋亡率,并提高放射引起的细胞内活性氧簇(ROS)的水平;放射处理后的极早期,甲氧基欧芹素可下调细胞BRCA1 mRNA的表达水平。
结论:甲氧基欧芹素是中药独活乙醇提取物抗肿瘤作用的主要活性物质,体外具有较强的抑制肿瘤细胞增殖作用,并对人胃癌细胞MKN28有放射增敏作用,其作用机制可能与诱导细胞凋亡、改变细胞周期时相分布、提高细胞内ROS水平以及下调DNA损伤修复相关基因BRCA1 mRNA表达水平下降有关。
第三部分中药延胡索抗肿瘤有效部位的提取、分离及其作用研究
目的:提取分离中药延胡索中具有抗肿瘤作用的有效部位;考察该有效部位的抗肿瘤作用及其机制。
方法:应用系统溶剂法中药延胡索的乙醇提取物中提取分离其抗肿瘤活性部位;运用四甲基偶氮唑蓝(MTT)法考察该有效部位的体外抗肿瘤作用;运用运用流式细胞术、荧光定量PCR等方法考察该化合物抗肿瘤的作用机制。
结果:
1.从中药延胡索中提取分离出其主要抗肿瘤有效部位——延胡索总生物碱。
2.延胡索总生物碱对不同组织来源的10种人源肿瘤细胞均有较强的增殖抑制作用,IC_(50)在18.39～40.24μg/ml。
3.延胡索总生物碱可诱导人肝癌HepG2细胞凋亡,并将HepG2细胞阻滞在S期,有浓度和时间依赖性;IC50浓度延胡索总碱作用于人肝癌HepG2细胞24或48h后,15种miRNAs均被检测到有不同程度的变化,其中尤以let-7a表达的上调和miR-221、miR-222表达的下调较为突出。
结论:延胡索总生物碱是中药延胡索乙醇提取物抗肿瘤作用的主要活性部位,体外具有较强的抑制肿瘤细胞增殖作用,其机制可能与诱导细胞凋亡、改变细胞周期时相分布、改变HepG2细胞miRNA表达谱有关。
PART I:BACKGROUD
Cancer is a kind of disease that extremely threatens human health. Treatment by drugs is one ofthe main methods on cancer. Nowaday, trends in anticancer drugs are personalized usage of chemotherapeutic agents and discovery on molecular targeted ones. However, those roles are limited in clinic by severe side effects and huge costs. It's an important task for us to find out some drugs that possess higher anticancer activity and lower toxicity and are even cheaper. Traditional Chinese medicine (TCM) may be a useful model for scientific research because of its standardized system of therapies and long-time practices. Chinese medicine provides a rich pool of novel and efficacious agents for treating a variety of cancer. So, isolation and identification of new effective constituents of Chinese medicine and investigation its mechanism using molecular biochemistry methods are ascendant aspects of drug-development in our country.
PART II ISOLATION OF ANTITUMOR-ACTIVE CONSTITUENTS FROM ANGELICAPUBESCENS AND INVESTIGATION ON THEIR EFFECTS
Objective To isolate the antitumor-active constituents from Angelica pubescens guided byactivity and investigate their effects on tumor in vitro.
Methods The effective extract of Angelica pubescens was separated by using several types ofcolumn chromtograpys and recrystallization to obtain the compounds, which structures weredetermined by spectroscopic methods, as well as comparing of their spectral data with those ofknown compounds. MTT assay was applied to detect the inhibitory effect of osthol. MTT andclonogenic assay were used to investigate radiosensitivity of osthol on human gastric cancer celllines MKN28 and MKN45. Apoptosis, cell cycle phases and ROS levels of MKN28 treated byosthol combining with radiotherapy were inspected by flow cytometery (FCM). And Realtime-PCR method was used to detect the mRNA expressions of BRCA1 after exposure to OSTand RT.
Results
1. Four compounds were isolated from the EtOH extract of this plant: osthol, columbianadin,lignoceric acid and columbianetin acetate. Among them, osthol is the main compound of thisherb to display antitumor activity.
2. Osthol has a significant cytotoxic effect on ten kinds of human cancer cells in vitro, and IC50 numerical values of OST are between 17.35 and 34.40μg/ml.
3. The enhanced radiosensitivity of gastric cancer cells MKN28 but not MKN45 has been observed. The sensitivity enhancement ratio (SER) at 37% survival level is 1.137 for 24h pretreatment of OST at dose of 10μg/ml.
4. Pretreatment of OST can increase apoptosis, cell cycle arrest at G_0/G_1 and intracellular ROS production induced by RT. And at the very early time after RT, OST can downregulate the expression of BRCA1 mRNA in MKN28.
Conclusions Osthol which is the main antitumor-active compound of Angelica pubescens has a significant cytotoxic effect in vitro and can sensitize MKN28 to radiotherapy. This sensitization may be associated with increasing apoptosis, cell cycle arrest at G_0/G_1 and intracellular ROS production and downregulating the expression of BRCA1 mRNA by OST.
PART III ISOLATION OF ANTITUMOR-ACTIVE PARTS FROM CORYDALISYANHUSUO AND INVESTIGATION ON THEIR EFFECTS
Objective To isolate the antitumor-active parts from Corydalis yanhusuo guided by activityand investigate their effects on tumor in vitro.
Methods The effective extract of Corydalis yanhusuo was separated by systematic solventextraction to obtain the parts. MTT assay was applied to detect the inhibitory effect of totalalkaloid fraction. Apoptosis and cell cycle phases of HepG2 treated by the total alkaloid wereinspected by flow cytometery (FCM). And the expression profile of microRNA in HepG2 wasdetermined by quantitative real-time PCR.
Results
1. The total alkaloid which was isolated from the EtOH extract of this plant is the main fraction displaying antitumor activity.
2. The total alkaloid fraction of C. yanhusuo has a significant cytotoxic effect on ten kinds of human cancer cells in vitro, and IC50 numerical values of it are between 18.39 and 40.24μg/ml.
3. The result of FCM in the range of 25μg/ml, 50μg/ml and 100μg/ml for 24h or 48h on HepG2 cells showed higher apoptosis rates. The ratio of S phase was significantly increased after exposure to the total alkaloid fraction for 24h or 48h compared with the control group. After exposure to the alkaloid for 24h or 48h, the expression of 15 cancer related microRNAs in HepG2 was altered, among which the up-regulation of let-7a and down-regulation of miR-221, mir-222 was the most significant.
Conclusions The total alkaloid fraction which is the main antitumor-active compound of C. yanhusuo has a significant cytotoxic effect in vitro. The mechanism is probably related with its regulatory effects on cell cycle, apoptosis and expression of miRNA in cancer cells.
引文
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