蕲艾鞣酸的提取分离、药理研究和结构鉴定
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摘要
艾叶(Artemisiae argyi)为菊科(Asteraceae)蒿属(Artemisia)植物,主要分布于亚洲东部地区,普遍生长于路旁荒野、草地。只要是向阳而排水顺畅的地方就能生长,但以湿润肥沃的土壤生长较好。本属植物资源极其丰富,近年来研究发现该属植物具有明显的抗氧化、抗癌等药理作用,引起了众多学者对该属植物研究的兴趣。其中对艾叶(Artemisiae argyi)的研究比较广泛,从该植物中分离得到了大量化学成分,并探索出各种活性能力。
本文以中药艾叶为研究对象,探索提取艾叶鞣酸的最佳工艺方法,并测定了不同地区艾叶中总鞣酸的含量,研究了蕲艾鞣酸清除自由基的能力﹑体外凝血作用﹑以及抗肿瘤等药理作用,并分析了蕲艾中的有效成分,鉴定其化合物的结构,。具体工作如下:
1.采用超声波法提取艾叶中鞣酸,通过四因素三水平正交试验探讨超声波提取艾叶中鞣酸的最佳工艺条件,并对湖北蕲春、安徽霍山、山东鄄城、江西樟树、河北安国五个产地艾叶中的总鞣酸含量进行分析比较,结果表明在超声温度60℃,超声功率150W,超声时间20min,固液比1:25,丙酮:水4:6的条件下鞣酸的提取率最高,其中湖北蕲春的艾叶总鞣酸含量达到13.29%,是所有研究品种中含量最高的,说明蕲春作为艾叶的地道产地具有实验依据。
2.通过Fenton反应产生·OH,连苯三酚自氧化产生O2-?模型,采用紫外分光光度法,研究蕲艾总鞣酸体外清除自由基的能力,结果表明蕲艾总鞣酸在1.0～10.0 mg/mL范围内对羟自由基和超氧阴离子自由基均具有清除作用,两者均呈现明显的量效关系,最有效的清除浓度均为8.0mg/mL,清除自由基能力均高于同等浓度的甘露醇,但低于同等浓度的抗坏血酸。
3.通过MTT法在体外检测不同浓度的蕲艾鞣酸对人肝癌细胞HepG2的细胞毒性作用,在此基础上以大孔树脂柱层析和薄层纯化对蕲艾总鞣酸正丁醇部位提取分离,得到6个不同极性的鞣酸馏分,并通过MTT法比较不同极性的各提取物对肝癌细胞的增殖影响。其结果显示蕲艾鞣酸使体外培养的人肝癌细胞HepG2的凋亡细胞数增加,增值效率下降且呈浓度与时间的依赖关系。6个不同极性的提取物均具有抑制肝癌细胞生长的作用,其中40%甲醇部位对肝癌细胞的生长抑制作用最强,是蕲艾鞣酸体外抗肝癌主要的活性部位。
4.将蕲春艾叶中不同组分按照传统方法进行炮制或者提取分离,得到六个不同组分,分别为:5一叔丁基连苯三酚、艾炭、艾灰、艾叶挥发油、鞣酸和艾焦油,使用家兔对以上六个组分分别进行了体外凝血试验,结果显示蕲春艾叶鞣酸的凝血效果最好,是主要的凝血物质。
5.针对蕲艾的活性成分进行了系统的提取分离和结构研究,在实验过程中使用大孔吸附树脂层析﹑硅胶柱层析﹑反相硅胶柱层析﹑薄层层析等方法,从湖北蕲春艾叶中提取分离出两个纯净化合物,结合红外﹑紫外和核磁等现代仪器鉴定分析单体化合物为:(A)5,6-二羟基-3’4’5’-三甲氧基黄酮,(B)丙基-2-丁烯醚。以上两种化合物均首次从该植物中分离得到。
The Artemisiae argyi belongs to Asteraceae Artemisia vegetation, mainly in the eastern region of Asia. It generally grows at the roadside wilderness and grass. The Artemisiae argyi can grow at any areas where there are enough sunshine and drainage. But it can grow better in the moist fertile soil. The Artemisia vegetation has rich resource in the world. The recent studies have found that the Artemisia vegetation has a significant antioxidant, anticancer and other pharmacological effects, this causing many scholars interested in the study of the Artemisia vegetation. Where more extensive research is done on the Artemisiae argyi, a large number of chemical components have been isolated from this plant, and found the ability of their various activities.
In this paper, we use the Artemisiae argyi as the research object in order to explore the optimal extraction process of tannins. Firstly we measured the total tannins content of the Artemisiae argyi in different areas and analyzed the active ingredients in Artemisiae argyi of Qichun. We identified the structure of the compounds and do some research on the free radical scavenging ability of the QiAis tannins. We also studied the pharmacological effects of the tannins in vitro blood coagulation and anti-tumor aspects. The specific work as follows:
1. We extract the tannins of the Artemisiae argyi by using the ultrasonic method and research the optimum technology conditions of ultrasonic extraction process for the Artemisiae argyi tannins extraction through the four factors and three levels experiment and orthogonal experiment. We analyzed and compared the total content of tannins in the Artemisiae argyi that come from five origin places (they are Qichun country in Hubei, Zhangshu country in Jiangxi , Huoshan country in Anhui , Juancheng country in Shandong , Anguo country in Hebei). The experimental results indicate that we can get the highest extraction rate under the conditions that the ultrasound at 60℃, ultrasonic power is 150W, ultrasonic time is 20min and the weight of ratio of solid to liquid was 1:25, the ratio of Acetone to Water was 4:6. The total tannin content of the Artemisiae argyi that come from HuBei Qichun is 13.29%.That is the highest content in the five research objects. That result indicates that we have trial basis to consider that the Qichun is the typical origin of the Artemisiae argyi.
2. We research the free radical scavenging ability in vitro of tannins in the Qichun Artemisiae argyi by using UV spectrophotometry, producing ?OH through the Fenton reaction and producing O2? model through Pyrogallol Autoxidation. The results show that the total tannins content have scavenging effect on the hydroxyl radical and superoxide radical in the range of 1.0～10.0 mg/mL. The tannins show a clear dose dependent mode to the hydroxyl radical and superoxide radical. The most effective scavenging concentrations are both 8.0 mg/mL. The free radical scavenging capacity of the tannins is higher than that of the mannitol with the same concentration but lower than that of the vitamin C with the same concentration.
3. We detect the cytotoxicity of the Qichun Artemisiae argyi tannins of different concentrations to the human hepatoma cell HepG2 by using MTT method in vitro. On this basis the butanol in the Qichun Artemisiae argyi tannins is extracted through the column chromatography of macroporous resin and TLC purification method. Then five tannin distillation fractions with different polarity are obtained. We compare the proliferation effectiveness of the extracts with different polarities on the hepatoma cell by MTT method. The result shows that the Qichun Artemisiae argyi tannins make the hepatoma cell HepG2 in vitro increase in the number of apoptosis cells. The value-added efficiency decrease at the same time the concentration and time express the dependent relationship. The five different polar extracts all can inhibit the growth of the hepatoma cell. There is 40% methanol has strongest inhibition effect for the hepatoma cell growth. It is the major activity part in the Qichun Artemisiae argyi tannins to antitumor in vitro.
4. We obtained six different components of compounds through extracting and separating the Qichun Artemisiae argyi with traditional method. The compounds are: 5 a-tert-butyl pyrogallol, Ai charcoal, Ai ash, essential oil, tannins and Ai tar. By using the above six compounds to do coagulation tests in vitro on rabbits we find that the Qichun Artemisiae argyi tannins have the best effect in the blood clotting, it is the main material for blood coagulation.
5. We extract and separate and do some structure research on the active ingredient of the Qichun Artemisiae argyi. During the experiment we use many methods such as Macroporous resin chromatography , Silica gel column chromatography,RP silica gel column chromatography and TLC. We extract and isolate two pure compounds from the Artemisiae argyi in Hubei Qichun, then we analyze and identify the two monomer compounds by using the modern equipment such as IR ,UV and NMR. We find the two compounds are (A) 5,6-Dihydroxy-3’4’5’-Trimethoxyflavone and (B)5-C butylene ether. These two compounds were first isolated from this plant.
本文以中药艾叶为研究对象,探索提取艾叶鞣酸的最佳工艺方法,并测定了不同地区艾叶中总鞣酸的含量,研究了蕲艾鞣酸清除自由基的能力﹑体外凝血作用﹑以及抗肿瘤等药理作用,并分析了蕲艾中的有效成分,鉴定其化合物的结构,。具体工作如下:
1.采用超声波法提取艾叶中鞣酸,通过四因素三水平正交试验探讨超声波提取艾叶中鞣酸的最佳工艺条件,并对湖北蕲春、安徽霍山、山东鄄城、江西樟树、河北安国五个产地艾叶中的总鞣酸含量进行分析比较,结果表明在超声温度60℃,超声功率150W,超声时间20min,固液比1:25,丙酮:水4:6的条件下鞣酸的提取率最高,其中湖北蕲春的艾叶总鞣酸含量达到13.29%,是所有研究品种中含量最高的,说明蕲春作为艾叶的地道产地具有实验依据。
2.通过Fenton反应产生·OH,连苯三酚自氧化产生O2-?模型,采用紫外分光光度法,研究蕲艾总鞣酸体外清除自由基的能力,结果表明蕲艾总鞣酸在1.0～10.0 mg/mL范围内对羟自由基和超氧阴离子自由基均具有清除作用,两者均呈现明显的量效关系,最有效的清除浓度均为8.0mg/mL,清除自由基能力均高于同等浓度的甘露醇,但低于同等浓度的抗坏血酸。
3.通过MTT法在体外检测不同浓度的蕲艾鞣酸对人肝癌细胞HepG2的细胞毒性作用,在此基础上以大孔树脂柱层析和薄层纯化对蕲艾总鞣酸正丁醇部位提取分离,得到6个不同极性的鞣酸馏分,并通过MTT法比较不同极性的各提取物对肝癌细胞的增殖影响。其结果显示蕲艾鞣酸使体外培养的人肝癌细胞HepG2的凋亡细胞数增加,增值效率下降且呈浓度与时间的依赖关系。6个不同极性的提取物均具有抑制肝癌细胞生长的作用,其中40%甲醇部位对肝癌细胞的生长抑制作用最强,是蕲艾鞣酸体外抗肝癌主要的活性部位。
4.将蕲春艾叶中不同组分按照传统方法进行炮制或者提取分离,得到六个不同组分,分别为:5一叔丁基连苯三酚、艾炭、艾灰、艾叶挥发油、鞣酸和艾焦油,使用家兔对以上六个组分分别进行了体外凝血试验,结果显示蕲春艾叶鞣酸的凝血效果最好,是主要的凝血物质。
5.针对蕲艾的活性成分进行了系统的提取分离和结构研究,在实验过程中使用大孔吸附树脂层析﹑硅胶柱层析﹑反相硅胶柱层析﹑薄层层析等方法,从湖北蕲春艾叶中提取分离出两个纯净化合物,结合红外﹑紫外和核磁等现代仪器鉴定分析单体化合物为:(A)5,6-二羟基-3’4’5’-三甲氧基黄酮,(B)丙基-2-丁烯醚。以上两种化合物均首次从该植物中分离得到。
The Artemisiae argyi belongs to Asteraceae Artemisia vegetation, mainly in the eastern region of Asia. It generally grows at the roadside wilderness and grass. The Artemisiae argyi can grow at any areas where there are enough sunshine and drainage. But it can grow better in the moist fertile soil. The Artemisia vegetation has rich resource in the world. The recent studies have found that the Artemisia vegetation has a significant antioxidant, anticancer and other pharmacological effects, this causing many scholars interested in the study of the Artemisia vegetation. Where more extensive research is done on the Artemisiae argyi, a large number of chemical components have been isolated from this plant, and found the ability of their various activities.
In this paper, we use the Artemisiae argyi as the research object in order to explore the optimal extraction process of tannins. Firstly we measured the total tannins content of the Artemisiae argyi in different areas and analyzed the active ingredients in Artemisiae argyi of Qichun. We identified the structure of the compounds and do some research on the free radical scavenging ability of the QiAis tannins. We also studied the pharmacological effects of the tannins in vitro blood coagulation and anti-tumor aspects. The specific work as follows:
1. We extract the tannins of the Artemisiae argyi by using the ultrasonic method and research the optimum technology conditions of ultrasonic extraction process for the Artemisiae argyi tannins extraction through the four factors and three levels experiment and orthogonal experiment. We analyzed and compared the total content of tannins in the Artemisiae argyi that come from five origin places (they are Qichun country in Hubei, Zhangshu country in Jiangxi , Huoshan country in Anhui , Juancheng country in Shandong , Anguo country in Hebei). The experimental results indicate that we can get the highest extraction rate under the conditions that the ultrasound at 60℃, ultrasonic power is 150W, ultrasonic time is 20min and the weight of ratio of solid to liquid was 1:25, the ratio of Acetone to Water was 4:6. The total tannin content of the Artemisiae argyi that come from HuBei Qichun is 13.29%.That is the highest content in the five research objects. That result indicates that we have trial basis to consider that the Qichun is the typical origin of the Artemisiae argyi.
2. We research the free radical scavenging ability in vitro of tannins in the Qichun Artemisiae argyi by using UV spectrophotometry, producing ?OH through the Fenton reaction and producing O2? model through Pyrogallol Autoxidation. The results show that the total tannins content have scavenging effect on the hydroxyl radical and superoxide radical in the range of 1.0～10.0 mg/mL. The tannins show a clear dose dependent mode to the hydroxyl radical and superoxide radical. The most effective scavenging concentrations are both 8.0 mg/mL. The free radical scavenging capacity of the tannins is higher than that of the mannitol with the same concentration but lower than that of the vitamin C with the same concentration.
3. We detect the cytotoxicity of the Qichun Artemisiae argyi tannins of different concentrations to the human hepatoma cell HepG2 by using MTT method in vitro. On this basis the butanol in the Qichun Artemisiae argyi tannins is extracted through the column chromatography of macroporous resin and TLC purification method. Then five tannin distillation fractions with different polarity are obtained. We compare the proliferation effectiveness of the extracts with different polarities on the hepatoma cell by MTT method. The result shows that the Qichun Artemisiae argyi tannins make the hepatoma cell HepG2 in vitro increase in the number of apoptosis cells. The value-added efficiency decrease at the same time the concentration and time express the dependent relationship. The five different polar extracts all can inhibit the growth of the hepatoma cell. There is 40% methanol has strongest inhibition effect for the hepatoma cell growth. It is the major activity part in the Qichun Artemisiae argyi tannins to antitumor in vitro.
4. We obtained six different components of compounds through extracting and separating the Qichun Artemisiae argyi with traditional method. The compounds are: 5 a-tert-butyl pyrogallol, Ai charcoal, Ai ash, essential oil, tannins and Ai tar. By using the above six compounds to do coagulation tests in vitro on rabbits we find that the Qichun Artemisiae argyi tannins have the best effect in the blood clotting, it is the main material for blood coagulation.
5. We extract and separate and do some structure research on the active ingredient of the Qichun Artemisiae argyi. During the experiment we use many methods such as Macroporous resin chromatography , Silica gel column chromatography,RP silica gel column chromatography and TLC. We extract and isolate two pure compounds from the Artemisiae argyi in Hubei Qichun, then we analyze and identify the two monomer compounds by using the modern equipment such as IR ,UV and NMR. We find the two compounds are (A) 5,6-Dihydroxy-3’4’5’-Trimethoxyflavone and (B)5-C butylene ether. These two compounds were first isolated from this plant.
引文
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