Study The Extraction,Separation,Pharmacology And Structural Identification Of Tannins From Artemisiae Argyi

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.