杜仲叶中绿原酸提取制备研究及绿原酸分子印记聚合物的合成和静态吸附性质初探
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摘要
绿原酸具有显著的清热解毒、抗菌消炎和抗衰老作用，对消化道的癌症有明显的抑制作用，具有抗生素作用及对免疫系统的调节作用。本文主要对从杜仲叶中提取、分离和纯化绿原酸，及产品的检测做了研究。结果如下：
1．绿原酸的提取
首先用氯仿除去杜仲叶中过多的脂类物质，然后根据影响中药有效成分提取的各种因素，采用单因素试验和均匀设计实验，分别考察了溶剂、温度、时间、料液比和提取次数对绿原酸提取效果的影响，得出最佳提取工艺条件：超声频率40Hz、乙醇浓度50％、料液比1／30、45℃下提取50mim、提取次数为4次，并验证了提取工艺条件是稳定可靠的，浸膏的得率为6.38％。
2．绿原酸的分离纯化及检测
将铅沉淀法应用到杜仲叶中绿原酸的分离，产品经重结晶后得到少量绿原酸晶体，晶体外观与标准品结晶体外观相似，产品晶体熔点(208～209℃)与文献相符合。
利用两次薄层色谱分离纯化杜仲叶中的绿原酸，产品在分析型硅胶G薄层板上条状点样，在甲醇～乙酸(4：0.2)溶液中展开，使绿原酸与难分离的黄色物质分离；再用硅胶H的制备型薄层板上条状点样后用乙酸丁酯～甲酸～水(7：2.5：2.5)的上层清液展开，得到绿原酸产品，产品的紫外光谱特征与文献相符，薄层色谱主斑与绿原酸标准品对应，HPLC的主峰t_R与绿原酸标准品的t_R吻合。HPLC外标法测得产品的纯度为91.6％，并讨论了两次薄层色谱展开条件和第2次薄板的处理。所建立的纯化制备方法简便、经济、直观、对绿原酸有很好的分离效果，适合于数百毫克级～克级的制备，并易于在一般实验室应用。
运用溶剂萃取—沉淀法得到了绿原酸沉淀，通过薄层进一步分离得到了绿原酸的针状沉淀，其紫外吸收与标准品相似，表明产品中含有绿原酸。
用沉淀聚合的方法合成了分子印迹聚合物，结果表明EDMA∶AIBN(77mg∶0.97mg)聚合得到白色粉末状固体。通过比较印迹聚合物和空白聚合物在不同乙腈浓度下对模板分子绿原酸的静态吸附，发现印迹聚合物对模板分子的吸附仅略高于空白聚合物对模板分子的吸附，结果不理想，原因还需要进一步的研究。还发现印迹聚合物在纯水中对绿原酸的吸附效果较好。
Chlorogenic acid has dramatic effects of heat-clearing and detoxifying, antibiosis and anti-inflammatory, and anti-aging, it has obvious inhibiting effects on digestive tract cancer, also has anti- and adjustment on immune system.This paper laid emphases on the study of extraction, separation and purification of chlorogenic acid from eucommia ulmodies leaves, and the detection of the products. The results are following.
1. The extraction of chlorogenic acid
First, the effects of factors such as solvent, tempreture, time, the radio of material to solvent, times of extraction on the extracion of chlorogenic acid were investigated, respectively, by single-factor experiments, and then by uniform design experiments. The obtained optimal process condition were, 50% of ethanol, 1/30 of the radio of material to solvent, extracting for 50min in ultrasonic bath of 40Hz frequency at 40℃, 4 times. The stability and reliability of the proposed process conditions were tested. The yield of ethonal extract of Eucommia Ulmodies Leaves was 6.38%.
2. Isolation, purification and detection of chlorogenic acid
Using lead precipitation as a method of separation of chlorogenic acid from eucommia ulmodies leaves. The crystalline morphology of the precipite was similar to that of chlorogenic acid standard, and the melting-point (208-209℃) agreed with the literature.
Alternatively, twice preparative TLC were applied to isolate and purify the ethonal extract. The first preparative TLC using a development solution of methanol-acetic acid (4:0.2) and a silica gel G plate. It was found that chlorogenic acid was seperated from yellow substances which were hard to separate. The second preparative TLC using a mixture of the supernatant of butyl-methanol-water solution(7:2.5:2.5) and a silica gel H plate.
The finall products of purifyication were analyzed by analytical TLC using the same condtions sa in the second preparative TLC. The ultraviolet spectrum of the product was similar to that given in literatures. The HPLC main peak and the TLC main spot of the procduct were in accord with that of chlorogenic acid standard. The purity of chlorogenic acid was larger than 91.6% by using HPLC external reference methods. The proposed method is simple, and suitable for commen laboratory to preparate several hundreds milligram to one gramme of chlorogenic acid.
The third method of isolation and purification was solvent extraction-precipitation Process. Needle-like sediment was obtained by further TLC separation, it's ultraviolet spectrum was similar to that of chlorogenic acid standard.
White power molecular imprinting polymer of chlorogenic acid was made by precipitation polymerization, under the condition of the ratio of EDMA to AIBN was 77mg : 0.97mg. The static adsorption experiment shown that the adsorptions of chlorogenic acid to the imprinting polymer were just little higher than those to the blank polymer in different concentration solutions of acetonitrile, and that water was better than aqueous solutions of acetonitrile for the adsorption of chlorogenic acid.
1．绿原酸的提取
首先用氯仿除去杜仲叶中过多的脂类物质，然后根据影响中药有效成分提取的各种因素，采用单因素试验和均匀设计实验，分别考察了溶剂、温度、时间、料液比和提取次数对绿原酸提取效果的影响，得出最佳提取工艺条件：超声频率40Hz、乙醇浓度50％、料液比1／30、45℃下提取50mim、提取次数为4次，并验证了提取工艺条件是稳定可靠的，浸膏的得率为6.38％。
2．绿原酸的分离纯化及检测
将铅沉淀法应用到杜仲叶中绿原酸的分离，产品经重结晶后得到少量绿原酸晶体，晶体外观与标准品结晶体外观相似，产品晶体熔点(208～209℃)与文献相符合。
利用两次薄层色谱分离纯化杜仲叶中的绿原酸，产品在分析型硅胶G薄层板上条状点样，在甲醇～乙酸(4：0.2)溶液中展开，使绿原酸与难分离的黄色物质分离；再用硅胶H的制备型薄层板上条状点样后用乙酸丁酯～甲酸～水(7：2.5：2.5)的上层清液展开，得到绿原酸产品，产品的紫外光谱特征与文献相符，薄层色谱主斑与绿原酸标准品对应，HPLC的主峰t_R与绿原酸标准品的t_R吻合。HPLC外标法测得产品的纯度为91.6％，并讨论了两次薄层色谱展开条件和第2次薄板的处理。所建立的纯化制备方法简便、经济、直观、对绿原酸有很好的分离效果，适合于数百毫克级～克级的制备，并易于在一般实验室应用。
运用溶剂萃取—沉淀法得到了绿原酸沉淀，通过薄层进一步分离得到了绿原酸的针状沉淀，其紫外吸收与标准品相似，表明产品中含有绿原酸。
用沉淀聚合的方法合成了分子印迹聚合物，结果表明EDMA∶AIBN(77mg∶0.97mg)聚合得到白色粉末状固体。通过比较印迹聚合物和空白聚合物在不同乙腈浓度下对模板分子绿原酸的静态吸附，发现印迹聚合物对模板分子的吸附仅略高于空白聚合物对模板分子的吸附，结果不理想，原因还需要进一步的研究。还发现印迹聚合物在纯水中对绿原酸的吸附效果较好。
Chlorogenic acid has dramatic effects of heat-clearing and detoxifying, antibiosis and anti-inflammatory, and anti-aging, it has obvious inhibiting effects on digestive tract cancer, also has anti- and adjustment on immune system.This paper laid emphases on the study of extraction, separation and purification of chlorogenic acid from eucommia ulmodies leaves, and the detection of the products. The results are following.
1. The extraction of chlorogenic acid
First, the effects of factors such as solvent, tempreture, time, the radio of material to solvent, times of extraction on the extracion of chlorogenic acid were investigated, respectively, by single-factor experiments, and then by uniform design experiments. The obtained optimal process condition were, 50% of ethanol, 1/30 of the radio of material to solvent, extracting for 50min in ultrasonic bath of 40Hz frequency at 40℃, 4 times. The stability and reliability of the proposed process conditions were tested. The yield of ethonal extract of Eucommia Ulmodies Leaves was 6.38%.
2. Isolation, purification and detection of chlorogenic acid
Using lead precipitation as a method of separation of chlorogenic acid from eucommia ulmodies leaves. The crystalline morphology of the precipite was similar to that of chlorogenic acid standard, and the melting-point (208-209℃) agreed with the literature.
Alternatively, twice preparative TLC were applied to isolate and purify the ethonal extract. The first preparative TLC using a development solution of methanol-acetic acid (4:0.2) and a silica gel G plate. It was found that chlorogenic acid was seperated from yellow substances which were hard to separate. The second preparative TLC using a mixture of the supernatant of butyl-methanol-water solution(7:2.5:2.5) and a silica gel H plate.
The finall products of purifyication were analyzed by analytical TLC using the same condtions sa in the second preparative TLC. The ultraviolet spectrum of the product was similar to that given in literatures. The HPLC main peak and the TLC main spot of the procduct were in accord with that of chlorogenic acid standard. The purity of chlorogenic acid was larger than 91.6% by using HPLC external reference methods. The proposed method is simple, and suitable for commen laboratory to preparate several hundreds milligram to one gramme of chlorogenic acid.
The third method of isolation and purification was solvent extraction-precipitation Process. Needle-like sediment was obtained by further TLC separation, it's ultraviolet spectrum was similar to that of chlorogenic acid standard.
White power molecular imprinting polymer of chlorogenic acid was made by precipitation polymerization, under the condition of the ratio of EDMA to AIBN was 77mg : 0.97mg. The static adsorption experiment shown that the adsorptions of chlorogenic acid to the imprinting polymer were just little higher than those to the blank polymer in different concentration solutions of acetonitrile, and that water was better than aqueous solutions of acetonitrile for the adsorption of chlorogenic acid.
引文
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