积雪草苷的提取、分离纯化与水解
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摘要
积雪草(Centella asiatica (L.) Urban)作为一种传统药材,应用历史悠久,现代医学研究表明积雪草可用于烧伤、抑郁症、胃溃疡等相关疾病的治疗。积雪草中起作用的药理成分主要是皂苷及其皂苷元类,包括积雪草苷、积雪草酸、羟基积雪草苷和羟基积雪草酸等,因此这些物质提取和纯化一直是积雪草研究的重点。近几年来的研究表明,以积雪草酸和羟基积雪草酸等苷元出发,合成的一系列苷元衍生物的药理作用明显优于其原始皂苷,可大大提高积雪草酸、羟基积雪草酸等苷元的利用率。但是由于种种原因,这些活性成分的提取率始终不高,从而造成相关产品价格不菲,限制了积雪草产品的开发和应用。因此,研究积雪草苷的提取、分离纯化及水解,有利于高值化利用积雪草资源。
针对以上情况,本文系统地介绍了积雪草中的活性成分利用现状,并综述了积雪草中活性物质的提取、纯化及分析方法的研究进展。本课题以积雪草为研究对象,对其活性成分积雪草苷和羟基积雪草苷的提取纯化方法进行了研究,并对从积雪草苷类活性物质出发制备积雪草酸以及羟基积雪草酸的工艺进行了探索,建立了一条关于积雪苷的提取、分离和水解制备积雪草酸和羟基积雪草酸的新工艺。主要内容如下。
1.积雪草中积雪草总苷提取工艺研究
探明了比色法测定积雪草总苷含量的显色反应条件,该条件下反应体系稳定,2 h内吸光度基本不变,以羟基积雪草苷为对照品、利用该显色条件测定积雪草总苷含量的方法线性关系良好,检测范围较广。最佳显色反应条件为：香草醛浓度为5%、高氯酸用量0.8 mL、反应温度为60℃、反应时间为15 min。
利用单因素及正交实验优化了浸渍法提取积雪草总苷的提取工艺,确定了最佳提取工艺条件：以70%乙醇水溶液为溶剂,料液比为1：10,室温下提取3次,每次提取时间为4h。
研究了积雪草中积雪草苷的富集部位,结果发现积雪草苷主要富集于积雪草的叶片中,积雪草总苷的含量高达6.12%,而茎部为2.43%,根部含量较低,仅含1.11%。
2.大孔吸附树脂纯化积雪草苷和羟基积雪草苷的工艺研究
通过大孔吸附树脂对积雪草苷和羟基积雪草苷两种皂苷的吸附和解吸性能以及吸附动力学数据,对HPD100、HPD300、X-5、D101和AB-8等5种大孔吸附树脂进行了筛选,并对最优树脂进行了吸附等温线进行了考察,用Langmuir和Freundlich方程对结果进行了拟合,最后通过动态吸附实验,建立了大孔吸附树脂纯化积雪草苷和羟基积雪草苷的工艺。工艺条件为：树脂HPD100为吸附剂,50%的乙醇水溶液为解吸剂,吸附上样流速为2 BV/h,动态解吸流速为2 BV/h,积雪草苷通过梯度洗脱后(洗脱液浓度10-50%的范围内),回收率可达72.0%,含量从上样前的2.0%提高到21.5%；羟基积雪草苷回收率可达70.4%,含量由上样前的3.9%提高到39.7%(洗脱液浓度10-50%的范围内),可以有效达到富集和纯化积雪草苷和羟基积雪草苷的目的。
3.积雪草苷的水解工艺研究
建立了一种同时分析测定积雪草苷、羟基积雪草苷、积雪草酸和羟基积雪草酸的HPLC法,该方法线性关系良好,稳定性强,精密度较高,适用检测范围较广。色谱条件为：Eclipse XDB-C18色谱柱(4.6 mm×250mm,5μm, Agilent);采用动态洗脱,流速为0.6 mL/min;柱温为35℃；检测波长为210 nm。此条件下积雪草苷、积雪草酸、羟基积雪草苷、羟基积雪草酸精密度的RSD分别为0.35%、0.24%、0.17%、0.18%,稳定性的RSD分别为0.54%、0.46%、0.44%、0.35%,平均加样回收率分别为101.09%、99.57%、100.86%、99.99%,均符合我国药典对药物分析的要求。可以应用于积雪草苷、羟基积雪草苷、积雪草酸、羟基积雪草酸的含量测定。
研究了积雪草苷和羟基积雪草苷的水解工艺。通过对催化剂、反应溶剂、皂苷初始浓度、反应时间、反应温度对水解的影响研究发现：最适于积雪草苷的水解的为强碱NaOH;反应溶剂为50%的乙醇水溶液；在本实验所选择的皂苷浓度范围内(积雪草苷0.22～1.10mg/mL;羟基积雪草苷0.40～1.20 mg/mL)水解反应的变化不明显；随着反应温度的升高,皂苷水解的反应速率明显增加,并且在实验温度范围内羟基积雪草苷的水解快于同等条件下积雪草苷的水解。用Arrhenius方程对两种皂苷的水解过程进行拟合,表明积雪草苷和羟基积雪草苷的水解过程符合一级反应,其反应活化能分别为70.1 kJ/mol和83.3 kJ/mol.
通过以上研究,本文建立了一套完整的包括从积雪草原草出发,提取积雪草总苷,大孔树脂富集和纯化积雪草苷和羟基积雪草苷,最后水解制备积雪草酸和羟基积雪草酸的流程,该工艺有效地避免了常规萃取法生产积雪草活性物质高耗能、重污染的缺点,经过该工艺制备的积雪草酸的纯度达91.3%,收率为54.8%,为高效地生产制备积雪草三萜化合物,合成新的积雪草酸或羟基积雪草酸衍生物提供了基础。
Centella asiatica (L.) Urban, as a traditional herbal medicine, has been used in many countries and areas for hundreds of years. According to the reports, Centella asiatica and its active components can be used in treatment of various diseases such as burning wounds, depression and gastric ulcer, etc. The main active components of Centella asiatica are saponins and aglycons, including asiaticoside, madecassoside, asiatic acid and madecassic acid, and their extraction and purification methods have always been one of the main research directions. Recently, some researches showed that under the order conditions asiatic acid and madecassic acid can be synthesized to a series of aglycon derivatives, and these derivatives had better pharmacological actions than their initial saponins. Such results greatly improved the aglycons'utilization. However, due to various reasons, the lower extraction rate of these active components resulted in related products expensive and limited Centella asiatica products development and applications. Therefore, the process of extraction, separation and purification, and hydrolysis of asiaticoside was important for better utilizing Centella asiatica materials.
This dissertation presented the current utilizing situation of active components of Centella asiatica, and introduced the research progress of extraction, purification and analyzing method of them. In order to use these active components efficiently, this study selected Centella asiatica for exploring extraction and purification method of the active components of asiaticoside and madecassoside, and the production progress of asiatic acid and madecassic acid. The research contents and achievements are included as following:
1. Study on extraction of saponins from Centella asiatica
A colorimetric method with selected madecassoside as the standard was established to examine the content of total saponins in Centella asiatica. The optimal reaction conditions of this colorimetric method were measured and listed as follows:the content of vanillin was 5%, the content of perchloric acid was 0.8 mL, the reaction temperature was at 60℃and the reaction time was 15 min. The investigation of the stability of reaction system indicated that the absorbency had no obvious variation during 2 h after the reaction. The result showed that this method had a good linear relationship and had a wider detection range.
Successively, a dipping method to extract total saponins from Centella asiatica was optimized by the single factor and the orthogonal test. The optimal extraction conditions were achieved as follows:the solvent was 70% ethanol-water solution; the ratio of material and solvent volumes was 1:10; extraction times were 3 times at room temperature and each extraction process lasted for 4h.
Finally, the contents of total saponins of different part of Centella asiatica were tested. According to the results, the content of total saponins in the leaf of Centella asiatica was the highest (6.12%), then in the stick (2.43%), and the lowest in the root (1.11%).
2. Enrichment and purification of madecassoside and asiaticoside with macroporous resins.
The adsorption and desorption properties of five macroporous resins including HPD100, HPD300, X-5, AB-8 and D110 for the enrichment and purification of asiaticoside and madecassoside from Centella asiatica extracts have been evaluated. Equilibrium adsorption isotherms were constructed at different temperatures and the model of Langmuir and Freundlich was used to describe the adsorption behavior of the selected optimal resin. Column packed with HPD100 resin was used to separate asiaticoside and madecassoside from Centella asiatica extracts, and the optimal conditions were as follows:resin HPD100 showed the best adsorption and desorption capacity among 5 resins; 50% ethanol-water solution was selected as the eluting solvent; the adsorption and desorption flow rate was both 2 BV/h.
After the treatment with gradient elution on HPD100 resin, the content of madecassoside in the product increased from 3.9 to 39.7%, with the high recovery yield of 70.4%; for asiaticoside the content increased from 2.0 to 21.5%, with the high recovery yield of 72.0%. The results showed that HPD100 resin revealed a good ability to separate madecassoside and asiaticoside.
3. Hydrolysis of asiaticoside and madecassoside
A HPLC method was developed to detect asiaticoside, madecassoside, asiatic acid and madecassic acid synchronously at the same time. The examination of precision, stability and average recovery indicated that this method had a good linear relationship, high stability and precision, and had a wide investigation rang. The chromatography conditions were as following:Eclipse XDB-C18 reversed-phase column (4.6 mm×250 mm,5μm, Agilent); the mobile phase was acetonitrile-acetic acid (0.3%)-water, dynamic elution; the flow rate was 0.6 mL/min:the column was 35℃; the detection wavelength was at 210 nm. The RSD values of precision of asiaticoside, madecassoside, asiatic acid and madecassic acid were 0.35%,0.24%,0.17%and 0.18%, respectively; the RSD values of stability were 0.54%,0.46%,0.44%and 0.35%, respectively; the average recovery were 101.09%,99.57%,100.86%and 99.99%, respectively. All the results above accorded with demands of pharmacopoeia, and could be used in the content detection of asiaticoside, madecassoside, asiatic acid and madecassic acid.
The hydrolysis of asiaticoside and madecassoside was studied by the examination of catalyst, reaction solvent, initial concentration of saponins, reaction time and temperature. NaOH was chosen as the catalyst to hydrolyze asiaticoside and madecassoside to produce asiatic acid and madecassic acid. The ethanol concentration of reaction solution fitting for hydrolysis reaction was 50%. There was no obvious influence on hydrolysis reaction when the contents of NaOH were between 0.1-2.0 mol/mL. The influences of different initial contents of saponins in reaction solution were also investigated, and under the conditions of asiticoside (0.22～1.10 mg/mL) and madecassoside (0.40-1.20 mg/mL) the recovery of saponins varied a little. The reaction temperature had a remarkable effect on hydrolysis reaction, the reaction rate enhanced obviously with the reaction temperature increasing. Arrhenius equation was used to describe the process of saponins hydrolysis, and results revealed that the hydrolysis processes of asiaticoside and madecassoside were both first order reaction process and the active energy was 70.1 kJ/mol and 83.3 kJ/mol, respectively.
In this research, a whole process was established including the production of total saponins of Centella asiatica, enrichment and purification of asiaticoside and madecassoside with macroporous resins, and the production of asiatic acid and madecassic acid. This process effectively avoided the shortcoming of high energy consumption and heavy pollution with using the conventional technique of solvent extraction to produce active components. For asiatic acid, the final purity could reach 91.3% with the recovery of 54.8% through this process. This research offered good methods for the production of new asiatic acid and madecassic acid derivatives.
针对以上情况,本文系统地介绍了积雪草中的活性成分利用现状,并综述了积雪草中活性物质的提取、纯化及分析方法的研究进展。本课题以积雪草为研究对象,对其活性成分积雪草苷和羟基积雪草苷的提取纯化方法进行了研究,并对从积雪草苷类活性物质出发制备积雪草酸以及羟基积雪草酸的工艺进行了探索,建立了一条关于积雪苷的提取、分离和水解制备积雪草酸和羟基积雪草酸的新工艺。主要内容如下。
1.积雪草中积雪草总苷提取工艺研究
探明了比色法测定积雪草总苷含量的显色反应条件,该条件下反应体系稳定,2 h内吸光度基本不变,以羟基积雪草苷为对照品、利用该显色条件测定积雪草总苷含量的方法线性关系良好,检测范围较广。最佳显色反应条件为：香草醛浓度为5%、高氯酸用量0.8 mL、反应温度为60℃、反应时间为15 min。
利用单因素及正交实验优化了浸渍法提取积雪草总苷的提取工艺,确定了最佳提取工艺条件：以70%乙醇水溶液为溶剂,料液比为1：10,室温下提取3次,每次提取时间为4h。
研究了积雪草中积雪草苷的富集部位,结果发现积雪草苷主要富集于积雪草的叶片中,积雪草总苷的含量高达6.12%,而茎部为2.43%,根部含量较低,仅含1.11%。
2.大孔吸附树脂纯化积雪草苷和羟基积雪草苷的工艺研究
通过大孔吸附树脂对积雪草苷和羟基积雪草苷两种皂苷的吸附和解吸性能以及吸附动力学数据,对HPD100、HPD300、X-5、D101和AB-8等5种大孔吸附树脂进行了筛选,并对最优树脂进行了吸附等温线进行了考察,用Langmuir和Freundlich方程对结果进行了拟合,最后通过动态吸附实验,建立了大孔吸附树脂纯化积雪草苷和羟基积雪草苷的工艺。工艺条件为：树脂HPD100为吸附剂,50%的乙醇水溶液为解吸剂,吸附上样流速为2 BV/h,动态解吸流速为2 BV/h,积雪草苷通过梯度洗脱后(洗脱液浓度10-50%的范围内),回收率可达72.0%,含量从上样前的2.0%提高到21.5%；羟基积雪草苷回收率可达70.4%,含量由上样前的3.9%提高到39.7%(洗脱液浓度10-50%的范围内),可以有效达到富集和纯化积雪草苷和羟基积雪草苷的目的。
3.积雪草苷的水解工艺研究
建立了一种同时分析测定积雪草苷、羟基积雪草苷、积雪草酸和羟基积雪草酸的HPLC法,该方法线性关系良好,稳定性强,精密度较高,适用检测范围较广。色谱条件为：Eclipse XDB-C18色谱柱(4.6 mm×250mm,5μm, Agilent);采用动态洗脱,流速为0.6 mL/min;柱温为35℃；检测波长为210 nm。此条件下积雪草苷、积雪草酸、羟基积雪草苷、羟基积雪草酸精密度的RSD分别为0.35%、0.24%、0.17%、0.18%,稳定性的RSD分别为0.54%、0.46%、0.44%、0.35%,平均加样回收率分别为101.09%、99.57%、100.86%、99.99%,均符合我国药典对药物分析的要求。可以应用于积雪草苷、羟基积雪草苷、积雪草酸、羟基积雪草酸的含量测定。
研究了积雪草苷和羟基积雪草苷的水解工艺。通过对催化剂、反应溶剂、皂苷初始浓度、反应时间、反应温度对水解的影响研究发现：最适于积雪草苷的水解的为强碱NaOH;反应溶剂为50%的乙醇水溶液；在本实验所选择的皂苷浓度范围内(积雪草苷0.22～1.10mg/mL;羟基积雪草苷0.40～1.20 mg/mL)水解反应的变化不明显；随着反应温度的升高,皂苷水解的反应速率明显增加,并且在实验温度范围内羟基积雪草苷的水解快于同等条件下积雪草苷的水解。用Arrhenius方程对两种皂苷的水解过程进行拟合,表明积雪草苷和羟基积雪草苷的水解过程符合一级反应,其反应活化能分别为70.1 kJ/mol和83.3 kJ/mol.
通过以上研究,本文建立了一套完整的包括从积雪草原草出发,提取积雪草总苷,大孔树脂富集和纯化积雪草苷和羟基积雪草苷,最后水解制备积雪草酸和羟基积雪草酸的流程,该工艺有效地避免了常规萃取法生产积雪草活性物质高耗能、重污染的缺点,经过该工艺制备的积雪草酸的纯度达91.3%,收率为54.8%,为高效地生产制备积雪草三萜化合物,合成新的积雪草酸或羟基积雪草酸衍生物提供了基础。
Centella asiatica (L.) Urban, as a traditional herbal medicine, has been used in many countries and areas for hundreds of years. According to the reports, Centella asiatica and its active components can be used in treatment of various diseases such as burning wounds, depression and gastric ulcer, etc. The main active components of Centella asiatica are saponins and aglycons, including asiaticoside, madecassoside, asiatic acid and madecassic acid, and their extraction and purification methods have always been one of the main research directions. Recently, some researches showed that under the order conditions asiatic acid and madecassic acid can be synthesized to a series of aglycon derivatives, and these derivatives had better pharmacological actions than their initial saponins. Such results greatly improved the aglycons'utilization. However, due to various reasons, the lower extraction rate of these active components resulted in related products expensive and limited Centella asiatica products development and applications. Therefore, the process of extraction, separation and purification, and hydrolysis of asiaticoside was important for better utilizing Centella asiatica materials.
This dissertation presented the current utilizing situation of active components of Centella asiatica, and introduced the research progress of extraction, purification and analyzing method of them. In order to use these active components efficiently, this study selected Centella asiatica for exploring extraction and purification method of the active components of asiaticoside and madecassoside, and the production progress of asiatic acid and madecassic acid. The research contents and achievements are included as following:
1. Study on extraction of saponins from Centella asiatica
A colorimetric method with selected madecassoside as the standard was established to examine the content of total saponins in Centella asiatica. The optimal reaction conditions of this colorimetric method were measured and listed as follows:the content of vanillin was 5%, the content of perchloric acid was 0.8 mL, the reaction temperature was at 60℃and the reaction time was 15 min. The investigation of the stability of reaction system indicated that the absorbency had no obvious variation during 2 h after the reaction. The result showed that this method had a good linear relationship and had a wider detection range.
Successively, a dipping method to extract total saponins from Centella asiatica was optimized by the single factor and the orthogonal test. The optimal extraction conditions were achieved as follows:the solvent was 70% ethanol-water solution; the ratio of material and solvent volumes was 1:10; extraction times were 3 times at room temperature and each extraction process lasted for 4h.
Finally, the contents of total saponins of different part of Centella asiatica were tested. According to the results, the content of total saponins in the leaf of Centella asiatica was the highest (6.12%), then in the stick (2.43%), and the lowest in the root (1.11%).
2. Enrichment and purification of madecassoside and asiaticoside with macroporous resins.
The adsorption and desorption properties of five macroporous resins including HPD100, HPD300, X-5, AB-8 and D110 for the enrichment and purification of asiaticoside and madecassoside from Centella asiatica extracts have been evaluated. Equilibrium adsorption isotherms were constructed at different temperatures and the model of Langmuir and Freundlich was used to describe the adsorption behavior of the selected optimal resin. Column packed with HPD100 resin was used to separate asiaticoside and madecassoside from Centella asiatica extracts, and the optimal conditions were as follows:resin HPD100 showed the best adsorption and desorption capacity among 5 resins; 50% ethanol-water solution was selected as the eluting solvent; the adsorption and desorption flow rate was both 2 BV/h.
After the treatment with gradient elution on HPD100 resin, the content of madecassoside in the product increased from 3.9 to 39.7%, with the high recovery yield of 70.4%; for asiaticoside the content increased from 2.0 to 21.5%, with the high recovery yield of 72.0%. The results showed that HPD100 resin revealed a good ability to separate madecassoside and asiaticoside.
3. Hydrolysis of asiaticoside and madecassoside
A HPLC method was developed to detect asiaticoside, madecassoside, asiatic acid and madecassic acid synchronously at the same time. The examination of precision, stability and average recovery indicated that this method had a good linear relationship, high stability and precision, and had a wide investigation rang. The chromatography conditions were as following:Eclipse XDB-C18 reversed-phase column (4.6 mm×250 mm,5μm, Agilent); the mobile phase was acetonitrile-acetic acid (0.3%)-water, dynamic elution; the flow rate was 0.6 mL/min:the column was 35℃; the detection wavelength was at 210 nm. The RSD values of precision of asiaticoside, madecassoside, asiatic acid and madecassic acid were 0.35%,0.24%,0.17%and 0.18%, respectively; the RSD values of stability were 0.54%,0.46%,0.44%and 0.35%, respectively; the average recovery were 101.09%,99.57%,100.86%and 99.99%, respectively. All the results above accorded with demands of pharmacopoeia, and could be used in the content detection of asiaticoside, madecassoside, asiatic acid and madecassic acid.
The hydrolysis of asiaticoside and madecassoside was studied by the examination of catalyst, reaction solvent, initial concentration of saponins, reaction time and temperature. NaOH was chosen as the catalyst to hydrolyze asiaticoside and madecassoside to produce asiatic acid and madecassic acid. The ethanol concentration of reaction solution fitting for hydrolysis reaction was 50%. There was no obvious influence on hydrolysis reaction when the contents of NaOH were between 0.1-2.0 mol/mL. The influences of different initial contents of saponins in reaction solution were also investigated, and under the conditions of asiticoside (0.22～1.10 mg/mL) and madecassoside (0.40-1.20 mg/mL) the recovery of saponins varied a little. The reaction temperature had a remarkable effect on hydrolysis reaction, the reaction rate enhanced obviously with the reaction temperature increasing. Arrhenius equation was used to describe the process of saponins hydrolysis, and results revealed that the hydrolysis processes of asiaticoside and madecassoside were both first order reaction process and the active energy was 70.1 kJ/mol and 83.3 kJ/mol, respectively.
In this research, a whole process was established including the production of total saponins of Centella asiatica, enrichment and purification of asiaticoside and madecassoside with macroporous resins, and the production of asiatic acid and madecassic acid. This process effectively avoided the shortcoming of high energy consumption and heavy pollution with using the conventional technique of solvent extraction to produce active components. For asiatic acid, the final purity could reach 91.3% with the recovery of 54.8% through this process. This research offered good methods for the production of new asiatic acid and madecassic acid derivatives.
引文
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