夏枯草黄酮的提取、纯化及其生物活性研究
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摘要
夏枯草为唇形科夏枯草属植物夏枯草的干燥成熟果穗,因“此草夏至后即枯”得名,具有消肿止痛、清火明目、散结等功效,是我国传统的中草药。夏枯草中的主要活性成分为黄酮类化合物、甾醇类及其苷类化合物、香豆素化合物、三萜及其苷类化合物、挥发油、苯丙素类化合物、有机酸等。
到目前为止,对夏枯草中的黄酮类化合物提取、分离纯化以及产品的生物活性未见有系统的报道,影响了夏枯草活性成分的开发和利用。
本文对夏枯草中的黄酮化合物进行了较为系统的研究,旨在充分利用这一丰富的药材资源,为研究开发我国具有自主知识产权的新药提供科学的依据。主要研究内容及实验结果如下：
1.对夏枯草的常规成分进行了分析。结果如下(g/100 g,干重)：总灰分含量为12.55,蛋白质含量为9.77,总糖含量为10.21,还原糖含量为1.32,脂肪含量为4.91,总多酚含量为2.78。采用火焰原子吸收法测定了夏枯草中的Mn、Cu、Mg、Fe、Ca、Zn、Cd等元素。结果表明,夏枯草中含有较为丰富的微量元素。
2.通过3种不同的提取方法(溶剂回流法、超声辅助提取法、微波辅助提取法)的比较,同时结合响应曲面的优化对夏枯草中的黄酮化合物进行提取。结果发现,溶剂回流法优于其他两种提取方法,其最佳提取条件为：乙醇体积分数为20%、提取温度为97.8℃、提取时间为3.2 h、液固比40：1,在此条件下夏枯草黄酮得率为5.72%。
3.通过静态吸附、静态解吸及吸附动力学研究,对D101、AB-8、NKA-9和NKA-Ⅱ4种大孔吸附树脂进行筛选,发现AB-8树脂对夏枯草黄酮有较好的吸附和解析能力,同时对AB-8树脂分离纯化夏枯草中总黄酮的工艺条件进行了优化。结果表明,弱极性树脂AB-8的吸附率为90.29%,解吸率为81.29%,是性能良好的总黄酮吸附剂,其最佳条件为：上样浓度3.732 mg/mL,pH为5左右,上样流速2 mL/min,洗脱液乙醇体积分数40%,解吸液体积5 BV,解吸流速1.5 mL/min。经AB-8大孔树脂纯化后,夏枯草乙醇提取物中总黄酮的纯度由46.25%提高到81.58%。
4.通过测定纯化后的夏枯草黄酮对DPPH和羟基自由基的清除能力、总还原能力、总抗氧化能力、β-胡萝卜素的漂白实验来研究其抗氧化能力。结果表明,纯化后的夏枯草黄酮具有良好的抗氧化能力,可作为一种新型的天然抗氧化剂。
5.选取大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、黑曲霉、米曲霉等5种菌种,用纯化后的的夏枯草黄酮进行体外抑菌实验,对最低抑菌浓度、最低致死浓度和抑菌圈进行测定。实验结果表明,夏枯草黄酮对细菌有较强的抑制作用,而对于霉菌则效果不明显。夏枯草黄酮对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌的最低抑菌浓度分别为1.5625 mg/mL、1.5625 mg/mL.3.125 mg/mL,最低致死浓度分别为3.125 mg/mL、3.125 mg/mL、6.25 mg/mL。因此,夏枯草黄酮可以作为一种理想的抑菌添加剂使用。
Prunella vulgaris Linn is the dry spike of Prunella vulgaris Linn which belongs to self-heal category. Its name comes from the saying "the grass will die away after the Summer Solstice". Prunella vulgaris L. is a traditional Chinese herbal medicine that has detoxification and detumescence effects. The main active components of Prunella vulgaris L. are triterpenes, steroide, phenylpropanoids, flavonoids, coumarone, essential oils, organic acids etc.
Up to now, there is no systematic and detailed studies are found on its extraction, separation, purification and bioactivity, which bring negative effects of utilities and exploitation of active components in Prunella vulgaris L.
Our focus in this paper is on the studying of flavonoids, which can enhance the application of Prunella vulgaris L. and provide a useful guideline for investigation of new drugs. The main investigations and experimental results in this paper were listed as follows, respectively:
1. The nutrients of Prunella vulgaris L. are analyzed. The results are shown as follows (g/100 g, dry mass):ash 12.55, protein 9.77, total sugar 10.21, reduced sugar 1.32, fats 4.91, total phenols 2.78%. The seven trace elements (Mn、Cu、Mg、Fe、Ca、Zn、Cd) were determined by flame atomic absorption spectrometry (FAAS). The results indicated that Prunella vulgaris L. has plenty of trace elements.
2. The Box-Behnken design combined with response surface methodology (RSM) was used to optimize three different extraction methods for total flavonoids from Prunella vulgaris L. The results indicated that the highest extraction yield of flavonoids by heat reflux extraction (HRE) could arrive 5.74% using 20% ethanol as solvent and 1:40 solid to liquid ratio for 3.2 h at the temperature of 97.8℃, which was more efficient than that by ultrasonic-assisted extraction (UAE) and microwaves-assisted extraction(MAE).
3. The separating and purifying purpose of the flavonoids from Prunella vulgaris L. was studied and compared with four kinds of macroporous resins including D101, AB-8, NKA-Ⅱand NKA-9. The adsorption rate, desorption rate and adsorption kinetics of each resin were studied, and some technical conditions were sptimized via static and dynamic experiments. The results showed that the weak polar AB-8 macroporous resin was good sorbent of the flavonoids, its adsorption rate and desorption rate were 90.29%and 81.29%, respectively. The optimum conditions for separating and purifying the flavonoids by AB-8 resin were:injecting concentration 3.732 mg/mL, pH=5, injecting velocity 2.0mL/min,40%(v/v) alcohol as desorption solvent, desorption velocity of flow 1.5 mL/mL and elution volume 5 BV. The content of flavonoids in the purified extracts reached 81.58%from about 46.25%in the crude extracts after AB-8 resin in the optimized conditions.
4. The antioxidant activities of the purified flavonoids were evaluated in vitro by scavenging capability of DPPH free radical and hydroxyl free radical, reducing power, total antioxidant capability and (3-carotene bleaching test. The results showed that the flavonoids have significant antioxidant activities, which can be used as a source of potential antioxidant.
5. The bacillus (Escherichia coli, Staphylococcus aureus and Bacillus subtilis) and fungus (Aspergillus niger and Aspergillus oryzae) were selected to in bacteriostasis experiments in vitro. The effect of inhibiting the microorgansims was determined by the inhibition zone diameter, MIC, MBC test. The results showed that the inhibition of bacillus is better than fungus. It has strong inhibition of Escherichia coli, Staphylococcus aureus and Bacillus subtilis, for MIC are 1.5625 mg/mL,1.5625 mg/mL and 3.125 mg/mL, MBC are 3.125 mg/mL,3.125 mg/mL and 6.25 mg/mL The Prunella vulgaris L. flavonoids were perfect additives which could inhibit some microorganisms.
到目前为止,对夏枯草中的黄酮类化合物提取、分离纯化以及产品的生物活性未见有系统的报道,影响了夏枯草活性成分的开发和利用。
本文对夏枯草中的黄酮化合物进行了较为系统的研究,旨在充分利用这一丰富的药材资源,为研究开发我国具有自主知识产权的新药提供科学的依据。主要研究内容及实验结果如下：
1.对夏枯草的常规成分进行了分析。结果如下(g/100 g,干重)：总灰分含量为12.55,蛋白质含量为9.77,总糖含量为10.21,还原糖含量为1.32,脂肪含量为4.91,总多酚含量为2.78。采用火焰原子吸收法测定了夏枯草中的Mn、Cu、Mg、Fe、Ca、Zn、Cd等元素。结果表明,夏枯草中含有较为丰富的微量元素。
2.通过3种不同的提取方法(溶剂回流法、超声辅助提取法、微波辅助提取法)的比较,同时结合响应曲面的优化对夏枯草中的黄酮化合物进行提取。结果发现,溶剂回流法优于其他两种提取方法,其最佳提取条件为：乙醇体积分数为20%、提取温度为97.8℃、提取时间为3.2 h、液固比40：1,在此条件下夏枯草黄酮得率为5.72%。
3.通过静态吸附、静态解吸及吸附动力学研究,对D101、AB-8、NKA-9和NKA-Ⅱ4种大孔吸附树脂进行筛选,发现AB-8树脂对夏枯草黄酮有较好的吸附和解析能力,同时对AB-8树脂分离纯化夏枯草中总黄酮的工艺条件进行了优化。结果表明,弱极性树脂AB-8的吸附率为90.29%,解吸率为81.29%,是性能良好的总黄酮吸附剂,其最佳条件为：上样浓度3.732 mg/mL,pH为5左右,上样流速2 mL/min,洗脱液乙醇体积分数40%,解吸液体积5 BV,解吸流速1.5 mL/min。经AB-8大孔树脂纯化后,夏枯草乙醇提取物中总黄酮的纯度由46.25%提高到81.58%。
4.通过测定纯化后的夏枯草黄酮对DPPH和羟基自由基的清除能力、总还原能力、总抗氧化能力、β-胡萝卜素的漂白实验来研究其抗氧化能力。结果表明,纯化后的夏枯草黄酮具有良好的抗氧化能力,可作为一种新型的天然抗氧化剂。
5.选取大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、黑曲霉、米曲霉等5种菌种,用纯化后的的夏枯草黄酮进行体外抑菌实验,对最低抑菌浓度、最低致死浓度和抑菌圈进行测定。实验结果表明,夏枯草黄酮对细菌有较强的抑制作用,而对于霉菌则效果不明显。夏枯草黄酮对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌的最低抑菌浓度分别为1.5625 mg/mL、1.5625 mg/mL.3.125 mg/mL,最低致死浓度分别为3.125 mg/mL、3.125 mg/mL、6.25 mg/mL。因此,夏枯草黄酮可以作为一种理想的抑菌添加剂使用。
Prunella vulgaris Linn is the dry spike of Prunella vulgaris Linn which belongs to self-heal category. Its name comes from the saying "the grass will die away after the Summer Solstice". Prunella vulgaris L. is a traditional Chinese herbal medicine that has detoxification and detumescence effects. The main active components of Prunella vulgaris L. are triterpenes, steroide, phenylpropanoids, flavonoids, coumarone, essential oils, organic acids etc.
Up to now, there is no systematic and detailed studies are found on its extraction, separation, purification and bioactivity, which bring negative effects of utilities and exploitation of active components in Prunella vulgaris L.
Our focus in this paper is on the studying of flavonoids, which can enhance the application of Prunella vulgaris L. and provide a useful guideline for investigation of new drugs. The main investigations and experimental results in this paper were listed as follows, respectively:
1. The nutrients of Prunella vulgaris L. are analyzed. The results are shown as follows (g/100 g, dry mass):ash 12.55, protein 9.77, total sugar 10.21, reduced sugar 1.32, fats 4.91, total phenols 2.78%. The seven trace elements (Mn、Cu、Mg、Fe、Ca、Zn、Cd) were determined by flame atomic absorption spectrometry (FAAS). The results indicated that Prunella vulgaris L. has plenty of trace elements.
2. The Box-Behnken design combined with response surface methodology (RSM) was used to optimize three different extraction methods for total flavonoids from Prunella vulgaris L. The results indicated that the highest extraction yield of flavonoids by heat reflux extraction (HRE) could arrive 5.74% using 20% ethanol as solvent and 1:40 solid to liquid ratio for 3.2 h at the temperature of 97.8℃, which was more efficient than that by ultrasonic-assisted extraction (UAE) and microwaves-assisted extraction(MAE).
3. The separating and purifying purpose of the flavonoids from Prunella vulgaris L. was studied and compared with four kinds of macroporous resins including D101, AB-8, NKA-Ⅱand NKA-9. The adsorption rate, desorption rate and adsorption kinetics of each resin were studied, and some technical conditions were sptimized via static and dynamic experiments. The results showed that the weak polar AB-8 macroporous resin was good sorbent of the flavonoids, its adsorption rate and desorption rate were 90.29%and 81.29%, respectively. The optimum conditions for separating and purifying the flavonoids by AB-8 resin were:injecting concentration 3.732 mg/mL, pH=5, injecting velocity 2.0mL/min,40%(v/v) alcohol as desorption solvent, desorption velocity of flow 1.5 mL/mL and elution volume 5 BV. The content of flavonoids in the purified extracts reached 81.58%from about 46.25%in the crude extracts after AB-8 resin in the optimized conditions.
4. The antioxidant activities of the purified flavonoids were evaluated in vitro by scavenging capability of DPPH free radical and hydroxyl free radical, reducing power, total antioxidant capability and (3-carotene bleaching test. The results showed that the flavonoids have significant antioxidant activities, which can be used as a source of potential antioxidant.
5. The bacillus (Escherichia coli, Staphylococcus aureus and Bacillus subtilis) and fungus (Aspergillus niger and Aspergillus oryzae) were selected to in bacteriostasis experiments in vitro. The effect of inhibiting the microorgansims was determined by the inhibition zone diameter, MIC, MBC test. The results showed that the inhibition of bacillus is better than fungus. It has strong inhibition of Escherichia coli, Staphylococcus aureus and Bacillus subtilis, for MIC are 1.5625 mg/mL,1.5625 mg/mL and 3.125 mg/mL, MBC are 3.125 mg/mL,3.125 mg/mL and 6.25 mg/mL The Prunella vulgaris L. flavonoids were perfect additives which could inhibit some microorganisms.
引文
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