金银花中绿原酸的提取分离及其抑菌作用研究
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摘要
由于绿原酸对许多疾病都具有明显的疗效,日益受到医学及药学界的重视,社会需求巨大,但目前国内绿原酸的相关研究尚非常薄弱,尚无成熟的绿原酸纯品生产工艺。本研究的主要目的是为了建立稳定有效的金银花绿原酸提取纯化物的检测鉴定方法;明确绿原酸的化学稳定性为其生产和储存工艺提供参考;通过工艺优化寻找更经济高效的绿原酸提取及分离纯化方法;通过对几种食品中常见致病菌的绿原酸抑制活性研究,进一步明确绿原酸的抑菌活性,完善绿原酸的抗菌谱。研究结果如下:
绿原酸鉴定检测方面:硅胶薄层层析经紫外成相分析得知,乙酸乙酯:水:乙酸(v/v,10:3:2)为流动相配比,保留因子值达到0.68;由紫外可见光光度仪在400 nm-200 nm波长扫描得知绿原酸在紫外327 nm波长处有最大吸收,以浓度C(x轴)对吸光度A (y轴)作图绘制UV标准曲线,标准曲线回归方程:y =0.0553x-0.0034,R=0.9996; 0～15μg/ml线性关系良好;经实验优化,高效液相分析参数为:色谱柱ZOBAX SB(150 mm×4.6 mm,5μm);流动相为甲醇:水:乙酸(18:81:1),流速:1 mL/min,检测波长:327 nm,柱温:30℃,进样量:20μL;此条件下,样品中绿原酸与相邻成分基线分离,以浓度C(x轴)对峰面积V(y轴)作图,得到标准曲线回归方程为:y = 51813 x-5832,R=0.9991,0～13μg/ml范围内线性关系良好;采用外标法确认提取的绿原酸的保留时间为8.832 min;同法测得原料金银花绿原酸含量为3.3 %(n=3,RSD=2.0 %),回收率98 %;而原料金银花浸膏的绿原酸含量为10.15 %(n=3,RSD=1.8 %)。
绿原酸的稳定性实验表明:相同存储条件下,绿原酸在pH 3.0时比pH 5.6时更稳定;而同样溶剂条件下,绿原酸于4℃恒温冰箱避光放置时比其25℃恒温储藏室自然光放置更稳定,储存30天后,经单因素Duncan多重函数检验比较得出二组实验数据在置信区间5 %水平上均表现为显著差异。
本文通过对醇回流法、超声波提取法、微波辅助提取法等方法的考察发现微波-超声波联合提取法的绿原酸提取率最高,且提取绿原酸的提取率比目前最普遍采用的70 %乙醇回流法的提取率71.15 %高20 %以上,远高于单独的超声法或微波辅助提取法50 %的水平。绿原酸经微波破壁后继续使用20Hz的低频率超声波温和提取,2次重复绿原酸的提取率即达到了92.66 %;微波-超声波联合提取法正交实验得出其最佳工艺参数为:在250 W功率下微波预处理3次,30 s/次;按料液比(v/v,1/20)加入70 %乙醇,在功率18 W下超声提取20 min,一次性提取率即达73 %以上;各因素的影响从大到小为:超声功率>溶剂倍量>超声时间。
以重庆金银花浸膏为原料,以绿原酸提取率为指标,采用紫外光谱法、高效液相法定性定量,着重研究比较了金属离子沉淀法与柱层析(比较聚酰胺、大孔树酯D101、硅胶G三种填料),结果表明,三种预分离方法中聚酰胺柱层析分离获得的绿原酸纯度最高,绿原酸的纯度从15%一次性提高至45.2 %,增幅达30 %,此时绿原酸的回收率达到81.6 %;硅胶G柱层析法的产物得率最高,达到60.6 %,但产物中绿原酸含量最低,仅为18.8 %;D101大孔树脂法产物中绿原酸的含量与Zn离子沉淀法相当,达到31 %,但是Zn离子沉淀法绿原酸回收率相对较低,仅为50.0 %。综合考虑绿原酸回收率及绿原酸纯度两个关键因素,最终确定聚酰胺柱层析为绿原酸的初步纯化方法。将聚酰胺柱层析的纯化物,进行乙酸乙酯萃取,萃取4次即基本萃取完全,浓缩结晶后,经HPLC测定,纯度达到92 %,绿原酸平均回收率为88.4 %(RSD=2.15 %);从将浸膏进行聚酰胺柱层析到乙酸乙酯萃取完全的绝对回收率达到72.1 %(81.6 %×88.4 %=72.1 %)。
本文分别选取两种革兰氏阴性菌(志贺氏菌、沙门氏菌)和革兰氏阳性菌(金黄色葡萄球菌和李斯特菌)进行绿原酸抑菌活性研究,实验结果表明,绿原酸对革兰氏阴性菌的抑菌活性比革兰氏阳性菌更强,绿原酸水溶液对志贺氏菌、沙门氏菌的最小抑菌浓度均为0.125 mg/ml,抑菌效果与0.1 mg/ml的卡那霉素的抑菌效果相当。
Because the Clorogenic acid(CHA) has a significant effect to many diseases, academic medicine attention and enormous social needs is increased, but the current domestic chlorogenic acid research is still very weak, there is not satisfactory production process of the CHA. So the purpose of this study is to establish a stable and effective identification and detection methods of CHA in honeysuckle, analyze chemical stability of the CHA for the production and storage of it, find more efficient economy extraction and purification methods by the optimization process, further explore the CHA antibacterial activity and the chlorogenic acid antimicrobial spectrum.
The best flowing ratio of the thin lyar chromatograph (TLC) was abtained:ethyl acetate / water / ethanoic acid (v/v,10/3/2) , the retention facter(Rf) value achieved 0.68; CHA had the biggest absorption in the ultraviolet 327 nm by the ultra-violet spectrograph(UV) scanning during 400-200 nm wave length, the specification curve regression equation: y =0.0553x-0.0034,R=0.9996, it’s good in 1~15μg/ml scope; after the high performance liquid chromatograph(HPLC) analyzes experiment, we obtained the baseline separating parameter: column ZOBAX SB (150 mm×4.6 mm,5μm), the flowing ratio was: methyl alcohol/water/ethanoic acid (v/v,18/81/1), the speed of flowing: 1 mL/min, detection wave length: 327 nm, oven tempreature: 30℃, injecting volum: 20μL, these conditions reached; the curve equation: y = 51813 x-5832, R=0.9991, it’s good 1~13μg /ml scope. The CHA retention time was 8.832 min, the CHA content of the raw material for extraction was 3.3 % (n=3, RSD=2.0 %), the recovery-ratio was 98 %; the CHA content of the material for purification was 10.15 % (n=3, RSD=1.8 %)
The CHA stability trial indicated that: under the same store condition:30 days later, the CHA pH 3.0 was stabler than pH 5.6; under the same resolver condition: the CHA in 4℃temperature refrigerators was stabler than in 25℃natural light laying storeroom. After the single factor Duncan multiple function examination comparison in the confidence interval 5 % levels the performance was remarkable difference.
This article compared ethylalcohol reflux condensation(ERC), ultrasonic wave extraction (UE), Microwave extraction (ME), microwave-ultrasonic wave extraction (MUE) . MUE’s CHA extraction rates was the highest. We made the CHA breaking through cell wall by microwave heating, then used ultrasonicwave(20 Hz), 2 times. Under this condition:chlorogenic acid's extraction rates achieved 92.66 %, the chlorogenic acid's extraction rate of MUE (92.66 %) was higher than UE or ME (50 %) levels and ERC (71.15 %), the average growth rate was more than 30 %. The MUE orthogonal experiment obtained the best extaction parameter: microwave pre-treated material under 250 W,3 times, 30 s/per time,then joined 70 % ethyl alcohol according to the material/fluid (v/v,1/20), ultrasonic wave extracted 20 min under 18 W. Under this condition:the extraction rate reached above 73 % by the first time extracting, Various factors' influence decrease is: ultrasonic power > resolver quantity > ultrasonic time.
With the UV and the HPLC detecting the content of CHA, the metallic ion precipitation isolation and the column chromatographic (three kind of padding: gathers amide, D101, silica gel G)pre-isolation CHA acid was compared, then ethyl acetate extraction further purification was used. The results showed that:the gathers amide column chromatographic was the best isolation method, the content of CHA increased from 15 % to 45.2 %, the growth rate is 30 %,the CHA recovery-ratio was 81.6 %; the silica gel G column chromatographic raw product rate was high, achieved 60.6 %, but the CHA content of the product to be lowest, only 18.8 %; both the D101 chromatographic and the Zn ion precipitation method chromatographic achieved 31 %, but the CHA recovery-ratio of Zn ion was low, only 50 %. After overall evaluation , we selected gathers amide column chromatographic to pre-isolate the CHA, and used the ethyl acetate further extracting 4 times ,then collected the crystallization completely.After the product detected by HPLC, the content of CHA achieved 92 %, the average recovery -ratio of the CHA was 88.4 % (RSD=2.15 %) and the absolute recovery-ratio was 72.1 % (81.6 %×88.4 %=72.1 %).
This article studied the CHA bacteriostasis activeness of different kinds of Gram-negative bacteria (Shigella, Salmonella) and the Gram-positive bacteria (golden yellow Staphylococcus and the Lister), the results showed that: compared to the Gram-positive bacteria, the CHA to Gram-negative bacteria's bacteriostasis activeness was stronger;the minimum inhibitory concentration of CHA to shigella and salmonella was 0.125 mg/ml, almost the same to 0.1 mg/ml kanamycin.
绿原酸鉴定检测方面:硅胶薄层层析经紫外成相分析得知,乙酸乙酯:水:乙酸(v/v,10:3:2)为流动相配比,保留因子值达到0.68;由紫外可见光光度仪在400 nm-200 nm波长扫描得知绿原酸在紫外327 nm波长处有最大吸收,以浓度C(x轴)对吸光度A (y轴)作图绘制UV标准曲线,标准曲线回归方程:y =0.0553x-0.0034,R=0.9996; 0～15μg/ml线性关系良好;经实验优化,高效液相分析参数为:色谱柱ZOBAX SB(150 mm×4.6 mm,5μm);流动相为甲醇:水:乙酸(18:81:1),流速:1 mL/min,检测波长:327 nm,柱温:30℃,进样量:20μL;此条件下,样品中绿原酸与相邻成分基线分离,以浓度C(x轴)对峰面积V(y轴)作图,得到标准曲线回归方程为:y = 51813 x-5832,R=0.9991,0～13μg/ml范围内线性关系良好;采用外标法确认提取的绿原酸的保留时间为8.832 min;同法测得原料金银花绿原酸含量为3.3 %(n=3,RSD=2.0 %),回收率98 %;而原料金银花浸膏的绿原酸含量为10.15 %(n=3,RSD=1.8 %)。
绿原酸的稳定性实验表明:相同存储条件下,绿原酸在pH 3.0时比pH 5.6时更稳定;而同样溶剂条件下,绿原酸于4℃恒温冰箱避光放置时比其25℃恒温储藏室自然光放置更稳定,储存30天后,经单因素Duncan多重函数检验比较得出二组实验数据在置信区间5 %水平上均表现为显著差异。
本文通过对醇回流法、超声波提取法、微波辅助提取法等方法的考察发现微波-超声波联合提取法的绿原酸提取率最高,且提取绿原酸的提取率比目前最普遍采用的70 %乙醇回流法的提取率71.15 %高20 %以上,远高于单独的超声法或微波辅助提取法50 %的水平。绿原酸经微波破壁后继续使用20Hz的低频率超声波温和提取,2次重复绿原酸的提取率即达到了92.66 %;微波-超声波联合提取法正交实验得出其最佳工艺参数为:在250 W功率下微波预处理3次,30 s/次;按料液比(v/v,1/20)加入70 %乙醇,在功率18 W下超声提取20 min,一次性提取率即达73 %以上;各因素的影响从大到小为:超声功率>溶剂倍量>超声时间。
以重庆金银花浸膏为原料,以绿原酸提取率为指标,采用紫外光谱法、高效液相法定性定量,着重研究比较了金属离子沉淀法与柱层析(比较聚酰胺、大孔树酯D101、硅胶G三种填料),结果表明,三种预分离方法中聚酰胺柱层析分离获得的绿原酸纯度最高,绿原酸的纯度从15%一次性提高至45.2 %,增幅达30 %,此时绿原酸的回收率达到81.6 %;硅胶G柱层析法的产物得率最高,达到60.6 %,但产物中绿原酸含量最低,仅为18.8 %;D101大孔树脂法产物中绿原酸的含量与Zn离子沉淀法相当,达到31 %,但是Zn离子沉淀法绿原酸回收率相对较低,仅为50.0 %。综合考虑绿原酸回收率及绿原酸纯度两个关键因素,最终确定聚酰胺柱层析为绿原酸的初步纯化方法。将聚酰胺柱层析的纯化物,进行乙酸乙酯萃取,萃取4次即基本萃取完全,浓缩结晶后,经HPLC测定,纯度达到92 %,绿原酸平均回收率为88.4 %(RSD=2.15 %);从将浸膏进行聚酰胺柱层析到乙酸乙酯萃取完全的绝对回收率达到72.1 %(81.6 %×88.4 %=72.1 %)。
本文分别选取两种革兰氏阴性菌(志贺氏菌、沙门氏菌)和革兰氏阳性菌(金黄色葡萄球菌和李斯特菌)进行绿原酸抑菌活性研究,实验结果表明,绿原酸对革兰氏阴性菌的抑菌活性比革兰氏阳性菌更强,绿原酸水溶液对志贺氏菌、沙门氏菌的最小抑菌浓度均为0.125 mg/ml,抑菌效果与0.1 mg/ml的卡那霉素的抑菌效果相当。
Because the Clorogenic acid(CHA) has a significant effect to many diseases, academic medicine attention and enormous social needs is increased, but the current domestic chlorogenic acid research is still very weak, there is not satisfactory production process of the CHA. So the purpose of this study is to establish a stable and effective identification and detection methods of CHA in honeysuckle, analyze chemical stability of the CHA for the production and storage of it, find more efficient economy extraction and purification methods by the optimization process, further explore the CHA antibacterial activity and the chlorogenic acid antimicrobial spectrum.
The best flowing ratio of the thin lyar chromatograph (TLC) was abtained:ethyl acetate / water / ethanoic acid (v/v,10/3/2) , the retention facter(Rf) value achieved 0.68; CHA had the biggest absorption in the ultraviolet 327 nm by the ultra-violet spectrograph(UV) scanning during 400-200 nm wave length, the specification curve regression equation: y =0.0553x-0.0034,R=0.9996, it’s good in 1~15μg/ml scope; after the high performance liquid chromatograph(HPLC) analyzes experiment, we obtained the baseline separating parameter: column ZOBAX SB (150 mm×4.6 mm,5μm), the flowing ratio was: methyl alcohol/water/ethanoic acid (v/v,18/81/1), the speed of flowing: 1 mL/min, detection wave length: 327 nm, oven tempreature: 30℃, injecting volum: 20μL, these conditions reached; the curve equation: y = 51813 x-5832, R=0.9991, it’s good 1~13μg /ml scope. The CHA retention time was 8.832 min, the CHA content of the raw material for extraction was 3.3 % (n=3, RSD=2.0 %), the recovery-ratio was 98 %; the CHA content of the material for purification was 10.15 % (n=3, RSD=1.8 %)
The CHA stability trial indicated that: under the same store condition:30 days later, the CHA pH 3.0 was stabler than pH 5.6; under the same resolver condition: the CHA in 4℃temperature refrigerators was stabler than in 25℃natural light laying storeroom. After the single factor Duncan multiple function examination comparison in the confidence interval 5 % levels the performance was remarkable difference.
This article compared ethylalcohol reflux condensation(ERC), ultrasonic wave extraction (UE), Microwave extraction (ME), microwave-ultrasonic wave extraction (MUE) . MUE’s CHA extraction rates was the highest. We made the CHA breaking through cell wall by microwave heating, then used ultrasonicwave(20 Hz), 2 times. Under this condition:chlorogenic acid's extraction rates achieved 92.66 %, the chlorogenic acid's extraction rate of MUE (92.66 %) was higher than UE or ME (50 %) levels and ERC (71.15 %), the average growth rate was more than 30 %. The MUE orthogonal experiment obtained the best extaction parameter: microwave pre-treated material under 250 W,3 times, 30 s/per time,then joined 70 % ethyl alcohol according to the material/fluid (v/v,1/20), ultrasonic wave extracted 20 min under 18 W. Under this condition:the extraction rate reached above 73 % by the first time extracting, Various factors' influence decrease is: ultrasonic power > resolver quantity > ultrasonic time.
With the UV and the HPLC detecting the content of CHA, the metallic ion precipitation isolation and the column chromatographic (three kind of padding: gathers amide, D101, silica gel G)pre-isolation CHA acid was compared, then ethyl acetate extraction further purification was used. The results showed that:the gathers amide column chromatographic was the best isolation method, the content of CHA increased from 15 % to 45.2 %, the growth rate is 30 %,the CHA recovery-ratio was 81.6 %; the silica gel G column chromatographic raw product rate was high, achieved 60.6 %, but the CHA content of the product to be lowest, only 18.8 %; both the D101 chromatographic and the Zn ion precipitation method chromatographic achieved 31 %, but the CHA recovery-ratio of Zn ion was low, only 50 %. After overall evaluation , we selected gathers amide column chromatographic to pre-isolate the CHA, and used the ethyl acetate further extracting 4 times ,then collected the crystallization completely.After the product detected by HPLC, the content of CHA achieved 92 %, the average recovery -ratio of the CHA was 88.4 % (RSD=2.15 %) and the absolute recovery-ratio was 72.1 % (81.6 %×88.4 %=72.1 %).
This article studied the CHA bacteriostasis activeness of different kinds of Gram-negative bacteria (Shigella, Salmonella) and the Gram-positive bacteria (golden yellow Staphylococcus and the Lister), the results showed that: compared to the Gram-positive bacteria, the CHA to Gram-negative bacteria's bacteriostasis activeness was stronger;the minimum inhibitory concentration of CHA to shigella and salmonella was 0.125 mg/ml, almost the same to 0.1 mg/ml kanamycin.
引文
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