金银花药效组分的鉴定方法研究
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摘要
目的：在确定金银花抗病毒有效部位的基础上，寻找和药效相关的化学鉴定特征，建立基本能够表述金银花有效性的鉴定方法，此鉴定方法具有广泛的适用性，可以用来鉴定药材、有效部位(提取物)及制剂。
方法：采用体外抗病毒试验确定有效部位；采用正交设计法对提取工艺进行筛选；采用方差分析和直观分析法对正交试验结果进行统计学处理；采用大孔树脂吸附法富集有效部位；采用单因素设计法对大孔树脂吸附的各项参数进行考察；采用HPLC法和分光光度法对有效组分进行含量测定；采用HPLC和HPLC-MS法进行鉴定特征的研究。
结果：
1．金银花抗病毒有效部位的制备工艺
最佳的提取工艺为A_1B_3C_1，即加8倍量70％乙醇溶液，提取3次，每次1h。大孔树脂吸附的参数：上样液浓度为每1毫升含100mg中药；上样液pH值为3；吸附流速为0.8ml／mim径高比1：11；上样液和树脂体积比1：3；水洗脱流速为1ml／min，洗脱至流出液无色澄明；有效部位部分是用50％乙醇以1ml／min流速洗至流出液近无色。照上法制备三份样品，结果绿原酸的平均含量为20.23％±0.37％，总黄酮的平均含量为75.15％±1.55％。
2．金银花药材的HPLC指纹图谱特征
最终建立的色谱条件：色谱柱为Waters Spherisorb 5μm ODS2 4.6×250mm Analytical Column；流动相为乙腈-0.35％磷酸溶液(11.5：88.5→21：79，梯度洗脱60min)；检测波长为240nm；流速为1.5mL／min；进样量为5μl。在此色谱条件下，精密度、稳定性和重复性试验中各共有峰相对保留时间和峰面积比值的RSD值均＜3％。稳定性试验结果显示供试品溶液在22h内是稳定的。对10批样品进行分析，共得到14个共有指纹峰，以绿原酸峰为参照峰，计算相对保留时间，分别0.2432±0.0059，RSD值为2.44％；0.2879±0.0068，RSD值为2.37％；0.5187±0.0035，RSD值为0.67％；0.6594±0.0122，RSD值为1.85％；0.7344±0.0085，RSD值为1.15％：0.8209±0.0157，RSD值为1.92％；1.0000±0.0000，RSD值为0％；1.0913±0.0118，RSD值为1.08％；1.4691±0.0156，RSD值为1.06％：1.5444±0.0256，RSD值为1.66％：4.2805±0.0721，RSD值为1.68％；4.701 9±0.1054，RSD值为2.24％；5.0998±0.0899，RSD值为1.76％：6.1591±0.1209，RSD值为1.96％。单峰占总峰面积＞10％的峰共3个，峰面积比分别为0.4930±0.0403，RSD值为8.18％；1.0000±0.0000，RSD值为0％；0.4955±0.0313，RSD值为6.31％。
3．通用的鉴定方法
对比药材、有效部位及制剂的HPLC色谱图，共得到5个共有峰，以峰2绿原酸为参照峰，计算相对保留时间，分别为0.6282±0.0063，RSD值1.00％；1.0000±0.0000，RSD值0％；1.3754±0.0154，RSD值1.12％：4.3924±0.0935，RSD值2.13％；4.9200±0.0347，RSD值0.70％。
对比药材、有效部位及制剂的HPLC-MS谱图，共得到4个共有峰，以峰2绿原酸为参照峰，计算相对保留时间，分别为0.7016±0.0099，RSD值1.40％；1.0000±0.0000，RSD值0％：1.5251±0.0084，RSD值0.55％；4.8072±0.0474，RSD值0.99％。最终推定共有峰1可能为马钱素(Loganin)，共有峰2为绿原酸(Chlorogenic acid)，共有峰3可能为金吉苷(金银花苷，Kingiside)，共有峰4可能为3，5-二咖啡酰奎尼酸或3，4-二咖啡酰奎尼酸(二咖啡酰鸡纳酸，3，5-Dicaffeoyl-quinic acid或3，4-Dicaffeoyl-quinic acid)。
结论：本文所建立的鉴定方法是在药效研究基础上，从金银花药材、有效部位和多种市售金银花制剂中提炼出来的共性特征，因此，此鉴定方法能够表述金银花的有效性，而且具有广泛的适用性，可以用来鉴定金银花药材、有效部位(提取物)及制剂。
Objective: Find the chemical identification mark of Lonicera japonica, related to its pharmacodynamic action, to establish an identification method which can present its efficacy, on the basis of ascertaining the antiviral part of Lonicera japonica. The identification method should possess extensive applicability to be used to identify the dried medical herb, the active part (or the extract) and the preparation of Lonicera japonica.Methods: Ascertain the active part by the antivirus test in the vitro; screen the abstraction technology by the orthogonal design; handle the data by the analysis of variance and direct-viewing; enrich the active part by the macroporous resin adsorptive process; investigate each parameter of macroporous resin adsorbing by one-factor design; determine the contents of the active compounds alignment by HPLC and spectrophotometry; research the identification mark by HPLC and HPLC-MS.Results: 1 the preparative method of the active antivirus part of Lonicera japonicaThe optimal extraction technology was A1B3C1 that the Lonicera japonica was extracted by 70% ethanol (8 times of the dried medical herb) 3 times, 1h per time. The parameter of macropotous resin adsorptive process was as followed: the extract (the concentration of the solution was 100mg dried medical herb per milliliter, pH=3) was subjected to macropotous resin column (diameter/high=1:11) with the adsorbing current velocity 0.8mL/min, the ratio of loading capacity to resin volume was 1:3. After eluted with water (twice of resin volume) until the effluent was colorless, the resin was eluted by 50% ethanol under the flow rate 1mL/min until the effluent was colorless, and the effluent eluted by 50% ethanol was the active part. Prepare 3 samples as the method above, and the average content of Chlorogenic acid was 20.23%±0.37%, the total flavonoids 75.15%±1.55%.
2 The HPLC fingerprint of dried medical herb of Lonicera japonica
The chromatographic condition: Waters Spherisorb ODS2 (5μtm, 4.6×250mm) as Analytical Column; ACN-0.35%H_3PO_4 (11.5:88.5→21:79, gradient elution for 60min) as mobile phase; wave length for determination 240nm; flow rate 1.5mL/min; and sample size 5μl. Under the condition above, RSD of the average rate of the relative retention time to the peak area was below 3% in the tests of precision, stability and reproducibility. The result of the stability test showed that the solution was stable in 22h. 14 common peaks were found after analyzing 10 samples, and the relative retention time,calculated with Chlorogenic acid peak as the reference peak,were respectively 0.2432±0.0059, RSD=2.44%; 0.2879±0.0068, RSD=2.37%; 0.5187±0.0035, RSD=0.67%; 0.6594±0.0122, RSD=1.85%; 0.7344±0.0085, RSD=1.15%; 0.8209±0.0157, RSD=1.92%; 1.0000±0.0000, RSD=0%; 1.0913±0.0118, RSD=1.08%; 1.4691±0.0156, RSD=1.06%; 1.5444±0.0256, RSD=1.66%; 4.2805±0.0721, RSD=1.68%; 4.7019±0.1054, RSD=2.24%; 5.0998±0.0899, RSD=1.76%; 6.1591±0.1209, RSD=1.96%.There were 3 single peaks whose relative area were all above 10%, and the proportion of their relative areas were respectively 0.4930±0.0403, RSD=8.18%;1.0000±0, RSD=0%; 0.4955±0.0313, RSD=6.31%。
3 The identification method for Lonicera japonica and its preparations
5 common peaks were found after contrasting the HPLC chromatograms of the medical material, the active part and the praeparatum. The relative retention time, calculated with Chlorogenic acid peak(peak 2) as the reference peak, were respectively 0.62824±0.0063, RSD=1.00%; 1±0.0, RSD=0%; 1.37544±0.0154, RSD=1.12%; 4.39244±0.0935, RSD=2.13%; 4.92004±0.0347, RSD=0.70%.
4 common peaks were found after contrasting the HPLC-MS chromatograms of the medical material, the active part and the praeparatum. The relative retention time, calculated with Chlorogenic acid peak(peak 2) as the reference peak, were respectively 0.70164±0.0099, RSD=1.40%; 1.0±0.00, RSD=0.0%; 1.52514±0.0084, RSD=0.55%; 4.80724±0.0474, RSD=0.99%. The final presumption was that possibly the common peak 1 was Loganin, the common peak 2 was Chlorogenic acid, the common peak 3 was Kingiside and the common peak 4 was 3, 5-Dicaffeoyl-quinic acid or 3, 4-Dicaffeoyl-quinic acid.
Conclusion: The identification method established was based on research of the drug efficacy. The commonness characteristics of Lonicera japonica were brought up from the dried medical herb, the active part and many kinds of the preparations on the market. So the method was able to present the efficacy of Lonicera japonica and could be used to identify the dried medical herb, the active part (or the extract) and the preparations of Lonicera japonica with extensive applicability.
方法：采用体外抗病毒试验确定有效部位；采用正交设计法对提取工艺进行筛选；采用方差分析和直观分析法对正交试验结果进行统计学处理；采用大孔树脂吸附法富集有效部位；采用单因素设计法对大孔树脂吸附的各项参数进行考察；采用HPLC法和分光光度法对有效组分进行含量测定；采用HPLC和HPLC-MS法进行鉴定特征的研究。
结果：
1．金银花抗病毒有效部位的制备工艺
最佳的提取工艺为A_1B_3C_1，即加8倍量70％乙醇溶液，提取3次，每次1h。大孔树脂吸附的参数：上样液浓度为每1毫升含100mg中药；上样液pH值为3；吸附流速为0.8ml／mim径高比1：11；上样液和树脂体积比1：3；水洗脱流速为1ml／min，洗脱至流出液无色澄明；有效部位部分是用50％乙醇以1ml／min流速洗至流出液近无色。照上法制备三份样品，结果绿原酸的平均含量为20.23％±0.37％，总黄酮的平均含量为75.15％±1.55％。
2．金银花药材的HPLC指纹图谱特征
最终建立的色谱条件：色谱柱为Waters Spherisorb 5μm ODS2 4.6×250mm Analytical Column；流动相为乙腈-0.35％磷酸溶液(11.5：88.5→21：79，梯度洗脱60min)；检测波长为240nm；流速为1.5mL／min；进样量为5μl。在此色谱条件下，精密度、稳定性和重复性试验中各共有峰相对保留时间和峰面积比值的RSD值均＜3％。稳定性试验结果显示供试品溶液在22h内是稳定的。对10批样品进行分析，共得到14个共有指纹峰，以绿原酸峰为参照峰，计算相对保留时间，分别0.2432±0.0059，RSD值为2.44％；0.2879±0.0068，RSD值为2.37％；0.5187±0.0035，RSD值为0.67％；0.6594±0.0122，RSD值为1.85％；0.7344±0.0085，RSD值为1.15％：0.8209±0.0157，RSD值为1.92％；1.0000±0.0000，RSD值为0％；1.0913±0.0118，RSD值为1.08％；1.4691±0.0156，RSD值为1.06％：1.5444±0.0256，RSD值为1.66％：4.2805±0.0721，RSD值为1.68％；4.701 9±0.1054，RSD值为2.24％；5.0998±0.0899，RSD值为1.76％：6.1591±0.1209，RSD值为1.96％。单峰占总峰面积＞10％的峰共3个，峰面积比分别为0.4930±0.0403，RSD值为8.18％；1.0000±0.0000，RSD值为0％；0.4955±0.0313，RSD值为6.31％。
3．通用的鉴定方法
对比药材、有效部位及制剂的HPLC色谱图，共得到5个共有峰，以峰2绿原酸为参照峰，计算相对保留时间，分别为0.6282±0.0063，RSD值1.00％；1.0000±0.0000，RSD值0％；1.3754±0.0154，RSD值1.12％：4.3924±0.0935，RSD值2.13％；4.9200±0.0347，RSD值0.70％。
对比药材、有效部位及制剂的HPLC-MS谱图，共得到4个共有峰，以峰2绿原酸为参照峰，计算相对保留时间，分别为0.7016±0.0099，RSD值1.40％；1.0000±0.0000，RSD值0％：1.5251±0.0084，RSD值0.55％；4.8072±0.0474，RSD值0.99％。最终推定共有峰1可能为马钱素(Loganin)，共有峰2为绿原酸(Chlorogenic acid)，共有峰3可能为金吉苷(金银花苷，Kingiside)，共有峰4可能为3，5-二咖啡酰奎尼酸或3，4-二咖啡酰奎尼酸(二咖啡酰鸡纳酸，3，5-Dicaffeoyl-quinic acid或3，4-Dicaffeoyl-quinic acid)。
结论：本文所建立的鉴定方法是在药效研究基础上，从金银花药材、有效部位和多种市售金银花制剂中提炼出来的共性特征，因此，此鉴定方法能够表述金银花的有效性，而且具有广泛的适用性，可以用来鉴定金银花药材、有效部位(提取物)及制剂。
Objective: Find the chemical identification mark of Lonicera japonica, related to its pharmacodynamic action, to establish an identification method which can present its efficacy, on the basis of ascertaining the antiviral part of Lonicera japonica. The identification method should possess extensive applicability to be used to identify the dried medical herb, the active part (or the extract) and the preparation of Lonicera japonica.Methods: Ascertain the active part by the antivirus test in the vitro; screen the abstraction technology by the orthogonal design; handle the data by the analysis of variance and direct-viewing; enrich the active part by the macroporous resin adsorptive process; investigate each parameter of macroporous resin adsorbing by one-factor design; determine the contents of the active compounds alignment by HPLC and spectrophotometry; research the identification mark by HPLC and HPLC-MS.Results: 1 the preparative method of the active antivirus part of Lonicera japonicaThe optimal extraction technology was A1B3C1 that the Lonicera japonica was extracted by 70% ethanol (8 times of the dried medical herb) 3 times, 1h per time. The parameter of macropotous resin adsorptive process was as followed: the extract (the concentration of the solution was 100mg dried medical herb per milliliter, pH=3) was subjected to macropotous resin column (diameter/high=1:11) with the adsorbing current velocity 0.8mL/min, the ratio of loading capacity to resin volume was 1:3. After eluted with water (twice of resin volume) until the effluent was colorless, the resin was eluted by 50% ethanol under the flow rate 1mL/min until the effluent was colorless, and the effluent eluted by 50% ethanol was the active part. Prepare 3 samples as the method above, and the average content of Chlorogenic acid was 20.23%±0.37%, the total flavonoids 75.15%±1.55%.
2 The HPLC fingerprint of dried medical herb of Lonicera japonica
The chromatographic condition: Waters Spherisorb ODS2 (5μtm, 4.6×250mm) as Analytical Column; ACN-0.35%H_3PO_4 (11.5:88.5→21:79, gradient elution for 60min) as mobile phase; wave length for determination 240nm; flow rate 1.5mL/min; and sample size 5μl. Under the condition above, RSD of the average rate of the relative retention time to the peak area was below 3% in the tests of precision, stability and reproducibility. The result of the stability test showed that the solution was stable in 22h. 14 common peaks were found after analyzing 10 samples, and the relative retention time,calculated with Chlorogenic acid peak as the reference peak,were respectively 0.2432±0.0059, RSD=2.44%; 0.2879±0.0068, RSD=2.37%; 0.5187±0.0035, RSD=0.67%; 0.6594±0.0122, RSD=1.85%; 0.7344±0.0085, RSD=1.15%; 0.8209±0.0157, RSD=1.92%; 1.0000±0.0000, RSD=0%; 1.0913±0.0118, RSD=1.08%; 1.4691±0.0156, RSD=1.06%; 1.5444±0.0256, RSD=1.66%; 4.2805±0.0721, RSD=1.68%; 4.7019±0.1054, RSD=2.24%; 5.0998±0.0899, RSD=1.76%; 6.1591±0.1209, RSD=1.96%.There were 3 single peaks whose relative area were all above 10%, and the proportion of their relative areas were respectively 0.4930±0.0403, RSD=8.18%;1.0000±0, RSD=0%; 0.4955±0.0313, RSD=6.31%。
3 The identification method for Lonicera japonica and its preparations
5 common peaks were found after contrasting the HPLC chromatograms of the medical material, the active part and the praeparatum. The relative retention time, calculated with Chlorogenic acid peak(peak 2) as the reference peak, were respectively 0.62824±0.0063, RSD=1.00%; 1±0.0, RSD=0%; 1.37544±0.0154, RSD=1.12%; 4.39244±0.0935, RSD=2.13%; 4.92004±0.0347, RSD=0.70%.
4 common peaks were found after contrasting the HPLC-MS chromatograms of the medical material, the active part and the praeparatum. The relative retention time, calculated with Chlorogenic acid peak(peak 2) as the reference peak, were respectively 0.70164±0.0099, RSD=1.40%; 1.0±0.00, RSD=0.0%; 1.52514±0.0084, RSD=0.55%; 4.80724±0.0474, RSD=0.99%. The final presumption was that possibly the common peak 1 was Loganin, the common peak 2 was Chlorogenic acid, the common peak 3 was Kingiside and the common peak 4 was 3, 5-Dicaffeoyl-quinic acid or 3, 4-Dicaffeoyl-quinic acid.
Conclusion: The identification method established was based on research of the drug efficacy. The commonness characteristics of Lonicera japonica were brought up from the dried medical herb, the active part and many kinds of the preparations on the market. So the method was able to present the efficacy of Lonicera japonica and could be used to identify the dried medical herb, the active part (or the extract) and the preparations of Lonicera japonica with extensive applicability.
引文
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