甘肃省道地药材甘草质量评价与标准提高
摘要
甘草是常用的中药材,性甘,味平；具有补脾益气,清热解毒,祛痰止咳,缓急止痛,调和诸药的功效。《中国药典》2010版一部规定为甘草(Glycyrrhiza uralensis Fisch.)、胀果甘草(Glycyrrhiza inflata Bat.)或光果甘草(Glycyrrhiza glabra L.)的干燥根及根茎,并对甘草药材采用了性状、显微鉴别、薄层色谱鉴别、检查(新增加了重金属及有机氯残留的限定)、甘草苷及甘草酸含量的测定来进行质量控制。甘草是甘肃省几大道地药材之一,但现有的指标测定仍然未全面反映甘草的质量状况,因此本论文在原有标准基础上,进行了标准提高研究及质量评价。
主要研究内容包括以下方面：
1甘草药材薄层鉴别方法的优化：选取了6种中成药的薄层色谱鉴别法来探讨甘草药材的薄层色谱条件,得出不同产地的甘草药材的薄层色谱图基本一致,分离较好。《中国药典》2010版中甘草的薄层鉴别提取了其醇溶性成分,但根据口炎清颗粒成药的薄层色谱结果,应在甘草的薄层鉴别方法中增加水溶性成分。
2甘草的含量测定及化学模式分析：HPLC法测定甘草中甘草苷、甘草酸、甘草素、异甘草素、甘草查尔酮A与甘草次酸的含量,并用主成分分析来评价甘草药材的质量。流动相为乙腈-0.1%的磷酸水溶液,室温下0.6mL·min-1的流速进行梯度洗脱,SPSS19.0进行数据分析。结果样品色谱峰的分离良好,且六种成分在各自浓度的范围内均具有良好的线性关系；运用主成分分析对样品进行排序后的结果与甘草药材的传统的评价结果基本相符,本方法准确、简便,可以作为甘草质量控制的方法。
3土壤理化因子与甘草成分的相关系分析：运用相关性分析及灰色关联度分析对甘草的六种成分及土壤因子(有机质,PH,全氮,铵态氮,硝态氮,全磷,速效磷,全钾,速效钾)进行综合评价并筛选出影响其质量的主导因子。相关性分析的结果表明土壤中速效磷对甘草酸含量的积累呈显著正相关；甘草次酸与铵态氮呈显著负相关；灰色关联度分析的结果表明速效钾、铵态氮及PH对甘草有效成分的合成起到了关键性的作用。因此我们在土壤及肥料的选择和使用中应重视N、P、K和PH。
4QAMS法测定甘草有效成分的方法学研究运用相关系数法及夹角余弦法对一测多评法测得的甘草中六种成分的结果进行了比较,得出以甘草酸为内标物的一测多评法测定六种成分的结果与外标法测得的结果没有显著性差异,可以作为多种指标含量测定方法的一种补充,用于甘草药材及饮片的质量控制。
5HPCE法测定甘草有效成分的方法学研究HPCE法测定甘草药材中6种有效成分的含量,以70mmol·L-1的硼酸钠缓冲液(PH为9.2)为电泳介质,未涂层弹性石英毛细管(75um×64.5cm,有效分离长度56cm)为分离通道,压力进样(150Mpa·S),20kV恒压电泳(20℃)分离,不同的波长段检测,整个分析在15min内完成。且六种成分均得到良好的分离且在各自浓度范围内均具有良好的线性关系。结果表明本方法准确、简便,可作为甘草质量控制的方法。
6甘草药材中有机氯残留的考察通过对15批甘草样品中有机氯的残留测定,除1批总BHC超标外,其余符合《中国药典》2010年版一部对甘草、黄芪的规定,超标率6.7%。总BHC的检出率53%,总DDT的检出率66.7%,PCNB的检出率80%,总体上看,甘肃各地种植甘草的有机氯残留低于中国药典的相关规定,安全性较高。
7甘草药材中重金属残留的考察通过对5种有害重金属元素的检测,结果表明甘肃出产甘草中5种有害重金属均符合《中国药典》的规定。铅、砷、镉、汞和铜的检出率分别为52%、65%、61%、78%和87%,检出率很高,仍然不能忽视种植、加工等环境的影响。
Licorice is a commonly-used traditional Chinese medicine, it is sweet, plain, with Spleen Qi, heat antidotes, dispelling phlegm to relieve cough, stoping pain, and moderating the propertyof herbs effect.10th edition of the Chinese Pharmacopoeia defined licorice as Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L.'s dried root and rhizome. The quality control of licorice mainly concentrated in the following aspects:character, microscopic identification, thin layer chromatography, The check items add the limitation of heavy metals and organochlorine residues, the content of glycyrrhizin and glycyrrhizic acid. Licorice is one of famous-region drugs in Gansu Province, but the existing indicators still do not fully reflect the quality condition of licorice. Therefore, this paper was aimed at the improvement of quality standard. Therefore, the main content of this paper is as follows:
1Optimization of licorice TLC identification method:Based on the method of extraction, solvent and developing solvent, we selected six TLC identification methods, the results showed that thin layer chromatogram of different origin licorice herbs were identical, and the separation was good. Alcohol-soluble ingredients were extracted in the TLC identification of licorice according to the Chinese Pharmacopoeia, but according to the results of the the Kouyanqing medicine TLC identification, the TLC identification of the water-soluble components should be considered at the same time.
2Determination of the content of licorice and chemical pattern analysis:six active ingredients of licorice content were determined by HPLC, and the quality of licorice was evaluated by principal component analysis method. The mobile phase were acetonitrile and0.1%aqueous solution of H3PO4with gradient elution, the column temperature was25℃. the rate of flow was0.6mL min-1, the data was analyzed by SPSS19.0software. The results showed that the six active ingredients of licorice were separated well, and had good linear relationships in their respective range of concentration. Evaluation results of samples using principal component analysis were almost consistent with the traditional evaluation. So the established method was accurate and simple, and it could be used for the quality control of the Licorice.
3The effect of soil indicators to licorice index components:the correlation between soil factors (including organic matter, pH, total nitrogen, ammonium nitrogen, nitrate nitrogen, total phosphorus, available phosphorus, total potassium, available potassium)and Six active ingredients of Licorice was studied, of which the leading affecting factor was also screened out. The application of correlation analysis and grey relational analysis was made to a comprehensive analysis. The results showed that correlation coefficient of glycyrrhizin and available phosphorus was significantly positively related; glycyrrhetinic acid and ammonium nitrogen was significantly negative related; Available potassium, ammonium nitrogen and pH played the most important role in the synthesis and metabolism in licorice. Therefore, special attentions should be paid to the effects of N-, K and PH conditions on the quality of radix in the choice of soil and fertilization.
4Determination of active ingredient of licorice by quantitative analysis of multi-components by single-marker(QAMS):Six active ingredients of licorice were determinated by QAMS method, and compared with correlation coefficient method and cosine method. The results showed that there is no significant difference between external standard method and QAMS method (glycyrrhizic acid as the internal standard substance), this method can be used as a supplement of multi-indicators ingredients determination for the licorice quality control.
5Determination of active ingredient of licorice by HPCE method:Six active ingredients of licorice were determinated by HPCE method,70mmol/L sodium borate buffer (pH9.2) was the electrophoresis media, uncoated fused silica capillary (75um x64.5cm, effective length56cm) was adopted in the separation channel, the voltage of sample was150MPa, the separation and detection of the sample was under the constant pressure of20KV with a Multi-Wavelength method detection. The analysis successfully completed in15min, and all had good linear relationship in their respective range of concentration. The results showed that the determination of the active ingredient content in the licorice by HPCE method was accurate and simple, which could be used as one of the methods of licorice quality control.
6Investigation of organochlorine residues for licorice:Organochlorine residues in the sample of15batches of licorice were detected, in addition to a batch of licorice was total BHC excessive, the rests were up to the2010version of "Chinese Pharmacopoeia" provision, excessive rate of licorice was6.7%. Total BHC detection rate of licorice was53%, total DDT detection rate of licorice was66.7%, the PCNB detection rate of licorice was80%. Overall, the organochlorine residues of licorice produced in Gansu Province were far below the relevant provision of the Chinese Pharmacopoeia.
7Investigation of the heavy metal residues for Licorice:Five heavy metals content was detected to evaluate the safety of licorice, the results indicated that the heavy metals of licorice produced in Gansu Province were up to the standard. The detection rate of lead, arsenic, cadmium. mercury and copper were52%,65%,61%,78%and87%. respectively. The detection rate is very high, so we can not ignore the influence of cultivation, processing environment.
主要研究内容包括以下方面：
1甘草药材薄层鉴别方法的优化：选取了6种中成药的薄层色谱鉴别法来探讨甘草药材的薄层色谱条件,得出不同产地的甘草药材的薄层色谱图基本一致,分离较好。《中国药典》2010版中甘草的薄层鉴别提取了其醇溶性成分,但根据口炎清颗粒成药的薄层色谱结果,应在甘草的薄层鉴别方法中增加水溶性成分。
2甘草的含量测定及化学模式分析：HPLC法测定甘草中甘草苷、甘草酸、甘草素、异甘草素、甘草查尔酮A与甘草次酸的含量,并用主成分分析来评价甘草药材的质量。流动相为乙腈-0.1%的磷酸水溶液,室温下0.6mL·min-1的流速进行梯度洗脱,SPSS19.0进行数据分析。结果样品色谱峰的分离良好,且六种成分在各自浓度的范围内均具有良好的线性关系；运用主成分分析对样品进行排序后的结果与甘草药材的传统的评价结果基本相符,本方法准确、简便,可以作为甘草质量控制的方法。
3土壤理化因子与甘草成分的相关系分析：运用相关性分析及灰色关联度分析对甘草的六种成分及土壤因子(有机质,PH,全氮,铵态氮,硝态氮,全磷,速效磷,全钾,速效钾)进行综合评价并筛选出影响其质量的主导因子。相关性分析的结果表明土壤中速效磷对甘草酸含量的积累呈显著正相关；甘草次酸与铵态氮呈显著负相关；灰色关联度分析的结果表明速效钾、铵态氮及PH对甘草有效成分的合成起到了关键性的作用。因此我们在土壤及肥料的选择和使用中应重视N、P、K和PH。
4QAMS法测定甘草有效成分的方法学研究运用相关系数法及夹角余弦法对一测多评法测得的甘草中六种成分的结果进行了比较,得出以甘草酸为内标物的一测多评法测定六种成分的结果与外标法测得的结果没有显著性差异,可以作为多种指标含量测定方法的一种补充,用于甘草药材及饮片的质量控制。
5HPCE法测定甘草有效成分的方法学研究HPCE法测定甘草药材中6种有效成分的含量,以70mmol·L-1的硼酸钠缓冲液(PH为9.2)为电泳介质,未涂层弹性石英毛细管(75um×64.5cm,有效分离长度56cm)为分离通道,压力进样(150Mpa·S),20kV恒压电泳(20℃)分离,不同的波长段检测,整个分析在15min内完成。且六种成分均得到良好的分离且在各自浓度范围内均具有良好的线性关系。结果表明本方法准确、简便,可作为甘草质量控制的方法。
6甘草药材中有机氯残留的考察通过对15批甘草样品中有机氯的残留测定,除1批总BHC超标外,其余符合《中国药典》2010年版一部对甘草、黄芪的规定,超标率6.7%。总BHC的检出率53%,总DDT的检出率66.7%,PCNB的检出率80%,总体上看,甘肃各地种植甘草的有机氯残留低于中国药典的相关规定,安全性较高。
7甘草药材中重金属残留的考察通过对5种有害重金属元素的检测,结果表明甘肃出产甘草中5种有害重金属均符合《中国药典》的规定。铅、砷、镉、汞和铜的检出率分别为52%、65%、61%、78%和87%,检出率很高,仍然不能忽视种植、加工等环境的影响。
Licorice is a commonly-used traditional Chinese medicine, it is sweet, plain, with Spleen Qi, heat antidotes, dispelling phlegm to relieve cough, stoping pain, and moderating the propertyof herbs effect.10th edition of the Chinese Pharmacopoeia defined licorice as Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L.'s dried root and rhizome. The quality control of licorice mainly concentrated in the following aspects:character, microscopic identification, thin layer chromatography, The check items add the limitation of heavy metals and organochlorine residues, the content of glycyrrhizin and glycyrrhizic acid. Licorice is one of famous-region drugs in Gansu Province, but the existing indicators still do not fully reflect the quality condition of licorice. Therefore, this paper was aimed at the improvement of quality standard. Therefore, the main content of this paper is as follows:
1Optimization of licorice TLC identification method:Based on the method of extraction, solvent and developing solvent, we selected six TLC identification methods, the results showed that thin layer chromatogram of different origin licorice herbs were identical, and the separation was good. Alcohol-soluble ingredients were extracted in the TLC identification of licorice according to the Chinese Pharmacopoeia, but according to the results of the the Kouyanqing medicine TLC identification, the TLC identification of the water-soluble components should be considered at the same time.
2Determination of the content of licorice and chemical pattern analysis:six active ingredients of licorice content were determined by HPLC, and the quality of licorice was evaluated by principal component analysis method. The mobile phase were acetonitrile and0.1%aqueous solution of H3PO4with gradient elution, the column temperature was25℃. the rate of flow was0.6mL min-1, the data was analyzed by SPSS19.0software. The results showed that the six active ingredients of licorice were separated well, and had good linear relationships in their respective range of concentration. Evaluation results of samples using principal component analysis were almost consistent with the traditional evaluation. So the established method was accurate and simple, and it could be used for the quality control of the Licorice.
3The effect of soil indicators to licorice index components:the correlation between soil factors (including organic matter, pH, total nitrogen, ammonium nitrogen, nitrate nitrogen, total phosphorus, available phosphorus, total potassium, available potassium)and Six active ingredients of Licorice was studied, of which the leading affecting factor was also screened out. The application of correlation analysis and grey relational analysis was made to a comprehensive analysis. The results showed that correlation coefficient of glycyrrhizin and available phosphorus was significantly positively related; glycyrrhetinic acid and ammonium nitrogen was significantly negative related; Available potassium, ammonium nitrogen and pH played the most important role in the synthesis and metabolism in licorice. Therefore, special attentions should be paid to the effects of N-, K and PH conditions on the quality of radix in the choice of soil and fertilization.
4Determination of active ingredient of licorice by quantitative analysis of multi-components by single-marker(QAMS):Six active ingredients of licorice were determinated by QAMS method, and compared with correlation coefficient method and cosine method. The results showed that there is no significant difference between external standard method and QAMS method (glycyrrhizic acid as the internal standard substance), this method can be used as a supplement of multi-indicators ingredients determination for the licorice quality control.
5Determination of active ingredient of licorice by HPCE method:Six active ingredients of licorice were determinated by HPCE method,70mmol/L sodium borate buffer (pH9.2) was the electrophoresis media, uncoated fused silica capillary (75um x64.5cm, effective length56cm) was adopted in the separation channel, the voltage of sample was150MPa, the separation and detection of the sample was under the constant pressure of20KV with a Multi-Wavelength method detection. The analysis successfully completed in15min, and all had good linear relationship in their respective range of concentration. The results showed that the determination of the active ingredient content in the licorice by HPCE method was accurate and simple, which could be used as one of the methods of licorice quality control.
6Investigation of organochlorine residues for licorice:Organochlorine residues in the sample of15batches of licorice were detected, in addition to a batch of licorice was total BHC excessive, the rests were up to the2010version of "Chinese Pharmacopoeia" provision, excessive rate of licorice was6.7%. Total BHC detection rate of licorice was53%, total DDT detection rate of licorice was66.7%, the PCNB detection rate of licorice was80%. Overall, the organochlorine residues of licorice produced in Gansu Province were far below the relevant provision of the Chinese Pharmacopoeia.
7Investigation of the heavy metal residues for Licorice:Five heavy metals content was detected to evaluate the safety of licorice, the results indicated that the heavy metals of licorice produced in Gansu Province were up to the standard. The detection rate of lead, arsenic, cadmium. mercury and copper were52%,65%,61%,78%and87%. respectively. The detection rate is very high, so we can not ignore the influence of cultivation, processing environment.
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