中国山楂果实中生理活性成分的HPLC分析研究
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摘要
山楂是一种重要的药食两用植物资源，在我国种植栽培面积广阔，产量可观。药理研究表明，山楂具有多种重要的生理活性，以往研究表明，这些生理活性与山楂中所含的黄酮、前花青素、酚酸以及三萜酸等活性成分有关。本研究采用国产高效液相色谱仪建立了一套分析山楂果实中这些成分的HPLC分析方法，并用这种方法对中国8个省市的37个有代表性的山楂品种的绿原酸、表儿茶素、前花青素B2、B5、C1、金丝桃苷、Isoquercitrin、熊果酸和齐墩果酸等主要活性成分进行了分析，同时还考察了果实生长期这些成分的含量变化。所使用的高效液相色仪为大连依利特有限公司生产，由两台P200Ⅱ高压恒流泵、UV200Ⅱ紫外可变波长检测器以及Echrom98色谱工作站等部分组成。多酚分析的色谱条件为：色谱柱：HYPERSIL BDS C_(18)分析柱(200mm×4.6mm ID，5μm)；流动相A：20％甲醇(磷酸调pH=3.20)；B：30％乙腈(pH=3.20)，梯度程序(B％／min)：10/0，10/6，30/13，60/22，100/27，100/32，10/35；流速：0.6ml/min；温度：室温；检测波长：280nm。实验结果表明各主要活性成分分离较好，各组分回归方程的相关系数r为0.9991～0.9999，最低检出限为2.51～35.56ng，相对标准偏差RSD＜5.0％，回收率在93.9％～105.8％，35min完成一次分析。熊果酸和齐墩果酸的色谱分析条件为：色谱柱：HYPERSIL BDS C_(18)分析柱(200mm×4.6mm ID，5μm)；流动相：甲醇-水=90：10(pH=3.0)；流速：0.6ml/min；温度：室温；检测波长：210nm。熊果酸和齐墩果酸回归方程的相关系数分别为0.9999，0.9997；最低检限：2.80ng，2.81ng；回收率：98.33％，98.69％。这些方法具有操作简单，快速准确的优点，可作为山楂生药、提取物、制剂以及各种加工产品进行质量控制和分析检测方法。含量分析结果显示，中国山楂果实中的黄酮类物质主要为金丝桃苷和Isoquercitrin两种，含量一般不超过100mg/kg；三萜酸类物质中以熊果酸含量较高，平均为952mg/kg；而前花青素类物质的平均含量达到4000mg/kg，可以认为是山楂果实活性物质的代表性成分；不同品种之间活性成分含量差异显著，各种成分含量的相对标准偏差均在30％以上，反映了中国山楂种质资源的多样性；同一品种山楂中各种活性成分的含量之间具有相关性。另外发现山楂果实中活性成分含量随地理纬度的降低多酚含量有升高的趋势。山楂果实生长过程活性成分的含量变化分析结果表明，含量随生长发育呈规律性变化，7月上旬多酚含量达到最大值，前花青素总含量可达果实鲜重的1.36％以上，之后逐渐下降，10月上中旬果实成熟时，各活性成分含量略有回升。此外，在中国山楂果实中还发现一些未知物质，有待进一步研究。
Hawthorn (Crataegus) had a long history of use in traditional Chinese medicine and European herbal medicine. It was used for both medicinal materials and edibles in China, and was a very important botanic resource. It had planting far and wide, year' yield was very large. Primary studies indicates that hawthorn have many important pharmacological activities in the prevention and treatment of some cardiovascular diseases. These benefits were attributed to several active components such as flavonoids, procyanidins, phenolic acids and triterpene acids etc. In the present research, a suit of analysis methods for those active components was established by using a homemade high performance liquid chromatographic (HPLC) apparatus. By these methods, the contents of chlorogenic acid, epicatechin, procyanidin B2, procyanidin B5, procyanidin Cl, hyperoside, isoquercitrin, ursolic acid and oleanolic acid of 37 representative varieties of Crataegus pinnatifida from 8 provinces were determined. The changes of those active components in the fruits during development were monitored by the developed HPLC methods. The HPLC system consisted of two P200II high-pressure permanent flow pump, HYPERSIL BDS C18 (200mm x 4.6mm ID, 5um) column, UV200 II detector and Echrom 98 chromatography workstation. Chromat: graphic condition for polyphenols: solvent A was 20% memanol (v/v) (pH=3.20) and B was 30% acetonitrile (v/v) (pH=3.20), the gradient elution condition (B%/min): 10/0, 10/6, 30/13, 60/22, 100/27, 100/32, 10/35; flow rate was 0.6ml/min at room temperatuie and detection wavelength at 280nm. The correlation coefficients of active substances were between 0.9991 and 0.9999, the detection limits ranged from 2.51ng to 35.56ng and RSDs were <5.0%, the recovery was 93.9%~105.8%, analytic time for one sample was 35 minutes. The chromatographic condition for ursolic acid and oleanolic acid: mobile phase: methanol: water=90:10 (pH=3.0), flow rate was 0.6ml/min at room temperature and detection wavelength was at 210nm. The correlation coefficients of ursolic acid and oleanolic acid were 0.9999 and 0.9997; the detection limits were 2.80ng and 2.81ng; the recoveries were 98.3% and 98.7%, respectively. These methods were briefness, rapid and accurate and would be used for quality control of hawthorn crude herb, extracts, and preparations. The analysis indicated that "hyperoside and isoquercitrin were primary flavonoids in Chinese Hawthorn fruits, the contents were not
more than 100mg/kg. Ursolic acid was predominance in triterpene acids in Chinese Hawthorn fruits; it averaged 952mg/kg. However the procyanidins was more than 4000mg/kg, so it was regarded as representative substances in Chinese Hawthorn fruits; different varieties had remarkable diversity, their standard deviation of content were 30% at the least, this reflected multifamily of idioplasmatic resource in Chinese Hawthorn fruits; content of diversified components in congener hawthorn had relativity. In addition, contents of polyphenols in Chinese Hawthorn had elevator trend along with descend of latitude. Dynamic change of content of active components in Chinese Hawthorn fruits were determinate, it changed disciplinarian along with growth, contents of polyphenols reached maximum at the first ten days of July, procyanidins had 1.36% of fruit weight upwards, following gradually descended, when fruit mature at the middle ten days of October, content of each active component ascended appreciably. Furthermore, some unknown substances in Chinese Hawthorn fruits were found, this needed farther research.
Hawthorn (Crataegus) had a long history of use in traditional Chinese medicine and European herbal medicine. It was used for both medicinal materials and edibles in China, and was a very important botanic resource. It had planting far and wide, year' yield was very large. Primary studies indicates that hawthorn have many important pharmacological activities in the prevention and treatment of some cardiovascular diseases. These benefits were attributed to several active components such as flavonoids, procyanidins, phenolic acids and triterpene acids etc. In the present research, a suit of analysis methods for those active components was established by using a homemade high performance liquid chromatographic (HPLC) apparatus. By these methods, the contents of chlorogenic acid, epicatechin, procyanidin B2, procyanidin B5, procyanidin Cl, hyperoside, isoquercitrin, ursolic acid and oleanolic acid of 37 representative varieties of Crataegus pinnatifida from 8 provinces were determined. The changes of those active components in the fruits during development were monitored by the developed HPLC methods. The HPLC system consisted of two P200II high-pressure permanent flow pump, HYPERSIL BDS C18 (200mm x 4.6mm ID, 5um) column, UV200 II detector and Echrom 98 chromatography workstation. Chromat: graphic condition for polyphenols: solvent A was 20% memanol (v/v) (pH=3.20) and B was 30% acetonitrile (v/v) (pH=3.20), the gradient elution condition (B%/min): 10/0, 10/6, 30/13, 60/22, 100/27, 100/32, 10/35; flow rate was 0.6ml/min at room temperatuie and detection wavelength at 280nm. The correlation coefficients of active substances were between 0.9991 and 0.9999, the detection limits ranged from 2.51ng to 35.56ng and RSDs were <5.0%, the recovery was 93.9%~105.8%, analytic time for one sample was 35 minutes. The chromatographic condition for ursolic acid and oleanolic acid: mobile phase: methanol: water=90:10 (pH=3.0), flow rate was 0.6ml/min at room temperature and detection wavelength was at 210nm. The correlation coefficients of ursolic acid and oleanolic acid were 0.9999 and 0.9997; the detection limits were 2.80ng and 2.81ng; the recoveries were 98.3% and 98.7%, respectively. These methods were briefness, rapid and accurate and would be used for quality control of hawthorn crude herb, extracts, and preparations. The analysis indicated that "hyperoside and isoquercitrin were primary flavonoids in Chinese Hawthorn fruits, the contents were not
more than 100mg/kg. Ursolic acid was predominance in triterpene acids in Chinese Hawthorn fruits; it averaged 952mg/kg. However the procyanidins was more than 4000mg/kg, so it was regarded as representative substances in Chinese Hawthorn fruits; different varieties had remarkable diversity, their standard deviation of content were 30% at the least, this reflected multifamily of idioplasmatic resource in Chinese Hawthorn fruits; content of diversified components in congener hawthorn had relativity. In addition, contents of polyphenols in Chinese Hawthorn had elevator trend along with descend of latitude. Dynamic change of content of active components in Chinese Hawthorn fruits were determinate, it changed disciplinarian along with growth, contents of polyphenols reached maximum at the first ten days of July, procyanidins had 1.36% of fruit weight upwards, following gradually descended, when fruit mature at the middle ten days of October, content of each active component ascended appreciably. Furthermore, some unknown substances in Chinese Hawthorn fruits were found, this needed farther research.
引文
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