猫人参药材质量评价与质量控制关键技术的研究
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摘要
抗肿瘤中草药猫人参为华东地区治疗各类消化道肿瘤的习用中草药,是目前该地区抗肿瘤处方中最常用的品种之一。为了更好地开发利用该药用资源,本论文对猫人参药材的质量评价与质量控制进行了以下研究：
1)首次建立了猫人参药材乙酸乙酯提取液的HPLC指纹图谱,确定了3种指标成分(2α,3a-二羟基-12,18-二烯-28-乌苏酸、积雪草酸、熊果酸),并进行了含量测定方法学研究。HPLC的条件为：Aglient HC-C18柱(250 mm×4.6 mm,5μm)色谱柱；柱温35℃；以乙腈-水为流动相进行梯度洗脱,检测波长210 nm；进样量20μL,流速0.8 ml/min。试验证明,本方法可操作性强,重现性好,为猫人参药材质量标准从定性走向定量从方法学上提供了有力的依据。同时,我们提出猫人参药材质量不仅与指标成分的绝对含量有关,而且很可能与其相对比例也相关。
2)通过对不同基原植物、入药部位的指纹图谱、指标成分、离体抗肿瘤活性的分析比较,我们发现对萼猕猴桃的根与大籽猕猴桃的根差异显著,因此我们认为猫人参的基原植物应该仅为大籽猕猴桃,而不应包括对萼猕猴桃。而大籽猕猴桃的根与粗茎在3个指标上均无显著差异,因此二者均可作为入药部位。
3)不同类型猫人参药材的比较分析表明,不同产地的猫人参在3种指标成分的绝对含量上差异相对较小,但是三者的比例差异显著,结合药理实验,初步认为富阳产药材的质量最优(积雪草酸、熊果酸、2α,3a-二羟基-12,18-二烯-28-乌苏酸三者的比例12：31：57)。3种指标成分含量和比例随着物候期和月份波动,结合药理实验和传统采挖观点,我们认为11-12月份植株枯叶至落叶期是猫人参的最佳采收时期。虽然搭架栽培和匍地栽培的药材在指标成分方面差异不大,但考虑到药材产量,搭架栽培更合适。
4)供试产区的土壤理化性质与猫人参药材的指标成分含量关系不显著,因此在栽培基地选址时对于土壤可不做严格要求。
本文研究结果基本澄清了猫人参在实际生产和应用中长期困扰的基原和入药部位问题,大幅度补充和完善了该药材的质量评价标准,将为猫人参资源的开发、利用奠定良好的基础。
Actinidia macrosperma C. F. Liang (Cat Ginseng) is one of the most frequently applied traditional Chinese medicine (TCM) for antitumor therapy, especially against alimentary canal cancers in East China. In order to exploit this resource in China with quality assessment and quality control, the following studies were carried out.
1) HPLC method was established for quantify determine of TCM Cat Ginseng determining the ursolic acid, asiatic acid and 2a,3a-dihydroxy-urs-12,18-dien-28-oic acid in rude medical material of Cat Ginseng for the first time, which were successf-ully separated on an Aglient HC-C18 column (250 mm×4.6 mm,5μm) at 35℃. The mobile phase was acetonitrile-water employing a gradient elution at a flow rate of 0.8 ml/min, and the detection wavelength was set at 210 nm. All results showed that the method of HPLC fingerprinting analysis was good and satisfactory. Simultaneously, we found the quality of Cat Ginseng not only related to absolute content of the reference components but also related to their relative proportions.
2) Through analysis and comparison of fingerprint, reference components and anti-tumor activity in vitro from different Actinidia species and medicinal parts, we found between A.macrosperma and A. valvata had significant differences. Therefore, we believe that the original plant of Cat Ginseng should only A. macrosperma, not including A. valvata. Moreover, the three reference components of stems and roots from A.macrosperma were not significantly different, so the two parts could be used as medicinal parts.
3) Through analysis and comparison of different types of Cat Ginseng, we found the absolute content of the three reference components from different regions had small differences, but existed significant differences in their relative proportions. Taking into account pharmacological experiments, preliminarily, we suggested that the best quality medicines produced in Fuyang (relative proportions of asiatic acid, ursolic acid and 2a,3α-dihydroxy- urs- 12,18- en- 28-oic acid were 12:31:57). The content and proportions of three reference components fluctuated with the month and phenology, combining with the view of pharmacological experiments and traditional excavation, we believe that 11-12 month period is the best harvest period for Cat Ginseng. Although displayed only small difference in the reference components of different cultivation, considering yield, scaffolding was more appropriate for Cat Ginseng.
4) The relationship between the content of reference components and physical and chemical properties of soil from different tested areas was not significant, so there was no strict requirements for the soil of cultivation.
In the present study, an efficient strategy is proposed for quality control of A.macrosperma as raw materials in the pharmaceutical and natural product industries, especially, solved the problem of the original plant and medicinal parts of A.macrosperma which troubled people for a long time, which forms a foundation for further exploitation and use of A.macrosperma resource.
1)首次建立了猫人参药材乙酸乙酯提取液的HPLC指纹图谱,确定了3种指标成分(2α,3a-二羟基-12,18-二烯-28-乌苏酸、积雪草酸、熊果酸),并进行了含量测定方法学研究。HPLC的条件为：Aglient HC-C18柱(250 mm×4.6 mm,5μm)色谱柱；柱温35℃；以乙腈-水为流动相进行梯度洗脱,检测波长210 nm；进样量20μL,流速0.8 ml/min。试验证明,本方法可操作性强,重现性好,为猫人参药材质量标准从定性走向定量从方法学上提供了有力的依据。同时,我们提出猫人参药材质量不仅与指标成分的绝对含量有关,而且很可能与其相对比例也相关。
2)通过对不同基原植物、入药部位的指纹图谱、指标成分、离体抗肿瘤活性的分析比较,我们发现对萼猕猴桃的根与大籽猕猴桃的根差异显著,因此我们认为猫人参的基原植物应该仅为大籽猕猴桃,而不应包括对萼猕猴桃。而大籽猕猴桃的根与粗茎在3个指标上均无显著差异,因此二者均可作为入药部位。
3)不同类型猫人参药材的比较分析表明,不同产地的猫人参在3种指标成分的绝对含量上差异相对较小,但是三者的比例差异显著,结合药理实验,初步认为富阳产药材的质量最优(积雪草酸、熊果酸、2α,3a-二羟基-12,18-二烯-28-乌苏酸三者的比例12：31：57)。3种指标成分含量和比例随着物候期和月份波动,结合药理实验和传统采挖观点,我们认为11-12月份植株枯叶至落叶期是猫人参的最佳采收时期。虽然搭架栽培和匍地栽培的药材在指标成分方面差异不大,但考虑到药材产量,搭架栽培更合适。
4)供试产区的土壤理化性质与猫人参药材的指标成分含量关系不显著,因此在栽培基地选址时对于土壤可不做严格要求。
本文研究结果基本澄清了猫人参在实际生产和应用中长期困扰的基原和入药部位问题,大幅度补充和完善了该药材的质量评价标准,将为猫人参资源的开发、利用奠定良好的基础。
Actinidia macrosperma C. F. Liang (Cat Ginseng) is one of the most frequently applied traditional Chinese medicine (TCM) for antitumor therapy, especially against alimentary canal cancers in East China. In order to exploit this resource in China with quality assessment and quality control, the following studies were carried out.
1) HPLC method was established for quantify determine of TCM Cat Ginseng determining the ursolic acid, asiatic acid and 2a,3a-dihydroxy-urs-12,18-dien-28-oic acid in rude medical material of Cat Ginseng for the first time, which were successf-ully separated on an Aglient HC-C18 column (250 mm×4.6 mm,5μm) at 35℃. The mobile phase was acetonitrile-water employing a gradient elution at a flow rate of 0.8 ml/min, and the detection wavelength was set at 210 nm. All results showed that the method of HPLC fingerprinting analysis was good and satisfactory. Simultaneously, we found the quality of Cat Ginseng not only related to absolute content of the reference components but also related to their relative proportions.
2) Through analysis and comparison of fingerprint, reference components and anti-tumor activity in vitro from different Actinidia species and medicinal parts, we found between A.macrosperma and A. valvata had significant differences. Therefore, we believe that the original plant of Cat Ginseng should only A. macrosperma, not including A. valvata. Moreover, the three reference components of stems and roots from A.macrosperma were not significantly different, so the two parts could be used as medicinal parts.
3) Through analysis and comparison of different types of Cat Ginseng, we found the absolute content of the three reference components from different regions had small differences, but existed significant differences in their relative proportions. Taking into account pharmacological experiments, preliminarily, we suggested that the best quality medicines produced in Fuyang (relative proportions of asiatic acid, ursolic acid and 2a,3α-dihydroxy- urs- 12,18- en- 28-oic acid were 12:31:57). The content and proportions of three reference components fluctuated with the month and phenology, combining with the view of pharmacological experiments and traditional excavation, we believe that 11-12 month period is the best harvest period for Cat Ginseng. Although displayed only small difference in the reference components of different cultivation, considering yield, scaffolding was more appropriate for Cat Ginseng.
4) The relationship between the content of reference components and physical and chemical properties of soil from different tested areas was not significant, so there was no strict requirements for the soil of cultivation.
In the present study, an efficient strategy is proposed for quality control of A.macrosperma as raw materials in the pharmaceutical and natural product industries, especially, solved the problem of the original plant and medicinal parts of A.macrosperma which troubled people for a long time, which forms a foundation for further exploitation and use of A.macrosperma resource.
引文
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