救必应高效薄层色谱指纹图谱及组织化学研究
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摘要
救必应,又名龙胆仔、白木香、白沉香、米碎木、山冬青、白皮冬青、大叶冬青、白山叶等。为冬青科冬青属植物铁冬青(Ilex rotunda Thunb.)的干燥树皮或根皮,是我国南方常用的中药。味苦,性寒,具有清热解毒、利湿、行气止痛、凉血止血等功效,临床上常用于感冒、扁桃体炎、咽喉炎、急性肠胃炎、痢疾、骨痛等。
救必应作为药用,始载于《岭南采药录》,称“白木香、味苦、清热毒”。《中华人民共和国药典》(1977年版一部、2010年版一部)也收载了该药材,其质量控制方法主要为药材性状鉴别、粉末显微鉴别,标准药材为对照的薄层色谱鉴别及运用高效液相色谱法进行的代表性化学成分的含量测定。但这些控制方法比较片面,不能系统的为救必应的化学成分进行质量标准的制定。除此之外,目前市场上的救必应药材不仅茎皮与根皮混用,并且由于生长年限、采收部位或者产地的不同等因素导致茎皮药材横切面厚度相差较大,质量优劣不等,甚至出现有同属近缘种植物药材混用的现象,影响救必应药材的临床使用标准及药效。
为此,本课题对救必应进行了化学成分分离鉴定,在此基础上,选用适宜的指标成分,构建救必应薄层色谱指纹图谱,并将救必应不同用药部位以及同属近缘种植物不同部位的薄层色谱图与救必应指纹图谱进行对比,为完善救必应质量标准提供科学依据。同时因救必应中皂苷类成分含量大且多数为其有效成分,如其中含量较大的具栖冬青苷等,本课题通过组织化学的定位研究来探讨皂苷类成分在救必应组织中分布的情况,为救必应药材的合理开发和应用提供了新的理论基础,也为其进一步的研究提供了新方向。研究结果如下：
1.采用薄层色谱法,建立了救必应薄层色谱指纹图谱,以多批次样品中稳定出现的11个峰构建其指纹特征。与随行对照品进行比对,辨认其中的3个特征峰分别为：丁香苷(syringin),具栖冬青苷(peduncu-loside),铁冬青酸(rotundic acid)。应用Chromafinger2005色谱指纹图谱系统解决方案软件(珠海科曼中药研究有限公司)将薄层色谱图像转化成轮廓曲线图并进行相似度评价,结果除了一份样品相似度为0.84以外,其余所有样品的相似度均在0.9以上,而相似度较低那一份样品经观察后发现整体药材厚度大,与其余样品差别较大。利用HPTLC指纹图谱可以为救必应建立一个简便的、系统的、专属性强的质量控制方法。
2.利用所建立的救必应指纹图谱,考察因生长年限等因素所导致的不同横切面厚度的救必应药材的薄层色谱差异,同时对比救必应不同部位(茎皮、根皮、木心和叶)的薄层色谱以及救必应两种同属近缘种植物药不同部位的薄层色谱差异。结果不同厚度救必应药材的薄层色谱差异较明显,与救必应药材共有模式相比较,厚度为0.5-2mm的救必应药材相似度最高,厚度为2-7mm的救必应药材次之,而厚度为9-12mm的救必应药材薄层色谱明显的与其他厚度药材的薄层色谱有较大分别,丁香营和具栖冬青苷处没有对应的斑点,且大部分的斑点位置和亮度与其他样品相对比也有较大差异。另外,随着厚度的增加,图谱中丁香苷所对应的斑点亮度呈现逐渐减弱现象,说明药材厚度对化学成分的含量有一定的影响。
而不同部位的薄层色谱图差异也较大,其中与救必应药材商品薄层色谱图极为相似的为救必应茎皮部位的色谱图,而在救必应根皮及木心部位的薄层图谱中,丁香苷所对应的斑点不明显,且木心部位图谱可以明显看出化学成分以及含量的差异,叶片部位差异更甚,丁香苷及具栖冬青苷的含量和其他部位相比明显较少。同属近缘种植物药之间的薄层图谱差异也较大,不能将药材混用之。
3.应用植物解剖学和组织化学技术,研究救必应植物中皂苷成分的累积分布状态。将救必应不同部位的显微切片用5%香草醛-冰醋酸和高氯酸混合试剂进行染色后放置于显微镜下观察并拍照。阴性对照品为用FAA固定液浸泡1个月的切片组织。结果显示救必应中的皂苷成分主要分布在营养器官的细胞组织中,其中在根中主要分布在次生韧皮部和皮层薄壁细胞中；在茎中主要分布在次生韧皮部细胞中；在叶中则主要分布在栅栏组织中,海绵组织有少量分布。
本课题为救必应建立了以高效薄层色谱指纹图谱为核心的救必应药材分析方法,结果表明,救必应植物不同药用部位以及药材不同横切面厚度对救必应药材质量影响较大,与同属近缘种植物间的差异也较大,应该加以区别应用。出于药用植物资源的可持续发展来看,将救必应不同部位的化学成分作对比研究也是具有长远的意义的。因此,以救必应主要成分化合物为指标建立救必应指纹图谱评价质量标准的技术平台具有重要意义。同时,本课题通过组织化学的定位研究来探讨皂苷类成分在救必应组织中分布的情况,为救必应进一步的研究提供了新方向。但由于课题经费以及时间问题,本实验暂时无法对药材进行定点采收并加以系统的比较,只是对于市场上收集到的药材进行初步对比,对于救必应药材不同生长年限等因素所照成的化学成分差异还需进一步深入的研究,救必应中其他类型化学成分的组织化学研究也有待探讨。
Ilex rotunda Thunb., which has other names as longdanzai, baimuxiang, baichenxiang, misuimu, shandongqing, baipidongqing, dayedongqing, baishanye, et al. It's from the cortex and velamen of Ilex rotunda Thunb., bitter taste and cold-natured, which has been a remedy for clearing heat and removing toxicity, removing dampness through diuresis, promoting the circulation of qi, relieving pain and hemostasis in Southern China. Modern pharmacology study showed that it possessed actions as cold, tonsillitis, pharyngolaryngitis, acute gastroenteritis, dysentery and ostalgia.
As a traditional Chinese medicine, Ilex rotunda Thunb. has been recorded in Lingnan Cai Yao Lu, it said that Baimuxiang, bitter taste and clear the heat poison. It also has been recorded in Chinese Pharmacology(1977and2010Edition), the quality of Ilex rotunda Thunb. are mostly controlled by medicinal material properties identification, powder microscopic identification, TLC contrasted with standard medicine and HPLC contrasted with representative chemical composition. But these control method is unilateral, can not set the quality standards for chemical composition of Ilex rotunda Thunb. systematically. In addition, not only cortex and velamen of Ilex rotunda Thunb. combine on the market, but also has large differencens of thickness and quality, even combine with its related species, influence the clinical standard and effects of Ilex rotunda Thunb.
Therefore, systematic separation and identification were studied on this medicine. On the basis, we screened suitable index components to establish quality evaluation method using HPTLC chromatographic fingerprint in the material medicine, and compare the differences of HPTLC between different position of Ilex rotunda Thunb. and its related species, which may help to establish the quality standard of this medicine. At the same time, Ilex rotunda Thunb. has large content of saponins and most of them are the effective constituent, just like peduncu-loside. Our study used anatomical, histochemical methods to investigate the saponins localization in Ilex rotunda Thunb., it may help the rational exploitation and application of Ilex rotunda Thunb., also improve the study of Ilex rotunda Thunb.
The results showed as follows:
1. The determination method of HPTLC fingerprinting of Ilex rotunda Thunb.. The fingerprint characteristics have been constructed by eleven stable peaks of thirteen batches drug materials collected from different areas. Comparing with reference substances, we could identify syringin, peduncu-loside and rotundic acid, respectively. The fingerprint characteristics have been obtained through " ChromafingerTM2005" solution software, and authentication and quality assessment have been analyzed by similarity analysis. The similarities of samples are all above0.9, except one sample is0.84, there have bigger thickness of rinds in that sample, which is easily recognized from other samples. HPTLC for determination the fingerprinting of Ilex rotunda Thunb. is simple, systematic, specificity, and is beneficial for identification of different plant plant parts and quality control of the material medicine.
2. HPTLC fingerprinting of Ilex rotunda Thunb. was used to compare the differences between different thickness of Ilex rotunda Thunb., at the same time to compare the differehces between different positions(including bark, velamen, core of stem and leaf) and its related species. The result shows that there are notable differences between different position and different thickness. Compare with the common pattern, the samples with thickness from0.5mm to2mm have the highest similarity, samples with thickness from2mm to7mm take second place. But sample with thickness from9mm to12mm can be easily differentiated from other samples, syringin and peduncu-loside have no corresponding spots in this sample, the position and lightness of other spots are all different from other samples. In addition, as the thickness increases, the lightness of syringin spot would trail away, the result explain that thickness have an affect on content of chemical composition.
The chromatogram of bark has the highest similarity with common pattern; velamen has less content of syringin because the spot is not obvious; all the spots of the core are have less lightness, the chromatogram of leaf has the lowest similarity with common pattern, the content of syringin and peduneu-loside are less than other positions obviously. The other relatives can be easily differentiated from Ilex rotunda too. They can not be used as Ilex rotunda Thunb.
3. Anatomical and histochemical methods were used to investigate the saponins localization in Ilex rotunda Thunb. Use5%vanillin-glacial acetic acid and perchloric acid as derivatization reagent to dye different cross-section of Ilex rotunda Thunb., The negative control product were the biopsy tissue which socked with FAA for1months. Histochemical localization results showed that saponins accumulated mainly in parenchyma cell of vegetative organ of Ilex rotunda Thunb., saponins distributed in secondary phloem and cortex of root, in secondary phloem and phloem of stem, mainly in palisade tissue of leaf.
In this paper, establishing chromatographic fingerprint in the material medicine can be used to identify and evaluate the quality of Ilex rotunda Thunb.. The result showed that different parts or different thickness may affect the quality of the medicine, The other relatives can be easily differentiated from Ilex rotunda Thunb. too. They can not be used as Ilex rotunda Thunb.. At the same time, our study used anatomical, histochemical methods to investigate the saponins localization in Ilex rotunda Thunb.
救必应作为药用,始载于《岭南采药录》,称“白木香、味苦、清热毒”。《中华人民共和国药典》(1977年版一部、2010年版一部)也收载了该药材,其质量控制方法主要为药材性状鉴别、粉末显微鉴别,标准药材为对照的薄层色谱鉴别及运用高效液相色谱法进行的代表性化学成分的含量测定。但这些控制方法比较片面,不能系统的为救必应的化学成分进行质量标准的制定。除此之外,目前市场上的救必应药材不仅茎皮与根皮混用,并且由于生长年限、采收部位或者产地的不同等因素导致茎皮药材横切面厚度相差较大,质量优劣不等,甚至出现有同属近缘种植物药材混用的现象,影响救必应药材的临床使用标准及药效。
为此,本课题对救必应进行了化学成分分离鉴定,在此基础上,选用适宜的指标成分,构建救必应薄层色谱指纹图谱,并将救必应不同用药部位以及同属近缘种植物不同部位的薄层色谱图与救必应指纹图谱进行对比,为完善救必应质量标准提供科学依据。同时因救必应中皂苷类成分含量大且多数为其有效成分,如其中含量较大的具栖冬青苷等,本课题通过组织化学的定位研究来探讨皂苷类成分在救必应组织中分布的情况,为救必应药材的合理开发和应用提供了新的理论基础,也为其进一步的研究提供了新方向。研究结果如下：
1.采用薄层色谱法,建立了救必应薄层色谱指纹图谱,以多批次样品中稳定出现的11个峰构建其指纹特征。与随行对照品进行比对,辨认其中的3个特征峰分别为：丁香苷(syringin),具栖冬青苷(peduncu-loside),铁冬青酸(rotundic acid)。应用Chromafinger2005色谱指纹图谱系统解决方案软件(珠海科曼中药研究有限公司)将薄层色谱图像转化成轮廓曲线图并进行相似度评价,结果除了一份样品相似度为0.84以外,其余所有样品的相似度均在0.9以上,而相似度较低那一份样品经观察后发现整体药材厚度大,与其余样品差别较大。利用HPTLC指纹图谱可以为救必应建立一个简便的、系统的、专属性强的质量控制方法。
2.利用所建立的救必应指纹图谱,考察因生长年限等因素所导致的不同横切面厚度的救必应药材的薄层色谱差异,同时对比救必应不同部位(茎皮、根皮、木心和叶)的薄层色谱以及救必应两种同属近缘种植物药不同部位的薄层色谱差异。结果不同厚度救必应药材的薄层色谱差异较明显,与救必应药材共有模式相比较,厚度为0.5-2mm的救必应药材相似度最高,厚度为2-7mm的救必应药材次之,而厚度为9-12mm的救必应药材薄层色谱明显的与其他厚度药材的薄层色谱有较大分别,丁香营和具栖冬青苷处没有对应的斑点,且大部分的斑点位置和亮度与其他样品相对比也有较大差异。另外,随着厚度的增加,图谱中丁香苷所对应的斑点亮度呈现逐渐减弱现象,说明药材厚度对化学成分的含量有一定的影响。
而不同部位的薄层色谱图差异也较大,其中与救必应药材商品薄层色谱图极为相似的为救必应茎皮部位的色谱图,而在救必应根皮及木心部位的薄层图谱中,丁香苷所对应的斑点不明显,且木心部位图谱可以明显看出化学成分以及含量的差异,叶片部位差异更甚,丁香苷及具栖冬青苷的含量和其他部位相比明显较少。同属近缘种植物药之间的薄层图谱差异也较大,不能将药材混用之。
3.应用植物解剖学和组织化学技术,研究救必应植物中皂苷成分的累积分布状态。将救必应不同部位的显微切片用5%香草醛-冰醋酸和高氯酸混合试剂进行染色后放置于显微镜下观察并拍照。阴性对照品为用FAA固定液浸泡1个月的切片组织。结果显示救必应中的皂苷成分主要分布在营养器官的细胞组织中,其中在根中主要分布在次生韧皮部和皮层薄壁细胞中；在茎中主要分布在次生韧皮部细胞中；在叶中则主要分布在栅栏组织中,海绵组织有少量分布。
本课题为救必应建立了以高效薄层色谱指纹图谱为核心的救必应药材分析方法,结果表明,救必应植物不同药用部位以及药材不同横切面厚度对救必应药材质量影响较大,与同属近缘种植物间的差异也较大,应该加以区别应用。出于药用植物资源的可持续发展来看,将救必应不同部位的化学成分作对比研究也是具有长远的意义的。因此,以救必应主要成分化合物为指标建立救必应指纹图谱评价质量标准的技术平台具有重要意义。同时,本课题通过组织化学的定位研究来探讨皂苷类成分在救必应组织中分布的情况,为救必应进一步的研究提供了新方向。但由于课题经费以及时间问题,本实验暂时无法对药材进行定点采收并加以系统的比较,只是对于市场上收集到的药材进行初步对比,对于救必应药材不同生长年限等因素所照成的化学成分差异还需进一步深入的研究,救必应中其他类型化学成分的组织化学研究也有待探讨。
Ilex rotunda Thunb., which has other names as longdanzai, baimuxiang, baichenxiang, misuimu, shandongqing, baipidongqing, dayedongqing, baishanye, et al. It's from the cortex and velamen of Ilex rotunda Thunb., bitter taste and cold-natured, which has been a remedy for clearing heat and removing toxicity, removing dampness through diuresis, promoting the circulation of qi, relieving pain and hemostasis in Southern China. Modern pharmacology study showed that it possessed actions as cold, tonsillitis, pharyngolaryngitis, acute gastroenteritis, dysentery and ostalgia.
As a traditional Chinese medicine, Ilex rotunda Thunb. has been recorded in Lingnan Cai Yao Lu, it said that Baimuxiang, bitter taste and clear the heat poison. It also has been recorded in Chinese Pharmacology(1977and2010Edition), the quality of Ilex rotunda Thunb. are mostly controlled by medicinal material properties identification, powder microscopic identification, TLC contrasted with standard medicine and HPLC contrasted with representative chemical composition. But these control method is unilateral, can not set the quality standards for chemical composition of Ilex rotunda Thunb. systematically. In addition, not only cortex and velamen of Ilex rotunda Thunb. combine on the market, but also has large differencens of thickness and quality, even combine with its related species, influence the clinical standard and effects of Ilex rotunda Thunb.
Therefore, systematic separation and identification were studied on this medicine. On the basis, we screened suitable index components to establish quality evaluation method using HPTLC chromatographic fingerprint in the material medicine, and compare the differences of HPTLC between different position of Ilex rotunda Thunb. and its related species, which may help to establish the quality standard of this medicine. At the same time, Ilex rotunda Thunb. has large content of saponins and most of them are the effective constituent, just like peduncu-loside. Our study used anatomical, histochemical methods to investigate the saponins localization in Ilex rotunda Thunb., it may help the rational exploitation and application of Ilex rotunda Thunb., also improve the study of Ilex rotunda Thunb.
The results showed as follows:
1. The determination method of HPTLC fingerprinting of Ilex rotunda Thunb.. The fingerprint characteristics have been constructed by eleven stable peaks of thirteen batches drug materials collected from different areas. Comparing with reference substances, we could identify syringin, peduncu-loside and rotundic acid, respectively. The fingerprint characteristics have been obtained through " ChromafingerTM2005" solution software, and authentication and quality assessment have been analyzed by similarity analysis. The similarities of samples are all above0.9, except one sample is0.84, there have bigger thickness of rinds in that sample, which is easily recognized from other samples. HPTLC for determination the fingerprinting of Ilex rotunda Thunb. is simple, systematic, specificity, and is beneficial for identification of different plant plant parts and quality control of the material medicine.
2. HPTLC fingerprinting of Ilex rotunda Thunb. was used to compare the differences between different thickness of Ilex rotunda Thunb., at the same time to compare the differehces between different positions(including bark, velamen, core of stem and leaf) and its related species. The result shows that there are notable differences between different position and different thickness. Compare with the common pattern, the samples with thickness from0.5mm to2mm have the highest similarity, samples with thickness from2mm to7mm take second place. But sample with thickness from9mm to12mm can be easily differentiated from other samples, syringin and peduncu-loside have no corresponding spots in this sample, the position and lightness of other spots are all different from other samples. In addition, as the thickness increases, the lightness of syringin spot would trail away, the result explain that thickness have an affect on content of chemical composition.
The chromatogram of bark has the highest similarity with common pattern; velamen has less content of syringin because the spot is not obvious; all the spots of the core are have less lightness, the chromatogram of leaf has the lowest similarity with common pattern, the content of syringin and peduneu-loside are less than other positions obviously. The other relatives can be easily differentiated from Ilex rotunda too. They can not be used as Ilex rotunda Thunb.
3. Anatomical and histochemical methods were used to investigate the saponins localization in Ilex rotunda Thunb. Use5%vanillin-glacial acetic acid and perchloric acid as derivatization reagent to dye different cross-section of Ilex rotunda Thunb., The negative control product were the biopsy tissue which socked with FAA for1months. Histochemical localization results showed that saponins accumulated mainly in parenchyma cell of vegetative organ of Ilex rotunda Thunb., saponins distributed in secondary phloem and cortex of root, in secondary phloem and phloem of stem, mainly in palisade tissue of leaf.
In this paper, establishing chromatographic fingerprint in the material medicine can be used to identify and evaluate the quality of Ilex rotunda Thunb.. The result showed that different parts or different thickness may affect the quality of the medicine, The other relatives can be easily differentiated from Ilex rotunda Thunb. too. They can not be used as Ilex rotunda Thunb.. At the same time, our study used anatomical, histochemical methods to investigate the saponins localization in Ilex rotunda Thunb.
引文
[1]广东省食品药品监督管理局.广东省中药材标准第一册[M].广州：广东科技出版社,2004：172-173.
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