药用石韦的孢子扫描电镜鉴定及黄酮化合物的组织化学定位和测定
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摘要
石韦为水龙骨科(Polypodiaceae),石韦属(Pyrrosia Mirbel)多种植物,广布于东北、华北、华中、西北和西南各省区。其地上部分可作药用,味苦、甘,性微寒,具有利尿通淋、清肺止血的功效。2005年《中华人民共和国药典》将同属3种植物石韦[Pyrrosia lingua(Thunb.)Fatwell]庐山石韦[Pyrrosia sheareri(Bak.)Ching]和有柄石韦[Pyrrosia petiolosa(Christ)Ching]的干燥叶作为石韦药材使用,同属多种植物也在全国不同地区应用。
为了使各种药源石韦易于区别,提供药材的亚显微结构资料,更有利于区分外观形态较相似的不同种石韦,对石韦的药材鉴定及质量控制是非常必要的。对中药石韦中的成分分析方法进行研究,并测定石韦中不同种、不同部位成分的含量,可为石韦药材的质量控制及合理使用提供化学依据。我们对5种药用石韦进行了较系统的研究,得到了以下结果:
(1)利用扫描电镜对所采毡毛石韦Pyrrosia drakeana(Franch.)Ching、有柄石韦Pyrrosiapetiolosa(Christ)Ching、华北石韦Pyrrosia davidii(Bak.)Ching、石韦Pyrrosia lingua(Thunb.)Fatwell孢子的表面饰纹观察,为以后的药材鉴定提供了可靠的亚显微结构资料。
(2)组织化学染色结果显示:药用石韦的黄酮类化合物基本分布于石韦药材叶的表皮、叶肉细胞以及星状毛;叶柄的皮层厚壁组织、髓细胞、维管束鞘及韧皮部;在根状茎中主要分布在表皮、髓细胞及韧皮部。在实际应用中,星状毛往往被弃而不用,这无形中损失了黄酮化合物提取的有效部位。
(3)采用正交实验设计法,筛选优化提取参数,探究石韦总黄酮的提取工艺,并利用最佳提取工艺提取5种不同石韦材料总黄酮,采用分光光度法进行测定后加以比较。结果表明,5种药用石韦的叶总黄酮含量明显高于根状茎的黄酮含量。总黄酮含量由高到低依次为:毡毛石韦117.508mg/g,石韦药材103.449mg/g,有柄石韦97.576mg/g,石韦68.994mg/g,华北石韦53.548mg/g。不同种石韦总黄酮含量存在显著差别,提示不同种石韦可能具有不同的药用功效。所以把总黄酮作为石韦中药质量控制的指标成分之一,对不同种石韦分别制定评价标准,区别对待,是很有必要的。
(4)利用薄层色谱及薄层扫描对5种石韦材料乙酸乙酯提取部位进行检测。结果显示,不同药用石韦所含的化学成分种类虽较相似,但也有一些差别。5种石韦样品中,地下部分均没有检测到槲皮素及山奈酚。只有毡毛石韦的地上部分含有槲皮素。另外,除华北石韦不含山奈酚外,其余材料均检测到了山奈酚。5种所测样品的叶中可能均有齐墩果酸的分布。
Pyrrosia leaf are the plants of Pyrrosia Mirbel,Polypodiaceae.they distribute in large area of China,the central area,the north,the northeast,the northwest and the southwest provinces.The parts on the ground are traditional medicine.taste of bitter and stweet.The plants have the function of diuresis and hemostasis.In the 2005 pharmacopoeia of the people,s republic of china,the dry leaves of three species from Pyrrosia Mirbel are used for medicinal material,they are Pyrrosia lingua(Thunb.) Fatwell,Pyrrosia sheareri(Bak.) Ching and Pyrrosia petiolosa(Christ) Ching.some other species from Pyrrosia Mirbel are also used for medicinal material in many area.
In order to differentiate the Pyrrosia materials,provide sub-microstructure information of the materials is very important.it is also significant in identification and quality control of medicinal materials.So the study of extraction and content mensuration of chemical component in Pyrrosia materials,can provide chemical data for quality control and valid applications.We have done some studies about Pyrrosia materials systematicly.
(1) By examining spore morphology of the Pyrrosia materials under ESEM,we authenticated and differentiate the different materials and provide sub-microstructure information of the materials for medicinal materials identification.The 4 species we herborized are Pyrrosia drakeana(Franch.) Ching,P.lingua(Thunb.) Fatwell,P.petiolosa(Christ) Ching,P.davidii(Bak.) Ching.
(2) The result of chromogenic reaction:Flavonoids of Pyrrosia materials distribute in of leaf, mesophyll cell and epidermal hair;sclerenchyma cell,Medullocell,bundle sheath and phloem; epidermis,Medullocell and phloem of rhizome.In practical application,the epidermal hair is always chucked away,this made the effective part of Flavonoids has been lost.
(3)The optimum extraction condition for total flavonoids was investigated by orthogonal design. Using this method,the total flavonoids from 5 different Pyrrosia materilals were extracted and determined by UV spectrophotometer.The determination of 5 different Pyrrosia leaves and rhizomes showed that the total flavonoids content of 5 different materials leaves is higher than the total flavonoids content of 5 different materials rhizomes.Chlorophyl of leaves may be bring some false positive effects to the result,but the effect is not marked.From high to low,the content of the total flavonoids is:Pyrrosia drakeana(Franch.) Ching117.508mg/g,Pyrrosia medicinal materials 103.449mg/g,P.petiolosa(Christ) Ching 97.576mg/g,P.lingua(Thunb.) Fatwell 68.994mg/g,P. davidii(Bak.) Ching 53.548mg/g.Flavonoids content of different species has large distance,this distance should bing on different officinal effect.So the total flavonoids should be one of the evaluated target of medicinal materials quality control,and different species of Pyrrosia should have different evaluated standard,it is important to differentiate them.
(4) The TLC and TLCS result of acetidin extractive from 5 different Pyrrosia leaves and rhizomes showed that the chemical components of 5 Pyrrosia materials are very alike,but the chromatograms have obvious differents.There aren't Quercetin and Kaempferol detected in the undergound parts of the 5 Pyrrosia materials.The overground part of Pyrrosia drakeana(Franch.) Ching had detected the Quercetin.In addition,there is no kaempferol in P.davidii(Bak.) Ching. Oleanolic is likely to distribute in all the 5 materials.By the detection of some effective components from 5 Pyrrosia materials,the chemical characteristic would be knowm.We shoule suit the remedy to the case and utilize the resource effectively and adequately.
为了使各种药源石韦易于区别,提供药材的亚显微结构资料,更有利于区分外观形态较相似的不同种石韦,对石韦的药材鉴定及质量控制是非常必要的。对中药石韦中的成分分析方法进行研究,并测定石韦中不同种、不同部位成分的含量,可为石韦药材的质量控制及合理使用提供化学依据。我们对5种药用石韦进行了较系统的研究,得到了以下结果:
(1)利用扫描电镜对所采毡毛石韦Pyrrosia drakeana(Franch.)Ching、有柄石韦Pyrrosiapetiolosa(Christ)Ching、华北石韦Pyrrosia davidii(Bak.)Ching、石韦Pyrrosia lingua(Thunb.)Fatwell孢子的表面饰纹观察,为以后的药材鉴定提供了可靠的亚显微结构资料。
(2)组织化学染色结果显示:药用石韦的黄酮类化合物基本分布于石韦药材叶的表皮、叶肉细胞以及星状毛;叶柄的皮层厚壁组织、髓细胞、维管束鞘及韧皮部;在根状茎中主要分布在表皮、髓细胞及韧皮部。在实际应用中,星状毛往往被弃而不用,这无形中损失了黄酮化合物提取的有效部位。
(3)采用正交实验设计法,筛选优化提取参数,探究石韦总黄酮的提取工艺,并利用最佳提取工艺提取5种不同石韦材料总黄酮,采用分光光度法进行测定后加以比较。结果表明,5种药用石韦的叶总黄酮含量明显高于根状茎的黄酮含量。总黄酮含量由高到低依次为:毡毛石韦117.508mg/g,石韦药材103.449mg/g,有柄石韦97.576mg/g,石韦68.994mg/g,华北石韦53.548mg/g。不同种石韦总黄酮含量存在显著差别,提示不同种石韦可能具有不同的药用功效。所以把总黄酮作为石韦中药质量控制的指标成分之一,对不同种石韦分别制定评价标准,区别对待,是很有必要的。
(4)利用薄层色谱及薄层扫描对5种石韦材料乙酸乙酯提取部位进行检测。结果显示,不同药用石韦所含的化学成分种类虽较相似,但也有一些差别。5种石韦样品中,地下部分均没有检测到槲皮素及山奈酚。只有毡毛石韦的地上部分含有槲皮素。另外,除华北石韦不含山奈酚外,其余材料均检测到了山奈酚。5种所测样品的叶中可能均有齐墩果酸的分布。
Pyrrosia leaf are the plants of Pyrrosia Mirbel,Polypodiaceae.they distribute in large area of China,the central area,the north,the northeast,the northwest and the southwest provinces.The parts on the ground are traditional medicine.taste of bitter and stweet.The plants have the function of diuresis and hemostasis.In the 2005 pharmacopoeia of the people,s republic of china,the dry leaves of three species from Pyrrosia Mirbel are used for medicinal material,they are Pyrrosia lingua(Thunb.) Fatwell,Pyrrosia sheareri(Bak.) Ching and Pyrrosia petiolosa(Christ) Ching.some other species from Pyrrosia Mirbel are also used for medicinal material in many area.
In order to differentiate the Pyrrosia materials,provide sub-microstructure information of the materials is very important.it is also significant in identification and quality control of medicinal materials.So the study of extraction and content mensuration of chemical component in Pyrrosia materials,can provide chemical data for quality control and valid applications.We have done some studies about Pyrrosia materials systematicly.
(1) By examining spore morphology of the Pyrrosia materials under ESEM,we authenticated and differentiate the different materials and provide sub-microstructure information of the materials for medicinal materials identification.The 4 species we herborized are Pyrrosia drakeana(Franch.) Ching,P.lingua(Thunb.) Fatwell,P.petiolosa(Christ) Ching,P.davidii(Bak.) Ching.
(2) The result of chromogenic reaction:Flavonoids of Pyrrosia materials distribute in of leaf, mesophyll cell and epidermal hair;sclerenchyma cell,Medullocell,bundle sheath and phloem; epidermis,Medullocell and phloem of rhizome.In practical application,the epidermal hair is always chucked away,this made the effective part of Flavonoids has been lost.
(3)The optimum extraction condition for total flavonoids was investigated by orthogonal design. Using this method,the total flavonoids from 5 different Pyrrosia materilals were extracted and determined by UV spectrophotometer.The determination of 5 different Pyrrosia leaves and rhizomes showed that the total flavonoids content of 5 different materials leaves is higher than the total flavonoids content of 5 different materials rhizomes.Chlorophyl of leaves may be bring some false positive effects to the result,but the effect is not marked.From high to low,the content of the total flavonoids is:Pyrrosia drakeana(Franch.) Ching117.508mg/g,Pyrrosia medicinal materials 103.449mg/g,P.petiolosa(Christ) Ching 97.576mg/g,P.lingua(Thunb.) Fatwell 68.994mg/g,P. davidii(Bak.) Ching 53.548mg/g.Flavonoids content of different species has large distance,this distance should bing on different officinal effect.So the total flavonoids should be one of the evaluated target of medicinal materials quality control,and different species of Pyrrosia should have different evaluated standard,it is important to differentiate them.
(4) The TLC and TLCS result of acetidin extractive from 5 different Pyrrosia leaves and rhizomes showed that the chemical components of 5 Pyrrosia materials are very alike,but the chromatograms have obvious differents.There aren't Quercetin and Kaempferol detected in the undergound parts of the 5 Pyrrosia materials.The overground part of Pyrrosia drakeana(Franch.) Ching had detected the Quercetin.In addition,there is no kaempferol in P.davidii(Bak.) Ching. Oleanolic is likely to distribute in all the 5 materials.By the detection of some effective components from 5 Pyrrosia materials,the chemical characteristic would be knowm.We shoule suit the remedy to the case and utilize the resource effectively and adequately.
引文
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