地黄基于HPLC指纹图谱的质量评价及种质资源利用研究
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摘要
本研究主要目的是建立和完善地黄三种常用的药用形式—鲜地黄、生地黄和熟地黄的高效液相色谱（HPLC）指纹图谱技术，并利用建立的指纹图谱技术评价和控制地黄药材的质量，对地黄的属内近缘种的药用价值等可利用性进行评价，同时利用指纹图谱技术进行地黄新品种的选育。
主要研究内容包括地黄指纹图谱方法探索及鲜地黄、生地黄和熟地黄指纹图谱的建立，鲜地黄、生地黄和熟地黄的质量评价，鲜地黄、生地黄和熟地黄HPLC指纹差异及熟地黄炮制质量控制和炮制新方法探讨，地黄属内近缘种化学指纹特征、可利用性和生物学特征研究，地黄新材料选育等。
为了建立和完善地黄指纹图谱技术，采用单因素分析方法，对地黄HPLC指纹图谱建立中的样品溶液制备方法，色谱条件进行研究，逐渐建立了用甲醇冷浸后超声波提取，蒸干后用少量低浓度甲醇溶解获得浓缩样品溶液。采用乙腈－磷酸溶液线性梯度洗脱方法，建立了鲜地黄、生地黄205nm HPLC指纹图谱和205nm单波长或205nm和284nm双波长熟地黄指纹图谱。同时建立了地黄中主要检测成分梓醇、5-羟甲基糠醛（5-HMF）和麦角甾苷含量随指纹图谱同时检测的单波长和多波长方法。鲜地黄HPLC指纹图谱可精确的反映地黄的品种特征，生地黄指纹图谱除呈现品种的信息外，还带有产地气候特征、栽培方式以及加工、贮存等信息，而熟地黄指纹图谱主要呈现加工信息，反映熟地的炮制熟化程度。
为了评价地黄品质，采用已建立的地黄指纹图谱技术和主成分分析中的综合主成分值对生地黄、熟地黄和鲜地黄进行质量评价，研究显示生地黄和鲜地黄主成分分析中主成分类型主要以色谱峰的化学性质划分，而熟地黄主成分划分主要与熟地炮制过程中色谱峰的变化规律有关。生地主成分分析中综合主成分值高的，即质量好的生地黄是来自地黄主产区河南、山西的材料和新制材料，综合主成分值与梓醇含量相关。熟地综合主成分值与5-羟甲基糠醛含量有关，而鲜地黄综合主成分值与麦角甾苷含量相关。同时简化了地黄综合质量评价方法，仅利用包括检测成分在内的少数色谱峰就可对地黄综合质量进行评价。
鲜地黄、生地黄和熟地黄药性不同，为揭开药性差异的本质，对鲜地黄、生地黄和熟地黄的指纹图谱进行了比较，研究了熟地炮制过程中指纹的变化。结果显示鲜地黄和生地黄指纹差异主要在色谱图后半部，即苯乙醇苷所在的区域，鲜地黄在干制成生地黄过程中苯乙醇苷类明显降低。其他次生成分大多降低，少数衍生物质峰出现并升高。而生地和熟地指纹图谱在前半部明显不同，炮制成熟地过程中梓醇等环烯醚萜苷类以及多种次生成分降低或消失，衍生物质5-羟甲基糠醛等含量上升。这些变化可能是从鲜地黄到熟地黄药性由寒性变为温性的原因之一。
为了充分利用地黄属种质资源，利用建立的鲜地黄指纹图谱技术研究了地黄属内部分近缘种的化学指纹特征（除天目地黄外），并利用普通杂交技术研究近缘种之间传粉特性和杂交亲和性。结果显示由于地黄属种间亲缘关系较近，从其化学指纹特征上很难判定亲缘关系特征。地黄的三个近缘种根部梓醇含量低于地黄，而裂叶地黄和茄叶地黄麦角甾苷含量较高，但地黄的这些近缘种不具备代替地黄药用的潜力。杂交结果显示地黄属均为虫媒植物。茄叶地黄为不育种，并对其起源和分类地位进行推测。部分地黄属近缘种之间正反交显示，裂叶地黄和湖北地黄之间杂交可获得有发芽力的种子，其他组合仅获得极少种子或种子不能发芽。
为了充分利用地黄种质资源，培育适应本地气候特征的药用、食用地黄新材料，利用不同品种等自然杂交种子进行新材料选育，利用建立的鲜地黄指纹图谱技术和主成分分析综合主成分值的大小初步选择21个药用地黄材料。对二甲氨基苯甲醛显色方法测定总环烯醚萜苷的相对含量，根据材料间总环烯醚萜苷相对含量大小初步选出10个食用材料。
The purpose of the research is to establish and improve HPLC fingerprint technology ofthree kind of rehmannias, i.e. fresh rehmannia, dry rehmannia and prepared rehmannia, and toassess and control the quality of the crude medicine, as well as to assess the value of the closerelative species of Rehmannia glutinosa for medicine and to breed new varieties with thefingerprint technology.
The contents of the research include method exploring of rehmannia HPLC fingerprints,establishing the HPLC fingerprints for fresh, dry and prepared rehmannia. Quality assessmentof the three types of rehmannia, the difference of fingerprint among them, quality control andnew ways to processing prepared rehmannia are also concerned. Chemical fingerprint,usability and biologic characters of close relative species of Rehmannia glutinosa andbreeding new materials are discussed in the end.
In order to establish and improve HPLC fingerprint method, single-factor analysis wasadopted. Preparation methods of sample solution and HPLC condition for fingerprint werestudied. The methods were gradually set up. After being soaked in methanol, the sample wasabstracted by ultrasonic. The solution was dried. Residues were dissolved with a little lowconcentration methanol solution and the concentrated sample solution resulted. Acetonitrileand phosphoric acid solution was used as mobile phase for linear gradient elution. HPLCfingerprints at205nm for fresh rehmannia and dry rehmannia, fingerprints at205nm ordouble wavelength fingerprints at205nm and284nm for prepared rehmannia were acquired.At the same time, methods for content determination of catalpol,5-hydroxymethyl furfural(5-HMF) and acteoside, the major constituents in criteria, at single or multiple wavelengthswith fingerprint determination were established. HPLC fingerprints of fresh rehmannia canaccurately reflect species characters. Besides species characters, dry remannia fingerprint canalso show information of climate of producing areas, cultivation mode and storage status etc.But prepared rehmannia fingerprints mainly carry processing information. They reflect theprepared degree of processing.
To assess the quality of rehmannia, Well-established HPLC fingerprint technology andsynthesis value of major components in principal component analysis (PCA) were applied toevaluate the quality of dry rehmannia, prepared rehmannia and fresh rehmannia. The resultindicated that the principal components type in PCA of fresh rehmannia and dry rehmanniawas classified on chemical property of chromatographic peaks. While in prepared rehmannia,it was related to the variation characters of peaks in processing. In PCA, dry rehmanniasscored high in synthesis value, i.e. with high quality, were almost from the major producing areas of rehmannia, Henan and Shanxi, or the materials freshly processed. And the synthesisvalue of major components was related to content of catalpol. But it was related to5-HMFand acteoside in prepared rehmannia and fresh rehmannia respectively. And also the methodfor assessing integrated quality of rehmannia was simplified. Only a few of peaks includingprescriptive constituents in criteria were needed to assess integrated quality of rehmannia.
The medical nature is different among fresh rehmannia, dry rehmannia and preparedrehmannia. To unveil the essence of the difference, the HPLC fingerprints of three typerehmannia were compared and variation of fingerprint in processing prepared rehmannia wasstudied. The results showed that fingerprint differences between fresh rehmannia and dryrehmannia were mainly in posterior part. It was the area of phenethanol glycosides. Thecontents of phenethanol glycosides were signaficantly decreased from fresh rehmannia to dryrehmannia. Meanwhile, most other secondary components also declined and a few ofderivative peaks appeared and increased. Fingerprint differences between dry rehmannia andprepared rehmannia were almost in forepart. During processing prepared rehmannia, catalpoland some other iridoid glycosides declined or disappeared, while the derivatives such as5-HMF increased. These changes might be one of the reasons for that the medical natureshifted from cold of fresh rehmannia to warm of prepared rehmannia.
In order to make the best use of germplasm resource of genus Rehmannia, chemicalfingerprint characters of a part of close relatives of R. glutinosa in the genus, exceptR．chingii, was studied with fresh rehmannia fingerprint technology, as well as pollinationcharacter of species and cross compatibility between them with common cross method. Theresult indicated that it was difficult to determine the relationship in the genus by chemicalfingerprint for their intimate relations. The content of catalpol in the root of three relatives islower than that in Rehmannia glutinosa. But acteoside in R．piasezkii and R．solanifolia arehigher. Even though, the relatives have little possibility to take the place of R. glutinosa inmedicine. Cross results showed that the species were all entomophilous pollination plants.R．solanifolia was infertility species and its origination and classification position wasdiscussed. Reciprocal crosses between the species showed that the cross between R．henryiand R．piasezkii could give germinative seeds. The other cross pairs gave few germinativeseeds.
In order to make the best use of germplasm resource of R. glutinosa and to breed newmedical or food materials adapting to local climate, breeding was conducted with naturalcross seeds of varieties and special materials.21materials were selected for medical use byfresh rehmannia fingerprint method and on synthesis value of major components in PCA.10materials were chosen for food use on relative content of total iridoid glycoside which wasdetermined by colorimetry with paradime thylaminobenzaldehyde to present color.
主要研究内容包括地黄指纹图谱方法探索及鲜地黄、生地黄和熟地黄指纹图谱的建立，鲜地黄、生地黄和熟地黄的质量评价，鲜地黄、生地黄和熟地黄HPLC指纹差异及熟地黄炮制质量控制和炮制新方法探讨，地黄属内近缘种化学指纹特征、可利用性和生物学特征研究，地黄新材料选育等。
为了建立和完善地黄指纹图谱技术，采用单因素分析方法，对地黄HPLC指纹图谱建立中的样品溶液制备方法，色谱条件进行研究，逐渐建立了用甲醇冷浸后超声波提取，蒸干后用少量低浓度甲醇溶解获得浓缩样品溶液。采用乙腈－磷酸溶液线性梯度洗脱方法，建立了鲜地黄、生地黄205nm HPLC指纹图谱和205nm单波长或205nm和284nm双波长熟地黄指纹图谱。同时建立了地黄中主要检测成分梓醇、5-羟甲基糠醛（5-HMF）和麦角甾苷含量随指纹图谱同时检测的单波长和多波长方法。鲜地黄HPLC指纹图谱可精确的反映地黄的品种特征，生地黄指纹图谱除呈现品种的信息外，还带有产地气候特征、栽培方式以及加工、贮存等信息，而熟地黄指纹图谱主要呈现加工信息，反映熟地的炮制熟化程度。
为了评价地黄品质，采用已建立的地黄指纹图谱技术和主成分分析中的综合主成分值对生地黄、熟地黄和鲜地黄进行质量评价，研究显示生地黄和鲜地黄主成分分析中主成分类型主要以色谱峰的化学性质划分，而熟地黄主成分划分主要与熟地炮制过程中色谱峰的变化规律有关。生地主成分分析中综合主成分值高的，即质量好的生地黄是来自地黄主产区河南、山西的材料和新制材料，综合主成分值与梓醇含量相关。熟地综合主成分值与5-羟甲基糠醛含量有关，而鲜地黄综合主成分值与麦角甾苷含量相关。同时简化了地黄综合质量评价方法，仅利用包括检测成分在内的少数色谱峰就可对地黄综合质量进行评价。
鲜地黄、生地黄和熟地黄药性不同，为揭开药性差异的本质，对鲜地黄、生地黄和熟地黄的指纹图谱进行了比较，研究了熟地炮制过程中指纹的变化。结果显示鲜地黄和生地黄指纹差异主要在色谱图后半部，即苯乙醇苷所在的区域，鲜地黄在干制成生地黄过程中苯乙醇苷类明显降低。其他次生成分大多降低，少数衍生物质峰出现并升高。而生地和熟地指纹图谱在前半部明显不同，炮制成熟地过程中梓醇等环烯醚萜苷类以及多种次生成分降低或消失，衍生物质5-羟甲基糠醛等含量上升。这些变化可能是从鲜地黄到熟地黄药性由寒性变为温性的原因之一。
为了充分利用地黄属种质资源，利用建立的鲜地黄指纹图谱技术研究了地黄属内部分近缘种的化学指纹特征（除天目地黄外），并利用普通杂交技术研究近缘种之间传粉特性和杂交亲和性。结果显示由于地黄属种间亲缘关系较近，从其化学指纹特征上很难判定亲缘关系特征。地黄的三个近缘种根部梓醇含量低于地黄，而裂叶地黄和茄叶地黄麦角甾苷含量较高，但地黄的这些近缘种不具备代替地黄药用的潜力。杂交结果显示地黄属均为虫媒植物。茄叶地黄为不育种，并对其起源和分类地位进行推测。部分地黄属近缘种之间正反交显示，裂叶地黄和湖北地黄之间杂交可获得有发芽力的种子，其他组合仅获得极少种子或种子不能发芽。
为了充分利用地黄种质资源，培育适应本地气候特征的药用、食用地黄新材料，利用不同品种等自然杂交种子进行新材料选育，利用建立的鲜地黄指纹图谱技术和主成分分析综合主成分值的大小初步选择21个药用地黄材料。对二甲氨基苯甲醛显色方法测定总环烯醚萜苷的相对含量，根据材料间总环烯醚萜苷相对含量大小初步选出10个食用材料。
The purpose of the research is to establish and improve HPLC fingerprint technology ofthree kind of rehmannias, i.e. fresh rehmannia, dry rehmannia and prepared rehmannia, and toassess and control the quality of the crude medicine, as well as to assess the value of the closerelative species of Rehmannia glutinosa for medicine and to breed new varieties with thefingerprint technology.
The contents of the research include method exploring of rehmannia HPLC fingerprints,establishing the HPLC fingerprints for fresh, dry and prepared rehmannia. Quality assessmentof the three types of rehmannia, the difference of fingerprint among them, quality control andnew ways to processing prepared rehmannia are also concerned. Chemical fingerprint,usability and biologic characters of close relative species of Rehmannia glutinosa andbreeding new materials are discussed in the end.
In order to establish and improve HPLC fingerprint method, single-factor analysis wasadopted. Preparation methods of sample solution and HPLC condition for fingerprint werestudied. The methods were gradually set up. After being soaked in methanol, the sample wasabstracted by ultrasonic. The solution was dried. Residues were dissolved with a little lowconcentration methanol solution and the concentrated sample solution resulted. Acetonitrileand phosphoric acid solution was used as mobile phase for linear gradient elution. HPLCfingerprints at205nm for fresh rehmannia and dry rehmannia, fingerprints at205nm ordouble wavelength fingerprints at205nm and284nm for prepared rehmannia were acquired.At the same time, methods for content determination of catalpol,5-hydroxymethyl furfural(5-HMF) and acteoside, the major constituents in criteria, at single or multiple wavelengthswith fingerprint determination were established. HPLC fingerprints of fresh rehmannia canaccurately reflect species characters. Besides species characters, dry remannia fingerprint canalso show information of climate of producing areas, cultivation mode and storage status etc.But prepared rehmannia fingerprints mainly carry processing information. They reflect theprepared degree of processing.
To assess the quality of rehmannia, Well-established HPLC fingerprint technology andsynthesis value of major components in principal component analysis (PCA) were applied toevaluate the quality of dry rehmannia, prepared rehmannia and fresh rehmannia. The resultindicated that the principal components type in PCA of fresh rehmannia and dry rehmanniawas classified on chemical property of chromatographic peaks. While in prepared rehmannia,it was related to the variation characters of peaks in processing. In PCA, dry rehmanniasscored high in synthesis value, i.e. with high quality, were almost from the major producing areas of rehmannia, Henan and Shanxi, or the materials freshly processed. And the synthesisvalue of major components was related to content of catalpol. But it was related to5-HMFand acteoside in prepared rehmannia and fresh rehmannia respectively. And also the methodfor assessing integrated quality of rehmannia was simplified. Only a few of peaks includingprescriptive constituents in criteria were needed to assess integrated quality of rehmannia.
The medical nature is different among fresh rehmannia, dry rehmannia and preparedrehmannia. To unveil the essence of the difference, the HPLC fingerprints of three typerehmannia were compared and variation of fingerprint in processing prepared rehmannia wasstudied. The results showed that fingerprint differences between fresh rehmannia and dryrehmannia were mainly in posterior part. It was the area of phenethanol glycosides. Thecontents of phenethanol glycosides were signaficantly decreased from fresh rehmannia to dryrehmannia. Meanwhile, most other secondary components also declined and a few ofderivative peaks appeared and increased. Fingerprint differences between dry rehmannia andprepared rehmannia were almost in forepart. During processing prepared rehmannia, catalpoland some other iridoid glycosides declined or disappeared, while the derivatives such as5-HMF increased. These changes might be one of the reasons for that the medical natureshifted from cold of fresh rehmannia to warm of prepared rehmannia.
In order to make the best use of germplasm resource of genus Rehmannia, chemicalfingerprint characters of a part of close relatives of R. glutinosa in the genus, exceptR．chingii, was studied with fresh rehmannia fingerprint technology, as well as pollinationcharacter of species and cross compatibility between them with common cross method. Theresult indicated that it was difficult to determine the relationship in the genus by chemicalfingerprint for their intimate relations. The content of catalpol in the root of three relatives islower than that in Rehmannia glutinosa. But acteoside in R．piasezkii and R．solanifolia arehigher. Even though, the relatives have little possibility to take the place of R. glutinosa inmedicine. Cross results showed that the species were all entomophilous pollination plants.R．solanifolia was infertility species and its origination and classification position wasdiscussed. Reciprocal crosses between the species showed that the cross between R．henryiand R．piasezkii could give germinative seeds. The other cross pairs gave few germinativeseeds.
In order to make the best use of germplasm resource of R. glutinosa and to breed newmedical or food materials adapting to local climate, breeding was conducted with naturalcross seeds of varieties and special materials.21materials were selected for medical use byfresh rehmannia fingerprint method and on synthesis value of major components in PCA.10materials were chosen for food use on relative content of total iridoid glycoside which wasdetermined by colorimetry with paradime thylaminobenzaldehyde to present color.
引文
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温学森,李先恩,赵华英,霍德兰,杨世林.2005.地黄常见种质的染色体观察.中草药,36(1):124~125
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薛建平,葛德燕,张爱民,柳俊.2003.地黄组织培养研究进展.中国中药杂志,28(12):1114~1117
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