金莲花及其复方的化学表征研究

【作者】 潘艳丽；

【导师】 肖培根； 张贵君；

【作者基本信息】 北京中医药大学 ， 中药学， 2006， 博士

【摘要】 研究金莲花及其复方的药效组分指纹特征及其化学表征,探讨中药材与复方鉴定特征的相关性和质量综合评价新的方法学体系。采用X射线衍射、光谱、色谱以及色谱-质谱联用技术建立金莲花及复方制剂的指纹鉴定特征标准;在药效组分的三个层次上进行指纹特征解析,辅助计算机建立药效组分品质评价数据库。以金莲花药典品种为基本材料,并对商品金莲花进行考察和比较鉴定;分别对金莲花及其复方样品的XRD、IR、HPLC、LC-MS的指纹特征进行测定,并对其化学表征进行解析,分析原料药和复方鉴定特征的相关性、鉴定方法的适用性、品质标准的可信性和科学性。研究结果:1商品金莲花主要为毛茛科植物Trollius chinensis Bunge的干燥花,即药典品种。2XRDF鉴定①内蒙古的野生金莲花样品有5个特有的衍射特征标记峰:8.17/14,7.19/15,5.42/21,3.57/20,2.781/21;黑龙江的野生金莲花样品有7个专属性衍射特征标记峰:10.05/15,5.13/38,4.94/19,3.43/24,3.11/18,2.644/27,2.479/20。而栽培品无d=3.22的标记峰。据此,可鉴定不同产地以及不同生态环境的金莲花。②复方金莲花XRDF特征标记峰有5个（ 11.51/25,9.27/33,6.64/25,5.55/46,5.13/38）,Px=22.73%; P=77.27%。原料配伍组分解析结果:金莲花（Flos Trollii Chinensis）专属性标记峰有3个（9.94/17,8.17/14,5.42/21）,Px =17.65%;在复方中专属性晶体晶面间距变化区域为1.986⁴.25。野菊花（Flos Chrysanthemi Indici）专属性标记峰1个（8.38/35）, Px=3.57%;在复方中专属性晶体晶面间距变化区域为1.971⁴.63;吻合晶面间距d=2.907A。金银花（FlosLoniceraJaponica ）专属性标记峰2个（4.95/15,4.50/16）, Px=9.09%;在复方中专属性晶体晶面间距变化区域为2.055 4.95;吻合晶面间距d₁=3.93A,d₂=3.05A。黄芩苷专属性标记峰9个（10.34/62,8.59/23,6.49/10,6.05/34,5.23/44,3.86/28, 2.713/20,1.881/10,1.752/10）, Px =18.37%;在复方中专属性晶体晶面间距变化区域为2.016⁴.44;吻合晶面间距d=2.910A。3FT-IR鉴定①金莲花红外光谱可以鉴别不同产地金莲花。内蒙古产金莲花具有1419cm^-1与760cm^-1吸收峰,黑龙江省产金莲花无760cm^-1峰,而河北省产金莲花则无1419cm^-1峰与760cm^-1吸收峰。二阶导数光谱可以鉴别金莲花的贮藏期,1737cm^-1峰的强度随着贮藏期的增加而增加;同时在1500¹ 480cm^-1波数区间,贮藏期4年以下的金莲花体现1499cm^-1与1485cm^-1特征峰,但1485cm^-1的峰强度随着时间的增长而呈减弱趋势,所以贮藏期4年以上的金莲花仅表现1499cm^-1峰。②复方金莲花有15个红外特征指纹峰(νcm^-1),即3735,2931,1664,1614,1450,1408,1 360,1291,1191,1067,915,768,687,597,417;二阶导数光谱可以鉴别复方金莲花与其原料药,复方二阶导数光谱的表征峰是1416cm^-1。通过红外光谱可以鉴别复方的原料药与提取物。原料配伍组分解析结果(νcm^-1):金莲花标识特征峰为2926,1609;野菊花标识特征峰为1660,1609;金银花标识特征峰为767。药效组分表征峰(νcm^-1):金莲花提取物为1614,1360,1291,1191,687,597;野菊花提取物为915,768,417。4HPLC鉴定①金莲花HPLC指纹图谱有26个共有峰（n=20）,确定了指纹谱中可标识的3个化学成分组分,即2′′-O-β-L-galactopyranosylorientin,orientin和vitexin。大鼠灌胃给药,可检出6个组分,峰保留时间均集中于30min前。不同产地金莲花HPLC指纹谱无显著性差异（RSD<1%）。②复方金莲花HPLC指纹图谱有12个共有峰（占总峰面积大于99%）,2个非共有峰（占总峰面积小于1%）,相似度（时间宽度0.25min,n=10）为0.996～1.000。原料药配伍组分解析:金莲花与复方对应的药效组分保留时间在15～38min和50～55min区段,共有8个色谱峰。野菊花与复方对应的药效组分保留时间在5～10min和25～40min两个区段,共有7个色谱峰。金银花与复方对应的药效组分保留时间在5～10min和23～38min两个区段,共有9个相对应色谱峰。化合物类别组分解析:黄酮类化合物保留时间位于15～50min。酚酸类化合物保留时间位于5～15min。化学单体解析:金莲花与复方金莲花对应的药效组分3个,2′′-O-β-L-galactopyranosylorientin（16.333min）、orientin（17.746min）和t_R=52.18min,其中2′′-O-β-L-galactopyranosylorientin和t_R=52.18min峰为金莲花药效组分特征峰,其峰面积比例为1:3。野菊花与复方对应的药效组分2个,cholorogenicacid（7.901min）,caffeicacid（10.384min）。金银花与复方对应的药效组分2个,cholorogenicacid（7.901min）,caffeicacid（10.384min）。baicalin（42.211min）。5 LC-MS鉴定通过质谱数据进行化学单体解析:①确认金莲花中的3个成分:2′′-O-β-L-galactopyranosylorientin t_R=11.927min,[M-H]^- 609;orientin t_R=13.949min,[M-H]^-447;vitexint_R=16.589min,[M-H] -431。对金莲花中所含的化学组分质量推测8个色谱峰的归属: Orientin-2′′-O-β-D-pyranxyloside t_R=12.935min , [M-H] - 579 ;2′′-O-β-L-galactopyranosyl vitexin t_R=13.791min , [M-H] - 593 ;2′′-O-（3′′′,4′′′dimethoxylbenzoyl） vitexin t_R=14.723min , [M-H] - 595 ; isoswertisint_R=26.382min,[M-H]^- 447;2′′-O-（2′′′methylbutyryl）orientint_R=30.729min,[M-H]^- 531;quercetin t_R=34.332min,[M-H]^- 301;2′′-O-（2′′′methylbutyryl）vitexin t_R=35.315min,[M-H] -515;2′′-O-（2′′′methylbutyryl）isoswertisin t_R=43.583min,[M-H] -529。②确认野菊花中的5个成分: acacetin t_R=57.54min,[M-H]^- 283;lianarin t_R=31.50min,[M-H]^- 591;luteolin t_R=33.21min,[M-H] -285;caffeicacidt_R=10.16min,[M-H]^- 179;cholorogenicacid t_R=7.41min,[M-H]^- 393。通过对野菊花中所含的化学组分质量分析色谱峰的归属推测3个成分:isoquercitrint_R=18.59min,[M-H]^- 463; cosmosiint_R=22.67min,[M-H]^-431;luteolin-7-O-glu t_R=18.16,[M-H] -⁴47。③确认金银花中的4个成分:hyperint_R=17.98min,[M-H]^- 463;luteolint_R=33.08min,[M-H]^- 285; caffeicacidt_R=9.95min,[M-H]^- 179;cholorogenic acid t_R=7.39min,[M-H]^- 393。通过对金银花中所含的化学组分质量分析色谱峰的归属推测1个成分:luteolin-7-O-glu t_R=18.08min,[M-H] -447。④确认复方中8个成分:cholorogenic acid t_R=7.47min,cafeeic acid t_R=10.027min,2′′-O-β-L-galactopyranosyl orientin t_R=12.38min , orientin t_R=13.763min , vitexint_R=16.431min,hyperin t_R=18.991min,baicalin t_R=28.83min,acacetin（t_R=57.77min）。并指认了3个色谱峰的归属。金莲花及其复方的指纹谱均能表述其相应的专属性,其中XRD指纹谱可用于成药及其原料配伍组分鉴定,并可用于不同产地和不同生态环境的同品种金莲花鉴别;能从原料药配伍组分上对复方进行品质评价。IR指纹法可用于同品种不同产地金莲花的鉴定;二阶导数红外光谱法可用于不同贮藏期的金莲花及其复方的品质鉴定;即IR可从原料药配伍组分及化学单体组分二个层面进行复方品质评价。XRD与IR可以在没有对照品的情况下,借助金莲花及复方金莲花的整体组分表征进行品质评价。同时通过对复方的表征分析,可以鉴定复方的组方中药。HPLC指纹谱可用于评价金莲花及其复方的品质,并可对主要药效组分的变化规律进行跟踪,建立依据药效组分为主要指标的质量标准;HPLC从原料药配伍组分、化合物类别组分及化学单体组分三个层面进行复方品质评价,评价方式最为全面。LC—MS质谱联用可以将液相色谱指纹峰与质谱数据相关联,对其化学表征给予更多的解析,为金莲花及其复方品质评价提供更多的信息;LC-MS从化学单体组分对复方进行品质评价。该论文创新点在于:首次建立了金莲花及其复方药效组分品质评价的4种指纹鉴定特征。提出了中药化学药效组分特征的三个层次,即原料药配伍组分、化合物类别组分和化学单体组分;并建立了对药效组分化学表征进行定位的基本方法。揭示了中药品质评价的原料药和复方制剂、质量标准和药效组分的相关性问题,为中药品质的本质特征和建立与药效组分相关的质量标准提供了新的研究方法和思路。在XRD指纹鉴别特征研究方面,创立了中药共有特征专属标记峰和晶面间距的吻合峰值为综合指标的鉴定方法;在IR指纹鉴别特征研究方面,采用一维光谱鉴别不同产地药材和二阶导数光谱鉴定不同贮藏期药材;在HPLC指纹鉴别特征研究方面,从原料药配伍组分、化合物类别组分和化学单体3个层次上对指纹特征解析,提出了以液相色谱监测药效组分的分离及复方工艺的方法和以色谱指纹峰面积的比率进行中药品质评价的方法。获相关发明专利2项（主要发明人之一）,专利已公开。更多还原

【Abstract】 Study the chemical character of the active components alignment on Flos Trollii Chinensis and its recipe discovery the associativity of the crude drug and its recipe and the new methodology on synthetic evaluating quality.Adopt XRD, IR, LC and LC-MS to set up the finger identified standard of Flos Trollii Chinensis and its recipe, analysis the character on three orders of the active components alignment and set up the database on evaluating the active components alignment assisting PC. First, we investigated and identify the kind of the commodity Flos Trollii Chinensis and based on the main species, Trollii Chinensis Bunge, to develop research. Then, we alone use XRD, IR, HPLC and LC-MS to analysis the chemical character on Flos Trollii Chinensis and its recipe. At last, we can analysis the associatibity between the crude drug and the patent, know the applicability of every identified method and the reliability and the science of quality standard.Results: 1 The origin of the commodity Flos Trollii Chinensis is mainly came from Trollius chinensis Bunge that is the rated kind in Pharmacopeia. 2 XRDF indicated that Flos Trollii Chinensis in Neimeng has five special marks that are 8.17/14, 7.19/15, 5.42/21, 3.57/20 and 2.781/21, Flos Trollii Chinensis in Heilongjiang has seven special marks that are 10.05/15, 5.13/38, 4.94/19, 3.43/24, 3.11/18, 2.644/27 and 2.479/20 and the cultivated Flos Trollii Chinensis has not 3.22. So we can identify Flos Trollii Chinensis from the different producing place and the different ecological environment. Recipe Jinlianhua have three five special peaks （11.51/25,9.27/33,6.64/25,5.55/46, 5.13/38） that have not expressed in medicinal materials, so Px is 22.73 %, P is 77.27 %（ this is the total rate of the recipe and the medicinal materials） by calculating. The special XRDF mark peaks of medicinal materials, Flos Trollii Chinensis has three special peaks （9.94/17, 8.17/14,5.42/21）, and Px is 17.65 %. And its special interplanar spacing in the prescription is from 1.986 to 4.25. Flos Chrysanthemi Indici has one special peaks（8.38/35）,and Px is 3.57 %. Its special interplanar spacing in patent is from 1.971 to 4.63, and the coincided interplanar spacing is 2.907 . Flos Lonicera Japonicae has two special peaks （4.95/15,4.50/16）, Px is 9.09 %. And its special interplanar spacing in patent is from 2.055 to 4.95, the first coincided interplanar spacing is 3.93A and the second is 3.05A。The special XRDF mark peaks of Baicalin have nine peaks（10.34/62,8.59/23,6.49/10,6.05/34,5.23/44,3.86/28, 2.713/20, 1.881/10,1.752/10）, and Px is 18.37 %. Its special interplanar spacing is from 2.016 to 4.44, and the coincided interplanar spacing is 2.016 .3 FT-IR indicated that it can identify Flos Trollii Chinensis of the different producing place and the second derivate spectrum can identify the different storage time.Recipe Jinlianhua have fifteen IR peaks and can be identified by the second derivate spectrum with 1 416 cm^-1.4 HPLC fingerprint indicated that Flos Trollii Chinensis fingerprint owns 26 peaks （n=20） and assure 3 peaks by comparing the reference compounds. After oral the aqua extract of Flos Trollii Chinensis by rats, we can detect 6 compounds. There is no significant difference between the different places of production（RSD<1%）. By the prolonged storage time, the contents of 2′′-O-β-L-galactopyranosyl orientin in Flos Trollii Chinensis present the depress trend and the contents of orientin present the heighten trend, but the together contents have no significant difference.The HPLC fingerprint of Recipe Jinlianhua owns twelve peaks by both and has two peaks only bodied in some samples. The former is more than 99% in the total area and the latter is less than 1%. The similarity scale is from 0.996 to 1.000.By analyzing the composition of the prescription, we can get the below conclusions. The character of the active constituents that the corresponding constituents of Flos Trollii Chinensis lies the retention time between 15min and 30min, which have seven peaks and mainly are flavanoid. The character of the active constituents that the corresponding constituents of Flos Chrysanthemi Indici lies the retention time between 5min and 10min and between 25min and 40min, which have seven peaks and mainly are phenolic acid and flavanoid. The character of the active constituents that the corresponding constituents of Flos Lonicerae Japonica lies the retention time between 5min and 10min and between 25min and 40min, which have nine peaks.Analyzing the classification of the compounds the flavanoid lies the retention time from 15min to 50min and the phenolic acid from 5min to 15min. Through analyzing the compound, we find that 2′′-O-β-L-galactopyranosyl orientin （t_R=16.333min）, orientin（t_R=17.746min） and another peak（t_R=52.18min） came from Flos Trollii Chinensis. The peak area ratio of 2′′-O-β-L-galactopyranosyl orientin and the peak（t_R=52.18min） is 1/3 and they are the special active peaks from Flos Trollii Chinensis in the recipe. Chlorogenic acid that its retention time is 7.901min and caffeic acid （t_R=10.384min） came from Flos Chrysanthemi Indici and Flos Lonicera Japonica. The retention time of Baicalin is 42.211 min.5 LC-MS fingerprint indicated that 2′′-O-β-L-galactopyranosyl orientin （t_R=11.927min, M+ is 609）, orientin（t_R=13.949min,[M-H]^- is 447） and vitexin （t_R=16.589min, [M-H]^- is 431） are came from Flos Trollii Chinensis. And we inferred eight peaks by analyzing the mass data that they are Orientin-2′′-O-β-D-pyranxyloside （t_R=12.935min, [M-H]^- is 579）, 2′′-O-β-L-galactopyranosyl vitexin （t_R=13.791min, [M-H]^- is 593）, 2′′-O-（3′′′,4′′′dimethoxylbenzoyl） vitexin （t_R=14.723min, [M-H]^- is 595）, isoswertisin （t_R=26.382min, [M-H]^- is 447）, 2′′-O-（2′′′methylbutyryl）orientin （t_R=30.729min, [M-H]^- is 531）, quercetin （t_R=34.332min, [M-H]^- is 301）, 2′′-O-（2′′′methylbutyryl） -vitexin （t_R=35.315min, [M-H]^- is 515） and 2′′-O-（2′′′methylbutyryl） isoswertisin （t_R=43.583min, [M-H]^- is 529）。We assured five compounds in LC-MS of Flos Chrysanthemi Indici that are acacetin（t_R=57.54min, [M-H]^- is 283）, lianarin（t_R=31.50min, [M-H]^- is 591）, luteolin （t_R=33.21min, [M-H]- is 285）, caffeic acid（t_R=10.16 min, [M-H]^- is 179） and cholorogenic acid（t_R=7.41min, [M-H]^- is 393） and inferred three compounds by MS data that are isoquercitrin （t_R=18.59min, [M-H]^- is 463）, cosmosiin （t_R=22.67min, [M-H]^- is 431） and luteolin-7-O-glu（t_R=18.16, [M-H]^- is 447）. By analyzing the mass data, we assured five compounds of Flos Lonicera Japonica that are hyperin（t_R=17.98min, [M-H]^- is 463）, luteolin（t_R=33.08min, [M-H]^- is 285）, caffeic acid（t_R= 9.95min, [M-H]^- is 179） and cholorogenic acid （t_R=7.39min, [M-H]- is 393） and inferred luteolin-7-O-glu （t_R=18.08min, [M-H]- is 447）.Last we assured eight compounds that are cholorogenic acid（t_R=7.47min）, cafeeic acid（t_R=10.027min）, 2′′-O-β-L-galactopyranosylorientin（t_R=12.38min）, orientin （t_R=13.763min）, vitexin（t_R=16.431min）, hyperin（t_R=18.991mi）, baicalin（t_R=28.83min）, acacetin（t_R=57.77min）.The fingerprint of Flos Trollii Chinensis and its recipe can present the corresponding specificity. XRDF can identify the recipe and its composition that is crude drug, and can be used to identify the different producing place and the different ecological environment of the same species. IR fingerprint can identify Flos Trollii Chinensis came from the different producing place and the second derivate IR spectrum can be used to identify the storage time of Flos Trollii Chinensis and the quality of Jinlianhua Recipe. XRD and IR can evaluate the quality of Flos Trollii Chinensis and Jinlianhua Recipe by analyzing the whole compositions under that we have not full standards. HPLC fingerprint can evaluate the quality of Flos Trollii Chinensis and its recipe, trail the change rule of the active components alignment and can set the quality specification based on the active components alignment. LC-MS fingerprint can connect LC with MS and obtain more chemical character for evaluating the quality on Flos Trollii Chinensis and its recipe.On evaluating the quality, XRD can evaluate in the composition from the crude drug, IR in the crude drug and the compound, HPLC in the crude drug, the classification of chemical compounds and the compound and LC-MS in the compound.The new ideas of the paper as follows:1 We first set up four kinds of fingerprint character to evaluating quality of Flos Trollii Chinensis and its recipe.2 Propose the three orders of the active constituents on Chinese materia medica Chemistry what are the crude drug, the kinds of chemical compounds and the compound and set up the positioning mode of the active components. 3 Propose the associativity of the crude drug and the recipe, the quality criteria and the active constituents that used in evaluating the quality of Chinese materia medica.4 In the studying the character of XRDF identification, we raise that establishing the quality synthetic standard is base of the owned by both special marks in the medicinal materials of the patent and the coincidence peak data. By studying the IR fingerprint, we realize that identify the different producing place by one dimension IR and the different storage time by the second derivative spectrum. On HPLC fingerprint, we proposed that can analysis the chemical character in the crude drug, the kinds of chemical compounds and the compound. We raise that can monitor the separation of the active constituents and the technology of Jinlianhua Recipe and evaluate the quality by calculating the peak area. 5 Obtain 2 invented patents on Jinlianhua Recipe（one of the main inventors） and the patent is open.更多还原