氢核磁共振法测定中药茜草中大叶茜草素和羟基茜草素的含量
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摘要
综合运用一维和二维核磁共振波谱技术,对茜草提取物中大叶茜草素、羟基茜草素的信号进行指认归属,并建立氢核磁共振法同时测定茜草中大叶茜草素、羟基茜草素含量的方法。在综合考虑茜草提取物的信号分离度及溶解性的基础上,最终选定氘代二甲基亚砜为测试溶剂,以大叶茜草素的烯烃信号(δ_H5.77,H-2')和羟基茜草素的H-3信号(δ_H6.67)为定量峰,以顺丁烯二酸为定量内标。该方法具有较好的稳定性、重复性、精密度和准确性,且测定结果与HPLC-UV方法所得数据基本一致。所建立的氢核磁定量方法操作简单,测试时间短,且定量过程中不需要对照品,可用于中药茜草的质量控制及评价。
The1 H chemical-shift values of mollugin and purpurin in extract from Rubiae Radix et Rhizoma were assigned on the basis of 1D and 2D NMR,and a specific,simple,sensitive1 H NMR method has been developed and validated for the simultaneous quantitative determination of mollugin in extract from Rubiae Radix et Rhizoma When the solvent effects on the resolution of target signals and the solubility of the crude extracts were taken into account,dimethyl sulfoxide-d6 was selected as an optimal1 H NMR solvent. Quantitative determination was carried out by using the signal of the olefinic proton of mollugin( δH5.77,H-2') and H-3 of purpurin( δH6. 67),which were not interfere with other signals. Maleic acid was selected as internal standard because of its stable physical and chemical properties and having simple resonance signal.The improved method possessed good stability,repeatability,precision and accuracy for the quantification of mollugin and purpurin and the determination results agreed with the data obtained by HPLC-UV method. Due to its simple operation,short test time and without any reference compound,the proposed quantitative1 H NMR method can alternatively be used for the quality control of Rubiae Radix et Rhizoma.
The1 H chemical-shift values of mollugin and purpurin in extract from Rubiae Radix et Rhizoma were assigned on the basis of 1D and 2D NMR,and a specific,simple,sensitive1 H NMR method has been developed and validated for the simultaneous quantitative determination of mollugin in extract from Rubiae Radix et Rhizoma When the solvent effects on the resolution of target signals and the solubility of the crude extracts were taken into account,dimethyl sulfoxide-d6 was selected as an optimal1 H NMR solvent. Quantitative determination was carried out by using the signal of the olefinic proton of mollugin( δH5.77,H-2') and H-3 of purpurin( δH6. 67),which were not interfere with other signals. Maleic acid was selected as internal standard because of its stable physical and chemical properties and having simple resonance signal.The improved method possessed good stability,repeatability,precision and accuracy for the quantification of mollugin and purpurin and the determination results agreed with the data obtained by HPLC-UV method. Due to its simple operation,short test time and without any reference compound,the proposed quantitative1 H NMR method can alternatively be used for the quality control of Rubiae Radix et Rhizoma.
引文
[1]国家药典委员会.中华人民共和国药典2015年版一部[M].北京:中国医药科技出版社,2015:234.
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[6]陈星,王侃,单鸣秋,等.UPLC测定茜草炭中4种醌类成分的含量[J].中国中药杂志,2012,37(19):2922-2925.
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[11]WANG Z W,WANG J S,YANG M H,et al.Developmental changes in the composition of five anthraquinones from Rheum palmatum as quantified by1H-NMR[J].Phytochem Anal,2013,24(4):329-335.
[12]HOLZGRABE U.Quantitative NMR spectroscopy in pharmaceutical applications[J].Prog Nucl Magn Reson Spectrosc,2010,57(2):229-240.
[13]NAPOLITANO J G,GODECKE T,RODRIGUEZ-BRASCO M F,et al.The tandem of full spin analysis and q HNMR for the quality control of botanicals exemplified with Ginkgo biloba[J].J Nat Prod,2012,75(2):238-248.
[14]PAULI G F,GODECKE T,JAKI B U,et al.Quantitative1H NMR:Development and potential of an analytical method:An update[J].J Nat Prod,2012,75(4):834-851.
[15]BURTON I W,QUILLIAM M A,WALTER J A.Quantitative1H NMR with external standards:Use in preparation of calibration solutions for algal toxins and other natural products[J].Anal Chem,2005,77(10):3123-3131.
[2]ITOKAWA H,MIHARA K,TAKEYA K.Studies on a novel anthraquinone and its glycosides isolated from Rubiae Radix et Rhizoma and R.akane[J].Chem Pharm Bull,1983,31(7):2353-2358.
[3]ITOKAWA H,QIAO Y F,TAKEYA K,et al.New triterpenoids from Rubiae Radix et Rhizoma var.pratensis(Rubiaceae)[J].Chem Pharm Bull,1989,37(6):1670-1672.
[4]CHUNG M I,JOU S J,CHENG T H,et al.Antiplatelet constituents of formosan Rubia akane[J].J Nat Prod,1994,57(2):313-316.
[5]JEONG G S,LEE D S,KIM D C,et al.Neuroprotective and anti-inflammatory effects of mollugin via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells[J].Eur J Pharmacol,2011,654(3):226-234.
[6]陈星,王侃,单鸣秋,等.UPLC测定茜草炭中4种醌类成分的含量[J].中国中药杂志,2012,37(19):2922-2925.
[7]TANAKA R,NITTA A,NAGATSU A.Application of a quantitative1H-NMR method for the determination of amygdalin in Persicae semen,Armeniacae semen,and Mumefructus[J].J Nat Med,2014,68(1):225-230.
[8]HOHMANN M,FELBINGER C,CHRISTOPH N,et al.Quantification of taurine in energy drinks using1H NMR[J].J Pharm Biomed Anal,2014,93:156-160.
[9]PIERI V,BELANCIC A,MORALES S,et al.Identification and quantification of major steviol glycosides in Stevia rebaudiana purified extracts by1H NMR spectroscopy[J].J Agric Food Chem,2011,59(9):4378-4384.
[10]LI C Y,XU H X,HAN Q B,et al.Quality assessment of Radix Codonopsis by quantitative nuclear magnetic resonance[J].Journal of Chromatography A,2009,1216(11):2124-2129.
[11]WANG Z W,WANG J S,YANG M H,et al.Developmental changes in the composition of five anthraquinones from Rheum palmatum as quantified by1H-NMR[J].Phytochem Anal,2013,24(4):329-335.
[12]HOLZGRABE U.Quantitative NMR spectroscopy in pharmaceutical applications[J].Prog Nucl Magn Reson Spectrosc,2010,57(2):229-240.
[13]NAPOLITANO J G,GODECKE T,RODRIGUEZ-BRASCO M F,et al.The tandem of full spin analysis and q HNMR for the quality control of botanicals exemplified with Ginkgo biloba[J].J Nat Prod,2012,75(2):238-248.
[14]PAULI G F,GODECKE T,JAKI B U,et al.Quantitative1H NMR:Development and potential of an analytical method:An update[J].J Nat Prod,2012,75(4):834-851.
[15]BURTON I W,QUILLIAM M A,WALTER J A.Quantitative1H NMR with external standards:Use in preparation of calibration solutions for algal toxins and other natural products[J].Anal Chem,2005,77(10):3123-3131.
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