木兰花碱的荧光性质及其在中药分析中的应用研究
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摘要
研究了木兰花碱(Magnoflorine,MAG)的荧光光谱和吸收光谱特征,考察了pH值等环境因素对荧光和吸光的影响,探讨了光谱性质与分子结构的关系。木兰花碱水溶液的三维荧光图谱中呈现3个荧光峰,激发波长λ_(ex)为230、275和315 nm,发射波长λ_(em)均为420 nm。随溶液pH值升高,激发光谱中的荧光峰红移并出现等荧光点,吸收光谱中的吸收峰红移并形成等色点,表明木兰花碱分子中的1个羟基发生了质子电离,用pH值-吸光法测得电离常数pK_a=4.77。以L-色氨酸为参比,测得木兰花碱水溶液的荧光量子产率Y=0.19。青风藤等多种中药材的三维荧光图谱中呈现木兰花碱的特征荧光峰,λ_(ex)/λ_(em)=315 nm/420 nm处的荧光峰不受共存组分的影响,且荧光强度稳定,据此建立了中药青风藤中木兰花碱的荧光分析新方法。在0.04～1.25μg/mL范围内,体系荧光强度I_F与木兰花碱浓度c呈线性关系,回归方程为:I_F=6146.8c+24.4(R=0.999,n=11),检出限0.52 ng/mL。采用本方法测得青风藤对照药材中MAG的含量为0.63%,加标回收率为101.2%～102.7%。LC-MS/MS法测定结果为0.61%,与荧光法基本一致。本方法简便快速,结果可靠,有良好的实际应用价值。
Fluorescence and absorption spectra of magnoflorine(MAG) were studied. The effects of environmental factors such as pH on fluorescence and absorbance were investigated, and the relationship between spectral properties and molecular structure was revealed. Three fluorescent peaks appeared in the three-dimensional(3 D) fluorescence spectra of the aqueous solution of MAG, with excitation wavelengths(λ_(ex)) of 230, 275 and 315 nm, and the same emission wavelength(λ_(em)) of 420 nm. As the pH of the solution increased, the fluorescence peak in the excitation spectrum red-shifted and an equal fluorescence point appeared, and the absorption peak in the absorption spectrum also red-shifted and an isosbestic point formed, indicating that proton ionization occurred in one hydroxyl group in the MAG molecule. The proton ionization constant(pK_a=4.77) of MAG was determined by a pH-absorption method. Using L-tryptophan as a reference, the fluorescence quantum yield of the aqueous solution of MAG was determined to be Y=0.19. The characteristic fluorescence peaks of MAG appeared in the 3 D fluorescence spectra of various Chinese herbal medicines. Using methanol-water mixed solvent containing 60% methanol, the extract of Sinomenium acutum(Qingfengteng) was prepared and diluted with water, then 3 D fluorescence spectra were obtained. It was observed that the fluorescence peak at λ_(ex)/λ_(em)=315 nm/420 nm in the spectrum of the Qingfengteng neutral aqueous solution was not affected by the coexisting components. Based on this, a method for the determination of MAG in Qingfengteng was established. In the range of 0.04-1.25 μg/mL, the regression equation for fluorescence intensity(I_F) and MAG concentration(c) was I_F=6146.8c + 24.4,(R=0.999, n=11), with a detection limit of 0.52 ng/mL. The content of MAG in the Qingfengteng reference material was determined to be 0.63% by the method, and the recoveries were 101.2%-102.7%. The content of MAG(0.61%) in the same sample was also determined by LC-MS/MS method, which was consistent with the fluorescence method.
Fluorescence and absorption spectra of magnoflorine(MAG) were studied. The effects of environmental factors such as pH on fluorescence and absorbance were investigated, and the relationship between spectral properties and molecular structure was revealed. Three fluorescent peaks appeared in the three-dimensional(3 D) fluorescence spectra of the aqueous solution of MAG, with excitation wavelengths(λ_(ex)) of 230, 275 and 315 nm, and the same emission wavelength(λ_(em)) of 420 nm. As the pH of the solution increased, the fluorescence peak in the excitation spectrum red-shifted and an equal fluorescence point appeared, and the absorption peak in the absorption spectrum also red-shifted and an isosbestic point formed, indicating that proton ionization occurred in one hydroxyl group in the MAG molecule. The proton ionization constant(pK_a=4.77) of MAG was determined by a pH-absorption method. Using L-tryptophan as a reference, the fluorescence quantum yield of the aqueous solution of MAG was determined to be Y=0.19. The characteristic fluorescence peaks of MAG appeared in the 3 D fluorescence spectra of various Chinese herbal medicines. Using methanol-water mixed solvent containing 60% methanol, the extract of Sinomenium acutum(Qingfengteng) was prepared and diluted with water, then 3 D fluorescence spectra were obtained. It was observed that the fluorescence peak at λ_(ex)/λ_(em)=315 nm/420 nm in the spectrum of the Qingfengteng neutral aqueous solution was not affected by the coexisting components. Based on this, a method for the determination of MAG in Qingfengteng was established. In the range of 0.04-1.25 μg/mL, the regression equation for fluorescence intensity(I_F) and MAG concentration(c) was I_F=6146.8c + 24.4,(R=0.999, n=11), with a detection limit of 0.52 ng/mL. The content of MAG in the Qingfengteng reference material was determined to be 0.63% by the method, and the recoveries were 101.2%-102.7%. The content of MAG(0.61%) in the same sample was also determined by LC-MS/MS method, which was consistent with the fluorescence method.
引文
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29 Bao G H,Qin G W,Wang R,Tang X C.J.Nat.Prod.,2005,68(7):1128-1130
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31 Min Y D,Choi S U,Lee K R.Arch.Pharm.Res.,2006,29(8):627-632
2 Sarma D N K,Koul S,Khosa R L.J.Pharm.Sci.Res.,2009,1(1):26-27
3 Li C,Wang M H.Korean J.Plant Resour.,2014,27(3):223-228
4 Morris J S,Facchini P J.J.Biol.Chem.,2016,291(45):23416-23427
5 Hung T M,Lee J P,Min B S,Choi J S,Na M,Zhang X,Ngoc T M,Lee I,Bae K.Biol.Pharm.Bull.,2007,30(6):1157-1160
6 Sakumoto H,Yokota Y.J.Nat.Med.,2015,69(3):441-448
7 Xue B J,Zhao Y Y,Su J,Miao Q,Miao P P,Chen N,Wang Z J,Zhang Y J,Ma S C.Eur.J.Drug Metab.Pharmacokinet.,2017,42(2):281-293
8 Kukula-Koch W,Mroczek T.Anal.Bioanal.Chem.,2015,407(9):2581-2589
9 Doslovkokorus Z R,Ivanovic I D,Simic M R,Vajs V E.J.Serb.Chem.Soc.,2006,71(3):251-255
10 Avula B,Wang Y H,Rumalla C S,Ali Z,Smillie T J,Khan I A.J.Pharm.Biomed.Anal.,2011,56(5):895-903
11 Wang S C,Zhang Z,He L C,Li H.Anal.Lett.,2010,43(9):1534-1542
12 Wang L L,Xu H F,Ye H Z,Yu L S,Lin Z Y,Liu X X,Chen G N.Anal.Methods,2013,5(19):5267-5271
13 Tian X T,Li Z X,Lin Y F,Chen M C,Pan G Y,Huang C G.Anal.Bioanal.Chem.,2014,406(3):841-849
14 SHI Xun-Li,WEI Yong-Ju,ZHANG Ying-Hua,WANG Ya-Min,LIU Cui-Ge.Spectroscopy and Spectral Analysis,2007,27(4):769-772史训立,魏永巨,张英华,王亚敏,刘翠格.光谱学与光谱分析,2007,27(4):769-772
15 Yang F,Liu C G,Wei Y J.Acta Pharm.Sin.B,2012,2(3):294-299
16 LI Wen-Hong,SUN Chong-Mei,WEI Yong-Ju.Acta Pharm.Sin.,2015,50(10):1324-1329李文红,孙冲梅,魏永巨.药学学报,2015,50(10):1324-1329.
17 Wu H L,Nie J F,Yu Y J,Yu R Q.Anal.Chim.Acta,2009,650:131-142
18 Yu Y J,Wu H L,Nie J F,Zhang S R,Li S F,Zhu S H,Yu R Q.Chemometr.Intell.Lab.Sys.,2011,106:93-107
19 Bai X M,Liu T,Liu D L,Wei Y J.Spectrochim.Acta A,2018,191:195-202
20 Liu T,An X N,Liu D L,Wei Y J.Spectrochim.Acta A,2019,208:172-178
21 WEI Yong-Ju,LI Na,QIN Shen-Jun.Spectroscopy and Spectral Analysis,2004,24(6):647-651魏永巨,李娜,秦身钧.光谱学与光谱分析,2004,24(6):647-651
22 XIE Tong,CHENG Xue-Fang.Chinese Journal of Clinical Pharmacology and Therapeutics,2017,22(9):984-991谢彤,程学芳.中国临床药理学与治疗学,2017,22(9):984-991
23 Lakowicz J R.Principles of Fluorescence Spectroscopy.Springer US,2006:8
24 WEI Yong-Ju.3D Fluorescent Fingerprint of Chinese Herbal Medicine.Beijing:Science Press,2012:40,114,131,151,165,295,363魏永巨.中药三维荧光检验法.北京:科学出版社,2012:40,114,131,151,165,295,363
25 Racková L,Májeková M,Kost'álová D,tefek M.Bioorg.Med.Chem.,2004,12(17):4709-4715
26 Rib昣r B,Lazar D,Ga?ic' O,Kanyó I.Acta Crystallogr.C,1992,48(5):945-947
27 Zhao X X,Peng C,Zhang H,Qin L P.Pharm.Biol.,2012,50(8):1053-1061
28 Lv H,Zhao M,Jiang Y,Tu P.J.Chin.Pharm.Sci.,2017,26(6):440-446
29 Bao G H,Qin G W,Wang R,Tang X C.J.Nat.Prod.,2005,68(7):1128-1130
30 Jin H Z,Wang X L,Wang H B,Wang Y B,Lin L P,Ding J,Qin G W.J.Nat.Prod.,2008,71(1):127-129
31 Min Y D,Choi S U,Lee K R.Arch.Pharm.Res.,2006,29(8):627-632