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Puerarin as an antioxidant fluorescence probe
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Chemical Physics Letters 452 (2008) 253–258 www.elsevier.com/locate/cplett
Puerarin as an antioxidant fluorescence probe Yu-Xi Tian a,b, Rui-Min Han b, Peng Wang b, Yi-Shi Wu a, Jian-Ping Zhang b,*, Leif H. Skibsted c,* a
Beijing National Laboratory for Molecular Science (BNLMS), State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China b Department of Chemistry, Renmin University of China, No. 59, ZhongGuanCun Street, Beijing 100872, China c Food Chemistry, Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1058 Frederiksberg C, Denmark Received 18 October 2007; in final form 28 December 2007 Available online 5 January 2008
Abstract Diphenolic isoflavonoid puerarin fluoresces in aqueous solution with maximal intensity at pH 8.5 (Ufl = 0.042, sfl = 1.91 ns). For acidic solutions, weak fluorescence is attributed to fluorescent 7-monophenolate formed via excited-state deprotonation of neutral puerarin. For pH > 8.5, fluorescence decreases monotonically with an unchanged lifetime, suggesting that excited-state acidity of 40 -hydroxyl remains similar to the ground-state one, and that the diphenolate is non-fluorescent. The crucial role of A-ring 7-phenolate for fluorescence of puerarin is substantiated by absence (presence) of fluorescence for the 7-propylpuerarin (40 -propylpuerarin). Puerarin and its derivatives with the unusual properties may be explored to be antioxidant fluorescence probes. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction Epidemiological studies have clearly shown beneficial health effects of antioxidants from fruits and vegetables [1]. The results of human intervention studies, however, are less clear and the supplementation with vitamin antioxidants has shown little if any protective effects on cardiovascular diseases and on cancers [2]. In contrast to supplementation with one or a few antioxidants, a change in diet involves a change in the intake of many bioactive compounds as present in plant food with the perspective of synergistic interaction between the individual antioxidants. Further development in the evaluation methods should accordingly focus on antioxidant interaction [3]. Antioxidants are classified according to their nutritive value (vitamin vs non-vitamin) and their hydrophilic–lipo-
*
Corresponding authors. Fax: +86 10 6251 6444 (J.-P. Zhang). E-mail addresses: [email protected] (J.-P. Zhang), ls@life. ku.dk (L.H. Skibsted).. 0009-2614/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2007.12.072
philic balance (soluble in water vs in lipid), and six possibilities for the binary interactions among the four groups of antioxidants then become possible. Synergistic antioxidation effects were recently demonstrated at a water–lipid interface for two non-vitamin antioxidants, i.e. the watersoluble puerarin and the lipid-soluble b-carotene [4]. Puerarin, a C-glycoside of the isoflavonoid daidzein from the root of Pueraria lobata used in traditional Chinese herbal medicine, has only two phenols (Scheme 1) and shows moderate water solubility. The pH-dependent fluorescence of puerarin and flavonoids has been utilized in the mechanistic studies of antioxidation at water–lipid interface [5,6]. In this work, we have carried out, in combination with theoretical calculations, detailed characterizations of the fluorescence properties of puerarin in aqueous solution at varying pH. The roles of the A-ring 7-hydroxyl in dominating the fluorescence of puerarin, as well as in the excited-state deprotonation of neutral puerarin have been revealed. The fluorescence properties of puerarin and derivatives may facilitate to explore their uses as fluorescent antioxidant probes for the study of antioxidant interaction at water–lipid interfaces.
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Y.-X. Tian et al. / Chemical Physics Letters 452 (2008) 253–258
tion was 0.2 nm. Measurements were done at room temperature.
OH CH2OH
HO O
HO 7
2.4. Quantum chemical calculations
8
O
A
C
R1O 6
2 2'
3
3'
4
5
O
B 6' 5'
4'
OR2
Scheme 1. Molecular structures of puerarin (R1 = R2 = H) and its derivatives (R1 and R2 = H or C3H7).
2. Materials and methods
The molecular geometries for neutral, mono- and dianionic puerarins in the ground state (S0) were performed with the GAUSSIAN 03 package [10] by using the B3LYP density functional (DFT) [11,12] and the 6-31G(d) basis set [13,14]. Geometries in the lowest-lying singlet excited state (S1) were optimized on the basis of the TURBOMOLE program suite [15] with the DFT method, the B3LYP functional and the SV (P) basis, in which the fine quadrature grids 4 was employed [16].
2.1. Sample preparation 3. Results and discussion Puerarin crude product (Huike Plant Exploitation Inc., Shanxi, China) was twice recrystallized in a 1:1 (v/v) binary solvent of acetic acid (>99.5%; dried and refluxed by the use of P2O5 and KMnO4, respectively) and methanol (>99.5%; Beijing Chemical Plant, Beijing, China). 7-Propylpuerarin and 40 -propylpuerarin (Scheme 1) were synthesized as described previously [7]. Aqueous solutions of puerarin, 7-propylpuerarin and 40 -propylpuerarin (each 2 105 M) were prepared using the Britton–Robinson buffer (pH 2.0–13.0) [8], for which water was supplied by a Milli-Q apparatus (Millipore Corp., Billerica, MA). The ionic strength was controlled at 0.10 using NaCl. The pH values were measured with the pH-211 meter (Hanna Instruments Inc., Woonsocket, RI).
3.1. Steady-state absorption and fluorescence spectra The absorption spectra of neutral (pH 2.0), monoanionic (pH 8.5) and dianionic (pH 13.0) puerarins are shown in Fig. 1a, and their fractional distribution against pH calculated from the pKa values are depicted in Fig. 1b. The fluorescence spectra for puerarin at varying pH are shown in
2.2. Steady-state fluorescence The steady-state absorption and fluorescence spectra, respectively, were recorded on a Cary50 absorption spectrometer (Varian, Inc. Palo Alto, CA) and a LS55 luminescence spectrophotometer (Perkin Elmer Inc., Foster, CA). Fluorescence quantum yield was determined by the use of 1,4-bis-(5-phenyl-2-oxazolyl)benzene (POPOP; Exciton Inc., Dayton, OH) as a reference (Ufl = 0.97 in cyclohexane) [9]. Sample solutions were thermostated at 25 °C with a water bath (RTE-110, Neslab Instruments Inc., Newington, NH). 2.3. Fluorescence lifetime The excitation laser pulses (315 nm, 120 fs) for timeresolved fluorescence were supplied by an optical parametric amplifier (OPA-800CF, Spectra Physics), which was pumped by the output from a regenerative amplifier (Spitfire, Spectra Physics, Mountain View, CA). The excitation pulse energy was 100 nJ/pulse. Fluorescence collected with a 90°-geometry was dispersed by a polychromator (250is, Bruker Optics, Billerica, MA), and detected with a streak camera (C5680, Hamamatsu Photonics, Hamamatsu, Japan). The time resolution was 2–100 ps depending on the delay-time-range settings, and the spectral resolu-
Fig. 1. (a) Steady-state absorption spectra of puerarin in aqueous solution at pH 2.0 (neutral form, >99.9%), pH 8.5 (monoanion, 91.0%) and pH 13.0 (dianion, >99.9%), arrows indicate excitation wavelengths. (b) Fractions of neutral, monoainoic and dianionic puerarins in aqueous solutions against pH.
Y.-X. Tian et al. / Chemical Physics Letters 452 (2008) 253–258
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Fig. 2. Fluorescence emission spectra of puerarin (2 105 M) at pH ranges of (a) 2.0–8.5 and (b) 8.5–13.0 in aqueous solution (kex = 315 nm).
Figs. 2a and b, which appear structureless and rather broad (3700 cm1) with a peak wavelength of kmax 463 nm independent of pH. The fluorescence intensity increases with increasing pH (2.0–8.5), and decreases with further increase in pH (8.5–13.0). Note that, at pH 8.5, monoanionic puerarin has its maximal fraction (91.0%) with a fluorescence quantum yield of Ufl = 0.042 ± 0.001. The fractions of neutral and dianionic puerarins at the pH values of 2.0 and 13.0 both are >99.9%. Fig. 3 exhibits the absorption, fluorescence excitation and emission spectra of puerarin in buffer. The emission spectra are similar to each other, and all of them exhibit large Stokes shifts, i.e. 11700 cm1 for the neutral and 7000 cm1 for the anionic puerarins. The emission spectrum at pH 13.0 in Fig. 3c was obtained by subtracting the background emission of buffer from the extremely weak emission of sample solution and, therefore, the shoulder around 400 nm is most likely caused by interference from scattered actinic light and/or emission from an adventitious impurity. In the wavelength range of 300–400 nm, the excitation spectra agree well with the absorption spectra in the cases of neutral (Fig. 3a) and monoanionic (Fig. 3b) puerarins, whereas obvious deviation is seen for dianionic puerarin (Fig. 3c). Interestingly, the excitation spectrum of the dianion agrees well with the absorption band of the monoanion. In Fig. 3a–c, substantial discrepancies between the excitation and the absorption spectra are found for the shorter-wavelength band, which may be ascribed to the deactivation of the higher-lying electronic excited state (e.g., via photo-ionization) before relaxing to the lowestlying fluorescent state. The pH-dependence of fluorescence intensity for puerarin under the excitation wavelengths of 315 nm and 355 nm
Fig. 3. Fluorescence excitation (solid line, kem = 463 nm) and emission (dotted line, kex = 315 nm) spectra of puerarin (2 105 M) in aqueous solution at indicated pH values for (a) neutral, (b) monoanionic and (c) dianionic forms. Absorption spectra (dashed line, cf. Fig. 1a) are shown for comparison. Excitation spectra are normalized with absorption spectra at lower-energy absorption maxima.
are shown in Fig. 4a. Actinic light at 355 nm selectively excited the anionic forms (cf. Fig. 1a), and the acid–base equilibrium constants for the ground-state puerarin, pK a1 7:0 and pK a2 9:6, could be obtained by fitting the data in Fig. 4a to the relation I fl ¼ a=ð1 þ ½Hþ =K a1 þ K a2 =½Hþ Þ;
ð1Þ
where Ifl represents fluorescence intensity, K a1 (7-hydroxyl) and K a2 (40 -hydroxyl) are the acid–base equilibrium constants, [H+] is the proton concentration and a is simply a proportionality constant. The pKa values obtained from the fluorescence data are close to the values of pK a1 ¼ 7:20 and pK a2 ¼ 9:84 previously derived from the pH-dependent absorption spectra [4].
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Scheme 2. Deactivation and ionization processes for neutral and anionic puerarins. For the S0 and the S1 states, dihedral angles (a) are shown behind the notations, pKa values are indicated above or below bidirectional arrows. DH 01 (3500 cm1) and DH 02 (4700 cm1) are standard molar ionization enthalpies in the S0-state calculated from the pKa values. The S1-state of neutral puerarin is shown in dashed line (exact energetic location unknown owing to the unobservable fluorescence emission). Vertical solid arrows indicate the absorptive (upward) and emissive (downward) transitions; dashed arrows represent the nonradiative deactivation processes. Vertical solid arrows indicate the absorptive (upward) and emissive (downward) transitions; dashed arrows represent the nonradiative deactivation processes.
Fig. 4. Normalized fluorescence intensity–pH profiles of (a) puerarin and (b) 40 -propylpuerain in aqueous solutions for excitation at 315 nm (filled circle) or 355 nm (open circle), solid lines were obtained by fitting the 355nm data to Eq. (1) for (a) and to Eq. (3) for (b), respectively. Insets show the difference between fluorescence intensities on 315-nm and 355-nm excitation, and the solid lines are just for guiding the eyes. (c) Experimental data (filled circle) showing the concentration effect on fluorescence intensity, solid and dashed lines are fitting curves according to different models (see text for details).
The inset of Fig. 4a illustrates the difference of normalized fluorescence intensity between the cases of 315nm and 355-nm excitations (pH 2.0–6.0). Here, it is worth noting that the 355-nm excitation did not induce any detectable fluorescence at pH < 4.0 although fluorescent monoanion, if any, would have been selectively excited. Therefore, the weak fluorescence under acidic condition must originate from the S1-state monoanion formed via deprotonation of the S1-state neutral puerarin (Scheme 2), which is further proven by the sub-linear dependence of the fluorescence intensity on the concentration of puerarin at pH 2.0 (Fig. 4c), i.e. the data strictly follow the equation: I fl ¼ a I 0 ð1 10CeL Þ
ð2Þ
where Ifl is the fluorescence intensity, I0 stands for the intensity of actinic light, C for the concentration and e (2400 L mol1 cm1 at 315 nm) for the extinction coefficient of neutral puerarin; L is the length of detected optical path and a is a proportionality constant. On the other hand, the contribution of singlet excitation energy transfer from the neutral puerarin to the monoanion is negligible, which would otherwise lead to the completely different concentration dependence (/C2) as shown by the dashed line in Fig. 4c. It is known that the acidity increases upon excitation for aromatic compounds with hydroxyl substituents, e.g., the pKa of 2-naphthol was found to decrease from 9.5 to 2.8 on going from the S0 to the S1 state [17]. For puerarin at the acidic condition (pH 2.0–6.0), the S1-state pK a1 can be determined by fitting the data in the inset of Fig. 4a using the following relation (parameters defined as in Eq. (1)) provided that an excited-state equilibrium is established, I fl ¼ a K a =ð½Hþ þ K a Þ:
ð3Þ
However, a pK a1 value of 2.0 thus estimated is considered to be unreliable because the equilibrium was indeed not reached: The intrinsic S1-state lifetime of neutral puerarin was calculated to be 39 ns from its absorption spectrum; on the other hand, although direct fluorescence emission from neutral puerarin at pH 2.0 was experimentally unobservable, an upper limit of its Ufl can be set as 0.001 on the basis of the Ufl of monoanion (maximum 0.042 at pH 8.5). Therefore, the S1-state lifetime of neutral puerarin could be <40 ps, which is too short to allow an excited-state ionization equilibrium.
Y.-X. Tian et al. / Chemical Physics Letters 452 (2008) 253–258
In Fig. 4a under 315 nm excitation, the intensity–pH profile in the range of pH > 6.0, in contrast to the cases of pH < 4.0, can be fully accounted for by the curve calculated using the S0-state pKa values. In addition, the absorption maximum of puerarin diphenolate in Fig. 1a is slightly blue-shifted (350 cm1) with respect to the monophenolate, indicating that pK a2 of puerarin can be similar to its pK a2 (9.8, Fig. 4a, Scheme 2). Fig. 4b displays the intensity–pH dependence of 40 -propylpuerarin under photo-excitation at 315 nm and 355 nm. Although 7-propylpuerarin shows no fluorescence at any pH in aqueous solution, 40 -propylpuerarin shows increasing fluorescence intensity for pH up to 8.5 with an intensity–pH profile similar to that of puerarin; the fluorescence quantum yield of 40 -propylpuerarin at pH 8.5 was determined to be Ufl = 0.043 ± 0.001. A pKa value of 6.9 was determined by using Eq. (3), which is close to pKa = 7.23 previously derived from the absorption data [4]. The marked difference in fluorescence properties between the two derivatives substantiates the role of the A-ring 7-phenolate in dominating the fluorescence properties of puerarins. 3.2. Fluorescence lifetime The fluorescence decay kinetics of puerarin at different pH are shown in Fig. 5. At pH 8.5 and 13.0 for mono-
257
aninoic and dianionic puerarins, respectively, the kinetics follow a single exponential decay with the lifetimes of 1.91 ± 0.01 ns and 1.94 ± 0.02 ns which are comparable with that of some simple phenols [18]. The similarity in lifetime strongly suggests that monoanionic puerarin is the fluorescent species at either pH. On the other hand, the decay of fluorescence at pH 2.0 for neutral puerarin is complicated and cannot be adequately described with a single exponential function; this aspect needs to be further investigated. 3.3. Ground- and excited-state geometries The molecular geometry optimization for neutral, monoanionic and dianionic puerarins in their S0 and the S1 states yielded the dihedral angles (a) between the planes of AC and the B rings as indicated in Scheme 2. It is seen that a angles of the S0-state molecules are similar (35–38°). With respect to the S0-state geometries, the S1-state monoanion becomes considerably less twisted (a = 14°), whereas changes in the S1-state geometries of neutral and dianionic puerarins are not significant. The smaller twist of the S1-state monoanion may explain its substantially larger Ufl in contrast to neutral and dianionic puerarins. The 7-phenolate, present in the S0 state or formed via deprotonation of the S1-state neutral puerarin, is mandatory for establishing an extended conjugate system conducive to fluorescence emission. In contrast, the 40 -phenolate, present in puerarin or in 7-propylpuerarin at higher pH, reduces the extension of conjugation and thereby the probability of emission. 4. Conclusion The present work demonstrates the unusual fluorescence properties of puerarin owing to the marked difference in the sensitivity of acid strength to optical excitation between the A-ring and B-ring phenols, as well as the critical roles of A-ring 7-phenolate in dominating the molecular and electronic structures. Since the B-ring phenol is located in the more hydrophobic part of the molecule, and is less acidic but more reducing than the A-ring phenol, puerarin at water–lipid interface may serve as an antioxidant fluorescent probe, as well as a radical shuttle in the presence of water-soluble and lipid-soluble antioxidants. Acknowledgements
Fig. 5. Fluorescence decay kinetics (open circle) of puerarin at pH values of (a) 2.0, (b) 8.5 and (c) 13.0 in aqueous solutions. Solid lines are fitting curves based on (a) bi-exponential or (b, c) single exponential model functions, decay time constants (s) are indicated.
This work has been supported by grants from Natural Science Foundation of China (#20673144, #20433010, #20703067) and Ministry of Science and Technology of China (#2006BAI08B04-06). Support from LMC, Centre for Advanced Food Studies to the Food Chemistry group at University of Copenhagen is acknowledged. We are grateful for the stimulating dis-
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cussion with Dr. G.-J. Zhao at Dalian Institute of Chemical Physics. References [1] B.N. Ames, L.S. Gold, W.C. Willett, Proc. Natl. Acad. Sci. 92 (1995) 5258. [2] D.P. Vivekananthan, M.S. Penn, S.K. Sapp, A. Hsu, E.J. Topol, Lancet 361 (2003) 2017. [3] E.M. Becker, L.R. Nissen, L.H. Skibsted, Eur. Food Res. Technol. 219 (2004) 561. [4] R.M. Han, Y.X. Tian, E.M. Becker, M.L. Andersen, J.P. Zhang, L.H. Skibsted, J. Agric. Food Chem. 55 (2007) 2384. [5] A.P. Nifli et al., J. Agric. Food Chem. 55 (2007) 2873. [6] J. Xi, R. Guo, J. Pharm. Biomed. Anal. 43 (2007) 111. [7] R.M. Han, Y.X. Tian, P. Wang, J.F. Xiang, X.C. Ai, J.P. Zhang, Chem. J. Chinese Univ. 9 (2006) 1716.
[8] H.A. McKenzie, R.M.C. Dawson, in: R.M.C. Dawson, D.C. Elliot, W.H. Elliot, K.M. Jones (Eds.), Data for Biological Research, second edn., Clarendon Press, Oxford, 1974, p. 475. [9] N. Boens et al., Anal. Chem. 79 (2007) 2137. [10] M.J. Frisch et al., GAUSSIAN 03W, Revision B.01, Gaussian Inc., Pittsburgh, PA, 2003. [11] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785. [12] A.D. Becke, J. Chem. Phys. 98 (1993) 5648. [13] A.D. McLean, G.S. Chandler, J. Chem. Phys. 72 (1980) 5639. [14] R. Krishnan, J.S. Binkley, R. Seeger, J.A. Pople, J. Chem. Phys. 72 (1980) 650. [15] Dr. Ahlrichs et al., Turbomole 5.71, The Quantum Chemistry Group, University of Karlsruhe, Germany, 2005. [16] G.-J. Zhao, K.-L. Han, J. Phys. Chem. A 111 (2007) 2469. [17] N. Agmon, J. Phys. Chem. A 109 (2005) 13. [18] O. Brede, S. Naumov, R. Hermann, Chem. Phys. Lett. 355 (2002) 1.
Chemical Physics Letters 452 (2008) 253–258 www.elsevier.com/locate/cplett
Puerarin as an antioxidant fluorescence probe Yu-Xi Tian a,b, Rui-Min Han b, Peng Wang b, Yi-Shi Wu a, Jian-Ping Zhang b,*, Leif H. Skibsted c,* a
Beijing National Laboratory for Molecular Science (BNLMS), State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China b Department of Chemistry, Renmin University of China, No. 59, ZhongGuanCun Street, Beijing 100872, China c Food Chemistry, Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1058 Frederiksberg C, Denmark Received 18 October 2007; in final form 28 December 2007 Available online 5 January 2008
Abstract Diphenolic isoflavonoid puerarin fluoresces in aqueous solution with maximal intensity at pH 8.5 (Ufl = 0.042, sfl = 1.91 ns). For acidic solutions, weak fluorescence is attributed to fluorescent 7-monophenolate formed via excited-state deprotonation of neutral puerarin. For pH > 8.5, fluorescence decreases monotonically with an unchanged lifetime, suggesting that excited-state acidity of 40 -hydroxyl remains similar to the ground-state one, and that the diphenolate is non-fluorescent. The crucial role of A-ring 7-phenolate for fluorescence of puerarin is substantiated by absence (presence) of fluorescence for the 7-propylpuerarin (40 -propylpuerarin). Puerarin and its derivatives with the unusual properties may be explored to be antioxidant fluorescence probes. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction Epidemiological studies have clearly shown beneficial health effects of antioxidants from fruits and vegetables [1]. The results of human intervention studies, however, are less clear and the supplementation with vitamin antioxidants has shown little if any protective effects on cardiovascular diseases and on cancers [2]. In contrast to supplementation with one or a few antioxidants, a change in diet involves a change in the intake of many bioactive compounds as present in plant food with the perspective of synergistic interaction between the individual antioxidants. Further development in the evaluation methods should accordingly focus on antioxidant interaction [3]. Antioxidants are classified according to their nutritive value (vitamin vs non-vitamin) and their hydrophilic–lipo-
*
Corresponding authors. Fax: +86 10 6251 6444 (J.-P. Zhang). E-mail addresses: [email protected] (J.-P. Zhang), ls@life. ku.dk (L.H. Skibsted).. 0009-2614/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2007.12.072
philic balance (soluble in water vs in lipid), and six possibilities for the binary interactions among the four groups of antioxidants then become possible. Synergistic antioxidation effects were recently demonstrated at a water–lipid interface for two non-vitamin antioxidants, i.e. the watersoluble puerarin and the lipid-soluble b-carotene [4]. Puerarin, a C-glycoside of the isoflavonoid daidzein from the root of Pueraria lobata used in traditional Chinese herbal medicine, has only two phenols (Scheme 1) and shows moderate water solubility. The pH-dependent fluorescence of puerarin and flavonoids has been utilized in the mechanistic studies of antioxidation at water–lipid interface [5,6]. In this work, we have carried out, in combination with theoretical calculations, detailed characterizations of the fluorescence properties of puerarin in aqueous solution at varying pH. The roles of the A-ring 7-hydroxyl in dominating the fluorescence of puerarin, as well as in the excited-state deprotonation of neutral puerarin have been revealed. The fluorescence properties of puerarin and derivatives may facilitate to explore their uses as fluorescent antioxidant probes for the study of antioxidant interaction at water–lipid interfaces.
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Y.-X. Tian et al. / Chemical Physics Letters 452 (2008) 253–258
tion was 0.2 nm. Measurements were done at room temperature.
OH CH2OH
HO O
HO 7
2.4. Quantum chemical calculations
8
O
A
C
R1O 6
2 2'
3
3'
4
5
O
B 6' 5'
4'
OR2
Scheme 1. Molecular structures of puerarin (R1 = R2 = H) and its derivatives (R1 and R2 = H or C3H7).
2. Materials and methods
The molecular geometries for neutral, mono- and dianionic puerarins in the ground state (S0) were performed with the GAUSSIAN 03 package [10] by using the B3LYP density functional (DFT) [11,12] and the 6-31G(d) basis set [13,14]. Geometries in the lowest-lying singlet excited state (S1) were optimized on the basis of the TURBOMOLE program suite [15] with the DFT method, the B3LYP functional and the SV (P) basis, in which the fine quadrature grids 4 was employed [16].
2.1. Sample preparation 3. Results and discussion Puerarin crude product (Huike Plant Exploitation Inc., Shanxi, China) was twice recrystallized in a 1:1 (v/v) binary solvent of acetic acid (>99.5%; dried and refluxed by the use of P2O5 and KMnO4, respectively) and methanol (>99.5%; Beijing Chemical Plant, Beijing, China). 7-Propylpuerarin and 40 -propylpuerarin (Scheme 1) were synthesized as described previously [7]. Aqueous solutions of puerarin, 7-propylpuerarin and 40 -propylpuerarin (each 2 105 M) were prepared using the Britton–Robinson buffer (pH 2.0–13.0) [8], for which water was supplied by a Milli-Q apparatus (Millipore Corp., Billerica, MA). The ionic strength was controlled at 0.10 using NaCl. The pH values were measured with the pH-211 meter (Hanna Instruments Inc., Woonsocket, RI).
3.1. Steady-state absorption and fluorescence spectra The absorption spectra of neutral (pH 2.0), monoanionic (pH 8.5) and dianionic (pH 13.0) puerarins are shown in Fig. 1a, and their fractional distribution against pH calculated from the pKa values are depicted in Fig. 1b. The fluorescence spectra for puerarin at varying pH are shown in
2.2. Steady-state fluorescence The steady-state absorption and fluorescence spectra, respectively, were recorded on a Cary50 absorption spectrometer (Varian, Inc. Palo Alto, CA) and a LS55 luminescence spectrophotometer (Perkin Elmer Inc., Foster, CA). Fluorescence quantum yield was determined by the use of 1,4-bis-(5-phenyl-2-oxazolyl)benzene (POPOP; Exciton Inc., Dayton, OH) as a reference (Ufl = 0.97 in cyclohexane) [9]. Sample solutions were thermostated at 25 °C with a water bath (RTE-110, Neslab Instruments Inc., Newington, NH). 2.3. Fluorescence lifetime The excitation laser pulses (315 nm, 120 fs) for timeresolved fluorescence were supplied by an optical parametric amplifier (OPA-800CF, Spectra Physics), which was pumped by the output from a regenerative amplifier (Spitfire, Spectra Physics, Mountain View, CA). The excitation pulse energy was 100 nJ/pulse. Fluorescence collected with a 90°-geometry was dispersed by a polychromator (250is, Bruker Optics, Billerica, MA), and detected with a streak camera (C5680, Hamamatsu Photonics, Hamamatsu, Japan). The time resolution was 2–100 ps depending on the delay-time-range settings, and the spectral resolu-
Fig. 1. (a) Steady-state absorption spectra of puerarin in aqueous solution at pH 2.0 (neutral form, >99.9%), pH 8.5 (monoanion, 91.0%) and pH 13.0 (dianion, >99.9%), arrows indicate excitation wavelengths. (b) Fractions of neutral, monoainoic and dianionic puerarins in aqueous solutions against pH.
Y.-X. Tian et al. / Chemical Physics Letters 452 (2008) 253–258
255
Fig. 2. Fluorescence emission spectra of puerarin (2 105 M) at pH ranges of (a) 2.0–8.5 and (b) 8.5–13.0 in aqueous solution (kex = 315 nm).
Figs. 2a and b, which appear structureless and rather broad (3700 cm1) with a peak wavelength of kmax 463 nm independent of pH. The fluorescence intensity increases with increasing pH (2.0–8.5), and decreases with further increase in pH (8.5–13.0). Note that, at pH 8.5, monoanionic puerarin has its maximal fraction (91.0%) with a fluorescence quantum yield of Ufl = 0.042 ± 0.001. The fractions of neutral and dianionic puerarins at the pH values of 2.0 and 13.0 both are >99.9%. Fig. 3 exhibits the absorption, fluorescence excitation and emission spectra of puerarin in buffer. The emission spectra are similar to each other, and all of them exhibit large Stokes shifts, i.e. 11700 cm1 for the neutral and 7000 cm1 for the anionic puerarins. The emission spectrum at pH 13.0 in Fig. 3c was obtained by subtracting the background emission of buffer from the extremely weak emission of sample solution and, therefore, the shoulder around 400 nm is most likely caused by interference from scattered actinic light and/or emission from an adventitious impurity. In the wavelength range of 300–400 nm, the excitation spectra agree well with the absorption spectra in the cases of neutral (Fig. 3a) and monoanionic (Fig. 3b) puerarins, whereas obvious deviation is seen for dianionic puerarin (Fig. 3c). Interestingly, the excitation spectrum of the dianion agrees well with the absorption band of the monoanion. In Fig. 3a–c, substantial discrepancies between the excitation and the absorption spectra are found for the shorter-wavelength band, which may be ascribed to the deactivation of the higher-lying electronic excited state (e.g., via photo-ionization) before relaxing to the lowestlying fluorescent state. The pH-dependence of fluorescence intensity for puerarin under the excitation wavelengths of 315 nm and 355 nm
Fig. 3. Fluorescence excitation (solid line, kem = 463 nm) and emission (dotted line, kex = 315 nm) spectra of puerarin (2 105 M) in aqueous solution at indicated pH values for (a) neutral, (b) monoanionic and (c) dianionic forms. Absorption spectra (dashed line, cf. Fig. 1a) are shown for comparison. Excitation spectra are normalized with absorption spectra at lower-energy absorption maxima.
are shown in Fig. 4a. Actinic light at 355 nm selectively excited the anionic forms (cf. Fig. 1a), and the acid–base equilibrium constants for the ground-state puerarin, pK a1 7:0 and pK a2 9:6, could be obtained by fitting the data in Fig. 4a to the relation I fl ¼ a=ð1 þ ½Hþ =K a1 þ K a2 =½Hþ Þ;
ð1Þ
where Ifl represents fluorescence intensity, K a1 (7-hydroxyl) and K a2 (40 -hydroxyl) are the acid–base equilibrium constants, [H+] is the proton concentration and a is simply a proportionality constant. The pKa values obtained from the fluorescence data are close to the values of pK a1 ¼ 7:20 and pK a2 ¼ 9:84 previously derived from the pH-dependent absorption spectra [4].
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Scheme 2. Deactivation and ionization processes for neutral and anionic puerarins. For the S0 and the S1 states, dihedral angles (a) are shown behind the notations, pKa values are indicated above or below bidirectional arrows. DH 01 (3500 cm1) and DH 02 (4700 cm1) are standard molar ionization enthalpies in the S0-state calculated from the pKa values. The S1-state of neutral puerarin is shown in dashed line (exact energetic location unknown owing to the unobservable fluorescence emission). Vertical solid arrows indicate the absorptive (upward) and emissive (downward) transitions; dashed arrows represent the nonradiative deactivation processes. Vertical solid arrows indicate the absorptive (upward) and emissive (downward) transitions; dashed arrows represent the nonradiative deactivation processes.
Fig. 4. Normalized fluorescence intensity–pH profiles of (a) puerarin and (b) 40 -propylpuerain in aqueous solutions for excitation at 315 nm (filled circle) or 355 nm (open circle), solid lines were obtained by fitting the 355nm data to Eq. (1) for (a) and to Eq. (3) for (b), respectively. Insets show the difference between fluorescence intensities on 315-nm and 355-nm excitation, and the solid lines are just for guiding the eyes. (c) Experimental data (filled circle) showing the concentration effect on fluorescence intensity, solid and dashed lines are fitting curves according to different models (see text for details).
The inset of Fig. 4a illustrates the difference of normalized fluorescence intensity between the cases of 315nm and 355-nm excitations (pH 2.0–6.0). Here, it is worth noting that the 355-nm excitation did not induce any detectable fluorescence at pH < 4.0 although fluorescent monoanion, if any, would have been selectively excited. Therefore, the weak fluorescence under acidic condition must originate from the S1-state monoanion formed via deprotonation of the S1-state neutral puerarin (Scheme 2), which is further proven by the sub-linear dependence of the fluorescence intensity on the concentration of puerarin at pH 2.0 (Fig. 4c), i.e. the data strictly follow the equation: I fl ¼ a I 0 ð1 10CeL Þ
ð2Þ
where Ifl is the fluorescence intensity, I0 stands for the intensity of actinic light, C for the concentration and e (2400 L mol1 cm1 at 315 nm) for the extinction coefficient of neutral puerarin; L is the length of detected optical path and a is a proportionality constant. On the other hand, the contribution of singlet excitation energy transfer from the neutral puerarin to the monoanion is negligible, which would otherwise lead to the completely different concentration dependence (/C2) as shown by the dashed line in Fig. 4c. It is known that the acidity increases upon excitation for aromatic compounds with hydroxyl substituents, e.g., the pKa of 2-naphthol was found to decrease from 9.5 to 2.8 on going from the S0 to the S1 state [17]. For puerarin at the acidic condition (pH 2.0–6.0), the S1-state pK a1 can be determined by fitting the data in the inset of Fig. 4a using the following relation (parameters defined as in Eq. (1)) provided that an excited-state equilibrium is established, I fl ¼ a K a =ð½Hþ þ K a Þ:
ð3Þ
However, a pK a1 value of 2.0 thus estimated is considered to be unreliable because the equilibrium was indeed not reached: The intrinsic S1-state lifetime of neutral puerarin was calculated to be 39 ns from its absorption spectrum; on the other hand, although direct fluorescence emission from neutral puerarin at pH 2.0 was experimentally unobservable, an upper limit of its Ufl can be set as 0.001 on the basis of the Ufl of monoanion (maximum 0.042 at pH 8.5). Therefore, the S1-state lifetime of neutral puerarin could be <40 ps, which is too short to allow an excited-state ionization equilibrium.
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In Fig. 4a under 315 nm excitation, the intensity–pH profile in the range of pH > 6.0, in contrast to the cases of pH < 4.0, can be fully accounted for by the curve calculated using the S0-state pKa values. In addition, the absorption maximum of puerarin diphenolate in Fig. 1a is slightly blue-shifted (350 cm1) with respect to the monophenolate, indicating that pK a2 of puerarin can be similar to its pK a2 (9.8, Fig. 4a, Scheme 2). Fig. 4b displays the intensity–pH dependence of 40 -propylpuerarin under photo-excitation at 315 nm and 355 nm. Although 7-propylpuerarin shows no fluorescence at any pH in aqueous solution, 40 -propylpuerarin shows increasing fluorescence intensity for pH up to 8.5 with an intensity–pH profile similar to that of puerarin; the fluorescence quantum yield of 40 -propylpuerarin at pH 8.5 was determined to be Ufl = 0.043 ± 0.001. A pKa value of 6.9 was determined by using Eq. (3), which is close to pKa = 7.23 previously derived from the absorption data [4]. The marked difference in fluorescence properties between the two derivatives substantiates the role of the A-ring 7-phenolate in dominating the fluorescence properties of puerarins. 3.2. Fluorescence lifetime The fluorescence decay kinetics of puerarin at different pH are shown in Fig. 5. At pH 8.5 and 13.0 for mono-
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aninoic and dianionic puerarins, respectively, the kinetics follow a single exponential decay with the lifetimes of 1.91 ± 0.01 ns and 1.94 ± 0.02 ns which are comparable with that of some simple phenols [18]. The similarity in lifetime strongly suggests that monoanionic puerarin is the fluorescent species at either pH. On the other hand, the decay of fluorescence at pH 2.0 for neutral puerarin is complicated and cannot be adequately described with a single exponential function; this aspect needs to be further investigated. 3.3. Ground- and excited-state geometries The molecular geometry optimization for neutral, monoanionic and dianionic puerarins in their S0 and the S1 states yielded the dihedral angles (a) between the planes of AC and the B rings as indicated in Scheme 2. It is seen that a angles of the S0-state molecules are similar (35–38°). With respect to the S0-state geometries, the S1-state monoanion becomes considerably less twisted (a = 14°), whereas changes in the S1-state geometries of neutral and dianionic puerarins are not significant. The smaller twist of the S1-state monoanion may explain its substantially larger Ufl in contrast to neutral and dianionic puerarins. The 7-phenolate, present in the S0 state or formed via deprotonation of the S1-state neutral puerarin, is mandatory for establishing an extended conjugate system conducive to fluorescence emission. In contrast, the 40 -phenolate, present in puerarin or in 7-propylpuerarin at higher pH, reduces the extension of conjugation and thereby the probability of emission. 4. Conclusion The present work demonstrates the unusual fluorescence properties of puerarin owing to the marked difference in the sensitivity of acid strength to optical excitation between the A-ring and B-ring phenols, as well as the critical roles of A-ring 7-phenolate in dominating the molecular and electronic structures. Since the B-ring phenol is located in the more hydrophobic part of the molecule, and is less acidic but more reducing than the A-ring phenol, puerarin at water–lipid interface may serve as an antioxidant fluorescent probe, as well as a radical shuttle in the presence of water-soluble and lipid-soluble antioxidants. Acknowledgements
Fig. 5. Fluorescence decay kinetics (open circle) of puerarin at pH values of (a) 2.0, (b) 8.5 and (c) 13.0 in aqueous solutions. Solid lines are fitting curves based on (a) bi-exponential or (b, c) single exponential model functions, decay time constants (s) are indicated.
This work has been supported by grants from Natural Science Foundation of China (#20673144, #20433010, #20703067) and Ministry of Science and Technology of China (#2006BAI08B04-06). Support from LMC, Centre for Advanced Food Studies to the Food Chemistry group at University of Copenhagen is acknowledged. We are grateful for the stimulating dis-
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