Singlet oxygen quenching and the redox properties of hydroxycinnamic acids

Free Radical Biology & Medicine, Vol. 26, Nos. 9/10, pp. 1202–1208, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(98)00313-X

Original Contribution SINGLET OXYGEN QUENCHING AND THE REDOX PROPERTIES OF HYDROXYCINNAMIC ACIDS SARAH FOLEY,* SUPPIAH NAVARATNAM,† DAVID J. MCGARVEY,* EDWARD J. LAND,† T. GEORGE TRUSCOTT* ‡ and CATHERINE A. RICE-EVANS *Department of Chemistry, Keele University, Keele, Staffordshire, UK; †CRC Section of Drug Development and Imaging, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK; and ‡Free Radical Research Group, Division of Biochemistry and Molecular Biology, UMDS–Guy’s Hospital, St. Thomas’s Street, London, UK (Received 19 June 1998; Revised 9 October 1998; Accepted 26 October 1998)

Abstract—The singlet oxygen quenching rate constants (kq) for a range of hydroxycinnamic acids in acetonitrile and D2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity. The magnitude of kq observed depends on both the nature of the substituent groups and solvent polarity. The variations in kq depend on the energy of the hydroxycinnamic acid/molecular oxygen charge transfer states, (Od22...HCAd1). In D2O the values of kq range from 4 3 107 M21 s21 to 4 3 106 M21 s21 for caffeic acid and o-coumaric acid respectively. In acetonitrile, the charge transfer energy levels are raised and this is reflected in lower singlet oxygen quenching rate constants with a kq value of 5 3 106 M21 s21 for caffeic acid. The phenoxyl radical spectra derived from the hydroxycinnamic acids were determined using pulse radiolysis of aqueous solutions and the reduction potentials were found to range from 534 to 596 mV. A linear correlation is observed between reduction potential, and hence free energy for electron transfer, and log kq. These correlations suggest a charge transfer mechanism for the quenching of singlet oxygen by the hydroxycinnamic acids. © 1999 Elsevier Science Inc. Keywords—Hydroxycinnamic acids, Singlet oxygen, Free radicals, Redox potentials

INTRODUCTION

of caffeic acid), o-, m-, p-coumaric acids, ferulic acid and sinapic acid and are produced from the shikimate pathway from either phenylalanine or L-tyrosine (Fig. 1). They most commonly occur as simple esters with quinic acid or glucose and are present in a wide variety of fruit and vegetables including tomatoes, spinach, broccoli, asparagus, white grapes, pears and peaches. The antioxidant properties of the HCA have been demonstrated with regard to their ability to scavenge radicals generated in the aqueous phase [4] to increase the resistance of low density lipoprotein (LDL) to lipid peroxidation [5,6]. Their anti-inflammatory and antimutagenic abilities have also been demonstrated [7,8]. The uptake and excretion of hydroxycinnamates from a tomato rich diet have recently been demonstrated in human nutritional studies [9,10]. In this paper the antioxidant potential of the HCA with respect to their ability to quench singlet molecular oxygen, a reactive oxygen species that appears to play a role in tumour promotion and carcinogenesis, has been

A growing amount of evidence suggests that the dietary intake of phytochemicals plays an important role in maintaining health and protecting against degenerative processes including cardiovascular disease and certain cancers. The protective effects of the antioxidant constituents of fruit and vegetables have been attributed to the carotenoids, vitamins C and E and the flavonoids [1,2]. In recent years antioxidant studies have been extended and now include simple phenolic acids or phenylpropanoids that are present in plants at greater concentrations than the polyphenolic flavanoids. The hydroxycinnamic acids (HCA) are amongst the most widely distributed phenolic acids in plant tissue [3]. They include caffeic acid, chlorogenic acid (the quinic acid ester Address correspondence to: Professor T. G. Truscott, Department of Chemistry, Keele University, Keele, Staffordshire, ST5 5BG, UK. Tel: 144 01782 583038; Fax: 144 01782 712378; E-Mail: [email protected]. 1202

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Instrumentation Kinetic measurements were made using a JK System 2000 Q switched Ruby laser operating on the fundamental at 694 nm. Time-resolved singlet oxygen phosphorescence (Eq. 1): O 2~ 1D g! 3 O 2~ 3( 2 g ! 1 h n ~1270nm!

(1)

was detected using a Judson germanium photodiode (G050, active diameter 5 0.5 cm) coupled to a Judson preamplifier. Pulse radiolysis measurements were made using a 9 –12 MeV Vickers linear accelerator as described previously [12] with pulses of 10 –100 ns duration and doses of between 1 and 10 Gy. Irradiation was in quartz flow through cells with a monitoring optical path length of 2.5 cm. Absorption measurements were recorded using a Perkin-Elmer Lambda-2 UV/Vis spectrophotometer.

Methods

Fig. 1. The synthetic pathway of the hydroxycinnamic acids in plants.

studied [11]. In addition, the reduction potentials of the phenoxyl radicals derived from the HCA have been estimated via pulse radiolysis and these show the quenching of singlet oxygen by the HCA occurs via a charge transfer mechanism.

Singlet oxygen quenching rate constant measurements. The rate constants (kq) for the quenching of singlet oxygen by the HCA were determined in acetonitrile and D2O by monitoring the time-resolved phosphorescence following laser excitation of methylene blue (MB) at 694 nm. This choice of wavelength precludes any absorption by the HCA. At each concentration of HCA the recorded luminescence trace was obtained by signal averaging ten single shots. The averaged traces were fitted with a single exponential. Linear plots of HCA concentration versus the observed pseudo first order rate constant, k2, for singlet oxygen deactivation were obtained, where k2 5 k1 1 kq[HCA], k1 being the intrinsic first order decay constant of singlet oxygen in the absence of HCA. The photosensitized production of singlet oxygen involves energy transfer from the excited triplet state of MB to molecular oxygen (Eq. 2): 3

k ET MB* 1 O 2 ~ 3 ( 2 ¡ MB 1 O 2 ~ 1 D g ! g! O

(2)

MATERIALS AND METHODS

Materials Methylene blue, potassium thiocyanate and sodium azide (British Drug Houses Ltd., Poole, Dorset, UK), caffeic acid, chlorogenic acid, m-, o-, p-coumaric acids, ferulic acid and sinapic acid (Sigma Chemical Co., Poole, Dorset, UK, Aldrich Chemical Co., Gillingham, Dorset, UK), Trolox (Aldrich), acetonitrile (spectroscopic grade, British Drug Houses Ltd.) and D2O (Goss Scientific Instruments Ltd., Chelmsford, Essex, UK) were all used as received.

Generation of phenoxyl radicals The method of production of phenoxyl radicals and their kinetic behaviour has been described previously [13]. The HCA (1 3 1024 M) were dissolved in phosphate buffer (pH 5 7) solutions containing NaN3 (0.1 M) purged of oxygen and saturated with N2O in order to convert hydrated electrons into hydroxyl radicals. The hydroxyl radicals react with the azide to produce the less oxidizing azide radical. The resulting ion radical reacts

S. FOLEY et al.

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with the HCA to produce its phenoxyl radical cation that rapidly deprotonates (Eqs. 3–7). H 2 O MM3 e 2 ~aq! 1 • OH

(3)

N 2 O 1 H 2 O 1 e 2 ~aq! 3 N 2 1 • OH 1 OH 2

(4)

OH 1 N 32 3 OH 2 1 N •3

(5)

HCA 1 N •3 3 HCA •1 1 N 32

(6)

HCA •1 3 HCA • 1 H 1

(7)

•

Determination of radical absorption coefficients Absorption coefficients were obtained using thiocyanate dosimetry. The absorbed doses were determined from transient (SCN)2•2 formation in air-saturated 1022 M KSCN solution. In such solutions, the primary •OH radicals are converted into (SCN)2•2 via reactions (Eq. 8) and (Eq. 9): •

OH 1 SCN 2 3 OH 2 1 SCN •

(8)

SCN • 1 SCN 2 3 ~SCN! 2 •2

(9)

The absorption coefficient of (SCN)2•2 was taken to be 7100 M21 cm21 at 500 nm assuming a G value of 2.9 [14]. In determining the HCA radical absorption coefficients, it was assumed that, under the low dose (up to 6 Gy) conditions employed, complete scavenging of N3• by the HCA occurred (G(N3•) 5 6.13) in N2O5saturated solutions [15].

Determination of reduction potentials The reduction potentials of the phenoxyl radicals derived from the HCA were determined at pH 7 with reference to the Trolox redox couple E 5 1480 mV, vs. the hydrogen electrode [16], the only exception being the

Table 1. Singlet-Oxygen Quenching Rate Constants (k q) for the HCA in Acetonitrile and D2O HCA

Acetonitrile k q/105 M21 s21

Fig. 2. Observed pseudo first order rate constants, k2, for decay of singlet oxygen as a function of the concentrations of hydroxycinnamic acids, from which the second order quenching rate constants, kq, were obtained in acetonitrile (A) and in D2O (B).

reduction potential for chlorogenic acid that was determined against ferulic acid. The electron transfer reactions between electron acceptor radicals and the electron donor e.g., HCA • 1 Trolox º HCA 1 Trolox •

Table 2. Phenoxyl Radical Properties for the HCA in Phosphate Buffer (pH 5 7)

D2O k q/106 M21 s21 HCA

Caffeic acid Chlorogenic acid m-Coumaric acid o-Coumaric acid p-Coumaric acid Ferulic acid Sinapic acid

51 — 6 — 7 36 47

40 34 7 4 6 20 30

(10)

Caffeic acid Chlorogenic acid o-Coumaric acid p-Coumaric acid Ferulic acid Sinapic acid

lmax/nm

«/103 M21 cm21

k(N3• 1 HCA)/ 109 M21 s21

380 390 360 340 360 360

9.6 8.6 3.4 27 13.2 20.2

3.9 3.7 0.2 1.0 4.3 3.3

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Fig. 3. Changes in absorption resulting from the formation of the corresponding phenoxyl radicals at pH 7 by N3•-induced oxidation of (A) caffeic acid, (B) chlorogenic acid, (C) o-coumaric acid, (D) p-coumaric acid, (E) ferulic acid, and (F) sinapic acid.

were followed by kinetic spectroscopy. The absorbance of each of the radicals was determined separately, and the absorbance at equilibrium allowed the determination of the radical concentrations. It should be noted that at

pH 7 the forms of the hydroxycinnamic acids, Trolox and the relatively stable semiquinones, are referred to as HCA, Trolox, HCA•, and Trolox•, respectively, without any implication as to the net charge on such species.

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D2O than acetonitrile whilst, the dihydroxyl substituted caffeic acid is the most efficient quencher and the monosubstituted coumaric acids are least efficient. All of these observations are rationalized on the basis of the electron donating properties of the substituents (hydroxyl groups being more electron donating than methoxy groups). Moreover, the results also show [19] that the position of the OH group has little effect on the quenching efficiency, as kq for o-, m-, and p-coumaric acids are very similar. In contrast, the number of methoxyl groups plays an important role as can be seen from the comparison of kq for p-coumaric, ferulic, and sinapic acids.

Fig. 4. Time profiles of absorption changes at 380 nm showing the establishment of equlibrium in a typical case. Because some decay of the absorption observed from the caffeic acid alone, and in the presence of Trolox, occurs during equilibrium, the slow decays were extrapolated back to the time of the pulse (as shown by fine lines).

RESULTS AND DISCUSSION

Singlet oxygen quenching by the HCA The measured values for the quenching of singlet oxygen by the series of HCA in acetonitrile and D2O are collected in Table 1. In all experiments the emission observed at 1270 nm decayed exponentially with k1, the intrinsic first order decay rate constant, being solvent dependent. The average lifetimes of 67 ms and 54 ms compare with the published values of 54 – 68 ms and 68 ms [17,18] for these two solvents, respectively. Plots of k2, the observed pseudo first order rate constant for the deactivation of singlet oxygen in the presence of the HCA were plotted against the concentration of the quencher producing straight line graphs as illustrated in Fig. 2A and 2B, the gradient of that is equal to the quenching rate constant kq and the intercept is k1. Similar plots were obtained for all the HCA. As Table 1 illustrates, the quenching rate constants are sensitive to both the nature of the substituent and the polarity of the solvent. Because the values of kq descend the group of HCA in the same order irrespective of the solvent, the observed differences are due to differences in substituents. Quenching rate constants are higher in

Absorption spectra of the radicals Table 2 gives a summary of the phenoxyl radical properties for each of the HCA studied. The maxima in the phenoxyl radical spectra range from 340 –390 nm and all exhibit a well defined peak as shown in Figs. 3A–F. The rate constants of quenching of azide radical and were measured by following the kinetics of formation of the HCA radical at the corresponding absorption maxima. With the exception of the o- and p-coumaric acids they are all approximately 3– 4 3 109 M21 s21 and approach diffusion control. Reduction potentials The reduction potentials of the phenoxyl radicals derived from the HCA studied were determined at pH 7 with reference to Trolox (Eq. 10). The determination of the equilibrium constant K from the absorbance at equilibrium is demonstrated by the system for Trolox vs. caffeic acid in Fig. 4 and Table 3. The absorbance of each radical is determined separately and the absorbance at equilibrium allows the determination of the ratio of radical concentrations at equilibrium. The equilibrium constant K is then calculated from this ratio and the initial concentrations of the parent compounds in solution (Eq. 11). As Table 3 illustrates the values of K are constant within experimental error and over a range of concentrations. K5

Table 3. Absorbances at 380 nm (Normalized to a Dose of 1.3 Gy) at Equilibrium for Varying Concentrations of Caffeic Acid and Trolox, From Which the Constant K Was Determined

[Caffeic Acid]/mM 8 8 10

[Trolox]/mM

Absorbance at 380 nm at equilibrium/1023

K

1 6 5

8.69 3.96 12.0

6.35 7.64 10.56

[Trolox•]z[HCA] [HCA•]z[Trolox]

(11)

Figure 4 shows an example of a kinetic trace that clearly shows the establishment of an equilibrium and that allows determination of the absorbance. The difference in reduction potentials between the two systems is given by the Nernst equation. DE 5 0.059 log K

(12)

Hydroxycinnamic acid antioxidant properties Table 4. Reduction Potentials at pH7 and Free Energy of Electron Transfer for the Various HCA HCA Caffeic acid Chlorogenic acid o-Coumaric acid p-Coumaric acid Ferulic acid Sinapic acid

K

E7/mV

DG/kJ mol21

8.18 15.5 92.3 72.2 90.7 19.1

534 550 596 590 595 556

210.6 29.1 24.6 25.2 24.7 28.5

The results of the various systems studied are given in Table 4 with E values ranging from 534 mV to 596 mV for caffeic acid and o-coumaric acid, respectively. The value of 534 mV for caffeic acid is in good agreement with the value of 540 mV obtained by Jovanovic and coworkers [20]. There is a linear correlation between log kq and the reduction potential at pH 7, the rate constants decreasing as the reduction potentials increase, as shown in Figure 5. Similar linear relationships have previously been observed for other series of phenolic compounds and amines [21–24]. Such correlations suggest that the quenching of singlet oxygen occurs via a charge transfer reaction mechanism with the following scheme (Eq. 13) being proposed. 1

O 2 1 HCA^ 1 ~O 2d 2 ...HCA d 1 ! ^ 3 ~O 2d 2 ...HCA d 1 ! 3 3 O 2 1 HCA

(13)

The free energy change, DG, that is associated with the electron transfer process is represented by Eq. 14: DG 5 F~E~D •1 /D! 2 E~ A/A •2 !! 2 E *

(14)

Fig. 5. Plot of the logarithm of the O2, 1Dg quenching rate constants for the HCA in D2O vs. the reduction potential in phosphate buffer at pH 7.

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where F is the Faraday constant (96.5 C mol21), E(D•1/D) is the reduction potential of the electron donor, the HCA, E(A/A•2) is the reduction potential of oxygen (E O2/O•2 5 2330 mV) [25] and E* is the 2 energy of the excited state acceptor (94 kJ mol21). For electron transfer reactions between members of the same homologous series of donors (the HCA) and the same acceptor (O2, 1Dg) a linear relationship exists between the logarithm of the quenching rate constant kq and DG, the free energy change for the reaction. Because all terms relating to the acceptor remain constant then log kq is linearly dependent on E(D•1/D) as illustrated in Fig. 5. Such behaviour is consistent with current theories on electron transfer reactions.

CONCLUSIONS

The antioxidant ability of the hydroxycinnamic acids has been demonstrated with respect to their capability to quench singlet molecular oxygen, an example of a reactive oxygen species. The results have shown that the electron donating ability of the substituents and the polarity of the solvent influences the quenching rate constant kq. It has also been shown that there is an inverse correlation between log kq and the reduction potentials of the hydroxycinnamic acids. The above results are evidence for the participation of charge transfer interaction during the quenching of singlet oxygen by the hydroxycinnamic acids. Acknowledgements — The pulse radiolysis experiments were performed at the Paterson Institute for Cancer Research Free Radical Research Facility, Christie Hospital NHS Trust, Manchester. The facility is supported by the European Commission T.M.R. Programme— Access to Large-Scale Facilities. E.J.L. thanks the Cancer Research Campaign for financial support. We also thank Dr. R. V. Benasson for useful discussions.

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