Physico-Chemical Properties and Antioxidant Activities of Methoxy Phenols

Free Radical Biology & Medicine, Vol. 24, No. 6, pp. 933–941, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00

PII S0891-5849(97)00382-1

Original Contribution PHYSICO-CHEMICAL PROPERTIES AND ANTIOXIDANT ACTIVITIES OF METHOXY PHENOLS K. I. PRIYADARSINI,* S. N. GUHA,*

and

M. N. A. RAO†

*Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India; and †Divi’s Laboratories Ltd., Dharam Karam Road, Ameerpet, Hyderabad, India (Received 19 May 1997; Revised 19 August 1997; Accepted 3 October 1997)

Abstract—A few structurally related phenols, dehydrozingerone (DZ), bromopentenone (BP), eugenol (EG) and isoeugenol (IEG), derived from plant products show antioxidant properties by inhibiting lipid peroxidation in membrane models. The phenoxyl radicals produced by the scavenging of free radicals during the inhibition of lipid peroxidation, were also generated by specific one-electron oxidants using pulse radiolysis. The radical lifetimes (second order rate constants for radical-radical reactions), reactivities with hydroxyl and model peroxyl radicals and the one-electron reduction potentials with respect to the standard couples were quantified. These results along with their lipophilicity data were correlated with their antioxidant activity (IC50 values). © 1998 Elsevier Science Inc. Keywords—Methoxy phenols, Free radicals, Pulse radiolysis, Antioxidants

INTRODUCTION

them have a methoxy group ortho (position 2) to the phenolic OH and a side chain at the para position (position 4). DZ and BP possess a carbonyl side chain, whereas EG and IEG have an alkyl side chain with a double bond and they are structural isomers. BP in addition has bromo group attached ortho (position 6) to the phenolic OH. These structurally related species were examined for their antioxidant activity and were studied in terms of their effect to inhibit in vitro lipid peroxidation.18 Further, the physico– chemical properties of the transient intermediates of these antioxidants generated by the scavenging of several oxidising free radicals were quantified using pulse radiolysis technique. Some aspects of the pulse radiolysis of BP and DZ were recently reported by us.17 We have used the data of these studies for comparing the antioxidant properties of all the related compounds together. In addition to the pulse radiolysis work, data on the lipophilicity (affinity towards lipids with respect to water) of the antioxidants were also determined.

Several naturally occurring phenols, derived from plants have been shown to be good antioxidants as they are efficient scavengers of free radicals.1–5 Oxygen free radicals are generally oxidants and are produced by both exogenous agents like radiation, drugs, toxins, chemical air pollutants etc., and endogenously by enzymatic reactions. Such reactive oxygen radicals have been implicated in several disorders such as cancer, aging etc.6 –9 Antioxidants especially from diet are capable of offsetting this elevated oxidative challenges. Well known among these natural phenols are flavonoids, tocopherols etc. Other phenolic compounds which are gaining importance as antioxidants include curcumin from turmeric, dehydrozingerone, eugenol etc. All of them are known to possess pharmacological properties.10 –21 Here we examined the antioxidant efficiency of a few structurally related phenols and correlated with the physico-chemical properties of their phenoxyl radicals. The phenolic antioxidants used for this study are dehydrozingerone (DZ,), bromopentenone (BP), eugenol (EG) and isoeugenol (IEG), vide Scheme 1 for their structure. They have many structural similarities. All of

EXPERIMENTAL

DZ and BP were synthesised by the method reported earlier.19 Their structures were confirmed by NMR, IR, mass spectra and elemental analysis. Eugenol from Sigma and Isoeugenol from Industrial Perfumes Ltd.

Address correspondence to: Dr. Mrs. K. Indira Priyadarsini, Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai400085, India; Fax: 91-22-5560750. 933

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K. I. PRIYADARSINI et al.

OH MeO

1 6

Rπ

2 3

5 4

Dehydrozingerone, DZ

R O

‡ Rπ = H; R = CH®CH©C©CH‹ Bromopentenone, BP

O

‡ Rπ = Br; R = HC®CH©C©CH¤©CH‹ Eugenol, EG‡ Rπ = H; R = CH¤©CH®CH¤

Rate constants for the reaction of the phenols with the radicals were determined by following the pseudo-first order time-dependent rise in the transient absorption with the phenol concentration. The slope of the linear plot gives the bimolecular rate constant. Estimation of the one-electron reduction potential of the phenoxyl radicals was made pulse radiolytically using equilibrium method. The details of the method have been given in references 28 –30. A reversible redox equilibrium is achieved in a few microseconds between two couples (A1/A and B1/B) one of them being a standard couple with a known reduction potential and the other an unknown couple under investigation. A 1z 1 B º A 1 B 1z

(1)

Isoeugenol, IEG‡ Rπ = H; R = CH®CH©CH‹ Scheme 1.

Mumbai, were used as received. Their purity was checked by HPLC and was found to be satisfactory. For in vitro study of lipid peroxidation, rat brain homogenates were used and lipid peroxidation was initiated by ferric chloride, ferrous sulfate and cumene hydroperoxide and was assayed by following the malonaldehyde product as thiobarbituric acid reactive substances (TBARS).18 Promethazine, chlorpromazine, sodium azide, phosphate salts and all other reagents were of the best quality available and were used without further purification. Pulse radiolysis studies were carried out with 7 MeV electrons of 50 ns pulse width, obtained from a linear accelerator, the details of which have been described in references 22,23. The transients were detected by kinetic spectrophotometry with optical path length of 1 cm. The slowest detectable response of the pulse radiolysis unit was limited to 5 ms. The absorbed dose was determined by the standard thiocyanate dosimeter.24 Doses employed for these studies were 8 –10 Gy. The dose variation from pulse to pulse is ;5%. Reactions of OH with the phenols were studied in N2O saturated solution containing a phenol in the pH range 4 –11. At pH 4 and above the reducing e-aq is quantitatively converted into OH radical by reaction with N2O. The radiation chemical yield for OH radical under N2O saturated conditions is taken to be 0.6 mmol dm23 J21. Azide radical (Nz3), a one-electron specific oxidant with a reduction potential, E(Nz3/N2 3 ) of 11.33 V vs. NHE was produced from N2 by its reaction with OH 3 radical in N2O saturated solution. Halo carbon peroxy radicals (CCl3Oz2) were generated on pulse-radiolysis of oxygen saturated aqueous solutions containing 48% (v/v) 2-propanol and 4% CCl4. Details for the generation of these radicals can be seen in references 25–27.

The approach to the equilibrium and the reversibility of the reaction was confirmed by following the change in kinetics of either the radical decay or formation. The equilibrium constant K, was estimated either by kinetic method or by following the equilibrium radical concentration (absorbance, D), according to the eqns (2) and (3): K 5 kf/kb and kobs 5 kf[B] 1 kb[A]

(2)

Deq 5 [DA 1 KDB([B]/[A])]/[1 1 K[B]/[A]]

(3)

where kf and kb are the rate constants for the forward and backward reactions for equilibrium (2) and in eqn (3), Deq is the absorbance of the radicals at a particular wavelength under equilibrium. DA and DB are the values obtained in the individual reactions. Applying Nernst’s equation, DE can be estimated from the known value of K as: DE 5 E(A1/A) 2 E(B1/B) 5 0.059 log K

(4)

For determining the reduction potential of these phenols, phenothiazines such as promethazine (PMZ1) and chlorpromazine (CPZ1) were found to be suitable as standards. The one-electron oxidised phenothiazines have strong absorption in the visible region, where the phenoxyl radicals have negligible absorption. Phenothiazine radicals have long lifetimes and the reduction potentials are , 1 V. Partition coefficient (log P) was calculated by using Hansch hydrophobic constants (p) as described by Hansch and Leo.31 RESULTS

Absorption spectra and PKA of the parent phenol The absorption spectra of the phenols at different pHs are significantly different due to their involvement in the

Properties of free radicals of phenolic antioxidants

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Table 1. Properties of Phenoxyl Radicals

Phenol (pKas)#

emax** (corrected) dm3 mol21 cm21

2k dm mol21 s21

Red. Potential* (mV vs NHE) PhOz, H1/PhOH

Standard Couple

DZ (8.3) BP (7.5) EG (9.5) IEG (9.5)

32000 6 1000 28000 6 900 8400 6 500 11500 6 800

1.3 6 0.6 3 109 1.1 6 0.7 3 109 5.5 6 0.9 3 108 3.8 6 0.8 3 108

1090 6 10 910 6 15 810 6 15 716 6 18

N3z /N2 3 PMZ12/PMZ1 12 PMZ /PMZ1 CPZ12/CPZ1

3

#

pKas correspond to the parent phenol; * Determined at pH 6; ** Corrected for parent absorption.

prototropic equilibrium as shown in eqn (5). The phenolic forms are mostly colorless and show absorption maximum in the range 250 –330 nm and the absorption due to the phenoxide ion is red shifted to 300 – 450 nm region and is yellow in color. By following the change in absorbance at appropriate wavelengths, the pKas of the parent compounds were determined and they are listed in Table 1. PhOH º PhO2 1 H1

(5)

Pulse radiolysis studies: reaction with azide radicals N3z Figure 1 gives the absorption spectra of the phenoxyl radicals of DZ, BP, EG and IEG produced 10 –30 ms after pulse radiolysing N2O saturated buffered aqueous solutions at pH 6, containing 100 mmol dm23 of the test compounds and 50 –100 mmol dm23 sodium azide. Inset of Fig. 1 shows a typical formation trace of the phenoxyl radical of BP under these conditions. The formation is complete in ;20 ms, and there is a negligible decay of radicals in this time scale, hence absorption reached in the plateau region of the formation signal is considered to be close to the true DODmax. Using this, transient spectra have been plotted. In case of IEG, the solutions also contained 10% acetonitrile to dissolve it in water. We studied the reactions of the phenols at pH 6 and 10. Although the rates of reactions with phenoxide ions (pH 10) are slightly faster than the phenols (pH 6), only phenoxyl radicals were generated from both the phenolic and phenoxide ion forms as shown in eqns (6) and (7). The absorption spectra of the phenoxyl radicals of DZ, BP and IEG show maximum absorption in the wavelength region 350 –370 nm, but those of EG have a maximum at much lower wavelength (300 nm) as the alkyl side chain is not in conjugation with the phenyl ring. Their absorption cross-sections for the phenoxyl radicals are very high and the extinction coefficients of the radicals at their absorption maxima after correcting for the ground state absorption at pH 6 are given in Table

1. The absorption spectra of the phenoxyl radicals produced by the same reaction remained the same at pH . 10, as the phenoxyl radicals have pKa at pH # 0.32 The phenoxyl radicals decay by second order kinetics by radical-radical reactions. The second order bimolecular rate constants (2k values) are included in Table 1. A few typical transient absorbance-time plots for the decay of phenoxyl radicals of DZ, BP, EG and IEG near the absorption maximum produced by reaction with Nz3 radicals at pH 6 are given in Fig. 2. From the 2k values obtained, it can be seen that for a radical concentration of one mmol dm23, the half lives of the radicals of DZ, BP, EG and IEG range from 760 ms to 2.6 ms. PhOH 1 Nz3 3 PhOz 1 H1 1 N2 3

(6)

PhO2 1 Nz3 3 PhOz 1 N2 3

(7)

Reaction with hydroxyl radical Hydroxyl radicals generated by water radiolysis reacted with the phenols with rate constants near diffusion controlled rates. The reactions are dependent on pH. The phenoxide ion (pH . pKa) reacts mainly by oxidation to form a phenoxyl radical, whereas the phenolic form in most of the cases reacts mainly by electrophillic addition to the ring double bonds in addition to minor oxidation.17 The rate constants for the reaction of OH to form the respective phenoxyl radicals at pH 6 are listed in Table 2. Reaction with haloperoxyl radicals Haloperoxyl radicals are the model peroxyl radicals and are often used to understand the scavenging ability of an antioxidant towards the lipid peroxyl radicals. The phenols under study were subjected to these radicals. The phenoxide ion forms (at pH 10) reacted with the radicals yielding phenoxyl radicals, while with the phenolic forms (pH 6) reaction was insignificant. The phenoxyl radical formation was confirmed by the radical absorption spectrum and decay kinetics (a typical example for

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K. I. PRIYADARSINI et al.

Fig. 1. Difference absorption spectra of the phenoxyl radicals at pH 6 of the phenols under study produced after pulse radiolysing an N2O saturated solution containing 100 mmol dm23 phenol, 0.05 mol dm23 azide and 0.005 mol dm23 phosphate. In case of IEG the solution also contained 10% acetonitrile. Inset shows absorption-time plot for the formation of the phenoxyl radical of BP at 370 nm.

the reaction with BP is shown in Fig. 3), which compared well with that obtained from Nz3 reaction. The rate constants as determined by following the growth of phenoxyl radicals as a function of the concentration of parent phenoxide ion (pH 10) are listed in Table 2. One-electron reduction potential The energetics for the formation of phenoxyl radical from the phenol in aqueous solutions can be measured in terms of their one-electron reduction potentials. Reversible electron transfer was achieved between the phenol/ phenoxyl radical and a standard couple and according to the eqns (2) and (3), the equilibrium constant and the one-electron reduction potentials were determined as described in the experimental section of this paper. For this purpose phenothiazine radical couples were used as re-

dox standards except in the case of DZ, where Nz3/N2 3 couple was used. Fig. 4 gives least square fitting of the data to eqn (3) for the redox equilibrium for couples involving isoeugenol and chlorpromazine. Inset of Fig. 4 shows the time variation of radical absorptions at equilibrium for the decay of chlorpromazine radical at 500 nm and formation of isoeugenol radical at 355 nm. Similar plots were obtained for other systems but are not shown here. From the equilibrium constants, using Nernst’s equation, the reduction potentials of different phenoxyl radicals were determined and listed in Table 1. Inhibition of lipid peroxidation The effects of dehydrozingerone, eugenol and isoeugenol on lipid peroxidation in rat brain homogenates induced by ferric ion, ferrous ion and cumene

Properties of free radicals of phenolic antioxidants

937

Fig. 2. Absorption-time plots showing the decay of the phenoxyl radicals produced under the conditions of Fig. 1 at their absorption maxima. (a) DZ at 360 nm, (b) BP at 370 nm, (c) EG at 300 nm and (d) IEG at 355 nm. Absorbed dose ; 9 Gy. In case of (a) and (b) negative absorption indicates bleaching of the parent absorption.

hydroperoxide were studied. The peroxidation was estimated in terms of TBARS. The detailed analysis of the work is published in reference 18. Under normalized identical conditions, the IC50 values (i.e., the amount the phenol required to inhibit 50% of the initial peroxidation is listed in Table 3 for ready reference). The data for bromopentenone is not included as the models are different. However, in rat mitochondria model, it has been shown to be more active than dehydrozingerone.

DISCUSSION

The phenols show high reactivity with several reactive oxidising free radicals (Table 2). All the phenols studied have almost similar reactivity with hydroxyl radicals. The reaction with hydroxyl radicals are pH dependent. The phenoxide ion in general gives a phenoxyl radical, but the phenolic form in reaction with hydroxyl radicals gives mainly OH-adduct in addition

938

K. I. PRIYADARSINI et al. Table 2. Rate Constants (dm3 mol21 s21) for the Reaction of Phenols With Oxidants

Phenol DZ BP EG IEG

OH (pH 6) 9.5 1.0 5.6 2.7

6 6 6 6

0.5 0.5 0.9 0.3

3 3 3 3

109 1010 109 109

4.7 6.5 4.8 2.9

N3z (pH 6)

CCl3O2z (pH 10)

6 6 6 6

1.4 1.2 7.5 2.2

0.8 0.5 0.2 0.1

3 3 3 3

109 109 109 109

6 6 6 6

0.4 0.4 0.5 0.6

3 3 3 3

109 109 108 107

to small oxidation product. The phenoxyl radicals produced by specific one-electron oxidant, Nz3 show strong absorption with lmax around 350 –370 nm. Only the phenoxyl radical of eugenol, due to lack of conjugation of the side chain in para position absorbs at lower wavelength (maximum at 300 nm). The phenoxyl radicals, decay by second order radical-radical reactions (probably by dismutation process) with 2k values of about 109 dm3 mol21 s21 and hence their

lifetimes are dependent on the initial concentration of the radicals produced. However, under cellular conditions, where the radical production is very low, the radical lifetimes are expected to be much longer and they decay by reaction with oxyl and peroxyl radicals of the lipids. The energetics to form the phenoxyl radicals from the phenols were estimated in terms of their one-electron reduction potentials (Table 1). Thus, the reduction potential indicates the OOH bond strength in solution phase. The values range from 700 –1090 mV vs NHE for these phenols at pH 6. The reduction potentials are pH dependent. For every pH unit below the pKaz the reduction potential increases by 59 mV, or as the pH increases above the pKaz they are more easily oxidisable. This is responsible for the increased reactivity of phenoxide ions as compared to the phenols towards electron transfer reactions. From the structure of the phenols, it is apparent that while all of them share a methoxy group at 2-position, they

Fig. 3. Difference absorption spectrum of the phenoxyl radical of BP produced by reaction with CCl3O2 radical at pH 10. Inset a and b respectively indicate absorption-time plots for the formation and decay of BP radical under these conditions.

Properties of free radicals of phenolic antioxidants

939

Fig. 4. Electron-transfer equilibrium involving the radicals of chlorpromazine (CPZ1) and isoeugenol (IEG) at pH 6. The equilibrium absorbance after 150 –200 ms at 355 nm vs [IEG]/[CPZ1] was fitted to eqn (3) to obtain the equilibrium constant K. Inset: (a) Decay of CPZ12 radical at 500 nm and (b) formation of phenoxyl radicals of IEG at 355 nm in a solution containing 5.7 3 1024 mol dm23 CPZ1 and 5 3 1025 mol dm23 IEG, 10% acetonitrile, 0.01 mol dm23 phosphate and 0.15 mol dm23 sodium azide.

differ in substitution in four and six positions. Substitution of an electron withdrawing b-keto group in para position as in DZ and BP, increases the OOH bond strength, whereas electron donating alkyl group as in EG and IEG makes the OOH bond easy to break. In Table 3. Partition Coefficient and Antioxidant Activity of Phenols IC50/ (mmol dm23) TBARS, Inhibition of lipid peroxidation* Phenol DZ BP EG IEG

Log P

Fe13

Fe12

Cumene hydroperoxide

1.76 3.26 2.89 3.12

. 500 — 74.2 13.2

29.8 — 11.3 3.4

243.5 — 136.8 16.7

* Data from Reference

18

.

case of BP as compared to DZ, substitution of a bulky bromo group in six position makes the molecule easy to be oxidised. This may most probably be due to the decrease in hydrogen bonding of the phenolic OH with ortho hydrogens. The reduction potentials of all these phenoxyl radicals are higher than that of trolox C radical (480 mV). Trolox c is a water soluble analog of a-tocopherol, ‘‘a golden standard’’ for all phenolic antioxidants. In all these cases conjugation in para position increases the resonance stability of the radical.33 With reference to the inhibition of lipid peroxidation in rat brain homogenates stimulated by ferric and ferrous ions, isoeugenol was the most active followed by eugenol and dehydrozingerone. Its activity in inhibiting ferrous ion induced lipid peroxidation (IC50 5 3.4 mmol

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dm23) was comparable to that of the standard, butylated hydroxy toluene, BHT (IC50 5 2.7 mmol dm23). Both ferric and ferrous ions stimulate lipid peroxidation through radical mechanisms. Most of these radicals are oxidising in nature. Phenols owe their antioxidant property to the scavenging of the free radicals mostly by electron transfer. Hence the antioxidant activity of a phenol depends on the efficiency with which it can transfer one electron to these oxidising radicals. Both thermodynamic and kinetic parameters should be such that the electron transfer is favorable. Thus, the reduction potential of a phenol, a quantity measured in solution and related to the first ionization potential has to be directly correlated with the antioxidant activity. Data listed in Tables 1 and 3 confirm this. Among the three, IEG is the most easily oxidisable and is also a very effective antioxidant. However, its slow reaction with haloperoxyl radicals is still a puzzle and needs to be examined in detail. At pH 10 where the haloperoxyl radical reaction was studied, a considerable fraction of the phenolic form is present and hence a slow reaction is expected. The phenoxyl radicals are unreactive towards many molecules, however, they can be regenerated by electron transfer from other antioxidants like ascorbate and a-tocopherol.17b The high reduction potentials of DZ and BP may leave a doubt that they may help in propagating the chain reaction by oxidising the polyunsaturated fatty acids. This may also be one of the reasons for the reduced antioxidant efficiency of DZ as compared to isoeugenol. However, in absence of HPLC analysis, exact stoichiometry of the reaction of phenols with haloperoxyl radicals could not be given. The log P data in Table 3 suggests that isoeugenol is the most lipophilic of all except BP. This is one of the important characteristics of an antioxidant to make them good substitute for vitamin-E. Because of their ability to interact with lipids, lipophilic antioxidants offer greater protection against peroxidation. This may be contributing further to the better antioxidant properties of isoeugenol, (despite its slow reactivity with peroxyl radicals), since the log P data in table 3 shows that isoeugenol is the most lipophilic of all except BP. Acknowledgements—The authors express their sincere thanks to Drs J. P. Mittal, C. Gopinathan and T. Mukherjee for constant encouragement and support.

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