ELSEVIER Pharmaceutics Acta Helvetiae 7 I (1996) 22 I -224

Hydroxyl radical scavenging by ethyl gallate and related compounds: a method for rapid evaluation

G. Hall, T.T.T. Le, J.B. Stanford, J.K. Sugden *

Department of Pharmaceutical Sciences, School of Applied Scirnces, De Mowfort (/nicer.+?, The Gatewu~, Leicestrr LEl 9RH, l/K

Received 5 August 1995: accepted 13 September 1995

Abstract

Ethyl gallate and two related compounds, ethyl 4-hydroxybenzoate and ethyl 3,4-dihydroxybenzoate were evaluated as hydroxyl radical scavengers in a model system using dequalinium chloride and hydrogen peroxide irradiated with simulated sunlight to generate the radicals. All of the compounds showed some hydroxyl radical scavenging properties, with ethyl 3,4-dihydroxybenzoate being the most active under the conditions of test. The molecular implications of these results are considered. The potential radical scavengers were assessed individually for their reaction with photogenerated hydroxyl radicals and the results are discussed. The potential applications of this method for evaluating potential preservatives are also discussed.

Kewordst Hydroxyl radical: Hydroxybenaoate; Scavenger: Photogenerated

1. Introduction

Medicines, cosmetics and foods are all subject to oxida- tion, leading to the formation of unpleasant flavours, odours and the formation of toxic substances all of which consti-

tute product spoilage. It is normal good manufacturing practice to incorporate antioxidants to reduce the instance of these spoilage reactions (Scott, 1965; Stuckey, 1972).

Antioxidants can be classified as either reducing agents or free radical scavengers. In the latter case, the antioxidant acts as a chain breaking agent in the three stage process of

initiation, propagation and termination in which products are oxidised by a free radical reaction (Chamsi, 1986; Kirby, 1988). Antioxidants react with free radicals to form stable products which prevent the reaction progressing, a primary stage in the free radical mediated decomposition of fatty material is either electron or hydrogen donation

and that the antioxidant forms a loose complex with the

I Corresponding author.

fatty chain. Bollard and Ten Have (1947) reported that the reaction between hydroquinone and ethyl linoleate was a two stage process involving the formation of a semi- quinone radical, two of which combined to give quinone

and hydroquinone, thus acting as an antioxidant. The antioxidant effects of ring substituted phenols in

food oils have been investigated (Wu et al., 1993; Namiki

et al., 1993) leading to the conclusion that ring substitution with bulky alkyl groups yields products which are oil soluble, less volatile and toxic as well as being reasonably

effective antioxidants. Recent work has shown that pheno- lit acids are relatively ineffective at retarding the rates of

autoxidation at 100°C (Marinova and Yanishlieva, 1994). The objective of the present work is to extend the

investigation of ring substituted phenols to encompass aqueous systems with particular reference to hydroxyl radicals which can be generated in aqueous systems, par- ticularly in the presence of substances, which can act as

sensitizers. Hydroxyl radicals can also be generated in aqueous solutions in which there are transition metal ions, derived from manufacturing equipment. that could cause

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222 G. Hall et al. /Pharmaceutics Acta Helvetiae 71 (1996) 221-224

Fenton reactions (Fenton, 1894), which lead to product spoilage. Previous workers (Pate1 and Sugden, 1992; Ho et al., 1994; Allman et al., 1995) have shown that dequalin- ium chloride in the presence of hydrogen peroxide is decomposed in visible light by a hydroxyl radical mediated reaction. This reaction has been used as a model system in which to determine the effect of potential hydroxyl radical scavengers by measuring the rate constant of this reaction with and without the test compounds.

2. Procedures

2.1. Chemicals

Dequalinium chloride (Sigma Chemical Co., batch no.

124F06081, ethyl gallate (NIPA Co., batch no. 8001, ethyl 3,4_dihydroxybenzoate (Aldrich Chemical Co., batch no 5063), ethyl 4-hydroxybenzoate (Aldrich Chemical Co., batch no 44703) hydrogen peroxide (Aldrich Chemical Co., batch no. 28141). Ethyl N-methyl-4-hydroxy-5-oxo-

3-pyrroline-3-carboxylate (De Montfort University, Leices- ter).

Evans et al. (1975) and absorbance readings were taken at 326 nm at time zero and then every fifteen minutes for a further 2 h to measure the residual dequalinium chloride. The irradiated control solutions were used as the blank in the spectrophotometric assay of the residual dequalinium chloride. Another series of solutions of the hydroxyben- zoates was made in which each compound (30 mg) was dissolved in deionised water and made up to 250 ml. Aliquots of 220 ml were taken and hydrogen peroxide (1 ml, 30% v/v> added. A 1 in 10 dilution of these solutions was made and the diluted solutions irradiated as described

above; uv/visible scans being taken at time zero and at hourly intervals up to 7 h. Attempts to repeat this work

with more concentrated solutions were unsuccessful since the rate of reaction was too slow to be measured in reasonable time.

2.5. Treatment of results

2.2. Apparatus

Photoreactor Unit (Evans et al., 1975), Cecil Uv/visi- ble spectrophotometer Model CE 272, Beckman Uv/visi-

ble spectrophotometer Model DU 70.

Linear regression analyses were made of plots of the percentage residual dequalinium chloride, the log of the

percentage residual dequalinium chloride and the recipro- cal of the percentage residual dequalinium chloride against time. The plot giving the largest linear regression coeffi-

cient was deemed to represent the order of reaction (Pate1

and Sugden, 1992). The rate constants were calculated from the slopes of the graphs (Florence and Attwood,

1981).

2.3. Beer Lambert plot of dequalinium chloride 3. Results and discussion

A stock solution of dequalinium chloride (50 mg/lOO ml) was prepared in deionised water. Serial dilutions for

this solution were prepared (5, 10, 20, 30, 40, 50 mg/lOO ml> and the absorbance measured at 326 nm. Linear re- gression analysis of the mean of two sets of readings gave

a regression coefficient of 0.9994 (p = 0.0001).

The results of the irradiation of the solutions of de- qualinium chloride with the various substituted phenols are shown in Table 1.

Irradiation of the additives alone in deionised water gave no evidence of photochemical degradation.

2.4. Preparation and irradiation of solutions

Solutions of dequalinium chloride in deionised water (O.O25%w/v) were prepared and filled into Dreschel bot- tles (300 ml>. A second series of solutions was prepared and hydrogen peroxide (0.03% v/v> added before the Dreschel bottle was made up to volume. Separate solutions of dequalinium chloride (0.025% w/v> in deionised water were prepared containing selected amounts of the test compounds with respect to dequalinium chloride and hy- drogen peroxide (0.03% v/v>. A separate series of con- trols were prepared in which the dequalinium chloride was omitted. All the solutions were irradiated by the method of

Ethyl N-methyl-4-hydroxy-5-oxo-3-pyrroline-3-

carboxylate was used since it has a planar structure and an acidic hydroxy group like those of phenols. The evidence from the use of ethyl N-methyl-4-hydroxy-5-oxo-3-pyrro- line-3-carboxylate shows that this compound does not act as a hydroxyl radical scavenger which indicates that the presence of an aromatic ring rather than a n cloud of electrons in C=C and C =0 bonds incorporated in a ring system is essential for hydroxyl radical scavenging in the

test system. Examination of the results (Table 1) shows that of the

hydroxybenzoates tested ethyl 3,4_dihydroxybenzoate has the greatest effect in scavenging hydroxyl radicals, ethyl gallate (ethyl 3,4,5_trihydroxybenzoate) is less active which suggests that the hydroxyl radical scavenging effect is not

G. Hall et al. / Pharmaceutics Acta Helvetiae 71 (1996) 221-224 223

solely related to the number of hydroxyl groups on the

aromatic ring but also to their position with respect to the ester group

Raghaven and Steenken (1980) have reported that phe- nols are usually attacked by hydroxy radicals in the 2 and 4 positions in acid or neutral solutions (deionised water

used in this work had a pH of 6.1) to give a dihydroxy compound. Consideration of ethyl 4-hydroxybenzoate shows that hydroxylation can occur at C3 and C5 which

are activated by the phenolic hydroxy group. In the case of ethyl 3,4_dihydroxybenzoate (II) the 3-hydroxy group will activate C2 and C6, whilst the 4-hydroxy group will activate C5. In the case of ethyl gallate (III) only C2 and

C6 are available for hydroxylation and both of these are subject to steric hindrance from the ester group at Cl and also deactivation due to the ester carbonyl group. Conse- quently, some explanation of the greater hydroxyl scaveng- ing properties of (II) can be offered from consideration of

the availability of accessible hydroxylation sites. Examination of Table 2 shows that on irradiation with

hydrogen peroxide in deionised water the ethyl hydroxy- benzoates decompose, the reactions following first order

kinetics. The rate constants show that ethyl gallate decom- poses at the greatest rate, followed by that of ethyl 4-hy- droxybenzoate and the slowest reaction being that of ethyl

Table I Photodegradation of dequalinium chloride (DC) (0.025% w/v)

Table 2

Photodegradation of ethyl hydroxybenzoates (O.O012%‘c) + hydrogen per-

oxide

Compounds Reaction order Rate constant min-’

Ethyl 4-hydroxybenzoate 1st 4.82~ lo-’

Ethyl 3,4-dihydroxybenzoate 1st 2.12x lo-?

Ethyl gallate 1st 7.42~ lo-’

3,4-dihydroxybenzoate. Ethyl 4-hydroxybenzoate has been shown to be the least effective hydroxyl radical scavenger of those tested. The relative positions of ethyl 3,4-dihy- droxybenzoates and ethyl gallate do not relate precisely with the results from the tests on the model system with dequalinium chloride, which could be due to a number of

factors, including insensitivity of the analytical method. However, the uv/visible spectra of the mixtures showed considerable changes from those of the pure compounds in deionised water. In the case of ethyl 4-hydroxybenzoate

there were shifts in the primary aromatic peak from 216 nm to below 200 nm and in the wavelength maxima of the

second absorption band from 256 to 255 nm. The spectrum of ethyl 3,4_dihydroxybenzoate showed slight shifts in the primary aromatic band and in the wavelength maximum of

the other absorption band from 260 nm to 255 nm. The spectrum of ethyl gallate showed a slight shift of the primary aromatic band and in every case these changes in

wavelength were accompanied by increases in absorbance. During irradiation with simulated sunlight all of the ab-

sorption bands showed a progressive reduction in intensity. Raghaven and Steenken (1980) have shown that hydroxyl

radicals convert phenols to the corresponding phenoxy radical which has increased absorption in the uv/visible

spectrum. The phenoxy radical undergoes further reactions leading ultimately to dihydroxybenzenes. This would sug- gest that the rates of the reaction between the hydroxyben-

zoates and hydroxyl radicals would need to be faster than

Compounds Reaction order Rate constant min-’ d

DC alone

DC+H,O,

DC + H;O; + ethyl gallate (0.025% w/v)

DC+ H,O? +ethyl gallate (0.05% w/v)

DC + H,O, + ethyl 3,4_dihydroxybenzoate (0.025% w/v)

DC + H,O, +ethyl 3,4_dihydroxybenzoate (0.05% w/v)

DC + H;Oi + ethyl 4-hydroxybenzoate (0.025% w/v)

DC + HZOZ + ethyl 4-hydroxybenzoate (0.05% w/v)

DC + Hz02 + ethyl N-methyl-4-hydroxy-5-oxo-3-pyrroline-3-carboxylate (0.025% w/v)

DC + H202 + ethyl N-methy1-4-hydroxy-5-oxo-3-pyrroline-3-carboxylate (0.05% w/v)

li Mean of three sets of results

I St 1 St

1st

1st

I St

1st

1st

1st

1st

1 St

0.32~ IO-’

2.390x IO_’

0.392~ IO-’

0.191 x IO_ 1

0.186X IO_ J

0.160~ lo-’

0.560~ IO-’

0.520~ lo-’

1.48X lo-?

1.07x IF2 -

224 G. Hall et al. / Pharmaceutics Acta Heluetiae 71 (1996) 221-224

that between the dequalinium chloride and hydroxyl radi- cals for the hydroxybenzoates to act as antioxidants. The hydroxyl radical scavenging properties of ethyl gallate are in the system tested inferior to those of ethyl 3,4-dihy-

droxybenzoate but they show a much greater concentration

effect. In the cases of ethyl 3,4_dihydroxybenzoate and ethyl 4-hydroxybenzoate the effect of doubling the concen- tration is not nearly so marked as it is with ethyl gallate. The main advantage of using this technique is that results can be obtained within a working day and it can be used to

assess a number of potential antioxidants in an aqueous system using the same drugs and additives as are intended for the product formulation with consequent cost savings.

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