Effects of Different Functional Group on Antioxidant Activity

Transcription

1 Effects of Different Functional Group on Antioxidant Activity Kwek S.Q. 1 and Leong L. P. 2 and Bettens R. 3 Department of Chemistry, Faculty of Science, National University of Singapore 10 Kent Ridge Road, Singapore ABSTRACT Diphenylpicrylhydrazyl (DPPH) is widely used for quickly accessing the ability of polyphenols to transfer labile H atoms to radicals, a likely mechanism of antioxidant protection. The rate constants of H abstraction by DPPH of 4 different 3,4-dihydroxy substituted antioxidants were determined from the kinetic analysis of the decay of the DPPH visible band following the addition of the antioxidants. The 4 antioxidants showed different kinetic rates. The reactions of methylcatehol reached steady state at around 100 seconds while 3, 4-dihydroxybenzoic acid took about 4000 seconds. The kinetic rate constant was determined for each reaction by using curve fitting method into the second order rate equation. It was found that rate constant is very sensitive to changes in DPPH/Antioxidant molar ratio. The results were compared with the Bond Dissociation Energy (BDE) of the antioxidants. BDE varies inversely with the rate constant with the antioxidants except 3,4-dihydroxybenzaldehyde. The unexpected results might be due to the solvent (methanol) effect. INTRODUCTION Antioxidants are often used to delay auto-oxidation in food lipids, a process responsible for off-flavours and formation of constituents that might pose a potential health risk. Lipid autoxidation is a free radical chain reaction and could be described in terms of initiation, propagation and termination processes. According to the mode of action, antioxidants may be classified as free radical terminators, chelators of metal ions capable of catalysing lipid oxidation, or as oxygen scavengers that react with oxygen in closed system. For this reason it is of interest to assess antioxidant activity of specific antioxidant. It is used to indicate the ability if the antioxidant to scavenge some radicals. One of the tests proposed for the assessment of antioxidant activity is a free radical colorimetry that relies on the reaction (DPPH + AH DPPH-H + A ) of specific antioxidant (AH) with a stable free radical 2,2-diphenyl-1-picryl-hydrazyl DPPH dissolved in methanol. As a result of reduction of DPPH by the antioxidant, the optical absorbance at 515nm of this purple colored solution of DPPH in MeOH decreases. This change is detected by means of a UV spectrophotometer. (Brand-Williams et al., 1995). 1 Student 2 Supervisor 3 Supervisor

2 In this experiment, the effects of functional groups on activity of the diphenolic antioxidants were observed. Bond Dissociation Energy (BDE) is the amount of energy needed to break a given bond to produce two fragments (in this case AO-H AO + H). BDE is an important factor in determining the efficacy of an antioxidant since the weaker the OH bond, the faster will be the reaction with the free radicals. According to previous studies, the addition of electron-donating substituents to an aromatic ring can increase radical scavenging activity as a result of increased electron density at carbon atoms in the ring. In contrast, the presence of electron-withdrawing substituents decreases electron density around the ring, hence decreasing its ability to scavenge free radicals due to poor aromatic ring resonance of the phenoxyl radical. However, it must be emphasized that the stoichiometric value does not explain the antioxidant efficiency (kinetically). An antioxidant (e.g. butylated-hydroxy-toluene) can reduces about 3 moles of DPPH even though it reacts slowly. Conversely, another antioxidant (e.g. isoeugenol) can reduce only 1 mole of DPPH but with a much higher rate. (V.Bondet et al., 1997). MATERIALS AND METHODS Reagents Antioxidants studied here included 3,4-dihydroxybenzoic acid (MW=154.12g), 3,4- dihydroxybenzaldedye (M.W. =138.12g), catechol (M.W.=110.11g) and methylcatechol (M.W.=124.14g). Methanol which was used as a solvent was of spectrophotometric grade. 2,2-diphenyl-1-picrylhydrazyl (DPPH ) was used as the free radical. All reagents used were purchased from Aldrich except catechol which is from BHD and methanol from MERCK. Spectrophotometric measurements The H-transfer reactions from an antioxidant to DPPH radical were monitored using UV visible spectrophotometer 1601 (Shimadzu). Glass cuvettes (1 x 1 x 4.5cm) were used for visible absorbance measurement. The temperature in the cell chamber was kept at 15 C by means of a temperature control. DPPH and Sample Preparations 1 x 10-3 M of freshly prepared solution of DPPH in methanol was diluted about 50 times to get the absorbance readings from 1.90 to A stock solution of x 10-2 M of antioxidant was freshly prepared, and diluted in 25mL of volumetric flasks to get three other different concentrations. Procedure To 3mL of a freshly prepared solution of DPPH in methanol, 40µL of a freshly prepared solution of the antioxidant in the same solvent was added. Spectra were recorded every second until the absorbance readings reached a plateau (steady state) for the determination of rate constants and stoichiometries. Each run was done in duplicates. 4 different molar ratio (DPPH/AH) ranging from 1 to 7 of a given antioxidant were used.

3 Data Analysis The stoichiometry, n, for each antioxidant was determined from the following equation. n = (A o A f ) / εc where A o = initial absorbance, A f = final absorbance, ε = molar absorption coefficient = =12,509 M -1 cm -1, c= initial concentration of antioxidant The curve fittings of the concentrations of DPPH at different times versus time plots were carried out using a simple second order reaction equation with Mircosoft Excel. Curve fittings were achieved through least square methods and yielded optimized values for kinetic rate constant (for the fast step) for the 4 different antioxidants. The rate constants for molar ratio (DPPH/AH) =7 were used as a basis of comparison with the respective Bond Dissociation Energy (BDE) (Foo Sook Yee et al., 2003)to make sure that the H-donating ability of the antioxidant were exhausted. RESULTS AND DATA ANALYSIS The H-transfer reactions from the catechols to DPPH were monitored by UV spectroscopy by recording the decay of the DPPH visible absorbance (λ max = 515nm in MeOH) that follows the addition of the antioxidant to the DPPH solution. With the 4 antioxidants, the visible absorbance quickly decayed over at most for 500 seconds as a result of the transfer of the most labile H atoms of the antioxidants (fast step). This step was followed by a much slower decrease of the visible absorbance. This might most probably be due to the residual H-donating ability of the antioxidant degradation products (slow step). Only the fast step was subjected to the kinetics analysis. Kinetic Model Different kinetic models were used for analyzing the H-atom transfer reaction between DPPH and the antioxidant during the fast step. The simplest model makes no hypothesis about the mechanism of antioxidant degradation. An antioxidant of stoichiometry n is simply regarded as n independent antioxidant subunits, AH, which transfers a single H atom to DPPH with the same second order rate constant. Hence, equation 1 can be used in the curve fitting of the concentration vs. time plots. d [DPPH] / d t = -k [DPPH] [AH] Integrating k t = {1/([AH] 0 -[DPPH] 0 ) }x Ln { [AH]/ [AH] 0 x [DPPH]/ [DPPH] 0 } Assuming [AH] = [AH] 0 -( [DPPH] 0 - [DPPH] ) Then, [DPPH] = {[AH] 0 [DPPH] 0 - [DPPH] 2 0 } / [AH] 0 exp{([ah] 0 -[DPPH] 0 )k t}- [DPPH] 0 Equation 1 Here, the curve fitting using this model were carried out with k as the floating parameter. The optimized value was determined by using the least square method.

4 Rate Constant/ M-1 s DPPH/AH Figure 1 Rate Constant vs DPPH/AH for methylcatechol BDE kj/mol Series1 rate constant/m-1s-1 3,4-dihydroxybenzaldehyde Figure 2 BDE vs second order rate constant for the 4 antioxidants R OH OH Stiochiometric Study Plateau time(s) Stoichiometry, n R COOH COH H CH ± ± ± ± 0.14 Kinetic Study DPPH/AH Molar Ratio ± 4 37 ± 3 65 ± ± ± 1 44 ± 1 55 ± ± ± ± ± ± ± ± ± ± 48 Bond Dissociation Energy/kJmol -1 From figure 1, it is shown that the rate constant turned out to be rather sensitive to DPPH/AH ratio. From the stoichiometric study, it was shown that 3,4-dihydroxy substituted ring typically give a n value of close to 2, in agreement with the stepwise formation of semiquinone radicals and quinones. However, 3,4-dihydroxybenzaldehdye give relatively higher stiochiometry. It might be due to the regeneration of catechol nucleus during the slow steps. Hence subsequent H abstractions by DPPH become possible. For the kinetic study, from figure 2, BDE varies inversely with the second order rate constant. Therefore, it can be concluded that the stability of the aryloxyl radical increases according to the reactivity of the substiuent groups. In other words, as the substituents possess stronger electron-donating properties, the kinetics of the reaction with DPPH

5 increases. Absence of electron donating group gives poor aromatic ring resonance of the aryloxyl radical and thus lowers the antiradical efficiency considerably. However the behavior of 3,4-dihydroxybenzaldehdye deviates from the trend. It has higher BDE value compared to 3,4-dihydroxybenzoic acid although the latter has a higher electron-withdrawing effect on the aromatic ring. This effect might be due to aldehyde s greater affinity to form hydrogen bonding with methanol, thus stabilizing its structure (Jos P. M. Lommerse,et al., 1996). The higher BDE value has led to the slow transfer of the H atoms to DPPH. That might explain why both antioxidants have comparable second order rate constant at the fast step. DISSCUSSION The results can be much further improved upon by use of stopped-flow experiments. In this method, commercially available apparatus is used to mix two solutions in 1 millisecond using an appropriately designed mixing jet. After mixing, the solution flows into an observation chamber, and the flow stops. At this point, data collection (usually an optical signal such as light absorption or fluorescence) is collected in a time-resolved manner. This has enhanced the possibility of making measurements from reactions on the millisecond timescale. Special mixing techniques, however, are required to ensure the sample reaches the excitation beam before the reaction has taken place. In the case of polyphenols having a single 1,2-dihydroxy substitution, the semiquinone radicals formed were assumed to form quinone via direct H transfer from the semiquinone radical to DPPH. However, this reaction may take place in competition with semiquinone disproportionation. This would complicate the rate law used for curvefitting. Thus, better kinetic models should be used. In principal, k values could be directly estimated under pseudo-first order conditions where concentration of antioxidant is about ten times that of the DPPH radicals. However, under such conditions, the decay of DPPH absorption band becomes too fast to be accurately monitored by conventional UV-visible spectroscopy. CONCLUSION Phenolic compounds can act as free radical scavengers by virtue of their hydrogendonating ability, forming phenoxyl radicals. The stabilization of such radicals by other functional groups in the structure enhances the antioxidant activity. This experiment had shown that para substitution with electron donor (alkyl) groups increases the stability of the aryloxyl radical and thus the antioxidant activity. Substitutions with electron withdrawing groups (COOH) have the opposite effect. However, the effect of solvent should not be overlooked. It has its importance in determining the rate constants and BDE values of antioxidants. REFERENCES Brand-Williams W, Cuvelier M E, Berset C 1995 Use of a free radical method to evaluate antioxidant activity. L ebensm Wiss T echnol 28 (1) 25È30. V. Bondet, Brand-Williams W, Berset C 1997 Kinetics and mechanisms of antioxidant activity using DPPH free radical method. L ebensm Wiss T echnol 28 (1) 25È30. Jos P. M. Lommerse, Sarah L. Price, Robin Taylor 1996 Hydrogen Bonding of Carbonyl, Ether, and Ester Oxygen Atoms with Alkanol Hydroxyl Groups Foo Sook Yee, 2003 Effects of electronegativity substituents on antioxidant activity. A computational study.