Effect of salt (zinc chloride) on antioxidant activity of gallic acid

The first 100s of reaction between gallic acid (GA) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical follows the second order reaction. The addition of zinc (Zn) significantly enhanced the rate of reaction, such that the rate constant when GA:Zn is 1:1 was significantly higher, (P<0.05), 120 ± 12 Ms as compared to 92 ± 6 Ms when zinc was absent. However, the addition of excess zinc, GA:Zn 1:2, decreased the second order rate constant to 75 ± 9 Ms Therefore, the presence of catalytic amount of zinc enhanced gallic acid scavenging properties, while excess zinc inhibited the rate of reaction. The mechanism by which GA scavenge for DPPH radical was probably that of a two step process, electron transfer, followed by proton transfer. INTRODUCTION Gallic acid, as well as its derivatives has important antioxidant activities by displaying radical scavenging abilities. They are polyphenols, which scavenge for radicals by donating hydrogen to radical, while itself oxidizes to a quinone, which is stable. Other than stabilizing radicals, which are products of cell metabolism, phenolic compounds can also combine with copper and iron metal ions, which can induce the radical production via Fenton reaction. Hynes et al. (2001) showed that complexation between ferric ions and gallic acid can result in the oxidation of gallic acid to hydroxyquinone and reduction of ferric ions to ferrous ions. However, some transition metal salts render protective effect to cells against harmful radicals. In a recent experiment, Kagay et al. (2002) demonstrated that zinc increased the hepatoprotectivity of epigallocatechin gallate in isolated rat hepatocytes. Zinc is an important metal, which is present in human bodies in large quantities, approximately 3 g in a 70 kg human and it is found in all six classes of enzymes important for physiological activities (Dunn, 1995). Zinc exhibits both hard and soft acid properties and is able to bind favourably to oxygen, nitrogen and sulphur compounds. For example, zinc plays a stabilizing role in metallothioneins by forming zinc-sulphur clusters. Other than structural role, zinc has a regulatory function in the body. Zinc is the active metal centre in the enzyme alcohol dehydrogenase and the primary function of the enzyme is to catalyse detoxification of alcohol to acetaldehyde. Therefore, based on these known structural, as well as catalytic function of zinc, we aim to determine if zinc exhibits a similar effect on the reaction between GA and DPPH radical. The reaction between GA and DPPH can be interpreted as the oxidation of a phenol to a quinone, which is analogous to the oxidation of alcohol to a carbonyl, thus a similar catalytic mechanism by zinc could thus take place. From this experiment, we also aim to elucidate if gallic acid donate a hydrogen atom to DPPH radical via a direct hydrogen transfer mechanism, or via an electron transfer, followed by proton transfer mechanism. 1 Student 2 Senior Lecturer MATERIALS AND METHODS Materials 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 1,1-diphenyl-2-picrylhydrazine (DPPH2) were purchased from Sigma. Gallic acid was obtained from Acros Organics. Zinc chloride, >98% was purchased from Fluka and methanol of HPLC grade was obtained from Merck. A stock solution of 2x10 M DPPH was prepared freshly. Gallic acid (GA) and zinc chloride (Zn) was made up to 0.06 M and diluted with methanol to make up the required ratio of GA and Zn mixture. Spectral and kinetics measurements UV-1601 spectrophotometer was used in the kinetic and spectral measurements. CPS-240A was used to set the cell temperature at 10C. Spectral scan of DPPH, DPPH2, GA and Zn mixture were carried out from 280-800 nm. A calibration curve for DPPH was made from 1x10 5 to 30x10 M at 515 nm. Thus the concentration of DPPH, CDPPH is Abs515 = 12443 x (CDPPH) – 0.0746 determined by linear regression, where, ε = 12443 M cm. Disposable cuvettes were filled with 3 ml of DPPH solution and placed in the spectrophotometer for 10 minutes at 10C. After that, 10 μl of 0.012 M GA was added to make up a GA:DPPH 1:5 ratio. The rate of reaction was followed at Abs515 for 1200s and a plateau has not reached. A mixture of GA and Zn was prepared according to the ratio 1:0.5, 1:1 and 1:2, and five repeats was carried out. The rate constant for the reaction was calculated based on the first and second order rate law given by Atkins (2000). Microsoft Excel 2000 was used to calculate the rate constant. Statistics Results were presented as means of rate constants ± SE. Student’s t-test was used to compare differences between means of rate constants. Differences with p < 0.1 were regarded as statistically significant. RESULTS When different concentration of Zn was added to GA in methanol, there was only slight hypsochromic shift, approximately 2 nm for GA+1Zn. GA+2Zn displayed the greatest drop in absorbance from 290 to 320 nm, as compared to the rest of the GA and Zn mixture. When GA and Zn are added to 2x10 M DPPH at 10C, both Abs515 and Abs324 changes with time. However, Zn alone did not cause a change (Fig 1). The various time dependent spectra for all three reaction conditions do not intercept with the initial DPPH spectrum at a common isosbestic point. However, these spectra have common isosbestic points with other spectra, such that they can be approximately grouped into 4,6 and 5 sets with common isosbestic points. It is evident from the presence of different isosbestic points in the course of reaction that there are different absorbing species, inclusive of DPPH, present in the reaction mixture.