In vitro antioxidant actions of sulfur-containing amino acids

2.2.1 Ferric reducing antioxidant power (FRAP) assay

Ferric reducing antioxidant power (FRAP) assay was conducted using published method of Oyaizu (1986) with slight modifications. Briefly, different concentrations (1000, 2000, and 4000 ppm) of each amino acid were prepared in distilled water as stock solutions. One mL of the stock solution was mixed with 2.5 mL of sodium phosphate buffer (0.2 M at pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was placed at 50 °C for 20 min. After the incubation, 2.5 mL of 10% trichloroacetic acid was added to the mixture. The upper layer of the mixture was taken. It was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl₃. The absorbance value of the solution was measured at wavelength of 700 nm using a spectrophotometer.

2.3.1 Ferrous ions (Fe²⁺) chelating activity

The ferrous ion-chelating ability of sulfur compound was measured using the method of Dinis et al. (1994). Briefly, 0.4 mL of the stock solution of each sulfur amino acid (20 µg/mL) was mixed with 0.2 mL of 2 mM FeCl₂. Then 0.4 mL of ferrozine (5 mM) was added to the mixture to initiate the reaction. The reacted mixture was shaken for 10 min at room temperature. The absorbance of the mixture was measured at wavelength of 562 nm. The ferrous ion-chelating activity was calculated using the following equation: $$\text{Chelating ability}(\%)=\{1-({\text{A}}_{\text{sample 562 nm}}/{\text{A}}_{\text{control 562 nm}})\}\times 100$$ where A_{control 562 nm} was the absorbance of the control reaction containing distilled water instead of sample and A_{sample 562 nm} was the absorbance of the sample in the presence of sulfur-containing amino acid.

2.3.2 Copper (Cu²⁺) and zinc (Zn²⁺) chelating activity

Copper (Cu²⁺) and zinc (Zn²⁺) chelation activities was determined by the method of Asakura et al. (1990), the sulfur-containing amino acids were diluted into 20 µg/mL in distilled water. CuSO4 and ZnCl2 metal solutions were prepared at concentrations of 0.24 mM and 0.8 mM in the same buffer. One mL of the sample solution was mixed with 1 mL of metal solution followed by the addition of 0.1 mL of 1 mM tetramethylmurexide solution. Absorbance value was then recorded at A282/A530 for Cu²⁺ and A462/A530 for Zn²⁺ to obtain bound metal (%) using the following equation: $${\text{Bound metal}}^{2+}(\%)=(1-(\text{Concentration of free metal}/\text{Concentration of total metal}))\times 100$$

In order to calculate the percentage of metal chelating activity, standard curves of copper and zinc were prepared. The range of Cu²⁺ was from 0.025 to 0.125 mM/L. The range for Zn²⁺ was from 0.2 to 1.0 mM/L.

2.5.1 DPPH free radical scavenging activity

DPPH free radical scavenging activity assay was carried out using the method of Blois (1958) with slight modifications. Briefly, 0.5 mL of 0.1 mM DPPH in ethanol was added to 1.5 mL of sulfur compound at different concentrations in ethanol. This mixture was reacted in a dark room for 30 min. The absorbance values of samples were recorded at wavelength of 517 nm. A standard curve was prepared using different concentrations of DPPH (r²: 0.9985). The DPPH scavenging effect (%) was calculated using the following equation according to Gülcin et al. (2010). $$\text{DPPH scavenging effect}(\%)=\{1-({\text{A}}_{\text{sample 517 nm}}/{\text{A}}_{\text{control 517 nm}})\}\times 100$$ where A_{control 517nm} was the absorbance value of the control reaction containing distilled water instead of sample and A_{sample 517nm} was the absorbance value of the sample in the presence of sulfur-containing amino acid.

2.5.2 ABTS radical cation scavenging activity

The ABTS radical scavenging activity was analyzed using the method of Re et al. (1999). Briefly, 2 mM of ABTS in ethanol was reacted with 2.45 mM potassium persulfate (K₂S₂O₈) to generate ABTS⁺ in a dark room for 4 h. The ABTS⁺ solution was diluted to obtain an absorbance of 0.75 at wavelength of 734 nm by using 0.1 M sodium phosphate buffer (pH 7.4). One mL of diluted ABTS⁺ solution was then reacted with 3 mL of sulfur compound in distilled water for 30 min. ABTS⁺ scavenging effect (%) was calculated using the following equation: $${\text{ABTS}}^{+}\text{scavenging effect}(\%)=(1-{\text{A}}_{\text{sample 734 nm}}/{\text{A}}_{\text{control 734 nm}})\times 100$$ where A_{control 734nm} was the absorbance of the control reaction containing distilled water instead of the sample and A_{sample 734nm} was the absorbance value of the sample in the presence of sulfur-containing amino acid.

2.5.3 Superoxide anion radical scavenging activity

Superoxide anion radical scavenging activity was measured using the method of Li et al. (2006). Briefly, 0.2 mL of sulfur compound stock solution was mixed with 0.4 mL of 150 µM of nitro blue tetrazolium (NBT) in phosphate-buffered solution (100 mM, pH 7.4), 0.4 mL of 460 µM β-nicotinamide adenine dinucleotide (β-NADH), and 0.05 mL of 60 μM phenazine methosulphate (PMS). After mixing well, the mixture was then placed at room temperature. After standing for 5 min, the absorbance value of the mixture was recorded at wavelength of 560 nm. Superoxide anion radical scavenging activity (%) was calculated using the following formula: $$\text{Superoxide anion radical scavenging effect}(\%)=\{1-({\text{A}}_{\text{sample 560nm}}/{\text{A}}_{\text{control 560nm}})\}\times 100$$ where A_{control 560nm} was the absorbance value of the control reaction containing distilled water instead of the sample and A_{sample 560nm} was the absorbance value of the sample in the presence of sulfur-containing amino acid.

3.1.1 DPPH and ABTS radical scavenging assays

DPPH and ABTS free radical scavenging activities of cysteine were linearly increased with increasing concentrations. However, Met and Tau did not possess DPPH or ABTS free radical scavenging activity (Fig. 2). Kavlentis (1988) has reported that cysteine can inhibit the formation of DPPH complex whereas methionine does not affect the DPPH experiment. Ripoll et al. (2012) have shown the taurine does not have ABTS radical scavenging ability. The addition of taurine is unable to decrease ROS production or inhibit lipid peroxidation in glutathione-depleted cell culture (Heidari et al., 2014). In the present study, ascorbic acid has more powerful DPPH radical scavenging activity than cysteine at the same concentrations (50–400 µg/mL). At 400 µg/mL, cysteine and ascorbic acid showed 84.42% and 94.94% of DPPH radical scavenging activity, respectively. However, the ABTS radical scavenging activity of cysteine was significantly higher than that of ascorbic acid at concentrations from 10 to 100 µg/mL. For instance, cysteine and ascorbic acid at 100 µg/mL had 96.77% and 91.78% ABTS radical scavenging activities, respectively. In general, ascorbic acid is considered as a representative antioxidant that can remove water-soluble free radical. Ascorbic acid is converted into ascorbate radical when electron of ascorbate radical supplies lipid radical to terminate the oxidative chain reaction (Nimse and Pal, 2015). In general, DPPH assay is used for ethanol-soluble free radicals, whereas ABTS assay is used to measure water-soluble free radicals. Kaviarasan et al. (2007) have found that ABTS assay is more sensitive than DPPH assay since DPPH radical is only involved in hydrogen (H⁺) transfer [DPPH^• to DPPH-H] while ABTS radical is involved in the electron transfer pathway [ABTS^• to ABTS]. According to the radical scavenging activity of cysteine, the presence of proteins containing disulfide bonds (Cys-Cys) might potentially increase the antioxidant ability of food.

Close

Fig. 2

Radical scavenging activities of sulfur amino acids. (a) DPPH radical scavenging activity and (b) ABTS radical scavenging activity of different concentrations of sulfur-containing amino acids (cysteine, methionine and taurine) and ascorbic acid (reference antioxidant). Radical scavenging activities of methionine and taurine was not detected.

Export to PPT

3.1.2 Superoxide anions radical scavenging activity

The principle of superoxide anion radical scavenging activity assay has been explained by Gülcin et al. (2010). The addition of PMS will induce the reduction of NBT with the generation of superoxide radicals (O₂⁻) in PMS-NADH system. Blue formazan at 560 nm is then formed through the NBT reduction. Therefore, inhibition of blue formazan production can be expressed as a decrease in absorbance at 560 nm due to O₂⁻ scavenging. At the same concentration (1000 ppm), cysteine, taurine, methionine, and BHT exhibited 31.32, 22.27, 17.8, and 11.29% superoxide anion radical scavenging activities, respectively (Fig. 3). Cysteine showed the highest (p < .001) superoxide radical scavenging activity. The excellent superoxide radical scavenging activity of cysteine has been reported by Hussain et al. (1996). Likewise, methionine also possessed higher (p < .001) superoxide radical scavenging activity than BHT. It has been reported that methionine can be easily oxidized to methionine sulfoxide by some oxidants such as oxygen, ozone, hydrogen peroxide, and superoxide (Levine et al., 2000). In a previous study, the superoxide radical scavenging activity (the reduction of NBT to form the blue formazan at 560 nm) of taurine has been observed at concentrations from 30 mM to 60 mM (Oliveira et al., 2010). Kim and Cha (2009) have found that taurine can reduce oxidative damage by altering superoxides into taurine chloramines.

Close

Fig. 3

Effect of sulfur-containing amino acids on superoxide anion radical scavenging (%). Different letters within the same concentration indicate significant differences (p < .05).

Export to PPT