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Laccase-Aided Antioxidant Activity Assay and Antioxidant Activity of Selected Thai Vegetables



S. Khammuang and R. Sarnthima
 
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ABSTRACT

This study aimed to develop the enzymatic preparation of ABTS•+ radical cation using laccase from Trametes versicolor. The result revealed that ABTS•+ preparation with the aid of the enzyme was reasonable easy, quick and high yield. The radical yields were controlled by concentration of ABTS substrate and enzyme amount. These ABTS•+ radicals were used for antioxidant activity assay of 31 Thai vegetables. The dried sample powders extracted by ethyl alcohol showed as high TEAC values as in Spondias pinnata, Eugenia grata and Polygonum odoratum. TEAC values determined using ABTS•+ and DPPH radical decolourisation methods were strongly consistency and correlate with total phenolic content in the samples. The quick and easy of the method as well as stability of the radical and reliable of the antioxidant capacity including environmental friendly suggest it to be a potential method for a total antioxidant activity measurement in biological samples.

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  How to cite this article:

S. Khammuang and R. Sarnthima, 2008. Laccase-Aided Antioxidant Activity Assay and Antioxidant Activity of Selected Thai Vegetables. Journal of Applied Sciences, 8: 2718-2724.

DOI: 10.3923/jas.2008.2718.2724

URL: https://scialert.net/abstract/?doi=jas.2008.2718.2724
 

INTRODUCTION

Fungal laccases (p-diphenol: dioxygen oxidoreductases, EC 1.10.3.2) are oxidoreductases which contain four Cu-atoms in an active centre. These enzymes catalyze the four-electron reduction of dioxygen to water concomitantly through the oxidation of substrate molecules (Baldrian, 2006; Couto and Toca-Herrera, 2007). Laccases have broad substrate specificity, ranging from various phenolic compounds to aromatic compounds. Laccases are industrially interesting enzymes with several application potentials in detergents, pulp bleaching, adhesives, fibre functionalization, detoxification, denim bleaching, textile dye decolourisation and baking (Couto and Toca-Herrera, 2006) as well as biosensors (Shleev et al., 2006) and biofuel cells (Chen et al., 2001; Zheng et al., 2008).

Antioxidants have attracted considerable attention over the past two decades, because of their great potential for combating Reactive Oxidative Species (ROS) that is ubiquitous in nature. In current scientific literature, antioxidant is among the most frequently occurring terms. One can find numerous papers associated with antioxidants. There are several of the commonly used methods for in vitro determination of antioxidant capacity. Those methods based on biological oxidants or specific ROS including peroxyl radical (ROO), superoxide radical anion (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (HO-), hypochlorous acid (HOCl) and singlet oxygen (1O2). Another method based on non-biological assays including scavenging of 2,2-azinobis-(3-ethyl benzothiazoline-6-sulphonate) (ABTS) radical cation (TEAC assay), scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH assay), ferric reducing antioxidant power (FRAP assay), Folin-Ciocalteu reducing capacity (FC assay), electrochemical total reducing capacity (Magalhaes et al., 2008). One most popular method for antioxidant screening and assay is TEAC assay. In this method, ABTS•+ radical cation is reduced by antioxidants, causing absorbance decrease at 414 or 734 nm, which first developed by Miller et al. (1993). It was based on the activation of metmyoglobin, acting as peroxidase, with H2O2 to generate ferrylmyoglobin radical, which then reacted with ABTS to form the ABTS•+ radical cation. In that method, the tested sample is added before the ABTS•+ generation and the sample having antioxidant activity is one that reduce or inhibit the ABTS•+ radicals formation. The method cost is still very expensive and has a major disadvantage as some antioxidants can react with H2O2 and/or with derivated oxidizing species that inhibit the ABTS•+ radical formation (Re et al., 1999). To prevent the interference of antioxidant compounds with radical formation, a post-addition assay or decolourisation approach was proposed.

ABTS•+ radical normally chemically generated by an oxidizing agent, potassium persulfate (Re et al., 1999) or by enzymatic reaction using metmyoglobin (Miller et al. (1993) or horseradish peroxidase (Cano et al., 1998). In this study, we interested to generate ABTS•+ radical cation by another oxidoreductase enzyme namely laccase. Laccase study is being conducted in our laboratory a few years ago. We accidental triggered that this enzyme might be able to use for antioxidant determination. When laccases catalyze the oxidation of its substrates, corresponding free radicals are generated as a product. Substrate, such as ABTS is normally used for laccase activity determination and ABTS•+ radical could be observed. Therefore, in this research we aimed to generate ABTS•+ radical cation by laccase from Trametes versicolor and assess the use of the radical for determination of total antioxidant activity of selected thai vegetables compared with another common method,DPPH radical decolourisation. Free radicals generation with enzymatic reactions is considerably more environmental friendly.

MATERIALS AND METHODS

Chemicals: Glacial acetic acid, HCl, sodium acetate, sodium carbonate, sodium citrate and Folin-ciocalteu reagent were purchased from BDH, England. ABTS was obtained from Sigma, Germany. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Aldrich, Germany. Gallic acid was a product of Fluka, Switzerland.

Laccase activity assay: The laccase from Trametes versicolor was purchased from Fluka, Germany (specific activity 0.53 U mg-1) and was used without further purification. The enzyme assay was based on the oxidation of ABTS as previous described (Khammuang and Sarnthima, 2007).

ABTS•+ radical preparation by laccase: The ABTS•+ cation radical solution was prepared by incubating 1.0 mM ABTS with 0.02 U mL-1 laccase (Trametes versicolor) in distilled water or 0.1 M sodium acetate buffer (pH 4.5) at room temperature (approximately 25°C) for 10 min. The radical solution (about 4 absorbance units at 734 nm) was filtered through a Nanosep 10 K Omega (PALL Life Sciences) to remove the enzyme. The ABTS•+ radical filtrate was diluted with distilled water or ethyl alcohol to the initial absorbance about 0.7 and studied for its stabilization or using for antioxidant activity determination.

Preparation of standard curve of trolox for TEAC assay:

Trolox, a water-soluble form of vitamin E was prepared as a 2.0 mM stock solution. The standard curve was performed using various amounts of Trolox (0-0.2 μmole) reacted with 1 mL of ABTS•+ radical at room temperature. The decrease in absorbance at 734 nm was monitored for 2 min by a UV-visible spectrophotometer (Perkin Elmer, Lamda25, USA). The inhibition percentage of the radical was calculated according to the equation (Eq. 1).

(1)

where, ODi is initial absorbance of ABTS•+ radicals and ODf is a final absorbance after 2 min of incubation. The standard curve was a plot of inhibition percentages and micromole of Trolox.

Preparation of standard curve of gallic acid for total phenol content determination: Gallic acid at various concentrations (0-0.01 mg) was used as a standard phenolic compound for total phenolic content determination. The experiments were performed according to the adapted method of Singleton and Rossi (1965).

Antioxidant activity analysis of selected Thai vegetable samples: The various fresh vegetables were obtained from local markets in Maha Sarakham province during July 2007 to December 2007. The samples were washed by tap water, rinsed with distilled water, left to shred out of water at room temperature. Some of each sample was left air-dried. The extraction of dried samples was followed by the method of Kulys and Bratkovskaja (2007). After filtration through sheet cloth and centrifugation, the ethyl alcohol extract was subjected to antioxidant activity assay. All experiments were performed in the Protein and Enzyme Technology Research Unit, Faculty of Science, Mahasarakham University.

RESULTS AND DISCUSSION

ABTS•+ radical preparation: The objective of this study was to generate ABTS•+ radical by the oxidation reaction of ABTS by laccase from T. versicolor comparison to those prepared by potassium persulfate. The results showed that the ABTS-+ radical prepared by the latter was very much lower in absorbance unit when reaction time and same starting concentration of ABTS compared with those prepared with the aid of laccase. The absorbance spectrum from both reactions was similar as shown in Fig. 1. ABTS-+ radicals could be generated by the enzyme concentration manner as shown in Fig. 2. We could use the enzyme as low activity (0.02 U mL-1) and the reaction finished within 10 min at room temperature. The spectrum of ABTS-+ radical cation prepared by laccase in our study was unsurprisingly similar to those generated by potassium persulfate from Re et al. (1999).

Fig. 1:
ABTS•+radical cation spectrum after reaction was reached to 16 h comparison between prepared with the aids of Tramates versicolor laccase and with potassium persulfate (4.9 mM) at room temperature (approximately 25°C). ABTS final concentration in both reactions was 1.0 mM. Laccase used in the reaction was 0.02 U mL-1 and was diluted 10 times with distilled water before scanning. Reaction prepared by potassium persulfate was non-diluted

Fig. 2:
Influence of enzyme concentration on ABTS radical formation. Reactions were prepared using distilled water in a quart cuvette at room temperature (~ 25°C). Concentration of ABTS in each reaction was 1.0 mM. The reaction was initiated by the adding of laccase. Control reaction (Ctrl) was a solution of 1.0 mM ABTS without the enzyme

Major advantage of using laccase over potassium persulfate is quick, high yield and environmental friendly. The laccase-prepared ABTS-+ radical (about 4 absorbance units at 734 nm, 1 mL) were investigated for its stability after dilution with distilled water or ethyl alcohol. The radicals were well stable after dilution with either distilled water or ethyl alcohol at least 30 min (data not shown). ABTS-+ radical decolourisation assay could be done within a minute as shown in Fig. 3.

Fig. 3:
Absorbance monitoring of ABTS•+ radicals scavenging reactions of standard Trolox solution in various concentrations (from 0-1.0 mM). The initial absorbance in each experiment was around 0.7 absorbance unit at 734 nm. Reactions were performed in a quart cuvette at room temperature and initiated by the adding of standard Trolox solution

This result was accordance with the previous report (Re et al., 1999). The decrease absorbance at 2 min was chosen for calculating the inhibition percentage. The results suggest that ABTS•+ radical preparation with the laccase enzyme is reasonably quick and easy. This might be suitable for antioxidant activity determination of biological samples.

More environmental friendly of enzymatic radical generation is one important advantage over chemical synthesis. This also supports the idea of green chemistry of the application of laccases (Riva, 2006). The radical generated by the enzymatic catalysis and used for antioxidant activity determination has been reported by horseradish peroxidase (HRP) catalyzed oxidation of luminol by hydrogen peroxide (Minioti and Georgiou, 2008). Another example of using HRP and hydrogen peroxide to oxidize ABTS has been reported as the application of interdigitated array microelectrodes as electrochemical sensors (Milardovic et al., 2007). Using laccases in the enzymatic method of antioxidant activity measurement can be used practically if the application of other peroxidases is unfavourable. Radical formation by laccase has been firstly reported by Kulys and Bratkovskaja (2007). In their report, recombinant laccase Polyporus pinsitus (rPpL) and Myceliophthora thermophila (rMtL) with several high reactive laccase substrates including ABTS, 2-phenoxazin-10-yl-ethanol (PET), 3-phenoxazin-10-yl-propane-1-sulfonic acid (PPSA) and 3-phenoxazin-10-yl-propionic acid (PPA) have been used. They showed that the method permits to measure sub-micromole concentration of an antioxidant and suggested that the optimization of the method may significantly increase the sensitivity.

Fig. 4:
Inhibition percentage by standard Trolox determined by using ABTS•+ radical prepared by the laccase reaction and determined by using DPPH- radicals. Data were averaged from a triplicate experiment

We here show the support results with available substrate ABTS. Antioxidant activity of tested vegetable samples as reported in term of TEAC values by laccase-prepared ABTS-+ assay was also in the sub-micromole concentration. This reveals a reasonable good sensitivity of the method. Moreover, the ABTS-+ radical cations were relative stable after diluted with distilled water or ethyl alcohol. This characteristic suggests the possibility of using these radicals for measurement of the total antioxidant activity in biological samples of both aqueous and ethyl alcoholic extracts.

Antioxidant activity analysis of selected Thai vegetables: Standard curve of Trolox for TEAC assay by ABTS•+ radical and DPPH- radical scavenging methods revealed very similar in their slopes and allowed measuring an antioxidant in sub-micromole concentration (0-0.02 μmole Trolox equivalent) as shown in Fig. 4, whereas a standard curve of gallic acid for total phenol content was linear in the range of 0-0.01 mg mL-1 as shown in Fig. 5. According to both standard curves of Trolox and gallic acid, inhibition percentage of 31 Thai vegetable extracts were calculated in terms of trolox equivalent antioxidant capacity (TEAC; μmole Trolox equivalent/mg dried weight of sample) by ABTS•+ and DPPH- radicals assays and total phenolic content as mg gallic acid equivalent (mg GAE/g dried weight of sample).

When laccase-aided generated ABTS•+ was using for antioxidant activity determination in biological samples, the results were as shown in Table 1.

Fig. 5:
Concentration-response curve for the absorbance at 725 nm for total phenolic content as a function of concentration of standard Gallic acid solution. (separately prepared stock standard solutions ± SD)

Among 31 plant samples tested, interesting high TEAC values were obtained from Eugenia grata (0.335), Polygonum odoratum (0.251) and Spondias pinnata (0.220), respectively. Present results from DPPH assay was relative low compared to those from ABTS•+ assay. This is in agreement with the study of Stratil et al. (2007). They suggested that the DPPH- method gives several times lower values for extracts than TEAC. This phenomenon could be explained by a relatively higher stability of the DPPH- radical which may result in significantly lower reactivity. Due to all assays for the assessment of phenolic compounds and antioxidant capacity are based on redox properties, there should have some correlation between content of phenolic compounds and antioxidant capacity as measured by each method. It was found that the TEAC values determined using ABTS•+ and DPPH radical decolourisation method were strongly consistency as shown in Fig. 6A (with r = 0.85). Thaipong et al. (2006) have observed high correlation results for the antioxidant activity measured in methanol extract of guava fruit extracts as determined by ABTS, DPPH, FRAP and ORAC assays. The result was in the same trends as reported by Stratil et al. (2007). In this study, antioxidant activity resulted in less correlate with total phenolic content in the samples Fig. 6B (with r = 0.67). A good correlation was found between TEAC and FC reducing capacity (R>0.9) for red wines, herbal and tea infusions and beers (Magalhaes et al., 2007). TEAC values correlated well with results found by elimination of DPPH- and both values revealed a linear relationship with the concentration of phenolics obtained with the Folin-Ciocalteu phenol test (Zalibera et al., 2008) .

Table 1: Total antioxidant activity and total phenolic contents of 31 selected thai vegetable extracts
a++++ (TEAC ≥0.4); +++ (TEAC 0.39-0.3); ++ (TEAC 0.29-0.2); + (TEAC 0.19-0.001) μmole Trolox/mg dried weight, b++++ (TEAC≥0.1); +++ (TEAC 0.099-0.05); ++ (TEAC 0.049-0.01); + (TEAC 0.009-0.001) μmole Trolox/mg dried weight, c+++++ (GAE≥2); ++++ (GAE 1.5-1.99); +++ (GAE 1-1.499); ++ (GAE 0.5-0.999); + (GAE 0.001-0.499) mg gallic acid/g dried weight, All analysis were averaged from triplicate

Fig. 6:
Correlation between TEAC values in μmole Trolox/mg dried weight determined by DPPH- and ABTS-+ radical assays (A) and correlation between total phenol content (GAE) in mg gallic acid equivalent/g dried weight and TEAC value in μmole Trolox/mg dried weight (B) of 31 selected Thai vegetables. Each data were averaged from triplicate

In this study, due to relatively less correlation between total phenolic content and antioxidant activity, it might possible that there are some phenolic compounds other than antioxidant substances present in the plant extracts. Meanwhile, antioxidant substances in these samples might be other chemical groups apart from phenolic compounds. Antioxidant compounds in those three vegetables are interesting to identify and further deeply characterize. That is because they might be vegetables of choice for thai people and the rest as great functional vegetables and fruits.

CONCLUSION

Preparation of ABTS•+ radical using the oxidation reaction of ABTS by T. versicolor laccase was a quick and easy procedure. The radicals were stable after diluted with either distilled water or ethyl alcohol and were suitable for antioxidant activity determination in dried-vegetable samples. The results of scavenging activity are reliable when compared to those values determined by DPPH- method. The benefits of the radicals; quick, easy and environmental friendly preparation, as well as good stability, suggest it should be very useful to a total radical scavenging capacity screening of various kinds of samples including biological sources.

ACKNOWLEDGMENTS

This research was supported by Mahasarakham University (Fiscal year 2007) and PERCH-CIC. The authors thank Ms. Anchalee Phonpuak, our undergraduate student for her technical assistance. We also thank Faculty of Science and Department of Chemistry for instrument supports.

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