Fungal laccases (p-diphenol: dioxygen oxidoreductases, EC 184.108.40.206)
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
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
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,
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
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).
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
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).
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
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.
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
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
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.
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.
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 ±
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) .
||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
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
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.
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
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.