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Extracts of Trametes elegans were examined for their total phenolic and flavonoid contents and in vitro antiradical potentials. Varying concentrations of the extracts were assessed against 1, 1-diphenyl-2-picrylhydrazyl (DPPH), Nitric Oxide (NO), hydrogen peroxide (H2O2) and hydroxyl (OH) radicals. The result of the total phenolic content expressed in mg gallic acid equivalents per gram of dried extract (mg GAE g-1) revealed that the acetone extract had higher phenolic content (4.79 mg GAE g-1) than the methanol extract. However, the methanol extract had higher total flavonoid (2.27 mg RE g-1) compared to the acetone extract. The extracts displayed appreciable radical scavenging activity to DPPH, NO and H2O2 radicals. The scavenging activities of the extracts were, however, lower to that of the positive control (butylhydroxytoluene). On the other hand, the acetone extract displayed better scavenging activity than the positive control at a concentration of 2 mg mL-1. Result from this study suggest that this underutilized indigenous macrofungus, Trametes elegans, can be exploited as a source of bioactive compounds with antioxidant potentials that can neutralize the damaging effects of free radicals.
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International Journal of Biological Chemistry 9 (5): 249-259, 2015
ISSN 1819-155X / DOI: 10.3923/ijbc.2015.249.259
© 2015 Academic Journals Inc.
In vitro Antiradical Activities of Trametes elegans Collected from
Osengere, Ibadan, Nigeria
S.I. Awala and V.O. Oyetayo
Department of Microbiology, Federal University of Technology, P.M.B. 704, Akure, Nigeria
Corresponding Author: S.I. Awala, Department of Microbiology, Federal University of Technology, P.M.B. 704, Akure,
Nigeria
ABSTRACT
Extracts of Trametes elegans were examined for their total phenolic and flavonoid contents and
in vitro antiradical potentials. Varying concentrations of the extracts were assessed against 1,
1-diphenyl-2-picrylhydrazyl (DPPH), Nitric Oxide (NO), hydrogen peroxide (H2O2) and hydroxyl
(OH) radicals. The result of the total phenolic content expressed in mg gallic acid equivalents per
gram of dried extract (mg GAE gG1) revealed that the acetone extract had higher phenolic content
(4.79 mg GAE gG1) than the methanol extract. However, the methanol extract had higher total
flavonoid (2.27 mg RE gG1) compared to the acetone extract. The extracts displayed appreciable
radical scavenging activity to DPPH, NO and H2O2 radicals. The scavenging activities of the
extracts were, however, lower to that of the positive control (butylhydroxytoluene). On the other
hand, the acetone extract displayed better scavenging activity than the positive control at a
concentration of 2 mg mLG1. Result from this study suggest that this underutilized indigenous
macrofungus, Trametes elegans, can be exploited as a source of bioactive compounds with
antioxidant potentials that can neutralize the damaging effects of free radicals.
Key words: Antiradical, extracts, butylhydroxytoluene, macrofungus, bioactives
INTRODUCTION
Free radicals are defined as molecules having an unpaired electron in the outer orbit and are
generally unstable and very reactive (Gilbert, 2000; Fang et al., 2002). All aerobic life forms existing
on earth are associated with oxidation processes, which are vital for their survival (Fang et al.,
2002). For example, oxygen radicals exert critical actions such as signal transduction, gene
transcription and regulation of soluble guanylate cyclase activity in cells (Zheng and Storz, 2000).
However, this vital mechanism may also lead to cell and tissue damages and mediate the
pathogenesis of many chronic diseases (McCord, 2000; Thetsrimuang et al., 2011). In other word,
there are “Two faces” of free radicals in biology in that they serve as signalling and regulatory
molecules at physiologic levels but also serves as highly deleterious and cytotoxic oxidants at
pathologic levels (Fridovich, 1999).
These damaging effects of these free radicals can be prevented by natural and synthetic
antioxidants. Although every organism has natural endogenous defence mechanisms to eliminate
free radicals, habitually excess production of Reactive Oxygen Species (ROS) overwhelms the
system (Tibuhwa, 2012). The restriction in the use of synthetic antioxidants such as such as
butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) due to their toxicity and
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carcinogenicity has raised an increased interest towards natural antioxidant. Since nature is the
master chemist, exploration of natural sources for novel bioactive agents may provide leads or
solutions for drug discovery and development (Debbab et al., 2012).
Mushrooms have been reported to be prolific producers of several bioactive metabolites and
have been used for centuries as medicine to treat or prevent different diseases (Wasser, 2011;
De Silva et al., 2012). The antioxidant properties of wild mushrooms have been studied and their
antioxidant activity, has been mainly related to their phenolic, tocopherols, ascorbic acid and
carotenoids contents, which could be extracted for the purpose of being used as functional
ingredients against chronic diseases related to oxidative stress (Mohsin et al., 2011). Mushrooms
belonging to the genus Trametes are polyporoid white rot fungi widely distributed in various
biotopes and have been the subject of many physiological and biochemical studies (Koroleva et al.,
2002).
This study is aimed at providing information on the total phenol and flavonoid contents, as well
as the antiradical activities of Trametes elegans collected fr o m I badan, Ni g e r ia using va r i ous
in vitro methods.
MATERIALS AND METHODS
Collection of macrofungus: Fresh fruit bodies of macrofungus suspected to be Trametes species
were collected from rotten cocoa woods from farms in Osengere, Egbeda Local government area of
Ibadan, Oyo State (Latitude: 7.39814N, Longitude: 4.00051E) in the month of August 2013. The
fruiting bodies were kept dry by wrapping in tissue paper and kept in a polythene paper containing
silica gel. The polythene bags containing the samples were well labelled for easy identification and
taken to the Department of Microbiology, Laboratory of the Federal University of Technology,
Akure, for further examination. The molecular identification of the macrofungus was carried out
at the Key Laboratory of Mycology and Lichenology, Institute of Microbiology, Beijing. This was
done by amplifying and sequencing the Internal Transcribed Spacer (ITS 4 and ITS 5) of the
nuclear ribosomal DNA (nrDNA). Basic Local Alignment Search Tool (BLAST) search revealed that
the macrofungus was 98% related to Trametes elegans from the National Center for Biotechnology
Information (NCBI) GenBank.
Preparation of mushrooms extracts: The powdered mushroom sample (100 g) was extracted
with 2000 mL of 95% acetone and methanol separately in an Erlenmeyer flask. The flasks were
covered with aluminium foil and allowed to stand for 3 days for extraction with occasional stirring.
The extracts were then filtered through Whatman filter paper (0.45 μm) using vacuum pump. The
filtrates were evaporated to dryness at 50°C in a rotary evaporator (RE-52A; Union Laboratory,
England) with 90 rpm under reduced pressure. The obtained concentrated extracts were stored in
dark at 4°C until further analysis.
Determination of total phenolic content: The total phenolic content of the extracts was
determined by the method of Singleton et al. (1999). About 0.2 mL of each of the extracts was mixed
with 2.5 mL of 10% folin ciocalteau’s reagent and 2 mL of 7.5% sodium carbonate (Na2CO3). The
reaction mixture was incubated at 45°C for 40 min and the absorbance was measured at 700 nm
in a spectrophotometer (Gulfex Medical and Scientific England, Spectrum Lab 23A, model number
23A08215). A calibration curve was earlier plotted as standard gallic acid curve. Total phenolic
content was expressed as mg gallic acid equivalents per gram of dried extract (mg GAE gG1) using
the linear equation obtained from standard gallic acid calibration curve.
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Determination of total flavonoid content: The total flavonoid content of the extracts was
determined using a colorimeter assay developed by Bao et al. (2005). The extract (0.2 mL) was
added to 0.3 mL of 5% sodium Nitrate (NaNO3) at zero time. After 5 min, 0.6 mL of 10% aluminium
chloride (AlCl3) was added and after 6minutes, 2 mL of 1 M NaOH (sodium hydroxide) was added
to the mixture followed by the addition of 2.1 mL of distilled water. Absorbance was read with a
spectrophotometer (Gulfex Medical and Scientific England, Spectrum Lab 23A, model number
23A08215) at 510 nm against the reagent blank and the calibration curve was prepared by using
rutin methanolic solutions at concentrations of 12.5-100 μg mLG1. Total flavonoid was expressed
as mg rutin equivalents per gram of dried extract (mg RE gG1).
DPPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging activity: The DPPH radical scavenging
ability of the extracts was determined by the method of Gyamfi et al. (1999), with slight
modification (Awah et al., 2010). Briefly, a 2.0 mL solution of the extract at different concentrations
diluted two-fold in methanol was mixed with 1.0 mL of 0.3 mM DPPH in methanol. The mixture
was shaken vigorously and allowed to stand at room temperature in the dark for 25 min. Blank
solutions were prepared with each test sample solution (2.0 mL) and 1.0 mL of methanol while the
negative control was 1.0 mL of 0.3 mM DPPH solution plus 2.0 mL of methanol.
Butylatedhydroxytoluene (BHT) was used as positive control. Thereafter, the absorbance of the
assay mixture was measured at 518 nm against each blank with a UV-visible spectrophotometer
(Gulfex Medical and Scientific England, Spectrum Lab 23A, model number 23A08215). Test was
carried out in triplicate. The DPPH radical inhibition was calculated using the equation:
Abs control (Abs sample Abs blank)
Inhibition (%) 100
Abc control


where, Abs control is the absorbance of the control (DPPH without sample), Abs sample is the
absorbance of the test sample (the sample test and DPPH solution) and Abs blank is the absorbance
of the sample blank (Sample without the DPPH solution).
Nitric oxide scavenging activity: The method of Ebrahimzadeh et al. (2008) was used to
determine the antiradical activity of the mushroom extracts against nitric oxide radical. A volume
of 2 mL of sodium nitroprusside prepared in 0.5 mM phosphate buffer saline (pH 7.4) was mixed
with 0.5 mL of mushroom extract and control (butylatedhydroxytoluene) at various concentrations
(0.5-2.0 mg mLG1). The mixture was incubated at 25°C for 150 min. An aliquot of 0.5 mL of the
solution was added to 0.5 mL of Griess reagents (1.0 mL of sulfanilic acid reagent 0.33% prepared
in 20% glacial acetic acid at room temperature for 5 min with 1 mL of naphthyethylenediamine
chloride (0.1% w/v)). The mixture was incubated at room temperature (26±2°C) for 30 min. The
absorbance was then measured at 540 nm with a spectrophotometer (Gulfex Medical and Scientific
England, Spectrum Lab 23A, model number 23A08215). The amount of nitric oxide radical
scavenging ability was calculated using the equation:
Absorbance of control Absorbance of sample
NO radical scavenging activity 100
Absorbance of control

where, Abs control is the absorbance of NO radical+methanol, Abs sample is the absorbance of NO
radical+sample extract or standard.
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Scavenging effect of hydroxyl radical: The ability of the extract to prevent Fe2+/H2O2 induced
decomposition of deoxyribose was carried out using the method of Halliwell and Gutteridge (1981).
Briefly, freshly prepared extract (0-100 μL) was added to a reaction mixture containing 120 μL of
20 mM deoxyribose, 400 μL of 0.1 M phosphate buffer (pH 7.4), 40 μL of 20 mM hydrogen peroxide
and 40 μL of 500 μM FeSO4 and the volume was made to 800 μL with distilled water. The reaction
mixture was incubated at 37°C for 30 min and the reaction was stopped by the addition of 0.5 mL
of 2.8% trichloroacetic (TCA). This was then followed by the addition of 0.4 mL of 0.6%
thiobarbituric acid (TBA) solution. The tubes were incubated in boiling water for 20 min and the
absorbance measured at 532 nm with a spectrophotometer (Gulfex Medical and Scientific England,
Spectrum Lab 23A, model number 23A08215). The amount of hydroxyl radical scavenging ability
was calculated using the equation:
Abs references Abs sample
Hydroxyl radical scavenging activity (%) 100
Abs reference

where, Abs reference is absorbance of the reference (reacting mixture without the test sample) and
Abs sample is absorbance of reacting mixture with the test sample.
Hydrogen peroxide scavenging assay: Hydrogen peroxide scavenging potential of the
mushrooms extract was determined using the method described by Jayaprakasha et al. (2004). A
solution of hydrogen peroxide (20 mM) was prepared in Phosphate Buffer Saline (PBS, pH 7.4).
Different concentrations of the extract (0.5-2 mg mLG1) in ethanol (1 mL) were added to 2 mL of
hydrogen peroxide solution in PBS. After 10 min the absorbance was measured at 230 nm against
a blank solution that contained hydrogen peroxide solution without the extract. The percentage of
H2O2 scavenging of the mushroom extract was calculated as follows:
Abs references Abs sample
Hydrgen peroxide scavenging activity (%) 100
Abs reference

where, Abs reference is absorbance of the reference (reacting mixture without the test sample) and
Abs sample is absorbance of reacting mixture with the test sample.
Statistical analysis: All experiments were carried out in triplicates. Data obtained was analyzed
by one way analysis of variance (ANOVA) and means were compared by New Duncan’s Multiple
Range Test (SPSS version 16). Differences were considered significant at p = 0.05.
RESULTS
Table 1 shows the result for the total phenol and total flavonoid contents of extracts. The
acetone extract gave higher amount of total phenol and lower amount of total flavonoid, while the
methanol extract produced higher total flavonoid and lower amount of total phenol.
Table 1: Total phenolic and flavonoid content of Trametes elegans extracts
Solvent Total phenolic (mg GAE gG1) Total flavonoid (mg RE gG1)
Acetone 4.79±0.06 0.97±0.00
Methanol 4.37±0.06 2.27±0.16
Each value is expressed as mean±standard error (n = 3), GAE: Gallic acid equivalent, RE: Rutin equivalent
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0.0 0.5 1.0 1.5 2.0 2.5
Concentration of extract (mg mL )
G
1
120
100
80
60
40
20
0
Radical scavenging activity (%)
TEA
TEM
BHT
0.5 1.0 1.5 2.0 2.5
TEA
TEM
BHT
0.0
Concentration (mg mL )
G
1
120
100
80
60
40
20
0
Radical scavenging activity (%)
Fig. 1: Scavenging effect of Trametes elegans extracts on DPPH radicals. Each value is
expressed as mean of triplicate results (n = 3), TEA: Acetone extract of Trametes elegans,
TEM: Methanol extract of Trametes elegans, BHT: Butylatedhydroxy toluene
Fig. 2: Scavenging effect of Trametes elegans extracts on nitric oxide radicals. Each value is
expressed as mean of triplicate results (n = 3), TEA: Acetone extract of Trametes elegans,
TEM: Methanol extract of Trametes elegans, BHT: Butylatedhydroxy toluene
Figure 1 shows the result of the DPPH radical scavenging activity of the extracts of
Trametes elegans. The extracts displayed concentration dependent free radical scavenging
ability (0.5-2 mg mLG1). At a concentration of 2 mg mLG1, the acetone extract displayed an activity
of 65%, while the methanol extract displayed 66.46% scavenging activity. The positive control
(BHT) displayed activity that was higher (99.46%) and significantly different from that of the
extracts.
The extracts of Trametes elegans displayed moderate and concentration dependent nitric oxide
(NO) scavenging ability as shown in Fig. 2. The ability of the extracts to scavenge NO radicals were
56.18 and 58.87% for acetone and methanol extracts respectively at 2 mg mLG1. The positive control
(BHT) exhibited a higher and significantly different (p<0.05) NO scavenging effect when compared
to the extracts (Fig. 2).
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0.0 0.5 1.0 1.5 2.0 2.5
Concentration of extract (mg mL )
G
1
90
80
70
60
50
40
30
20
10
0
-10
Ra
d
ical
s
ca
v
e
nging acti
v
it
y
(%)
TEA
TEM
BHT
0.0 0.5 1.0 1.5 2.0 2.5
Concentration of extract (mg mL )
G
1
120
100
80
60
40
20
0
Radical scavenging activity (%)
TEA
TEM
BHT
Fig. 3: Scavenging effect of Trametes elegans extracts on hydroxyl radicals. Each value is
expressed as mean of triplicate results (n = 3), TEA: Acetone extract of Trametes elegans,
TEM: Methanol extract of Trametes elegans, BHT: Butylatedhydroxy toluene
Fig. 4: Scavenging effect of Trametes elegans extracts on hydrogen peroxide radicals. Each
value is expressed as mean of triplicate results (n = 3), TEA: Acetone extract of
Trametes elegans, TEM: Methanol extract of Trametes elegans, BHT: Butylatedhydroxy
toluene
The hydroxyl radical scavenging effect of the extract of Trametes elegans is shown in Fig. 3. The
scavenging effects of extracts at 2 mg mLG1 were above 50% and well pronounced. Acetone extract
of Trametes elegans produced an effect of 84.18% that was slightly higher than that of the positive
control (BHT) at a concentration of 2 mg mLG1.
Figure 4 shows the scavenging effect of the extracts of Trametes elegans on hydrogen peroxide
radicals. The extracts displayed concentration dependent activities (0.5-2 mg mLG1). At a
concentration of 2 mg mLG1, acetone extract of Trametes elegans exhibited higher activity of
76.03%. The activity of the positive control (BHT) was not concentration dependent and it produced
higher scavenging effect than the extracts at all tested concentrations.
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DISCUSSION
Several studies have reported the antioxidative potency of wild mushrooms (Kosanic et al.,
2013; Pennerman et al., 2015). These antioxidant activities have been attributed to the presence
of various bioactives (Chowdhury et al., 2015). The present study investigated the in vitro
antiradical activities of extracts obtained from Trametes elegans from Osengere, Ibadan, Nigeria.
Varying amount of total phenol and total flavonoids were found in the extracts. This observation
might be connected with the solvent and the chemical nature of the bioactives. It has been reported
that the chemical nature of phytochemicals, extraction method, sample particle size, solvent used,
as well as the presence of interfering substances affects extraction efficiency (Stalikas, 2007).
The presence of phenolics and flavonoids in wild mushrooms has been reported by many
authors (Chowdhury et al., 2015; Pennerman et al., 2015). Phenolic compounds present in
mushrooms possess strong antioxidants properties, with a strong positive correlation between the
phenolic content and antioxidant activity (Reis et al., 2011). They are capable of playing a protective
role in preventing radical related disease, cancer and cardiovascular disease (Ferreira et al., 2010).
Flavonoids are a large group of polyphenolic compounds having a benzo-γ-pyrone structure
(Kumar and Pandey, 2013). The chemical nature of flavonoids depends on their structural class,
degree of hydroxylation, other substitutions and conjugations and degree of polymerization
(Heim et al., 2002). They are capable of scavenging free radicals and chelating metal ions
(Kumar et al., 2013). The presence of these phenolic compounds in extracts of Trametes elegans
used in this study, suggest that the macrofungus could be a potential source of natural
antioxidants.
The result from this study shows that the extracts of Trametes elegans were able to scavenge
for DPPH (Fig. 1). Several authors have also reported the DPPH radical scavenging activities of
wild mushrooms (Reis et al., 2011; Vamanu and Nita, 2013). When a solution of DPPH is mixed
with that of a substance that can donate a hydrogen atom, it gives rise to the reduced form of the
DPPH compound, leading to the reduction of the violet colour. Substances which are able to
perform this reaction can be considered as antioxidants and therefore radical scavengers
(Ebrahimzadeh et al., 2010). This implies that Trametes elegans extracts contain bioactive
compounds capable of donating hydrogen atom to DPPH.
It has been found that phenolics, flavonoids and tocopherols reduce the DPPH radicals by their
hydrogen donating ability (Zhao et al., 2006). Results from several studies have shown positive
correlation between Total Phenol Content (TPC) assay and DPPH radical scavenging activity assay
(Luther et al., 2007; Silva et al., 2007). It was however noted from this study that though the
acetone extract had higher TPC value than the methanol extract, yet it displayed lower radical
scavenging activity. It is therefore suggested that other biologically active substances could be
present and this might have contributed to the higher radical scavenging activity displayed by the
methanol extract of T. elegans. Similar observation was reported by Othman et al. (2011) while
carrying out a comparative study on the antioxidant activity of red onion and garlic. From their
study, it was observed that there was a negative correlation between the result of TPC assay and
DPPH radical scavenging assay. Khamsiah et al. (2006) suggested that free radical scavenging
activity is not due to the phenolics only because they found that the antioxidant activity of
methanol extract of Orthosiphon stamineus was not solely caused by phenolic compounds.
The extracts of T. elegans were capable of scavenging nitric oxide (Fig. 2). The nitric oxide
scavenging effect of various wild mushrooms has also been reported (Menaga et al., 2013;
Vamanu and Nita, 2013). Nitric Oxide (NO) is classified as a free radical because of its unpaired
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electron and displays important reactivity with certain types of proteins and other free radicals.
The toxicity of NO becomes adverse when it reacts with superoxide radical, forming a highly
reactive peroxynitrite anion (ONOOG) (Nagmoti et al., 2012).
In this study, the methanol and acetone extracts of T. elegans at different concentrations were
assessed for their nitrite free radical scavenging activity in an in vitro model. In this method, nitric
oxide generated from sodium nitroprusside reacts with oxygen to form nitrite. The nitrite ions
diazotize with sulphanilamide acid. This couples with naphthylethylenediamine to form a pink
colour which is measured at 540 nm. As antioxidants donate protons to the nitrite radical, the
absorbance is decreased. The decrease in absorbance is used to measure the extent of nitrite radical
scavenging (Turkoglu et al., 2007).
The ability of the extracts to scavenge nitric oxide suggests that the extracts contain
substances that can directly compete with oxygen in the reaction with nitric oxide. It was observed
that the methanol extracts of T. elegans when compared to the acetone extracts of both mushrooms
exhibited higher scavenging ability. Boora et al. (2014) attributed the higher NO scavenging
potency of Parinari curatellifolia as compared to that of Combretum zeyheri and Combretum
platypetalum to the presence of flavonoids and saponins. The higher scavenging ability of NO by
the methanol extract of T. elegans when compared to the acetone extract might be attributed to the
higher flavonoids content. Trametes elegans, can therefore be a useful source of natural antioxidant
that can help combat the detrimental effect of nitric oxide generated in the body.
Potent hydroxyl radical scavenging effect was observed in the extracts of T. elegans (Fig. 3).
Several authors have reported the hydroxyl radical scavenging effect of various mushrooms
(Oyetayo, 2009; Khatua et al., 2015). Hydroxyl radical is an extremely reactive oxygen species that
can propagate a chain reaction, wreaking havoc on many biological molecules such as DNA and
lipids (Uttara et al., 2009; Kumar et al., 2011). The removal of hydroxyl radicals may be due to the
presence of substances capable of donating hydrogen or electron. Phenolic compounds possess the
ability to scavenge free radicals to the presence of hydroxyl groups that can give up its hydrogen
atom to the hydroxyl group and form a stable phenoxyl radicals (Ozgen et al., 2010). Several studies
have reported strong correlation between the total phenolic contents and hydroxyl radical
scavenging activity (Olabinri et al., 2010; Aksoy et al., 2013). Higher OH radical scavenging effect
of the acetone extract of T. elegans might be related to its higher total phenolic contents. Results
from this study suggest that extracts of T. elegans could be promising candidates in the prevention
of DNA damage.
The extracts were also able to scavenge for hydrogen peroxide (Fig. 4). The ability of the
extracts to scavenge hydrogen peroxide may be attributed to the presence of phenolics, which can
donate electron to H2O2, thus converting it to water. It was observed that the acetone extract of
T. elegans which displayed the higher TPC, also exhibited higher scavenging ability at all
concentrations. Kim et al. (2013) in his finding observed a strong linear correlation between the
TPC and scavenging of H2O2 while evaluating the antioxidant activity of the water and ethanol
extract of dried citrus fruit peel (Citrus unshiu).
Hydrogen peroxide (H2O2) is not a highly reactive oxygen species. Its low reactivity gives it
enough time to penetrate biological membranes. In the presence of transition metals, it might form
highly reactive hydroxyl radical which might wreak havoc on macromolecules such as lipids and
DNA (Sahreen et al., 2011). The removal of H2O2 in biological and food system is of outmost
importance for antioxidant defence.
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CONCLUSION
Extracts of Trametes elegans displayed effective in vitro radical scavenging activity against
DPPH, NO and H2O2 radicals. Natural antioxidants that can neutralize the damaging effects of free
radicals can therefore be sourced for from this macrofungus. However, further studies are needed
to establish the pharmacological effects of these natural extracts in the treatment or prevention of
radical and age associated diseases.
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... The antioxidant effect correlated with the cytotoxicity of an extract is the maximum expressed value of such a product because it shows the differences between the tested mushrooms and their degree of involvement in these mechanisms [57]. These capacities were best demonstrated for lyophilized/atomized extracts, where in vitro antiradical protection showed how the molecules present in the extracts could react with the physiological factors involved in oxidative stress and with the gut microbial fingerprint [58]. ...
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