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Antioxidant properties and polyphenol

contents of trembling aspen bark extracts

Article in Wood Science and Technology · August 2009

DOI: 10.1007/s00226-009-0240-y

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Wood Sci Technol (2009) 43:457–470
DOI 10.1007/s00226-009-0240-y

ORIGINAL

Antioxidant properties and polyphenol contents

of trembling aspen bark extracts

Papa Niokhor Diouf Æ Tatjana Stevanovic Æ

Alain Cloutier

Received: 7 November 2007 / Published online: 10 February 2009

Ó Springer-Verlag 2009

Abstract Trembling aspen (Populus tremuloides Michx) bark was extracted with
water and the crude extract fractionated with tert-butyl-methyl ether (TBME), ethyl
acetate (EtOAc) and n-butanol (BuOH) to obtain four different fractions. The
antioxidant properties of the bark hot water extracts and its fractions were deter-
mined by three in vitro experiments: DPPH assay, phosphomolybdenum assay and
canola oil thermoxidation assay by DSC analysis. Most of the results of the reported
tests showed that the crude hot water extract and its fractions exhibited a strong
antioxidant activity, higher than the synthetic antioxidant BHT. The results of this
study confirm that antioxidant activity is a property that strongly depends on the
oxidation conditions used in the particular oxidation test. Among the fractions
separated from the aqueous extracts of bark, BuOH, TBME and EtOAc soluble
fractions exhibited the best antioxidant efficiency, in phosphomolybdenum assay,
DPPH assay and canola oil thermoxidation, respectively. Total phenol, flavonoid
and flavanol contents were also evaluated and the results confirmed that the poly-
phenols contained in the hot water extract of this bark are mainly composed of non-
flavonoid compounds.

Introduction

The interest for polyphenols is ever growing especially due to the beneficial health
effects that are attributed to several classes of polyphenols (Arts and Hollman
2005). The role of polyphenols as natural antioxidants is inspiring the study
regarding their role in the prevention and treatment of cancer, inflammatory,
cardiovascular and neurodegenerative illnesses. Antioxidants are molecules that

P. N. Diouf
T. Stevanovic (&)
A. Cloutier

Département des Sciences du Bois et de la Forêt, Faculté de Foresterie et Géomatique,
Centre de Recherche sur le Bois, Université Laval, Quebec G1K7P4, Canada
e-mail: [email protected]

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458 Wood Sci Technol (2009) 43:457–470

delay or prevent the oxidation of other chemicals. The food industry initially used
these natural compounds or/and synthesized substances with higher antioxidant
activity. However, previous research has reported that some synthetic antioxidants,
such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are
carcinogenic substances (Parke and Lewis 1992; Kahl and Kappus 1993).
Finding new natural, safe and economical antioxidant substances, especially from
abundant and low-value raw materials (bark wastes from forest industry) is a real
challenge nowadays, especially to develop the sustainability concept (Moure et al.
2001). In a recent research, the health benefits of the molecules from the forest
biomass, such as the inhibition of reactive oxygen species ROS by natural
antioxidants found in bark, have been studied (Goncalves et al. 2005; Cui et al.
2005; Pilarski et al. 2006). In order to identify the antioxidant-rich fractions, various
approaches are adopted. One of them consists of fractionating the crude extracts
through immiscible solvent–solvent partitioning to obtain different fractions (Gao
et al. 2006; Sanchez et al. 2006; Jia et al. 2007; Ku and Mun 2008).
The Canadian forest industry generates high quantities of trembling aspen bark
which is mainly used as fuel for thermal energy production. Trembling aspen bark
was investigated extensively between the 1950s and 1970s (Chang and Mitchelle
1955; Hossfeld and Hunter 1958; Faber 1960; Pearl et al. 1961; Thieme 1967;
Pronin and Vaughan 1968; Pearl and Darling 1971). Native Americans used its bark
as food and medicine: root bark tea for excessive menstrual flow; and a tea made of
the inner bark for venereal disease treatment, stomach pain, urinary ailments,
worms, colds, and fevers. The tincture of the bark contains the salicin which is the
remedy for fevers, rheumatism, arthritis and diarrhoea. But the antioxidant activities
of trembling aspen bark extracts have not been studied yet. The present paper
reports on the determination of the antioxidant properties of trembling aspen bark
hot water extract and its fractions in three in vitro models. Total phenol, total
flavonoid and total flavanol contents of the studied extracts have also been
determined.

Materials and methods

Raw material

Fresh trembling aspen bark was supplied on the 26 June 2005 by the Oriented
Strand Board mill of Louisiana Pacific Canada, Québec-Chambord OSB division,
located in Chambord, Quebec, Canada. After air drying, the bark was milled,
successively sieved to select particles between 40–60 mesh, and then stored at -4°C
in darkness prior to the experiments.

Extraction and fractionation

After fat removal with petroleum ether, 15 g of ground material (13.8 g eq. o.d.
material) was extracted with 150 mL of distilled water for 1 h under reflux (85°C).
The extract was filtered through a Whatman No. 4 filter paper and rinsed with

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Wood Sci Technol (2009) 43:457–470 459

50 mL of hot distilled water. Extraction of the residue was repeated twice under the
same conditions. The three filtrates were combined and freeze-dried to yield crude
extract.

Solvent partitioning of the crude extract of Populus tremuloides

The crude hot water extract (CHWE) was partitioned with solvents authorised in
food industry, in increasing order of polarity, namely tert-butyl-methyl ether, ethyl
acetate and n-butanol. One gram of CHWE was suspended in 50 mL of distilled
water, defatted with petroleum ether and sequentially partitioned with (3 9 50 mL)
each of tert-butyl-methyl ether (TBME), ethyl acetate (EtOAc) and n-butanol
(BuOH) to obtain four different fractions. The combined organic layers for each
solvent were concentrated to dryness in vacuo at 40°C using a rotary evaporator.
The left-over aqueous layer was freeze-dried to yield the aqueous fraction (H2O).
Dry extracts were stored in glass bottles at -20°C until testing.

Method I: total antioxidant capacity by phosphomolybdenum method

The total antioxidant capacities (TAC) of the crude extract and its fractions were
evaluated by the method of Prieto et al. (1999). This assay is based on the reduction
of Mo (VI) to Mo (V) by the sample and the subsequent formation of a green
phosphate/Mo (V) complex at acidic pH. A total of 0.3 mL of methanolic sample
solution was mixed with 2.7 mL of the reagent solution (0.6 M sulphuric acid,
28 mM sodium phosphate and 4 mM ammonium molybdate). The effective
concentration of the sample was 100 lg/mL in the reaction mixture. For the blank,
0.3 mL methanol was mixed with 2.7 mL of the reagent. The absorbance of the test
sample was measured at 695 nm. Trolox was used as a reference standard and TAC
results were expressed as Trolox equivalents (mg TE/g of dry sample). BHT was
used as a reference for comparison.

Method II: free radical scavenging activity on DPPH radical

The free radical scavenging activity (FRSA) of the crude extract and its fractions
was evaluated according to the method described by Wang et al. (1998) with some
modifications. Equal volumes of the methanolic solution of the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) free radical (200 lM) and of the extracts (200 lg/mL) were
rapidly mixed in rapid kinetic accessory SFA-22 (Hi-Tech Scientific, Salisbury,
England). The temperature was kept at 30°C. The radical scavenging effect was
continuously followed by monitoring the change of absorbance at 516 nm for
15 min, against methanol as the blank. The scavenging percentage was calculated
according to:


% Scavenging ¼ Acontrol  Asample =Acontrol  100
where Acontrol is the absorbance at 516 nm of 100 lM DPPH without adding the
extract/fractions, Asample is the absorbance at 516 nm of 100 lM DPPH with

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460 Wood Sci Technol (2009) 43:457–470

100 lg/mL of sample. Trolox was used as a reference standard and FRSA results
were expressed as Trolox equivalents (mg TE/g of dry sample). BHT was used as a
reference for comparison.

Method III: thermoxidative stability of canola oil by DSC analysis

Differential Scanning Calorimetry (DSC) is commonly used to investigate processes

which involve enthalpy changes. With lipid oxidation being an exothermic process,
the DSC is a convenient method for recording the released heat (Suja et al. 2004;
Gouveia et al. 2006; Garcı́a-Pérez et al. 2008). The time of the onset point (OIT) is
determined as the time at which the oxidation process starts. A Mettler Toledo DSC
822e was used to carry out the tests. The equipment was calibrated with pure indium
and the baseline was obtained with an empty open aluminium pan. Canola oil (CO)
obtained from local market, with and without extract, was studied at a heating rate
of 10°C/min from 25 to 275°C under an oxygen flow of 50 mL/min. A total of
0.1 mL methanolic solution containing 1 mg dry extract/fraction was added to 1 mL
of CO; 0.1 mL of methanol in 1 mL of canola oil was used as a control and
10.5 ± 0.5 mg of each sample were analysed in a standard 40-ll aluminium pan.
The antioxidant activity of each extract was evaluated by the protection factor (PF),
calculated according to:
PF = OITsample =OITcontrol
BHT, at 200 lg/mL (limited legal maximum of its addition to food, fats and oils),
was used as a reference.

Total phenol content

The total phenols contents of the crude extract and its fractions were determined
spectrophotometrically by the Folin-Ciocalteu method following the procedure
described in the literature (Singleton et al. 1999). A total of 0.5 mL of methanolic
solution of sample at 0.2 mg/mL was mixed with 2.5 mL of the Folin-Ciocalteu
reagent (diluted 10 times by distilled water) and 2.0 mL of an aqueous sodium
carbonate solution (75 mg/mL). The final mixture was heated at 50°C for 10 min
and after that the absorbance at 765 nm was read. Results are expressed in gallic
acid equivalents (mg GAE/g of dry extract).

Total flavonoid content

The AlCl3 method according to Brighente et al. (2007) was used for the determination
of total flavonoid content of extracts. To 2 mL of sample at 1 mg/mL in methanol
contained in PTFE screw capped vial, the equal volume of the 2% AlCl3
6H2O
solution (2 g in 100 mL methanol) was added. The mixture was vigorously shaken,
and absorbance was read at 415 nm after 1 h of incubation at 20°C. Results are
expressed in quercetin equivalents (mg QE/g of dry extract).

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Wood Sci Technol (2009) 43:457–470 461

Total flavanol content

The content of flavanols was determined by vanillin assay as described by Scalbert

et al. (1989). To 1 mL (1 mg/mL) of the water solution of the sample, 2.0 mL of the
vanillin reagent (1% vanillin in 70% sulphuric acid) are added and the reaction is
performed for 15 min at 20 ± 0.5°C (in water bath). The reaction is stopped by
cooling in ice bath and the absorbance is read at 500 nm. Results are expressed in
(?)-catechin equivalents (mg CE/g of dry extract).

Statistical analysis

Data are presented as means of three values ± SE. All experimental results were
analysed statistically by ANOVA and Turkey HSD test was used to determine the
differences of means. Significance was defined at P value \0.05.

Results and discussion

Method I

This assay is based on the reduction of Mo (VI) to Mo (V) by the sample and the
subsequent formation of a green phosphate/Mo (V) complex at acidic pH which is
then determined spectrophotometrically. In this method, a higher TAC value
corresponds to a higher antioxidant activity. The crude extract and its fractions
demonstrate all antioxidant capacity. All fractions show higher antioxidant activity
than the crude extract. This observation allows to confirm that solvent partitioning
of the crude extract may concentrate antioxidant compounds in each fraction.
Figure 1 shows the total antioxidant capacity of CHWE and its fractions compared
with BHT. The best antioxidant efficiency has been determined for the BuOH
fraction (352.1 mg TE/g) followed in decreasing order by EtOAc (320.0 mg TE/g),
TBME (317.0 mg TE/g; not significantly different to EtOAc), the crude extract
CHWE (277.0 mg TE/g), and the water fraction H2O (205.0 mg TE/g). The water
fraction that presents therefore the least important antioxidant capacity is more
effective than the synthetic antioxidant BHT (171.9 mg TE/g).

Method II

The method frequently used to determine the antioxidant/antiradical activity of a

given compound is based on the reaction of that compound with the free radical
DPPH, stable in its crystalline form and in solution, coloured deep violet and stable
for several days. The effect of the antioxidants on DPPH is based on their ability to
donate one hydrogen atom to DPPH, thus converting this radical into a stable
molecule according to the following equation:

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462 Wood Sci Technol (2009) 43:457–470

c
350
b b

300
a

250
mg TE/g dry sample

200
e

150

100

50

0
CHWE TBME EtOAc BuOH H2O BHT

Fig. 1 Total antioxidant capacity of trembling aspen hot water extract and its fractions. Different letters
represent significant differences at 0.05 probability level

DPPH þ RH ! DPPH  H þ R
:
The more efficiently the antioxidant compound ‘‘consumes’’ the DPPH [which
correspond to higher free radical scavenging activity (FRSA) values of Trolox
equivalents], the better is the antioxidant/antiradical power. CHWE as well as its
different fractions demonstrate all radical scavenging activity and one more
effective than BHT (36.2 mg TE/g). Figure 2 shows the free radical scavenging
activity of CHWE and its fractions compared with BHT. The best efficiency is
determined for the fraction TBME (115.9 mg TE/g), followed by the fractions
EtOAc (94.1 mg TE/g), BuOH (93 mg TE/g; not significantly different to EtOAc),
CHWE (72.2 mg TE/g), and finally the fraction H2O with the least antiradical
efficiency (45.0 mg TE/g) as also determined by method I.

Method III

Canola oil is the vegetable oil with the highest selling rate and consumption per
person in Canada. CO consists, on average, of 61% monounsaturated fatty acids and
the major fatty acid is oleic acid (total content 56%) (Romero et al. 2007). In
addition, it has a low content of saturated fatty acids (8.1%) and a medium content
of polyunsaturated fatty acids (30.7%), mainly represented by linoleic (21.5%) and
linolenic acids (8.0%). Therefore, the study of its oxidative stability without/with
antioxidants for high temperature application (e.g. deep frying, microwave) was
undertaken here. The use of natural compounds to prevent degradation during frying
has been reported earlier (Yanishlieva et al. 1997; Lalas and Dourtoglou 2003; Su

123
Wood Sci Technol (2009) 43:457–470 463

120 b

100 c c
mg TE/g dry sample

80 a

60
d

e
40

20

0
CHWE TBME EtOAc BuOH H2O BHT

Fig. 2 Free radical scavenging activity of trembling aspen hot water extract and its fractions. Different
letters are significantat 0.05

et al. 2004; Romero et al. 2007). DSC has many advantages compared to other
accelerated stability methods like Schaal Oven Test (SOT), Active Oxygen Method
(AOM), Rancimat, etc. including small sample size (less than 20 mg), minimal
sample preparation, simplicity of operation and results in less than 1 h (Tan and
Man 2002). Oil samples which require 15 days by SOT could be evaluated in less
than 1 h by DSC. Unsaturated lipid substrates of CO, conveniently presented here as
LH, particularly polyunsaturated fatty acids are prime targets of oxidation which
proceeds via a chain reaction, including induction (i), propagation (p) and
termination (t) steps as follows (Rousseau-Richard et al. 1988):
Inducer ! 2 L
ði1 Þ
L
þ O2 ! LO2
ði2 Þ
LO2
þ LH ! LO2 H þ L
ði3 Þ
L
þ O2  LO2
ðp1 Þðp1 Þ
LO2
þ LH ! LO2 H þ L
ðp2 Þ
LO2
þ LO2
! ðt1 Þ
LO2
þ L
! non radical products ðt2 Þ
L
þ L
! ðt3 Þ
The induction period is a preparatory stage where the chemical compounds
needed for the full development of oxidation are formed. This period is considered
as a relative measure of oxidative stability. PF value higher than 1 indicates an

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464 Wood Sci Technol (2009) 43:457–470

antioxidant activity, while PF value lower than 1 indicates a pro-oxidant activity of

the tested extract. The higher the PF factor value, the better the antioxidant activity.
The PF factors of the canola oil by adding trembling aspen bark extract/fractions
and BHT as a standard are presented in Fig. 3. The PF determined for canola oil
with the addition of the fractions TBME, EtOAc and BuOH are around 1 but
significantly different from the control (P \ 0.05) and from the rest of the oil
samples with the fractions CHWE and H2O, which for their part, demonstrate the
pro-oxidant properties. The best antioxidant activities were determined for the oil
sample containing the EtOAc and BuOH fractions of the trembling aspen bark
extract but lower than BHT.

Polyphenol contents

As phenolic compounds constitute one of the major groups of compounds acting as

primary antioxidants or free radical scavengers, it was reasonable to determine their
total amount in the hot water extract and its fractions. Total phenols, flavonoids and
flavanols were determined spectrophotometrically using Folin-Ciocalteu assay,
Al3? complexation reaction and vanillin-HCl assay, respectively. The data on
yields, total phenols, total flavonoids and flavanol content of all extracts are shown
in Table 1. The amount of extractable components from the CHWE ranged from
15.3 mg/g (TBME fraction extract) to 76.4 mg/g (water extract). According to the
data presented in Table 1, the amounts of total phenol contents decrease with
polarity of the solvent, while the total extraction yields increased. These results

1.04

e
1.03

1.02

c
c
1.01
b
Control PF = 1
PF

1.00

d
0.99 a

0.98

0.97

0.96
CHWE TBME EtOAc BuOH H2O BHT

Fig. 3 Effect of trembling aspen hot water extract and its fractions on canola oil thermoxidation stability
by DSC analysis. Different letters represent significant differences at 0.05 probability level

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Wood Sci Technol (2009) 43:457–470 465

Table 1 Total phenols, flavonoids and flavanols of the trembling aspen bark crude water extract and its
various fractions
Extract/fractions Yield Total phenols Total flavonoids Total flavanols
(mg extract/g (mg gallic (mg quercetin/g (mg catechin/g
o.d. bark) acid/g extract) extract) extract)

CHWE 140.1 ± 6.0 113.5 ± 6.1 11.5 ± 0.2 10.2 ± 0.4

TBME 15.3 ± 0.7 218.0 ± 7.6 12.5 ± 0.2 18.3 ± 0.5
EtOAc 20.1 ± 0.9 159.9 ± 6.9 9.3 ± 0.0 9.7 ± 0.1
BuOH 28.3 ± 1.2 139.1 ± 5.4 10.0 ± 0.1 9.1 ± 0.3
H2O 76.3 ± 3.3 21.1 ± 0.2 9.9 ± 0.0 5.2 ± 0.1

CHWE crude hot water extract, TBME tert-butyl methyl ether fraction, EtOAc ethyl acetate fraction,
BuOH butanolic fraction, H2O aqueous extract

(Table 1) seem to indicate that the major phenolic constituents of the trembling
aspen bark are phenolics other than flavonoids. Similar results have already been
presented in the literature. The phenolic glycosides represent an important class of
the extractives found in this species (Thieme 1967). Studies on the hot water
extractives of Populus tremuloides bark have identified the phenylglycosides
salicin, tremuloidin and salireposide (Pearl et al. 1961; Pearl and Darling 1971). In
addition to these glucosides, salicyl alcohol, gentisyl alcohol, benzoic acid and some
sugars were identified as important components of these extractives. The major
flavonoids of trembling aspen bark seem to be, according to our results, the
flavanols. This remains a challenge for future identification work.

Discussion

In this study, the antioxidant activities of the trembling aspen bark hot water extract
and its fractions were measured by different methods, i.e. DPPH assay, phospho-
molybdenum assay and canola oil thermoxidation assay. The extract/fractions were
found to have different levels of antioxidant activity in different systems tested.
These experimental results seem to indicate that the hot water extract and its
fractions could contain different antioxidants, since they are demonstrating varying
reactivity in the three in vitro models tested in this work (Figs. 1, 2, 3). However,
the most efficient extracts BuOH, EtOAc and TBME for a chosen method remain
efficient when tested by the other two methods, but not in the same order. Measuring
the total antioxidant activity using only one assay seems therefore to be rather
unrealistic, yet there are numerous published results based on application of only
one selected assay claiming to measure total antioxidant activity in vitro. As
phenolic content of a sample is an important factor determining its antioxidant
capacity, it was correlated with the results of the antioxidant assays performed on
different extract fractions. The results of different antioxidant assays were also
compared and correlated between themselves, using linear regression analysis. The
same procedure has been used by several authors (Anagnostopoulou et al. 2006;
Chen et al. 2006; Smejkal et al. 2007; Nsimba et al. 2008). Correlation between the

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466 Wood Sci Technol (2009) 43:457–470

results of different antioxidant assays is shown in Table 2. Flavonoid and flavanol

contents are significantly lower than phenol contents, and on the other hand it was
impossible to establish the correlations between the antioxidant activities and the
flavonoids and flavanols contents, these relations are not presented here.
Antioxidant activity of an extract is often associated with their redox properties,
which allows them to act as reducing agents (Yildirim et al. 2000; Siddhuraju et al.
2002). Jayaprakasha et al. (2003) reported that the antioxidant activity by method I
of different extracts may mainly depend on the presence of polyphenols which may
act as reducing agents, by donating the electrons and reacting with free radicals to
convert them to more stable products, thus terminating the free radical chain
reaction. The results of the present study could be interpreted in a similar way.
TBME, EtOAC, BuOH fractions which contain the highest amounts of total phenols
present the highest reduction potential, as determined by method I. This is
demonstrated by the relatively high correlation determined between total phenol
contents and antioxidant activity by method I (Table 2).
The antioxidant activity of a given compound or extract is also often associated
to its radical scavenging activity. In general, it is considered that the antioxidant
capacity of a molecule is equivalent to its capacity to react with free radicals. DPPH
is a commercially available free radical reagent and relatively stable compared to
the highly reactive superoxide and hydroxyl species that are primarily responsible
for oxidative damage in biological systems, and is widely used to test the ability of
compounds to act as free radical scavengers or hydrogen donors (Brand-Williams
et al. 1995; Gao et al. 2006; Garcı́a-Pérez et al. 2008). The results of the present
study indicate that phenolic compounds are powerful scavengers of free radicals as
demonstrated by a high correlation of DPPH free radical scavenging activity with
the total phenol contents of the extract/fractions (r = 0.98). Similar results were
obtained by Nsimba et al. (2008). It seems likely that the free scavenging activity of
the crude hot water trembling aspen bark extract and its fractions may be attributed
to phenolic compounds. The data obtained revealed that CHWE and the fractions
isolated from trembling aspen bark are free radical scavengers and primary
antioxidants that react with DPPH radical, which may be attributed to its proton
donating ability.
The DSC method is an available method for assessing oxidative deterioration of
vegetable oils with or without added antioxidant (Tan and Man 2002; Gouveia et al.
2006; Garcı́a-Pérez et al. 2008). In the present study, method III measures the effect
of trembling aspen bark hot water extract and its fractions on the thermoxidation of
CO. Food processing involving heat (e.g. deep-fat frying and microwave heating) is

Table 2 Comparison between different antioxidant assays as represented by correlation coefficient (r)
Variable Total phenols Method I Method II Method III

Total phenols – 0.79 0.98 0.58

Method I 0.79 – 0.86 0.74
Method II 0.98 0.86 – 0.69
Method III 0.58 0.74 0.69 –

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Wood Sci Technol (2009) 43:457–470 467

not beneficial for the chemical changes and nutritional value of vegetable oils. Lipid
oxidation is one of the major deteriorative reactions in heated oils, and often results
in a significant loss of quality (Tan and Man 2002). The role of phenolic compounds
in this measurement is to break the oxidation chain reaction by acting in the phase of
propagation. When a phenolic compound (AH) is present, it donates its mobile H
atom to free radicals; if the A
radical produced is unreactive, it stops the kinetic
chain of the oxidation (and therefore it is called a chain breaking antioxidant) and it
reacts only (or mainly) in new termination steps (Rousseau-Richard et al. 1988):
LO2
þ AH ! LO2 H þ A
ðp3 Þ
L
þ AH  LH þ A
ðp4 Þðp4 Þ
LO2
þ A
! ðt4 Þ
L
þ A
! non radical products ðt5 Þ
A
þ A
! ðt6 Þ
In this way, phenolic compounds increase the thermoxidative stability of the oil
until complete consumption of the antioxidant agent. However, the moderate
correlation obtained (r = 0.58) between the antioxidant activities of the studied
extracts by method III and total phenol contents indicates that other important
factors should be taken into consideration. Nevertheless, as found by the two other
methods, TBME, EtOAC, BuOH fractions which contain the highest amounts of
total phenols increase the thermoxidative stability of CO, whereas CHWE and H2O
which contain the lowest total phenols enhance it. Contrary to the methods I and II,
BHT which is a very good fat-soluble phenolic compound protects better CO
thermoxidation than the more efficient fractions. One should also consider the oil
solubility of the extracts/fractions having in mind their hydrophilic nature. On the
other hand, this suggested that the potent antioxidant components present in these
fractions (TBME, EtOAc and BuOH) seemed to be more soluble in a nonpolar
solvent. Similar results were mentioned by Jia et al. (2007) for the methanolic
extracts from Cassia tora L. Another factor which should be considered is the
stability of the extracts at high temperature. The oxidative stability test by DSC is
conducted at high temperature. Some authors (Yen and Hung 2000; Xu et al. 2007)
reported that some phenolic extracts are unstable and can lose their antioxidant
efficiency at high temperatures. This observation was confirmed by the moderate
antioxidant activity in canola oil tests at high temperature for the best antioxidant
extracts (TBME, EtOAc, and BuOH fractions) with PF values near unity but
significantly different from the control. Additionally, two hypotheses can explain
the pro-oxidant effects for the CHWE and H2O bark extract fractions. In order that a
given phenolic compound is an efficient inhibitor of oil oxidation, the free radical
formed should be poorly reactive and should not participate in the chain reactions.
On the contrary, if A
radical produced is reactive (reaction p-4 of the propagation
process), one would have a pro-oxidant system. The other factor that can explain the
pro-oxidant activity of CHWE and H2O may be attributed to the presence of
inorganic extractives. The hot water extraction will not only solubilise the
phenolics, but also the sugars and some mineral constituents which are likely to also

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468 Wood Sci Technol (2009) 43:457–470

contain the transition metal cations. Ngueho Yemele et al. (2008) have reported
5.1% of ash content in the bark of P. tremuloides. Transition metals enhance the rate
of oxidation of edible oils by increasing the rate of generation of free radicals from
fatty acids or hydroperoxides (Nawar 1996).
All these considerations indicate the need to apply several methods for the
determination of the antioxidant activity which cannot be determined unambigu-
ously by just one prescribed method. In that way, method I enables the
determination of the total antioxidant activity based on all reducing compounds
present in the extracts, while method II allows determining the antioxidant activity
of phenolic compounds present in the extracts. These two methods are therefore
complementary (r = 0.86). Method III allows to determine the global influence of a
given compound or extract, either inhibitory (antioxidant) or accelerating (pro-
oxidant) and seems to better translate the reality of the support-organic substrate
exposed to the phenomenon of oxidation and the complexity of the reactions taking
place.

Conclusion

The present results demonstrate that the trembling aspen bark could serve as a
source for natural antioxidant agents under certain conditions. The application of
different methods of antioxidant activity determination has shown the interesting
antioxidant potential of trembling aspen bark hot water extract and its various
fractions. The properties of the studied bark extract and its fractions, determined in
this research, such as their polyphenol contents, combined with the important
antioxidant activities of these materials demonstrate the potential of their
application like free radical scavengers, reducing agents and lipid thermoxidation
inhibitors. For their future utilization, further research on qualitative/quantitative
chemical composition as well as their toxicity should be carried out. Research is in
progress to isolate and identify the antioxidant components in TBME, EtOAc and
BuOH fractions.

Acknowledgments The authors are very grateful to the Fonds québécois de recherche sur la nature et
les technologies (FQRNT) and Fonds de la recherche forestière du Saguenay–Lac Saint Jean for the
financial support to this project and to Mr. Yves Bédard for his technical support.

References

Anagnostopoulou MA, Kefalas P, Papageorgiou VP, Assimopoulou AN, Boskou D (2006) Radical
scavenging activity of various extracts and fractions of sweet orange-peel (Citrus sinensis). Food
Chem 94:19–25
Arts ICW, Hollman PCH (2005) Polyphenols and disease risk in epidemiologic studies. Am J Clin Nutr
81:317S–325S
Brand-Williams W, Cuvelier ME, Berset C (1995) Use of a free radical method to evaluate antioxidant
activity. Lebensm Wiss Technol 28:25–30
Brighente IMC, Dias M, Verdi LG, Pizzolatti MG (2007) Antioxidant activity and total phenolic content
of some Brazilian species. Pharm Biol 45:156–161

123
Wood Sci Technol (2009) 43:457–470 469

Chang YP, Mitchelle RL (1955) Chemical composition of common North American pulpwood barks.
Tappi 38:315–320
Chen FA, Wu AB, Shieh P, Kuo DH, Hsieh CY (2006) Evaluation of the antioxidant activity of Ruellia
tuberosa. Food Chem 94:14–18
Cui YY, Xie H, Wang JF (2005) Potential biomedical properties of Pinus massoniana bark extract.
Phytother Res 19:34–38
Faber HB Jr (1960) The methanol extractable aromatic materials in the inner bark of P. tremuloides.
Tappi 43:406–413
Gao H, Shupe TF, Hse CY, Eberhardt TL (2006) Antioxidant activity of extracts from the bark of
Chamaecyparis lawsoniana (A. Murray) Parl. Holzforschung 60:459–462
Garcı́a-Pérez ME, Diouf PN, Stevanovic T (2008) Comparative study of antioxidant capacity of yellow
birch twigs extracts at ambient and high temperatures. Food Chem 107:344–351
Goncalves C, Dinis T, Batista MT (2005) Antioxidant properties of proanthocyanidins of Uncaria
tomentosa bark decoction: a mechanism for anti-inflammatory activity. Phytochemistry 66:89–98
Gouveia AF, Duarte C, da Costa MLB, Bernardo-Gil MG, Moldão-Martins M (2006) Oxidative stability
of olive oil flavoured by Capsicum frutescens supercritical fluid extracts. Eur J Lipid Sci Technol
108:421–428
Hossfeld RL, Hunter WT (1958) The petroleum ether extractives of aspen bark. Tappi 41:359–362
Jayaprakasha GK, Selvi T, Sakariah KK (2003) Antibacterial and antioxidant activities of grape (Vitis
vinifera) seed extracts. Food Res Int 36:117–122
Jia ZB, Tao F, Guo L, Tao GJ, Ding XL (2007) Antioxidant properties of extracts from juemingzi (Cassia
tora L.) evaluated in vitro. Lebensm Wiss Technol 40:1072–1077
Kahl R, Kappus H (1993) Toxicology of the synthetic antioxidants BHA and BHT in comparison with the
natural antioxidant Vitamin E. Z Lebensm Unters Forsch 196:329–338
Ku CS, Mun SP (2008) Antioxidant properties of monomeric, oligomeric, and polymeric fractions in hot
water extract from Pinus radiata bark. Wood Sci Technol 42:47–60
Lalas S, Dourtoglou V (2003) Use of rosemary extract in preventing oxidation during deep-fat frying of
potato chips. J Am Oil Chem Soc 80:579–583
Moure A, Cruz JM, Franco D, Dominguez JM, Sineiro J, Dominguez H, Nunez MJ, Parajo JC (2001)
Natural antioxidants from residual sources. Food Chem 72:145–171
Nawar WW (1996) Lipids. In: Fennema OR (ed) Food chemistry. Marcel Dekker, New York, pp 225–319
Ngueho Yemele MC, Koubaa A, Diouf PN, Blanchet P, Cloutier A, Stevanovic T (2008) Effects of hot
water treatment of black spruce and trembling aspen bark raw material on the physical and
mechanical properties of bark particleboard. Wood Fiber Sci accepted 24 January 2008 (in press)
Nsimba RY, Kikuzaki H, Konishi Y (2008) Antioxidant activity of various extracts and fractions of
Chenopodium quinoa and Amaranthus spp. seeds. Food Chem 106:760–766
Parke DV, Lewis DFV (1992) Safety aspects of food preservatives. Food Addit Contam 9:561–577
Pearl IA, Darling SF (1971) Studies on barks of family of salicaceae.27. Hot water phenolic extractives of
bark and leaves of diploid Populus tremuloides. Phytochemistry 10:483–484
Pearl IA, Darling SF, De Haas H, Loving BA, Scott DA, Turley RH, Werth RE (1961) Preliminary
evaluation for glycosides of barks of several species of the genus Populus. Tappi 44:475–478
Pilarski R, Zielinski H, Ciesiolka D, Gulewicz K (2006) Antioxidant activity of ethanolic and aqueous
extracts of Uncaria tomentosa (Willd.) DC. J Ethnopharmacol 104:18–23
Prieto P, Pineda M, Aguilar M (1999) Spectrophotometric quantitation of antioxidant capacity through
the formation of a phosphomolybdenum complex: specific application to the determination of
vitamin E. Anal Biochem 269:337–341
Pronin D, Vaughan CL (1968) A literature survey of Populus species with emphasis on P. tremuloides.
Revised edition ed Forest Service Research Note FPL-0180 Madison, WI US Department Of
Agriculture, pp 67
Romero N, Robert P, Masson L, Ortiz J, Gonzalez K, Tapia K, Dobaganes C (2007) Effect of alpha-
tocopherol, alpha-tocotrienol and Rosa mosqueta shell extract on the performance of antioxidant-
stripped canola oil (Brassica sp.) at high temperature. Food Chem 104:383–389
Rousseau-Richard C, Richard C, Martin R (1988) Étude cinétique de l’influence complexe, pro ou
antioxydante, de dérivés phénoliques sur l’oxydation induite d’un substrat polyinsaturé. I. Analyse
du mécanisme réactionnel. J Chim Phys 85:167–173
Sanchez J, Melchor G, Martinez G, Escobar A, Faure R (2006) Antioxidant activity of Rhizophora
mangle bark. Fitoterapia 77:141–143

123
470 Wood Sci Technol (2009) 43:457–470

Scalbert A, Monties B, Janin G (1989) Tannins in wood—comparison of different estimation methods. J

Agric Food Chem 37:1324–1329
Siddhuraju P, Mohan PS, Becker K (2002) Studies on the antioxidant activity of Indian Laburnum
(Cassia fistula L.): a preliminary assessment of crude extracts from stem bark, leaves, flowers and
fruit pulp. Food Chem 79:61–67
Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Analysis of total phenols and other oxidation
substrates and antioxidants by means of Folin-Ciocalteu reagent oxidants and antioxidants.
Academic, San Diego, pp 152–178
Smejkal K, Holubova P, Zima A, Muselik J, Dvorska M (2007) Antiradical activity of Paulownia
tomentosa (Scrophulariaceae) extracts. Molecules 12:1210–1219
Su YL, Xu JZ, Ng CH, Leung LK, Huang Y, Chen ZY (2004) Antioxidant activity of tea theaflavins and
methylated catechins in canola oil. J Am Oil Chem Soc 81:269–274
Suja KP, Abraham JT, Thamizh SN, Jayalekshmy A, Arumughan C (2004) Antioxidant efficacy of
sesame cake extract in vegetable oil protection. Food Chem 84:393–400
Tan CP, Man YBC (2002) Recent developments in differential scanning calorimetry for assessing
oxidative deterioration of vegetable oils. Trends Food Sci Tech 13:312–318
Thieme H (1967) Über die Phenolglykoside der Gattung Populus. Planta Med 15:35–40
Wang MF, Li JG, Rangarajan M, Shao Y, Lavoie EJ, Huang TC, Ho CT (1998) Antioxidative phenolic
compounds from sage (Salvia officinalis). J Agric Food Chem 46:4869–4873
Xu GH, Ye XQ, Chen JC, Liu DH (2007) Effect of heat treatment on the phenolic compounds and
antioxidant capacity of citrus peel extract. J Agric Food Chem 55:330–335
Yanishlieva NV, Marinova EM, Marekov IN, Gordon MH (1997) Effect of an ethanol extract from
summer savory (Saturejae hortensis L) on the stability of sunflower oil at frying temperature. J Sci
Food Agric 74:524–530
Yen GC, Hung CY (2000) Effects of alkaline and heat treatment on antioxidative activity and total
phenolics of extracts from Hsian-tsao (Mesona procumbens Hemsl.). Food Res Int 33:487–492
Yildirim A, Mavi A, Oktay M, Kara AA, Algur OF, Bilaloglu V (2000) Comparison of antioxidant and
antimicrobial activities of tilia (Tilia argentea desf ex DC), sage (Salvia triloba L.), and black tea
(Camellia sinensis) extracts. J Agric Food Chem 48:5030–5034

123
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