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Zimniewska M. Antioxidant Activity of Fibres Originating from Traditional Varieties of Polish Flax Plants.
FIBRES & TEXTILES in Eastern Europe
2015; 23, 6(114): 41-47.
DOI:
10.5604/12303666.1167418
Antioxidant Activity of Fibres Originating
from Traditional Varieties of Polish Flax Plants
Malgorzata Zimniewska
Institute of Natural Fibres & Medicinal Plants,
Poznan, Poland
E-mail: gosiaz@iwnirz.pl
Abstract
This paper describes the examination of raw ax bres in terms of their antioxidant activity.
Fibres originating from ve varieties of ax plants cultivated in Poland: Artemida, Modran,
Sara, Nike and Luna were extracted with the application of different methods: dew retting
and water retting. The extraction method has an inuence on the bre chemical composi-
tion, resulting in different levels of bre antioxidant properties. The antioxidant activity
of ax bres was evaluated with the application of ferric ion reducing antioxidant power
(FRAP). The results of the study indicated differences in the bioactivity of ax bres linked
with the method of their extraction applied as well as the lignin and phenolic acid content
in the bre chemical composition.
Key words: ax bres, antioxidant activity, dew retting, water retting, lignin, ferulic acid,
coumaric acid.
n Materials and methods
Flax bres extracted from straw with the
application of two different methods e.g.
dew retting and water retting were used
as the material for the investigation. The
bres obtained from ve varieties: Arte-
mida, Modran, Sara, Nike and Luna by
dew retting and water retting were tested
in order to determine differences in their
chemical composition and biological ac-
tivity. All ax varieties mentioned were
developed by the Institute of Natural Fi-
bres and Medicinal Plants, differing in
their resistance against fungi and some
harmful climatic conditions.
Generally dew retting is applied after
the mechanical pulling of stalks and de-
seeding. Flax stems are spread evenly in
a eld and left for 3 - 7 weeks, depend-
ing on climatic conditions. In this time,
microorganisms, mainly fungi, secrete
enzymes that degrade the following sub-
stances: pectin, proteins, sugars, starch,
fats and waxes, tannins, and minerals
[13, 14]. Water retting is a traditional ret-
ting method where the ax stalks are held
below the water surface in dams, ditches
or slow running streams and rivers. Now
water retting is conducted in modern ret-
ting tanks. In water-retting, a variety of
ax bres and to nd an explanation of
this phenomenon by the assessment of
lignin as well as phenolic acid contents
in dew retted and water retted ax bres.
Phenolic acids are known as strong anti-
oxidants, [4 - 8], especially ferulic acid,
which is used, apart from other applica-
tions, as a cosmetic ingredient to protect
the skin against the unfavorable effect of
the environment and to act as an antiag-
ing agent [6]. Figure 1 presents oxidis-
ing phenolic compounds. Based on an
effectively scavenging chain reaction
and deleterious radicals and suppressing
radiation induced oxidative reactions,
phenolic acids serve for preserving the
physiological integrity of cells exposed
to both air and for impinging UV radia-
tion [9, 10].
The presence of phenolic substances in
ax bres has been studied by a few re-
searchers [11, 12], but there is no analy-
sis of bres antioxidant activity.
The aim of the current study was to in-
vestigate the antioxidant activity of bres
extracted from different ax plant varie-
ties in relationship to the bre extraction
method applied, which has an effect on
the bre chemical composition.
DOI: 10.5604/12303666.1167411
n Introduction
Flax bres are known as favorable in
contact with human skin. A study on
linen clothing’s inuence on the human
body conducted in steady conditions
proved their positive effect in ensuring
well-being, without causing the desyn-
chronisation of muscle motor units, and
thus without increasing of the tendency
to tiredness of users [1]. Another study
showed that wearing of natural bre py-
jamas with hydrophilic properties inu-
ences the activity of sebaceous glands
positively, which improves resistance
to skin diseases [2]. Researchers proved
that linen clothes characterised by hydro-
philic properties do not cause an increase
in reactive oxygen species and oxidative
stress as opposed to garments made of
synthetic bres characterised by hydro-
phobic properties [3]. Linen garments
are known as very breathable and provide
optimal comfort for users, especially in
hot climate conditions. It allows for the
exchange of air captured in the skin-
clothing area through the fabric with the
outer environment, giving the rapid es-
cape of moisture (sweat) from the skin
and thereby hindering bacteria growth.
This is related to the higher water reten-
tion of ax in comparison to other bres
and particularly ax ability for moisture
management.
There is no information in research litera-
ture on the original antioxidant properties
of ax bres obtained from traditional
non-modied varieties in relationship to
the bre extraction method.
The current study was conducted to de-
termine the inherent bioactivity of raw Figure 1. Oxidizing phenolic compounds.
FIBRES & TEXTILES in Eastern Europe 2015, Vol. 23, 6(114)
42
anaerobic bacteria is present and they are
considered as the primary agents respon-
sible for bre release [15]. Several spe-
cies of bacteria have been identied and
investigated during tank retting, of which
spore-forming Clostridium spp. have
been shown to contribute considerably to
pectin-degrading activity and, therefore,
to retting.
All bre types used for the study were
extracted from ax plants grown in the
same vegetation season. The parameters
of processes applied for bre extraction
are presented in Table 1.
Evaluation of ax chemical
composition
Flax bres contain in their chemical
composition natural polymers like cel-
lulose, lignin, hemicelluloses, pectin
as well as waxes, fats, and others. The
chemical composition of all the types of
bres extracted was evaluated with the
use of relevant standards:
n Wax and fat content: Branch Standard
BN – 86/7501-10
n Pectin content: a method developed at
INF&MP
n Lignin content: Branch Standard BN –
86/7501-11
n Cellulose content: PN – 92/P-50092
n Hemicelluloses content: Branch
Standard BN – 77/7529-02
n Phenolic acids, e.g. coumaric and fer-
ulic acid, in ax bres was assessed
by the high-performance liquid chro-
matography (HPLC) method.
Additionally lignin extracted from dew
retted bres coming from different varie-
ties of ax plants were tested to show the
presence of phenols. Lignin was extract-
ed from bres according to the method
described in Standard: BN – 86/7501-11.
Fourier Transform Infrared Spectros-
copy was used to determine the absorp-
tion spectra for lignin. The trials were
performed with a spectrophotometer
- FT-IR NICOLET iS10, (Thermo Sci-
entic, USA) at infrared wavelengths of
350 – 4000 cm-1
Assessment of bre antioxidant
activity
Antioxidants are a large group of natural
and synthetic compounds with the abil-
ity to reduce free radicals and prevent
some amount of oxidative damage that
destroys and depletes the skin function
and structure while also preventing some
of the degenerative effects in skin caused
by sun exposure. In this study, two meth-
ods were used for the testing of ax bre
antioxidant capacity:
n Ferric Ion Reducing Antioxidant Pa-
rameter (FRAP)
10 mmol/L of 2,4,6-tripyridyl-s-tria-
zine (TPTZ) in 40 mmol/L of HCL and
20 mmol/L of FeCl3·6H2O. The working
FRAP reagent was freshly prepared by
mixing 25 ml of acetate buffer, 2.5 ml of
TPTZ solution and 2.5 ml of FeCl3·6H2O.
The freshly prepared mixture was incu-
bated at 37 °C in a water bath for ve
minutes and then a blank reading was
taken spectrophotometrically at 593 nm.
After that, 30 µl of the extract or standard
and 90 µl of distilled water were added
to 900 µl of the working FRAP reagent.
Absorbance was measured at 0 min im-
mediately upon addition of the working
FRAP reagent after vortexing. Thereafter
an absorbance reading was taken after
four minutes [18, 19]. Three repetitions
of the antioxidant test were conducted for
each bre sample.
n Results
Chemical composition of ax bres
The results of tests of the chemical com-
position of the materials used in the cur-
rent study i.e. ax bres extracted from
different varieties of brous plants with
the use of dew and water retting are
shown in Table 2.
The results of evaluation of the chemi-
cal composition of ax bres indicates
diversity in the share of particular com-
ponents in different plant varieties, which
is strongly related to the bre extraction
method.
All varieties of water retted ax bres
tested were characterised by a higher
content of cellulose and lower content
of lignin and hemicellulose in compari-
son with the relevant dew retted bres.
It means that the process of water retting
caused better removal of non-cellulosic
compounds (excluding waxes) from -
bres, giving them properties that were
more suitable for textile processing i.e.
better quality and spinability.
On the other hand, all the varieties of
dew retted ax bres showed a higher
share of lignin, the compound related to
the bioactivity of the bres. Analysis of
ax varieties indicated that water retted
Artemida showed the highest cellulose
content, but dew retted Modran and Sara
contained the largest amount of lignin.
The statistical analysis of results of ax
chemical composition was conducted to
verify if the differences are statistically
Table 1. Conditions of the processes applied for bre extraction
Fibre extraction method Time Temperature Equipment
Dew retting 43 days Ambient weather conditions Field
Water retting 74 h Water temp. 32 – 33 °C Lab-scale tank
Figure 2. DPPH radical.
O2N
O2N
NO2
N N
o
n 2,2-Diphenyl-1-picrylhydrazyl Radi-
cal Scavenging Capacity Assay
(DPPH) [16]
The DPPH* (Figure 2) radical is one of
the few stable organic nitrogen radicals,
which bears a deep purple colour. This
assay is based on the measurement of
the reducing ability of antioxidants to-
ward DPPH*. The ability was evaluated
by measuring the decrease in its absorb-
ance. Antioxidant assays are based on
measurement of the loss of DPPH colour
at 515 nm after a reaction with test com-
pounds, with the reaction being moni-
tored by a spectrometer. The percentage
of remaining DPPH* (DPPH*REM) is
proportional to the antioxidant concen-
tration, and the concentration that causes
a decrease in the initial DPPH* con-
centration by 50% is dened as EC50.
The time needed to reach a steady state
with EC50 is dened as TEC50.
The Ferric Ion Reducing Antioxidant
Power (FRAP) assay takes advantage
of electron-transfer reactions, Figure 3.
The ferric reducing activity (FRAP) of
the bres extracts were estimated accord-
ing to the method developed by Benzie
and Strain [17]. The reaction mixture
contained 300 mmol/L of acetate buffer,
43
FIBRES & TEXTILES in Eastern Europe 2015, Vol. 23, 6(114)
similar levels of absorbance in that
band. The level of phenolic acid absorb-
ance shown by the FTIR test conrmed
the investigation of Holser, who evalu-
ated ferulic and p-coumaric acid spec-
tra in the 650 - 1500 cm−1 region [23].
The FTIR spectra of lignin extracted
from bres coming from different vari-
eties of ax plant conrmed the results
of lignin from other ax varieties in this
band were inconsiderable. C-O stretch-
ing vibration in band 1140 – 1230 cm-1
conrmed the presence of phenols. In
this case, the absorbance was the high-
est for lignin obtained from the Modran
variety of ax, while the lowest was
observed for lignin extracted from the
Luna variety. Other ax varieties showed
signicant. Analysis with the use of Sha-
piro–Wilk and U Mann–Whitney and T-
Student tests for a condence interval of
95% conrmed signicant differences in
the share of particular components of -
bres for p < 0.05.
The test for phenolic substance content in
ax bres proved that all varieties of dew
retted ax bres contained some amount
of ferulic acid (Figure 4) and coumaric
acid (Figure 5), which are known as
strong antioxidants. The phenolic acid
content in ax bres is shown in Table 3.
In that test, phenolic acids could be de-
tected only in dew retted bres, because
in the case of water retted bres traces of
the acids were extremely weak and their
detection by the HPLC method applied
was impossible.
Apart from the fact that all varieties of
dew retted ax bres contained a higher
amount of lignin and hemicellulose than
in comparison to water retted bres,
HPLC analysis of the bre proved that
the dew retted bres contained some
amount of phenolic acids, while higher
amounts of phenolic acid occurred in
Modran and Sara varieties. It is known
that ferulic acid is a component of a pri-
mary cell wall and is bonded with lignin
and hemicellulose in plants [7, 8]. Fig-
ure 6 (see page 44) shows the FTIR spec-
tra determined for lignin extracted from
dew retted bres coming from different
ax varieties. Characteristic absorption
bands in infrared shown in Table 4 al-
lowed to nd the phenols at the spectra
of extracted lignin.
Lignin extracted from all varieties of
ax showed a broad band at 3410 –
3460 cm−1, attributed to the hydroxyl
groups in phenolic structures. The high-
est absorbance in that band was observed
in the case of lignin extracted from Sara
ax. Differences between the absorbance
Table 2. Chemical composition of ax bres extracted from brous plants.
Fibre Fibrous plant
variety
Lignin Cellulose Waxes and fats Hemicelluloses Pectin
content, % SD, % content, % SD, % content, % SD, % content, % SD, % content, % SD, %
Dew retted
ARTEMIDA 6.33 0.41 64.86 0.64 1.30 0.02 19.31 0.14 1.32 0.17
MODRAN 7.96 0.11 64.16 1.22 1.72 0.03 21.64 0.13 1.44 0.09
SARA 7.42 0.22 62.21 0.22 1.63 0.02 22.30 0.14 1.20 0.13
NIKE 6.40 0.41 64.31 0.87 1.19 0.03 20.91 0.02 1.72 0.16
LUNA 5.18 0.17 69.61 0.14 1.06 0.13 16.86 0.04 2.12 0.19
Water retted
ARTEMIDA 3.51 0.04 74.01 0.49 4.40 0.00 14.57 0.05 0.58 0.15
MODRAN 3.52 0.02 72.42 1.00 4.25 0.02 15.30 0.31 1.43 0.23
SARA 3.18 0.13 70.76 0.14 4.43 0.11 17.31 0.17 2.15 0.08
NIKE 3.05 0.13 71.31 0.91 4.41 0.12 15.55 0.19 1.35 0.07
LUNA 2.56 0.21 72.77 0.35 4.49 0.02 13.91 0.08 0.65 0.02
Figure 3. FRAP - electron-transfer reaction.
Figure 4. Ferulic acid.
Table 3. Phenolic acid content in ax bres (*the non-determined trace amounts of the
acids equal to detection limits are shown in unit mg/ml of the calibration curve according
to the test method).
Fibre Fibrous plant
variety
Content of coumaric
acid, mg/100 g
SD coumaric
acid
Content of ferulic
acid, mg/100 g
SD ferulic
acid
Dew
retted
ARTEMIDA 5.580 0.118 4.645 0.092
MODRAN 6.136 0.448 5.128 0.385
SARA 6.021 0.394 5.014 0.329
NIKE 5.688 0.293 4.739 0.241
LUNA 5.708 0.984 4.859 0.803
Water
retted
ARTEMIDA
< 0.0022 mg/ml* - < 0.0019 mg/ml* -
MODRAN
SARA
NIKE
LUNA
Figure 5. p-coumaric acid.
[Fe(III)(TPTZ)2]3+ [Fe(II)(TPTZ)2]2+, lmax = 593 nm
+ antioxidant
- e
FIBRES & TEXTILES in Eastern Europe 2015, Vol. 23, 6(114)
44
of HPLC bre analysis that proved that
Modran and Sara ax bres contained
the highest level of phenolic acids.
The relationship between phenolic acid
and lignin content in the dew retted ax
bres tested was analysed and presented
in Figure 7. The relationship between
lignin and phenolic acid content was
high (Figure 7), as conrmed by corre-
lation analysis - the Pearson correlation
coefcient was 0.76. The analysis of cor-
relation was conducted regardless of the
low number of observations in order to
have preliminary information about the
relationship between the main bre com-
ponents and bioactivity. All varieties of
water retted ax bres were character-
ised by lower amounts of lignin, as well
as they had only trace amounts of phenol-
ic acid, unable to detect precisely by the
method used. The reason for this is clear
because ferulic acid is easily soluble in
water and can be readily removed from
bres by water during the retting process.
Coumaric acid is poorly soluble in water
and its removal could be partial.
Antioxidant activity of ax bres
FRAP tests were conducted for differ-
ent sample concentrations. It is known
that the value of FRAP increases with
increasing sample concentration.
The results of the test of the antioxi-
dant activity of ax bres assessed with
the use of the FRAP method shown in
Figure 8 proved that dew retted bres
Figure 6. FTIR spectra of lignin extracted from bres obtained from different varieties of ax plant: 1. – lignin extracted from Artemida
bre, 2. - lignin from Modran bre, 3. – lignin from Sara bre, 4. – lignin from Nike bre, 5. – lignin from Luna bre.
Table 4. Characteristic absorption bands in infrared [20 - 22].
Bonds Compound type Band range, cm-1
C-H stretching vibrations aromatic rings 2800 - 3000
C-H bending vibrations (in-plane) aromatic rings 1000 - 1100
C-H bending vibrations (off-plane) aromatic rings 675 - 870
C-H bending vibrations methyl group -CH3
methylene group -CH2-
1430 - 1470 and 1375
1430 - 1470
C-C stretching vibrations aromatic rings 1500 - 1600
C-C stretching vibrations quaiacyl ring 1270
C-C stretching vibrations syringyl ring 1330
C-O stretching vibrations phenols
carboxylic acids
1140 - 1230
1250
C=O stretching vibrations aldehydes and ketones
carboxylic acids
1675 - 1725
1680 - 1725
O-H stretching vibrations phenols
carboxylic acids
3200 - 3600
2500 - 3000
O-H bending vibrations carboxylic acids 1400 and 920
A
A
BB
1166.06
1166.06
1204.81
0.15
0.10
0.05
0.01
Absorbance
4000 3000 2000 1000
Wavenumber, cm-1
1. Artemida
2. Modran
3. Sara
4. Nike
5. Luna
Figure 7. Relationship between lignin and phenolic acid content in dew retted ax bres.
y = 8,675 + 0,305x
A r te mi da
Modran
Sara
Nike
Luna
5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5
Lignin content
10,0
10,2
10,4
10,6
10,8
11,0
11,2
11,4
Content of phenolic ac id [mg/100g]
A r te mi da
Modran
Sara
Nike
Luna
y = 8.675 + 0.305x
Content of phenolic acid, mg/100 g
Lignin content
45
FIBRES & TEXTILES in Eastern Europe 2015, Vol. 23, 6(114)
The coefcient of the Pearson linear cor-
relation reached the following values:
n FRAP and both phenolic acid contents
- 0.89
n FRAP and ferulic acid – 0.88
n FRAP and cumaric acid is 0.9
The correlation analysis conrmed very
strong relationship between phenolic
acid content and antioxidant activity of
dew retted ax bres.
The results of FRAP tests of ax bres
indicated an inherent antioxidant activity
of raw ax bres; however, it was not the
same for all ax plant varieties and was
strongly related to the ax variety and
method of bre extraction. The value of
the ferric reducing antioxidant power of
the dew retted Modran bres tested was
the highest in comparison to the activ-
ity of the other varieties of ax plants.
The dew retted Modran ax bres tested
tent in the bre extracts. From all samples
tested, dew retted bres extracted from
the Modran variety showed the highest
content of lignin as well as coumaric and
ferulic acid, which resulted in the highest
ferric reducing antioxidant power. The
second best was Sara variety.
The relationship between the phenolic
acid content and antioxidant activity of
the dew retted ax bres tested is pre-
sented in Figure 9 (see page 46). The line
of trend drawn in the diagram indicated
a strong relationship between the bre
ability to reduce free radicals and the
phenolic acid content in the bres.
Statistical analysis conducted for pre-
liminary evaluation of the relationship
between the antioxidant activity of ax
bres tested by the FRAP method and the
phenolic acid content indicated a strong
correlation.
extracted from the following varieties:
Modran, Sara and Luna have higher abil-
ity for Fe ion reduction from Fe+3 to Fe+2
in comparison to relevant water retted -
bres. However, it must be noted that the
value of FRAP for dew retted ax from
the Modran variety was very high in
comparison to other bres. Dew retted -
bres extracted from the Sara variety also
showed a high FRAP value. The ability
of bres extracted from Luna, Nike and
Artemida to reduce the Fe ions was very
low and differences between dew and
water retted bres were similar (Fig-
ures 8.c - 8.e). The variations between
the antioxidant activity of dew and water
retted bres are the most visible for ax
varieties with high FRAP values, e.g. for
Modran and Sara varieties (Figures 8.a,
8.b).
The antioxidant activity of ax bres was
strongly related to the phenolic acid con-
0
50
100
150
200
250
0123
Sample concentration, mg/ml
FRAP, mmol/l
0
50
100
150
200
250
0 0.5 1 1.5 2 2.5
Sample concentration, mg/ml
FRAP, mmol/l
0
50
100
150
200
250
0 0.5 1 1.5 2 2.5
Sample concentration, mg/ml
FRAP, mmol/l
0
50
100
150
200
250
0 0.5 1 1.5 2 2.5
Sample concentration, mg/ml
FRAP, mmol/l
0
50
100
150
200
250
0.0 0.5 1.0 1.5 2.0 2.5
Sample concentration, mg/ml
FRAP, mmol/l
Figure 8. Antioxidant activity of ax bres extracted
from: a) Modran, b) Sara, c) Nike, d) Luna, e) Artemida
variety - FRAP method.
Dew retting
Water retting
Log. (Dew retting)
Log. (Water retting)
Dew retting
Water retting
Log. (Dew retting)
Log. (Water retting)
a) b)
c) d)
e)
FIBRES & TEXTILES in Eastern Europe 2015, Vol. 23, 6(114)
46
showed strong antioxidant properties and
can be used for textile manufacture and
serve as a barrier against free radicals
coming from air and UV radiation and
reaching human skin. Dew retted Sara
bres could also act for the neutralisation
of free radicals; but their activity was not
so strong as the Modran variety.
Results of the DPPH test were not so
signicant, reaching low values in the
range from 1.21 (for Nike) to 3.28 (for
Modran). Generally, in the FRAP meth-
od, the reaction runs very quickly due to
employing metal ions and covering the
largest number of components, whereas
in the case of the DPPH radical method,
the reaction is much slower and covers
only the most reactive substances. For
this reason, the values of the DPPH re-
sults are signicantly lower in compari-
son to the FRAP method [24].
n Conclusions
The current study conducted on ax -
bres extracted from ve varieties of -
brous plants: Artemida, Modran, Sara.
Nike and Luna with the use of dew and
water retting methods proved that un-
modied ax bres can show inherent
antioxidant properties. The biological
activity of the bres tested was strongly
related to the lignin content and related
with the phenolic acid content in their
chemical composition. The bre anti-
oxidant power was related to the method
of their extraction applied, especially in
case of the Modran and Sara varieties,
where the dew retted bres showed the
highest activity, whereas water retted -
bres show very weak biological activity
in the tests conducted. The results of the
study obtained allowed to draw the fol-
lowing conclusions:
1. The study proved that the antioxi-
dant activity of the ax bres tested
were strongly related to the method
of retting and to the ax plant variety,
which had an effect on the chemical
composition of the bres.
2. Dew retted ax bres obtained from
three varieties tested were character-
ised by better antioxidant properties
in comparison with respective water
retted bres. Two other ax varieties
showed low antioxidant power and
differences between dew and water
retted bres were slight.
3. Dew retted bres contained a higher
amount of lignin and, hence, phenolic
acids in comparison to water retted
bres. The study proved there is a
strong relationship between the con-
tent of lignin and phenolic acids bond-
ed with it as well as the bioactivity of
dew retted ax bres.
4. Water retted ax bres showed lower
biological activity because the most
active phenolic substances are water
soluble and were removed during ret-
ting. The water retting process allows
for removal of more non-cellulosic
compounds, excluding waxes to ob-
tain purer bres. Water retted ax -
bres extracted from all the ax plants
tested were characterised by the high-
est cellulose content, which allowed
to conclude that the quality of textile
made of the bres would be better
than of respective dew retted bres.
5. Flax bres extracted from the Modran
variety with the use of dew retting
showed the highest antioxidant activ-
ity. The second best was the Sara va-
riety, which is strongly related to the
highest content of lignin and phenolic
acids in their structure in comparison
to other ax varieties.
6. Fibres were obtained from Artemida
and Luna varieties with the highest
cellulose content, which predisposes
them as the best for textile processing,
were characterised by weak biological
activity due to low lignin and phenolic
acid content.
7. The biological activity of ax bres is
inversely proportional to their textile
quality, because the higher lignin con-
tent in ax bres negatively inuences
textile processing.
Acknowledgment
The work was supported by the Polish Mini-
stry of Science and Higher Education.
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retted ax bres.
Phenolic acid content, mg/100 g
FRAP, mmol/l
10.0 10.2 10.4 10.6 10.8 11.0 11.2 11.4
140
120
100
80
60
40
20
0
Artemida
Modran
Sara
Luna
Nike
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