Antioxidant activity of extract from Polygonum aviculare L

Abstract

Free radicals induce numerous diseases by lipid peroxidation, protein peroxidation, and DNA damage. It has been reported that numerous plant extracts have antioxidant activities to scavenge free radicals. Whether Polygonum aviculare L. (Polygonaceae) has antioxidant activity is unknown. In this study, dried Polygonum aviculare L. was extracted by ethanol, and the extract was lyophilized. The antioxidant activities of extract powder were examined by free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays. The results show that the IC50 value of Polygonum aviculare L. extract is 50 microg/ml in free radical scavenging assays, 0.8 microg/ml in superoxide radical scavenging assays, and 15 microg/ml in lipid peroxidation assays, respectively. Furthermore, Polygonum aviculare L. extract has DNA protective effect in hydroxyl radical-induced DNA strand scission assays. The total phenolics and flavonoid content of extract is 677.4 +/- 62.7 microg/g and 112.7 +/- 13 microg/g. The results indicate that Polygonum aviculare L. extract clearly has antioxidant effects.

Full text

281
HSU Biol Res 39, 2006, 281-288
Biol Res 39: 281-288, 2006
BR
Antioxidant activity of extract from Polygonum
aviculare L.
CHIN-YUAN HSU
Department of Life Science, Chang Gung University, Tao-Yuan, Taiwan
ABSTRACT
Free radicals induce numerous diseases by lipid peroxidation, protein peroxidation, and DNA damage. It has
been reported that numerous plant extracts have antioxidant activities to scavenge free radicals. Whether
Polygonum aviculare L. (Polygonaceae) has antioxidant activity is unknown. In this study, dried Polygonum
aviculare L. was extracted by ethanol, and the extract was lyophilized. The antioxidant activities of extract
powder were examined by free radical scavenging assays, superoxide radical scavenging assays, lipid
peroxidation assays and hydroxyl radical-induced DNA strand scission assays. The results show that the IC50
value of Polygonum aviculare L. extract is 50 µg/ml in free radical scavenging assays, 0.8 µg/ml in
superoxide radical scavenging assays, and 15 µg/ml in lipid peroxidation assays, respectively. Furthermore,
Polygonum aviculare L. extract has DNA protective effect in hydroxyl radical-induced DNA strand scission
assays. The total phenolics and flavonoid content of extract is 677.4 ± 62.7 mg/g and 112.7 ± 13 mg/g. The
results indicate that Polygonum aviculare L. extract clearly has antioxidant effects.
Key terms: antioxidant activity, free radical, phenolics, lipid peroxidation, DNA damage Polygonum
aviculare L.
Corresponding author: Dr. Chin-Yuan Hsu, Department of Life Science, Chang Gung University, 259, Wen-Hwa 1st Road,
Kwei-Shan, Tao-Yuan 333, Taiwan, Tel: (886-3) 211-8800, ext 3402, E-mail: hsu@mail.cgu.edu.tw
Received: July 19, 2005. In revised form: September 26, 2005. Accepted: October 7, 2005
INTRODUCTION
Reactive oxygen species produced by
ultraviolet light, ionizing radiation,
chemical reactions, and metabolic processes
have numerous pathological effects, such as
causing lipid peroxidation, protein
peroxidation, DNA damage, and cellular
degeneration related to cardiovascular
disease, ageing, cancer, inflammatory
diseases, and a variety of other disorders (2,
6, 9, 16, 20, 28). They include superoxide
radical anion (
•
O
2-
), hydroxyl radicals
(
•
OH), singlet oxygen (
1
O
2
), and hydrogen
peroxide (H
2
O
2
). In cellular oxidation
reactions, superoxide radical normally is
formed first, and its effects can be
magnified because it produces other kinds
of cell-damaging free radicals and oxidizing
agents. The damaging action of the
hydroxyl radical is the strongest among free
radicals (18).
Phenolics have been reported to have a
capacity to scavenge free radicals. They are
commonly found in both edible and non-
edible plants and have multiple biological
effects, including antioxidant activity (13,
27). The antioxidant activity of phenolics is
mainly due to their redox properties, which
allow them to act as reducing agents,
hydrogen donators, and singlet oxygen
quenchers. In addition, they have a metal
chelation potential (22). Phenolics, such as
flavonoids, phenolic acids, stilbenes,
lignans, lignin, and tannins, are especially
common in leaves, flowering tissues, and
woody parts, such as stems and barks (17).
They have been suggested to play a
preventive role in the development of
cancer, heart disease, and ageing-related
diseases.
The importance of the antioxidant
constituents of plant materials in the
maintenance of health and protection from
ageing-related diseases has intrigued
scientist for a long time. I have screened the

HSU Biol Res 39, 2006, 281-288
282
antioxidant activity of a variety of wild
plants and other substances used in
traditional Oriental medicine by 1,1-
Diphenyl-2-picrylhydrazyl (DPPH) free
radical scavenging assays. The results show
that Polygonum aviculare L. (Polygonaceae)
exhibits a DPPH free radical scavenging
effect. Polygonum aviculare L. is used in
traditional Oriental medicine and belongs to
the “li-shui-shen-shih” category of drugs. It
is used traditionally to treat ailments caused
by high humidity, because of its diuretic
property. In the present study, I wish to
study the antioxidant effects of Polygonum
aviculare L. on superoxide radical
scavenging, lipid peroxidation, and DNA
damage.
METHODS
Chemicals
1,1-Diphenyl-2-picrylhydrazyl (DPPH),
nitroblue tetrazolium chloride (NBT), 2-
thiobarbituric acid (TBA), ΦX174 RF1
supercoiled DNA, Folin-Ciocalteu reagent,
L-ascorbic acid, and (+)-catechin were
purchased from Sigma Chemical Co. The
other chemicals and solvents used in this
experiment were of the highest quality
available. Dried Polygonum avicular was
purchased from a local drugstore.
Preparation of plant extract
Dried Polygonum aviculare L. was made
into powder form. 5 g of dried powder were
extracted in 50 ml 50% ethanol solution at
25
o
C for 30 min with shaking. The extract
was centrifuged at 15000 rpm for 3 min,
and the supernatant was collected. The
supernatant was concentrated in a rotary
evaporator and then lyophilized. The
resulting powder extract was used in this
study (4).
DPPH assay
1 mg extract powder was dissolved in 1 ml
of 50% ethanol solution to obtain 1000 µg/
ml sample solution. 1000 µg/ml solutions
were series diluted into 1 µg/ml, 5 µg/ml, 10
µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml, 200
µg/ml, 500 µg/ml, and 1000 µg/ml with 50%
ethanol. In each reaction, the solutions were
mixed with 1 ml of 0.1 mM 1,1-Diphenyl-2-
picrylhydrazyl (DPPH), 0.45 ml of 50 mM
Tris-HCl buffer (pH 7.4), and 0.05 ml
samples at room temperature for 30 min.
50% ethanol solution was used as control.
The reduction of the DPPH free radical was
measured by reading the absorbance at 517
nm. DPPH, a purple-colored, stable free
radical is reduced to the yellow-colored
diphenylpicrylhydrazine when antioxidants
are added. L-ascorbic acid and (+)-catechin
were used as positive controls. The
inhibition ratio (percent) was calculated
from the following equation: % inhibition =
[(absorbance of control – absorbance of test
sample)/absorbance of control] x 100%. The
antioxidant activity of each sample was
expressed in terms of IC50 (micromolar
concentration required to inhibit DPPH
radical formation by 50%), calculated from
the inhibition curve (5, 8, 30).
NBT (superoxide scavenging) assay
The superoxide anion radical scavenging
activity was performed by using the
methods of Liu and Ng (18). Superoxide
radicals were generated in 3.0 ml of Tris-
HCl buffer (16 mM, pH 8.0), which
contained 78 µM β-nicotinamide adenine
dinucleotide (reduced form, NADH), 50
µM nitroblue tetrazolium (NBT), 10 µM
phenazin methosulfate (PMS), and test
samples in 50% ethanol solution (final
concentrations were 1, 5, 10, 20, 50, and
100 µg/ml, respectively). The color reaction
of superoxide radicals and NBT was
detected at OD 560 nm. (+)-catechin was
used as a positive control. The inhibition
ratio (%) was calculated from the following
equation: % inhibition = [(absorbance of
control – absorbance of test sample)/
absorbance of control] x 100%.
Lipid peroxidation assay
The brain of young adult male Balb/c mice
were dissected and homogenized with a
homogenizer in ice-cold Tris-HCl buffer (20
mM, pH 7.4) to produce a 1/10 homogenate.

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HSU Biol Res 39, 2006, 281-288
The homogenate was centrifuged at 12000g
for 15 min at 4
o
C, and the supernatant was
used for in vitro lipid peroxidation assay. A
1 ml aliquot of liposome was incubated with
the test samples (final concentrations were 1,
5, 10, 20, 50, and 100 µg/ml, respectively) in
the presence of 10 mM FeSO
4
and 0.1 mM
ascorbic acid at 37
o
C for 1 h. The reaction
was terminated by the addition of 1.0 ml of
trichloroacetic acid (TCA; 28%, w/v) and
1.5 ml of TBA (1%, w/v), followed by
heating at 100
o
C for 15 min. The absorbance
of the malondialdehyde (MDA)-TBA
complex was measured at 532 nm. (+)-
catechin was used as a positive control. The
inhibition ratio (%) was calculated from the
following equation: % inhibition =
[(absorbance of control – absorbance of test
sample)/absorbance of control] x 100% (4).
DNA strand scission assay
The assay was performed according to the
method of Keum et al., with minor
modifications (15). The reaction mixture
(30 µl) contained 10 mM Tris-HCl, 1 mM
EDTA buffer (pH 8.0), ΦX174 RF1
supercoiled DNA (0.6 µg), and H
2
O
2
(0.04M). Various amounts of the test
extract samples dissolved in 10 µl of
ethanol (final concentrations of the plant
extract in each assay were 1, 10, 100, 500,
and 1000 µg/ml, respectively) were added
prior to H
2
O
2
addition. Hydroxyl radicals
were generated by irradiation of the
reaction mixtures at a distance of 5 cm with
a 12 W UV lamp. After incubation at room
temperature for 20 min, the reaction was
terminated by the addition of a loading
buffer (0.25% bromophenol blue tracking
dye and 40% sucrose), and the mixtures
were then analyzed by 0.8% submarine
agarose gel electrophoresis (50eV, 1.5 h).
The gel was stained with ethidium bromide,
destained in water, and photographed on a
transilluminator (4).
Determination of total flavonoid
1 mg samples were added in 1ml of 80%
ethanol. A aliquot of 0.5 ml was added to
test tubes containing 0.1 ml of 10%
aluminum nitrate, 0.1 ml of 1 M potassium
acetate, and 4.3 ml of 80% ethanol. The
absorbance of the supernatant was measured
at 415 nm after 40 min at room temperature.
Total flavonoid concentration was calculated
using quercetin as standard (19).
Determination of Total Phenolics
Total phenolics content was determined
according to the Folin-Ciocalteu method
(23), using gallic acid as a standards 1 mg
extract powders were dissolved in 1 ml 50%
methanol solution. 0.5 ml extract solution
was mixed with 0.5 ml of 50% Folin-
Ciocalteu reagent. The mixture was let sit
for 2-5 min before the addition of 1.0 ml of
20% Na
2
CO
3
. The mixture was centrifuged
at 150 g for 8 min after 10 min of incubation
at room temperature. The absorbance of the
supernatant was measured at 730 nm. The
total phenolic content was expressed as
gallic acid equivalents (GAE) in milligrams
per gram sample (4).
RESULTS
The free radical scavenging activity of
Polygonum aviculare L. extract was
assessed by 1,1-Diphenyl-2-picrylhydrazyl
(DPPH) assay. (+)-catechin and L-ascorbic
acid were used as controls. Both are well-
known antioxidant compounds. The result
is shown in Figure 1. The IC
50
values (the
concentration required to inhibit radical
formation by 50%) of Polygonum aviculare
L. extract are 50 µg/ml. The IC
50
values of
(+)catechin and ascorbic acid are 35 µg/ml
and 50 µg/ml, respectively; they exhibit a
similar curve of antioxidant activity
compared to (+)-catechin and L-ascorbic
acid. The IC
50
value of Polygonum
aviculare L. extract is lower than that of
(+)-catechin and is similar to that of L-
ascorbic acid. This result demonstrates that
Polygonum aviculare L. extract has an
inhibitory effect on the DPPH radical.
The superoxide scavenging activity of
Polygonum aviculare L. extract was
evaluated by NBT (Superoxide Scavenging)
assay. (+)-catechin served as a control. The
result is shown in Figure 2. The IC
50
value
of Polygonum aviculare L. extract is 0.8 µg/

HSU Biol Res 39, 2006, 281-288
284
ml. The IC
50
value of (+)-catechin is 40 µg/
ml. Almost all superoxide radicals were
inhibited by 10 µg/ml Polygonum aviculare
L. extract. The superoxide scavenging
activity of Polygonum aviculare L. extract is
apparently higher than that of (+)-catechin.
Therefore, Polygonum aviculare L. seems to
be a potential source of superoxide radicals
scavenging. This result shows that
Polygonum aviculare L. extract has a
significant superoxide scavenging activity.
The lipid peroxidation suppressing
activity of Polygonum aviculare L. extract
was estimated by TBA assay. (+)-catechin
was employed as control. The result is
shown in Figure 3. The IC
50
values of
Polygonum aviculare L. extract is 16 µg/
ml. The IC
50
value of (+)-catechin is 17 µg/
ml. Compared to (+)-catechin, they exhibit
the similar curve of antioxidant activity.
The lipid peroxidation suppressing activity
of Polygonum aviculare L. extract is
identical to that of (+)-catechin. This result
indicates that Polygonum aviculare L.
extract has suppressing activity on lipid
peroxidation.
Figure 1: Free-radical scavenging activity of
Polygonum aviculare L. extract are measured
by using the DPPH assay: (◊) (+)catechin; (O)
ascorbic acid; (L) Polygonum aviculare L.
extract. Results are mean ± SD (N=5).
Figure 2: Superoxide scavenging activity of
Polygonum aviculare L. extract are measured by
using the NBT assay: (◊) (+)catechin; (L)
Polygonum aviculare L. extract. Results are
mean ± SD (N =5).
Figure 3: Effects of Polygonum aviculare L.
extract on both ferric ion and ascorbic acid
induced lipid peroxidation on mouse brain
homogenates: (◊) (+)catechin; (L )
Polygonum aviculare L. extract. Results are
mean ± SD (N =5).
ΦX174 RF1 DNA strand scission
induced by UV photolysis of H
2
O
2
elevated the protective effect of DNA of
Polygonum aviculare L. extract. The result
is shown in Figure 4. ΦX174 RF1
supercoiled DNA was utilized as control
(lane 1). UV illumination alone did not
cause DNA strand cleavage (lane 2). The
treatment of supercoiled DNA with UV
plus H
2
O
2
led to the conversion of the
DNA to open circular form (lane 3). The
treatment of supercoiled DNA with UV,
H
2
O
2
plus the different concentration of

285
HSU Biol Res 39, 2006, 281-288
Polygonum aviculare L. extract led to the
maintenance of the DNA in the
supercoiled form (lanes 4-8). Almost
complete protection was expressed at a
dose of 1000 µg/ml. This protective effect
of DNA exhibits dose-dependency. This
result shows that Polygonum aviculare L.
extract has DNA protective activity under
oxidative stress.
TABLE 1
Total phenolic content and total flavonoid
content of ethanolic extract from
Polygonum aviculare
Extract Total phenolic Total flavonoid
(mg of GAE/g) (mg/g of samples)
Polygonum
aviculare 677.4 ± 62.7 (N=8) 112.7 ± 13 (N=10)
Total phenolics are expresed as gallic acid equivalent
(GAE).
Total flavonoid are expresed as mg of total flavonoid
content / g of samples based on quercetin as standard.
Values represent mean ± S.D.
DISCUSSION
Antioxidant activity of Polygonum
aviculare L. extract has been found by
means of free radical scavenging assays,
superoxide radical scavenging assays, lipid
peroxidation assays, and hydroxyl radical-
induced DNA strand scission assays. In
addition, Polygonum aviculare L. extract
has high phenolics and flavonoid contents.
This study indicates that Polygonum
aviculare L. extract obviously has
antioxidant effects.
DPPH is a stable radical that has been
used widely to evaluate the antioxidant
activity of various natural products (12). In
this study, DPPH scavenging activity has
been found in Polygonum aviculare L.
extract. The maximum inhibition of
Polygonum aviculare, (+)catechin and
ascorbic acid is about 80% in this study.
The maximum inhibition concentration of
Polygonum aviculare L. and (+)-catechin is
approximately 100 µg/ml. The maximum
inhibition concentration of ascorbic acid is
approximately 200 µg/ml. The inhibitory
curve of DPPH scavenging activity of
Polygonum aviculare L. is similar to that of
Acacia confusa (4), Cat’s claw (Uncaria
tomentosa) (24), and Anthriscus cerefolium
(5). However, the IC
50
value of Polygonum
aviculare L. (50 µg/ml) is less than that of
Acacia confusa (5 µg/ml), Cat’s claw
(Uncaria tomentosa) (18 µg/ml), and
Anthriscus cerefolium (45 µg/ml) (4, 24, 5).
Nevertheless, Polygonum aviculare L.
Figure 4: Protection effect of Polygonum
aviculare L. extract on DNA strand scission
induced by H
2
O
2
and UV. ΦX174 RF1
supercoiled DNA as control (lane 1), ΦX174
RF1 supercoiled DNA was exposed to UV
alone (lane 2), UV plus H
2
O
2
(lane 3), or plus
H
2
O
2
in the presence of final concentration of
1000 µg/ml (lane 4), 500 µg/ml (lane 5), 100
µg/ml (lane 6), 10 µg/ml (lane 7), 1 µg/ml
(lane 8) of Polygonum aviculare L. extract.
Lane 1 represents native ΦX174 RF1
supercoiled DNA without any treatment. OC:
Open circular; SC: Super coiled.
Plant phenolics are widely distributed in
plants. They are highly effective free
radical scavengers and exhibit strong
antioxidant activity. The content of total
phenolics in the Polygonum aviculare L.
was determined spectrometrically according
to the Folin-Ciocalteu procedure and
calculated as gallic acid equivalent
contents. The result is shown in Table 1.
The total phenolic content of Polygonum
aviculare L. extract is 677.4 ± 62.7 mg/g.
The content of total flavonoid in the
Polygonum aviculare L. also was
determined spectrometrically and calculated
as quercetin equivalents content. The result
is shown in Table 1. The total flavonoid
content of Polygonum aviculare L. extract
is 112.7 ± 13 mg/g. These results imply that
Polygonum aviculare L. extract contains a
high quantity of phenolics and flavonoids.

HSU Biol Res 39, 2006, 281-288
286
extract is a potential source of natural
antioxidants.
In cellular oxidation reactions,
superoxide radicals normally are formed
first, and their effects can be magnified
because they produce other kinds of free
radicals and oxidizing agents (18).
Additionally, xanthine oxidase is one of the
main enzymatic sources of those reactive
oxygen species in vivo. In this study,
superoxide radicals scavenging property has
been found in Polygonum aviculare L.
extract. The IC
50
value of Polygonum
aviculare L. extract is 0.8 µg/ml, whereas
the IC
50
value of (+)-catechin is 40 µg/ml.
The maximum inhibitory effect of
Polygonum aviculare L. is about 100%. The
maximum inhibition concentration of
Polygonum aviculare L. is approximately 10
µg/ml. However, the maximum inhibition
concentration of (+)-catechin is higher than
100 µg/ml. It is noteworthy that the
superoxide scavenging activity of
Polygonum aviculare L. extract is superior
to that of (+)-catechin. Moreover, the IC
50
value of Polygonum aviculare L. (0.8 µg/ml)
is larger than that of Paeonia suffruticosa
(50 µg/ml) (18). In other words, Polygonum
aviculare L. has better superoxide radicals
scavenging activity than Paeonia
suffruticosa. These results show that
Polygonum aviculare L. is an important
source for superoxide radical scavenging.
In the current study, lipid peroxidation
of mouse brain homogenates was induced
by ferric ion plus ascorbic acid. Lipid
peroxidation scavenging activity has been
found in Polygonum aviculare L. extract.
The IC
50
value of Polygonum aviculare L.
extract is about 16 µg/ml. The IC
50
value of
(+)-catechin is about 17 µg/ml. The
maximum inhibitory effect of Polygonum
aviculare L. is about 75%. The maximum
inhibition concentration of Polygonum
aviculare L. is approximately 20 µg/ml.
The inhibitory effect of Polygonum
aviculare L. is higher than that of
(+)catechin when the concentration is
higher than IC
50
values. In other words,
Polygonum aviculare L. extract has better
scavenging effect than (+)-catechin when
the concentration is higher than 17 µg/ml.
This result indicates that Polygonum
aviculare L. extract is a good source of
lipid peroxidation scavenging.
The cellular damage resulting from
hydroxyl radicals is strongest among free
radicals. Hydroxyl radicals can be
generated by biochemical reactions.
Superoxide radical is converted by
superoxide dismutase to hydrogen peroxide,
which subsequently can produce extremely
reactive hydroxyl radicals in the presence
of transition metal ions, such as iron and
copper or by UV photolysis. Hydroxyl
radicals can attack DNA to cause strand
scission. That is, incubation of ΦX174 RF1
supercoiled DNA with H
2
O
2
and then UV
radiation resulted in complete conversion of
supercoiled DNA to the open circular form.
In this study, the administration of
Polygonum aviculare L. extract to the
reaction mixture substantially decreased the
DNA strand scission induced by both H
2
O
2
and UV radiation. It shows a dose-
dependent protection of DNA under
oxidative stress. The higher the
concentration of Polygonum aviculare L.
extracts, the better the DNA protection.
There is almost complete protection at a
dose of 1000 µg/ml. The effect of DNA
protection of Polygonum aviculare L. is
similar to that of Acacia confusa (4). These
results reveal that Polygonum aviculare L.
extract is an excellent DNA protector.
Phenolics are found in large quantities in
the plant kingdom, and they have been
shown to have multiple biological functions,
including antioxidant activity (21, 25, 14). In
this study, we examined the content of
phenolics from the extract of Polygonum
aviculare. The result showed that
Polygonum aviculare L. extract contains
677.4 ± 62.7 mg/g phenolics. It indicated
that the Polygonum aviculare extract
contained a higher amount of phenolics than
the bark and heartwood extracts of Acacia
confusa based on Folin-Ciocalteu procedures
(4). Therefore, Polygonum aviculare L. is a
significant source of phenolics. The results
in this study suggest that the effectiveness of
the antioxidant activity of Polygonum
aviculare extract is probably related to the
high contents of phenolics, and the observed
antioxidant activities of the extract may be
due to the hydroxyl groups in phenolics (10).

287
HSU Biol Res 39, 2006, 281-288
A similar finding has been demonstrated in
the plant extracts of Eucommia ulmoides
(Du-zhong) and Acacia confusa in which
enriched phenolics correlated well with their
antioxidant activities (4, 29).
It also has been reported that Polygonum
aviculare L. can be employed supportively
in the therapy of gingivitis by oral rinse (7).
It was suggested that this phenomenon was
attributed to the flavonoid components that
decrease capillary fragility and exert a
cortisone-like effect on gingival tissues (7).
In this study, we examined the content of
flavonoids from the extract of Polygonum
aviculare. The result showed that
Polygonum aviculare L. extract contains
high flavonoids, 112.7 ± 13 mg/g. It has
been suggested that the therapeutic effect of
Polygonum aviculare L. on gingivitis is
derived from its high flavonoid.
Additionally, the flavonoid content of
Polygonum aviculare L. also is higher than
that of propolis (29).
It is well known that free radicals are the
principal cause of several diseases, including
Parkinson’s disease, coronary heart disease,
cancer, and Alzheimer’s disease (3, 1, 11,
26). This study demonstrated that
Polygonum aviculare L. has high phenolics
contents and excellent antioxidant activity. It
would be interesting to investigate further
the potential effectiveness of Polygonum
aviculare L. for treating diseases caused by
the overproduction of free radicals. Also, the
antimicrobial effect, bioavailability and
potential toxicity of Polygonum aviculare L
need to be studied in vivo.
ACKNOWLEDGEMENTS
This work was supported by NSC 94-2311-
B-182-008 grants from the National
Science Council, R. O. C.
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