Antioxidant activity and free radical scavenging capacity between

Korean medicinal plants and avonoids by assay-guided comparison

Chang W. Choi
a,b,
*, Sei C. Kim
a,b
, Soon S. Hwang
a
, Bong K. Choi
a
, Hye J. Ahn
a
,
Min Y. Lee
a
, Sang H. Park
b
, Soo K. Kim
c
a
Department of Biology and Medicinal Science, Pai Chai University, Doma 2-dong 439-6, Seo-gu, Daejeon 302-735, South Korea
b
Biomed RRC, Pai Chai University, Doma 2-dong 439-6, Seo-gu, Daejeon 302-735, South Korea
c
Department of Animal Sciences and Environment, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, South Korea
Received 12 July 2002; received in revised form 29 August 2002; accepted 6 September 2002
Abstract
Several Korean medicinal plants were selected to evaluate for free radical scavenging capacities and antioxidant activities using
commonly accepted assays. They were extracted with dichloromethane, methanol or ethanol, respectively and selected for the best
antioxidant results. Flavonoids, such as catechin, morin, naringenin, quercetin and rutin, were included and used as standards in this
study. Each sample under assay condition showed a dose-dependent free radical scavenging effect of DPPH (1,1-diphenyl-2-picryl
hydrazyl radical) and a dose-dependent inhibitory effect of xanthine oxidase and lipid peroxidation. Among plant extracts, the root
bark of Morus alba and the leaf of Saururus chinensis showed stronger SC
50
or ID
50
values than other plant extracts. They also
showed a protective effect on DNA damage caused by hydroxyl radicals generated from UV-induced photolysis of hydrogen
peroxide. A rapid evaluation for antioxidants using TLC screening and DPPH staining methods demonstrated each plant extract
having various free radical scavenging capacity. Stained silica layer revealed a purple background with yellow spots at the location
of drops, which showed radical scavenging capacity. The intensity of the yellow color depends upon the amount and nature of
radical scavenger present in the samples. This antioxidant potential corresponded with the results of DPPH spectrophotometric
assay.
# 2002 Published by Elsevier Science Ireland Ltd.
Keywords: Medicinal plants; Flavonoids; Free radical scavenging capacity; Antioxidant activity
1. Introduction
Plants contain a wide variety of free radical scaven-
ging molecules, such as flavonoids, anthocyanins, carte-
noids, dietary glutathionine, vitamins and endogenous
metabolites and such natural products are rich in
antioxidant activities [1/4]. These plant-derived antiox-
idants have been shown to function as singlet and triplet
oxygen quenchers, peroxide decomposers, enzyme in-
hibitors and synergists [1]. Electron acceptors, such as
molecular oxygen, react easily with free radicals to
become radicals themselves, also referred to as reactive
oxygen species (ROS). The ROS include superoxide
anions (O
+
2

), hydrogen peroxide (H
2
O
2
) and hydroxyl
radicals (
+
OH) [5]. There are increasing suggestions by
considerable evidence that the free radicals induce
oxidative damage to biomolecules (lipids, proteins and
nucleic acids), the damage which eventually causes
atherosclerosis, ageing, cancer, diabetes mellitus, inflam-
mation, AIDS and several degenerative diseases in
humans [6/10].
Several methods have been developed to measure the
free radical scavenging capacity (RSC), regardless of the
individual compounds which contribute towards the
total capacity of a plant product in scavenging free
radicals. The methods are typically based on the
inhibition of the accumulation of oxidized products,
since the generation of free radical species is inhibited by
the addition of antioxidants and this gives rise to a
reduction of the end point by scavenging free radicals.
The reliable method to determine RSC involves the
* Corresponding author. Tel./fax: /82-42-520-5617
E-mail address: choicw@mail.pcu.ac.kr (C.W. Choi).
Plant Science 163 (2002) 1161/1168
www.elsevier.com/locate/plantsci
0168-9452/02/$ - see front matter # 2002 Published by Elsevier Science Ireland Ltd.
PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 3 3 2 - 1
measurement of the disappearance of free radicals, such
as 2,2?-azino-bis (3-ethylbenzenthiazoline-6-sulphonic)
acid radical (ABTS
+
), the 2,2-diphenyl-1-picrylhydra-
zyl radical (DPPH
+
) or other colored radicals, with a
spectrophotometer [11,12].
Owing to the increasing demand for information
about the total RSC of all types of plant extracts, an
easy, rapid and reliable method for the determination of
RSC of various samples might be useful. The method
should not be time-consuming, but sensitive enough to
screen differences between plants parts used for herbal
medicine, which include the flower, top, aerial and
roots. In this paper, assay-guided comparison among
plant extracts was applied to evaluate the RSC or
antioxidant activity. In addition, the effects of flavo-
nol-type (morin, quercetin, rutin), flavonone-type (nar-
ingenin) and flavanol-type (catechin) flavonoids on
superoxide anion radical generating systems were also
compared. Even though the role of flavonoids is still
controversial, their antioxidant activity may be rendered
suitable as protective agents from ROS-related effects.
2. Materials and methods
2.1. Reagents
Catechin, morin, naringenin, quercetin, rutin,
DPPH
+
, xanthine oxidase, xanthine, nitrobluetetrazo-
lium (NBT), FX174 RF1 DNA and thiobarbituric acid
were purchased from Sigma Chemical Co. (St. Louis,
MO). Ethanol, methanol, dichloromethane, ethyl ace-
tate, chloroform and toluene of analytical grade were
purchased from Merck (Darmstadt, Germany).
2.2. Plant materials and extracts
All plants were collected from various locations in
Korea and naturally dried. Plants were extracted with
ethanol, dichloromethane or methanol and evaporated
to dryness under reduced pressure. The powdery ex-
tracts were solubilized in ethanol to a final concentra-
tion of 10 or 1.0 mg ml
1
.
2.3. Antioxidant assays
2.3.1. Superoxide anion generation by xanthine/xanthine
oxidase assay
Superoxide anions were generated by the xanthine/
xanthine oxidase system and measured by the slightly
modified NBT reduction method [13]. The reaction
mixture in a total volume of 1 ml contained 50 mM
potassium phosphate buffer (pH 7.8), xanthine (0.05
mM) and NBT (0.6 mM). Varying concentrations of
each plant extract or flavonoid, such as catechin, morin,
naringenin, quercetin and rutin, in ethanol was added
into the mixture. The final concentration of ethanol in
the reaction mixture did not exceed 1% (v/v), of which
concentration did not influence the activity of xanthine
oxidase. The reaction was initiated by the addition of
xanthine oxidase (25 mU ml
1
) in the same phosphate
buffer. The reaction was run at room temperature for 10
min, the absorbance (Ab) of formazan chromophore
was measured against a blank solution in which
xanthine oxidase was replaced by buffer solution. A
control solution was included, in which sample was
replaced by ethanol.
2.3.2. Linoleic acid peroxidation assay
The reaction mixture contained 500 ml linoleic acid
(20 mM), 500 ml Tris/HCl (100 mM, pH 7.5), 100 l
FeSO
4
+
7H
2
O (4 mM) and a varying concentration of
each plant extract or flavonoid. Linoleic acid peroxida-
tion was initiated by the addition of 100 ml of ascorbic
acid (2 mM), incubated for 30 min at 37 8C and
terminated by the addition of trichloroacetic acid
(5.5%). Some 1 ml of the mixture was added with 250
ml of thiobarbituric acid in 50 mM NaOH, followed by
heating for 10 min. The mixtures were centrifuged at
3500/g for 10 min and the absorbance of thiobarbi-
turic acid-reacting substances (TBARS) in the super-
natant was read at 532 nm and converted into the
percentage antioxidant activity using the following
equation: linoleic acid peroxidation inhibition (%) /
(Ac/As) /100/(Ac/An); Ac/Ab of control (without
extract), As/Ab of extract and An/Ab of blank
(without extract and FeSO
4
+
7H
2
O).
2.3.3. DPPH photometric assay
Each sample stock solution (1.0 mg ml
1
) was diluted
to final concentrations of 500, 250, 100, 50 and 10 mg
ml
1
, in ethanol. A total of 1 ml of a 0.3 mM DPPH
ethanol solution was added to 2.5 ml of sample solution
of different concentrations and allowed to react at room
temperature. After 30 min, the Ab values were measured
at 518 nm and converted into the percentage antioxidant
activity using the following equation described pre-
viously [14]: scavenging capacity %/100/[(Ab of
sample/Ab of blank)/100/Ab of control]. Ethanol
(1.0 ml) plus plant extract solution (2.5 ml) was used as a
blank, while DPPH solution plus ethanol was used as a
negative control. The positive controls were DPPH
solution plus each 1 mM flavonoid. The SC
50
values
were calculated by linear regression of plots, where the
abscissa represented the concentration of tested plant
extracts or flavonoids and the ordinate the average
percent of scavenging capacity from three replicates.
2.3.4. Rapid screening of antioxidant by dot-blot and
DPPH staining
An aliquot (3 ml) of each dilution of each plant extract
and flavonoid was carefully loaded on a 20/20 TLC
C.W. Choi et al. / Plant Science 163 (2002) 1161/1168 1162
layer (silica gel 60 F
254
; Merck) and allowed to dry.
Drops of each sample were loaded in order of decreasing
concentration along the column. The staining of the
silica plate was based on the procedure of Soler-Rivas et
al. [15]. The sheet bearing the dry spots was placed
upside down for 10 s in a 0.4 mM DPPH solution in
methanol. Stained silica layer revealed a purple back-
ground with yellow spots at the location of the drops,
which showed radical scavenger capacity. The intensity
of the yellow color depends upon the amount and nature
of radical scavenger present in the sample.
2.3.5. TLC analysis and DPPH staining
Drops (3 ml) of each stock solution from plant extracts
(1 or 10 mg ml
1
) were loaded individually onto the
baseline of the layer, which was then developed with
toluene: ethyl acetate (93:7, v/v) or chloroform/ethyl
acetate (60:40, v/v) depending upon the samples. The
layer was dried and stained with DPPH solution, as
mentioned above.
2.3.6. DNA strand scission by hydroxyl radicals
DNA strand breakage by hydroxyl radicals was
measured by agarose gel electrophoresis. The assay
was based on the conversion of supercoiled FX174
RF1 double-strand DNA to open circular and linear
forms by the DNA damaging agents. The standard
reaction mixture (20 ml) contained TE (10 mM Tris/
HCl and 1 mM EDTA, pH 8.0), FX174 RF1 super-
coiled DNA (0.5 mg) and hydrogen peroxide (20 mM).
Hydroxyl radical was generated by UV photolysis of
hydrogen peroxide under transilluminator. After incu-
bation at room temperature for 15 min, the mixtures
were electrophoresed at 100 V. The gels were then
stained with ethidium bromide and photographed on a
transilluminator.
3. Results and discussion
It has long been recognized that naturally occurring
substances in higher plants have antioxidant activity.
Among those substances, the flavonoids widely distrib-
uted in plants have the ability to scavenge free radicals,
superoxide and hydroxyl radicals by single-electron
transfer. In our experiments, we measured the total
antioxidation effect of some medicinal plants and
compared them with that of flavonoids. Because of the
complex nature of phytochemicals, the antioxidant
activities of plant extracts cannot be evaluated by only
a single method. Therefore, commonly accepted assays,
including enzymatic and non-enzymatic methods, were
employed to evaluate the total antioxidant effects of
some medicinal plants in Korea. The results should be
encouraged in future in vivo studies, which could
ultimately lead to the application of these medicinal
plants in pharmaceutical and cosmetic formulations.
3.1. Xanthine/xanthine oxidase assay
In a preliminary experiment, the extraction either by
ethanol or dichloromethane was more efficient in
antioxidant assay and RSC than that by methanol, but
for the uniform assay we selected the plant extracts by
dichloromethane. Table 1 reports the IC
50
of xanthine/
xanthine oxidase activity, IC
50
of linoleic acid peroxida-
tion and SC
50
values of DPPH for selected plant extracts
and flavonoids. Xanthine oxidase catalyses the oxida-
tion of hypoxanthine or xanthine to uric acid, during the
oxidation an equivalent rate of superoxide radical is
produced [16]. The following reaction is the oxidation of
NBT to water-soluble formazan by the superoxide
formed from xanthine by xanthine oxidase. Among
plant extracts, maximum inhibition of NBT reduction
by the root bark extract of Morus alba was steadily
reached at 100 mg ml
1
(Fig. 1A), though its value of
IC
50
is more than that of quercetin and less than those of
catechin, morin, naringenin and rutin. Minimum inhibi-
tion of NBT reduction by the root extract of Poly-
gonatum odoratum, but its value of IC
50
is still lower
than that of rutin. All tested flavonoids and plant
extracts inhibited the formation of reduced NBT in a
dose-related manner (Fig. 1A, B), as has been reported
for some flavonoids [17,18]. In our experiment, querce-
tin and morin were more efficient than other flavonoids,
while rutin was the least for inhibition of NBT reduc-
tion.
We have previously reported on the influence of
flavonoids on the toxicity of copper to plant pathogenic
fungi [19,20]. Quercetin and morin conferred substantial
protection against the inhibition of fungal growth by
copper, whereas naringenin and rutin offered less
effective protection, suggesting that the protection effect
of the flavonoids is highly dependent on the keto
function and hydroxyl group substitution of the flavo-
noid skeleton. Likewise, the result of the present study
indicates that the antioxidant activity of flavonoids may
be correlated to their structure [16,21], such as 3?,4?-
dihydroxy system of the B-ring in quercetin and 2?,4?-
dihydroxy system of the B-ring in morin [22].
During the oxidation of xanthine to uric acid, its
inhibition can also be detected by a decreased produc-
tion of uric acid. A xanthine oxidase inhibitor without
any additional superoxide scavenging activity will pro-
duce the reduction in the rate of NBT. In fact, many
flavonoids have been reported to be potent inhibitors of
xanthine oxidase rather than scavenging superoxide
anions [18,23]. In particular, catechin and rutin having
a 4-oxo function significantly inhibited uric acid forma-
tion, competing with the xanthine for the active site of
xanthine oxidase [18]. Therefore, the potential antiox-
C.W. Choi et al. / Plant Science 163 (2002) 1161/1168 1163
idant activities of plant extracts and flavonoids were
additionally measured with the following assays.
3.2. Linoleic acid peroxidation and DPPH
spectrophotometric assays
The inhibition of linoleic acid peroxidation was
assayed by the TBARS test. All tested flavonoids
showed a dose-dependent inhibition of linoleic acid
peroxidation (data not shown) and among those, rutin
was the most efficient by the lowest IC
50
value, while
naringenin was the least efficient (Table 1), the result
was similar to previous report [18]. It is noteworthy that
the leaf extracts of Saururi herba (Saururus chinensis
(Lour) Baill) produced the best inhibitory effect of
linoleic acid peroxidation among plant extracts, which
was also very active to inhibit xanthine/xanthine
oxidase reaction and to scavenge DPPH free radicals.
Unlike DPPH assay, the inhibition of linoleic acid
peroxidation by plant extracts is much more effective
than that in the range of pure flavonoids. Because of the
presence of various other phytochemicals, such as
phenolic compounds, amino acids, ascorbic acid, toco-
pherols and pigments, that might contribute some
antioxidant activity singly or in combination.
In order to verify the additional scavenging activity,
the RSC of tested samples were measured spectro-
photometrically with DPPH, a stable free radical, which
produce a violet solution in ethanol. It is reduced in the
presence of an antioxidant molecule, giving rise to no
color, which has been used to evaluate the antioxidant
activity of plant and microbial extracts [14,24/26].
When each plant extract was examined using stable
DPPH radicals, all extracts exhibited the various RSC.
Among those, the root bark extracts of M. alba showed
the most scavenging by the lowest value of SC
50
and a
dose-dependent manner in DPPH assay (Table 1 and
Fig. 2A).
The RSC of flavonoids was tested by their ability to
bleach the stable DPPH radical, as previously reported
[18,27]. During the assay, the reaction was very stable,
producing reliable values in repeated tests because
Table 1
Inhibitory activity of xanthine oxidation and linoleic acid peroxidation and scavenging capacity of DPPH by plant extracts and avonoids
Plant extracts and flavonoids Xanthine oxidase Linoleic acid peroxidation DPPH
I (%)
a
IC
50
b
I (%)
a
IC
50
b
S (%)
a
SC
50
b
Astragali membranaceus 72.1 59.2 100 15.3 16.9 1,152.1
Houttuynia cordata 92.1 15.5 100 6.5 30.1 434.8
Morus alba 100 5.9 100 15.0 55.4 90.3
Polygonatum odoratum 50.8 90.9 100 9.5 25.4 493.1
Saururus chinensis 85.7 25 100 6.2 34.7 216.6
Catechin 100 23.3 70.9 62.3 100 4.8
Morin 100 8.6 56.5 84.5 100 12.2
Naringenin 100 20.3 53.0 98.4
c
481.3
Quercetin 100 0.9 82.5 63.1 100 8.1
Rutin 42.9 116.6 63.7 59.7 95.5 18.7
a
Percent of inhibition and scavenging at 100 mg ml
1
as a mean of triplicate experiments.
b
Values obtained from regression lines with 95% of confidence level. IC
50
is defined as the concentration sufficient to obtain 50% of a maximum
inhibition and SC
50
is defined as the concentration sufficient to obtain 50% of a maximum scavenging capacity.
c
Negative data.
Fig. 1. Xanthine/xanthine oxidase inhibition activity of plant extracts
(A); Astragali membranaceus root (white bar), Houttuynia cordata leaf
(light grey bar), Morus alba root (checker bar), Polygonatum odoratum
root (dark grey bar), Saururus chinensis leaf (black bar) and avonoids
(B); catechin (white bar), morin (light grey bar), naringenin (checker
bar), quercetin (dark grey bar), rutin (black bar). Values are mean of
triplicate analysis.
C.W. Choi et al. / Plant Science 163 (2002) 1161/1168 1164
DPPH containing an odd electron gives a strong
absorption at 518 nm in visible spectrophotometer. As
this electron becomes paired off in the presence of a free
radical scavenger, the absorption fades and the resulting
decolorization is stoichiometric with respect to the
number of electrons taken up [28]. Except for narin-
genin, all tested flavonoids showed a significant and
dose-dependent DPPH scavenging capacity, among
those catechin was the most efficient by the lowest
SC
50
value. When a range of concentration of narin-
genin, similar to other flavonoids, was treated the value
was always negative. Its SC
50
among flavonoids was the
most, even 100 times more than that of catechin. We
obtained a range of DPPH scavenging capacity of 17/
77% when naringenin was treated at an increasing
concentration from 150 mg ml
1
through 1 mg ml
1
.
In addition, its reaction time with DPPH took a long
time, observing the fading purple color visibly. The
interaction of a potential antioxidant with DPPH
depends on its structural conformation and this struc-
tural requirement is correlated with the presence of
hydroxyl groups on the flavonoids [17,29,30].
3.3. Rapid screening of antioxidant by dot-blot and
DPPH staining
In order to visualize quantitatively the RSC of the
tested samples, measured spectrophotometrically, were
detected in the TLC by the DPPH staining method. For
the rapid screening of RSC, each diluted sample was
applied as a dot on a TLC layer that was later stained
with DPPH solution (Fig. 3). The appearance of yellow
color in the spots has a potential value for the indirect
evaluation of the RSC of the dot samples [15,31,32]. The
method is typically based on the inhibition of the
accumulation of oxidized products, since the generation
of free radicals is inhibited by the addition of antiox-
idants and the end point by scavenging the free radical.
When flavonoids were analyzed, fast-reacted and
strong intensities of white-yellow spots appeared up to
dilutions of 100 mg ml
1
of catechin, naringenin and
rutin (final concentration 300 ng) and 50 mg ml
1
of
morin and quercetin. However, initially faint spots
appeared and several hours later, weak spots could be
observed in naringenin-loaded, similar to the result of
DPPH spectrophotometric assay. It explains why we
had the negative result of DPPH scavenging capacity by
naringenin in spectrophotometric assay, measured 10
min after reaction. Appropriate dilutions of plant
extracts also gave a positive reaction with DPPH,
depending on their RSC and nature. Among plant
extracts, the extracts from the root of M. alba and the
leaf of Saururi herba were stronger in the RSC,
detecting up to 1 mg ml
1
dilution (final concentration
3 mg). Leaf extracts of Houttuynia cordata showed
intermediate RSC, while root extracts of Astragali
membranaceus and P. odoratum, respectively, showed
weak RSC by fading color intensity similar to the purple
Fig. 2. DPPH radical scavenging capacity of (A); Astragali membra-
naceus root (white bar), Houttuynia cordata leaf (light grey bar),
Morus alba root (checker bar), Polygonatum odoratum root (dark grey
bar), Saururus chinensis leaf (black bar) and avonoids (B); catechin
(white bar), morin (light grey bar), naringenin (checker bar), quercetin
(dark grey bar), rutin (black bar). Values are mean of triplicate
analysis.
Fig. 3. Dot blot assay of RSC on a silica sheet stained with a DPPH
solution in methanol. Each 3 ml dilution of avonoid (2 and 1 mg
ml
1
, 500, 250, 100 and 50 ml ml
1
) and plant extract (10, 5, 2.5 and 1
mg ml
1
, 500 and 100 ml ml
1
) applied from top to down. From left
to right dots are (avonoids): catechin (1), morin (2), rutin (3),
naringenin (4), quercetin (5), (plant extracts): Morus alba root (6),
Saururus chinensis leaf (7), Houttuynia cordata leaf (8), Astragali
membranaceus root (9) and Polygonatum odoratum root (10).
C.W. Choi et al. / Plant Science 163 (2002) 1161/1168 1165
background (Fig. 1). According to color intensities, the
overall order of the decreasing RSC was M. alba /
Saururus chinensis /H. cordata/P. odoratum/A.
membranaceus. These results correspond to the results
by DPPH spectrophotometric assay.
3.4. TLC analysis with DPPH staining
For the qualitative detection of RSC in the plant
extracts, the same procedures on TLC layer were
applied as mentioned above except developing in two
solvent systems (Table 2 and Fig. 4A, B). As expected,
both extracts of M. alba and Saururi herba show the
numerous migrated spots having strong intensities on
TLC plate that developed on toluene/ethyl acetate
solvent (Fig. 4A). The number of white spots and their
migration were various among plant extracts, depending
on TLC solvent system. As presented in Fig. 4(B), other
plant extracts developed on different organic solvent
system showed the clear differences in the number of
spots. Fruit peel extract from Citrus unshiu (lanes 1 and
2) and root extract from Platycodon grandiflorum (lanes
3 and 4), which developed on chloroform/ethyl acetate
(lanes 1 and 3) showed more migrating spots than single
spot without migration in toluene/ethyl acetate system
(lanes 2 and 4). Their intensity and reaction speed were
also various, suggesting that spots may contain indivi-
dually different characteristics (Table 2), some of which
have a fast RSC, reducing the DPPH radical very
rapidly, while others have a slower RSC, taking a longer
time to react. Antioxidants of several plant extracts do
not all operate in the same way and maybe more
effective against different free radicals.
3.5. DNA strand scission by hydroxyl radicals
The oxidation of DNA bases is produced by ROS,
generated both from endogenous sources and from
reactions of xenobiotics [33,34]. The cells possess
efficient DNA repair mechanisms for oxidative DNA
damage [35,36], but damaged forms of DNA oxidation
is persistent during replication of DNA, leading to
mutation. Fig. 5 shows the electrophoretic pattern of
DNA after UV-photolysis of H
2
O
2
in the presence of
flavonoids or plant extracts. DNA strand scission was
assessed by measuring the conversion of supercoiled
DNA to open circular and further to linear forms by
flavonoid [18] and extracts from Citrus species and
Ganoderma lucidum [37,38]. DNA derived from FX174
supercoiled double strand DNA showed two bands on
agarose gel electrophoresis, the faster-migrating band
corresponding to the native form of supercoiled circular
DNA (scDNA) and the slower-migrating band being the
open circular form (ocDNA). The UV irradiation of
DNA in the presence of H
2
O
2
caused the cleavage of
supercoiled DNA, indicating that OH generated by UV
photolysis of H
2
O
2
produced DNA strand scission and
breakage. Neither hydrogen peroxide nor UV alone
Table 2
DPPH scavenging capacity of extracts from various plant parts by DPPH staining
Plant Part Number of spots Reaction speed Intensity of spots
c
Astragali membranaceus
a
Root 1 Slow
Capsicum annuum
b
Green fruit 1 Fast
Yellow fruit 1 Fast
Red fruit 1 Fast
Carthamus tinctorius
b
Seed 1 Fast
Chrysanthemum coronarium
b
Leaves 2 Fast ,
Cichorium intybus
b
Leaves 1 Fast
Citrus unshiu
b
Fruit peel 4 Fast All
Dioscorea batatas
b
Root 1 Fast
Epimedium koreanum
b
Leaf 1 Fast
Houttuynia cordata
a
Leaf 1 Fast
Morus alba
a
Root bark 5 Fast All
Platycodon grandiflorum
b
Root 3 Fast 2 , 1
Polygonatum odoratum
a
Root 1 Slow
Saururus chinensis
a
Leaf 3 Fast All
Catechin 1 Fast
Morin 1 Fast
Naringenin 1 Slow
Quercetin 1 Fast
Rutin 1 Fast
a
Various ethanol extracts from Korean ethnophamaceutical plants loaded onto TLC plate, developing by toluene/ethyl acetate (93:7, v/v).
b
Various ethanol extracts from Korean ethnophamaceutical plants loaded onto TLC plate, developing by chloroform/ethyl acetate (60:40, v/v)
and staining by DPPH.
c
, Weak; , intermediate; , strong.
C.W. Choi et al. / Plant Science 163 (2002) 1161/1168 1166
induced any DNA damage (Fig. 5A, lanes 1 and 2), but
their combination of both resulted in almost complete
destruction of supercoiled form of phage DNA under
our experimental condition (lane 3). The presence of all
flavonoids under investigation suppressed the formation
of linear DNA (linDNA) and protected from damage
(lanes 4/8). Both extracts of M. alba and Saururi herba
seemed more efficiently protected FX174 supercoiled
double strand DNA from hydroxyl radical-induced
strand scission (Fig. 5B). In particular, when 50 mg
ml
1
extracts of Saururi herba was treated, we could
observe three forms of DNA resulting from scDNA to
ocDNA and linDNA. In the presence of an increasing
concentration of extracts of both plants, the proportion
of both the ocDNA and linDNA significantly decreased
while the amounts of the residual supercoiled DNA
recovered (Fig. 5B, lanes 1/3 and 5/7). Unexpectedly,
we could not observe the band on the gel when treated
concentration at 400 mg ml
1
of each extract. Our
assumption for this is that probably a higher concentra-
tion of extracts may disturb the DNA migration. The
DNA cleavage analysis also demonstrated the consis-
tently strong antioxidant properties of extracts of M.
alba and Saururi herba used in traditional medicine.
Acknowledgements
This research was supported by a grant (R12-1999-
002103-0) from MOST and KOSEF of Korea through
the Research Center for Biomedicinal Resources (RRC).
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DPPH solution. (A) The samples from left to right were extracts of
Morus alba root bark (lane 1), Saururus chinensis leaf (lane 2),
Houttuynia cordata leaf (lane 3), Astragali membranaceus root (lane 4)
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Fig. 5. Effects of avonoids and plant extracts on the protection of
DNA strand scission induced by H
2
O
2
plus UV. (A) Some 0.4 mM
each avonoid treated: FX174 RF supercoiled DNA (concentration:
0.5 ug per lane) was exposed at 20 mM H
2
O
2
alone (lane 1), UV alone
(lane 2), H
2
O
2
/UV (lane 3), catechin/H
2
O
2
/UV (lane 4),
naringenin/H
2
O
2
/UV (lane 5), morin/H
2
O
2
/UV (lane 6), quer-
cetin/H
2
O
2
/UV (lane 7), rutin/H
2
O
2
/UV (lane 8) and M (Mar-
ker). Some 0.4 mM each avonoid. (B) Extracts: lane 1: DNA/
Saururus chinensis 50 mg ml
1
/H
2
O
2
/UV; 2: DNA/Saururus
chinensis 100 mg ml
1
/H
2
O
2
/UV; 3: DNA/Saururus chinensis
200 mg ml
1
/H
2
O
2
/UV; 4: DNA/Saururus chinensis 400 mg
ml
1
/H
2
O
2
/UV; 5: DNA/Morus alba 50 mg ml
1
/H
2
O
2
/UV;
6: DNA/Morus alba 100 mg ml
1
/H
2
O
2
/UV; 7: DNA/Morus
alba 200 mg ml
1
/H
2
O
2
/UV; 8: DNA/Morus alba 400 mg ml
1
/
H
2
O
2
/UV.
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