Life Sciences 73 (2003) 167 – 179

www.elsevier.com/locate/lifescie

Screening of medicinal plant extracts for antioxidant activity

Si Eun Lee a, Hyun Jin Hwang b, Jung-Sun Ha a, Han-Seung Jeong c, Jeong Hee Kim a,*
a
Department of Biochemistry, College of Dentistry, Kyung Hee University, 1 Hoegi-Dong, Dongdaemoon-Ku,
Seoul 130-701, South Korea
b
Department of Chemistry, College of Natural Sciences, Kyung Hee University, Seoul 130-701, South Korea
c
R&D Center, Ahram Biosystems Inc., Seoul 130-103, South Korea
Received 24 June 2002; accepted 11 December 2002

Abstract

The methanol extracts of nine medicinal plants traditionally used in Chinese medicine were screened for
antioxidant activity versus resveratrol, which has been shown to protect cells from oxidative damage [Toxicol.
Lett. 102 (1998) 5]. Most of the plant extracts used in this study inhibited the H2O2-induced apoptosis of Chinese
hamster lung fibroblast (V79-4) cells. The extracts of Areca catechu var. dulcissima, Paeonia suffruticosa, Alpinia
officinarum, Glycyrrhiza uralensis and Cinnamomun cassia strongly enhanced viability against H2O2-induced
oxidative damage in V79-4 cells. Relatively high levels of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging activity were detected in extracts of Areca catechu var. dulcissima, Paeonia suffruticosa and
Cinnamomun cassia (IC50 < 6.0 Ag/ml). The activities of superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GPX) were dose-dependently enhanced in V79-4 cells treated with most of the plant
extracts. The extracts of Areca catechu var. dulcissima showed higher antioxidant activity than resveratrol in all
experiments. These results suggest that the plant extracts prevent oxidative damage in normal cells probably
because of their antioxidant characteristics.
D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Antioxidant activity; Antioxidant enzymes; Apoptosis; Radical scavenging

Introduction

Reactive oxygen species (ROS) are an entire class of highly reactive molecules derived from the
metabolism of oxygen. Moreover, ROS can cause extensive damage to cells and tissues, during

* Corresponding author. Tel.: +82-2-961-0915; fax: +82-2-960-1457.

E-mail address: jhkimh@khu.ac.kr (J.H. Kim).

0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0024-3205(03)00259-5
168 S.E. Lee et al. / Life Sciences 73 (2003) 167–179

infections and various degenerative disorders, such as cardiovascular disease, aging, and neurodegener-
ative diseases like Alzheimer’s disease, mutations and cancer (Ames, 1998; Cox and Cohen, 1996;
Finkel and Holbrook, 2000; Harman, 1994).
Mammalian cells possess elaborate defense mechanisms for radical detoxification. Key metabolic
steps are the superoxide dismutase (SOD) catalysis of the dismutation of superoxide to hydrogen
peroxide and oxygen, and the conversion of H2O2 into water and oxygen by catalase (CAT) and
glutathione peroxidase (GPX), which destroys toxic peroxides. In addition to antioxidant enzymes,
several small-molecule antioxidants play important roles in the antioxidant defense systems. These can
be divided into compounds made in vivo, and compounds obtained from diet. Glutathione, bilirubin, and
melatonin are examples of the former, and vitamins such as a-tocopherol, h-carotene, and ascorbic acid
and micronutrient elements such as zinc and selenium examples of the latter (Halliwell and Gutteridge,
1998).
In recent years, there has been a worldwide trend towards the use of the natural phytochemicals
present in berry crops, teas, herbs, oilseeds, beans, fruits and vegetables (Deiana et al., 1999; Kitts et
al., 2000; Lee and Shibamoto, 2000; Lie; Velioglu et al., 1998; Wang and Jiao, 2000). The medicinal
plants examined in this study have long been used as traditional Chinese medicines and were selected
from among 50 medicinal plant extracts that our group has already screened for antioxidant effects
(Kim et al., 2000). Earlier studies showed that extracts from Areca catechu var. dulcissima possess
antidepressant properties (Dar and Khatoon, 2000). Au et al. (2001) suggested that the methanol
extracts of Paeonia suffruticosa potently inhibit human immunodeficiency virus (HIV)-1 integrase.
Cinnamomun cassia was reported to have an antimutagenic effect (Sharma et al., 2001) and stimulated
human lymphocytes to proliferate (Shan et al., 1999). The anti-genotoxicity of galangin (Alpinia
officinarum) has been studied (Heo et al., 2001), and Areca catechu var. dulcissima showed strong
scavenging activity against the superoxide anion radical (Ohsugi et al., 1999). The aqueous extracts of
Paeonia suffruticosa showed the high potency at inhibiting hemolysis and lipid peroxidation (Liu and
Ng, 2000). A cyclic phenylacetamide of Solvia miltiorrhiza was found to be a scavenger of the DPPH
radical (Choi et al., 2001). However, the antioxidant activities of plant extracts have not been
comparatively analyzed and very little research has been done on the biological activity of these plant
extracts in vivo.
Therefore, we investigated the effect of these plants extracts on the activities of the antioxidant
enzymes SOD, CAT and GPX, and DPPH radical scavenging activity, lipid peroxidation inhibitory
activity and cell proliferation effects. Resveratrol was used as an antioxidant control. It is a constituent
of grapes and other food products, and has been shown to prevent carcinogenesis in murine models and
to protect cells from oxidative damage and apoptosis (Clement et al., 1998; Jang et al., 1997; Songsiri et
al., 1997). In addition, we measured the protective effect of these medicinal plants on the apoptotic cell
death induced by H2O2.

Methods

Plant material

Samples of medicinal plants were purchased at a local pharmacy in Seoul, Korea (Table 1). Each dried
plant (100 g) was extracted at 80 jC in 70% (v/v) methanol for 3 hr. The extract was then filtered and the
 S.E. Lee et al. / Life Sciences 73 (2003) 167–179 169

Table 1
Botanical species
Scientific name Family name Organ
Glycyrrhiza uralensis Leguminosae Root stock
Solvia miltiorrhiza Labiatae Root
Paeonia suffruticosa Ranunculaceae Root bark
Spirodela polyrrhiza Araceae Aerial parts
Areca catechu var. dulcissima Palmae Seed
Cornus officinalis Cornaceae Fruit
Alpinia officinarum Zingiberaceae Root
Nelumbo nucifera Nymphaceaceae Seed
Cinnamomun cassia Lauraceae Bark

filtrate was concentrated with a vacuum rotary evaporator (Eyela, Japan) under low pressure. The residue
was freeze-dried in a freezing-dryer (Ilsin, Korea) and stored at 70 jC. The powder obtained was
dissolved in dimethyl sulfoxide (DMSO) and diluted with phosphate buffered saline (PBS, pH 7.4) to
give a final extract concentration ranging from 0.8 to 100 Ag/ml.

Chemicals

The following chemicals were obtained from the Sigma Chemical Co. (St Louis, MO, USA): (–)-
epigallocatechin-3-gallate (EGCG), 3,4V,5-trihydroxy-trans-stilbene (Resveratrol), propidium iodide
(PI), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT), 1,1-diphenyl-2-picrylhy-
drazyl (DPPH), thiobarbituric acid (TBA), 2,2V-di-p-nitrophenyl-5,5V-diphenyl-3,3V-(3,3V-dimethoxy-4,4V-
diphenylene)-ditetrazolium chloride (NBT), nicotinamide adenine phosphate (NADPH), xanthine,
xantine oxidase, ethylenediaminetetraacetic acid sodium salt (Na-EDTA), pyridine, sodium azide,
glutathione, and glutathione reductase. Ethanol was purchased from Hayman Chemical Co. (Witham,
Essex, UK), and hydrogen peroxide from Fluka Chemical Co. (Buchs, SG, Swiss). Cell culture
materials were purchased from Gibco BRL (Gaithersburg, MD, USA). All other chemicals were of the
highest analytical grade and purchased from common sources.

Cell culture

Chinese hamster lung fibroblasts, V79-4 (ATCC CCL-93) cells, were maintained at 37 jC in an
incubator in a humidified atmosphere of 5% CO2 / 95% O2. Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS), 100 Ag/ml of streptomycin, 100
unit/ml of penicillin and 2 mM L-glutamine.

Cell viability

Cell viability was estimated by the MTT assay, which is based on the cleavage of a tetrazolium
salt by mitochondrial dehydrogenases in viable cells (Hansen et al., 1989). V79-4 cells were seeded
in a 96 well plate at 1.2  105 cells/ml. Sixteen hr after plating, cells were treated with various
concentrations of total extracts (4, 20, and 100 Ag/ml) and 1 hr later 1 mM of H2O2 was added to
170 S.E. Lee et al. / Life Sciences 73 (2003) 167–179

the culture. Cells were incubated for an additional 24 hr at 37 jC. During the last 4 hr, the cells
were incubated with 20 Al of MTT stock solution (5 mg/ml) in 200 Al medium at 37 jC. Samples
were then extracted with acidic isopropanol and absorbance was measured using an ELISA reader
(Bio-Rad, USA) at 570 nm. Relative cell viability was determined by the amount of MTT
converted to the insoluble formazan salt. The optical density of the formazan formed in the control
cells was taken as 100% viability. Data are mean percentages of viable cells versus the respective
controls.

DPPH free radical scavenging activity

In order to measure antioxidant activity, the DPPH free radical scavenging assay was carried out
according to the procedure described by Blosi (1958). Total extracts of each plant at various
concentrations (0.8, 4, 20, and 100 Ag/ml) were added to a 1.5  10 4 M solution of DPPH in
methanol and the reaction mixture was shaken vigorously. The amount of DPPH remaining was
determined at 520 nm, and the radical scavenging activity was obtained from the following equation:
Radical scavenging activity (%) = {(ODcontrol ODsample) / ODcontrol}  100. The antioxidant activity
of plants extracts was expressed as IC50, which was defined as the concentration (in Ag/ml) of extract
required to inhibit the formation of DPPH radicals by 50%.

Lipid peroxidation inhibitory activity

Lipid peroxidation was assayed by measuring malondialdehyde (MDA) according to the method of
Ohkawa et al. (1979). Cells were exposed to total extracts of each plant at various concentrations (4,
20 and 100 Ag/ml) for 60 min, followed by 1 mM H2O2 for 60 min. Cells were then washed with cold
PBS, scraped and homogenized in ice-cold 1.15% KCl. Samples containing 100 Al of the cell lysates
were combined with 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid adjusted to pH 3.5 and 1.5 ml of
0.8% TBA. The mixture was brought to a final volume of 4.0 ml with distilled water and heated at 95
jC for 120 min. After cooling to room temperature, 5.0 ml of a mixture of n-butanol and pyridine
(15:1, v/v) was added to each sample and the mixture shaken vigorously. After centrifugation at 1500
rpm for 10 min, the supernatant fraction was isolated and the absorbance was measured at 532 nm.
Lipid peroxidation inhibitory activity was expressed as IC50.

Nuclear staining with PI

Cells used in this study were constantly observed under an inverted phase-contrast microscope
(Olympus, Japan). V79-4 cells were placed in a 60 mm culture plate at a concentration of 1.2  105
cells/ml. Sixteen hr after plating, cells were treated with 100 Ag/ml of the plant extracts and after a
further incubation of 1 hr 1 mM H2O2 was added to the culture. After 24 hr, cells were harvested by
centrifugation and washed with phosphate buffered saline (PBS) twice. Cells were then resuspended in
PBS at a concentration of 4  105 cells/ml and placed on a microscope slide using a cytospin (Hanil,
Korea). The slide was left at room temperature to evaporate to dryness. Cells were fixed with cold
ethanol in the dark, washed with PBS and stained with 2.5 Ag/ml of propidium iodide and DNase-free
RNase (100 Ag/ml). The morphology of the stained cells was examined under a fluorescence microscope
(E800, Nikon, Japan).
 S.E. Lee et al. / Life Sciences 73 (2003) 167–179 171

Flow cytometry analysis

V79-4 cells were treated with 100 Ag/ml of plant extracts and then with 1 mM H2O2 for 24 hr and
harvested by centrifugation. The cell pellets obtained were rinsed with warm PBS, mixed with a 1:1
(v/v) mixture of PBS and 0.2 M Na2HPO4 –0.1 M citric acid (pH 7.5) and fixed in cold ethanol at 4
jC for 1 hr. Fixed cells were washed with PBS and resuspended in a staining solution containing PI
(10 Ag/ml) and DNase-free RNase (100 Ag/ml). Cell suspensions were incubated at 37 jC for 1 hr in
the dark and analyzed on a fluorescence-activated cell sorter flow cytometer (FACScaliber, Becton
Dickinson, USA).

Assays for antioxidant enzymes

Cells were treated with 4, 20 and 100 Ag/ml of total extracts of each plant for 60 min, and then lysed
in a lysis buffer appropriate for the requirements of each assay, as described below. Protein contents were
quantified by using Bradford’s method using bovine serum albumin as a standard (Bradford, 1976).
Results are expressed as relative enzyme activity per mg of protein compared with the corresponding
control cultures.

Superoxide dismutase (SOD) activity

SOD activity was assayed using the nitroblue tetrazolium (NBT) method of Beauchamp and
Fridovich (1971). NBT was reduced to blue formazan by O2 , which has a strong absorbance at 560
nm. However, the presence of SOD inhibits this reaction. The cells were homogenized in 0.05 M
sodium carbonate buffer (pH 10.2). The assay mixture consisted of 0.05 M sodium carbonate buffer
(pH 10.2) containing 3 mM xanthine, 0.75 mM NBT, 3 mM EDTA, 1.5 mg/ml BSA and 50 Al of
homogenate. The reaction was initiated by adding 50 Al of xanthine oxidase (0.1 mg/ml) and incubated
for 30 min at room temperature. The reaction was stopped by adding 6 mM of copper (II) chloride and
centrifuged at 1500 rpm for 10 min. The absorbance of formazan at 560 nm was then measured in the
supernatants.

Catalase (CAT) activity

The reaction mixture contained 12 Al of 3% (v/v) H2O2 and 100 Al of cell lysates in 50 mM phosphate
buffer (pH 7.0) to a final volume of 1.0 ml. Samples were incubated for 2 min at 37 jC and the
absorbances of the samples were monitored for 5 min at 240 nm. Changes in absorbance were taken to
be proportional to the breakdown of H2O2 (Carrillo et al., 1991).

Glutathione peroxidase (GPX) activity

GPX was assayed by the method of Paglia and Valentine (1967). The reaction mixture contained 0.1
M phosphate buffer (pH 7.0), 1 mM EDTA, 10 mM glutathione, 1 mM NaN3, 1 unit of glutathione
reductase, 1.5 mM NADPH and 0.1 ml of cell lysate. After incubating for 10 min at 37 jC, H2O2 was
added to each sample to a final concentration of 1 mM. GPX activity was measured as the rate of
NADPH oxidation at 340 nm.
172 S.E. Lee et al. / Life Sciences 73 (2003) 167–179

Statistics

All data represent means F S.E. Statistical analyses was performed using analysis of variance
followed by the Student’s t-test.

Results

Effect of plant extracts on cell proliferation

The effect of medicinal plant extracts on V79-4 cell proliferation was evaluated by using the
MTT assay (Fig. 1A). Treatment with most of the plant extracts slightly stimulated cell proliferation
but did not induce changes in cell morphology at concentration from 4 to 100 Ag/ml. In the same
concentration range, extracts of Spirodela polyrrhiza slightly reduced cell proliferation, and at the
higher concentration of 500 Ag/ml, most of the extracts, except Solvia miltiorrhiza, Nelumbo
nucifera and Cornus officinalis extracts, significantly inhibited V79-4 cell survival (data not
shown). Thus we decided to use the concentration range 4–100 Ag/ml in all subsequent experi-
ments.
We next evaluated the protective effect of the plant extracts on the viability of H2O2 treated cells.
Cells were treated with extracts for 1 hr prior to the addition of H2O2, whereas control cells were treated
with H2O2 in the presence of vehicle only. The final concentration of DMSO was below 0.1% in
throughout this study, and DMSO had no cyto-protective effect at this concentration. Relative cell
survival was determined 24 hr later by the MTT assay. As shown in Fig. 1B, treatment with total extracts
of Areca catechu var. dulcissima and Paeonia suffruticosa induced higher cell survivals than those of the
other plant extracts in H2O2 treated-cells. At a dose of 100 Ag/ml, the percentages of cell surviving after
treatment with these extracts were 61 and 50%, respectively. Treatment with 100 Ag/ml of the extracts of
Alpinia officinarum, Glycyrrhiza uralensis and Cinnamomun cassia induced 48, 42 and 40% increases
in cell survival, respectively.

DPPH radical scavenging activity of the plant extracts

The DPPH radical scavenging activities of the total extracts of the medicinal plants and of resveratrol
are shown in Fig. 2. Extracts of Areca catechu var. dulcissima had the highest DPPH radical scavenging
activity, with an IC50 value of 1.8 Ag/ml. This value was higher than that of resveratrol, with an IC50
value of 4.8 Ag/ml, and total extracts of Cinnamomun cassia, Paeonia suffruticosa and Alpinia
officinarum showed higher DPPH radical scavenging activity than the other extracts, with IC50 values
of 5.0, 5.9 and 6.7 Ag/ml, respectively. All samples showed DPPH radical scavenging activity in a dose-
dependent manner.

Lipid peroxidation inhibitory activity of plant extracts

We assessed the activity of plant extracts to inhibit lipid peroxidation in H2O2-treated V79-4 cells.
Samples of 4–100 Ag/ml were used for lipid peroxidation activity analysis and extracts of Alpinia
officinarum, Areca catechu var. dulcissima, Paeonia suffruticosa, Cinnamomun cassia and Nelumbo
 S.E. Lee et al. / Life Sciences 73 (2003) 167–179 173

Fig. 1. Effect on the cell proliferation of medicinal plants in V79-4 cells (A) and the protective effect of extracts of medicinal
plants on H2O2-induced oxidative damage (B) in V79-4 cells. Each experiment was performed at least 3 times and data are
expressed as average percent changes versus the control F S.D. Black, gray and white bars indicate 4, 20 and 100 Ag/ml of
samples, respectively. 1; Glycyrrhiza uralensis, 2; Solvia miltiorrhiza, 3; Paeonia suffruticosa, 4; Spirodela polyrrhiza, 5; Areca
catechu var. dulcissima, 6; Cornus officinalis, 7; Alpinia officinarum, 8; Nelumbo nucifera, 9; Cinnamomun cassia and 10;
control. * p < 0.05.

nucifera presented relatively higher inhibitory activity at 100 Ag/ml than that of the H2O2 treated control
cells (Fig. 3). Resveratrol had an IC50 value of < 4 Ag/ml (data not shown). The overall lipid
peroxidation inhibitory activity of samples used in this study revealed lower activity compared to other
data, such as the DPPH radical scavenging activity of these samples.
174 S.E. Lee et al. / Life Sciences 73 (2003) 167–179

Fig. 2. DPPH radical scavenging activity of medicinal plants. Total extracts of each plant were added to a methanolic solution of
DPPH and radical scavenging activity was measured at 520 nm. Each experiment was performed at least three times and data
are expressed as average percent changes versus the control F S.D. Black, gray, light gray and white bars indicate 0.8, 4, 20
and 100 Ag/ml samples, respectively. The numbers 1 – 9 are described in the legend to Fig. 1., the number 10 represents
resveratrol and 11 the control. * p < 0.05.

Fig. 3. Lipid peroxidation inhibitory effect of the medicinal extracts. Cells were incubated with 100 Ag/ml of each plant extract
for 1 hr, and this was followed by 1 mM of H2O2 for 1 hr. The amounts of MDA were measured at 532 nm. Each experiment
was performed at least 3 times and data are expressed as average percent changes versus the control F S.D. 1; P. suffruticosa,
2; A.C. var. dulcissima, 3; A. officinarum, 4; N. nucifera, 5; C. cassia. * p < 0.05.
 S.E. Lee et al. / Life Sciences 73 (2003) 167–179 175

Reduction of H2O2-induced nuclear fragmentation by plant extracts

In order to analyze the protective effect of the medicinal plant extracts on H2O2-induced apoptosis, we
used PI to stain the nuclei of V79-4 cells treated with H2O2 alone or with plant extract and H2O2. PI, a
widely used phenanthrene derivative, is a nonspecific DNA intercalating agent, and is excluded by the
plasma membrane of living cells, and hence trapped by dead cells. As a result, control cells revealed
intact nuclei, but cells treated with 1 mM of H2O2 showed significant nuclear fragmentation (data not
shown). However, treatment of most of the plant extracts prior to H2O2 treatment showed marked
reductions in nuclear fragmentation. In addition to the morphological evaluation, the protective effects of
the plant extracts were confirmed by flow cytometry. Analysis of the DNA contents following H2O2
treatment of V79-4 cells revealed an increase in the proportion of cells with sub-G1 DNA content, to
28.5% (Table 2). This result indicates that apoptosis was induced by H2O2. However, cells that were
pretreated with any plant extract showed significantly reduced sub-G1 DNA content. In particular,
treatment with 100 Ag/ml of Paeonia suffruticosa and Areca catechu var. dulcissima extract prior to
H2O2 treatment reduced the apoptotic sub-G1 population by more than 90%.

Effect of plant extracts on SOD, CAT and GPX

In order to investigate whether these antioxidant activities of plant extracts are mediated by an
increase in antioxidant enzymes, we measured SOD, CAT and GPX activities in V79-4 cells treated with
extracts of the medicinal plants, which had previously shown relatively high DPPH radical scavenging
activities (Fig. 4). In addition, we compared these results with those of resveratrol.
All samples dose-dependently increased the antioxidant enzyme activity of V79-4 cells. In particular,
treatment with total extracts of Areca catechu var. dulcissima induced higher antioxidant enzyme
activities than the other samples. At a dose of 100 Ag/ml, total extracts of Areca catechu var. dulcissima
activated SOD, CAT and GPX levels by 45, 51 and 48%, respectively; these values were similar to those

Table 2
Flow cytometric analysis of the protective effect of medicinal plants against H2O2-induced apoptosis in V79-4 cells
Sub G1 G1 S G2/M
Cells (%)
Control 0 54.9 30.4 14.7
H2O2 28.5 47.8 16.3 7.4
Glycyrrhiza uralensis 11.9 63.7 17.5 6.9
Solvia miltiorrhiza 13.6 57.5 18.9 10.0
Paeonia suffruticosa 0.5 63.2 23.2 13.1
Spirodela polyrrhiza 10.0 61.0 19.2 9.8
Areca catechu var. dulcissima 2.9 61.8 14.3 21.0
Cornus officinalis 9.2 50.5 22.6 17.7
Alpinia officinarum 4.2 51.6 11.0 33.2
Nelumbo nucifera 8.7 55.5 8.0 27.8
Cinnamomun cassia 3.8 67.7 12.3 16.2
V79-4 cells were treated with 100 AM H2O2 alone or 100 Ag/ml of extract for 1 hr prior to the addition of H2O2. Cells were
harvested at 24 hr and analyzed for cell cycle distribution by flow cytometry.
176 S.E. Lee et al. / Life Sciences 73 (2003) 167–179

Fig. 4. Effects of medicinal plants on SOD (A), CAT (B) and GPX (C) activities in V79-4 cells. Cells were treated with each
plant extract for 60 min and SOD, CAT, and GPX activities were measured at 560 nm, 240 nm and 340 nm, respectively. Each
experiment was performed at least 3 times and the data are expressed as average enzyme units per mg of protein from the
control F S.D. Black, gray and white bars indicate 4, 20 and 100 Ag/ml samples, respectively. 1; G. uralensis, 2; S.
miltiorrhiza, 3; P. suffruticosa, 4; S. polyrrhiza, 5; A. C. var. dulcissima, 6; C. officinalis, 7; A. officinarum, 8; N. nucifera, 9; C.
cassia, 10-resveratrol and 11-control. * p < 0.05.
 S.E. Lee et al. / Life Sciences 73 (2003) 167–179 177

of resveratrol. Treatment with 100 Ag/ml of resveratrol activated SOD, CAT and GPX by 45, 57 and
47%, respectively. Among other samples, total extracts of Paeonia suffruticosa, Alpinia officinarum and
Cinnamomun cassia induced high increases in antioxidant enzyme activity. Treatment with 100 Ag/ml of
total extracts of Paeonia suffruticosa induced 43, 28 and 45% increases in SOD, CAT and GPX levels,
respectively. Total extracts of Alpinia officinarum increased SOD, CAT and GPX activities by 40, 23 and
42%, respectively, at a dose of 100 Ag/ml. Treatment with total extracts of Cinnamomun cassia at a dose
of 100 Ag/ml induced SOD, CAT and GPX increases of 38, 40 and 41%.

Discussion

High levels of free radicals or active oxygen species create oxidative stress, which leads to a variety of
biochemical and physiological lesions and often results in metabolic impairment and cell death (Ames,
1998).
Various antioxidants have been detected in natural products. Borek (1991) reported upon the
antioxidative effects of aged garlic extract (AGE). AGE exerts its antioxidant action by scavenging
ROS, by enhancing the activities of the cellular antioxidant enzymes SOD, CAT and GPX and by
increasing glutathione in cells. Antioxidant effects in extracts of the medicinal herb Achyrocline
satureioides have been studied (Desmarchelier et al., 1998). The aqueous and methanolic extracts of this
plant were found to be able to reduce the production of TBA reactive substances in rat liver homogenates.
Extracts of the medicinal plants used in this study did not show cytotoxicity in the range of 4–100 Ag/
ml. Extracts of Areca catechu var. dulcissima, Paeonia suffruticosa and Alpinia officinarum more
effectively increased the viability of H2O2-treated cells, which is possibly because of the higher DPPH
radical scavenging activities of these plants.
Extracts of the medicinal plants dose-dependently increased DPPH free radical scavenging activity, in
vitro. The extracts, which showed strong DPPH radical scavenging activity (IC50 < 6.0 Ag/ml) were
those of Areca catechu var. dulcissima, Cinnamomun cassia, Paeonia suffruticosa and Alpinia
officinarum. The possible effect of the plant extracts on lipid peroxidation in H2O2-treated V79-4 cells
was also assessed. Oxidation of unsaturated fatty acids in biological membranes leads to the formation
and propagation of lipid radicals, the uptake of oxygen, the rearrangement of double bonds in
unsaturated lipids and the eventual destruction of membrane lipids, which produce breakdown products
such as malondialdehyde. Most of the plant extracts showed lower lipid peroxidation inhibitory activity,
with IC50’s > 100 Ag/ml, than DPPH radical scavenging activity.
Cells exposed to H2O2 exhibit distinct morphological features of programmed cell death, such as
nuclear fragmentation and an increase in the percentage of cells with a sub-G1 DNA content. However,
treatment with most of the plant extracts reduced the appearance of the morphological features
characteristic of apoptotic cells. Rather, they were similar morphologically and in terms of DNA profile
to the control cells. These data suggest that most of the plant extracts protect cells against H2O2-induced
apoptosis.
We also observed that treating cells with total extracts of medicinal plants increased the activity of all
antioxidant enzymes examined, including SOD, CAT and GPX. These enzymes are modulated in various
diseases by free radical attack (Ames, 1998). Thus, maintaining the balance between the rate of radical
generation and the rate of radical scavenging is an essential part of biological homeostasis. It is of
particular interest to note that SOD catalyzes the breakdown of O2
to O2 and H2O2, and thus prevents the
178 S.E. Lee et al. / Life Sciences 73 (2003) 167–179

formation of OH , and thereby, has been implicated as an essential defense against the potential oxygen
toxicity. The ROS scavenging activity of SOD is effective only when it is followed by the actions of
CAT and GPX, because the dismutase activity of SOD generates H2O2, which needs to be further
scavenged by CAT and GPX. Among the plant extracts selected in this study, extracts of Areca catechu
var. dulcissima and Cinnamomun cassia were found to activate CAT and GPX to a greater extent than
SOD, which suggests that the H2O2 formed by SOD can be effectively scavenged. Also, these results
suggest that the antioxidant activity of extracts of Areca catechu var. dulcissima and Cinnamomun cassia
may be due to the degradation of H2O2 and other peroxides. From these results, the DPPH radical
scavenging activity of Areca catechu var. dulcissima extracts was found to be higher than that of
resveratrol. Furthermore, treatment with total extracts of Areca catechu var. dulcissima induced a similar
increase in antioxidant enzyme activity to that induced by resveratrol. These results suggest that Areca
catechu var. dulcissima extracts have strong antioxidant activity. Most of the plant extracts enhanced
cellular antioxidant activities, such as those of SOD and GPX. In some plant extracts, CAT activity was
reduced compared to that of the control cells at suboptimal concentrations.
Some variation in the extent of the extract antioxidant activities was observed for each type of assay
used in this study. The extracts of Areca catechu var. dulcissima, Paeonia suffruticosa, Alpinia
officinarum and Cinnamomun cassia had strong effects in terms of cell survival and antioxidant activity.
Solvia miltiorrhiza, Cornus officinalis and Nelumbo nucifera extracts had relatively high DPPH radical
scavenging activities, but low lipid peroxidation inhibitory activities. These differences are probably due
to their different antioxidative mechanisms.
Our results further support the view that some medicinal plants are promising sources of potential
antioxidants. Future studies will be aimed at investigating the effects of Areca catechu var. dulcissima,
Paeonia suffruticosa, Alpinia officinarum and Cinnamomun cassia on the regulation of cellular
mechanisms and upon isolating and identifying the substances responsible for the antioxidant effects
of the plant extracts.

Acknowledgements

S. E. Lee is a post-doctoral researcher of Brain Korea 21 program supported by Ministry of

Education, Korea. H. J. Hwang and H.-S. Jeong acknowledge the agricultural R&D grant from Ministry
of Agriculture and Forestry, Korea.

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