Journal of Ethnopharmacology 93 (2004) 409–415

Antioxidant and anticancer activities of organic extracts

from Platycodon grandiflorum A. De Candolle roots
Ji-Young Lee a , Woo-Ik Hwang b , Seung-Taik Lim a,∗
aGraduate School of Biotechnology, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea
b Department of Biochemistry, College of Medicine, Korea University, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea
Received 21 May 2003; received in revised form 20 March 2004; accepted 26 April 2004

Abstract

This study examined the antioxidant and anticancer activities of the petroleum ether extracts from the roots of Platycodon grandiflorum
A. DC, which is a plant used as both a herbal medicine and food in Asia. Extracts from Platycodon grandiflorum in petroleum ether were
fractionated (fractions I–V) by silica gel column chromatography using gradient solvents (petroleum ether: ethyl ether, 9:1–5:5, v/v). The
antioxidant activities of the fractions were evaluated in terms of their inhibition of lipid peroxidation as well as their free radical scavenging
activity. Fraction II, which was extracted at an 8:2 mixture of petroleum ether and ethyl ether, exhibited the greatest antioxidant activity
among the fractions. On the other hand, the cytotoxicity of each fraction, which was evaluated by the MTT assay using human cancer cell
lines (HT-29, HRT-18 and HepG2), was greatest in fraction III, which was extracted with a 7:3 petroleum ether and ethyl ether mixture. Both
fractions, II and III, were sub-fractionated by thin layer chromatography, and the sub-fractions each were screened for their antioxidant and
anticancer activities. In addition, the antioxidant activity was closely related to the content of phenolic compounds, and the anticancer active
fraction exhibited a typical UV absorbance spectrum of polyacetylene.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Antioxidant activity; Cytotoxicity; Platycodon grandiflorum; DPPH; MTT

1. Introduction Anticancer agents, on the other hand, are mainly related to

Antioxidants are added to a variety of foods to prevent or
deter free radical-induced lipid oxidation, which is respon-
sible for the development of off-flavors and the undesirable
chemical compounds in food (Angelo, 1996). The free radi-
cals can also be generated in biological systems in the form
of reactive oxygen species (ROS), such as superoxide anion
radicals (O2 •− ), hydrogen peroxide (H2 O2 ), hydroxyl rad-
icals (OH• ), and the singlet oxygen (1 O2 ) (Halliwell et al.,
1995). These reactive ROS cause destructive and irreversible
damage to the components of a cell, such as lipids, proteins
and DNA (Lopaczyski and Zeisel, 2001). Although normal
cells possess antioxidant defense systems against ROS, the
continuous accumulation of damage to the cells induces dis-
eases such as cancer and aging (Matés and Sánchez-Jiménez,
2000). The continuous antioxidant dose also plays a pre-
ventive role against these diseases by removing the ROS in
biological systems (Sgambato et al., 2001).
∗ Corresponding author. Tel.: +82 2 3290 3435; fax: +82 2 927 5201.
E-mail address: limst@korea.ac.kr (S.-T. Lim).
their curative role in a damaged system. Under normal con-
ditions, the cells in which the DNA or other components are
irreversibly damaged by various causes undergo apoptotic
cell death, which is a self-destructive metabolism accord-
ing to the genetically encoded cell death-signal (Korsmeyer,
1995; Hooper et al., 1999). However, cancer cells, which are
already irreversibly developed, obtain the capability to evade
apoptosis by various ways. The aim of anticancer agents is
to trigger the apoptosis signaling system in these cancer cells
whilst disturbing their proliferation (Bold et al., 1997).
Plants have many phytochemicals with various bioactivi-
ties, including antioxidant, anti-inflammatory and anticancer
activities. For example, some studies have reported that ex-
tracts from natural products, such as fruits, vegetables and
medicinal herbs, have positive effects against cancer, com-
pared with chemotherapy or recent hormonal treatments
(Pezzuto, 1997; Wu et al., 2002). Therefore, many plants
have been examined to identify new and effective antioxi-
dant and anticancer compounds, as well as to elucidate the
mechanisms of cancer prevention and apoptosis (Pietta et al.,
1998; Kim et al., 1998; Swamy and Tan, 2000). In particu-

0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.04.017
410 J.-Y. Lee et al. / Journal of Ethnopharmacology 93 (2004) 409–415
lar, oriental medicinal plants are considered to be one of the
most promising sources due to their variety of species and
applications. In addition, their therapeutic effect has been
demonstrated by their clinical uses in Asia for many decades.
The roots of Platycodon grandiflorum A. De Candolle
(Korean name, ‘Doraji’, Japanese name, ‘Kikyo’, and Chi-
nese name, ‘Jiegeng’), which belongs to the Campanulaceae
family, have been used as either a food material or a tradi-
tional oriental medicine. The extracts from Platycodon gran-
diflorum have been reported to have a wide range of health
benefits. In particular, in Korea, the roots grown for 4 years
have been used to treat bronchitis, asthma, pulmonary tu-
berculosis, diabetes and inflammatory diseases (Takagi and
Lee, 1972; Lee, 1973). Recently, its immunopharmacologi-
cal effects have been studied (Nagao et al., 1986), and some
active compounds, such as triterpenoid (Nikaido et al., 1999)
and saponin (Ishii et al., 1984) have been identified. Some
studies even extended the cultivation period of Platycodon
grandiflorum to 22 years using a patented method (Lee,
1991), and reported that its aqueous extracts were effec-
tive in preventing hypercholesterolemia, hyperlipidemia, and
CCl4 -induced hepatotoxicity (Lee and Jeong, 2002). How-
ever, there are few reports on the antioxidant and anticancer
activity of the organic extract from Platycodon grandiflo-
rum.
In a previous study, it was reported that the crude
petroleum ether extract from Platycodon grandiflorum ex-
hibited strong inhibitory activity against human cancer cell
growth, and the activity of the organic extract was greater
than that of the aqueous extract (Lee et al., 1998). In this
study, the petroleum ether extract of Platycodon grandi-
florumroot was fractionated using gradient solvents, and
their antioxidant activity was compared with commercial
antioxidant agents. In addition, the anticancer activity of
the fractions was also examined.
2. Materials and methods
2.1. Material
The chopped and dried roots of Platycodon grandiflo-
rumA. DC (Campanulaceae family) cultivated in Youngju,
Korea, were purchased from the oriental herbal market,
and the Korea Molecular Medicine and Nutrition Re-
search Institute confirmed the identity. The human rectal
(HRT-18), hepatoma (HepG2) and colon (HT-29) can-
cer cell lines were purchased from the American Type
Culture Collection (Maryland, USA). Dulbecos Modi-
fied Eagle Medium (DMEM), fetal bovine serum and
trypsin–EDTA were acquired from Gibco BRL (Grand
Island, NY, USA), and the culture supplies such as the
48-well plates were obtained from Nunclon Brand Prod-
ucts. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)
were purchased from the Sigma-Aldrich Company (St.
Louis, MO, USA). Silicic acid (BIO-SIL A 100–200 mesh)
was a product from Bio-rad Lab. (Richmond, CA, USA),
and the analytical and preparative thin layer chromatogra-
phy (TLC) was performed using silica gel 60 F254 (Merck,
Darmstadt, Germany).
2.2. Extraction and fractionation
The dried roots of Platycodon grandiflorum (600 g) were
ground into powder, and extracted with petroleum ether (4 L)
by shaking for 72 h at room temperature. After the powder
particles had settled down, the clear yellow supernatant was
filtered with a 0.22 ␮m pore size PTFE filter (Milipore Co.,
Billerica, USA), and concentrated (10.3 g, dry weight) by
vacuum-evaporation. The concentrate was then fractionated
(Fractions I–V) using a silica gel column with a solvent
gradient of petroleum ether and ethyl ether (9:1–5:5), and
the fractions were concentrated under reduced pressure.
2.3. Measurement of the antioxidant activity
2.3.1. Ferric thiocyanate test (FTC)
The antioxidant activity analysis using ferric thiocyanate
was performed according to the method reported by Osawa
and Namiki (1981). Six hundred micrograms of the dried
solids from each fraction were dissolved in 0.12 mL of 98%
ethanol, and 2.88 mL of a 2.51% linoleic acid solution in
EtOH and 9 mL of a 40 mM phosphate buffer (pH 7.0)
were added. The mixture was incubated at 40 ◦ C in a dark
screw-cap vial. During the incubation, a 0.1 mL aliquot was
taken from the mixture, and diluted with 9.7 mL of 75%
ethanol, followed by the addition of 0.1 mL of 30% ammo-
nium thiocyanate. Precisely 3 min after adding the 0.1 mL
of 20 mM ferrous chloride in 3.5% hydrochloric acid, the
absorbance for the red color was measured at 500 nm. The
level of lipid peroxidation inhibition by each fraction was
calculated from the absorbance ratio to that of a blank with-
out any sample.
2.3.2. Thiobarbituric acid test (TBA)
This assay was performed according to the method re-
ported by Kikuzaki and Nakatani (1993). Two milliliters of
20% trichloroacetic acid and 2 mL of thiobarbituric acid so-
lutions were added to 1 mL of the mixture solution contain-
ing linoleic acid, which was prepared according to the FTC
procedure. The mixture was then placed in a boiling water
bath for 10 min. After cooling, the mixture was centrifuged
at 3000 rpm for 20 min, and the absorbance of the super-
natant was measured at 532 nm.
2.3.3. DPPH radical scavenging test
This test was measured as described by Blois (1958). One
milliliter of the fraction solutions (50, 100, and 200 ␮g/mL
in ethanol) was added to 1 mL of a DPPH solution (0.2 mM
in ethanol). After a 30 min of reaction at room temperature,
the absorbance of the solution was measured at 517 nm.
 J.-Y. Lee et al. / Journal of Ethnopharmacology 93 (2004) 409–415
The free radical scavenging activity of each fraction was
determined by comparing its absorbance with that of a blank
solution (no sample).
2.4. Cytotoxicity on cancer cells (MTT test)
This assay was performed according to a slight modifi-
cation of the procedure reported by Mosmann (1983). In
48-well plates, a human cancer cell suspension (3 × 104
cells/well) was incubated for 24 h at 37 ◦ C. The cells were
then rinsed and grown in a new DMEM containing each frac-
tion (300 ␮g/mL). After incubation for 24 or 48 h at 37 ◦ C,
the DMEM was removed, and the cells were again incubated
again with 0.25 mL of DMEM and 0.05 mL of a MTT solu-
tion (0.5 ␮g/mL) for 4 h. 0.7 mL of the lysing buffer (20%
sodium dodecyl sulfate in 50% N,N-dimethylformamide, pH
4.6) was then added to each well to dissolve the purple for-
mazan produced by the MTT, and the cells were incubated
for a further 2 h. The plate was read using a spectrophotome-
ter (Beckman model DU-64) at a wavelength of 540 nm.
The cytotoxicity was obtained by comparing the absorbance
between the samples and the control.
2.5. Total phenolic content
The total phenolic content was determined using a
Folin–Ciocalteu reagent according to the procedure reported
by Singleton and Rossi (1965). The fractions (400 ␮g/mL
in ethanol) were mixed with 0.75 mL of a Folin–Ciocalteu
reagent, which was diluted 10-fold with distilled water im-
mediately before use, which was followed by standing at
room temperature for 5 min. Subsequently, 0.75 mL of a
sodium bicarbonate solution (60 mg/mL) was added. This
mixture was stored for 2 h at room temperature, and its
absorbance was measured at 725 nm. The analysis was per-
formed in triplicate, and the results are expressed as the
ferulic acid equivalents.
2.6. Thin layer chromatography (TLC)
The fractions from the silica gel column were an-
alyzed by TLC with a mobile phase of petroleum
ether:chloroform:methanol (15:7:3, v/v/v) (A) or petroleum
ether:diethyl ether:acetic acid (80:20:1, v/v/v) (B), accord-
ing to a slight modification of the procedure reported by
Amarowicz et al. (2000). The separated spots on the TLC
plate were identified under UV lights of a short (254 nm)
and long (365 nm) wavelength, and also by spraying with
H2 SO4 . The phenolic compounds could be visualized by
spraying the plates with a 1% FeCl3 solution in 1 M HCl
(Reio, 1958). For the rapid detection of the antioxidant
activity of the spots, the plates were stained with a DPPH
solution (Soler-Rivas et al., 2000). After discerning the most
active spot on the plates, a preparative TLC plate was used
to scrape and collect—it’s a large amount for a quantitative
DPPH assay of the antioxidant activity.
411
3. Results and discussion
3.1. Yield and total phenolic content
Fractions I–V were separated from the crude petroleum
ether extract using a silica gel column and gradient solvents
(9:1–5:5), yielding 0.02–0.33 g/g, based on the initial weight
of the crude extract (Table 1). The total phenolic content
in the fractions ranged from 1.66 to 4.80 mg/g, and fraction
II, which was eluted with a 8:2 mixture of petroleum ether
and ethyl ether, contained the highest level of the phenolic
compounds.
3.2. Inhibition of lipid peroxidation
The antioxidant activity of the crude extract and its frac-
tions were measured using ferric thiocyanate (FTC) and thio-
barbituric acid (TBA) tests. In the FTC test, which deter-
mines the amount of peroxide produced at the initial stage
of lipid peroxidation, a lower absorbance indicates a higher
level of antioxidant activity. Fig. 1 shows the changes in
the absorbance for each fraction during 3 days of incuba-
tion at 40 ◦ C, in comparison with BHA and ␣-tocopherol.
The absorbance of the control increased in proportion to
the incubation time, and the absorbance of the other sam-
ples also increased with increasing incubation time. How-
ever, most samples, with the exception of fraction I, showed
a significantly lower increment in the rate compared to the
control after 48 h (P < 0.01). Although the differences be-
tween the samples were not so significant until 48 h, frac-
tion II showed a slightly higher inhibition effect after 72 h
than the others (P < 0.01). However, the differences in the
absorbance between the fractions and commercial antioxi-
dants, ␣-tocopherol and BHA, also increased as incubation
continued.
Different from the FTC test, which is related to the per-
oxide formation in the initial stage of lipid oxidation, the
TBA test measures the amount of malondialdehyde (MDA)
produced after the decomposition of the lipid peroxide dur-
ing the oxidation process. MDA is a very unstable com-
pound causing mutagenic and cytotoxic events (Zin et al.,
2002). At a low pH and high temperature (100 ◦ C), MDA
binds TBA to form a red complex that can be measured at
Table 1
Extraction yields and phenolic contents of each fraction from petroleum
ether extract of Platycodon grandiflorum
Fraction Yields (g/g)a Total phenolic content (mg/g)b
I (9:1) 0.33 1.66 ± 0.42
II (8:2) 0.11 4.80 ± 0.26
III (7:3) 0.26 2.91 ± 0.24
IV (6:4) 0.05 3.45 ± 0.17
V (5:5) 0.02 3.79 ± 0.10
ag fractions/g solids of petroleum ether extracts.
bg total phenolic/g fractions: Ferulic acid equivalents (mean ± S.D.;
n = 3).
412 J.-Y. Lee et al. / Journal of Ethnopharmacology 93 (2004) 409–415

1 fractions, which was slightly lower than the activity of BHA.

The slightly higher value of fraction I than the control in the
TBA test (P < 0.05) was expected from the co-oxidation of
oil or fat, which may have been eluted early from the silica
gel column.
Absorbance at 500 nm

3.3. Scavenging activity of free radical

Free radicals are known to be a major factor in bio-

0.5
Control
FI
F III
FV
-Tocopherol
0 increase in activity. Fraction II exhibited the strongest an-
0 24 48
Time (h)
Fig. 1. Absorbance of the fractions (F) from Platycodon grandiflorum by
FTC method with increasing incubation time, compared with BHA and
␣-tocopherol (mean ± S.D.; n = 3).
532 nm after incubation for 72 h. The results from the TBA
test (Fig. 2) strongly correlated with the FTC data, and most
fractions, except for fraction I, showed a significantly lower
absorbance than the control (P < 0.01). However, in the
FTC test, fraction II, III and V showed significantly higher
inhibition activities than ␣-tocopherol (P < 0.01), and frac-
tion II was second only to BHA in activity. Therefore, from
the results of both tests, fraction II was the most effective
in inhibiting the lipid peroxidation than the other separated
logical damages, and DPPH has been used to evaluate
the free radical-scavenging activity of natural antioxidants
(Yokozawa et al., 1998; Zhu et al., 2001). DPPH, which is
a radical itself with a purple color, changes into a stable
compound with a yellow color by reacting with an antioxi-
dant, and the extent of the reaction depends on the hydrogen
Crude donating ability of the antioxidant (Bondent et al., 1997).
F II
Fig. 3A shows the DPPH free radical scavenging activity
F IV
BHA
of each fraction at three different concentrations, 50, 100
and 200 ␮g/mL. All the fractions showed dose-dependant
72 tioxidant activity in accordance with the other tests (P <
0.01), and the activities decreased in the order of fraction
III > crude > V > I > IV at a concentration of 200 ␮g/mL.
The IC50 of fraction II for the DPPH radical scavenging
activity was calculated to be approximately 130 g/mL.
Since fraction II had the highest antioxidant activ-
ity in the three antioxidant activity tests, it was further
sub-fractionated by TLC. Five different spots from frac-
tion II were detected under the short and long wavelength
of UV light, and showed different colors by H2 SO4 : Rf
= 0.45 (yellow-brown), 0.56 (violet), 0.64 (green-yellow),
0.68 (dark brown) and 0.81 (brown) for sub-fraction II-1,
2, 3, 4 and 5, respectively. From a rapid DPPH test on the
TLC plate, all the sub-fractions, except for II-5, showed
a bright yellow color, and in particular, sub-fraction II-2
had a relatively stronger free radical scavenging activity

0.8

0.7
Absorbance at 532 nm

0.6

0.5

0.4

0.3

0.2

0.1

0
Control Crude FI F II F III F IV FV BHA
Toco-
pherol

Fig. 2. Absorbance at 532 nm of the fractions (F) from Platycodon grandiflorum extract by TBA method, compared with BHA and á-tocopherol (mean
± S.D.; n = 3).
 J.-Y. Lee et al. / Journal of Ethnopharmacology 93 (2004) 409–415
Fig. 3. DPPH free-radical scavenging activity (%) of each fraction (F)
with increasing concentrations (A), and of the sub-fractions (SF) isolated
from fraction II (B) compared with BHA and ␣-tocopherol (mean ±
S.D.; n = 3).
than the other sub-fractions. Since it was reported that the
antioxidant activity of a plant extract often originates from
phenolic compounds (Velioglu et al., 1998; Amarowicz
et al., 2000), a phenolic spot test was performed directly
on the TLC plate. Similar to the previous phenolic test re-
sults of fraction II, sub-fraction II-2 showed a strong blue
color, indicating that it has a high concentration of phenolic
compounds which are responsible for its high antioxidant
activity.
According to the results of analytical TLC, all the five
sub-fractions were obtained using preparative TLC, and the
DPPH free radical scavenging activities of the sub-fractions
were measured quantitatively (Fig. 3B). The radical scaveng-
ing activity of sub-fraction II-2 was significantly stronger
than those of the other sub-fractions and ␣-tocopherol, but
was still lower than that of BHA (P < 0.01). However, con-
sidering that the sub-fraction is not a pure compound, the
active compound can be expected to be comparable to the
strong antioxidant, BHA.
3.4. Cytotoxicity activity on cancer cells
The anticancer activities of the Platycodon grandiflorum
extracts and its fractions were investigated using a MTT
assay on three human cancer cell lines, HT-29, HepG2
and HRT-18. A mitochondrial enzyme in living cells,
succinate-dehydrogenase, cleaves the tetrazolium ring, con-
verting the MTT to an insoluble purple formazan. Therefore,
413
Fig. 4. Cytotoxicity of each fraction (F) (300 ␮g/mL) on three human can-
cer cells (HT-29, HRT-18 and HepG2) (A), and of the sub-fractions (SF)
from fraction III on human colon cancer cells (HT-29) with increasing
concentration (100 and 200 ␮g/mL) (B) after 24 h incubation, measured
by MTT test (mean ± S.D.; n = 4).
the amount of formazan produced is directly proportional
to the number of viable cells (Mosmann, 1983). Fig. 4A
shows that fractions II, III and V significantly inhibited
cancer cell growth at a concentration of 300 ␮g/mL (P <
0.01), and in particular fraction III exhibited the highest
cytotoxicity (>50%) in all three cell lines. Among the cell
lines, the cytotoxic susceptibility to fraction III was greater
in the HepG2 (69.1 ± 0.52%) than in HT-29 (53.0 ±
2.22%) or HRT-18 (56.2 ± 0.50%) cells (P< 0.01), but this
difference was not as significant for the other fractions. The
significant negative activities (P < 0.01) of some fractions
to specific cell lines means that they increased the growth
rate of cancer cell, but more confirmative studies will be
needed in the future to confirm this.
Fraction III was also examined by analytical TLC,
and six sub-fractions were obtained by preparative TLC
(III-1–III-6). The anticancer activities of the sub-fractions
were assayed using one of the cell lines previously used,
HT-29, at a concentration of 100 and 200 ␮g/mL after 24 h
incubation. As shown in Fig. 4B, sub-fractions III-1 and
III-2 had a significantly higher cytotoxicity on HT-29 than
the other sub-fractions (P < 0.01), in a dose-dependent man-
ner. The estimated IC50 for the cytotoxicity of sub-fractions
III-1 and III-2 were approximately 340 and 390 ␮g/mL,
respectively. The UV absorbance spectra of sub-fractions
III-1 and III-2 revealed that they have a similar λmax at
238, 253, 268 and 283 nm, which is a typical spectrum for
the diyn-ene chromophore of polyacetylene. Polyacetylenes
414 J.-Y. Lee et al. / Journal of Ethnopharmacology 93 (2004) 409–415
found in ginseng (Matsunaga et al., 1990) and other medic-
inal plants (Jung et al., 2002) have been reported to exhibit
anticancer activity. A few polyacetylene compounds have
also been reported from the Platycodon grandiflorum or-
ganic extracts (Tada et al., 1995), but they are believed to
be different from the polyacetylene in this study because of
the different UV spectra.
4. Conclusion
The petroleum ether extract of Platycodon grandiflo-
rum was confirmed to exhibit antioxidant and anticancer
activities. The lipid peroxidation and free radical scav-
enging assays on the fractions from the silica gel column
and TLC suggest that the antioxidant active and proba-
bly phenolic compound in this study has a high activity,
which is comparable to BHA. Their MTT assay revealed
that Platycodon grandiflorum also contains a strong poly-
acetylenic anticancer compound, which exhibited cyto-
toxicity on the three human cancer cell lines. However,
further studies will be needed to identify the exact molec-
ular structures of both the antioxidant and anticancer
compounds.
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