J Acupunct Meridian Stud 2009;2(4):306−308

B RI E F R E P ORT

Antioxidant Activity and Cytotoxicity of the

Traditional Indonesian Medicine Tahongai
(Kleinhovia hospita L.) Extract
Enos Tangke Arung1*, Irawan Wijaya Kusuma1, Sri Purwatiningsih1,
Seong-Soo Roh2, Chae Ha Yang2, Soohyeon Jeon3, Yong-Ung Kim3*,
Edi Sukaton1, Joko Susilo1, Yuli Astuti1, Britanto Dani Wicaksono4,
Ferry Sandra4, Kuniyoshi Shimizu5, Ryuichiro Kondo5
1
Wood Chemistry Laboratory, Department of Forest Product Technology, Forestry Faculty,
Mulawarman University, Samarinda, East Kalimantan, Indonesia
2
Department of Preparatory Oriental Medicine, College of Oriental Medicine, Daegu Haany
University, Daegu, South Korea
3
Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry,
Daegu Haany University, Gyeongsangbuk-do, South Korea
4
Stem Cell and Cancer Institute, Jakarta, Indonesia
5
Department Systematic Forest and Forest Products Sciences, Agriculture Faculty,
Kyushu University, Fukuoka, Japan

Received: Jul 1, 2009 Abstract

Accepted: Aug 23, 2009
KEY WORDS:
antioxidant activity;
cytotoxicity;
Kleinhovia hospita;
traditional medicine
We investigated the leaves of Kleinhovia hospita, a plant which has been tradition-
ally used in Indonesia as phytotherapy to cure liver disease, to describe antioxidant
materials from plant sources. K. hospita leaves were extracted with methanol and
further partitioned into n-hexane, diethyl ether, and ethyl acetate. The antioxi-
dant activity of each fraction and the residue was assessed using a 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging method and their cytotoxicity on HepG2
liver cancer cells was determined by a MTT assay. The K. hospita leaf methanol
extract showed strong antioxidant activity (96%) compared with vitamin C (98%) by
the DPPH method and the measured activity from the subsequent extracts of the
methanol extract were 48.9% for n-hexane, 74.0% for diethyl ether, 84.3% for ethyl
acetate, and 77.1% for the residue. The MTT assay showed the cytotoxicity of the
methanol extract on HepG2 cells at 14%, 76%, and 80% at concentrations of 50 μg/
mL, 87.5 μg/mL, and 125 μg/mL, respectively. Leaf extracts of the medicinal plant
K. hospita showed potent antioxidant activity and moderate cytotoxicity on HepG2
liver cancer cells.

*Corresponding authors. Wood Chemistry Laboratory, Department of Forest Product Technology, Faculty of Forestry,
Mulawarman University, Samarinda, 75123 East Kalimantan, Indonesia.
E-mail: tangkearung@fahutan.unmul.ac.id, tangkearung@yahoo.com
or
Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University,
Gyeongsangbuk-do, South Korea.
E-mail: ykim@dhu.ac.kr

©2009 Korean Pharmacopuncture Institute

Antioxidant activity and cytotoxicity of Tahongai (Kleinhovia hospita L.)
1. Introduction
Kleinhovia hospita L. has been used as a traditional
medicine in parts of Malaysia, Indonesia, and Papua
New Guinea to treat scabies. The bark and leaves
are used as shampoo against lice and the leaf juice
is used as eyewash. The leaves eaten as vegetables
and the bast fibers are used in ropes for tying or
for tethering livestock [1]. In Papua New Guinea
and the Solomon Islands, a preparation from the
cambium is used to treat pneumonia. The leaves
and bark of K. hospita contain cyanogenic com-
pounds that are assumed to help kill ectoparasites,
such as lice, and extracts of the leaves have shown
antitumor activity against mouse sarcomas [2].
Several compounds, such as scopoletin, quercetin
[3], rutin, and kaempferol [4] have been isolated
from the leaves.
In Indonesia, the leaves are traditionally used
by some tribes, including the Toraja, Bugis, and
Makasar, for curing liver disease. Unfortunately,
there are no scientific reports regarding this ap-
plication. In this paper, the antioxidant activity of
K. hospita leaf extracts were evaluated by a 1,1-
diphenyl-2-picrylhydrazyl (DPPH) reduction assay
and the extract’s cytotoxicity on HepG2 cells was
determined, as a cell model related to liver
disease.
2. Materials and Methods
2.1. Chemicals and reagents
The K. hospita leaves used here were collected in
Samarinda, East Kalimantan, Indonesia on February
2007. Methanol, n-hexane, diethyl ether, and ethyl
acetate (Merck, Germany) were used in leaf extrac-
tion and fractionation. DPPH (Tokyo Kasei Kogyo,
Japan) and ethanol and dimethyl sulfoxide (DMSO)
from Merck were used in the DPPH assay. Dulbecco’s
modified eagle’s medium (DMEM) and fetal bovine
serum from Invitrogen (Carlsbad, CA, USA) and so-
dium bicarbonate, streptomycin, and penicillin
from Sigma (St. Louis, MO, USA) were used in cell
culture. Phosphate buffered saline, 3-(4,5-dimethyl-2.
thiazolyl)-2,5-diphenyltetrazolium bromide (MTT)
from Sigma, and isopropanol and HCl from Merck
were used in the MTT assay.
2.2. Extraction and solvent fractionation
One kilogram of dried leaves of K. hospita was ex-
tracted with 15 L of methanol at room temperature
for 24 hours, filtered using filter paper (Whatman 2;
Sigma-aldrich, Germany) and the filtrate dried
under vacuum by rotary evaporation, producing
307
178.38 g of extract. A 90.95 g portion of the extract
was suspended in a ratio of 1:2 methanol:water (v/v)
and partitioned into n-hexane, diethyl ether, and
ethyl acetate. The rotary evaporated n-hexane, di-
ethyl ether, ethyl acetate soluble fractions, and the
water soluble residue massed 41.97 g, 2.95 g, 2.70 g,
and 34.97 g, respectively.
2.3. Antioxidant assay
Three mg of sample was first dissolved in 1L of
DMSO and used for experimentation in a concen-
tration of 100 μg/mL. An assay using a DPPH radical
scavenging method was performed as previously
described by Arung et al [5].
2.4. Cytotoxicity on HepG2 cells
HepG2 cells were grown and maintained in DMEM
supplemented with L-glutamine, 10% fetal bovine
serum, sodium bicarbonate, 100 μg/mL streptomy-
cin, and 100 U/mL penicillin at 37ºC in a humidi-
fied 5% CO2 atmosphere. To determine cell viability,
the MTT assay was used as described by Arung et al
[6] with minor modifications and with hydrogen
peroxide (H2O2) used as a positive control.
3. Results and Discussion
To best of our knowledge, this is the first time
that K. hospita leaf extract has been evaluated for
antioxidant activity and cytotoxicity. The antioxi-
dant activity of the methanol extract (96%) was
similar than that of vitamin C (98%) as a positive
control. The n-hexane, diethyl ether, ethyl ace-
tate, and residue fractions all showed scavenging
activity (Figure 1), with the ethyl acetate fraction
120
100
DPPH scavenging (%)
80
60
40
20
0
C Vc FH FD FE Re
Concentration (100 μg/mL)
Figure 1 Effect of Kleinhovia hospita leaf extracts on
DPPH scavenging activity. C = control; Vc = vitamin C; FH =
n-hexane fraction; FD = diethyl ether fraction; FE = ethyl
acetate fraction; RE = residue fraction.
308 E.T. Arung et al

140 140
Viable cells (% vs. control)

Viable cells (% vs. control)

120 120
100 100
80 80
60 60
40 40
20 20
0 0
C DMSO 0.07 50 87.5 125 C DMSO 0.07 FH FD FE
H2O2 KH extact H2O2 100 μg/mL
(%) (μg/mL) (%)
Figure 2 Cytotoxicity effect of Kleinhovia hospita metha- Figure 3 Cytotoxicity of soluble fractions of Kleinhovia
nol extracts on HepG2 cells. C = control; DMSO = vehicle; hospita on HepG2 cells. C = control; DMSO = vehicle; FH =
KH = Kleinhovia hospita. n-hexane fraction; FD = diethyl ether fraction; FE = ethyl
acetate fraction.

showing scavenging activity (84% at 100 μg/mL)
higher than the n-hexane (48%), diethyl ether
(74%), and residue (77%) fractions. This result in-
dicated that scavenging activity decreased after
fractionation of the methanol extract and that
components of the extract appeared to work syner-
gistically to produce the overall methanol extract
scavenging activity. Further experiments are in
progress to clarify the active compounds in these
extracts.
Assessment of the cytotoxic effect of the metha-
nol extract on HepG2 liver cancer cells showed that
induced cytotoxicity on HepG2 cells increased with
concentration, at 50 μg/ml, 87.5 μg/mL, and 125 μg/
mL to produce effects of 14%, 76%, and 80%, respec-
tively (Figure 2), using for a positive control H2O2,
an established cytotoxicity inducer. These results in-
dicate that the extract could be described as a
hepatoprotector, correlating with the traditional use
in Indonesia of this plant for treating liver disease.
Assessment of the cytotoxic effect of the soluble
fractions on HepG2 cells showed that the diethyl
ether fraction (63%) was superior in induced cyto-
toxicity compared with the n-hexane and ethyl ace-
tate fractions (10% and 0%, respectively, Figure 3).
The isolation of active compounds from these frac-
tions is in progress.
Considering the present results, K. hospita leaf
methanol extract was found to be a good, poten-
tial candidate for future use as an antioxidant and
a medicine for curing liver cancer, as has been ex-
perienced by some tribes in Indonesia.
Acknowledgments
This work was funded by DIKTI of Indonesian
Education Ministry with grant 026/SP2H/PP/DP2M/
III/2008. This work was supported by the Korea
Research Foundation Grant funded by the Korean
Government (MOEHRD, Basic Research Promotion
Fund, KRF-2008-331-E00467).
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