Received 1 July 2009; accepted 23 August 2009.
Abstract
We investigated the leaves of
Kleinhovia hospita, a plant which has been traditionally 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 antioxidant 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 50μg/mL, 87.5μg/mL, and 125μg/mL, respectively. Leaf extracts of the medicinal plant
K. hospita showed potent antioxidant activity and moderate cytotoxicity on HepG2 liver cancer cells.
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 compounds 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 application. 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 extraction 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 sodium 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 extracted 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 evap oration, producing 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, diethyl 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 concentration of 100μg/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, 100μg/mL streptomycin, and 100 μ/mL penicillin at 37ºC in a humidified 5% CO
2 atmosphere. To determine cell viability, the MTT assay was used as described by Arung et al [
6] with minor modifications and with hydrogen peroxide (H
2O
2) 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 antioxidant activity of the methanol extract (96%) was similar than that of vitamin C (98%) as a positive control. The
n-hexane, diethyl ether, ethyl acetate, and residue fractions all showed scavenging activity (
Figure 1), with the ethyl acetate fraction showing scavenging activity (84% at 100μg/mL) higher than the
n-hexane (48%), diethyl ether (74%), and residue (77%) fractions. This result indicated that scavenging activity decreased after fractionation of the methanol extract and that components of the extract appeared to work synergistically 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 methanol extract on HepG2 liver cancer cells showed that induced cytotoxicity on HepG2 cells increased with concentration, at 50μg/ml, 87.5μg/mL, and 125μg/mL to produce effects of 14%, 76%, and 80%, respectively (
Figure 2), using for a positive control H
2O
2, an established cytotoxicity inducer. These results indicate 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 cytotoxicity compared with the
n-hexane and ethyl acetate fractions (10% and 0%, respectively,
Figure 3). The isolation of active compounds from these fractions is in progress.
Considering the present results,
K. hospita leaf methanol extract was found to be a good, potential candidate for future use as an antioxidant and a medicine for curing liver cancer, as has been experienced 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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PII: S2005-2901(09)60073-X
doi:10.1016/S2005-2901(09)60073-X
© 2009 Korean Pharmacopuncture Institute. Published by Elsevier Inc. All rights reserved.
Source: http://www.jams-kpi.com/article/S2005-2901%2809%2960073-X/fulltext
