Amoebicidal Activities of Indonesian Medicinal Plants in
Balikpapan, East Kalimantan
Fendi Yoga Wardana
1
, Defi Kartika Sari
2
, Myrna Adianti
2,3
, Adita Ayu Permanasari
2
, Lidya
Tumewu
2
, Tomoyoshi Nozaki
4
, Aty Widyawaruyanti
2,5
and Achmad Fuad Hafid
2,5
*
1
Master's Program of Pharmacy, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60286, Indonesia;
2
Institute
of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia;
3
Department of Health, Faculty of Vocational, Universitas Airlangga, Surabaya 60115, Indonesia;
4
Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, 113-0033, Japan;
5
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60286,
Indonesia.
Keywords: Amoebiasis, anti-amoebic, Indonesian medicinal plants, Entamoeba histolytica.
Abstract: Entamoeba histolytica is a protozoan agent causing human amoebiasis, which is responsible for
100,000 deaths annually throughout the world. The recommendation in the treatment of
amoebiasis using metronidazole has been reported to be less effective, because of the drug
resistance effect by Entamoeba histolytica. Therefore, the search of new drugs with amoebicidal
activity is important. The natural substances from medicinal plants are potentially a good object
to be studied. The aim of this study was to evaluate Indonesian medicinal plants for their anti-
amoebic activities. The hexane, dichloromethane and methanol extracts of 114 samples derived
from 22 species of medicinal plants explored in the Balikpapan forest, East Kalimantan had been
tested. Their anti-amoebic activity was determined by in vitro cell-based assay against
Entamoeba histolytica HM-1:IMSS (clone 6) strain. According to cell-based assay, five of 114
samples tested showed anti-amoebic activities. The highest anti-amoebic activity was obtained
from the dichloromethane extract of Cratoxylum sumatranum stembark with 50% inhibitory
concentration (IC
50
) of 22.07 ± 0.84 μg/ml. Subsequently, the dichloromethane extract of leaves
and the dichloromethane extract of stem from Garcinia parviflora with IC
50
of 38.59 ± 9.46
μg/ml and 68.34 ± 0.4 μg/ml, respectively. The hexane extract of stembark and the
dichloromethane extract of stem from Cratoxylum sumatranum had IC
50
of 69.79 ± 16.58 μg/ml
and 118.49 ± 15.26 μg/ml, respectively. The dichloromethane extracts of Cratoxylum
sumatranum stembark and Garcinia parviflora leaves are the most potential candidates in the
development of anti-amoebic drugs.
1 INTRODUCTION
Amoebiasis is an infection of the gastrointestinal
tract in humans caused by the protozoa Entamoeba
histolytica (E. histolytica). Protozoa parasites are
able to attack the intestinal mucosa and can spread to
other organs especially the liver. When the amoeba
infection has reached the liver it will cause an
amoebic liver abscess (Samuel et al., 2001).
Amoebiasis infection is responsible for 100,000
deaths annually throughout the world. It is therefore
considered to be the third most medically important
parasitosis after malaria and schistosomiasis
(Tanyuksel & Petri, 2003).
At present the types of antiamoebic drugs used
in medical treatment are divided into two classes:
luminal and tissue amoebicides. Iodoquinol and
paromomycin are used for the treatment of luminal
amoebicides (Singh et al., 2009), while the
medications used for the treatment of tissue
amoebicides is metronidazole (Towson et al., 1994).
However, several studies have reported that drug
resistance is cause by E. histolytica
(Samarawickrema et al. Wassmann et al., 1999).
Other studies have also reported that metronidazole
Wardana, F., Sari, D., Adianti, M., Permanasari, A., Tumewu, L., Nozaki, T., Widyawaruyanti, A. and Hafid, A.
Amoebicidal Activities of Indonesian Medicinal Plants in Balikpapan, East Kalimantan.
DOI: 10.5220/0008357700770082
In Proceedings of BROMO Conference (BROMO 2018), pages 77-82
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
77
is less effective against infections occurring in the
intestinal lumen tissues (Bhopale et al., 1995).
Since humans are among the main hosts that
place this parasitic life cycle, then proper treatment
for amoebiasis infection is necessary to stop the
development of the parasite. The search for an
effective new drug for anti-amoebic activity with
small side effects is needed at this time. In this case
the selection of natural ingredients as a drug has
advantages based on its long-term use by humans.
The natural substances obtained from medicinal
plants are potentially a good object to be studied and
are expected to have low toxicity on humans
(Newman & Cragg, 2012).
According to the WHO (World Health
Organization) report, around 80% of community in
less developed countries almost completely rely on
traditional medicine for their health treatment
(Bansal et al., 2004). Extracts from various plants
have been isolated and explored for their anti-
amoebic activity (Ciba foundation Symposium 185,
1994). A wide variety of active phytochemicals,
such as flavonoids, terpenoids, polyphenols,
coumarin, saponins, alkaloids, xanthone and
thiophenes, had been identified as inhibiting the
growth of various protozoa (Alanís et al., 2003).
Moreover, a number of bioflavonoid compounds,
such as apigenin, galangin, kaempferol, narigenin,
pinocembrin and quercetin showed biological
activity against E. histolytica and G. lamblia
(Calzada et al., 1999). Indonesia is said to have the
second largest biodiversity in the world, with around
40,000 species of endemic plants including 6,000
medicinal plants (Nugraha & Keller, 2011). A
further study aimed at finding new anti-amoebic
agents for the treatment of amoebiasis was
conducted identifying of 22 medicinal plants
obtained from forest exploration in Balikpapan, East
Kalimantan, Indonesia. The selected plants were
evaluated for the activity of their crude extract in
inhibiting the growth of E. histolytica according to
in vitro cell-based assay. The selection of these plant
species is primarily based on the follow-up of the
use of ethnobotany for the treatment or relief of
symptoms of infectious diseases.
2. EXPERIMENTAL
2.1. Plants Materials
The plants used in this study were the results of
Balikpapan's forest exploration (East Kalimantan,
Indonesia). The plants used have been verified by
licensed botanists at the Balikpapan Botanical
Gardens, Balikpapan, Indonesia. The plant species,
botanical names, families, and parts of plants used to
obtain the extract are presented in Table 1.
2.2. Extraction of Medicinal Plants
The dried plant materials (100 g) were pulverized
and then subjected to solvent extraction with
different polarities sequentially in ascending order
starting with hexane, dichloromethane (DCM) and
ultimately methanol. The extraction process was
carried out by using an ultrasonic system for each
solvent. The filtrates were evaporated using an
evaporator at a temperature not more than 40 °C.
The extracts for bioactivity assay were dried in
vacuum before being used.
2.3. Sample Stock Preparations
Each of the dry extract was weighed for 10 mg and
dissolved in 1 mL of dimethyl sulfoxide (Merck) to
get stock solutions at a concentration of 10 mg/mL.
The stock solutions were stored at
-30 °C until being used.
2.4. Culture of Entamoeba histolytica
The cells of HM-1:IMSS (clone 6) Entamoeba
histolytica strain, were kindly provided by Prof. T.
Nozaki, The University of Tokyo, cultivated in
Bisate-Iron-Serum (BI-S) medium (Sigma) that was
supplemented with 10% (v/v) bovine serum (Sigma)
and 1% (v/v) Diamond Vitamin-Tweena solution
(JRH Biosciences, USA) at 37 °C. The cell was
conditioned for 2 days to reach a confluence 80%.
2.5. Analysis of Anti-amoeba Activities of
Plant Extracts
The Entamoeba histolytica cells were seeded in 98-
well plates. 200 µL of cells and BI-S medium were
added into each well, then the wells were incubated
2 hours at 35.5 °C. After 2 hours of incubation, they
were replaced with mixture of medium and extract
(used 2.5 µL extract and 247.5 µL medium), then
incubated 24 hours. The medium was replaced with
10 % WST-1 reagent (Roche, Germany) in warmed
OPTI-MEM medium (Gibco-Life Technologies).
After that they were incubated for 30 minutes at 37
°C and the absorbance at 560 nm was measured
using Elisa reader. The percent inhibition of cells
growth by the samples was calculated by comparing
to the control by using probit analysis, and IC
50
values were determined.
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
78
Table 1: Anti-amoebic activity against Entamoeba histolytica of Balikpapan medicinal plants tested in this study
No.
Plant Species
Family
% growth inhibition
a
Leaves
Stem
Hexane
DCM
Methanol
Hexane
DCM
Methanol
Hexane
DCM
Methanol
1
Melicope glabra
Rutaceae
0
4.36
0
16.09
12.78
0
-
c
-
-
2
Luvunga scandens
Rutaceae
4.20
3.31
0
6.17
0.63
0.89
-
-
-
3
Artocarpus sericicarpus
Moraceae
13.14
22.79
0
0
0
21.36
-
-
-
4
Artocarpus anisophyllus
Moraceae
0
0.89
0
0
26.63
2.06
-
-
-
5
Artocarpus dadah
Moraceae
0
0.54
0
0
0
0
-
-
-
6
Scorodocarpus borneensis
Olaccaceae
0
0
0
0
28.87
0
-
-
-
7
Eusideroxylon zwageri
Lauraceae
0
1.07
0
-
-
-
-
-
-
8
Fagraea racemosa
Loganiaceae
0
6.43
12.78
-
-
-
16.89
16.09
0
9
Pternandra galeata
Melastomataceae
0
3.31
4.02
-
-
-
0.54
19.66
6.97
10
Goniothalamus
macrophyllus
Annonaceae
0
10.36
12.96
-
-
-
32.71
40.66
16.89
11
Fordia splendidissima
Fabaceae
0
3.49
3.40
-
-
-
9.56
23.15
11.17
12
Garcinia parviflora
b
Clusiaceae
20.46
53.71
b
10.99
-
-
-
49.33
49.87
b
7.33
13
Aglaia lawii
Meliaceae
5.11
34.67
22.61
-
-
-
-
-
-
14
Cratoxylum sumatranum
b
Hypericaceae
0
41.20
12.78
53.80
b
97.23
b
29.67
2.40
59.96
b
27.61
15
Gonocaryum littorale
Icacinaceae
0
0
26.72
-
-
-
-
-
-
16
Orophea hexandra
Lauraceae
5.99
23.68
30.56
-
-
-
-
-
-
17
Alstonia angustiloba
Apocynaceae
0
29.58
26.90
0
2.49
31.81
-
-
-
18
Gymnacranthera
farguhariana
Lauraceae
0
29.13
0
-
-
-
-
-
-
19
Alseodaphne elmeri
Lauraceae
0
0
8.67
15.1
11.89
0
-
-
-
20
Neolistsea cassiaefolia
Lauraceae
0
0
0
-
-
-
-
-
-
21
Vernonia arborea
Asteraceae
6.34
2.75
0.48
7.30
15.08
0
-
-
-
22
Ficus geocaris
Moraceae
0
0
27.49
-
-
-
-
-
-
a
Adjusted to a concentration of 100 μg/ml and positive control using cells in the BI-S medium
b
The plant extracts with growth inhibition of ≥ 50 % and potentially high anti-amoebic activity
C
Not computed
Amoebicidal Activities of Indonesian Medicinal Plants in Balikpapan, East Kalimantan
79
2.6. Cytotoxicity Assay
The cytotoxicity of the samples was assessed by
MTT assay (Wahyuni et al., 2013). In brief, Huh7.it
cells in 96-well plates were treated with serial
dilutions of the medicinal plant extracts or control
for 48 hours. The medium was replaced with MTT
reagent containing medium and incubated for 4
hours. The MTT solution was removed and 100
µL/well of DMSO 100% was then put for
dissolution. The absorbance at 560 nm was
measured using Elisa reader. The percentages of cell
viability was calculated by comparing to the control,
and (CC
50
) values were determined.
3 RESULTS AND DISCUSSION
A total of 22 species of medicinal plants from
Balikpapan's forest exploration (East Kalimantan,
Indonesia) were tested as anti-amoebic. The 22
samples were extracted using different polarity
solvents, resulting in a total of 114 extracts being
used in this study. In the screening, each extract was
tested for inhibitory activity of Entamoeba
histolytica HM-1:IMSS (clone 6) strain using
concentration doses of 100 μg/mL with an
incubation period of 24 hours. The results in the
form of percent inhibitions of extract on cell-based
assay against E. histolytica are presented in Table 1.
Among 114 tested extracts, only five extracts
showed anti-amoebic activities higher or equal to
50% mortality. Five extracts were obtained from two
plants species, namely Garcinia parviflora and
Cratoxylum sumatranum. The highest anti-amoebic
activity (% mortality = 97.23) was obtained from the
dichloromethane extract of C. sumatranum
stembark. This showed that the chemical compound
from stembark of C. sumatranum had very strong
amoebic cell inhibition activity according to cell-
based assay.
At the end of the first screening, five extracts
were obtained from the dichloromethane (DCM)
extracts from leaf and stem of G. parviflora, the
DCM extract from stem of C. sumatranum, and the
hexane and DCM extracts from the stembark of C.
sumatranum. For the five extracts tested for anti-
amoebic activities and from cytotoxic test to obtain
50% inhibitory concentration (IC
50
), 50% cytotoxic
concentration (CC
50
) and selectivity index (SI:
CC
50
/IC
50
), the results are shown in Table 2.
According to the results of anti-amoebic
activities, the DCM extract of C. sumatranum
stembark showed the highest values of IC
50
= 22.07
± 0.84 μg/mL and SI = 1.35. Subsequently, the
hexane extract of stembark and the DCM extract of
stem from C. sumatranum had IC
50
of 69.79 ± 16.58
μg/mL and 118.49 ± 15.26 μg/mL, respectively.
The chemotaxonomy approach of plants from
hypericaceae family, it had potential as an anti-
amoebic. The methanol extract from Harungana
madagascariencis (hypericaceae) has been reported
to have good inhibitory activity against growth of E.
Histolytica with IC
50
of 82.05 μg/mL (Moundipa et
al., 2005).
Furthermore, anti-amoebic activities of the DCM
extract of leaves and the DCM extract of stem from
G. parviflora gave IC
50
of 38.59 ± 9.46 μg/mL and
68.34 ± 0.4 μg/mL, respectively. The ethanol extract
from G. mangostana belongs to the genus Garcinia
in the Clusiaceae family, within the same genus as
G. parviflora, has been reported to possess minimal
inhibitory concentration (MIC) against E. histolytica
of 500 μg/mL (Hounkong et al., 2014).
Chemical compounds of C. sumatranum and G.
parviflora that possess anti-amoebic activities have
not yet been reported. The chloroform and acetone
extracts from air-dried roots of C. sumatranum, was
reported to possess antibacterial activities against
Staphylococcus aureus and Micrococcus luteus
(Tantapakul et al., 2014). The authors identified
several compounds contained in the extract
including xanthone and benzophenone compounds.
Meanwhile, the methanol extract from twigs G.
parvifolia, a plant genetically close to G. parviflora,
has also been reported to have antibacterial activities
against methicillin-resistant Staphylococcus aureus.
The chemical compounds contained in the extract
are phloroglucinol, depsidone and xanthone
(Rukachaisirikul et al., 2006). Therefore, further
research is still required to isolate compounds that
take a role in anti-amoebic activity, and the study is
still under way.
4 CONCLUSION
The results obtained by dichloromethane extract
from stembark of Cratoxylum sumatranum and
leaves of Garcinia parviflora have better anti-
amoebic activity than other extracts. This suggests
that these plants are the most potential candidates in
the development of anti-amoebic drugs, especially to
confirm the correct amoebicidal activity and
biochemical anti-amoebic inhibitory mechanism.
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
80
Table 2. Anti-amoebic activity (IC
50
) and cytotoxicity (CC
50
) of Garcinia parviflora and Cratoxylum sumatranum
Plant Species
Parts
Solvent
IC
50
(μg/ml)
a
CC
50
(μg/ml)
a
SI
Garcinia parviflora
Leaves
DCM
38.59 ± 9.46
39.29 ± 0.21
1.02
b
Stem
DCM
68.34 ± 0.40
40.16 ± 0.39
0.59
Cratoxylum sumatranum
Stem bark
Hexane
69.79 ± 16.58
28.26 ± 0.16
0.41
Stem bark
DCM
22.07 ± 0.84
29.69 ± 1.57
1.35
b
Stem
DCM
118.49 ± 15.26
25.93 ± 0.28
0.22
a
Data represent mean ± SD of data from two repetitions experiment
b
Plant extracts with the high SI values
ACKNOWLEDGEMENTS
This part of research was supported by Japan
International Cooperation Agency (JICA), World
Class Professor (WCP) program from the Indoneisa
Ministry of Research Technology and Higher
Education, Natural Product Management Research
and Development (NPMRD) from Institute Tropical
Disease (ITD) Universitas Airlangga Indonesia.
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