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, 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 [1]. 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 [2].
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 [3], while the medications used for the
treatment of tissue amoebicides is metronidazole [4].
However, several studies have reported that drug
resistance is cause by E. histolytica [5,6], thus
requiring increased doses to treat infection [7] and
thereby increasing the risk of discomfort from the
drug's side effects [8,9]. Other studies have also
reported that metronidazole is less effective against
infections occurring in the intestinal lumen tissues
[10].
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
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: 10.5220/0009842300002406
In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1
ISBN: 978-989-758-347-6
Copyright
c
 2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
1

plants are potentially a good object to be studied and
are expected to have low toxicity on humans [11].
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. [12].
Extracts from various plants have been isolated and
explored for their anti-amoebic activity [13]. 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
[14]. Moreover, a number of bioflavonoid
compounds, such as apigenin, galangin, kaempferol,
narigenin, pinocembrin and quercetin showed
biological activity against E. histolytica and G.
lamblia [15]. 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 [16]. 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.
2.6 Cytotoxicity Assay
The cytotoxicity of the samples was assessed by MTT
assay [17]. 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.
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
2

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
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 Bark
Stem
Hexan
e
DCM
Metha-
nol
Hexan
e
DCM
Methano
l
Hexan
e
DCM
Methano
l
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
Melastomata
ceae
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
3

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 [18].
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 [19].
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 [20].
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 [21]. Therefore, further research is still
required to isolate compounds that take a role in anti-
amoebic activity, and the study is still under way.
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
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
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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.
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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