JKGT VOL.

2, NOMOR 1, JULY (2020) 1-4

(Penelitian)
Effect Of Ethanol, Hexane And Water Extracts Of Clinacanthus Nutans
Leaves On Hone-1 Proliferation
Moehamad Orliando Roeslan1, 3, Siti Ladia Fatima2
1
Department of Histology, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia
2
Undergraduate Student at Faculty of Dentistry, Trisakti University, Jakarta, Indonesia
3
Corresponding author
Email: orliando.roeslan@trisakti.ac.id, orliandoichol@gmail.com

ABSTRACT

Background: Mortality from cancer has increased especially in the developing country. Global pattern shows the half of
mortality occurred in Asia. One type of cancer occurring in Indonesia is nasopharyngeal cancer. Clinacanthus nutans is a
species of herbal plants in the Acanthaceae family that grows in tropical Asia, especially Indonesia. This plant is considered as
one of traditional medicinal plants, which is effective in treating several diseases, including cancer. Different polarity of solvent
that used to extract this plant will have different dissolved compounds.
Objective: To determine the effect of ethanol, hexane, and water extract of C. nutans leaves on HONE-1 cell proliferation.
Method: This study conducted using experimental research in vitro laboratory. Three different extract, ethanol, hexane, and
water extract of C. nutans leaves treated to HONE-1 cell. Cell proliferation was measured using MTT assay.
Result: The results showed significant differences (p <0.05) of treatments group compared to negative control. Ethanol extract
showed the highest inhibition of HONE-1 cell proliferation.
Conclusion: Ethanol, hexane, and water extract of C. nutans leaves could inhibit the HONE-1 cell proliferation.

Keywords: Clinacanthus nutans, ethanol, hexane, water, HONE-1 cell, proliferation.

INTRODUCTION differentiation and high metastasis.9 Typically

Nasopharyngeal cancer is one of cancer in
head and neck that are most frequently occur. 1 The
distribution of this cancer in Asia is quite high,
namely 81%, while in Africa only reached 9 %.
Countries in southeast asia give a large
contribution, up to 67%.2 In indonesia, this disease
is number 4 tumor that occured most frequently
after cervical cancer, breast cancer and skin cancer.
The incidence is expected at around 12,000 new
cases per year, but many of the cases was not
detected because of a shortage of consciousness
and lack of hospital facilities in indonesia. 3 Men are
at risk 3 times higher than women to suffer this
disease. Incidence is the highest in persons with the
age of 50 to 60 years old.4 Elderly people have
higher risk to relapse and have low survival rate.5
Risk factor of nasopharyngeal cancer include
smoking, salted fish consumption, 6 nitrosamine in
some of the Chinese traditional food, 7 exposure of
formaldehyde, wood dust and smoke. 8
Nasopharynx cancer clinically characterized by low
diagnosed at advanced stadium.10 Early
identification of this disease is effecting the
prognosis. At advanced stadium, the prognosis is
very poor.1
Management of nasopharyngeal cancer is
currently carried out by surgery accompanied by
radiation and chemotherapy. However, this therapy
causes various side effects on normal tissue with
various symptoms such as nausea, vomiting,
anorexia, diarrhea, oral mucositis and numbness.
Based on these side effects, as well as the relatively
expensive costs, so far research on cancer therapies
continues to be obtained from natural sources. 11
One of the natural source which has
anti-cancer activity is Dandang gendis
(Clinacanthus nutans).
Clinacanthus nutans is a plant species in the
Acanthaceae family that grows in tropical Asia,
especially Indonesia. The Acanthaceae family is
considered as one of the sources of medicinal
plants which provides effective traditional

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medicines for several diseases.12 Until now this
plant attracted many researchers because of the
potential and pharmacological effects it has. All
parts of the plant have been reported to have
antiviral activity,13 anti-inflammatory properties,
antibiofilms,14 antioxidants,15 and anti cancer.16
In previous studies, ethanol extract of C.
nutans leaves showed antitumor activity by
increasing the regulation of the immune response.17
In addition, ethanol extract also showed anti-
inflammatory,18 antioxidant19 and anti-dengue
activities.18 Whereas hexane extract of C. nutans
leaves showed anti-proliferation activity against
A549 (lung cancer cell line), CNE1
(nasopharyngeal cancer cell line) and HepG2 (liver
cancer cell line).16 The water extract of this plant
also has been reported to have anti-angiogenic
activity.20 The aim of this study is to determine the
effect of ethanol, hexane and water extract of C.
nutans leaves on HONE-1 (epithelial tumor cell
lines) cell proliferation.
METHODS
This study is an in vitro experimental
laboratory. Clinacanthus nutans leaves were
extracted using ethanol, hexane and water, after
which the three extracts were treated to HONE-1
cells.
Plant Material Preparation
Clinacanthus nutans leaves was washed
thoroughly using water to remove dust particles
attached. The leaves are dried for approximately 2
weeks. The dried leaves are then turned into a
powder form with a blender. One hundred grams of
C. nutans powder was extracted with 500 mL
ethanol, hexane and water by maceration method
using for 72 hours. After that, the extract residue is
filtered and continued by concentrating with a
rotary evaporator. The ethanol extract, hexane, and
water obtained were then stored at -20°C until the
extract was ready for use.
Cell Culture
HONE-1 cells were cultured in RPMI medium
(Gibco), supplemented with 10% fetal bovine
serum (Gibco), 250 IU/ml penicillin (Invitrogen)
under standard culture conditions at 37°C in 5%
CO2 humidified incubator. The medium was
changed regularly and the cells were sub-cultured
every 3-4 days.
Proliferation Assay
Confluent cells in 96 wellplates were treated
with ethanol, hexane and water extracts at
concentration of 100 μg/mL. After 24 hours, each
well was given 50 μL MTT (mitochondrial-
dependent reduction of 3- (4,5-dimethyl-2-
thiazolyl) -2,5-diphenyl-2H-tetrazoliumbromide)
then incubated for 4 hours. Each well was added
with 100 μL acidified isopropanol and placed in an
incubator for 1 hour. The absorbance was measured
at 570 nm using a microplate reader (Tecan,
Salzburg, Austria). The cell viability (%) relative to
the control wells containing cell culture medium
without test samples as a vehicle was calculated
using [A] test / [A] control x 100. Where [A] test is the
absorbance of the test sample and [A] control is the
absorbance of the control.
Statistical Analysis
Shapiro-Wilk test was used for normality test.
The differences between experimental groups were
analyzed using a one-way ANOVA with Post-Hoc
test. A p-value (p  0.05) was considered
statistically significant.
RESULTS
The positive control group showed a decrease
in proliferation by 91.8% ± 2.68%, ethanol extract
showed a decrease in proliferation by 89% ±
4.85%, hexane extract showed a decrease in
proliferation by 61.8% ± 27.2% and water extract
showed a proliferation decreased by 56.7% ±
26.8% (Figure 1). The results of the normality test
(Shapiro-Wilk) obtained a value p > 0.05, which
means normal data distribution, with these results it
can be followed by a one-way ANOVA test.
One-way ANOVA test results showed a
significant difference between groups (p < 0.05).
Then continued with the Dunnet post-hoc test, the
results showed a significant difference between
each group (positive control, ethanol, hexane and
water) with negative control group (p <0.05).
Figure 1. Cell proliferation after treated with ethanol, hexane
and water extracts of C. nutans leaves at concentrations 100
μg/mL. Doxorubicin 3 M served as positive control. The data
are expressed as mean (n =3). *Indicates a significant difference
compared to untreated cells (p < 0.05).
DISCUSSION
Clinacanthus nutans is a traditional medicinal
plant that is often used by people in Asia. The
ability of C. nutans leaves extract as a natural
remedy is often used because of its efficacy and
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benefits mainly due to the content of bioactive
compounds in it. One of them is purpurin-18 phytyl
ester isolated from chloroform extract of C. nutans
leaves which has the potential to be a therapeutic
agent for gingivitis and ulceration. 14
In previous studies, C. nutans was shown to
have antitumor activity against Hela cells, MCF-7,
A549,21 K-562, IMR-32.22 HSC-4, and HONE-1.23
The results of this study indicate that ethanol
extracts have the highest inhibitory activity against
HONE-1 cell proliferation compared to two other
solvents, hexane and water. Ethanol extract was not
significantly different from positive control,
meaning ethanol extract’s ability to inhibit HONE-
1 cell proliferation is equivalent to doxorubicin 3
μM. Meanwhile, there is no significant difference
between hexane and water extract. Hexane extract
could inhibit HONE-1 cell proliferation up to 61%,
while water extract up to 56%. It also showed that
the ability of both extracts is good enough, because
it could inhibit proliferation up to 50%. Since water
is easily obtained to extract C. nutans leaves, water
extracts can be easily obtained in remote areas.
People in rural areas can extract C. nutans leaves
with water for the treatment of nasopharyngeal
cancer, but still must be under the supervision of an
authorized doctor.
The high ability of ethanol extract to inhibit
priliferation is thought due to the high ethanol
polarity, so that it is able to dissolved active
compounds. Ethanol solvents are effectively able to
dissolved flavonoid compounds, phenols, alkaloids,
terpenoids, saponins, tannins, anthocyanins. 24 The
possibility is these compounds have high anticancer
activity. Based on previous research, flavonoids are
the main active compounds in C. nutans.12
Hexane is the solvent with the lowest polarity
index among the three solvents used in this study.
Hexane is able to dissolve non-polar compounds
such as lignin, waxes, lipids, aglycon, sterols and
terpenoids.25 Terpenoids are probably compounds
that have anticancer activity in hexane extracts.
Terpenoids is the largest class of natural products.
There are about 25,000 chemical structures of
terpenoid that so far have been used in various
fields, including medicine. Parts of terpenoids
which are known to have anticancer activity are
vincristine, irinotecan, etoposide, paclitaxel,
limonene, cantharidin, artemisinin,
andrographolide, alisol, lycopene, celastrol and
many more.26
Water has the highest polarity index, which
is 9. Water is able to dissolve alkaloids and
glycoside compounds,24 proteins, sugars, tannins.27
In previous studies, tannins showed anticancer
activity against HeLa cells, MCF-7 (breast cancer
cell lines), HCT-116 (intestinal tumor cell strain),
CCD-18Co (non-tumor intestinal cell strain), M220
(pancreatic cancer cell strain) and many more. 28
This study is not in line with research conducted by
Ng et al., which states that water extracts of C.
nutans leaves are unable to inhibit A549 cell
proliferation (lung cancer cell lines), CNE1 cells
(nasopharyngeal cell lines) and HepG2 cells (liver
cancer cell lines).16 However, compared to the
research of Ng et al., there is a fundamental
differences with current study, namely in terms of
the extraction method. Although both use
maceration techniques, Ng's study used warm
water, while this study uses water at room
temperature. This can cause differences in soluble
compounds.
CONCLUSION
Ethanol, hexane and water extract of C.
nutans leaves can inhibit HONE-1 cell
proliferation. Ethanol extract showed the highest
activity, even showed activity that equivalent to
positive control (doxorubicin 3 μM). However,
further research is still needed to find out what
compounds are responsible for the anticancer
activity in each extract.
ACKNOWLEDGMENT
The author would like to thank Sheila Sutanto,
S.Si. for her technical support.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of
interest.
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