Journal of Applied Pharmaceutical Science Vol. 12(02), pp 133-143, February, 2022
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2021.120213
ISSN 2231-3354
In vitro cytotoxicity of ethanolic extract of the leaf of Calotropis
gigantea from Ie Jue Geothermal Area, Aceh-Indonesia, and its
mouthwash formulation against dental pulp cells
Diana Setya Ningsih
1
, Rinaldi Idroes
2
*, Boy M. Bachtiar
3
, Khairan Khairan
2
, Trina Ekawati Tallei
4
, Muslem Muslem
5
1
Graduate School of Mathematics and Applied Sciences, Universitas Syiah Kuala, Banda Aceh, Indonesia.
2
Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh, Indonesia.
3
Department of Oral Biology, Faculty of Dentistry, Universitas Indonesia, Jakarta, Indonesia.
4
Department of Biology, Faculty of Mathematics and Natural Sciences, Sam Ratulangi University, Manado, Indonesia.
5
Department of Chemistry, Faculty of Science and Technology, Universitas Islam Negeri Ar-Raniry, Banda Aceh, Indonesia.
ARTICLE INFO
Received on: 29/05/2021
Accepted on: 10/09/2021
Available Online: 05/02/2022
Key words:
Calotropis gigantea extract,
geothermal area, mouthwash
formulation, cytotoxicity,
antibacterial.
ABSTRACT
The ethanolic Calotropis gigantea leaf extract (ECGLE) from Ie Jue geothermal area, Aceh-Indonesia, and ECGLE-
based mouthwash formulation has been prepared. The formulation was prepared with various extract concentrations
ranging from 0 to 25% of ECGLE. Both the extract and formulation were evaluated for antibacterial and in vitro
cytotoxic activity in order to determine their potential medicinal value in the oral cavity. Antibacterial tests were carried
out against Gram-negative bacteria (Porphyromonas gingivalis), Gram-positive bacteria (Solobacterium moorei), and
a mix of both Gram-negative and Gram-positive bacteria (P. gingivalis + S. moorei). The cytotoxic activity was
evaluated against human dental pulp primary cells (hDPPC) by calorimetric assay using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide. All formulations passed the stability test with a pH of 5.35–5.92. Antibacterial
activity testing revealed that the higher the ECGLE concentration, the more effective it is against bacteria. In comparison
with other formulations, formulation-3 containing 3 gr of ECGLE demonstrated the highest activity. The minimum
inhibitory concentration (MIC) value and % inhibition of formulation-3 against P. gingivalis, S. moorei, and a mix of
both bacteria were 0.089, 0.075, and 0.083 µg/ml and 88.924%, 90.691%, and 89.72%, respectively. The cytotoxicity
activities (IC
50
) for both ECGLE and a formulation containing ECGLE were 6.44 and 0.27 gr/ml, respectively. The
ability of cells to undergo apoptosis showed a strong correlation between cell viability and the ECGLE extract (R
2
=
0.973) as well as ECGLE-based mouthwash formulation (R
2
= 0.897). The greater the concentration of ECGLE extract
or ECGLE-based mouthwash formulation, the lower the viability of hDPPCs, but the greater the antibacterial activity.
INTRODUCTION
Aceh has several geothermal sites which are contributed
by volcanic activities from Jaboi (Idroes et al., 2021b), Seulawah
Agam (Marwan et al., 2019a, 2019b), and Burni Geureudong
(Dharma et al., 2021; Marwan et al., 2021; Putri et al., 2019).
This condition has an effect on the biodiversity that exists in the
area (Idroes et al., 2016, 2017). Ie Jue is one of the geothermal
manifestations in Seulawah Agam volcano (Idroes et al., 2019a,
2019c; Marwan et al., 2021). This manifestation has higher
acidity and temperature than the others (Ie Seu’eum and Ie
Broek) (Idroes et al., 2018, 2019b). This situation contributes to
plants’ characteristics with thermal and dry resistances that grow
massively in the Ie Jue area. The extreme environment forces the
synthesis metabolism to produce more secondary metabolites
(Nuraskin et al., 2019, 2020). As a result, the plants that grow
there may have medicinal properties, such as antioxidants (Cane
et al., 2020; Suhartono et al., 2019), antimicrobial (Estevam et
al., 2015; Ningsih et al., 2019), antibacterial (Nuraskin et al.,
*Corresponding Author
Rinaldi Idroes, Department of Pharmacy, Faculty of Mathematics and
Natural Sciences, Universitas Syiah Kuala, Banda Aceh, Indonesia.
E-mail: rinaldi.idroes @ unsyiah.ac.id
© 2022 Diana Setya Ningsih et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://
creativecommons.org/licenses/by/4.0/).
133

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143134
2019, 2020; Rahmad et al., 2019; Tallei et al., 2020), antiviral
(Khairan et al., 2021; Tallei et al., 2020, 2021a, 2021b; Tumilaar
et al., 2021), antibiofilm (Pratiwi et al., 2015), and wound healing
(Earlia et al., 2019a, 2019b).
One of the plants which are plenteous in the Ie Jue area
is Calotropis gigantea. This plant is categorized as a weed that
can thrive in unfertilized soil, geothermal area, near the coastal
area, or places with direct sunlight exposure (Tezara et al.,
2011). Calotropis gigantea, from the family Asclepiadaceae, is
a unique plant from Southeast Asia. This plant is also known as
giant milkweed/swallow-wort (English), erukku (Indian), remiga
(Malaysian), and widuri or biduri (Indonesian), and in Aceh, it is
known as rubeek (Sampath Kumar et al., 2015).
Calotropis gigantea is commonly used as a therapy
for various diseases, such as antibacterial, anticancer, antitumor,
antioxidant, and wound healing (Bairagi et al., 2018; Deshmukh
et al., 2009; Idroes et al., 2021; Jacinto et al., 2011; Kar et al.,
2018; Seniya et al., 2011; Singh et al., 2010). All parts of this plant
can be used as herbal medicine, including its leaf (Bairagi et al.,
2018). Calotropis gigantea leaf has been reported to contain several
compounds, such as flavonoids, alkaloids, saponins, triterpenoids,
tannins, and polyphenols. The use of this plant in traditional therapy
tends to disregard the concentration and side effects which may
be caused by the compound content in the leaves. Usually, the
medicinal plant has biological and chemical activities that can
influence the cell of an organism, such as cytotoxic activity.
Based on Nguyen et al. (2017) and Jacinto et al. (2011),
the methanol and dichloromethane extracts of C. gigantea leaf
have high cytotoxicity against human pancreatic cancer cell lines,
colon carcinoma, lung non-small-cell adenocarcinoma, and liver
hepatocarcinoma (Bairagi et al., 2018; Deshmukh et al., 2009;
Idroes et al., 2021; Jacinto et al., 2011; Kar et al., 2018; Nguyen
et al., 2017; Seniya et al., 2011; Singh et al., 2010). However,
there is no investigation yet concerning the cytotoxic activities
of this plant extract against the oral cells such as human dental
pulp primary cells (hDPPCs). The exposure of C. gigantea leaves
extract or its formulation has the potential to produce positive or
negative effects against the hDPPCs, which can be investigated by
in vitro cytotoxicity test.
Niles et al. (2009) stated that the in vitro cytotoxicity
simplified the threshold test of drug safety and validated the target
for the basis of drug modification. Furthermore, cytotoxicity studies
play a role in safeguarding the safety, reducing the production cost,
observing the reaction of the cellular membrane, and penetration
ability of the drug to the body, including the oral cavity (Bácskay
et al., 2018; Niles et al., 2009). Investigation of cytotoxic activities
of C. gigantea leaves, utilized for oral cavity, is still limited,
especially in the form of herbal mouthwash formulation. Hence,
this research aimed to identify the antibacterial and cytotoxic
activities of the ethanolic C. gigantea leaf extract (ECGLE) and
its formulation in the form of mouthwash against the hDPPCs.
MATERIALS AND METHODS
Materials and apparatus
This research was a laboratory experiment with a posttest-
only control group design. It was conducted in the Laboratory
of Oral Biology, Faculty of Dentistry, Universitas Indonesia.
Calotropis gigantea leaves were collected from Ie Jue geothermal
area, Aceh Besar Regency (location coordinate: 5
o
30ʹ24″N
95
o
37ʹ46″E), Aceh-Indonesia. Leaf samples were extracted with
ethanol and their cytotoxicity against hDPPCs was determined.
The pulp cell was the sixth

generation maintained by Oral Biology
Laboratory, Faculty of Dentistry, Universitas Indonesia.
Preparation of ECGLE
The leaves of C. gigantea were washed and dried for 14
days at room temperature. The dry leaves were cut and ground using a
crusher to produce a simplicia. One part of dried simplicia was added
to a macerator, followed by the addition of 10 parts of ethanol 96%,
rinsed for 6 hours, and allowed to rest for 18 hours. The resulting
macerate was precipitated and filtered. The filtrate was evaporated
using a rotary evaporator to obtain a concentrated ECGLE.
Preparation of the ECGLE-contained formulation
The procedure for preparing the mouthwash formulation
was modified from a previously published study (de Paula et al.,
2014). The composition of this formulation can be seen in Table 1.
All materials were dissolved in distilled water up to 100 ml. Each
formulation is distinguished by the amount of ECGLE added to
the mouthwash formulation.
Quality control tests of the ECGLE-based mouthwash
formulation
The stability test of the ECGLE-based mouthwash
formulation was carried out using the shock thermal method
by combining high and low temperatures during storage. The
formulated mouthwash was put into a heat-resistant container and
then exposed to high temperature (60°C) for 1 day. Furthermore,
the mouthwash was exposed to low temperatures for 1 day
(−20°C). This process was carried out continuously for 6 days (3×
cycles) and then transferred to room temperature. During these
processes, physical conditions were observed, especially changes
in consistency, color, smell, and appearance (Ahmad et al., 2018).
Bacterial strains and culture conditions
Porhyromonas gingivalis ATCC 33277 and
Solobacterium moorei ATTC 22971 strains were used in this
research. Bacteria were spread separately on an agar brain
heart infusion medium (BHI-Himedia Laboratories, India) and
incubated for 24 hours under a microaerophilic atmosphere (10%
H
2
: 10% CO
2
: 80% N
2
).
Antibacterial activity test
Antibacterial tests were carried out using the microdilution
method. 100 µl of ECGLE extracts (25%, 12.5%, 6.25%, 3.13%,
and 1.56%) and ECGLE-based mouthwash formulation (F0, F1,
F2, and F3) were added to the culture plate 96 well (microplate).
Then 100 µl of bacteria (P. gingivalis, S. moorei, and a mix of
both P. gingivalis and S. moorei bacteria, which have been diluted
previously) was added. The microplate was closed and put in an
anaerobic jar at 37°C for 24 hours (10% H
2
:10% CO
2
:80% N
2
).
After incubation, the microplates were removed and read using a
microplate reader (M965+, Metertech Inc., Taipei, Taiwan) at 600
nm. The minimum inhibitory concentration (MIC) was defined as
the lowest concentration that completely inhibits the growth of
the microorganism or no visible microbial growth observed within

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143 135
the plate. The percentage of inhibition of the extract/formulation
against the tested bacteria was calculated by the following formula
(Saquib et al., 2019):
% inhibition =
Abs. Control − Abs. sample
× 100
Abs. Control
Cell culture
Preparation of alpha minimum essential medium (MEM)
Alpha MEM was prepared in a sterile condition by
dissolving fetal bovine serum 10% and AA 1% into a 50 ml
volumetric flask. The medium solution was filtered using a 0.2 µm
syringe filter (Laredo-Naranjo et al., 2016).
Cell inoculation/growing
Alpha MEM, which had previously been prepared, was
taken up to 2 ml, diluted with hDPPCs, and added to a 15 ml
Falcon tube. The solution was centrifuged for 5 minutes at 2,000
rpm. The supernatant was removed before adding 1 ml new MEM.
It was stirred and removed into a 25 cm
2
flask, followed by the
addition of another 4 ml MEM. The medium containing the cells
was incubated at 37°C with 5% CO
2
. The medium was replaced
every 2 days until it reached 70%–80% confluence (Naz et al.,
2019).
Cell harvesting, calculation, and seeding
The MEM was removed from the flask after the incubation.
Three ml of PBS was added to the flask, then discharged, and added
with 3 ml 0.25% Trypsin ethylenediaminetetraacetic solution,
followed by 5 minutes incubation. An inverted microscope (Carl
Zeiss type Axiovert 40 CFL) was used to ensure that all cells were
detached from the flask surface. The cell suspension was poured into
15 ml Falcon tube and added with MEM medium to reach 3 ml. The
procedure was followed by a 5-minutes centrifugation at 2,000 rpm
at room temperature. Afterward, the supernatant was discharged
and 1 ml of new MEM medium was added to the tube. The cell
suspension was then diluted by a factor of 10. A total of 10 µl of
suspension was taken, homogenized, and dropped dropwise onto
a hemocytometer for cell calculation using the following equation:
Total cell =
number of cell in
hemocytometer
× dilution factor × 10
4
(coef)
number of cell
counted
After that, the cell suspension was dripped into a 96-well
plate based on the calculation. The plate was reincubated at 37°C
with 5% CO
2
for 72 hours (Naz et al., 2019).
Exposure of the sample to cells
Ethanolic extract of C. gigantea leaves (30 µl) was added
to a 96-well plate containing cells after the medium had been replaced
with 70 µl new MEM. The microplate was incubated for 24 hours at
37°C with 5% CO
2
. The microscope observation was carried out after
24 hours incubation with 10× magnification. The same procedure was
applied to study the mouthwash formulation (Naz et al., 2019).
Determination of cytotoxicity using MTT
After the medium was renewed with 100 µl MEM, the
microplate was added with 50 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) solution and incubated for 3 hours
at 37°C with 5% CO
2
. Acidified isopropanol (100 µl) was added and
the microplate was incubated on the shaker at room temperature for
1 hour. The absorbance was measured using Elisa Reader (Metertech
type Accu Reader +) at 600 nm, where the cell viability was calculated
based on the following formulation (Tabari et al., 2017):
% viability cells =
(abs
sample
- abs
blank
)
× 100%
(abs
control
- abs
blank
)
Morphological analysis of hDPPC
Cell morphology was analyzed following ECGLE
exposure to determine the changes in these cells. These include cell
shrinkage, membrane decay, swelling, chromatin condensation,
and the formation of apoptotic bodies. The observation was
intended to predict the apoptosis mechanism. Meanwhile,
vacuolation of the cytoplasm and the formation of double vesicle
membranes containing organelles were observed in order to
ascertain autophagic cell death (Bustillo et al., 2009).
Results of the analysis
The data were analyzed using Microsoft
®
Excel (Office
SP, 2007). Data analysis included average, standard deviation
Table 1. Formulation composition with a variation of ECGLE concentration.
Formulation (gr)
Composition Formulation 0 Formulation 1 Formulation 2 Formulation 3
C. gigantea extract - 0.1 0.2 0.3
Sodium benzoate 0.1 0.1 0.1 0.1
Saccharin 0.1 0.1 0.1 0.1
Propylene 15 15 15 15
Mint aroma 0.5 0.5 0.5 0.5
Sodium fluoride 0.05 0.05 0.05 0.05
Distilled water 100 ml 100 ml 100 ml 100 ml

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143136
of absorbance, linearity, correlation, IC
50
of the ECGLE, and
ECGLE-based mouthwash formulation on hDPPC viability.
RESULTS
The stability test revealed that all mouthwash
formulations had the same organoleptic, viscosity, and temperature
parameters at 0 and 6 days (3× cycles). A slightly different
result was shown by the formulation acidity. The increase in pH
occurred with the increase in ECGLE concentration. The increase
in pH also occurred between before the cycle (0 days) and after
passing through three cycles (6 days). The pH before the cycle was
lower compared to that after three cycles. An increase in pH (more
alkaline) occurred at F3 (Table 2). However, the pH value of all
formulations was still in the neutral range (5.35–5.92).
Antibacterial activities
The MIC activity from both ECGLE- and ECGLE-based
mouthwash formulation showed that the smaller the concentration
used, the lower the MIC resulted (Fig. 1A). The lowest MIC value
was shown at a concentration of 1.56%, where P. gingivalis had
a lower MIC value of 0.059 mg/ml compared to other bacteria
(0.079 mg/ml for S. moorei and 0.055 mg/ml for a mix of both
bacteria).
However, when compared to the ECGLE-based
mouthwash formulation, the MICs were significantly lower (Fig.
1B) than ECGLE MIC (Fig. 1B). Overall, the resulting MIC value
ranges were 0.089–0.094 mg/ml for P. gingivalis, 0.075–0.800
mg/ml for S. moorei, and 0.083–0.096 mg/ml for a mix of both
bacteria (dual-species). ECGLE-based mouthwash formulation 3
had the lowest overall MIC values for each bacterium.
The small MIC value generated was inversely
proportional to the percentage of inhibition produced. The
smaller the MIC, the greater the percentage of inhibition. In
ECGLE, the percentage of inhibition was much smaller than
mouthwash ECGLE-based mouthwash formulation. The highest
percentage of inhibition produced by ECGLE was 63.361% for P.
gingivalis, 90.400 ± 0.498 for S. moorei, and 93.327 ± 1.199 for
dual-species. Meanwhile, the percentage of mouthwash ECGLE-
based mouthwash formulation inhibitors was 88.924 ± 2.531 (P.
gingivalis), 90.691 ± 2.342 (S. moorei), and 89.72 ± 2.417 (dual-
species). The results of statistical tests showed that there were
significant differences in MIC and the percentage of inhibition
produced in this study between ECGLE concentrations (Saquib
et al., 2019).
Value of IC
50
using MTT assay
The results indicated that the higher concentration of
ECGLE used, the higher the absorbance obtained. It also revealed
that the absorbance of ECGLE-based mouthwash formulation was
higher than ECGLE (Table 1).
Table 1 exhibits that the higher concentration of ECGLE,
the higher the number of living hDPPCs, or in other words, the cell
viability increased with R
2
= 0.973 (Fig. 1). The value of R
2
is
close to 1, which indicated a significant positive correlation.
On the other hand, the determination of linear correlation
obtained from that of ECGLE-based mouthwash formulation
gave a value of R
2
= 0.8968 (Fig. 3). This value showed a strong
correlation between the concentrations of each formulation with
the cell viability percentage. The correlation was negative, in
which if the formulation concentration is high, then the percentage
of cell viability is low, or vice versa.
The linear equation indicated that the ECGLE had a
greater IC
50
(6.44 gr/ml) in comparison with that of the ECGLE-
based mouthwash formulation (0.27 gr/ml). From LINEST, the
R
2
value generated from ECGLE or mouthwash formulation was
close to 1, allowing an assumption that the experimental values of x
and y will not be significantly different from that of the theoretical
values. Moreover, the slope reaches 0.8145 with an intersection on
the y-axis at 1.2112. Meanwhile, the formulation had a slope of R
2
= −58.668 with an intersection on the y-axis at 17.055 (Table 4).
Morphology of hDPPCs
The changes on hDPPCs after the exposure with the
ECGLE- and ECGLE-based mouthwash formulation were
observed using an inverted microscope with 10× magnification
(Figs. 4 and 5). Morphological observation of hDPPCs was as
much important as investigating the cytotoxicity effect of ECGLE.
Cell morphology analysis revealed that ECGLE
exposure resulted in visible bulges. This was due to the blebbing
of the cells, indicating an initial phase of cell apoptosis mechanism
(programmed cell death). It was marked by the cell image with
bulges as indicated by → and the living cells indicated by →.
Additionally, the images suggested the presence of cell shrinkage,
Table 2. The stability test of mouthwash formulation contained ECGLE.
Formulation test
F0 (day) F1 (day) F2 (day) F3 (day)
0 6 0 6 0 6 0 6
1. Organoleptic
a. Color Transparent,
green
Transparent,
green
Transparent,
green
Transparent,
green
Transparent,
green
Transparent,
green
Transparent,
green
Transparent,
green
b. Taste Sweet Sweet Sweet Sweet Sweet Sweet Sweet Sweet
c. Smell Unique Unique Unique Unique Unique Unique Unique Unique
2. pH 5.35 5.52 5,48 5,56 5,56 5,59 5.78 5.92
3. Viscosity Nd Nd Nd Nd Nd Nd Nd Nd
4. Temperature 25.8 25.8 25.8 25.8 25.8 25.8 25.8 25.8
Nd: not detected.

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143 137
as indicated by a change in cell morphology from a basil to a
spherical shape.
DISCUSSION
Stability tests conducted on the mouthwash formulation
using organoleptic, temperature, viscosity, and pH parameters
(Table 2) revealed that formulation 3 (F3) performed significantly
better than the other formulations. However, overall the entire
formulation was very non erosive to the teeth (Table 1). All
formulations showed a pH in the range 5.35–5.92. This highly
meets the recommended mouthwash pH standards according to
ISO 16408-2015, that is, 3.0–10.5 (ISO, 2015), and the standards
according to Collares, that is 4.11–7.0 (Collares et al., 2014).
Based on this fact, it was ascertained that mouthwash ECGLE-
based mouthwash formulation is safe to use (pH 5.35–5.92).
Based on in vitro analysis, both ECGLE- and ECGLE-
based mouthwash formulation showed excellent antibacterial
activity. The lowest MIC to inhibit the growth of all tested
bacteria occurred at the lowest ECGLE concentration (1.56%)
and ECGLE-based mouthwash formulation. It was assumed
that the active substance produced was much greater so that the
higher the extract concentration, the greater the ability to inhibit
bacterial growth. Both extract and ECGLE-based mouthwash
formulation were shown to be more able to inhibit Gram-positive
Figure 1. MIC activity at various concentrations: (A) EGCLE- and (B) ECGLE-based mouthwash
formulation.

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143138
Figure 2. Cell viability and linearity of ethanolic extract of C. gigantea leaves.
Figure 3. Cell viability and linearity of the ECGLE-based mouthwash formulation.
Table 3. The relationship between MIC and the % inhibition occurred in the absorbances of ECGLE and formulation containing ECGLE.
Concentration/
formulation
P. gingivalis S. moorei P. gingivalis + S. moorei
MIC (mg/ml) % inhibition MIC (mg/ml) % inhibition MIC (mg/ml) % inhibition
ECGLE
25% 0.221 ± 0.110 73.041 ± 13.190 0.273 ± 0.031 66.625 ± 3.431 0.208 ± 0.113 74.477 ± 14.126
12,50% 0.131 ± 0.050 84.039 ± 0.426 0.167 ± 0.035 79.546 ± 4.555 0.182 ± 0.297 77.756 ± 3.383
6,25% 0.093 ± 0.016 88.634 ± 1.775 0.211 ± 0.026 74.231 ± 3.490 0.182 ± 0.021 77.721 ± 2.845
3,13% 0.059 ± 0.005 92.847 ± 0.525 0.083 ± 0.003 89.848 ± 0.232 0.092 ± 0.008 88.811 ± 0826.
1.56% 0.059 ± 0.019 92.830 ± 2.416 0.079 ± 0.005 90.400 ± 0.498 0.055 ± 0.009 93.327 ± 1.199
CHX 0.065 ± 0.011 92.056 ± 1.295 0.075 ± 0.008 90.831 ± 0.935 0.069 ± 0.019 91.573 ± 2.327
ECGLE-based mouthwash formulation
F0 0.094 ± 0.002 88.304 ± 2.687 0.076 ± 0.004 90.684 ± 1.919 0.086 ± 0.002 89.231 ± 2.965
F1 0.096 ± 0.001 88.062 ± 3.173 0.8 ± 0.000 90.07 ± 2.498 0.093 ± 0.002 88.552 ± 2.625
F2 0.098 ± 0.001 87.909 ± 2.957 0.085 ± 0.002 89.479 ± 2.902 0.096 ± 0.001 88.107 ± 2.822
F3 0.089 ± 0.002 88.924 ± 2.531 0.075 ± 0.000 90.691 ± 2.342 0.083 ± 0.001 89.72 ± 2.417
CHX 0.475 ± 0.007 94.093 ± 1.570 0.069 ± 0.028 91.928 ± 1.284 0.099 ± 0.067 88.823 ± 5.262
The measurement was carried out in triplicate.

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143 139
Table 4. Absorbance of ECGLE- and ECGLE-based mouthwash formulation.
ECGLE (%) A
600
nm ± SD Mouthwash formulation A
600
nm ± SD
Control negative (medium) 0.088 ± 0.004 Control negative (medium) 0.088 ± 0.004
Control positive (cell) 0.501 ± 0.053 Control positive (cell) 0.501 ± 0.053
25 0.095 ± 0.035 F0 0.078± 0.022
12.5 0.042 ± 0.022 F1 0.040± 0.029
6.25 0.019 ± 0.017 F2 0.010± 0.002
3.13 0.018 ± 0.004 F3 0.008 ± 0.005
1.56 0.016 ± 0.008
The measurement was carried out in triplicate.
Figure 4. The effect of ECGLE addition on the morphology of hDPPCs. Living cells were indicated by the black arrow
(→) and dead cells were indicated by the red arrow (→). The cells were observed using an inverted microscope with 10x
magnification.
Figure 5. The effect of ECGLE-based mouthwash formulation addition on the morphology of hDPPCs. Living cells
were indicated by the black arrow (→) and dead cells were indicated by the red arrow (→). The cells were observed
using an inverted microscope with 10x magnification.

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143140
than Gram-negative bacteria. This was thought to be caused by
the lipopolysaccharide of Gram-positive bacteria being much
thinner than that of Gram-negative bacteria. This finding was
in line with Varposhti’s research which stated that the thickness
and composition of the bacterial membrane affected the bacteria
inhibition ability (Varposhti et al., 2014).
The results of this study produced different cytotoxicity
absorbance values, with ECGLE collected from the Ie Jue
geothermal area yielding higher absorbance than the ECGLE-based
mouthwash formulation (Table 4). It was based on the concentration
difference between ECGLE- and ECGLE-containing formulation,
with ECGLE having a higher concentration than ECGLE-based
mouthwash formulation. The concentration/color intensity
changed the produced absorbance from the real absorbance. It was
in agreement with Lambert–Beer’s law, which stated that there
is a correlation between absorbance and sample concentration.
Furthermore, the MTT method used in this research had a weakness.
It was difficult to remove the formazan solution attached to the cell
mitochondria, which affected the calculation results. This condition
was in line with the report by Aslanturk (2018), where undissolved
formazan used in the MTT method formed purple threads within the
cells, which are difficult to be removed (Aslantürk, 2018). Research
by Wang et al. (2010) suggested thorough evaluation on the method
to study the in vitro cell proliferation which relied on the chemical
properties of the studied plant supplemented (Wang et al., 2010).
These results on absorbance explained that the higher
the sample concentration, the higher the absorbance (Table 4).
Nonetheless, the contrary results were shown by the mouthwash
formulation with a high ECGLE concentration. It was ascribed to
the effect of interaction between the additives ingredients of the
formulation. The addition of ingredients other than the ECGLE
weakened or removed the ability of the mouthwash, affecting the
absorbance from the mouthwash formulation.
In this research, the cytotoxicity activities of ECGLE-
and the ECGLE-based mouthwash formulation against hDPPCs
were observable (Figs. 2 and 3). It was in line with Nguyen et
al. (2017) and Jacinto et al. (2011) reports that C. gigantea leaf
possessed high cytotoxicity (Bairagi et al., 2018; Deshmukh et al.,
2009; Idroes et al., 2021a; Jacinto et al., 2011; Kar et al., 2018;
Nguyen et al., 2017; Seniya et al., 2011; Singh et al., 2010). The
high cytotoxicity was attributed to the presence of secondary
metabolite contents of C. gigantea, such as cardiac glycoside.
This compound was similar to calotropone, possessing cytotoxic
activities against human chronic myelogenous leukemia cells and
gastric cancer, based on the in vitro studies using the MTT method
with IC
50
of 9.7 and 6.7 µg/ml, respectively (Wang et al., 2008).
Cardiac glycosides were known to be capable of
inducing apoptosis by disrupting the homeostasis and inducing the
mitochondrial pathway (Fig. 6) (Muti et al., 2016). This ability was
also in line with the increasing concentration percentage, leading to
the lower survival rate of the cell. The presence of the lysis process
was suspected of causing necrosis on cells due to its cytotoxicity.
The necrosis leads to cell swelling and cellular structure damage,
stopping the metabolism, and then releasing out its components.
This activity caused the cells to stop dividing and growing. In
vitro cells with necrosis did not have sufficient time or energy to
activate apoptosis and did not release apoptosis marker leading to
the disruption in intracellular communication (Muti et al., 2016).
Low IC
50
given by the formulation, in comparison with
the crude extract, was associated with the different ingredients
of each sample (Table 5). In the extract, the ingredients only
consisted of the ECGLE with concentration variation and
distilled water as the solvent. Meanwhile, in the formulation,
the ingredients consisted of other ingredients, including sodium
benzoate, saccharin, propylene glycol, disodium EDTA, sodium
fluoride, and flavoring and coloring agents (Table 1). The
additional ingredients were suspected to contribute to these higher
cytotoxic activities, indicated by the low surviving cell viability. It
was corroborated by Garland et al.’s report (1989) that saccharin
reduced cell viability. Jeng et al. (1998) reported that sodium
fluoride was toxic against the fibroblast cells from oral mucosa
and commonly used to prevent caries (Jeng et al., 1998). Park
Figure 6. The graphic illustration of cardiac glycoside influence against hDPPCs cell.

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143 141
et al. (2011) agreed that sodium benzoate possessed cytotoxic
activity in a mammalian cell by increasing the intracellular Ca
2+
concentration and mitochondrial transmembrane potential in a
dose-dependent manner.
By observing the viability value of <50%, C. gigantea
was concluded to possess high cytotoxicity. However, these
results should be compared with other cytotoxicity tests such as
LDH, BrdU, RT-CES, and agar overlay. Yet, Bácskay et al. (2018)
suggested not to compare the cytotoxicity test using MTT assay
with XTT test owing to the use of tetrazolium-based material,
which is predicted to have a similar limitation with MTT test
(Bácskay et al., 2018). Furthermore, plants with high cytotoxicity
levels cannot always be used in the body, in which the exposed
cell is not merely one cell but many other cells forming a colony.
It then contributes to the higher cell resistance to be protected from
the extract. ECGLE concentration used in further research should
be lower to be capable of reducing the toxic effect of the extract.
The cytotoxicity effect was observed further in Figures
3 and 4, where the higher the extract concentration administered,
the higher the cell death occurred. The presence of blebbing also
indicates one of the cell death patterns (autophagia). Blebbing
condition was more intense on the formulation exposure in
comparison with that on the extract. Thus, it was concluded that
the formulation has higher activities than the extract. These high
activities were associated with the presence of other ingredients
added in the mouthwash formulation. The formulation ingredient
could also be capable of causing cell shrinkage, and the swells
were observable on the surface, as seen in Figures 4 and 5.
Ghabanci et al. (2013), in similar research, reported the comparison
of three mouthwash having the cytotoxic effect on the cultured
cell. The commercial formulation was also revealed to have
high cytotoxicity (Ghabanchi et al., 2013). Research by Muller
et al. (2017) suggested that chlorhexidine 0.05% had moderate
cytotoxic activities, where chlorhexidine and cocamidopropyl
betaine 0.2 % showed strong cytotoxic and antibacterial activities
(Sun et al., 2016).
The ECGLE- and ECGLE-based formulation were used as
a mouthwash. The oral cavity contains many cells, not only single
cells such as hDPPCs, but also other cells, which interact with each
other to form cell layers. Logically, single cells tend to react on their
own to fight external conditions so that it is thought to be easier to die
than if several cells were combined together. This is in accordance
with Muller’s (2017) report, which described the ideal conditions
that exist in the oral cavity (which contains several cells such as oral
fibroblasts, epithelial cells, and immune system cells), which are
thought to affect the ability of cells to survive external stimulation.
Nevertheless, if we want to reduce the activity, it is suggested to use
the extract in a lower concentration than that reported in this research.
The most intriguing aspect of this research was the discovery that
ECGLE extract/ECGLE-based mouthwash formulation with high
cytotoxicity activity also has good antibacterial properties. This is
believed to be because of the high antioxidant content, which inhibits
antibacterial growth but is toxic to cells. The use of high antioxidants
tends to lead to other cancer/degenerative treatments.
CONCLUSION
The ECGLE and ECGLE-based mouthwash formulation
produced in this research had high cytotoxicity. As a result,
additional research is required to establish a safety standard for its
use on humans. The results revealed that the extract concentration
of C. gigantea leaves determined the level of cytotoxicity on the
dental pulp cell. In this research, it was observed that the ECGLE-
based mouthwash formulation with 0.3 g/ml extract of C. gigantea
and 25% extract was far more cytotoxic against the hDPPC. The use
of a lower concentration of active compounds of the C. gigantea
extract was recommended to reduce the cytotoxicity effect on
the hDPPC. ECGLE extract and ECGLE-based mouthwash
formulation have shown in vitro growth inhibition of hDPPC.
This was due to the induction of cell cycle arrest and apoptosis.
However, future study is needed to understand the mechanisms of
cytotoxicity of this plant extract.
ACKNOWLEDGMENTS
The authors appreciate the collaboration between
Universitas Syiah Kuala, Banda Aceh, Indonesia, and Universitas
Indonesia, Jakarta, Indonesia, who have contributed during the
research and the making of this article. This research is funded by
the Ministry of Education and Culture of the Republic of Indonesia
under grant No 38/UN11.2.1/PT.01.03/DPRM/2020.
AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception
and design, acquisition of data, or analysis and interpretation of
data; took part in drafting the article or revising it critically for
important intellectual content; agreed to submit to the current
journal; gave final approval of the version to be published; and
agree to be accountable for all aspects of the work. All the authors
are eligible to be an author as per the international committee of
medical journal editors (ICMJE) requirements/guidelines.
Table 5. Calculation of IC
50
from ECGLE- and ECGLE-based mouthwash formulation using linear regression.
Sample Value
ECGLE IC
50
6.44 gr/ml
Slope 0.814544921 1.211190355 Intercept
Sdslope 0.080912533 1.044061238 SD Intercept
R-square 0.971180289 1.542274121 Sdreg
Formulation IC
50
0.27 gr/ml
Slope −58.668 17.0552 Intercept
Sdslope 14.07108056 2.632458125 SD Intercept
R-square 0.896821686 3.146389264 Sdreg

Ningsih et al. / Journal of Applied Pharmaceutical Science 12 (02); 2022: 133-143142
CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of
interest in this work.
ETHICAL APPROVALS
This study does not involve experiments on animals or
human subjects.
PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional
claims in published institutional affiliation.
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How to cite this article:
Ningsih DS, Idroes R, Bachtiar BM, Khairan K, Tallei TE,
Muslem M. In vitro cytotoxicity of ethanolic extract of the
leaf of Calotropis gigantea from Ie Jue Geothermal Area,
Aceh-Indonesia, and its mouthwash formulation against
dental pulp cells. J Appl Pharm Sci, 2022; 12(02):133–143.