Scientia
Pharmaceutica Article
Antiproliferative Activity of Triterpenoid and Steroid
Compounds from Ethyl Acetate Extract ofCalotropis gigantea
Root Bark against P388 Murine Leukemia Cell Lines
Kartini Hasballah
1,
*, Murniana Sarong
2
, Renzavaldy Rusly
3
, Herdina Fitria
2
, Dewi Rara Maida
2
and Muhammad Iqhrammullah
2,4





Citation:Hasballah, K.; Sarong, M.;
Rusly, R.; Fitria, H.; Maida, D.R.;
Iqhrammullah, M. Antiproliferative
Activity of Triterpenoid and Steroid
Compounds from Ethyl Acetate
Extract ofCalotropis giganteaRoot
Bark against P388 Murine Leukemia
Cell Lines.Sci. Pharm.2021,89, 21.
https://doi.org/10.3390/
scipharm89020021
Academic Editor: Helen D. Skaltsa
Received: 9 April 2021
Accepted: 17 May 2021
Published: 19 May 2021
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4.0/).
1
Department of Pharmacology, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia
2
Department of Chemistry, Faculty of Mathematics and Sciences, Universitas Syiah Kuala,
Banda Aceh 23111, Indonesia; [email protected] (M.S.); [email protected] (H.F.);
[email protected] (D.R.M.); [email protected] (M.I.)
3
Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia; [email protected]
4
Graduate School of Mathematics and Applied Sciences, Universitas Syiah Kuala,
Banda Aceh 23111, Indonesia
*Correspondence: [email protected] or [email protected]
Abstract:
Calotropis giganteahas been known to produce bioactive secondary metabolites with
antiproliferative activities against cancer cells. Herein, we extracted the secondary metabolites using
ethyl acetate from its root bark and further tested its antiproliferative activities against P388 murine
leukemia cell lines. The subfractions from the ethyl acetate extract was obtained from Vacuum Liquid
Column Chromatography (VLCC), and followed by Gravity Column Chromatography (GCC). The
subfraction C
2and D
1were identied to contain triterpenoids and steroids with the most potent
cytotoxicity against Brine Shrimp Lethality Test (BSLT). A 3-(4,5-dimethylthiazol-2-yl) -2-5 diphenyl
tetrazolium bromide (MTT) assay suggested that ethyl acetate extract has the highest antiproliferative
activities against P388 murine leukemia cell lines (IC
50= 21.79g/mL), as opposed to subfraction
C
2(IC
50= 50.64g/mL) and subfraction D
1(IC
50= 49.33g/mL). The compound identied in
subfraction C
2and D
1are taraxerol acetate and calotropone, respectively. Though taraxerol acetate
and calotropone were active in inhibiting the leukemic cell lines, their IC
50s were lower than the
ethyl acetate extract, which is probably due to the synergism of the secondary metabolites.
Keywords:leukemia; calotropone; taraxerol acetate; anticancer;Calotropis gigantea
1. Introduction
Cancer is one of the most feared diseases in modern life and its treatment is usually
carried out by chemotherapy, radiation therapy, surgery and immunotherapy [1,2]. Those
treatments result in side effects, which eventually lead the researcher to look for alternative
cancer treatments using natural compounds with high bioactivities. Furthermore, it is
expected that the plant-derived compound may yield a lower risk of side effects in com-
parison with synthetic compounds [3]. One of the plants that is famous for its bioactive
compounds isCalotropis gigantea[4,5]. This plant comprises chemical compounds such as
alkaloids, resins, phenols, amyrin, sitosterol, isogiganterol, giganterol, avonol glycosides,
naphthalene, triterpenoids, tannins, saponins, sterols and steroids [6]. It also includes
calotropone, an emerging bioactive compound with anticancer properties that has been
extracted fromC. gigantearoot using an ethanol solvent [7].
Ethyl acetate extract ofC. gigantearoot has been reported to contain saponins, triter-
penoids and coumarins [8]. The existence of triterpenoids such as calotropursenyl acetate
and calotropfriedelenyl acetate could be shown from the isolation of root bark ofC. procera;
a species from the same family asC. gigantea[9]. Furthermore, one type of triterpenoid—
Sci. Pharm.2021,89, 21.

Sci. Pharm.2021,89, 21 2 of 11
lupeol—could be found in the latex ofC. gigantea, which has been reported to exhibit
antitumor, anti-inammatory and other benecial activities [10].
Our recent study revealed that the methanol ethyl acetate, and n-hexane extracts from
C. gigantearoot bark, are highly cytotoxic againstArtemia salina[11]. The highest median
lethal concentration (LC50) value of the aforementioned study reached 36.79g/mL for the
ethyl acetate extracts. Next, this root bark ethyl acetate extract ofC. giganteawas isolated
and grouped into ve combined fractions which have the same stain pattern. The two most
potent of these combined fractions have cytotoxic activity againstA. salinawith an LC50
of 20.3g/mL and 18.9g/mL [11]. A study had successfully isolated-taraxerol and
-sitosterol acetates fromC. gigantearoot bark showing cytotoxic activities againstA. salina
with LC50values of 29.56 and 23.61g/L, respectively [12].
Despite its bioactive potential, only a few studies have reported on the use of extracts
fromC. gigantearoot bark as an anticancer agent. The petroleum ether and chloroform
extracts fromC. gigantearoot bark have been suggested to possess anti-tumor activities
against Ehrlich ascites carcinoma in Swiss albino mice [13]. Pregnanone compounds,
identied in 2008 [14], were reported to be active against chronic myelogenous leukemia
K562 and human gastric cancer SGC-7901 cell lines. Herein, we have investigated the ethyl
acetate extract from root bark ofC. gigantea, which has never been reported for its activities
against cancer cell lines. Triterpenoids and steroids were expected to be isolated from the
extract. Furthermore, we determined each of the compound's structures whose potential
anticancer activities are the highest against P388 murine leukemic cell lines. A natural
compound—Artonin E—Was employed as the positive control because of its high activity
against various cancers [15,16]. Moreover, artonin E has been used as the standardized
positive control in the Research Center for Bioscience and Biotechnology, Institut Teknologi
Bandung, Bandung, Indonesia.
2. Materials and Methods
2.1. Materials
The root bark ofC. giganteawas collected from Musar Area, Alue Naga Village,
Syiah Kuala District, Banda Aceh, Indonesia during July–September, 2017. Identica-
tion ofC. giganteawas held on 2 October 2017 at the Laboratory of Biology, Faculty of
Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh, Indonesia (No.
943/UN11.1.28.1/DT/2017).
Solvents used in this study were methanol 95%, n-hexane, ethyl acetate, dichloromethane,
dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) and sodium dodecyl sulphate
(SDS). All chemicals used were analytical grade and purchased from Merck (Selangor,
Malaysia). Silica gel GF254for the Vacuum Liquid Column Chromatography (VLCC), silica
gel G60 70–230 mesh ASTM for Gravity Column Chromatography (GCC) and Thin Layer
Chromatography (TLC) plate were also purchased from Merck (Selangor, Malaysia).
Artemia salina(brine shrimp) eggs were purchased from Aquatic Animal Research
Center, Universitas Syiah Kuala, Indonesia and used in the Brine Shrimp Lethality Test
(BSLT). P388 cell lines were obtained from the collection of Research Center for Bio-
science and Biotechnology, Institut Teknologi Bandung, Indonesia and used in the 5
3-(4,5-dimethylthiazol-2-yl) -2-5 diphenyl tetrazolium bromide (MTT) assay. The posi-
tive control, artonin E, was purchased from Natural Product Chemistry Laboratory, Institut
Teknologi Bandung, Indonesia.
2.2. Extraction of C. gigantea Root Bark
The procedure followed the reported study with minor modications [11]. FreshC.
gigantearoot bark (9.7 kg) was washed with distilled water, cut into small pieces, and
dried. The sample was then made into a powder using a crusher (Miyako, Jakarta Barat,
Indonesia). The procedure was continued with maceration using methanol 95% for324 h
(3:7 kg/L). Next, macerate was ltered using lter paper and concentrated using a vacuum
rotary evaporator (Heidolph, Schwabach, Germany) to acquire concentrated methanolic

Sci. Pharm.2021,89, 21 3 of 11
extract. The extract was rstly suspended in distilled water and partitioned using n-
hexane, ethyl acetate and methanol solvents, respectively. Afterwards, each extract was
reconcentrated using a vacuum rotary evaporator (30–35

C; 200 rpm).
2.3. Selection of the Combined Fractions Based on Their Cytotoxicity
Concentrated ethyl acetate extract (7 g) was fractionated using the VLCC with 100 g
silica gel GF254as the stationary phase. The extract was eluted using gradient elution of
n-hexane:ethylacetate, and was followed by ethylacetate:methanol. The outgoing fraction
was stored in an Erlenmeyer 100 mL until it reached the maximum volume marker, then
concentrated. The procedure was followed by TLC plate (stationary phase: alumina) using
eluent n-hexane:ethyl acetate system (7:3). Fractions with the same stain pattern were
combined and concentrated to produce a series of combined fractions, namely A, B, C,
D and E, which later were phytochemically tested following the suggestions of previous
literature [11]. To identify which combined fraction had the highest cytotoxicity, BSLT was
conducted againstArtemia salina[17]. Each combined fraction of C and D exhibited the
highest cytotoxicity and were thus used for further investigations. Fraction C was eluted
with ethylacetate 100% and concentrated to obtain 0.3 g solid. Fraction D was eluted with
ethylacetate:methanol (9:1) and was concentrated, yielding 2.7 g solid.
2.4. Isolation of Triterpenoids and Steroids
The isolation procedures were in accordance with the reported studies [12]. To obtain
the triterpenoids, fraction C was chromatographed using a GCC method with silica gel G60
70–230 mesh ASTM and n-hexane:ethylacetate (7:3). The purer fractions were then recrys-
tallized using a combination of n-hexane:dichloromethane and dichloromethane:methanol
solvents with a gradient elution. From this GCC process, 20 subfractions (C1–20) of com-
bined fraction C were obtained and analyzed by TLC., of which a subfraction C2with a
yellowish solid form was taken for phytochemical testing; triterpenoids were positive. This
revealed that the subfraction containing triterpenoids had a Retention factor (Rf) of 0.62.
Purication was carried out by recrystallization by using a methanol:dichloromethane
solvent mixture, yielding 9 mg shiny yellowish crystals.
As for the steroids, a similar GCC method was employed against fraction D with gradi-
ent eluents of n-hexane:dichloromethane, and were followed by dichloromethane:methanol.
The re-chromatography process of fraction D produced 40 subfractions (D1-40) with differ-
ent stain patterns. Subfraction D1was eluted with dichloromethane 100% and concentrated
to obtained 120 mg solid. Subfraction D1formed an amorphous solid and was then reana-
lyzed by TLC using the chloroform:methanol eluent system (9.5:0.5). The phytochemical
tests revealed that subfraction D1contains secondary metabolites of steroids, and then
it was recrystallized using methanol:chloroform solvents to produce 50 mg yellowish-
white solids.
2.5. 3-(4,5-Dimethylthiazol-2-yl) -2-5 Diphenyl Tetrazolium Bromide (MTT) Assay
The P388 cell lines at an initial cell density of approximately 2.5–3.0104 cells/well
were harvested and inoculated into 96-well plates. After 24 h of incubation for cell attach-
ment and growth, the cells were washed with PBS and then inoculated and cultured with
and without samples (1 mg/mL each extract of n-hexane, ethyl acetate and methanol from
the root bark ofC. giganteaand also the subfractions C2and D1ofC. gigantearoot bark
ethyl acetate extract). For each sample, varying concentrations of samples were made by
dissolving the samples in DMSO at the required concentrations from 0.1, 0.3, 1, 3, 10, 30,
and 100g/mL and were added into the wells. The control well received only DMSO.
After 72 h of incubation, the medium was aspirated and 10L of MTT solution (5 mg/mL
in PBS pH 7.2) was added to each well and the plate was incubated for 4 h at 37

C. After
incubation, 100L of DMSO (<0.5%) was added to each well and then homogenized with
shakers for formazan stable colors for 15 min, at which point the MTT-stop solution con-
taining SDS was added and another 24 h incubation was conducted. The absorbance was

Sci. Pharm.2021,89, 21 4 of 11
read by using an ELISA reader at540 nm and the surviving cell fraction was calculated.
All of these testing processes were carried out three times. The number of cells that grew
was proportional to the amount of formazan formed. Artonin E (100 mg) was used as a
positive control. OriginPro 8 software (Northampton, MA, USA) was used to calculate cell
inhibition for each sample. The half-maximal inhibitory concentration (IC50) value was the
concentration required for 50% growth inhibition.
2.6. Structure Characterization
The isolated compounds of subfraction C2were then characterized using Fourier
Transform Infrared (FTIR) spectrometry (Shimadzu FT-IR 8400, Kyoto, Japan) and mass
spectrometry (Shimadzu GCMS-QP 2010 Ultra, Kyoto, Japan). Characterization using FTIR
spectrum was used to determine various types of functional groups contained in a com-
pound. Small absorption ranges can be used to determine each type of bond (wavenumber
range: 4000–400 cm
1
) [17]. Subfraction C2was also characterized with mass spectrometry
to identify the contained compounds [18]. For subfraction D1, the isolated compounds
were characterized using FTIR spectrometry (Shimadzu FT-IR 8400, Kyoto, Japan) and
Hydrogen Nuclear Magnetic Resonance (
1
H-NMR) Spectroscopy (JEOL ECA 500 Tokyo,
Japan). Subfraction C2was only characterized with mass spectrometry because the number
of the obtained sample was not adequate for
1
H-NMR.
3. Results
3.1. Extraction Yield and Phytochemical Properties
The methanol extract from maceration was concentrated with a vacuum rotary evap-
orator and produced concentrated methanol extract of 160.25 g (1.65%w/w). From the
partitioning the methanolic extract fromC. gigantearoot bark, the concentrated extracts ob-
tained were 20.47 g from n-hexane, 73.60 g—ethyl acetate and 66.18 g—methanol solvents.
The results of the phytochemical tests on the ethyl acetate extract fromC. gigantea
root bark reveal the content of secondary metabolites (such as steroids, triterpenoids,
saponins, phenols and coumarin). The positive presence of steroids and triterpenoids in
the sample after reacting using a Liebermann-Burchard reagent was indicated by changes
in color, to green and red, respectively. The positive test result of saponin was characterized
by the foam formation in the sample after it was shaken. The presence of phenol was
characterized by the formation of a blue color. The coumarin presence was characterized
by a greenish-yellow uorescence color under a UV lamp [19].
3.2. Fractions and Their Cytotoxicity against Brine Shrimp Lethality Test
After the VLCC process, the fractions obtained were 26 fractions which were then
analyzed by TLC. Those with the same stain pattern were combined, so that ve groups
of fractions were obtained, namely combined fractions A, B, C, D, and E. The same stain
pattern was predicted to have the same secondary metabolite compounds. Each group of
fractions was weighed and its components were analyzed using TLC.
A preliminary study using BSLT to examine the cytotoxic activity against A. salina
showed that fractions A, B, C, D and E have LC50of 593.8, 595.8, 20.3, 18.9 and 31.6g/mL
respectively. Fractions C and D have the most potent cytotoxic activity; hence, they were
selected to be further isolated by GCC, tested by MTT assay, and were elucidated for their
structures [17].
3.3. MTT Assay of the Extracts from C. gigantea Root Bark
Table 50of 21.79g/mL, which means
that it is more potent than its isolated compounds (Subfraction C2and D1) and other
extracts (n-hexane and methanol extracts). Ethyl acetate extract contains steroid, terpenoid,
coumarin, saponin and phenol compounds. These ve classes of compounds synergistically
contribute to an increase in the antiproliferative activity of the P388 cell lines.

Sci. Pharm.2021,89, 21 5 of 11
Table 1.
The result of an MTT assay of n-hexane, ethyl acetate, methanol extracts and subfractions C
2
and D
1(n = 3).
Sample IC 50(g/mL)
N-hexane extract 49.02 0.06
Ethyl acetate extract 21.79 0.03
Methanol extract 68.45 0.02
Subfraction C
2(ethyl acetate) 50.64 0.03
Subfraction D
1(ethyl acetate) 49.33 0.01
Artonin E (positive control) 0.74 0.27
3.4. Structure Characterization of Subfraction C2and D1
Table 2and D1. In subfraction C2
FTIR spectral data, the absorption at wavenumber 2920 cm
1
was observed to be assigned
to C-H moieties, and 1701 cm
1
—for C = O moieties. In addition, C=C at wavenumber
1462 cm
1
and C-O (ester) group at 1190 cm
1
were shown. As for subfraction D1, the
presence of O-H moieties was indicated by the vibrational band appearance at 3007 cm
1
.
Furthermore, spectral bands observed at 2934, 1715 and 1057 cm
1
were assigned for C-H
(alkane), C=O and C-O stretching vibrations.
Table 2.FTIR data from subfractions C
2and D
1.
Functional Group
Wavenumber (cm
1
)
Subfraction C2 Subfraction D1 Ref. [20]
O-H Not detected 3007 3000–3700
C-H (alkane) 2920 2934 2800–3000
C=O 1701 1715 1700–1725
C=C 1462 Not detected 1400–1600
C-O (ester) 1190 1057 1050–1260
Mass spectrometry was carried out on subfraction C2, where the results were revealed
in Figure. The data of the spectral peak appearances from mass spectrometry, based on
the compound's fragmentations, are presented in Table. The detected subfraction C 2
has peaks atm/z467, 457, 410, 357, 344, 325, 284, 257 and 200, which are consistent to the
reported MS prole of taraxerol acetate (C32H52O2, molecular weight = 468.76 g/mol) [21].
The fragmentation pattern of taraxerol acetate compounds can be seen in the previously
published report [21]. The structure was conrmed by the absence of hydroxyl group and
the presence of C-O esters and C=C double bound in the FTIR spectrum (Table).
As for the compound elucidation of subfraction D1,
1
H-NMR was employed, where
its spectrum has been presented (Figure). The presence of ve aromatic protons were
observed at: 7.94; 7.44 and 7.55g/mL were observed in the
1
H-NMR spectrum, which
are typical for calotropone (Table). FTIR data of D1 (Table) also conrms the presence
of aromatic uptake at wavenumber around 1500 cm
1
. Spectral peak at: 5.40g/mL
(1H, m) was assigned to the olenic proton. The appearance of three high eld methyl
singlets could be observed at1.39, 0.96 and 2.08g/mL. These chemical shift values of
the isolated compound have similarities to that of reported calotropone [14].

Sci. Pharm.2021,89, 21 6 of 11
Figure 1.Mass spectrometry spectrum of subfraction C
2.
Table 3.Data on taraxerol acetate fragment ion data.
Fragment ion (m/z)
Subfraction D1 Ref. [21]
467 468
457 453
410 409
397 -
383 -
370 -
357 359
344 344
325 329
- 269
284 284
257 257
200 204
- 189
179 -
127 -
- 121

Sci. Pharm.2021,89, 21 7 of 11
Figure 2.
1
H-NMR spectrum of subfraction D
1.
Table 4.Data of
1
H-NMR spectrum of subfraction D1 and reference of calotropone [14].
Position
H (g/mL)
This Study
(CDCl3, 500 MHz)
Ref. [14]
(CDCl3, 400 MHz)
1 1.74 (1H, m, H-1a), 1.75 (1H, m, H-1a),
2 1.12 (1H, m, H-1b) 1.13 (1H, m, H-1b)
3 1.82, 1.49 (each 1H, m) 1.81, 1.46 (each 1H, m)
4 3.54 (1H, m) 3.53 (1H, m)
5 2.31 (1H, m), 2.33 (1H, dd, 12.8, 3.6 Hz)
6 2.22 (1H, m, overlapped) 2.25 (1H, m, overlapped)
7 NA NA
8 5.40 (1H, m) 5.41 (1H, m)
9 2.16, 1.92 (each 1H, m) 2.20, 1.91 (each 1H, m)
10 1.81 (1H, m) 1.80 (1H, m)
11 1.32 (1H, m) 1.32 (1H, m)
12 NA NA
13 2.08, 1.47 (each 1H, m, overlapped) 2.06, 1.45 (each 1H, m, overlapped)
14 4.82 (1H, m) 4.80 (1H, dd, 11.3, 4.5 Hz)
15 NA NA
16 NA NA
17 2.13 (1H, m, H-15a), 2.12 (1H, m, H-15a),
18 1.94 (1H, m, H-15b) 1.92 (1H, m, H-15b)
19 2.89 (1H, m, H-16a), 1.89 (1H, m, H-16b) 2.90 (1H, m, H-16a), 1.88 (1H, m, H-16b)
20 NA NA
21 1.39 (3H, s) 1.41 (3H, s)
1
0
0,96 (3H, s) 0,98 (3H, s)
2
0
NA NA
3
0
2.08 (3H, overlapped) 2.06 (3H, overlapped)
4
0
NA NA
5
0
7.94 (1H, d, 7.3 Hz) 7.93 (1H, d, 7.5 Hz)
6
0
7.44 (1H, t, 7.3 Hz) 7.43 (1H, t, 7.5 Hz)
7
0
7.55 (1H, t, 7.5 Hz) 7.56 (1H, t, 7.5 Hz)
1 7.44 (1H, t, 7.3 Hz) 7.43 (1H, t, 7.5 Hz)
2 7.94 (1H, d, 7.7 Hz) 7.93 (1H, d, 7.5 Hz)
3 NA NA
NA: not applicable, determined by
13
C-NMR.

Sci. Pharm.2021,89, 21 8 of 11
4. Discussion
Calotropis giganteafrom Aceh grows wildly like weeds with very strong support-
ing roots in the Alue Naga area;100–500 m from the shoreline and0.8 m above sea
level. Some classes of chemical components of plants, such as triterpenoids, phenolic and
coumarin, are believed to have cancer prevention properties [22]. In our study, ve groups
of secondary metabolites were obtained. Based on the phytochemical tests, the ethyl acetate
extract was revealed to contain steroids, saponins, triterpenoids, phenols and coumarins.
In the other study in India, the phytochemical analysis of the extracts fromC. gigantearoots
revealed the presence of saponins, triterpenoids and steroids [23]. Varied results obtained
from the studies could be associated with the different geographical locations in which the
plants grow. It is known that the topography and climate of the environment where the
plants grow and develop could affect the production of their secondary metabolites [24].
Saponins have been reported for their antiproliferative effects by regulating the im-
mune system [25–27]. Paris saponin had shown an inhibition effect towards cervical cancer
and is a positive regulator of the immune system in tumor-bearing mice [25]. Further-
more, Paris saponin was revealed to have an inhibitory effect, signicantly against U14
cellsin vitro[26]. A mixture of monodesmoside saponins had been reported to be highly
cytotoxic against colon and P388 cancer cell lines [27].
Along with saponins, coumarins have been reported in many different researches for
having cytotoxic activities. One of the coumarin compounds obtained by multi-step syn-
thesis of hydroxy benzophenones had shown excellent antiproliferative potential against
murine EAC and dense DL tumors by triggering anti-angiogenesis and apoptosis [28]. A
natural coumarin, osthole, showed inhibitionin vitrotowards Hepatocellular Cell Carci-
noma (HCC) proliferation. In the G2 phase, it also causes cell accumulation and induces
apoptosis. This coumarin can signicantly suppress the growth of HCC tumorsin vivo[29].
Other studies had shown that the coumarin metabolite, 4-hydroxycoumarin, inhibits cell
proliferation in the gastric carcinoma cell line [30]. A derivative compound of coumarin,
scopoletin, isolated from theMacaranga gigantifoliaMerr. Leaves, show strong cytotoxic
activities against cell lines of P388 murine leukemia [31].
A previous study reported that phenol has antiproliferative effects on several types of
tumors. Phenol compounds ofZingiber ofcinaleR. rhizome had shown cytotoxic activity
in cancer cells through apoptosis [32]. The chemopreventive effects of phenol had been
reported for having inhibition of proliferation effects, cell cycle blocking and apoptosis
induction in different tumor cell lines [33,34]. One phenolic compound, named rosmarinic
acid, found in Labiatae herbs, had a signicant effect in inhibiting the cell proliferation in
both the G0–G1 and G1–S phases induced by TNF-and PDGF [35].
Triterpenoid compounds themselves have the property of inhibiting tumor growth.
C. gigantea-derived triterpenoids could inhibit tumor growth by increasing cell apoptosis,
as evidenced by the results of previous study [36]. A triterpenoid-rich extract of bamboo
shavings and its main component, friedelin, had effective antitumor activities to inhibit the
growth of P388 and A549 cancer cell lines [37].
Subfraction C2contained triterpenoid compounds, where similarities with taraxerol
acetate compounds were found based on the FTIR and mass spectrometry spectral proles.
Taraxerol acetate is a triterpenyl ester compound with the molecular formula C32H52O2,
which has a molecular weight of 468.76 g/mol [21]. The chemical structure of taraxerol
acetate has been presented in Figurea.
A study reported that steroid compounds may function as anti-inammatory agents,
and are useful in inhibiting prostate cancer [38]. A steroid fraction of Brassica campestrisL.
bee pollen chloroform extract displayed strong cytotoxicity in human prostate cancer PC-3
cells by triggering apoptosis [38]. Methanol extract of root bark ofC. giganteawas reported
to contain steroid group compounds, namely stigmasterol and-sitosterol [39]. The stig-
masterol antitumor activity towards Ehrlich Ascites Carcinoma (EAC) might be attributed
to activation of the protein phosphatase 2A that causes apoptosis [40].-sitosterol has

Sci. Pharm.2021,89, 21 9 of 11
shown a cytotoxic effect by interfering with multiple cell-signaling pathways, including cell
cycle, apoptosis, proliferation, invasion, angiogenesis, metastasis and inammation [41].
Figure 3.Structures of (a) taraxerol acetate and (b) calotropone.
Subfraction D1contained steroid compounds that produce yellowish-white solids
and are characterized using FTIR and
1
H-NMR, showing similarities with the calotropone
compound (Figureb) as found before, in which its structure was identied as 12 -O-
benzoyl-3,14,17-trihydroxypregnane-20-one and has 468.59 g/mol [14].
The results of the MTT Assay against P388 murine leukemia cell lines in this study
showed that subfraction C2and D1, respectively containing taraxerol acetate and calotro-
pone, had IC50of 50.64 and 49.33g/mL, respectively. Nonetheless, the ethyl acetate extract
is the most potent, with IC50as low as 21.79g/mL. Based on the National Cancer Institute
(NCI) provisions, an extract is declared to be highly active as in anticancer if it has an
IC50value of <30g/mL and is moderately active if30g/mLIC50100g/mL [42].
Hence, both subfractions have a moderate anticancer activity. Meanwhile, the ethyl acetate
is high. Higher antiproliferative activities in the ethyl acetate extract were ascribed to the
synergism among the secondary metabolites [43].
It is worth mentioning that taraxerol acetate has been reported to have antitumor
effects towards human glioblastoma cells [44]. Taraxerol acetate derived from methanolic
extract from Hibiscus rosa sinensis leaves and stems is also effective against the K562
Leukemia cell line [45]. The steroidal aglycone (pregnanone), also called calotropone,
isolated from ethanol extract fromC. giganteaL. root, has shown an inhibitory effect on the
cell lines of chronic myelogenous leukemia K562 and the human cancer SGC-7901 in the
previous study [14].
5. Conclusions
Ethyl acetate extract fromC. gigantearoot bark contains steroids, terpenoids, coumarins,
saponins and phenol compounds. Compounds from the aforementioned classes synergisti-
cally contribute to increase the antiproliferative activity against the P388 murine leukemia
cell lines. The synergism is responsible for a higher antiproliferative activity of ethyl acetate
than its isolates. We recommend further study into the ethyl acetate extract fromC. gigantea
root bark for the identication of its bioactive compound constituents.
Author Contributions:
Conceptualization, K.H. and M.S.; Data curation, R.R., K.H. and M.S.; Formal
analysis, M.S. and M.I.; Funding acquisition, K.H.; Investigation, H.F., D.R.M., and M.I.; Methodology,
K.H., M.S., and R.R.; Project administration, K.H., M.S., and R.R.; Resources, K.H.; Software, H.F., M.I.,
and D.R.M.; Supervision, K.H. and M.S.; Validation, R.R.; Writing—Original draft, K.H.; Writing—
Review and editing, M.S., R.R., and M.I. All authors have read and agreed to the published version
of the manuscript.

Sci. Pharm.2021,89, 21 10 of 11
Funding:
The authors wish to thank Universitas Syiah Kuala, Directorate of Research and Commu-
nity Service, Ministry of Research, Technology, and Higher Education for its nancial support under
thePenelitian Produk TerapanScheme No. 105/SP2H/LT/DPRM/IV/2017.
Institutional Review Board Statement:Not applicable.
Informed Consent Statement:Not applicable.
Data Availability Statement:Not applicable.
Acknowledgments:
We acknowledge the contributions made by Diva Rayyan Rizki, S. Ked., Hawa
Purnama Celala Ary Cane, and Agnia Purnama, during the making of this article.
Conicts of Interest:The authors declare no conict of interest.
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