DOI:10.31557/APJCP.2023.24.10.3345
Cytotoxic Activities from the Crude Extract of Cleome Rutidospermae

RESEARCH ARTICLE

Editorial Process: Submission:10/21/2021 Acceptance:10/22/2023

Cytotoxicity Activity, Metabolite Profiling, and Isolation
Compound from Crude Hexane Extract of Cleome rutidospermae
Budiman Yasir 1,2,3, Abdul Rahim 2, Subehan Lallo 2, Yohei Saito 4, Kyoko
Nakagawa-Goto4, Abdul Rohman5, Gemini Alam2*
Abstract
Objective: This study isolated the chemical compounds and evaluated the cytotoxic activity of the crude
hexane extract of Cleome rutidospermae herb (CRH). Methods: The isolate was purified using silica gel, column
chromatography, and preparative thin layer chromatography (PTLC). Furthermore, the structure of the compounds was
identified by spectroscopic methods using 1D, 2D NMR, and mass spectrometry. The cytotoxic activity of CRH at aconcentration of 20 ug/mL was also tested against MCF-7, A549, KB, KB-VIN, and MDA-MB-231 cancer cells using
the sulforhodamine B (SRB) method. Results: The CRH contained compounds of unsaturated fatty acid, saturated fatty
acid, lipid, glycerol, ω-3 fatty acid, and cholesterol. Two compounds were obtained from the plant, and their structures
were identified as (1) Stigmasta-5,22-dien-3-ol (STML) and (2) 1,2-Benzene dicarboxylic acid, 1,2-bis (2-Ethylhexyl)
esters (DEHP). These compounds were reported in this plant for the first time. In comparison, CRH had % growth
inhibition in the proliferation of MCF-7 cells up to 28.1%, with cancer cells A549, KB, KB-VIN, and MDA-MB-231
by >50% Compared to the negative DMSO of 0.20%, while the positive control could inhibit the growth of all cancer
cells (100%). Conclusion: Our findings suggested that crude herb from the plant CRH was the potential for breast
cancer treatment.
Keywords: Cleome rutidospermae- Stigmasta-5,22-dien-3-ol- Bis(2-Ethylhexyl) esters (DEHP)- Cytotoxic
Asian Pac J Cancer Prev, 24 (10), 3345-3352

Introduction
Medicinal plants have been proven to be an alternative
approach for treating various health conditions. Althoughsynthetic drugs have taken traditional healing to somedegree, the revival and attention of herbal medicines arevery important (Mirzaeian et al., 2021). Medicinal plants
are affordable and eco-friendly, with fewer side effects
than synthetic drugs. In fact, among the modern medicines
currently in use, approximately 40% come from nature,
while 60% of anticancer and 75% of infectious diseases
drugs are natural or derivatives. Furthermore, some of theseplants have attracted scientists’ interest in investigating
cancer treatment. Additionally, phytoconstituents havebeen essential in developing clinically valuable prospects
for treating neoplasms (Gupta et al., 2021).
Cleome rutidospermae is an annual herbaceous plant
known as the Fringed Spider Flower. Besides being a
weed, it is also an important medicinal plant widely foundin the tropics. Furthermore, it is ayurvedically, Greek,

and local Kabiraj used to manufacture bread. Its roots orseeds are used as a stimulant, antiscorbutic, anthelminthic,
rubefacient, vesicant, and carminative. The plant is also
an analgesic, antipyretic, anti-inflammatory, antimicrobial,
antioxidant, anti-seizure, anti-diabetic, and diuretic, withwound healing and laxative activity (Ghosh et al., 2019).
Its herb contains water (81.0 g), energy (239 kJ/57 kcal),protein (5.5 g), fat (0.9 g), carbohydrate (10.1 g), Fiber(1.7 g), Ca (454 mg), Mg (38 mg), P (59 mg), and Fe (2.7mg) per 100 g of edible portion, tannins, lipids, aminoacids, flavonoids, cardiac glycosides, alkaloids, steroids,
saponins, terpenoids, polyphenols, and phlorotannin
pentose and reducing sugars (Ghosh et al., 2019).
Natural ingredients are considered an essential
source of discovery for anticancer treatments, and manycytotoxic drugs in clinics and hospitals today are derived
from plants and other natural sources (Sammar et al.,2019). Phytochemicals from plant extracts are included
in important sources of natural products that haveshown excellent cytotoxic activity. However, plants of

Sekolah Tinggi Ilmu Farmasi Makassar, Makassar, 90242, Indonesia. 2Department of Pharmacognosy-Phytochemistry Laboratory,
Faculty of Pharmacy, Hasanuddin University, Makassar, 90245, Indonesia. 3Faculty of Health Sciences, Almarisah Madani
University, Makassar, 90245, Indonesia. 4School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health
Sciences, Kanazawa University, Kanazawa, 920-1192, Japan. 5Department of Pharmaceutical Analysis and Medicinal Chemistry,
Faculty of Pharmacy, Gadjah Mada University, Yogyakarta, 55281, Indonesia. *For Correspondence: daengta007@yahoo.com
1

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Budiman Yasir et al

different origins show diverse chemical compositions
and bioactivity. Therefore, discovering new plant-based
anticancer agents from other parts is always challenging(Khan et al., 2022).
In this study, two compounds were isolated, namely(1) Stigmasta-5,22-dien-3-ol (STML) and (2) 1,2-Benzene
dicarboxylic acid, 1,2-bis (2-Ethylhexyl) esters (DEHP)
using 1D, 2D NMR, and mass spectrometry. Meanwhile,
the activity of CRH was tested against several cancer cell
pathways using the SRB method. These compounds havenever been reported or isolated from this plant.

Materials and Methods
Plant material
The CRH was obtained from Makassar City,
Tamalanrea Sub-District, South Sulawesi Province,
Indonesia. The plant identification was conducted at
the Botanical Laboratory in the Department of Biology,
Mathematics, and Natural Sciences, Universitas Negeri
Makassar. Furthermore, the plant samples were washedwith running water, wet sorted, cut into small pieces, driedin a 40-50 oC oven for three days, dry sorted, and stored
in containers tightly sealed with silica gel.
Extraction and isolation
The extraction of Cleome rutidospermae herb (2 kg)with hexane solvent (1:10 ml) was conducted using themaceration method at room temperature for three days
(Tiwari et al., 2011; Yasir et al., 2022). It was stirred once
daily and filtered using a vacuum pump with whatmanTM
no 42-filter paper. Subsequently, the liquid extract wasevaporated to obtain a 35 g viscous extract using rotavapor
and a water bath. The extract was partitioned using
petroleum ether and methanol (1:1) three times as muchas 250 ml. The petroleum ether and methanol extracts
obtained were 29.2 g and 5.74 g, respectively.
The methanol extract was partitioned with hexaneand methanol-water comparison (1:1) 3 times as much as
300 ml to obtain methanol-water and hexane partitioned
extract of 0.4753 g and 5.3247 g, respectively. The hexane
partitioned extract was isolated using a normal phase (NP)silica chromatography column, with hexane and ethyl
acetate mobile phase (3:1) 3 times as much as 300 ml.
The partitioned using 200 ml ethyl acetate and 200 ml
methanol to give 1-5 sub-fraction, sub-fraction (2) 0.0514
g washed with hexane, and insoluble hexane (b) 0.0230 g.
Subsequently, the soluble hexane (a) 0.0284 g was isolatedusing an NP silica chromatography column with hexane
mobile phase and ethyl acetate (4:1). This aims to obtain(1) STML 0.0202 g amorphous powder. Sub-fraction(3) 0.0644 g was washed with acetone to obtain soluble
and insoluble acetone of (b) 0.0086 g and (a) 0.0438 g,
respectively. The insoluble acetone was isolated using
PTLC, NP silica with hexane mobile phase, and ethyl
acetate (5:1) to obtain (2) DEHP 0.0335 g yellowish oilcompound.
Cytotoxicity activity
The samples of CRH weighed as much as 1 mg
with a 20 μg/ml concentration. The test used five lines

3346 Asian Pacific Journal of Cancer Prevention, Vol 24

of human tumor cells, namely A549 (lung carcinoma),
MDA-MB-231 (estrogen receptor-negative, progesterone
receptor-negative, and HER2-negative breast cancer),
MCF-7 (estrogen receptor-positive, HER2-negative
breast cancer), KB (isolated initially from epidermoid
carcinoma of the nasopharynx), and KB-VIN (vincristine(VIN)-resistant KB subline showing MDR phenotype by
overexpressing P-gp). All the cell lines were obtained fromLineberger Comprehensive Cancer Center (UNC-CH) or
ATCC. Furthermore, 4,000−12,000 cells were seeded in
the microtiter plate of 96 wells, with each test sample
in DMSO (negative control). Subsequently, 10%trichloroacetic acid was added after 72 hours, followed by
the 0.04% sulforhodamine B method. DMSO substanceswith 0.1% v/v concentration showed no inhibitory
effect on cancer cells (Rahim et al., 2018). The growth(%) was calculated on a plate-by-plate basis for tested
wells compared to the control. It was also articulatedusing the following formula, Average absorbance oftest wells × 100 / Average absorbance of control wells(Houghton et al., 2007; Lamkanfi et al., 2008).

Results
Isolation compound
The CRH was checked profile using 1H NMR(Figure 1) and contained unsaturated fatty acids, saturated
fatty acids, lipids, glycerol, ω-3 fatty acids, cholesterol,and position of functional groups in Table 1. It waspartitioned using petroleum ether, and MeOH with theratio of (1:1). The soluble layer of MeOH was evaporated
and further partitioned into a combination of hexane,
MeOH-H2O (1:1) as much as 300 ml. A combinationof various chromatography techniques was also used toseparate the hexane layer from obtaining the following
compounds (1) STML and (2) DEHP with reported 1D
spectroscopic, 2D NMR, and mass spectrometry data.
The STML (1) is a white amorphous powder and steroid
group compound based on ESI+ MS m/z 413.18 [M +H]+ spectrometry mass data with the molecular formula
C29H48O Figure 3. According to the 1H NMR spectroscopy
in Table 2 showing the presence of 5 methyl group signals,
the proton methine, which appeared at position δH 5.35
(1H, br d, J=5.50 Hz), δH 5.17 (1H, dd, J=8.24 Hz), δH
4.99 (1H, dd, J=8.70 Hz), and δH 3.52 (1H, septet) were
emerging hydroxyl group signal positions or methylene
protons. That is observed in carbon positions using
DEPT 135 data. Furthermore, the 13C NMR showed
the compound had 29 carbons, and DEPT 135 data
indicated the function of carbon with 5 protons methyl
and methylene group. Meanwhile, the carbon position ofmethine was δC 71.82, which binds to hydroxyl groups,
δC 121.72, δC 138.32, δC 129.27 as a double bond, andcarbon without protons at position δC 140.75, δC 36.51,
and δC 42.21.
NMR’s 2-dimensional data, including HMQC and
HMBC, showed the correlation between hydrogen andcarbon in Figure 2, blue line, i.e., methyl proton δH 1.02
(3H, d, J = 7.79 Hz) correlated with carbon δC 36.51(C-10), δC 37.25 (C-1), δC 50.15 (C-15 9), and δC 140.75(C-5). Proton methyl δH 1.03 (3H, s) correlated with δC

DOI:10.31557/APJCP.2023.24.10.3345
Cytotoxic Activities from the Crude Extract of Cleome Rutidospermae

Figure 1. 1H NMR Spectra of Cleome Rutidospermae Herb Hexane Extract (CRH). Assignments (A) 1 to 17 arereported in table 1 (400 MHz, CDCl3). (A) = Major signal; (B) = (A) An expanded
40.51 (C-20), δC 55.94 (C-17), and δC 138.32 (C-22).
Furthermore, δH 0.68 (3H, m) correlated with δC 55.94(C-17), δC 42.21 (C-13), and δC 39.77 (C-12), while δH
0.80, δC 11.87 (3H, m) correlated with carbon δC 18.98(C-28) and δC 51.24 (C-24). In addition, δH 0.80, δC
12.05 (3H, m) correlated with δC 39.68 (C-25) and δC
51.24 (C-24).
The correlation data with protons using 1H-1H Cosyin figure 2, the thick red line showed proton H3 δH 3.52
(1H, septet) correlated with H2 and H4, H6 δH 5.35 (1H,
br d, J=5.50 Hz) with H7, H22 δH 5.17 (1H, dd, J=8.24
Hz) correlated with H20 and H23 δH 4.99 (1H, dd, J=8.70
Hz) and proton H24. Furthermore, 1D and 2D NMR data

indicated that this compound first reported in the plant
was steroid (1) STML.
The DEHP (2) is a yellow oil and phthalate groupcompound. According to spectrometry, mass data gave amolecular weight of ESI+ MS m/z 391.20 [M + H]+ with
the C24H38O4 formula Figure 3. The same compound waspreviously isolated from Aloe vera plants and discovered
to have antileukemic as well as antimutagenic effects(Lee et al., 2010). The 1H NMR spectroscopy data in
Table 2 showed the presence of 4 methyl group signals,
ten methylene protons, and four methines observed at
the proton position using DEPT 135 and HMBC data.
According to 13C NMR , this compound has 24 carbons,

Figure 2. Structure of Compound (1) Stigmasta-5,22-dien-3-ol (STML) and (2) 1,2-Benzene dicarboxylic acid,
1,2-bis (2-Ethylhexyl) ester (DEHP)
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Budiman Yasir et al

Table 1. 1H NMR Spectra Signal from Cleome Rutidospermae Herb Hexane Extract (CRH) Assignments (A) 1 to 17
Peak (A)δ (ppm)Multiplicity

Compound/proton

References δ (ppm)

1

8.1

s

Trans olefinic

8.1, d (Hasan et al., 2007)

2

7.24

s

Chloroform (solvent)/(CHCl3)

7.26 (de Combarieu et al., 2015; Gottlieb etal., 1997)

3

5.37-5.30m

All unsaturated fatty acids/(CH = CH)
Methylene protons/(CH2)

5.36 (de Combarieu et al., 2015)5.37 – 5.30, m 2H (Shirey et al., 2018)4

5.12-5.07

m

Glycerol (triacylglycerols)/CHOCOR,
Glycerol (sn-1,2-diacylglycerols)/CHOCOR
Methynic (CH), Aliphatic/(CH2)

5.27; 5.08 (de Combarieu et al., 2015)
5.12; 5.07 (Que et al., 2000; Sivakumar etal., 2013)

5

4.57

d, J=6.9 Hz

Glycerol (all acylglycerols)/CH2OCOR

4.30 (de Combarieu et al., 2015)

6

4.28

dd, J=11.9, 4.3
Hz

Vinyl protons
Glycerol (all acylglycerols)/CH2OCOR

4.28 (d, J = 3.2 Hz) (Breen et al., 2005)
4.15 (de Combarieu et al., 2015)

7

4.13

q, J=6.0 Hz

Glycerol (sn-1,3-diacylglycerols)/CHOH
Methylene group/(CH2O)

4.07 (de Combarieu et al., 2015)
4.13; q, J = 6 Hz (Reheim & Tolba, 2016)

8

3.64

s

Fatty alcohols/(CH2OH)
Polyethylene glycol/(OCH2CH2)

3.64 (de Combarieu et al., 2015)
3.64, s (Fang et al., 2014)

9

2.78

t

Linoleyl and linolenyl/(CH=CHCH2CH = CH)
Methylene/(CH2)2.81 (de Combarieu et al., 2015)
2.78, t (Flores et al., 2013)10

2.74-2.79t, m

ω-3 fatty acids,
Ethylene (CH2CH2)

2.74, t; 2.80−2.73, m (Li et al., 2018; Vicente et al., 2015)

11

2.25

m

Methylenic/(CH2)

2.25 (Durand et al., 2020)12

1.94-2.09mAllylic (CHCHCH2)

1.94–2.15 (Hwang et al., 2017)

13

1.65

s

All acyl chains/(CH2CH2COOH)
Methylene/(CH2)

1.64 (de Combarieu et al., 2015)
1.65 (Tshilanda et al., 2014)14

1.57-1.58

s-m

Lipids/(CH2CCO)

1.58 (Righi et al., 2014)15

1.2-1.4m

Saturated fatty acids/(CH)n1.2-1.4, m (Knothe & Kenar, 2004)

16

1.83-0.66m

Cholesterol and methyl groups/(CH3)0.66; 1.83 (Park et al., 2011; J. Zhao et al.,
2011)

17

0

s

Tetramethylsilane/(TMS)

0.02, s (Hoffman, 2006)

while DEPT 135 data showed a carbon position with two
methyl protons at δC 14.13 and δC 11.04, respectively,
with methylene carbon at δC 23.06, δC 29.00, δC 39.43,

δC 23.81. Each of the four methylene protons can beobserved at HMQC and HMBC positions, and the 2 protonacetolactone position δC 68.23 (CH2OCO). Furthermore,
413.18(1)(2)

391.20Figure 3. Data Mass Spectra of (1) Stigmasta-5,22-dien-3-ol (STML) ESI+ m/z 413.18 [M + H]+ and (2) 1,2-Benzene
dicarboxylic acid, 1,2-bis (2-Ethylhexyl) ester (DEHP) m/z 391.20 [M + H]+ using Electrospray Ionization (ESI)
mass spectrometry.

3348 Asian Pacific Journal of Cancer Prevention, Vol 24

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Cytotoxic Activities from the Crude Extract of Cleome Rutidospermae

Table 2. Data 1H NMR, 13C NMR, and DEPT 135 of (1) Stigmasta-5,22-dien-3-ol (STML) and (2) 1,2-Benzene
dicarboxylic acid, 1,2-bis (2-Ethylhexyl) ester (DEHP)
STML (1)
Position

DEPT

1

CH2

2

CH2

3
4
5

C (ppm)

DEHP (2)H (ppm)Position

DEPT

37.25

br d, 5.35 (1H) J=5.50 Hz

1

C

132.53septet, 3.51 (1H)

31.66

dd, 5.17 (1H) J=8,24 Hz

2

C

132.53br d, 5.33 (1H)

CH

71.82

dd, 4.99 (1H) J=8,70 Hz

3

CH

128.88dd, 5.22 (1H)

CH2

42.30

septet, 3.52 (1H)

4

CH

130.95dd, 5.16 (1H)C

140.75m, 0.82 (3H)

5CH

130.95

br q, 2.42 (1H)

6

CH

121.72m, 0.82 (3H)

6

CH

128.88br s, 4.68 (2H)

7

CH2

25.41

d, 1.02 (3H) J=7,79

7C

167.838

CH

31S.90s, 1.03 (3H)

8

CH2

68.23

9

CH

50.15

m, 0.80 (3H)

9

CH

38.8

10

C

36.51

m, 0.68 (3H)

10

CH2

23.81

11

CH2

21.09

11

CH3

11.04

12

CH2

39.77

12

CH2

30.43

13

C

42.21

13

CH2

29

14

CH

56.87

14

CH2

23.06

15

CH2

24.37

15

CH3

14.13

16

CH2

28.93

17

CH

55.94

18

CH3

12.26

19

CH3

19.41

20

CH

40.51

21

CH3

21.26

22

CH

138.32

23CH

129.27

24

CH

51.24

25

CH2

39.68

27

CH

56.76

28

CH3

18.98

26

CH3

12.05

29

CH3

11.87

13

1

the part of two carbon methines on the benzene framework
is located at δC 128.88 and δC 130.95 and had a proton
correlation with carbon at δC 130.95.
NMR’s 2-dimensional data including HMQC and
HMBC showed the correlation between hydrogen andcarbon at (Figure 2, blue line) i.e. 2 methyl protons δH
0.90 (3H, m) (C-15, C-15’) correlated with carbon δC
23.06 (C-14, C-14’), and δC 29.00 (C-13, C-13’). Also,
0.92 (3H, s) (C-11, C-11’) correlated with δC 23.81 (C-10,
C-10’), and δC 38.80 (C-9, C-9’), δH 4.22 (2H, n) (C-8)
with carbon carbonyl position δC 167.83 (C-7, C-7’).
Furthermore, two protons methine δH 1.42 (1H, m) (C-9,C-9’) correlated with carbon (C-11, C-11’, C-10, C-10’,
C-12, C-12’), methylene protons δH 1.32 (2H, m) (C10, C-10’) correlated with carbon δC 38.80 (C-9, C-9’)and δC 23.81 (C-10, C-10’). δH 1.33 (2H, m) (C-13,
C-13’) correlated with δC 30.41 (C-12, C-12’), and two
methylene protons 1.30 (2H, m) (C-14, C-14’) correlatedwith δC 29.00 (C-13, C-13’) and δC 14.13 (C-15, C-15’).
In addition, the proton methine H-3 δH 7.70 (1H, m)

13

C (ppm)H (ppm)1

correlated with carbon δC 130.95 (C-4), δC 132.53 (C-2),
and methine H-6 δH 7.70 (1H, m) with carbon δC 130.95(C-5), and δC 132.53 (C-1).
The correlation data with protons using 1H-1H Cosy
(Figure 2, thick red line) showed that proton methyl
H-15, H-15’ δH 0.90 (3H, m) correlated with H-14,
H-14’. Furthermore, H-11, H-11’ 0.92 (3H, s) correlatedwith H-10, H-10’ δH 1.32 (2H, m), while H-10, H-10’
correlated with methine H-9, H-9’ δH 1.42 (1H, m), andproton H-9, H-9’ with H-8, H-8’ δH 4.22 (2H, n).
Also, 1D and 2D NMR data indicated that thiscompound was a group of phthalate acid (2) DEHP
and was first reported in this plant. In addition, it was
isolated from Streptomyces bangladeshensis sp. Nov(Al-Bari et al., 2005), Aspergillus awamori (Lotfy et al.,2018), Aloe vera (Lee et al., 2010), Calotropis gigantea
flower (Linn.) (Habib and Karim, 2009), Penicillium
janthinellum (El-sayed et al., 2015), Acinetobacter sp.
SN13 (Xu et al., 2017), and Benincasa hispida flower(Du et al., 2006).
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Budiman Yasir et al

Cytotoxic study
The preliminary test results of the CRH against
cytotoxic cancer cells with 20 ug/mL showed that the %growth of MCF-7 cancer cells was 28.1%, A549 at 60.6%,
and MDA-MB-231 at 68.4%. Furthermore, KB 58.1%
and KB-VIN 86.3%, compared to the negative DMSO
of 0.20%, control the growth of all cancer cells (100%).
The inhibition of these cells correlated with thecontents which were successfully isolated in CRH, such as(1) STML that have inhibition of cancer cells >50% in ES2
and OV90 (Bae et al., 2020), SGC-7901, and MGC-803(Zhao et al., 2021), as well as MCF-7 and NIH/3T3 cancer
cells (Ayaz et al., 2019), which are chemopreventive (Aliet al., 2015). Also, (2) DEHP is effective as an anticancer.
Therefore, as the compound concentrations increase,
it inhibits the growth of cancer cells MCF-7, HEPG2,
HELA, and HCT116 (Lotfy et al., 2018). This compound
has potent cytotoxicity for MCF-7 cancer cells with IC50
of 6.941 μg/ml, moderate cytotoxicity for a HEPG2 cancer
cell, and IC50 of 26.73 μg/ml for HELA cancer cell andshowed no activity against colon carcinoma (HCT116)(Lotfy et al., 2018). Subsequently, it inhibits basal alveolar
epithelial cells A-549 370 μg/mL (El-sayed et al., 2015).

Discussion
Many studies have been published regarding the
anticancer potential of various plant extracts that canbe good candidates for cancer drug discovery anddevelopment one example was the paclitaxel compoundisolated from Taxus brevifolia for chemotherapy use(Liebmann et al., 1993). This STML (1) was first reported
to be found in this species. However, this compound has
been reported in several species of Cleome. STML, also
known as stigmasterol once isolated from the Cleome
gynandra (Ranjitha et al., 2009), Cleome paradoxa(Abdel-Monem, 2012), and Cleome viscosa. It wasreported that STML compounds have the potential to be
anticancer for new treatments in ovarian cancer (Bae etal., 2020), gastric cancer (Zhao et al., 2021), glycolysis
in triple-negative breast cancer cells (MDA-MB-231),
and lung cancer (Dong et al., 2021). This compound
has an anticancer mechanism through the induction ofcell apoptosis, ROS production, and excess calcium in
ES2 and OV90 cells. STML stimulates cell death byactivating ER-mitochondria, suppressing cell migration
and angiogenesis genes in human ovarian cancer cells(Bae et al., 2020). In addition, this compound also induces
apoptosis and autophagy by inhibiting the Akt/mTOR
pathway in gastric cancer cells (Zhao et al., 2021) andregulating retinoic acid associated with orphan C receptors(Dong et al., 2021).
The DEHP (2) compound is one of the compounds
successfully isolated in this plant and first reported inthis species. This compound has been reported to befound in Cleome serrata (Mcneil et al., 2012). In addition,
compounds 2-ethyl-cyclohexane-2-ene-6-hydroxymethylene-1- carboxylic acid and 3β-hydroxy-lup-20(29)en-28-oic acid were also found in this plant (Rahman etal., 2008). DEHP (2) is known to have the potential to be
an anticancer in the treatment of cancers such as breast

3350 Asian Pacific Journal of Cancer Prevention, Vol 24

cancer, liver cancer, cervical cancer, and colon cancer(Lotfy et al., 2018). Compound Bisphenol A combination
with DEHP mainly induced the expression of HDAC6,
inhibited tumor suppressor gene PTEN upregulated theexpression of oncogene c-MYC. Eventually, it elevatedthe susceptibility to thyroid tumors (Zhang et al., 2022).

Author Contribution Statement
BY and AR conceptualized and designed the study;
BY performed the experiments and collected the data; S,
YS, KNG, and BY analyzed and interpreted the results;
BY prepared the initial manuscript; AR and GM reviewed
and approved the final version of the manuscript.

Acknowledgements
The authors would like to thank the Schoolof Pharmaceutical Sciences, College of Medical,
Pharmaceutical and Health Sciences, Kanazawa
University, Kanazawa, 920-1192, Japan. And providing
the essential laboratory facilities, we are grateful to Dr.
Masuo Goto (The University of North Carolina at Chapel
Hill, US) for the cytotoxicity assay.
Funding Statement
The authors would like to thank this research was
supported scholarship by Kanazawa University ShortTerm Exchange Program for Science and Technology(KUEST) 2019 – 2020 from Japan Student Services
Organization (JASSO).
Statement of conflict Interest
The authors do not have any conflicts of interest to
declare.

References
Abdel-Monem AR (2012). A new alkaloid and a new diterpene
from Cleome paradoxa B.Br. (Cleomaceae). Nat Prod Res,26, 264–9.
Al-Bari MAA, Bhuiyan MSA, Flores ME, et al (2005).
Streptomyces bangladeshensis sp. nov., isolated from soil,
which produces bis-(2-ethylhexyl)phthalate. Int J Syst Evol
Microbiol, 55, 1973–77.
Ali H, Dixit S, Ali D, et al (2015). Isolation and evaluation of
anticancer efficacy of stigmasterol in a mouse model of
DMBA-induced skin carcinoma. Drug Des Devel Ther, 9,2793–800.
Ayaz M, Sadiq A, Wadood A, et al (2019). Cytotoxicity andmolecular docking studies on phytosterols isolated from
Polygonum hydropiper L. Steroids, 141, 30–5.
Bae H, Song G, Lim W (2020). Stigmasterol causes ovariancancer cell apoptosis by inducing endoplasmic reticulum
and mitochondrial dysfunction. Pharmaceutics, 12.
Breen LE, Schepp NP, Tan CHE (2005). Photochemical
generation and 1H NMR detection of alkyl allene oxides insolution. Can J Chem, 83, 1347–51.
de Combarieu E, Martinelli EM, Pace R, Sardone N (2015).
Metabolomics study of Saw palmetto extracts based on 1H
NMR spectroscopy. Fitoterapia, 102, 56–60.
Dong Y, Chen C, Chen C, et al (2021). Stigmasterol inhibits the
progression of lung cancer by regulating retinoic acid-related

DOI:10.31557/APJCP.2023.24.10.3345
Cytotoxic Activities from the Crude Extract of Cleome Rutidospermae
orphan receptor C. Histol Histopathol, 2021, 18388.
Du Q, Shen L, Xiu L, Jerz G, Winterhalter P (2006).
Di-2-ethylhexyl phthalate in the fruits of Benincasa hispida.
Food Addit Contam, 23, 552–5.
Durand PL, Grau E, Cramail H (2020). Bio-based thermoreversible aliphatic polycarbonate network. Molecules,
25, 1–12.
El-sayed O, Asker MMS, Shash SM, Hamed SR (2015).
Isolation, Structure elucidation and Biological Activityof Di-(2-ethylhexyl) phthalate Produced by Penicillium
janthinellum 62. Int J Chemtech Res, 2015, 58–66.
Fang DL, Chen Y, Xu B, et al (2014). Development of lipidshell and polymer core nanoparticles with water-soluble
salidroside for anticancer therapy. Int J Mol Sci, 15,3373–88.
Flores AFC, Blanco RF, Souto AA, Malavolta JL, Flores DC(2013). Synthesis of fatty trichloromethyl-β-diketones andnew 1H-pyrazoles as unusual FAMEs and FAEEs. J Braz
Chem Soc, 24, 2059–65.
Ghosh P, Chatterjee S, Das P, et al (2019). Natural Habitat,
Phytochemistry And Pharmacological Properties Of A
Medicinal Weed-Cleome Rutidosperma Dc. (Cleomaceae):
A Comprehensive Review. Int J Pharm Sci Res, 10, 1605.
Gottlieb HE, Kotlyar V, Nudelman A (1997). NMR Chemical
Shifts of Common Laboratory Solvents as Trace Impurities.
J Org Chem, 62, 7512–5.
Gupta PK, Saraff M, Gahtori R, et al (2021). Phytomedicines
targeting cancer stem cells: Therapeutic opportunities andprospects for pharmaceutical development. Pharmaceuticals,
14, 1-25.
Habib MR, Karim MR (2009). Antimicrobial andCytotoxic Activity of Di-(2-ethylhexyl) Phthalate andAnhydrosophoradiol-3-acetate Isolated from Calotropis
gigantea (Linn.) Flower. Mycobiology, 37, 31.
Hasan A, Rasheed L, Malik A (2007). Synthesis andcharacterization of variably halogenated chalcones andflavonols and their antifungal activity. Asian J Chem, 19,
937–48.
Hoffman RE (2006). Standardization of chemical shifts of TMS
and solvent signals in NMR solvents. Magn Reson Chem,
44, 606–16.
Houghton P, Fang R, Techatanawat I, et al (2007). The
sulphorhodamine (SRB) assay and other approaches totesting plant extracts and derived compounds for activities
related to reputed anticancer activity. Methods, 42, 377–87.
Hwang HS, Winkler-Moser JK, Liu SX (2017). Reliability of 1H
NMR Analysis for Assessment of Lipid Oxidation at Frying
Temperatures. J Am Oil Chem Soc, 94, 257–70.
Khan M, Khan M, Adil SF, Alkhathlan HZ (2022). Screeningof potential cytotoxic activities of some medicinal plants of
Saudi Arabia. Saudi J Biol Sci, 29, 1801-7.
Knothe G, Kenar JA (2004). Determination of the fatty acidprofile by 1H-NMR spectroscopy. Eur J Lipid Sci Technol,
106, 88–96.
Lamkanfi M, Kanneganti TD, van Damme P, et al (2008).
Targeted peptidecentric proteomics reveals caspase-7 asa substrate of the caspase-1 inflammasomes. Mol Cell
Proteom, 7, 2350–63.
Lee KH, Kim JH, Lim DS, Kim CH (2010). Anti-leukaemic
and Anti-mutagenic Effects of Di(2-Ethylhexyl)phthalate
Isolated from Aloe vera Linne. J Pharm Pharmacol, 52,
593–8.
Li R, Zhang P, Liu T, et al (2018). Use of Hempseed-Oil-Derived
Polyacid and Rosin-Derived Anhydride Acid as Cocuring
Agents for Epoxy Materials. ACS Sustain Chem Eng, 6,4016–25.
Liebmann JE, Cook JA, Lipschultz C, et al (1993). Cytotoxic

studies of pacfitaxel (Taxol®) in human tumour cell lines.
Br J Cancer, 1993, 1104–9.
Lotfy MM, Hassan HM, Hetta MH, El-Gendy AO, Mohammed
R (2018). Di-(2-ethylhexyl) Phthalate, a major bioactive
metabolite with antimicrobial and cytotoxic activity isolated
from River Nile derived fungus Aspergillus awamori.
Beni-Suef Univ J Basic Appl Sci, 7, 263–9.
Mcneil MJ, Porter RBR, Williams LAD (2012). ChemicalComposition and Biological Activity of the Essential Oilfrom Jamaican Cleome serrata. Nat Prod Commun, 7,
1231–2.
Mirzaeian R, Sadoughi F, Tahmasebian S, Mojahedi M (2021).
The role of herbal medicines in health care quality and therelated challenges. J HerbMed Pharmacol, 10, 156–65.
Park Y, Jones DP, Ziegler TR, et al (2011). Metabolic effects ofalbumin therapy in acute lung injury measured by proton
nuclear magnetic resonance spectroscopy of plasma: A pilot
study. Crit Care Med, 39, 2308–13.
Que NLS, Ribeiro AA, Raetz CRH (2000). Two-dimensional
NMR spectroscopy and structures of six lipid A species from
Rhizobium etli CE3: Detection of an acyloxyacyl residue ineach component and origin of the aminogluconate moiety.
J Biol Chem, 275, 28017–27.
Rahim A, Saito Y, Miyake K, et al (2018). Kleinhospitine e andCycloartane Triterpenoids from Kleinhovia hospita. J Nat
Prod, 81, 1619–27.
Rahman SMM, Munira S, Hossain MA (2008).
PHYTOCHEMICAL STUDY OF THE ARIAL PARTS OF
Cleome rutidosperma DC PLANT. Indo J Chem, 8, 459–62.
Ranjitha J, Bakiyalakshmi K, Anand M, Sudha PN (2009).
Phytochemical Investigation of n-Hexane Extract of Leavesof Cleome gynandra. Asian J Chem, 21, 3455–8.
Reheim MAMA, Tolba AMES (2016). Synthesis andSpectral Identification of Novel Stable Triazene: As Raw
Material for the Synthesis Biocompatible SurfactantsPyrazole-Isoxazole-Dihydropyrimidine-Tetrahydropyridine
Derivatives. Int J Org Chem, 6, 44–54.
Righi V, Apidianakis Y, Psychogios N, et al (2014). In vivo highresolution magic angle spinning proton NMR spectroscopyof Drosophila melanogaster flies as a model system toinvestigate mitochondrial dysfunction in Drosophila GST2
mutants. Int J Mol Med, 34, 327–33.
Sammar M, Abu Farich B, Rayan I, Falah M, Rayan A (2019).
Correlation between cytotoxicity in cancer cells and free
radical scavenging activity: In vitro evaluation of 57
medicinal and edible plant extracts. Oncol Lett, 18, 6563-71.
Shirey RJ, Globisch D, Eubanks LM, Hixon MS, Janda KD(2018). Noninvasive Urine Biomarker Lateral Flow
Immunoassay for Monitoring Active Onchocerciasis. ACS
Infect Dis, 4, 1423–31.
Sivakumar B, Murugan R, Baskaran A, et al (2013).
Identification and characterization of process-related
impurities of trans-resveratrol. Sci Pharm, 81, 683–95.
Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H (2011).
Phytochemical screening and Extraction: A Review. Int
Pharm Sci, 1, 98–106.
Tshilanda DD, Mpiana PT, Onyamboko DNV, et al (2014).
Antisickling activity of butyl stearate isolated from Ocimum
basilicum (Lamiaceae). Asian Pac J Trop Biomed, 4, 393–8.
Vicente J, de Carvalho MG, Garcia-Rojas EE (2015). Fatty
acids profile of Sacha Inchi oil and blends by 1H NMR and
GC-FID. Food Chem, 181, 215–21.
Xu J, Lu Q, de Toledo RA, Shim H (2017). Degradation of
di-2-ethylhexyl phthalate (DEHP) by an indigenous isolate
Acinetobacter sp. SN13. Int Biodeterior Biodegradation,
117, 205–14.
Yasir B, Astuti A, Raihan M, et al (2022). Optimization of
Asian Pacific Journal of Cancer Prevention, Vol 24

3351

Budiman Yasir et al
Pagoda (Clerodendrum paniculatum L.) Extraction Based
By Analytical Factorial Design Approach, Its PhytochemicalCompound, and Cytotoxicity Activity. Egypt J Chem, 65,
421-30.
Zhang X, Guo N, Jin H, et al (2022). Bisphenol A drives di(2ethylhexyl) phthalate promoting thyroid tumorigenesis via
regulating HDAC6/PTEN and c-MYC signaling. J Hazard
Mater, 425, 127911.
Zhao H, Zhang X, Wang M, Lin Y, Zhou S (2021). Stigmasterol
Simultaneously Induces Apoptosis and Protective Autophagy
by Inhibiting Akt/mTOR Pathway in Gastric Cancer Cells.
Front Oncol, 11.
Zhao J, Avula B, Joshi VC, et al (2011). NMR fingerprinting
for analysis of Hoodia species and Hoodia dietary products.
Planta Med, 77, 851–7.

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