The potential use of geraniol esters from citronella oil as anticancer agent

Article abstract of DOI:10.13005/ojc/350310

Geraniol which is mainly contained in citronella oil is one of the Indonesian natural products with anticancer potential. In this study, synthesis of geranyl butyrate, geranyl caproate, and geranyl caprylate from geraniol was conducted using sodium hydroxide as a catalyst.The aim was to enhance the anticancer activity of geraniol as a starting material. In order to achieve this, its esters were identified through the use of Gas Chromatography-Mass Spectroscopy (GCMS), Fourier Transform Infra Red (FTIR), Thin Layer Chromatography (TLC), and Proton Nuclear Magnetic Resonance (¹H-NMR).They were analyzed for their potential as anticancer agents through Brine Shrimp Lethality Test (BSLT) against Artemia salina Leach, Mosmann method against murine leukemia (P388) cells and normal (Vero) cells. It was found that geraniol esters have the potential to be anticancer compounds. This was indicated by LC₅₀ values of 0.96-1.46 μg/ml against A. salina L, IC₅₀ values of 22.34-32.29 μg/ml against P388 cells, and very less cytotoxic effect on Vero cells with IC₅₀ values of 116.08-172.93 μg/ml. Therefore, there is an expectation that acyclic ester compounds should be used in treating cancer.

Full text of DOI:10.13005/ojc/350310

ISSN: 0970-020 X
CODEN: OJCHEG
019, Vol. 35, No.(3):
Pg. 987-996
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Access, Peer Reviewed Research Journal
2
www.orientjchem.org
The Potential Use of Geraniol Esters From Citronella Oil
As Anticancer Agent
GALUH WIDIYARTI*, MEGAWATI MEGAWATI and MUHAMMAD HANAFI
Research Center for Chemistry, Indonesian Institute of Sciences (LIPI) Kawasan PUSPIPTEK,
Serpong, South Tangerang City, Banten 15314, Indonesia.
*
Corresponding author E-mail: galuh.laksmono@gmail.com
http://dx.doi.org/10.13005/ojc/350310
(Received: February 26, 2019; Accepted: May 02, 2019)
ABSTRACT
Geraniol which is mainly contained in citronella oil is one of the Indonesian natural products
with anticancer potential. In this study, synthesis of geranyl butyrate, geranyl caproate, and geranyl
caprylate from geraniol was conducted using sodium hydroxide as a catalyst.The aim was to enhance
the anticancer activity of geraniol as a starting material. In order to achieve this, its esters were
identified through the use of Gas Chromatography-Mass Spectroscopy (GCMS), Fourier Transform
Infra Red (FTIR), Thin Layer Chromatography (TLC), and Proton Nuclear Magnetic Resonance
1
(
H-NMR).They were analyzed for their potential as anticancer agents through Brine Shrimp Lethality
Test (BSLT) against Artemia salina Leach, Mosmann method against murine leukemia (P388)
cells and normal (Vero) cells. It was found that geraniol esters have the potential to be anticancer
compounds.This was indicated by LC50 values of 0.96-1.46 µg/ml against A. salina L, IC50 values of
22.34-32.29 µg/ml against P388 cells, and very less cytotoxic effect on Vero cells with IC50 values
of 116.08-172.93 µg/ml. Therefore, there is an expectation that acyclic ester compounds should be
used in treating cancer.
Keywords: Geraniol, Citronella oil, Ester, BSLT, Mosmann, Anticancer.
INTRODUCTION
analog compounds in treating cancer. It is expected
to provide a drug with lower toxicity, higher activity
and more selectivity.
Cancer has been discovered to be one
of the major reasons for human death through the
globe and in Indonesia it is ranked second after
heart disease with its patients increasing on a
yearly basis .However it can be treated by surgery,
radiation, chemotherapy, and through drugs but no
satisfactory result has been obtained from any of
these methods.Therefore, there is a need to conduct
research on the use of bioactive or synthesizing
In recent years, Plant extracts are used as
chemo-preventiveandchemotherapeuticcompounds
applied in the prevention of cancer because of their
1-3
4-7
usefulness in research . In Indonesia, Citronella is
an example of these compounds and it is isolates
from Lemongrass plant, a genus of Cymbopogon
and family of Poaceae. Different kinds of essential
This is an
Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
988
oils mostly utilized in cosmetics, perfume, sanitary,
food, and pharmaceuticals industries throughout the
world are made from it.It is, however, further divided
into three, based on source, namely Ceylon derived
from Cymbopogon nardus Lenabatu W. Wats.;
Java from Cymbopogon winterianus Jowit; and
Lemongrass oil derived from Cymbapogon citrates
Their potency as chemoprevention activity for cancer
was tested against nauplii of A. Salina using BSLT
method. This is generally considered as a bench
top pre-assay to discover the cytotoxic agent for the
21,22
anticancer compound .The MTT method was also
used to check their cytotoxic activity against murine
23-24
leukemia (P388) cells .The same method was also
8
25
DC Stapt . Indonesia is the third largest producer of
used to test them against normal (Vero) cells .
9
the oil in the world, after China and Vietnam and
almost all their products are dominated by the Java
type which are traditionally used as medicine, insects
repellent, toothache and body warmers.
MATERIALS AND METHODS
Materials
The starting materials include geraniol
Citronella oil and its compounds have
been observed to have the ability to be applied in
bioactivity processes as antimicrobial, antibacterial,
from Aroma Essence Prima with 90% purity, butyric
(Sigma Aldrich B1754), caproic, and caprylic acids
from Sumi Asih with 99% purity. The catalyst used
was NaOH (E. Merck & Co. 106469) with 1% of HCl
solution as a netraulizer. The standard procedure
was used in drying and distilling all the solvents
including n-hexane, ethyl acetate (EtOAc), and
water. Furthermore, mass and molecular structure
spectroscopy, In vitro cytotoxic activity and BSLT test
were conducted using analytical grade chemicals.
10
antifungal, antiviral, antiparasitic , antioxidant and
1
1-13
anti-inflammatory agents
. They mostly contain
monoterpene compounds such as citronellol,
14
citronellal, and geraniol which have been reported
to be effective against cancerous cell proliferation
and inhibition of DNA synthesis. This shows that
they are one of the new classes of cancer chemo-
1
3,15-17
preventive compounds
. Most of the raw
General procedure
The precoated silica gel plates were
analyzed through the use of the TLC (Kiesel gel
materials used in the production of synthetic drugs
in Indonesia are imported from other countries,
therefore, local natural materials like geraniol mostly
found in citronella oil are need to be utilized.
60F254 0.25 mm), and there was visualization of the
spots in them under the UV light (254 and 365 nm)
irradiation, sprinkled with 10% sulphuric acid solution
Fatty acids such as butyric (C ), caproic
4
o
under 110 C. A Merck & Co. 64271 (70-230 mesh)
(
C ), and caprylic (C ) acids have also been reported
6
8
was used to conduct column chromatography on the
to function in bioactivity as antiviral, antifungal,
antimicrobial and potentially as antineoplastic
silica gel.The measurement of IR spectra was done
21
on an FTIR Shimadzu prestige with the use of KBr
pellets while the evaluation of mass spectrum (MS)
was conducted through the use of GCMS Agilent
Technologies GC Agilent Technologies 7890B with
1
8,19
agents
. Hence the combination of the ester
compounds derived from geraniol with these
fatty acids could be used in the chemoprevention
of cancer. Furthermore, combining caproate
synthesized from citronellol and caproic acid in palm
kernel oil has also been reported to be effective
in inhibiting murine leukemia (P388) cells growth
through the increment of its cytotoxic activity. The
5
977A MSD. NMR Jeol Spectrophotometer was
1
used to record H-NMR spectrum for 500 MHz in
deuterio chloroform. It is important to point out that
the chemical shifts (d) were in ppm and downfield
from CDCl at d 7.26 ppm.
3
38.49 and 10.63 µg/ml concentration have also been
found to inhibit 50% of murine leukemia (P388) cells
Synthesis of geraniol ester compound
20
growth (IC ) .
10 mmol of geraniol, 12 mmol of butyric acid
5
0
(
or caproic acid or caprylic acid), and 5% (w/w) of
Therefore, this study was aimed at
synthesizing geranyl butyrate, geranyl caproate, and
geranyl caprylate using sodium hydroxide (NaOH)
as a base catalyst to enhance the anticancer activity
of geraniol derivatives. The geraniol esters were
NaOH were arranged in a 250 mL round-bottomed
flask. The mixture was stirred and heated at 80 C
for 8 hours. This was followed by the neutralization
of the reaction by 1% of HCl solution for the purpose
of separating it from the NaOH catalyst. EtOAc was
used to extract the reaction mixture and the organic
o
1
identified by using TLC, FTIR, GCMS, and H-NMR.

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
989
phase was washed through the use of distilled water,
dried over anhydrous Na SO and concentrated
water-soluble substrate 3-4,5-dimethyl thiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) into
an insoluble blue colored formazan product by the
mitochondria succinate dehydrogenase enzymes
in living cells.The measurement of the activity level
was conducted by using the cells viability because
of the fact that MTT can only be reduced in cells that
2
4
o
under reduced pressure at 45 C under vacuum
to produce the crude ester product. The analysis
of the products was conducted by TLC through
the use of eluent of n-hexane: EtOAc = 95:5 and
further purification was conducted through the use
of column chromatography eluted with n-hexane, a
gradient of EtOAc to 100%.The identification of pure
ester product was conducted through the application
26
are metabolically active.
The first step of the process was growing
the cell cultures. This was conducted by incubating
1
of spectroscopic methods (MS, IR, H-NMR).
the cells in RPMI 1640-fetal bovine serum (FBS) at
BSLT test
0
3
7 C with a flow of 5% of CO for 24 h and observed
2
Hatching of the brine shrimp was the first
thing to be conducted. This was done by placing
about 50-100 mg brine shrimp eggs (A. salina L) in
a vessel under light regime conditions and through
the application of artificial seawater from commercial
sea salt, they were hatched and grew to larvae
under an inverted microscope.The attachment of the
cells to the walls of flasks caused the replacement
of the medium with new serum, after which they
were primarily incubated before the confluent growth
was reached. This refurbishment was continuously
conducted until the sufficient number of cells needed
for testing was obtained.
with 48 h of incubation under room temperature
0
(
(
25-29 C). There was collection of the larvae
nauplii) through the use of pipette leaving their
The second step was the preparation of
cells for testing. The murine leukemia (P338) cells
line were provided from Meijo Seika Co., Ltd.,
Tokyo Japan, while kidney of an African green
monkey (Vero, ATCC CCL-81) with normal cells
was obtained from the Department of Biology
Universitas, Indonesia. Each cell line was cultured
in a suitable medium to obtain the desired growth
and their growth curve was plotted.The medium was
discarded, washed, and added to the RPMI medium
after the adequate number of cells needed for the
test has been. There was transfer of the cells into
sterile tubes where they were centrifugated for 5 min
at 1200 rpm. The supernatant was cast aside and
the sediment of the cell was put into the RPMI 1640
medium containing 10% FBS.The suspension of the
cell was taken and the calculation of their density
was conducted through the use of hemocytometer
under a microscope. The formula below was used
in calculating the number of cells.
shells behind.
The application of the Meyers method to
conduct the BSLT analysis was the second step
21,22
.
To do this, there was dissolution of four (4) mg of
the water-soluble samples in artificial seawater
to produce 2 mg/ml solution while the water-
insoluble compounds were dissolved in DMSO.The
preparation of 10, 100, 500, and 1000 µg/ml of the
solution followed this and there was the transfer of
about 100 µl of seawater with 10-11 shrimp larvae
of A. salina to the 96-well microplates. The DMSO
groups were used as the control of the experiment
while there was addition of approximately 100 µl
of each sample to the test wells and each of the
concentrations was made to be in three replicates.
There was incubation of the solutions for a period
of 24 h at room temperature under illumination after
which the live and dead larvae present in every well
were counted. The determination of the 50% lethal
concentration of the sample solutions (LC ) was
5
0
conducted through the use of Probit analysis.
In vitro anticancer analysis against P388 and
Vero cells using the MTT method
The calculated figure was used to dilute
the cells with medium RPMI-serum to obtain 2 X
0 cells/ml of cell suspension.
Cytotoxicity test against murine leukemia
4
1
(
P388) cells was conducted by using Mosmann
23
method through the application of the MTT reagent .
This was established on the reduction of yellow
The third step of the process was the
cytotoxicity test. This was conducted by disturbing

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
990
4
the cells with densities 1-2 X 10 cells/well in RPMI
Oily liquids were obtained as the products
and the analysis of the TLC revealed that there
are ester compounds in them as represented by a
spot with Rf value of 0.4, 0.4, and 0.3 for geranyl
butyrate, geranyl caproate, and geranyl caprylate
compounds, respectively. These spots were lower
1
640 culture medium in 96 well-plates and incubating
them in a 5% of CO incubator at 37 C for 24 h and
0
2
later stocked at the bottom wells. The next day, the
media was taken, washed with FBS, and 100 µl of
culture medium containing DMSO or sample with any
of 100, 50, 25, 10, 5, 2.5 and 1 µg/ml concentrations
were added and incubated for 48 hours.The sample
was dissolved in DMSO and diluted using phosphoric
acid buffer solution (PBS).Culture medium was then
removed, washed with 100 µl of FBS, and an addition
of 100 µl of a new culture medium containing MTT
mg/ml was made. It was incubated at 37 C in 5%
of CO for 4 hours. Since it has been established
that living cells will interact with MTT to form purple
formazan crystals. The medium containing MTT
was removed after 4 h it washed with PBS, and
than the 0.8 R value of geraniol when used as a
f
starting material. The crudes were further purified
by using chromatography column using silica gel
as the stationary phase and a solvent mixing ratio
for n-hexane and gradient of EtOAc at 100% as the
mobile phase.It obtained using a ratio 98:2, while pure
geranyl caprylate compound was obtained at 95:5.
The percentage yield of pure geranyl butyrate, geranyl
caproate and geranyl caprylate were 66.94% (0.75g),
76.72% (1.94g) and 54.92% (1.54g), respectively.
0
5
2
2
00 µl of isopropanoate solution was added and
The formation of a new group of geranyl
incubated at room temperature for 12 h to completely
dissolve the formazan crystals.The bio-reduction of
MTT was assessed by measuring the absorbance
of each well at 550 nm through the use of ELISA
reader. The regression equation and its IC50 (i.e.
the concentration that caused the death of 50% of
the population of the cells) were determined. The
cytotoxicity was conducted in three replicates. It is
important to point out that the IC50 value determines
how effective a compound can be in suppressing the
biological function of a sample that is still active.The
calculation of this value is conducted by using the
-1
butyrate with 1722.43 cm wave number (Vmax) was
showed by IR spectrum.This stretched the vibration
of carbonyl (C=O) of the ester formed with a variation
from the absorption of carbonyl (C=O) of butyric acid
-1
at 1707 cm wave number (Vmax).Furthermore, the
formation of a new group of geranyl caproate with
-
1
1
718.93 cm was displayed by the IR spectrum.
This stretched the vibration of carbonyl (C=O) of the
ester formed with a variation from the absorption of
-
1
carbonyl (C=O) of caproic acid at 1705.7 cm . The
formation of a new group of geranyl caprylate with
-
1
23-26
1718.53 cm was displayed by the IR spectrum.
This stretched the vibration of carbonyl (C=O) of the
ester formed with a variation from the absorption of
linear regression analysis
.
RESULTS AND DISCUSSION
-
1
carbonyl (C=O) of caprylic acid at 1702 cm .These
spectra can be seen in Figure 2.
As stated earlier, the synthesis of geraniol
estersfromgeraniolwithbutyricacid, caproicacid, and
The MS of geraniol esters was identified
using GCMS. For the geranyl butyrate, it showed
a dominant peak at 14.61 min of retention time
corresponding to a molecular formula of C H O
caprylic acid was conducted by using a base catalyst
0
(
NaOH) at 80 C for 8 h as shown in Figure 1.
14
24
2
of geranyl butyrate (the IUPAC name is butyric acid
,2-dimethyl-3,7-dimethyl-2,6-octadienyl ester)
2
at MS equals to 224.34 g/mol. For both geranyl
caproate and geranyl caprylate, it was found
that the MS of geranyl caproate (hexanoic acid
3,7-dimethyl-6-octenyl ester with molecular formula
of C H O ) and geranyl caprylate (octanoic acid
16
28
2
3
,7-dimethyl-2,6-octadienyl ester with molecular
formula of C H O ) were 252.39 and 280.45 g/mol
18
32
2
with the indication of dominant peaks at retention
times of 17.30 and 19.39 minutes, respectively.
Fig. 1. Esterification of geraniol (A) to produce geranyl
butyrate (B), geranyl caproate (C), and geranyl caprylate (D)

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
991
1
Figure 1 and Table 1 show the H-NMR
spectra data for both geraniol and geraniol ester
compounds with respect to the molecular structure
they possess. It reveals that new chemical shifts
are formed by methyl (CH ), methylene (CH ) and
(
A)
3
2
methyne (CH) groups of carboxylic acid or fatty acids
and another shift at dH = 4.13-4.59 ppm (2H, t, 7.2-
7.4 Hz).This involved 2 protons (H) of the methylene
(
CH ) group used in forming triplet adjacent to CH2
2
group and downfield due to its interaction with the
electronegativity of the oxygen and carbonyl (C=O)
of the ester formed. These are not the same with
that of 2 protons (2H) of CH group used in forming
2
triplet as adjacent to CH group and downfield due
to its interaction with the electronegativity of alcohol
oxygen (OH) in the geraniol at dH =3.65 ppm
(
B)
(
2H, t,) as it can be seen in Table 1.
The data for geranyl butyrate obtained
1
from the H-NMR spectrum showed that at dH =
4.56 ppm (2H, d, 7.2 Hz), a chemical shift occurred
for 2 protons (H) of the CH group used in forming
2
doublet as adjacent to CH group and downfield due
to its interaction with the electronegativity of oxygen
and ester carbonyl (C=O) group. Another shift was
experienced at dH = 5.41 and 5.31 ppm (1H, t,
7
.2 Hz) for one proton of 2 CH groups interacting
(
C)
with 2 protons from the CH group, and downfield
2
because of its double bond. A chemical movement
was observed at dH = 2.32 and 2.16 ppm 2(2H, t,
7
.2 Hz) for 2 protons of 2CH groups interacting with
2
CH group. Another shift was seen at dH = 1.51 and
2
1
.67 (3H, s) for 3 CH groups interacting with the
3
quaternary carbon atoms (C). At dH = 1.79 (2H, m,
.5 Hz), chemical shifts of 2 protons of CH group
7
2
used in forming multiplet were observed due to its
interaction with 3 protons of the CH group and
3
2
protons of the CH group while another was seen at
2
dH = 2.16 (2H, m, 7.2 Hz) for 2 protons of CH group
2
used to form quartet shifted due to its interaction with
Fig. 2. IR spectra of geranyl butyrate (A), geranyl caproate
B), and geranyl caprylate (C)
2
protons of the CH group and 1 proton of the CH
2
(
group, and at dH = 0.9 ppm (3H, t, 7.8 Hz), 3 protons
of CH group interacting with CH group shifted.
The GCMS chromatograms also showed
3
2
another peak at retention times of 10.92, 11.33 and
The data for geranyl caproate from the
11.21 min for geranyl butyrate, geranyl caproate and
1
H-NMR spectrum revealed that there was a chemical
shift at dH = 4.13 ppm (2H, d, 7.2 Hz) for 2 protons
H) of the methylene (CH ) group that formed doublet
geranyl caprylate, respectively.This can be attached
to the unreacted reactant residuals of the geraniol.
This shows that the esterification reaction is not
perfect yet, therefore, there was a need for further
optimization.
(
2
as adjacent to CH group and downfield because of
its interaction with the electronegative oxygen and
ester carbonyl (C=O) group. Another was observed

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
992
at dH = 5.09 and 5.39 ppm (1H, t, 7.3 Hz) for one
ppm (2H, d, 7.2 Hz), 2 protons (H) of the CH group
2
proton of 2 CH groups interacting with 2 protons
that formed doublet as adjacent to CH group shifted
and downfield because of its interaction with the
electronegative oxygen and ester carbonyl (C=O)
group. At dH = 5.40 and 5.18 ppm (1H, t, 7.2 Hz),
one proton of 2 CH groups interacting with 2 protons
from the CH group, and downfield because of its
2
double bond. The was observable chemical shift at
dH = 2.01 and 2.06 ppm (2H, t, 7.2 Hz), for 2 protons
of 2 CH groups that interacted with CH group and
2
2
at dH = 1.67 and 1.58 ppm (3H, s), 2 CH groups
from the CH group shifted and downfield because
3
2
that interacted with 2 protons from the quaternary
carbon atoms (C) shifted.A chemical movement was
observed at dH = 2.31 and 1.62 ppm (2H, t, 7.8 Hz)
of its double bond. At dH = 2.08 and 2.09 ppm
(2H, t, 7.2 Hz), 2 protons of 2 CH groups interacting
2
with CH group shifted. At dH = 1.67 and 1.61 ppm
2
for 2 protons of 2 CH groups used in forming triplet
(3H, s), 2 CH groups interacting with 2 protons
2
3
that interacted with 2 protons from the CH group.At
from the quaternary carbon atoms (C) shifted. At
2
dH = 1.29 and 1.31 ppm (2H, q, 7.8 Hz), 2 protons of
dH = 2.29 and 1.67 ppm (2H, t, 7.8 Hz), 2 protons
2
CH groups that formed quartet due to its reaction
of 2 CH group used to form triplet and interacting
2
2
with 2 protons of the CH group and one proton of
with 2 protons from the CH2 group shifted. At
2
the CH group shifted, and at dH = 0.88 ppm (3H, t,
dH = 1.29 and 1.28 ppm (2H, q, 7.8 Hz), 2 protons
7.2 Hz), there was a chemical shift of 3 protons of
of 3 CH groups used to form quartet because
2
CH group that interacted with the CH group.
of its interaction with 2 protons of the CH group
3
2
2
and 1 proton of the CH group shifted, and at
1
Furthermore, the H-NMR spectrum of
dH = 0.88 ppm (3H, t, 7.1 Hz), 3 protons of CH group
3
geranyl caprylate data revealed that at dH = 4.59
interacting with CH group shifted.
2
1
Table 1: H-NMR data for geraniol esters (at 500 MHz in CDCl , d in ppm, J in Hz)
3
Position
Geraniol
Geranyl
butyrate
Geranyl
Geranyl
(
H)
caproate
caprylate
3
.65 (alcohol)
4.18 (2, t)
5.39 (1, t)
1
2
4.56 (2, d, 7.1)
5.41 (1, t, 7.2)
4.13 (2, d, 7.2)
5.39 (1, t, 7.3)
4.59 (2, d, 7.2)
5.40 (1, t, 7.2),
3
4
5
6
-
-
-
-
2(2, t)
2(2, t)
5.2(1, t)
2.16 (2, t, 7.2)
2.16 (2, q, 7.2)
5.31 (1, t, 7.2)
2.06 (2, t, 7,1)
2.01 (2, t, 7,2)
5.09 (1, t, 7,3 )
2.09 (3, t, 7.2)
2.08 (3, t, .2)
5.18 (1, t, 7.2)
7
8
9
-
-
-
-
1.82(3, s)
1.67 (3, s,)
1.67 (3, s)
1.58 (3, s)
1.67 (3, s)
-
1.67 (3, s)
1.70(3, s)
1.51 (3, s)
1.61 (3, s)
1
0
1.82(3, s)
1.67 (3, s)
1.67 (3, s)
1
’
’
’
’
’
’
’
’
-
-
-
-
-
-
-
-
-
-
2
2.32 (2, t, 7.2)
2.31 (2, t, 7,8)
1.62 (2, t, 7,8)
1.29 (2, q,7,8)
1.31 (2, q, 7.8)
0.88 (3, t, 7.2)
-
2.29 (2, t, 7.8)
1.67 (2, t, 7.5)
1.29 (2, q, 7,8)
1.29 (2, q, 7.8)
1.29 (2, q, 7.8)
1.28 (2, q, 7.5)
0.88 (3, t, 7.1)
3
4
5
6
7
8
1.79 (2, m ,7.5)
0.9 (3, t, 7.8)
-
-
-
-
-
Note: s : singlet, d : doublet, t : triplet, q : quartet, m : multiplet, and chemical shift of spectrum
1
H-NMR of geraniol according to literature.
Cytotoxicity test is used in analyzing the
degree of cell toxicity using human or animal cells.It
is often used as a preliminary test in discovering new
chemoprevention agents for cancer or anticancer
drug candidates.For the purpose of this study, it was
conducted through the use of BSLT method because
it is easy, cheap, and reliable. The test showed that
geraniol and geraniol esters were very active against

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
993
A. salina with LC50 1.39 and 1.43-1.96 µg/ml as
shown in Fig. 3. They were active within LC50 value
The IC50 values of geranyl butyrate and
geranyl caproate were lower compared to geraniol.
This means that they have higher anticancer
activities, and through the esterification of geraniol,
its activity can also improve.This is possible because
of the increasing hydrophobicity or lipophilicity of
geranyl butyrate and geranyl caproate compared
to geraniol as indicated by increased log P of these
compounds which are 3.75 and 4.62 for geranyl
butyrate and geranyl caproate respectively and 2.49
for geraniol. Furthermore, increment in lipophilicity
results in the variation of physical properties, for
example, it increases its solubility in fat which
consequently improves its power to penetrate cell
membrane walls. It also causes an increment in the
solubility of fluids that are not inside the cells which
helps in improving how drugs are transported to the
receptors. This research revealed that anticancer
activity can be influenced by hydrophobicity or
lipophilicity. This is in agreement with the result of a
study conducted on the antitumoral properties of gallic
acid and its ester derivatives which reported that tumor
celllinesaremoreinfluencedbyalkylestersthangarlic
≤
30 µg/ml; therefore, they have the potentials of
21,22
being chemo-preventive or anticancer agents
.
Fig. 3. The preliminary cytotoxic activity test of geraniol
esters by using BSLT method
The American National Cancer Institute
(
NCI) stated that murine leukemia (P388) cells are
type of tumor cells that make use of cytotoxic test
protocol. They are mostly used for further testing
of compounds in order to check their suitability as
29
acid because of their hydrophobic mediety .
2
7,28
chemo-preventive agents or anticancer drugs
.
The cytotoxic activity of geranyl caprylate
Therefore, In vitro cytotoxic test of geraniol esters
was conducted against murine leukemia (P388)
cells using the MTT method.The result showed that
geraniol esters have a cytotoxic effect and are active
against murine leukemia (P388) cells (IC <40 µg/
(
IC50 = 32 µg/ml) is lower than geraniol (IC50 = 28 µg/
mL). This may be because of the high lipophilicity
of geranyl caprylate (log P = 5.46 > 5). This makes
it too soluble in fat, decrease its ability to penetrate
the walls of the cell membrane and bioactivity. This
is in accordance with the Lipinski’s rules five which
states that a bioactive compound will be orally active
50
2
8
mL ). The activity yielded IC 22.34-32.29 µg/mL
50
as shown in Fig.4.The IC50 shows the concentration
of geraniol esters inhibiting 50% of the growth of
murine leukemia (P388) cells, therefore, they have
the prospects of being anticancer agents.
30-31
if log P is less than 5 (Log P value of < 5)
.
Artonin E, commonly used as a positive
control of cytotoxic test against leukemia cancer
cells, was used as the standard in testing the
In vitro cytotoxic activity. The IC50 value of artonin
E inhibiting 50% the growth of murine leukemia
(
P388) cancer cells was 0.4 µg/mL as shown in
Fig. 4.The IC50 of geraniol esters was high but lower
than artonin E.This may be because of the low purity
of the products, and the difference in their structures
where geraniol esters are acyclic carbon compounds
without amine content and artonin E is an aromatic
carbon compound with amine content.Nevertheless,
the result showed that acyclic compounds such as
geraniol esters can be considered as new potential
anticancer agents.
Fig. 4. IC50 value of geraniol esters against murine leukemia
(
P388) cells

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
994
3
5,36
Furthermore, it was required to examine
the cytotoxic activity of these geraniol esters
against other cancer cells. Several previous studies
reported that ester compounds can be used in the
treatment of cancer. In the silico study of geraniol
ester compounds like geranyl isobutyrate, geranyl
benzoate, geranyl 3-methyl benzoate, geranyl
analgesic and anticancer
. The geranium oil
derived from Pelargonium geranium was also found
to be active against certain cancers because of
its ability to cause tumor regression. Citronellol,
geraniol, and natural ester compounds such as
citronellyl propanoate, geranyl propanoate, geranyl
n-butyrate and geranyl tiglate have all been observed
to engage in anticancer activities .Paclitaxel (taxol)
is also an anticancer compound derived from various
Taxus species while one of the most commonly used
chemotherapeutic agents against many types of
cancer is tricyclic diterpene with a complex ester on
37
4
-nitrobenzoate, and geranyl nicotinate, it was
reported that their anti-inflammation potential is one
of the causes of chronic diseases such as cancer in
32
humans .Citronellyl 5-aminolevulinate, a citronellol
ester compound derived from citronellol, has been
reported to be effective in inhibiting cells growth of
skin tumor. Cytotoxic activity of the compound on
mice was found by photodynamic therapy to have
increased significantly in deadly skin tumor cells
38-40
the side chain .Therefore, geraniolesterswhichare
acyclic compounds have the potentials of being the
new alternative chemoprevention agents for cancer.
33
about 10 times smaller than citronellol .
In vitro cytotoxicity against zero cells was
conducted through the use of the MTT method. The
concentration of geraniol esters used for cytotoxicity
test against zero cells was 31.25-1000 µg/ml and
three replicates were made in order to determine the
Natural monoterpene ester compounds of
essential oil such as linalyl acetate, geranyl acetate,
and eugenol acetate were also reported to have
the potential to be an antifungal, anesthetic, and
anti-inflammatory which is all important in treating
5
0% growth inhibition (IC ). This study showed that
50
34
cancer .Geranyl acetate from the biotransformation
of geraniol through esterase can be used as
geraniol esters do not have any significant cytotoxicity
on the normal zero cell line as shown in Table 2.
Table 2: Inhibition percentage and IC50 geraniol esters against zero cell line
Concentration of
sample (µg/ml)
Geranyl butyrate
Geranyl caproate
Geranyl caprylate
Inhibition (%)±SD
IC50 (µg/ml)
Inhibition (%)±SD
IC50 (µg/ml)
Inhibition (%)±SD
IC50 (µg/ml)
3
1.25
13.32±1.87
26.85±3.34
25.29±3.59
58.48±3.06
91.99±1.95
97.61±1.43
8.82±2.47
17.22±1.12
51.54±3.91
98.67±0.66
101.17±0.16
101.22±0.32
4.57±5.67
6.64±4.23
6
2.5
25
50
00
1
162.51
116.08
18.49±2.65
71.10±5.58
101.33±0.78
102.34±0.16
172.93
2
5
1
000
The result revealed that the inhibition of 92-
01% and 98-102% was found at 500 and 1000 µg/
as base catalyst produced geranyl butyrate, geranyl
caproate, and geranyl caprylate with a medium yield
(54-76%).The LC50 and IC50 values found against A.
salina L. and murine leukemia (P388) cancer cells
as well as their weak cytotoxic activity against zero
cells indicated their potential as anticancer agents.
It was also found that the esterification of geraniol
with butyric acid can enhance the anticancer activity
of the compounds. Hydrophobicity or lipophilicity
was also observed to have an influence on the
anticancer activity such that increasing lipophilicity
allows increasing anticancer activity, however in a
situation where it is too lipophilic (Log P > 5), it may
decrease. The study revealed that because of the
1
ml respectively.These values indicate the cytotoxicity
level of the geraniol esters such that the lower the IC50
values, the higher the toxicity, hence the cytotoxicity
level of the samples was divided into strong (<100µg/
ml), moderate (101-200 µg/ml) and weak (>200µg/ml).
Geraniol esters were found to be indicating a weak
26
inhibition against zero cell lines .
CONCLUSION
In conclusion, it was discovered that
synthesizing geraniol esters through the use of NaOH

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
995
ability of acyclic monoterpene esters to inhibit cancer,
geraniol esters have the potentials of being the new
alternative chemoprevention agents for cancer.
of the Republic of Indonesia. The authors express
their gratitude to Professor Soleh Kosela and Dr Emil
BudiantofromtheDepartmentofChemistry,Universitas
Indonesia for their contribution to this article, and to Mrs.
EviWulandariandMr.SalahudinoftheResearchCenter
for Chemistry, Indonesian Institute of Sciences for their
assistance in IR and GCMS analysis.
ACKNOWLEDGEMENT
The research was funded by (Insinas Riset
Pratama Individu (IRPI) Programme, grant number:
13/E/KPT/2018, cooperation agreement: 001/P/RPL-
LIPI/INSINAS-2/VII/2018) the Ministry of Research,
Technology, and Higher Education (Kemenristek-DIKTI)
CONFLICT OF INTEREST
Authors declare that there is no conflict of interest.
REFERENCES
1
2
3
4
5
.
.
.
.
.
Kamil, N.; Kamil, S. Systematic Rev. in
Pharm., 2015, 6(1), 13-17.
Biol. Interact., 2009, 179, 2(3), 160-168.
Bayala, B.; Bassole, I.; Simpore, J. Am. J.
Cancer Res., 2014, 4(6), 591-607.
17.
18.
19.
Bernard,S.;andChristopher,P.IARCNonserial
Publication, WHO Press., 2014, 630.
Anonymous.Pusat data daninformasiDepkes
RI., 2015, 1-7.
Fauser J. Anticancer Agent. Med. Chem.,
2014, 14, 49-65.
Narayanan, A.;Baskaran, S.A.;Amalaradjon,
M.; Venkitanarayanan. Int. J. Mol. Sci., 2015,
16, 5014-5027.
Newman, D.J.; and Cragg, G.M. J. of Nat.
Prod., 2016, 79, 629-661.
Qurishi, Y.; Hamid, A.; Majeed, R.; Hussain,
A.; Qazi, A.K.; Future Oncol., 2011, 7(8),
20.
21.
Widiyarti, G.;Hanafi, M.;Kosela, S.;Budianto,
E. IJAC., 2016, 12(3), 209-220.
1007-1021.
Meyer, B.R.; Ferrigni, N.R.; Putnam, J.E.:
Jacobsen, L.B.; Nichols, D.E.; Mc. Laughlin,
J.L. Planta Medica., 1982, 31-34.
6
7
.
.
Mandal, S.;Bandyopadhyay, S.;Ghosh M.K.;
Mandal, C. Anticancer Agent Med. Chem.,
2012, 12, 49- 75.
22.
Kamanja, I.T.; Mbaria, J.M.; Gathumbi, P.K.;
Mbaabu, M.B.;Kabasa, J.D.:Kiama, S.G. The
J. of Med. Res., 2018, 4(5), 249-255.
MosmannT.J.Immunol. Meth., 1983, 65, 55-63.
Colegate,S.M.;Molyneux,R.J.Bioactivenatural
products: detection, isolation and structural
Christodoulu, M.; Kontos, C.; Halabalaki, M.;
Scorilas, A.Anticancer Agent in Med.Chem.,
2
013, 13, 1-25.
Ganjewala D.J of Essential OilTherapeutics.,
009, 3, 56-65.
23.
24.
8
9
1
1
1
1
1
.
2
nd
.
Anonymous. National Agency for Export
Development (NAFED)., 2008, 1, 2.
determination. 2 . edition. CRC Press Taylor
and Francis Group. 2008, 338-385.
0.
1.
2.
3.
4.
Raut, J.S.;Karuppayil, S.M.Ind.Crops.Prod.,
25.
26.
Khushboo, J.; Dhara, B.; Maitreyi, Z. Int. J.
Pharm. Sci. Rev. Res., 2016, 37(2), 130-133.
Garbi, M.I.; Osman, E.E.; Kabbashi, A.S.;
Saleh, M.S. J. of Sci. and Innovative Res.,
2015, 4(2), 89-93.
2
014, 62, 250-264.
Cavar, S.;and Maksimovic, M. Food Control.,
012, 23, 263-267.
2
Madnooni, M.; Mansour, K.; Gholivand, M.
African J. Biotechnol., 2012, 11(2), 498-503.
Biblap, B.; and Jhinuk, C. APJCP., 2013, 14,
27.
28.
Dykes, D.J.; and Waud, W.R.; Cancer
Research., 1997, 25-40.
3735-3742.
Ito,C.; Itoigawa, M.; Takakura, T.; Enjo, F.;
Tokuda, H.;and Furukawa, H.J.of Nat.Prod.,
2003, 66, 200-205.
Can, B.K.H.; Buchbauer, G. Handbook of
Essensial oil. Printed in the United States of
America on acid-free paper., 2010, 56.
Chen, W.; Viljoen, A. South African J of
Botany., 2010, 643-651.
29.
30.
Locatelli, C.; Filippin, F.B.M. Eur. J. Med.
Chem., 2013, 60, 233-239.
Lipinski,C.A.;Lombardo,F.;Dominy,B.W.;Freeney,
F. J. Advanc. Drug Delivery Rev., 1997, 23, 3-25.
1
1
5.
6.
Sharma, P.; Mondhe, D.; Muthiah, S. Chem.

WIDIYARTI et al., Orient. J. Chem., Vol. 35(3), 987-996 (2019)
996
3
3
1.
2.
Siswandono. Public Lecture medicinal
chemistry: rational design of medicine and
molecular modeling, Labtiap. BPPT Jakarta.,
35.
Dipak, P.;Sikha, N.;Amit, B.;and Ahindra, N.;
Int. J. Med. Chem. Sci., 2008, 6(1), 11-16.
De Sousa, D.P.Molecules., 2011, 16, 2233-2252.
Saraswathi, J.; Venkatesh, K.; Nirmala, B.;
Majid, H.H.; Rani, A.R.; J. Med. Plant Res.,
2011, 5(13), 2587-2598.
36.
37.
2015, 1-62.
Ravi, S.; Sureshkumar, C.; Dhivya, S.;
Dhamodaran, S.; and Narasimhan, S. Int. J.
Curr.Res., 2012, 4(9), 40-44.
38.
39.
40.
Wang, Y.; and Tang, K. J. Biotechnol., 2011,
10(72), 16379-16386.
3
3
3.
4.
El-Far, M.;Elshal, M.;Bondock, S.;and Refaat,
M. Int. J. Med. Sci., 2009, 1(17), 278-288.
De Sousa, D.P.; Júnior, G.A.S.; Andrade,
L.N.; and Batista, J.S. Rec. Nat. Prod., 2011,
Priyadarshini, K.; and Keerthi, A.U.; Med.
Chem., 2012, 2, 1-7.
Barbuti, A.M.; and Chen, Z.S. Cancers.,
2015, 7, 2360-2371.
5
, 117-122.