Cytotoxicity Activity of Biotransformed Ethyl p-methoxycinnamate by Aspergillus niger

Abstract

The extraction of Kaempferiagalanga rhizome using steam distillation and supercritical fluid extraction (SFE) carried out. After fractionation, the major compound of the K. galanga, ethyl p-methoxycinnamate (EPMC) was Aspergillus ethyl p-hydroxycinnamate (EPHC). The biological activity of EPMC and its biotransformed product (EPHC) established by activity on human breast cancer (MCF-7) cell line using MTT assay. Ethyl p-hydroxycinnamate (EPHC) was most cytotoxic at 1000 μg/mL percentage cell viability was 9.87% IC50 was 340μg/mL. showed slight cytotoxicity activity compared to EPMC. that the biotransformation process was able to produce metabolite (EPHC) higher cytotoxicity activity compared to its parent compound (EPMC).

Full text

ORIENTAL JOURNAL OF CHEMISTRY
www.orientjchem.org
An International Open Free Access, Peer Reviewed Research Journal
ISSN: 0970-020 X
CODEN: OJCHEG
2016, Vol. 32, No. (5):
Pg. 2731-2734
Cytotoxicity Activity of Biotransformed
Ethyl p-methoxycinnamate by Aspergillus niger
MUHAMMAD NOR OMAR1*, NOR HAZWANI MOHD HASALI1 and MOHD AMBAR YARMO2
1Department of Biotechnology, Kulliyyah of Science,
International Islamic University Malaysia, Bandar Indera Mahkota, 25200 Kuantan Pahang Malaysia.
2School of Chemical Sciences and Food Technology, Faculty of Science and Technology Universiti
Kebangsaan Malaysia, 43600 Bangi Selangor Malaysia.
*Corresponding author E-mail: mnoromar@iium.edu.my
http://dx.doi.org/10.13005/ojc/320547
(Received: June 04, 2016; Accepted: September 19, 2016)
ABSTRACT
The extraction of Kaempferia galanga rhizome using steam distillation and supercritical fluid
extraction (SFE) was carried out. After fractionation, the major compound of the K. galanga extract, ethyl
p-methoxycinnamate (EPMC) was transformed using Aspergillus niger into ethyl p-hydroxycinnamate
(EPHC). The biological anticancer activity of EPMC and its biotransformed product (EPHC) was
established by cytotoxicity activity on the human breast cancer (MCF-7) cell line using MTT assay.
Ethyl p-hydroxycinnamate (EPHC) was most cytotoxic against MCF-7 at 1000 µg/mL where
percentage of cell viability was 9.87 %, while IC50 was 340 µg/mL. EPHC showed slightly higher
cytotoxicity activity compared to EPMC. The results of this study show that the biotransformation
process was able to produce a metabolite (EPHC) with higher cytotoxicity activity compared to its
parent compound (EPMC).
Keywords: Kaempferia galanga, ethyl p-methoxycinnamate, biotransformation, cytotoxic.
INTRODUCTION
Microbial transformation has been
extensively used to create new metabolites from
natural product constituents. This transformation
process can be used as an alternative to chemical
synthesis for the preparation of pharmacologically
active compounds1-4. Biotransformation using
Aspergillus niger has been used to transform
asiaticoside to produce a product with excellent
wound healing properties5. Other studies have
reported that the biotransformed product of ethyl
p-methoxycinnamate exhibited antimicrobial
properties against selected bacteria and fungus6,7.
Malaysian Zingiberaceae plants have been
studied extensively due to their pharmaceutical
properties. These include plant species from
Alpinia8, Zingiber9,10, Galanga10 and Kaempferia6,7.
Cytotoxicity studies show that Kaempferia galanga

2732 OMAR et al., Orient. J. Chem., Vol. 32(5), 2731-2734 (2016)
extracts inhibited the proliferation of human cervical
cancer C33A cell line11. In another study, the
methanolic extract of K. galanga rhizomes reportedly
contained ethyl-p-methoxycinnamate, which is highly
cytotoxic to HeLa cells12. Ethyl p-methoxycinnamate
has been reported to possess many biological
properties such as anticancer13 and anti-monoamine
oxidase activities14. Recently, Jagadish and his co-
researchers reported that successive ethyl acetate
extract of K. galanga showed selective toxicity
against four types of cancer cells15.
Thus, this study aims to evaluate the in
vitro cytotoxicity of ethyl p-methoxycinnamate and
ethyl p-hydroxycinnamate against the human breast
cancer (MCF-7) cell line in order to screen their
potential as anti-cancer agents.
MATERIALS AND METHOD
Chemicals for Cell Culture
Human breast cancer cell line (MCF-7)
was obtained from the Kulliyyah of Pharmacy, IIUM
Kuantan, Malaysia. Phosphate buffer saline (PBS,
GIBCO), Dulbecco’s modified eagle medium (DMEM,
GIBCO) and trypsin solution (GIBCO) were obtained
from Fisher Scientific Shah Alam Malaysia, while
fetal bovine serum (FBS), Thiazolyl blue tetrazolium
bromide (MTT) stock solution and 90% methanol
were obtained from Sigma-Aldrich Subang Jaya
Malaysia.
Plant Materials
K. galanga rhizomes were obtained from
Taman Pertanian Jubli Perak Sultan Haji Ahmad
Shah Kuantan Malaysia. The rhizomes were washed
and sliced before drying in the vacuum oven
(Memmert, Manchester) at 45°C for 5 days until the
samples were completely dry. Then, the samples
were ground using a blender and stored at -4°C prior
to further analyses.
Extraction and fractionation of ethyl
p-methoxycinnamate (EPMC)
The powdered rhizomes of K. galanga were
extracted using steam distillation and supercritical
fluid extraction (SFE) according to previously
reported methods6,7,9. For fractionation of ethyl
p-methoxycinnamate (EPMC), the essential oil was
stirred with boiling water and then recrystallized at
cold temperature ( -4°C). After crystallization, the
mixture was filtered and the crystal was kept in the
desiccator for 24 hours prior to further analysis.
Fungus culture preparation and biotransformation
procedure
The culture preparation and biotransformation
was carried out according to methods previously
reported6,7. The fungus A. niger was streaked on
SDA at 30°C for a week and stored at 4°C. After
cultivation, the well grown mycelia were placed
in a conical flask (250 mL) and inoculated with
10.0 mL of sterilized medium broth containing
glucose, glycerol, peptone, yeast extract, KH2PO4,
NaCl and distilled H2O. The flask was incubated at
30°C for 48 hr at 120 rpm. Ethyl p-methoxycinnamate,
EPMC (480 mg) was dissolved in dimethyl
sulfoxide (DMSO) (24 mL) and distributed among
48 flasks containing 48 h stage culture media and
continuously shaken for 24 h using a rotary shaker
(120 rpm) at 30°C. After incubation, the culture
media and mycelium were separated using cotton
in a funnel. Then, mycelium was washed with ethyl
acetate (1.5 L) while the culture media was extracted
3 times with ethyl acetate (1.5 L). The biotransformed
products were isolated by column chromatography
using silica gel column (200 - 300 mesh, Merck Ltd.)
with hexane : ethyl acetate as solvent7.
MTT assay
The human breast cancer cell line (MCF-7)
was maintained in DMEM containing 2 % FBS
and grown in 6 cm² tissue culture dishes until
confluent11,15. After confluence, th e cells were
washed using PBS to remove the FBS. Then, after
the addition of trypsin (1 mL), the dish was shaken
and incubated at 37°C under 5 % CO2 for 3 min to
detach the cells from the flask surface. After adding
4 mL of DMEM, the dish was shaken and the cells
were divided equally into two new 60-mm culture
dishes assigned to the EPMC and EPHC assays.
The volumes of the new petri dishes were made up
to 5 mL using DMEM and the cell was incubated
for 48 hr at 37°C under 5 % CO2. Finally, the cell
mixture (100 µL) was added into each well of a
96-well plate and incubated for 24 hr at 37 °C under
5 % of CO2.
The cytotoxic assay was carried out
using the two-fold broth microdilution method and

2733OMAR et al., Orient. J. Chem., Vol. 32(5), 2731-2734 (2016)
Fig. 1: Cell viability of the human breast cancer (MCF-7) cell line
at different concentrations of EPMC and EPHC using MTT assay
performed using sterile 96-well flat bottom plates.
7 µg of EPMC (or EPHC) was added to 1393 µL of
DMEM in the first well to a concentration of 0.5 %.
Then, the samples were diluted using two-fold serial
dilution to a final concentration of 1000, 500, 250 and
125 µg/mL. The diluted samples were transferred to
96-well plates containing MCF-7 cells and incubated
at 37°C for 24 hours. Colorimetric MTT assay was
carried out as described by Mosmann16. After 24 h,
30 µL of MTT solution was added to the wells and left
in the incubator at 37°C for 3-4 hr. This was followed
by the addition of 150 µL of DMSO into each well
to stop the reaction. The plate was then read using
a 96-well micro plate reader at a wavelength of 570
nm within 1 h after the addition of DMSO.
RESULTS AND DISCUSSION
MTT Assay and minimum inhibitory
concentration
Figure 1 shows the percentage of cell
viability using MTT assay and the half maximal
inhibitory concentration (IC50) of EPMC and EPHC
against the human breast cancer (MCF-7) cell line.
The IC50 value is the concentration of the sample
where the cell viability was at 50 %. Based on the
results shown in Figure 1, the IC50 of EPMC was
360 µg/mL, and in EPHC was 340 µg/mL. The ability
of microorganisms to modify natural products into
other more bioactive compounds that exhibit higher
activity compared to their parental compound has
attracted a great deal of attention in recent years.
Several reports of biotransformed products screened
for their activity and compared against the parental
compound are present in literature. For example, the
biotransformed product of asiaticoside was reported
to have better wound healing activity compared to
its parental compound5, and several biotransformed
products showed better antimicrobial properties
than their parental compound6. In this study, the
EPHC as the biotransformed product was compared
against its parent compound EPMC for activity.
MTT was carried out to determine the cytotoxicity
of ethyl p-methoxycinnamate (EPMC) and its
biotransformed product ethyl p-hydroxycinnamate
(EPHC) against the MCF-7 human breast cancer
cell line. The assay was carried out to identify the
minimum concentration of compound that could
inhibit cell growth or in this case cause cell viability
to decrease. Ethyl p-hydroxycinnamate (EPHC)
showed higher cytotoxicity against the MCF-7 cell
line since it lowered percentage of cell viability to
9.87 % compared to ethyl p-methoxycinnamate
(EPMC), where cell viability was 22.58 % at the
highest concentration (1000 µg/ml). However, both
compounds showed cytotoxic activity against the
MCF-7 cell line at all concentrations used in the
assay.

2734 OMAR et al., Orient. J. Chem., Vol. 32(5), 2731-2734 (2016)
The half maximal inhibitory concentration
(IC50) which is the measure of the effectiveness of
a compound in inhibiting biological or biochemical
functions was determined. The readings were taken
by measuring the concentration of the sample when
the cell viability was at 50 %. By extrapolating the
results obtained in Figure 1, it can be said that
EPHC is more cytotoxic than EPMC as its IC50
value against MCF-7 was 340 µg/mL, while the IC50
of EPMC was higher at 360 µg/mL. Therefore, from
the results obtained, it can be concluded that Ethyl
p-hydroxycinnamate (EPHC) is the more potent
cytotoxic agent since a lower concentration is
required to inhibit at least 50 % growth of the MCF-7
cancer cell line.
CONCLUSION
Both compounds (EPMC and EPHC) were
active and exhibited good inhibition potential against
MCF-7 cell lines. Ethyl p-hydroxycinnamate (EPHC)
resulted in lower cell viability against the MCF-7 cell
line at high concentrations of 1000 µg/mL compared
to ethyl p-methoxycinnamate (EPMC). The IC50 value
of EPHC was 340 µg/mL against the MCF-7 cell line.
Therefore, the results of cytotoxicity studies and
the IC50 values demonstrate the potent selective
toxicity property of ethyl p-hydroxycinnamate
against the breast cancer cell line. In conclusion, the
biotransformed product, ethyl p-hydroxycinnamate
(EPHC) has good potential as an anti-cancer agent
indicated by higher positive results compared to its
parental compound, ethyl p-methoxycinnamate.
ACKNOWLEDGEMENT
The authors would like to express
their greatest appreciation and gratitude to the
International Islamic University Malaysia (IIUM) for
financial support (RMGS).
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