Antimicrobial activity and microbial transformation of ethyl p-methoxycinnamate extracted from Kaempferia Galanga

Article abstract of DOI:10.13005/ojc/300315

Ethyl p-methoxycinnamate (EPMC), a major constituent of the Kaempferia galanga rhizome, was transformed to ethyl p-hydroxycinnamate (EPHC) using Aspergillus niger. The EPHC metabolite was elucidated by NMR spectroscopic technique. Antimicrobial microbial

Full text of DOI:10.13005/ojc/300315

ISSN: 0970-020 X
CODEN: OJCHEG
2014, Vol. 30, No. (3):
Pg. 1037-1043
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Antimicrobial Activity and Microbial Transformation of Ethyl
p-Methoxycinnamate Extracted from Kaempferia galanga
MUHAMMAD NOR OMAR1*, NOR HAZWANI MOHD HASALI1,
HELMIYOUSIF ALFARRA1, MOHD AMBARYARMO2 and AHMAD MUZAMMIL ZUBERDI1
1Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia (IIUM),
Jalan Sultan Hj. Ahmad Shah, 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/300315
(Received: July 28, 2014; Accepted: September 02, 2014)
ABSTRACT
Ethyl p-methoxycinnamate (EPMC), a major constituent of the Kaempferia galanga rhizome,
was transformed to ethyl p-hydroxycinnamate (EPHC) using Aspergillus niger.The EPHC metabolite
was elucidated by NMR spectroscopic technique. Antimicrobial microbial study found that EPHC
was active against all strains tested with a good minimum inhibitory concentration (MIC). It was
active against both Staphylococcus aureus and Bacillus cereus at MIC 333 µg/ml while against
Pseudomonas aeruginosa, Escherichia coli and Candida albicans at MIC 111 µg/ml. It was also
shown that EPHC exhibited more growth inhibition potential than EPMC. Besides that, EPHC has
shown the minimum bactericidal concentration (MBC) toward B. cereus, P. aeruginosa and E. coli
at the concentration of 1000 µg/mL while EPMC did not show killing potential toward the tested
microorganisms.
Key words: Biotransformation, Kaempferia galanga Linn, Ethyl p-methoxycinnamate,
Ethyl p- hydroxycinnamate, Aspergillus niger, Antimicrobial.
INTRODUCTION
Malaysian natural product plants have been
studied extensively. These include medicinal plant
species from Andrographis2,3,4,5, Musa6, Plumeria7,
Citrus8.9, Cymbopogon10, Artocarpus11, Alpinia12 and
Centella13.However, plant species of Zingiberaceae
continue to attract much phytochemical interests
due to their culinary uses, besides their biological
and pharmaceutical activities. Kaempferia galanga
Linn., belongs to the Zingiberaceae family, is one
The need for a new antibiotic against
pathogenic microorganism has increased due
to the current problems of resistance associated
with them1. Plant based antimicrobial agents
have enormous therapeutically potential as they
can serve the purpose without any side effect,
that often associated with synthetic agents.

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OMAR et al., Orient. J. Chem., Vol. 30(3), 1037-1043 (2014)
of the plants that mainly distributed in tropics and hydroxycinnamate (EPHC) produced by A. niger.
subtropics of Asia.Besides Malaysia, it can be found
in Southern China, Indochina and India14.It has been
known from ancient times that the essential oil of K.
galanga plays an important role in native medicine Materials
as it possesses interesting biological activities such
EXPERIMENTAL
The rhizomes of K. galanga L. were
as antimicrobial15, antihypertensive16, anticancer17 collected at different locations throughout Malaysia
and antiproliferative activity18.
in December 2013 and stored at -20 °C prior to
analysis.Five reference strains of human pathogens
The emergence of biotransformation used for antimicrobial assay were Staphylococcus
technique to produce new natural drug as an aureus (ATCC25923), Bacillus cereus (ATCC11778),
antimicrobial agent was successfully reported. Pseudomonas aeruginosa (ATCC27853) and
According to Muller19, fungi are effective in Escherichia coli (ATCC8739) while one fungal
increasing the chemical diversity by producing strains was Candida albicans (IMRC533/11 A). The
new derivatives with lower toxicity but improved bacteria were maintained on Muller-Hinton Agar
biological activity. While Kim et al20, described that (MHA) and Muller- Hinton Broth (MHB) and stored in
the biotransformation afforded another metabolite the refrigerator at 4°C.Meanwhile, C. albicans was
that has higher antimicrobial activity compared maintained on Sabouraud’s Dextrose Agar (SDA)
to the parent metabolite. Thus, this approach and stored in the refrigerator at 4°C. Both bacteria
successfully enhances the antimicrobial activity of and fungus C. albicans were subcultured onto fresh
the compound.For example, rhapontigenin produced media at regular interval.
from rhapontin by biotransformation showed 4-16
times higher antimicrobial activity than rhapontin. Extraction of K. galanga oil and purification of
The activity was higher against Gram-positive ethyl p-methoxycinnamate
strains than Gram-negative strains20. Another study
The fresh rhizomes of K. galanga were
by Srivastava et al21, indicates that A. flavus can washed and cut into smaller pieces. The rhizomes
transform artemisinin to deoxyartemisinin (Figure 1) were then extracted using steam distillation and
and shows antibacterial activity against S.aureus, S. supercritical fluid extraction (SFE) according to the
epidermidis and S. mutans at a minimum inhibitory previously reported method23. The essential oil was
concentration (MIC) of 1 mg/mL compared to collected and stored at -4°C for future analyses. For
artemisinin whose MIC was >2 mg/mL. Siddhardha purification of ethyl p-methoxycinnamate (EPMC, 1)
et al.22 showed biotransformation of (-)-α-santonin the essential oils were stirred with boiling water and
using A. parasiticus resulted in the production then recrystalized at cold temperature
(-4°C).
of 3,4-epoxy-α-santonin, 4,5-dihydro-α-santonin After crystallization, the mixture was filtered through
and 1,2-dihydro-α-santonin and evaluated for the Whatman No 1 and then the crystal was kept in the
biological activity of the transformed products. desiccator for 24 hours prior to further analysis.
3,4-epoxy-α-santonin exhibited higher degree of
antibacterial and antifungal activity. 4,5-dihydro-α- Fungusculturepreparationandbiotransformation
santonin showed slight increase in the activity, procedure.
compared to (-)-α-santonin. 1,2-dihydro-α- santonin
Aspergillus niger fungal culture preparation
is equipotent to the substrate (-)-α-santonin. Pure fungal culture of A. niger was obtained from
This is the first report on the biotransformation of Microbiology Laboratory Kulliyyah of Science,
(-)-α-santonin by A.parasiticus and evaluation of the International Islamic University Malaysia. The pure
biological activities of the biotransformed products. fungus was streaked on Sabouraud Dextrose Agar
slant (SDA) at 30 °C for a week and stored at 4
Therefore, this present study was initiated °C. After cultivation, the well grown mycelia on the
to screen the antimicrobial activity of ethyl p- agar slants were placed in the conical flask (250 ml)
methoxycinnamate (EPMC), extracted from K. and inoculated with 10.0 ml of sterilized medium
galanga Linn., and compare the bioactivity broth containing glucose, glycerol, peptone, yeast
with its biotransformation metabolite, ethyl p- extract, KH2PO4, NaCl and distilled H2O. The flask

OMAR et al., Orient. J. Chem., Vol. 30(3), 1037-1043 (2014)
1039
was incubated using an incubator shaker for 48 hr
at 120 rpm and 30°C. Then, mycelia suspensions
were transferred into 17 flasks containing sterile
broth medium (100 ml of each) and incubated for 24
hour using the similar condition as reported earlier24.
detector temperatures were 250 °C and 230 °C,
respectively.The other analytical conditions were as
follows: Temperature programming: 60 °C, as initial
temperature, for 5 min, 8 °C/min to 180 °C at 10 °C/
min to 240 °C, holding for 5 min. Mass spectroscopy
analysis was carried out using positive electron
ionization method in order to obtain fragments of the
compounds. The identification of compounds were
based on a comparison of their retention times with
those of authentic standards and by comparison of
their mass spectra with those of data in the Wiley
Registry of Mass Spectral Data and National Institute
of Standards andTechnology (NIST) libraries18.Low
resolution mass spectrometry (m/z) was carried
out on a VG Biotech Quattro II triple quadrupole
instrument.
Biotransformation of the bioactive compounds
Ethyl p-methoxycinnamate, 1 (480 mg,
) was dissolved in dimethyl sulfoxide (DMSO) (24
ml) and distributed among 48 flasks containing 24
hour stage culture (10 mg/0.5 ml in each flask) and
continuously shaken for 24 h using a rotary shaker
(120 rpm) at 30 °C. At the same time, a control flask
having substrate without fungus and a control flask
containing fungus without substrate were analysed in
order to check the substrate ability and to determine
the endogenous metabolite, respectively.
Nuclear Magnetic Resonance (NMR) analysis
The nuclear magnetic resonance (NMR)
Separation and purification of biotransformed
product
spectroscopy was used to elucidate the structure
of compound (1 & 2) using 1D (1H- and 13C-NMR)
and 2D (COSY 45°, HSQC, HMBC and NOESY)
experiments according to the standard protocols.
The NMR analysis was carried out at the Department
of Chemistry, Universiti Kebangsaan Malaysia. 1H
and 13C NMR spectra were recorded on a Bruker
400 MHz FT-NMR spectrometer. The crystal was
dissolved in CD2Cl2 and chemical shifts (α) are
reported in ppm downfield of tetramethylsilane.
Coupling constants (J) are quoted in Hz.
After incubation, the culture media and
mycelium were separated using cotton in a funnel.
Then, mycelium was washed with ethyl acetate (1.5
liter) while the culture media were extracted 3 times
with ethyl acetate (2 liter). The combined organic
extract was dried with anhydrous sodium sulphate
(Na2SO4) and evaporated using a rotary evaporator.
The similar procedures were employed for the control
group. The residues from both experimental and
control group were analysed by TLC to confirm
the presence of biotransformed product. The
biotransformed products were isolated by column
chromatography using silica gel column (200-300
mesh, Merck Ltd.) with hexane: ethyl acetate as
solvent.
Antimicrobial Assays
Minimum Inhibitory Concentration (MIC)
The test was performed using 96-well
plates and prepared in a three-fold dilutions manner,
where the concentration in each well is three times
less than the preceding well. The first well for each
sample (single compounds) was prepared in a 1000
µg/mL concentration by mixing 30 µL of the single
compounds (10 mg/mL concentration) with 270 µL
broth media.After mixing the samples with the broth,
the mixture (100 µL) was transferred to the next
well, in the same column, containing 200 µL broth
media. The similar procedure was repeated for the
subsequent well until the end of each column. The
final concentrations formed in each well after this
process were 1000 (first well), 333, 111, 37, 12.3,
4.1, 1.37, and 0.45 µg/mL25.
Structural elucidation of the biotransformed
product
Gas Chromatography/Mass Spectrometry (GC/
MS)
The crystal was further analyzed using an
Agilent 5975C GC-MSD system (Agilent, Avondale,
USA) equipped with a 30 m x 0.25 mm i.d. x 0.25
ìm , HP-5 capillary column (SGE, Australia) and
70 eV of the electron impact technique. The carrier
gas was helium at a flow rate of 1.3 mL/min. The
crystal was dissolved in dichloromethane (DCM)
at concentration of 1%. Then, 1 ìL of sample
was injected into the column. The injector and

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OMAR et al., Orient. J. Chem., Vol. 30(3), 1037-1043 (2014)
Minimum Bactericidal/Fungicidal Concentration and 13C-NMR data. The NMR spectral data of
(MBC)
compound 2 are summarized in the Table 1. These
The minimum bactericidal/fingicidal finding presented a clear correlation to support the
concentration (MBC/MFC) test was carried out proposed structure and identified by comparing their
according to Betts et al26, with slight modifications spectra with literature27.
in order to determine whether the MIC values only
inhibit the growth or even kill the microorganism.
The antimicrobial assay in terms of the
100 µL of each broth in the well that represents MIC minimum inhibitory concentrations (MIC) of of
values for all bacteria and fungus was spread in MHA ethyl p- methoxycinnamate (EPMC) and ethyl
and SDA, respectively. Plates were kept in incubator p-hydroxycinnmate (EPHC) against different
for 24 hr.The MBC/MFC could be determined when pathogenic bacteria and fungus are shown in Table
the microorganisms were killed and unable to regrow 2.
back.
MIC tests were done selectively against four
RESULTS AND DISCUSSION
types of bacteria which were B. cereus, S. aureus,
E. coli, P. aeruginosa and one fungus which was C.
Microbial transformation of ethyl albicans. The result showed that both compounds
p-methoxycinnamate (1) by A. niger afforded a main have good antimicrobial activities against all
compound, identified as ethyl p-hydroxycinnamate tested bacteria and fungus. The MIC values also
(2) (Figure 2) in good yield (24%). The molecular indicated that both compounds (EPMC & EPHC)
formula of 2 proved to be C11H12O3 from 1H-NMR were able to inhibit Gram-negative bacteria at lower
Table 1: 1H- NMR, 13C-NMR, COSY and HMBC Spectral Data of EPHC, 2
Position
13C-NMR
1H- NMR
COSY
HMBC
1
127.07, C
129.94, CH
115.80, CH
157.98, C
144.18, CH
115.55, CH
167.42, C
60.46, CH2
14.12, CH3
-
-
2, 6
3, 5
4
7
8
9
10
11
7.4 (2H, d, J5 = J9 = 8.4 Hz)
6.8 (2H, d, J6 = J8 = 9.0 Hz)
-
7.6 (1H, d, J = 15.6 Hz)
6.3 (1H, d, J = 16.2 Hz)
-
H3,5
H2,6
-
H8
H7
-
C 1,4,7
C 3,4, 5
C 1, 8, 9
C 1,7,9
4.2 (2H, q, J = 7.2 Hz)
1.3 (3H, t, J = 7.2 Hz)
H11
H10
C 11, 9
C 10
Table 2. Minimum Inhibitory Concentration (MIC) of ethyl
p-methoxycinnamate (EPMC) and ethyl p-hydroxycinnamate (EPHC)
Microorganisms
Minimum Inhibitory Concentration
(MIC) value (µg/mL)
EPMC,1
EPHC, 2
Staphylococcus aureus
Bacillus cereus
Escherichia coli
Pseudomonas aeruginosa
Candida albicans
1000
1000
333
333
333
333
333
111
111
111

OMAR et al., Orient. J. Chem., Vol. 30(3), 1037-1043 (2014)
1041
Table 3. Minimum Bactericidal/Fungicidal
Concentration (MBC/MFC) of ethyl
p-methoxycinnamate (EPMC) and ethyl
p-hydroxycinnamate (EPHC)
concentrations compared to Gram-positive bacteria.
The MIC value of ethyl p-hydroxycinnamate (EPHC)
against both E. coli and P. aeruginosa was 111
µg/mL, which were more active than the value
exhibited by ethyl p- methoxycinnamate (EPMC,
MIC = 333 µg/mL) (Table 2). Meanwhile, the MIC
values of EPHC against both B. cereus and S.
aureus was 333 µg/mL, which were also lower than
the value exhibited by EPMC (MIC=1000 µg/mL).
Furthermore, EPHC was able to inhibit the growth
of C. albicans with the concentration that exhibited
inhibition potential (MIC) was 111 µg/mL which were
lower than the value exhibited by EPMC (MIC=
333 µg/mL).
Microorganisms
MBF/MFC value
(µg/mL)
EPMC, 1
EPHC, 2
Staphylococcus aureus
Bacillus cereus
Escherichia coli
Pseudomonas aeruginosa
Candida albicans
-
-
-
-
-
1000
1000
1000
1000
333
Fig. 1: Biotransformation of artemisinin to deoxyartemisinin by Aspergillus flavus
Fig. 2: Biotransformation pathway of ethyl p-methoxycinnamate
into ethyl p-hydroxycinnamate acid by Aspergillus niger
In terms of minimum bactericidal
concentration (MBC), there were bactericidal effect
of EPHC against S. aureus, B. cereus, E. coli
and P. aeruginosa at 1000 µg/mL and fungicidal
effect (MFC) against Candida albicans at 333 µg/
mL (Table 3). However, there were no MBC was
obtained for ethyl p-methoxycinnamate (EPMC)
against all tested microorganisms. Hence, ethyl
p-hydroxycinnamate (EPHC) has great potential
to inhibit and killing the growth of Gram-positive

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OMAR et al., Orient. J. Chem., Vol. 30(3), 1037-1043 (2014)
bacteria, Gram-negative bacteria and fungus
compared to ethyl p- methoxycinnamate (EPMC).
The present of hydroxyl group in structure may
increase the antimicrobial activities of compound28.
The result has proved that the folkloric use of this
plant in treating microbial infections and suggested
that ethyl p-hydroxycinnamate (EPHC) could be
exploited in future as one of the new antibiotics in
line with the current drug development.
p-hydroxycinnamate (EPHC), which shown
antimicrobial activities.
Looking at the antimicrobial activities,
ethyl p-hydroxycinnamate (EPHC) has an inhibitory
potential effect against S. aureus, B. cereus, P.
aeruginosa, E. coli and C. albican better than the
effect by ethyl p-methoxycinnamate (EPMC). EPHC
has also shown bactericidal and fungicidal effects
against all tested bacteria and fungus while EPMC
did not show killing potential toward them.
CONCLUSION
Biotransformation has been successfully
utilized as a tool to generate pharmaceutical
compounds from natural products. Through this
process, ethyl p-methoxycinnamate (EPMC),
extracted from Kaempferia galanga plant, was
transformed using Aspergilus niger to ethyl
ACKNOWLEDGEMENTS
The authors would like to express greatest
appreciation and gratitude to the International
Islamic University of Malaysia (IIUM) and The
Ministry of Higher Education (MOHE) for financial
support (RMGS). Also thank to all Kulliyyah of
Science (KOS) and IIUM staff for technical supports.
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