Determination of Antibacterial Activity of Essential Oil of Myristica
fragrans Houtt. using Tetrazolium Microplate Assay and its Cytotoxic Activity
Against Vero Cell Line
Suthagar Pillai Piaru,
Essential oils and volatile constituents of Myristica fragrans
(nutmeg) are widely used as antioxidants, antidiabetic agents and for the prevention
and treatment of different human diseases such as cancer, cardiovascular diseases
and bacterial and viral infections. The aim of the study is to determine the
antimicrobial activity and in vitro cytotoxicity of essential oil of
nutmeg on normal cell line (Vero cell line). The Minimum Inhibitory Concentration
(MIC) of the antimicrobial activity was determined using tetrazolium microplate
assay. Eight gram negative and 2 g positive species of test microorganism were
selected to determine the MIC exhibited by the essential oil. Cytotoxicity assay
of the essential oil against normal cell line was conducted using Vero cell
line. The percentage of inhibition was calculated and expressed as IC50.
Essential oil exhibited variant antimicrobial activity against the tested strains.
The MIC values of nutmeg oil ranged from 0.031 to 1 mg mL-1 and showed
higher activity against Shigella dysenteriae with MIC value of 0.031
mg mL-1. The nutmeg oil possessed low cytotoxicity against Vero cell
line with IC50 at 24.83 μg mL-1. Nutmeg essential
oil exhibited strong antibacterial activity against the Shigella dysenteriae
with low cytotoxic effects. Thus, the essential oil can be further studied on
its action of mechanism and fractionation will be carried out to determine the
strength of its antibacterial activity.
to cite this article:
Suthagar Pillai Piaru, Roziahanim Mahmud and Shanmugapriya Perumal, 2012. Determination of Antibacterial Activity of Essential Oil of Myristica
fragrans Houtt. using Tetrazolium Microplate Assay and its Cytotoxic Activity
Against Vero Cell Line. International Journal of Pharmacology, 8: 572-576.
Received: December 29, 2011;
Accepted: May 26, 2012;
Published: September 06, 2012
Since long time back, aromatic herbs and spices have been added to various
foods as ingredients to improve the taste, flavor and organoleptic properties.
Essential oils are secondary metabolites which derived from plants, herbs and
spices are made up of volatile compounds comprising volatile constituents like
lipids, terpenoids, ketones, phenols and oxygenated derivatives. They are characterized
by a strong odor and are formed by aromatic plants (Bakkali
et al., 2008).
The antimicrobial activities of plant oils and extracts have formed the foundation
of many applications such as in raw and processed food preservation, pharmaceuticals,
alternative medicine and natural therapies (Lis-Balchin and
Nutmeg (Myristica fragrans Houttuyn) is the seed kernel of inside the
fruit and mace is the lacy covering (aril) on the kernel (Nadkarni,
1988) and cultivated in tropical regions and indigenous to the Maluku Province
of Indonesia. It has been studied to having anthelmintic, hepatoprotective anti-inflammatory,
aphrodisiac, insecticide properties and a treatment for rheumatism, diarrhea,
asthma, atherosclerosis and flatulence (Burkill, 1966;
Ozaki et al., 1989; Pooja
et al., 2012). In addition to that nutmeg mace was found to exhibit
strong antifungal and antibacterial activities (Singh et
al., 2005). Eugenol, a component of nutmeg, is widely used in dentistry
as root canal sealers reported to show antibacterial activity against oral bacteria
(Lai et al., 2001).
To our knowledge, though extensive studies on nutmeg have been conducted but
less research has been studied on the volatile mixtures of the plant species.
Therefore, the aim of this study is to investigate the antibacterial activity
and in vitro cytotoxicity of essential oil of nutmeg on normal cell line.
The cytotoxic study is important to determine the safety of the essential oil
for therapeutic application since few compounds especially myristicin, one of
chemical compounds of nutmeg is reported to be toxic (Pooja
et al., 2012).
MATERIALS AND METHODS
Plant material: The fresh fruits of nutmeg (Myristica fragrans)
were obtained from Balik Pulau, Penang, Malaysia. Voucher specimens (11253)
Myristica fragrans was authenticated by botanist, Mr. Shunmugam and deposited at the Herbarium
Unit of the School of Biological Sciences, Universiti Sains Malaysia.
Extraction of essential oil: The fresh fruits of the plants collected
were submitted to water distillation for 3 h using a modified Clevengers
apparatus. The ground samples (200 g) were boiled with water (200 mL) for 3
h in a 1 L round bottom flask fitted with a condenser. The extracted essential
oils were dried over anhydrous sodium sulphate and after filtration, stored
at 4°C until tested and analyzed.
Microbial strains, culture medium and inocula preparation: Thirteen
species of test microorganism which selected to study were originally clinical
isolates obtained from Department of Medical Microbiology and Parasitology (JTMP),
School of Medical Sciences, University Science Malaysia, Kelantan. Gram-negative
species which involved in this study were Enterobacter aerogenes, Escherichia
coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus
vulgaris, Pseudomonas aeruginosa, Salmonella typhi, Shigella
dysenteriae and Gram-positive species were Staphylococcus aureus and
Bacillus subtilis. Their identities were confirmed by culturing on the
specific media followed by biochemical test using API system as previously reported
(Mbaveng et al., 2008). Bacterial strains stock
cultures were kept on Nutrient Agar (Difco, USA) at 4°C. All the microbial
strains were sub-cultured on a fresh appropriate agar plate 24 h prior to antibacterial
test. Inocula were prepared by transferring several single colonies of microbes
to a sterile broth. The microbial cell suspension was mixed to homogeneity to
give a final density of 5x105 CFU mL-1 and these were
confirmed by viable counts. The infective dose for most microorganisms is 105
Preparation of crude extracts and antibiotics: The essential oil was
dissolved in 50% Dimethyl Sulfoxide (DMSO) in sterile Mueller Hinton Broth (MHB)
for bacterial isolates in order to obtain a stock concentration of 10 mg mL-1.
The stock solution was further diluted using respective broth in five-folds
to obtain working concentration of 2 mg mL-1. The final concentration
of DMSO in the well was ensured to be less than 2%. Preliminary analyses with
2% (v/v) DMSO affected neither the growth of the test organisms nor the change
of tetrazolium color due to this growth. Gentamicin, amoxycillin, vancomycin,
chloramphenicol and penicillin were prepared to a final concentration of 0.1
mg mL-1 and served as the positive drug control against bacterial
Tetrazolium microplate assay: The Minimum Inhibitory Concentration (MIC)
of test microorganisms and reference antibiotics were determined by using tetrazolium
microplate assay according to Eloff (1998) with slight
modification. This assay was performed using 96-well clear microtitre plates.
The wells in column A of each row were left blank and the last seven wells from
column B to H were filled with 100 μL of sterilized MHB (bacterial isolates).
Working solution of plant extracts were added to the wells in column A and B
of each row and an identical two-fold serial dilution were made from column
B to the column G. The last wells in column H was served as drug-free controls.
An appropriate solvent blanks (DMSO) were included as negative control. Then,
100 μL of bacterial inoculum were added in all the wells from column A
to H and mixed thoroughly to give final concentrations ranging from 1-0.015
mg mL-1. Tests were carried out in triplicates. The cultured microplates
were sealed with parafilm and incubated at 37°C for 24 h for bacterial were
incubated at 28°C for 48 h. The MIC of sample was detected following addition
(50 μL) of 0.2 mg mL-1 MTT in all the wells (MTT, Sigma-Aldrich,
USA) and incubated at 37°C for 30 min. Microbial growth were determined
by observing the change of color tetrazolium (MTT) in the microplate wells (purple
formazan when there is growth and clear solution when there is no growth). MIC
was defined as the lowest sample concentration showing no color change (clear)
and exhibited complete inhibition of bacterial growth. MIC value <0.5 mg
mL-1 was defined as potential strong activity.
Cytotoxicity screening Vero cell line: The Vero cell line was obtained
from kidney of a normal adult African green monkey on March 27th, 1962, by Yasummura
and Kawakita at the Chiba University, Japan (APHA, 1992).
Vero cells was maintained in RPMI-1640 medium supplemented with 10% FBS, glutamine
(2 raM), penicillin (100 units mL-1) and streptomycin (100 μg
mL-1). The cells were cultured at 37°C in a humidified 5% CO2
incubator. Vero cells were cultured and maintained in RPMI 1640 medium supplemented
with 10% FBS. The cells were cultured at 37°C in a humidified 5% CO2
Cytotoxicity assay: The essential oil of nutmeg tested for in vitro
cytotoxicity, using Vero cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay (Mosmann, 1983) with slight modifications.
Briefly, after being harvested from culture flasks, the cells were seeded at
1x106 cells in each well of 96-well plate containing 100 μL
of fresh growth medium per well and cells were permitted to adhere for 24 h.
The cells were treated with the nutmeg essential oil which were serially diluted
with growth medium to obtain various concentration (100, 50, 25, 12.5, 6.25
μg mL-1). One hundred microliter of each concentration was added
to each well. After 24 h of treatment the medium was aspirated and the cells
were washed once with sterile Phosphate Buffered Saline (PBS). 5 mg mL-1
of MTT in PBS was added to each well and the plate was incubated at 37°C
in 5% CO2 for 2-4 h, until a purple precipitate was clearly visible
under a microscope. The medium was discarded and 200 μL of Dimethyl Sulfoxide
(DMSO) was added to each well to dissolve the dark blue crystals of formazan
salt and the plates shaken for 5 min. After incubation at 37°C for 10 min,
the absorbance was measured at 540 nm using a Multiskan Ascent microplate reader.
Each plate contained the essential oil, negative control (0.1% DMSO) and blank.
Cytotoxicity is expressed as the percentage of inhibition against various concentrations
and concentration of oil inhibiting cell growth by 50% (IC50). All
tests and analyses were run in triplicate.
Statistical analysis: All values are expressed as Mean±standard
deviation. The MIC data for each microorganism were analyzed using One-way Analysis
of Variance (ANOVA). The p-value <0.05 was considered as significant. The
software SPSS Version 16 was used for the statistical analysis.
RESULTS AND DISCUSSION
Antibacterial assay: The antibacterial activity of the nutmeg essential
oil was studied using the tetrazolium microplate assay and the results were
shown in Table 1. This colorimetric assay represents an alternative
approach to determine MIC economically and yields greater reproducible result.
The application of tetrazolium salt in the assay as colorimetric indicator have
enhanced the sensitivity and accuracy of MIC determination since the formazan
derivatives produced by bacteria or fungi can be quantified (Masoko
et al., 2007).
Essential oil exhibited antibacterial activity against the tested strains but
in variable degree. The synergistic and/or antagonistic effects of essential
oil might be taken into consideration for the variation of antibacterial effects
observed. The variation in the degree of sensitivity of the bacterial strains
towards the extract may be due to the intrinsic tolerance of the bacterial and
the nature and combinations of phytochemical compounds present in the extract
as reported by Nanasombat and Lohasupthawee (2005).
The results obtained from this studies support the antimicrobial activity in
the previous reports by Dorman et al. (2000).
The MIC values ranged from 0.031 to 1 mg mL-1. The essential oil
showed higher activity against Shigella dysenteriae with MIC value of
0.031 mg mL-1 followed by Staphylococcus aureus, Enterobacter
aerogenes, Pseudomonas aeruginosa, Escherichia coli and
Proteus mirabilis with MIC value of 1 mg mL-1. Escherichia
coli and Pseudomonas aeruginosa have been known to be multi-drug
resistant and is very difficult to control by therapeutic means, respectively.
The oil exhibited the lowest MIC values against Klebsiella pneumonia,
Salmonella typhi, Bacillus subtilis and Proteus vulgaris
with value above 1 mg mL-1. The resistance Bacillus subtilis is
the ability of the bacteria to form resting spores and are more resistant to
environmental conditions than any other tested bacteria while Salmonella
typhi is a multi-drug resistant bacterium. Nutmeg essential oil showed lower
MIC value compared to chloramphenicol, a commercial antibiotic against Shigella
dysenteriae. This indicates that the essential oil shows significant and
potent antibacterial activity against Shigella dysenteriae. Control treatment
(DMSO) did not show an inhibitory effect on any of the tested bacteria. Sensitivity
of the microorganisms against amoxycillin, gentamicin, chloramphenicol and vancomycin
was presented in Table 1.
The constituents present in nutmeg essential oil such as p-cymene, α-pinene,
β-pinene, limonene, α-terpinene, α-terpinolene, caryophyllene
oxide and camphene reported by Piaru et al. (2011)
have also been studied for their antimicrobial activity (Sokmen
et al., 2004).
|| Minimum inhibitory concentration (mg mL-1) of
nutmeg essential oil and antibiotics
|All the samples were run in triplicate (n = 3), -: Not tested
||In vitro cytotoxic activity of nutmeg essential oil
against Vero cell line
|Values are Mean±SD, the sample was run in triplicate
(n = 3), *p<0.05, evaluated by one-way analysis of variance (ANOVA)
Based on a report, pinene-type monoterpene hydrocarbons (α-pinene and
β-pinene) had slight activity against number of microorganisms (Dorman
et al., 2000).
Gram-negative Pseudomonas aeruginosa is known to have a high level of
intrinsic resistance against all known antimicrobials and antibiotics, due to
a very restrictive outer membrane barrier which is highly resistant even to
synthetic drugs (Al-Howiriny, 2002). However, nutmeg
essential oil was able to inhibit growth of this bacterium. In most literature,
Gram-positive organisms appear to be more sensitive than Gram negative to essential
oil. However, according to Seyyednejad et al. (2008)
gram-positive bacteria have been found to be less or equally sensitive to gram-negative
bacteria. The studies are sound scientifically since our studies showed the
same pattern of sensitivity of nutmeg oil against gram positive bacteria.
Cytotoxicity assay: Essential oil of nutmeg was evaluated in vitro
for its cytotoxicity activity against Vero cells using MTT assay. The results
of cytotoxicity evaluation of the essential oil are shown in Table
2. The results showed that nutmeg essential oil possessed very low cytotoxicity
against Vero cell line with IC50 at 24.83 μg mL-1.
Even though in the presence of myristicin and elemicin (Pooja
et al., 2012), is often related to intoxication of nutmeg while safrole
has been studied to show carcinogenic effects (Zhou et
al., 2007), the lower cytotoxicity effect of nutmeg oil against the
Vero cell line (normal cell line) maybe due to the synergistic effect of presence
of other compounds in the extract. The interaction of the compounds would have
inhibited the cytotoxic effects of myristicin, elemicin and safrole against
the normal cell line. This suggests the antibacterial activity is not due to
the cytotoxic effects of the nutmeg essential oil.
Summarizing these results, it can be concluded that nutmeg essential oil exhibited
broad spectrum of antibacterial activity against the tested microorganisms with
low cytotoxic effects against normal cell line. Thus, the essential oil can
be further studied on its action of mechanism and fractionation will be done
to determine the strength of its antibacterial activity.
This project was funded by Universiti Sains Malaysia Research University (RU)
Grant (1001/PFARMASI/ 813021). The authors report no conflict of interest.
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