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JOURNAL OF PURE AND APPLIED MICROBIOLOGY, December 2014. Vol. 8(6), 4471-4475
* To whom all correspondence should be addressed.
Tel: 09-5492899, Fax: 09-5492889;
E-mail: jolius@ump.edu.my
Phenolic Content, Antioxidant Capacity and Antimicrobial
Activity of Essential Oil from Habbatus sauda Seed
Nur Farahin Ishak
1
, Noor Intan Shafinas Muhammad
2
, Nuraini Mohd Yusoff
1
,
Sook Fun Pang
1
, Mohd Arman Abd Kadir
1
, Sazwani Suhaimi
1
, Hafizah Ramli
1
,
Mohd Faried Abdullah
1
, Zainal Giman
1
and Jolius Gimbun†
1,3
1
Faculty of Chemical & Natural Resources Engineering,
2
Faculty of Technology,
3
Center for Advanced
Research in Fluid Flow (CARIFF), Universiti Malaysia Pahang, 26300 Gambang, Malaysia.
(Received: 10 August 2014; accepted: 31 October 2014)
This paper presents an extraction and antimicrobial study of essential oil obtained
from Nigella sativa seeds. Extraction using polar solvent resulted in higher recovery of
desired bioactive compounds, thymoquinone and thymol. It was found that extraction of
antioxidant (i.e. Agidol 7) favours aqueous solvent, i.e. 50% ethanol and 50% methanol.
The highest TPC, TFC and antioxidant activities were obtained at power 224W and
temperature of 50 °C. This study shows that N. sativa seed oil has an effective antibacterial
activity from the in-vitro studies. The result showed that the essential oil extracted using
n-hexane has the highest (30.17%) effectiveness against E. coli.
Key words: Nigella sativa, Ultrasonic assisted extraction, Flavonoid, Thymol,
Thymoquinone, Antimicrobial.
Nigella sativa is commonly known as
habbatus sauda belongs to a botanical family of
Rununculaceae. The N. sativa seed is rich with
medicinal value and has been used as a natural
remedy since antiquity, especially by people in the
Mediterranean region. Previous research revealed
that it contained an abundance of active ingredients
useful for anticancer and anti-inflammatory, anti-
dermatophyte, asthma, hypertension, diabetes,
cough, bronchitis, fever, dizziness and
gastrointestinal disturbances.
The first step to recover and purify
essential oil from plant materials involves an
extraction process. The yield of essential oil is
dependent on the solvent used, extraction method
and condition
1
. Conventional extractions such as
soxhlet extraction and maceration (ME) are normally
performed at high temperatures for several hours.
In recent years, a better extraction method has
been developed such as the ultrasonic-assisted
extraction (UAE), microwave-assisted extraction
(MAE) and supercritical extraction. Supercritical
extraction is less favorable owing to its energy
consumption and higher capital cost. The localize
superheating in microwave-assisted extraction
induces a rapid temperature rise thus possesses
challenge in temperature control. Extraction is a
mass transfer process involving solvent transport
to the solid phase (inner transport), dissolution of
the solutes (solubility) and release of solutes from
the solid matrix to the bulk phase (external
transport). The UAE technique reduces the inner
and external mass transfer limitation and hence
increases the yield of extraction. Furthermore,
ultrasonic wave can break the cell membranes
reducing control of inner mass transport. Therefore,
the UAE method was employed in this work.
Solvent type plays an important role in essential
oil extraction. A combined effect of different
extraction methods (ME, and UAE) and varying
solvent polarity to the polyphenol extraction from
N. sativa has never been studied previously, and
hence this is one of the objectives of this work.
J PURE APPL MICROBIO, 8(6), DECEMBER 2014.
4472 ISHAK et al.: STUDY OF ESSENTIAL OIL FROM Habbatus sauda SEED
MATERIALS AND METHODS
Chemicals and Plant Material
The HPLC grade solvents such as the n-
hexane, 2-propanol and methanol were purchased
from Merck (Darmstadt, Germany). The dimethyl
sulfoxide (DMSO), Thymoquinone, 1,1-diphenyl-
2-icrylhydrazyl (DPPH), aluminium chloride (AlCl
3
),
sodium hydroxide (NaOH), sodium nitrate (NaNO
3
),
Folin-Ciocalteu reagent, gallic acid and quecertin
were obtained from Sigma Aldrich (St. Louis, MO).
Solvent for extraction such as 95% ethanol (Copens
Scientific (M) Sdn Bhd), 99.8% methanol and 40-
50% n–hexane (KOFA Chemical (M) Sdn Bhd) was
obtained from a local supplier.
Essential Oil Extraction
The powdered plant material was weighed
(25 wt. %) and mixed with solvent in a 250 ml sealed
Erlenmeyer flask. UAE was carried out in an
ultrasonic bath (JK-DUCH-6210LHC, China) at
either 35 or 53 kHz for time ranged from 15 to 120
minutes and temperature was set at range from 30
to 60 °C. Maceration was performed at 50 °C in a
stirred vessel using a similar plant material to
solvent ratio. The temperature of 50 °C was chosen
based on the result from UAE extraction which
shows excellent extraction of essential oil at 50 °C.
The supernatant was then separated from the
residue by vacuum filtration through 0.45 µm nylon
membrane.
GC-MS Analysis of Essential Oil
Analysis of the volatile oil: The GC-MS
analysis was performed with a quadruple GC-MS
system, capillary column (30 m x 0.25 mm; 0.25 ¼m
film thickness). The carrier gas was helium and
column head pressure of 15 psi yielding a linear
flow rate of 0.8 m/min. The split ratio was 1: 10 and
the initial column temperature was held at 200 °C
for 15 min and then raised at 10 °C/min and
maintained at 260 °C until all components had
eluted. The components were identified by
matching their mass spectra in the NIST 05 library
and their retention indices were compared with
literature values.
Total Phenolic Content (TPC)
Total phenolic contents were assayed
using Folin-Ciocalteu reagent, following
Singleton’s method. An aliquot (0.125 ml) of a
suitable diluted extract was added to 0.5ml of
ultrapure water and 0.125 ml of the Folin-Ciocalteu
reagent. The mixture was vortex for 3 minutes,
before adding 1.25 ml of 7% Na
2
CO
3
solution. The
solution was the adjusted with ultrapure water to a
final volume of 3ml and mixed thoroughly. After
leave in dark for 90 minutes at 25 °C, the absorbance
versus prepared blank was read at 760nm. Total
phenolic contents of seeds (two replicates per
treatment) were expressed as mg gallic acid
equivalent per gram (mg GAE/ 100g) through
calibration curve with gallic acid. The calibration
curve range was 50-400 mg/ml (r
2
= 0.99). All
samples were performed in two replicates.
Total Flavonoid Content (TFC)
Total flavonoid content was measured
according colorimetric assay. A 250µl diluted extract
was mixed with 75µl NaNO
2
(5%). After 6 min
vortex, 150µl of 10% AlCl
3
and 500µl of NaOH (1M)
were added to the mixture. Finally, the mixture was
adjusted to 2.5ml with ultrapure water. The
absorbance versus prepared blank was read at
510nm. Total flavonoid contents of seeds (two
replicate per treatment) were expressed as mg
quercetin equivalents per gram (mg QE/ 100g)
through the calibration curve quercetin. The
calibration curve range was 50-500mg/ml.
DPPH Assay
The electron donation ability of the
obtained extracts and essential oils was measured
by bleaching of the purple coloured solution of
1,1-diphenyl-2-picryhydrazyl radical (DPPH)
according to the method of Hanato et al. [3].
Diluted essential oil prepared in methanol were
added to 0.5ml of a 0.2 mmol/l DPPH methanolic
solution. The mixture was shaken vigorously and
left standing in dark at room temperature for 30
minutes. The absorbance of the resulting solution
was then measured at 517 nm after 30 minutes. The
ability to scavenge the DPPH radical was calculated
using the following equation:
DPPH scavenging effect (%) = [(A
o
-A
1
)/A
o
] × 100
where A
o
is the absorbance of the control
at 30 minutes, and A
1
is the absorbance of the
sample at 30 minutes.
Antimicrobial Test
The essential oil from N. sativa was
individually tested against Escherichia Coli
(ATCC 8739). The species was obtained from
ISO17025 certified lab at Central Laboratory,
Universiti Malaysia Pahang. During this
investigation, the culture was maintained in
J PURE APPL MICROBIO, 8(6), DECEMBER 2014.
447
3ISHAK et al.: STUDY OF ESSENTIAL OIL FROM Habbatus sauda SEED
cryovials at -20°C. Broth subcultures were prepared
by inoculating, with one single colony from a plate,
into a test tube containing 10 ml of sterile nutrient
broth (Biolife, Italy). After inoculation, the tubes
were incubated overnight at 37 °C until it turbid.
The antibacterial and antifungal activity of N.
sativa essential oils was evaluated using the agar
diffusion method. The test was performed using
sterile petri dishes (100 mm diameter) with 100
microliters of prepared culture spread on the
surface of sterile Mueller–Hinton agar medium (25
ml, pH 7). A sterile filter paper (Whatman No. 1, 6
mm diameter) was immediately impregnated with
10 ul of N. sativa essential oil and aseptically placed
on the surface of the agar plate that previously
inoculated with a sterile microbial suspension (one
microorganism per petri dish). Spread plates were
then kept at ambient temperature for 30 min to allow
diffusion of extracts prior to incubation. After 20
min standing, the plate was turned upside down
and incubated at 37 °C for 24 h. All petri dishes
were sealed with sterile laboratory films to avoid
eventual evaporation of the test samples. The
microbial growth on plates was visualized directly
on the plate, captured using a digital camera for
further processing. The inhibition area was
obtained by processing the image as binary using
ImageJ software. The percentage of microbial
inhibition was calculated using the following
equation:
Microbial inhibition (%) = |Ac • As|/(Ac) × 100
where Ac and As are the areas of the
control dish solution without and microbial
solutions with N. sativa essential oil, respectively.
Statistical Analysis
Each experiment was repeated in
triplicates. Analysis of variance (ANOVA) was
performed by using the data analysis tools in
Microsoft Excel 2010, and a least significant
difference (LSD) test was used to compare the
means with a confidence interval of 95%.
RESULTS AND DISCUSSION
Influence of Solvent Type and Extraction Method
on Essential Oil Yield from N. sativa
Gas chromatography mass spectrometry
(GC-MS) analysis of the essential oil showed a
significant amount of thymoquinone (0.6~0.8%),
thymol (0.09~0.15%), agidol 7 (0.33~6.44%), p-
cymene (0.31~0.57%), and (E)-²-Ocimene
(0.11~0.13%). It was found that extraction of
antioxidant (Agidol 7) favours aqueous solvent,
i.e. 50% ethanol and 50% methanol (Fig. 1). The
highest yield of essential oil was obtained using
50% ethanol followed by n-hexane, while the
lowest is 50% methanol. Extraction using ethanol
shows highest simultaneous extraction of TFC (2.47
µg QE/g DW), TPC (0.23 mg GAE/g DW) and
antioxidant activities (60%). Methanol has a higher
yield of TPC (0.38 mg GAE/g DW) and antioxidant
activities (67%) but very low TFC (0.25 µg QE/g
DW). Extraction using n-hexane, 50% methanol and
50% ethanol is not notable, thus, ethanol was
employed for the remainder of this work. It was
found that there is no significant difference at P <
0.05 on the effect of residence time and ultrasonic
frequency after 30 minutes. However, the highest
TPC, TFC and antioxidant activities were obtained
at power 224W and temperature of 50 °C (Fig. 2).
Extraction at the higher temperature (60 °C) is not
an improvement due to thermal degradation of
bioactive compounds. It was found that UAE is
better than ME as it can provide much higher
extraction of TFC, TPC and antioxidant within 30
minutes compared to 4 hours for ME.
Table 1. GC-MS analysis of essential oil from Nigella Sativa seeds
VOC n-Hexane Ethanol Methanol 50% Ethanol 50% Methanol Harzallah et al
2
p-Cymene 0.57 0.43 0.31 ND ND 49.48
(E)-²-Ocimene 0.13 ND 0.11 ND ND ND
Thymoquinone 0.8 0.6 0.63 ND ND 0.79
Thymol 0.09 0.14 0.15 ND ND ND
Agidol 7 (AO 425) 0.33 0.45 1.14 6.44 2.43 ND
Sitosterol 0.05 0.07 0.07 ND ND ND
J PURE APPL MICROBIO, 8(6), DECEMBER 2014.
4474 ISHAK et al.: STUDY OF ESSENTIAL OIL FROM Habbatus sauda SEED
Assessment on Antimicrobial Properties of N.
Sativa Oil
Fig. 3 shows excellent antimicrobial
properties of the essential oil extracted from N.
sativa, which were studied using E. coli ATCC8739.
The inhibition area was obtained by processing
the image as binary using ImageJ software. The
highest inhibition effect of 30.17% was achieved
using oil extracted using hexane, meanwhile
ethanol extracted oil also showed some (7.25%)
Fig. 1. Effect of solvent to TFC, TPC and antioxidant extraction from N. sativa
Fig. 2. Effect of power and temperature to TFC, TPC and antioxidant extraction from N. sativa
Fig. 3. Antimicrobial properties of essential oil from N. sativa
antimicrobial activities. Oil obtained using ethanol
showed lower antimicrobial inhibition due to lower
concentration of thymoquinone as opposed to
those obtained using n-hexane.
CONCLUSIONS
The essential oil yield depends on the
solvent used for the extraction process. The
highest thymoquinone content (0.8%) was
J PURE APPL MICROBIO, 8(6), DECEMBER 2014.
4475
ISHAK et al.: STUDY OF ESSENTIAL OIL FROM Habbatus sauda SEED
obtained using n-hexane extracts while the highest
thymol concentration (0.15%) was obtained using
methanol extracts. It was found that extraction of
antioxidant (Agidol 7) favours aqueous solvent,
i.e. 50% ethanol and 50% methanol. The highest
TPC, TFC and antioxidant activities were obtained
at power 224W and temperature of 50 °C. This study
shows that N. sativa seed oil has effective
antibacterial activities in in-vitro studies. The
result showed that the essential oil obtained from
ethanol and hexane extracts are effective against
E. coli. Essential oil extracted using hexane is more
potent (30.17% inhibition) as opposed to those
obtained using ethanol (7.25% inhibition).
ACKNOWLEDGEMENTS
Funding from the Universiti Malaysia
Pahang (RDU120366) is gratefully acknowledged.
REFERENCES
1. Ncube, N.S., Afolayan, A.J. and Okoh, A.I.
Assessment techniques of antimicrobial
properties of natural compounds of plant origin:
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Biotechnol., 2008 ;7: 1797-1806.
2. Harzallah, H.J., Kouidhi, B., Flamini, G.,
Bakhrouf, A., and Mahjoub, T. Chemical
composition, antimicrobial potential against
cariogenic bacteria and cytotoxic activity of
Tunisian Nigella sativa essential oil and
thymoquinone. Food Chem., 2011; 129 : 1469–
1474.
3. Hanato, T., Kagawa, H., Yasuhara, T., and
Okuda, T. Two New Flavonoids and Other
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