International Food Research Journal 26(4): 1165-1172 (August 2019)

Journal homepage: http://www.ifrj.upm.edu.my

Characterising and optimising antioxidant and antimicrobial properties of clove

extracts against food-borne pathogenic bacteria

1*
Ishaq, A., 1Syed, Q. A., 1Khan, M. I. and 2Zia, M. A.

National Institute of Food Science and Technology, Faculty of Food Nutrition and Home Sciences,
1

University of Agriculture Faisalabad, Pakistan

2
Department of Biochemistry, University of Agriculture Faisalabad, Pakistan

Article history Abstract

Received: 18 September, 2018 Phenolic compounds present in clove extract have been reported to possess strong inhibitory
Received in revised form: effect against a vast range of microorganisms. The present work was aimed to optimise the
12 January, 2019 antioxidant and antimicrobial potentials of clove extract against major food-borne pathogenic
Accepted: 28 March, 2019
bacteria (Salmonella Typhimurium, Escherichia coli, Listeria monocytogenes). Results revealed
that clove extract obtained from n-hexane extraction had higher extraction yield (48.84%),
antioxidant potential (TPC = 54.05 mg/g of GAE, TFC = 15.54 mg/g, DPPH = 0.29 mg/mL,
FRAP = 0.69 mg/mL) and antimicrobial activity as compared to the extracts obtained from
Keywords ethanol, petroleum ether and steam distillation. Minimum inhibitory concentration of clove
extract (0.25%) was more effective against L. monocytogenes (ZI = 11.17 mm) in comparison
Clove extract
with S. Typhimurium (ZI = 8.8 mm) and E. coli (ZI = 9.27 mm). Additionally, clove extract was
Antioxidant potential
Antimicrobial activity also effective in disrupting cell membrane integrity and affecting membrane permeability of
Membrane permeability the assessed bacteria. It was also found that L. monocytogenes was more susceptible to clove
Cell membrane integrity phytochemicals as compared to the other bacteria. Hence, clove extract has strong potential to
be used as a natural antioxidant and antimicrobial agent in food industry.
© All Rights Reserved

Introduction plant genetics, climatic conditions and extraction

methods (Khalil et al., 2017).
Products obtained from plants have been Several methods have been used to extract
extensively used for their therapeutic applications clove oil. The most popular methods include
since ancient times, and the present demand for these steam distillation, solvent extraction (Guan et al.,
phytogenic products has increased. Recently, studies 2007), hydro-distillation (Jeyaratnam et al., 2016),
have been focused to extract the useful compounds microwave-assisted extraction (González-Rivera et
from plants for applications in pharmaceutical, al., 2016), supercritical carbon dioxide extraction
flavouring and food industries. Several plants (Chatterjee and Bhattacharjee, 2013) and ultrasound-
have been investigated for their antioxidant and assisted extraction (Alexandru et al., 2013). Each
antimicrobial effects due to the presence of various method has its own advantages and disadvantages,
functional ingredients in their essential oils. Similarly, and quantity of extracted functional ingredients
essential oil obtained from clove buds also possesses depends upon the type of method used.
several functional properties i.e., antibacterial, Essential oil obtained from clove represents
antifungal, antioxidant, antitumor, anti-inflammatory, considerable antioxidant properties. Antioxidant
insecticidal and flavour-imparting characteristics. potential of clove essential oil can be explained as
Clove oil has several active ingredients that describe formation of complexes with reduced metals which
its functionality. It primarily contains eugenol (48 inhibits peroxidation by eliminating free radicals and
- 89%), β-caryophyllene (5 - 22%) and eugenyl formation of iron-oxygen chelate complex (Ito et al.,
acetate (0.4 - 22%). Additionally, it also possesses 2005). In a trial, Gülçin (2001) revealed antioxidant
small quantity of α-humulene. Percentages of these properties of clove extracts and reported 96.7%
functional components depend upon several factors inhibition of linoleic acid emulsion peroxidation at
such as variety of clove, part of plant, type of soil, 15 µg/mL comparable to butylated hydroxytoulene

*Corresponding author.
Email: anum1797@yahoo.com
1166 Ishaq, A., Syed, Q. A., Khan, M. I. and Zia, M. A./IFRJ 26(4) : 1165-1172

(BHT). In another study, Gülçin et al. (2004) (Germany) and Sigma-Aldrich (USA). The other
demonstrated 93.3% and 97.9% inhibition of linoleic materials were purchased from reputed scientific
acid oxidation at 20 and 60 µg/mL, respectively. store of Faisalabad, Pakistan. Bacterial strains
Additionally, studies have revealed that clove (S. Typhimurium, E. coli and L. monocytogenes)
extracts possess significant antibacterial potential were provided by Institute of Food Microbiology,
against several bacteria such as Enterococcus faecalis, University of Agriculture Faisalabad.
Staphylococcus aureus, Listeria monocytogenes,
Bacillus cereus, Escherichia coli, Salmonella Experimental design
typhi and Pseudomonas aeruginosa. The active The experiments for characterisation, extraction,
compounds in clove extract, particularly eugenol, antioxidant assay and antimicrobial analyses were
damage bacterial membranes and cause leakage of performed under Controlled Randomized Design
intracellular materials (Oyedemi et al., 2009). Clove (CRD) while experiments related to bacterial cell
extract also has inhibitory effect on the biofilms, integrity and membrane permeability were conducted
and it is evident from studies that clove extract can under CRD using factorial arrangement.
reduce the formation of biofilms up to 99% (Raja
et al., 2015). In another investigation, El-Maati Sample preparation and characterisation of clove
et al. (2016) studied phenolic extracts of clove for buds
their antioxidant and antimicrobial properties, and For the characterisation of clove, clove buds were
concluded that clove extract can be used in food and firstly ground and then clove powder was subjected
pharmaceutical products as natural antimicrobial or to proximate analysis.
antioxidant agent.
Above mentioned discussion strongly supports Moisture contents
the antioxidant and antimicrobial potential of clove The moisture contents of clove powder were
extracts. Studies have revealed the effect of clove oil/ measured by drying the samples in conventional
extract on bacterial reduction, but no studies have forced air laboratory oven (Model: DO-1-30/02,
been conducted on exploring the effect of applying PCSIR, Pakistan) at 105 ± 2°C until constant weight
clove extract on bacterial cell integrity and membrane was achieved (AACC, 2000).
permeability. So, the present work aimed to elucidate
the effect of clove extracts on cell membrane Crude protein
permeability and integrity against different food- The percentage of crude proteins was estimated
borne pathogenic bacteria; Salmonella Typhimurium, through Kjeltech Apparatus (AACC, 2000). Briefly,
Escherichia coli and Listeria monocytogenes. 1.5 g sample was taken and mixed with 5 g digestion
Additionally, the present work also evaluated the mixture (K2SO4, CuSO4 and FeSO4) and 30 mL H2SO4
antioxidant potential of clove extract obtained from in digestion flask. The mixture was then subjected to
different extraction techniques. mild heating (40°C) for 10 min and then temperature
was raised to 60°C until the completion of digestion
Materials and methods process i.e., turning of mixture to greenish colour.
After cooling, the volume was made up to 250 mL
The present work was conducted in Meat by adding distilled water. Then, the sample was
Science and Technology Laboratory (MSTL) and subjected to neutralisation process in distillation
Food Microbiology and Biotechnology Laboratory assembly by mixing 10 mL sample with 10 mL NaOH
(FMBL), National Institute of Food Science and solution (40%) in distillation tube against boric acid
Technology (NIFSAT), University of Agriculture solution (4%) with methyl red (indicator). Following
Faisalabad, Pakistan. In the present work, locally distillation, titration was performed against 0.1 N
cultivated variety of cloves was exposed to H2SO4 and the volume of acid was noted.
characterisation, antioxidant potential estimation and
antimicrobial assay. Crude fat
Crude fat was determined by using Soxhlet
Materials and chemicals Extraction System (AACC, 2000). Briefly, 2 g
Clove buds were purchased from the local market sample (oven dried) was taken in thimble and crude
in Faisalabad, Pakistan. All the other reagents for fat was extracted by using n-hexane after completing
extraction (ethanol, n-hexane, petroleum ether), five siphon cycles. Next, n-hexane was evaporated
compositional analysis, antioxidant assay and using rotary evaporator and crude fat contents were
microbial analysis were purchased from Merck estimated based on differences in the weights of
sample.
 Ishaq, A., Syed, Q. A., Khan, M. I. and Zia, M. A./IFRJ 26(4) : 1165-1172 1167

Crude fibre Total flavonoid contents

Crude fibre contents of powdered clove samples The total flavonoid contents were determined by
were determined by digestion of sample (fat free) aluminium chloride colorimetric method (Chang et
with 1.25% H2SO4 (30 min) and then with 1.25% al., 2002). Briefly, 1 mL methanol was added to 50
NaOH solution using Labconco Fibertech apparatus µL extract followed by the addition of distilled water
(AACC, 2000). (4 mL). The solution was then allowed to incubate
for 5 min and 0.3 mL NaNO2 (5%) and 0.3 mL AlCl3
Ash (10%) were then added. After that, 6 min incubation
Determination of ash in clove powder was period was given and 2 mL NaOH solution (1 M) was
performed by charring the samples followed by added. Finally, volume of the solution was made up
placement in the muffle furnace at 550°C until the to 10 mL using distilled water and absorbance was
colour changed to greyish white (AACC, 2000). taken at 510 nm in UV/vis Spectro-photometer.

Nitrogen free extract Reducing ability

The nitrogen free extract was calculated through The ferric reducing antioxidant power (FRAP)
subtraction method using Equation 1: was estimated by following the method of Benzie
and Strain (1999). Briefly, sample (200 μL) was
mixed with equal quantities (500 μL each) of sodium
NFE% = 100 – (Moisture + Crude protein +
phosphate buffer (0.2 M, pH 6.6) and potassium
Crude fat + Crude fibre + Ash)
ferricyanide (1%). The solution was incubated for 20
(Eq. 1) min at 50°C and centrifuged at 2,200 g for 10 min
after adding 2.5 mL 10% trichloroacetic acid (TCA).
Extraction of clove oil Afterwards, 500 μL supernatant was taken and mixed
Extraction of clove oil was done through with distilled water (500 μL) and 0.1% solution of
Soxhlet extraction technique and steam distillation ferric chloride (100 μL). Finally, the absorbance was
as described by Quan et al. (2004) and Guan et al. measured at 700 nm using UV/vis spectrophotometer.
(2007), respectively. For solvent extraction, 30 g
sample was transferred to filter paper extraction DPPH activity
thimble and placed into the reflux flask (500 mL) The antioxidant capacity of resultant clove
followed by extraction with 250 mL organic solvents powder extracts was determined as free radical
(ethanol, n-hexane, petroleum ether) in Soxhlet scavenging potential through DPPH (2,2 diphenyl-
apparatus. After the completion of extraction process 1-picrylhydrazyl) assay (Jung et al., 2010). Briefly,
(3 - 4 h), the obtained extracts were concentrated on 4 mL extract was taken in cuvette and 1 mL DPPH
rotary evaporator at 50°C. For steam distillation, 100 solution was added to the sample. The solution was
g ground sample was taken in 500 mL distillation then allowed to incubate at 25°C for 30 min and
flask and subjected to steam distillation for 6 - 8 h absorbance was taken at 520 nm through UV-Vis
followed by dehydration and refrigeration (4 ± 0.5°C) Spectro-photometer. Equation 2 was used to calculate
until further analysis. percent inhibition:

Antioxidant activity of clove extracts Reduction in absorbance (%) = [{AB(s) –

AB(e)} / AB(s)] × 100 (Eq. 2)
Total phenolic contents (TPC)
The total phenolic contents of clove extracts were
determined by following the method described by where AB(s) = absorbance of blank sample (t = 0
Jang et al. (2008). Briefly, 125 µL clove extract was min), AB(e) = absorbance of tested extract solution
mixed with 500 µL distilled water and 125 µL Folin- (t = 30 min)
Ciocalteu reagent. The resultant mixture was kept
at room temperature for 5 min and then combined Antimicrobial activity of clove extracts
with 1.25 mL Na2CO3 solution (7%). Then, distilled Zones of inhibition (ZI) for clove extracts against
water was added to the solution to make the total S. Typhimurium, E. coli and L. monocytogenes were
volume 3 mL. Afterwards, the mixture was kept measured by disc diffusion method (Rahman et al.,
for 90 min before taking the absorbance at 765 nm 2017) using clove extracts obtained from different
by UV-Visible Spectro-photometer. Total phenolic extraction methods. Micro-wells were filled with 90
contents were determined and expressed as mg Gallic μL peptone water. After that, 10 μL of each extract was
acid equivalent (GAE/g).
1168 Ishaq, A., Syed, Q. A., Khan, M. I. and Zia, M. A./IFRJ 26(4) : 1165-1172

added in the wells followed by dipping of sterilised on the release of cytoplasmic β-galactosidase activity
discs having diameter of 6 mm for 1 h. Commercial from bacterial cells by using ONPG (O-nitrophenyl-
antibiotics (10 μL) against S. Typhimurium (nalidixic β-D-galactoside) substrate. Briefly, bacterial cells
acid), E. coli (nalidixic acid) and L. monocytogenes were harvested, washed and resuspended in sterilised
(chloramphenicol) were also added in some wells and NaCl solution (0.9%). The optical densities (OD)
served as positive controls. Bacterial cultures were were adjusted to 1.2 by taking absorbances at 420
inoculated on their respective growth media (XLT4 nm using UV-Vis Spectro-photometer. Next, 1.6 mL
for S. Typhimurium, MacConkey Sorbitol medium bacterial suspension was mixed with 1.6 mL clove
for E. coli, MOX for L. monocytogenes) and discs extract and 150 μL ONPG (30 mM). Finally, the
were placed on the plates followed by incubation at absorbances were taken at 420 nm after every 10 min
37°C for 24 h. After prescribed incubation time, the up to 120 min (Liu et al., 2015).
zones of inhibition were measured for each pathogen.
Statistical analysis
Selection of best treatment The obtained data were statistically analysed
Based on antioxidant activity and antimicrobial using Statistics 8.1 software. Analysis of variance
susceptibility tests, clove extract obtained from (ANOVA) was performed to measure the level of
n-hexane was chosen for further analysis on significance. All pair-wise comparison was made
antimicrobial activity and its effect on cell membrane through Tukey’s Test (Montgomery, 2008).
integrity and membrane permeability against
pathogenic bacteria. Results and discussion

Minimum inhibitory concentrations and zones of Compositional analysis of clove

inhibition The results of compositional analysis of clove
Minimum inhibitory concentration (MIC) of powder are presented in Table 1. The mean values
clove extract obtained from n-hexane was determined of moisture, crude protein, crude fat, crude fibre,
by using the Kirby Bauer Method (Bauer et al., 1966). ash and nitrogen-free extract were 10.36%, 7.62%,
Firstly, stock solution of clove extract was prepared. 16.67%, 3.14%, 3.57% and 58.64%, respectively.
Then, 50 µL distilled water was added to each well of
Table 1. Average composition of clove powder.
micro-titration plate with the help of micro-dispenser.
Parameter Average value (%)
Afterward, 50 µL solution was added to the first well.
Then 50 µL dilution was shifted to the next well and Moisture 10.36
so on. Then 2 filter paper discs having 6 mm diameter Crude protein 7.62
were soaked in each well. Bacterial suspensions were Crude fat 16.67
taken and inoculated as previously mentioned, and Crude fibre 3.14
MICs were determined after incubating the cultures at Ash 3.57
37°C for 24 h. Similarly, the zones of inhibition were Nitrogen-free extract 58.64
determined at different concentrations as previously
mentioned.
Extraction optimisation of clove essential oil
Cell membrane integrity
Bacterial cell membrane integrity of clove extract Extraction yield
against S. Typhimurium, E. coli and L. monocytogenes Different solvent systems; ethanol extraction
was determined by following the protocol of Chen system (EES), n-hexane extraction system (HES),
and Cooper (2002). After harvesting, bacterial cells petroleum ether extraction system (PES) and steam
were washed and resuspended in sterilised NaCl distillation extraction system (SES) were employed
solution (0.9%) followed by addition of 2 mL clove for the extraction of clove essential oil, and the
extract in each sample except control. Afterwards, 2 extraction yield of each system are shown in Table 2.
mL sample was taken from each tube at an interval of The maximum extraction yield was obtained by HES
1 h (up to 14 h), filtered with syringe filters (0.2 μm) (48.84%) followed by PES (43.86%), EES (41.47%)
and absorbances were taken at 260 nm using UV-Vis and SES (11.72%).
Spectrophotometer. Solvent extraction is a preferable method to extract
essential oils from plant materials due to its high
extraction efficiency and less energy consumption.
Membrane permeability
Hexane has maintained the dominant position
Membrane permeability was determined based
 Ishaq, A., Syed, Q. A., Khan, M. I. and Zia, M. A./IFRJ 26(4) : 1165-1172 1169

amongst all the solvents for many years. Hexane polyphenols which are effective in scavenging
has high ability to extract oil due to the presence of free radicals (Khalil et al., 2017). Clove extract
several isomers. It is preferred for solvent extraction principally contains eugenol and iso-eugenol which
due to its high volatility and low sensible heat (335 are majorly responsible for its antioxidant potential.
kJ/kg). Moreover, it can be easily removed from oil These phytochemicals form complexes with reduced
or extract with low use of energy. Additionally, the metals and impose inhibitory effect on lipid oxidation
azeotrope (95% hexane and 5% water) of hexane by eliminating free radicals and forming iron-oxygen
is quite efficient for the removal of contents from complexes (Ito et al., 2005).
solids at slightly lower boiling temperature (61.6°C) Ferrous (Fe2+) and ferric (Fe3+) ions are strong
by direct use of steam. Furthermore, hexane also pro-oxidants, and promote the generation of reactive
has good dissolving properties with oils which is oxygen species (ROS). Similarly, free radical chain
beneficial when washing off extracts from solids or reactions also promote lipid peroxidation. Minimising
fibrous materials (NFPA, 2009). the generation of ferrous ions and scavenging of
free radicals delay the process of lipid oxidation
Antioxidant activity of clove extracts in food and biological systems. Chelating agents
Different phenolic compounds present in plants disrupt the complex formation which results in the
possess strong therapeutic properties against several decrease in red colour. This discoloration is observed
life-style related ailments due to their antioxidant by spectrophotometer and chelating capacity of an
potential. The total phenolic and flavonoid contents, antioxidant is based on the extent of the discoloration.
FRAP and DPPH activities of clove extracts obtained Lower absorbance usually indicates high metal
from different extraction systems are shown in Table chelating activity of antioxidants. In the present work,
2. The TPCs of clove extracts ranged from 23.62 to the chelating ability of clove extract was described
54.05 mg/g GAE with HES exhibiting the highest by inhibiting the generation of ferrous and ferrozine
value and SES showing the least. The statistical complex by capturing ferrous ions before ferrozine. In
analyses revealed that extraction system had DPPH assay, antioxidants generally cause reduction
significant (p < 0.05) effect on TPCs of clove extract. of stable violet DPPH radicals followed by their
The differences in phenolic contents could be due to conversion to a yellowish compound i.e., diphenyl-
varied affinity of clove polyphenols towards different picrylhydrazine. The hydrogen-donating antioxidant
solvents. Similarly, the TFC of clove extracts were reduces alcoholic DPPH and converts it into non-
also significantly different. Maximum TFCs were radical form DPPH-H (Gülçin et al., 2012). Findings
recorded in HES (15.54 mg/g) followed by PES of the present work strongly support the power of
(14.63 mg/g), EES (12.46 mg/g) and SES (9.22 clove extract to reduce radical form of DPPH into
mg/g). non-radical form.
Table 2 also shows the results of FRAP and
DPPH analyses of clove extracts. It was revealed that Antimicrobial activity of clove extracts
clove extract of HES showed highest DPPH activity The antimicrobial activity of clove extracts
(0.29 mg/mL) followed by PES (0.24 mg/mL), EES obtained through different extraction systems (EES,
(0.21 mg/mL) and SES (0.15 mg/mL). Similar trend HES, PES, SES) against food-borne pathogenic
was also observed for FRAP. bacteria is demonstrated in Table 3. The highest
zones of inhibition were shown by HES; 24.27 mm
Table 2. Extraction yield and antioxidant activity of clove
extracts obtained from different extraction processes. for S. Typhimurium, 25.8 mm for E. coli and 29.67
TFC (mg FRAP DPPH
mm for L. monocytogenes. Results also suggested
Extraction TPC (mg that L. monocytogenes was more susceptible to
Treatments quercetin (mg/ (mg/
Yield (%) GAE/g)
/g) mL) mL) antimicrobial clove extract as compared to other
S1 41.47c 48.41c 12.46b 0.59b 0.21c pathogens. Other prepared extracts of clove also
S2 48.84 a
54.05 a
15.54 a
0.69 a
0.29a showed considerable antimicrobial potential, but
S3 43.86b 50.88b 14.63a 0.63b 0.24b their zones of inhibitions were smaller as compared
S4 11.72d 23.62d 9.22c 0.32c 0.15d to HES. Antimicrobial activity of clove extracts was
Different superscript letters in each column indicate significant compared with commercial antibiotics specific for
difference (p < 0.05). S1 = ethanol (95%); S2 = n-hexane (95%); S3 = each bacterial strain i.e., nalidixic acid was used
petroleum ether (95%); S4 = steam distillation.
for S. Typhimurium (31.57 mm) and E. coli (31.57
Several reports have demonstrated that plants mm) whereas chloramphenicol was used for L.
contain a variety of phytochemicals such as monocytogenes (38.8 mm).
1170 Ishaq, A., Syed, Q. A., Khan, M. I. and Zia, M. A./IFRJ 26(4) : 1165-1172

Table 3. Antimicrobial activity of clove extracts against Table 4 (Cont.)

Salmonella Typhimurium, Escherichia coli and Listeria C3 11.53c 14.53c 17.37c
monocytogenes.
C4 13.73 b
16.63 b
19.2b
Inhibition Zone (mm)
Different superscript letters in each column indicate significant
Treatment Salmonella Escherichia Listeria difference (p < 0.05). C0 = control; C1 = 95% n-hexane extract (0.25%
Typhimurium coli monocytogenes solution); C2 = 95% n-hexane extract (0.50% solution); C3 = 95%
n-hexane extract (1.00% solution); C4 = 95% n-hexane extract (1.50%
T0 31.57a 31.57a 33.8a
solution). For C0, nalidixic acid was used for S. Typhimurium and E.
T1 19.33 d
20.9d
26.6d coli whereas chloramphenicol was used for L. monocytogenes.
T2 24.27b 25.8b 29.67b
Integrity of bacterial plasma membranes
T3 22.33c 22.37c 27.53c
Results illustrated in Figure 1a show that amount
T4 14.33e 15.77e 19.73e of 260 nm absorbing materials released from S.
Different superscript letters in each column indicate significant Typhimurium and E. coli suspensions showed
difference (p < 0.05). T0 = control; T1 = ethanol (95%); T2 = n-hexane
(95%); T3 = petroleum ether (95%); T4 = steam distillation. For quite similar patterns whereas materials released
T0, nalidixic acid was used for S. Typhimurium and E. coli whereas from suspension of L. monocytogenes showed
chloramphenicol was used for L. monocytogenes.
different pattern. The OD-260 values of all bacterial
The principal polyphenol present in clove extract suspensions increased up to 10 h which indicated
(eugenol) possesses strong antimicrobial potential good inactivation kinetics of clove extract while
against a wide range of Gram-positive and Gram- these values tended to decrease after 10 h.
negative bacteria. Eugenol induces bacterial cell lysis The release of cytoplasmic components from
by damaging their cell wall and plasma membrane bacterial cells can only be monitored if plasma
resulting in the leakage of intracellular fluids along membrane of cells is compromised. When bacterial
with protein and lipid-based contents (Oyedemi et membranes interact with antimicrobial agents,
al., 2009). cellular materials such as phosphate and potassium
ions, DNA, RNA and other protein and lipid-based
Minimum inhibitory concentration compounds start coming out of the bacterial cells.
Based on the data obtained by extraction These intracellular constituents can be detected
efficiency, antioxidant activity and antimicrobial through UV-Vis spectrophotometer. The values of OD
potential, clove extract obtained by hexane-based taken by spectrophotometer indicate the damage of
extraction system was further assessed for its bacterial cell membrane (Franklin and Snow, 1981).
inhibitory effect. The MIC and zones of inhibition Inferences of the present work show the role of clove
of clove extract are depicted in Table 4. Findings extract in the quick release of OD-260 materials.
showed that the zones of inhibitions increased with However, the decrease in the amount of releasing
increasing concentration of clove extract solution. cellular constituents after a certain period (10 h) is
No zones of inhibition were observed below 0.25% mainly due to precipitation, adsorption of releasing
solution therefore this concentration was declared components to precipitates and denaturation of RNA
as MIC of clove solution against selected food- or DNA. It was also observed that the extent of leakage
borne pathogens. The maximum concentration i.e., of nucleotides was different in L monocytogenes
1.5% showed highest diameters of inhibition for S. as compared to S. Typhimurium and E. coli. This
Typhimurium (13.73 mm), E. coli (16.63 mm) and variation in nucleotide leakage was mainly attributed
L. monocytogenes (19.2 mm). Results of the present to the difference in cell membrane structure of Gram-
work undoubtedly described that clove extract has positive and Gram-negative bacteria (Denyer, 1995).
strong efficiency to inhibit the growth of pathogenic
microorganisms. Bacterial membrane permeability
The potential of clove extract to permeate
Table 4. Minimum inhibitory concentrations and
the membranes of S. Typhimurium, E. coli and L.
inhibition zones of clove extract using n-hexane as
solvent against Salmonella Typhimurium, Escherichia coli
monocytogenes was determined as a function of
and Listeria monocytogenes. releasing cytoplasmic β-galactosidase. After treating
Inhibition Zone (mm)
bacterial suspensions with clove extracts, a lag time
Treatment
of 10 min was given and after that a progressive
Salmonella Salmonella Salmonella
Typhimurium Typhimurium Typhimurium release of β-galactosidase was seen from bacterial
C0 31.3a 31.03a 34.37a
cells up to 100 min. However, the effect of clove
extract was more prominent on L. monocytogenes as
C1 8.8 d
9.27 e
11.17e
compared to S. Typhimurium and E. coli. However, a
C2 9.87d 11.6d 13.77d
non-significant and steady release of β-galactosidase
 Ishaq, A., Syed, Q. A., Khan, M. I. and Zia, M. A./IFRJ 26(4) : 1165-1172 1171

Figure 1: (a) Release of 260 nm absorbing materials and (b) Release of cytoplasmic β-galactosidase from cells of
Salmonella Typhimurium, Escherichia coli and Listeria monocytogenes with treatment of clove extract.

was observed in control bacterial suspensions (Figure Acknowledgement

1b). The results also revealed that the release of
β-galactosidase from bacterial membranes was time- The corresponding author is thankful to all co-
dependent. authors for their technical guidance in completing the
present work and preparing the manuscript.
Conclusion
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