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In this study, the antibacterial activity of methanol extract of henna (Lawsonia inermis) leaves, ethanol extract of pomegranate (Punica granatum) peel, volatile oil of sesame (Sesamum indicum) and peanut (Arachis hypogaea) were investigated against some Gram-positive and Gram-negative bacteria includingStaphylococcus aureus, Bacillus cereus, Escherichia coli and Acinetobacter sp. Henna extract was most effective substrate against all tested bacteria followed by pomegranate and peanut while sesame was less effective. All extracts were screened for their antibacterial activity in combination with commonly used antibiotics, including ciprofloxacin and erythromycin to evaluate synergistic effects using Minimum inhibitory concentrations (MIC) method which determined by microbroth dilution assays. Different interactions (synergistic and indifference) were observed between plant extracts and used antibiotics. The fractional inhibitory concentration (FIC) index ranged from 0.01 to 1.25 for B. cereus, 0.5 to 1 for P. aeruginosa, 0.01 to 0.3 for S. aureus and 0.06 to 0.25 for A. baumannii. The best synergistic capacity appeared between erythromycin and sesame. In vitro interaction between antimicrobial agents in combination with tested plant extracts showed synergistic effects. The MICs of each antibiotic was decreased to half when it is used in combination with tested plant extracts. This decreasing in MICs was observed in all plant extracts against tested bacteria as well as the extracts exhibited weak antibacterial activity alone.
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7
SYNERGISTIC EFFECT OF DIFFERENT PLANT EXTRACTS AND ANTIBIOTICS
ON SOME PATHOGENIC BACTERIA
Suzan A. Shareef a,
*, Abdulilah S. Ismaeil b, Akhter A. Ahmad b
a Dept. of General Sciences, College of Basic Education, Salahaddin University, Kurdistan Region, Iraq –
(suzan.sharif@su.edu.krd)
b Dept. of Biology, College of Science, Salahaddin University, Kurdistan Region, Iraq – (abdulilah.ismaeil; akhter.ahmed)
@su.edu.krd
Received: Nov., 2019 / Accepted: Feb., 2020 / Published: Mar.,2020 https://doi.org/10.25271/sjuoz.2020.8.1.653
ABSTRACT:
In this study, the antibacterial activity of methanol extract of henna (Lawsonia inermis) leaves, ethanol extract of pomegranate
(Punica granatum) peel, volatile oil of sesame (Sesamum indicum) and peanut (Arachis hypogaea) were investigated against some
Gram-positive and Gram-negative bacteria including Staphylococcus aureus, Bacillus cereus, Escherichia coli and Acinetobacter
sp. Henna extract was most effective substrate against all tested bacteria followed by pomegranate and peanut while sesame was
less effective. All extracts were screened for their antibacterial activity in combination with commonly used antibiotics, including
ciprofloxacin and erythromycin to evaluate synergistic effects using Minimum inhibitory concentrations (MIC) method which
determined by microbroth dilution assays. Different interactions (syne rgistic and indifference) were observed between plant
extracts and used antibiotics. The fractional inhibitory concentration (FIC) index ranged from 0.01 to 1.25 for B. cereus, 0.5 to 1
for P. aeruginosa, 0.01 to 0.3 for S. aureus and 0.06 to 0.25 for A. baumannii. The best synergistic capacity appeared between
erythromycin and sesame. In vitro interaction between antimicrobial agents in combination with tested plant extracts showed
synergistic effects. The MICs of each antibiotic was decreased to half when it is used in combination with tested plant extracts.
This decreasing in MICs was observed in all plant extracts against tested bacteria as well as the extracts exhibited weak antibacterial
activity alone.
KEYWORDS: Antibacterial activity; Combination; Minimum inhibitory concentration (MIC); Plant extract; Synergistic.
1. INTRODUCTION
Infectious diseases caused by bacteria and fungi affect
millions of people worldwide. Throughout the history of
mankind, infectious diseases have remained a significant
cause of death and disability; it accounts for one-third of all
deaths in the world (Us ha et al ., 2010). The discovery of
antibiotics was an essential part of combating bacterial
infections that once ravaged humankind (Usha et al., 2010).
Antibiotics are critical weapons in combating bacterial
infections and can be beneficial for human health (Berge and
Wierup, 2012). However, overtime, the effect of antibiotics
that routinely used have decreased against certain infections
due to production of toxic reactions and the development of
resistant strains of bacteria (DiMasi et al, 2016). The rapi d
development of drug-resistant bacteria is an important health
problem that occurred worldwide (Rouveix, 2007 and
Ahmed, 2013). The alarming growth of the number of
antibiotic resistant bacteria and difficulties in treatment of
infections, besides sometim es antibiotics use may cause
opposite effects, such as allergic reactions, immune
destruction, and hypersensitivity have initiated a search for
new antibacterial compounds and develop new alternative
strategies in combating bacterial infections (Agrawal et al.,
1996). Medicinal plants, with their long history of use in folk
medicine for the treatment of infectious diseases, have
become a promising innovative antimicrobial substances by
extraction of phytochemicals, which are active to prevent
infections (Abiramasundari et al., 2011 and Agrawal et al.,
1996). Plant -derived compounds could exhibit a direct
* Corresponding author
This is an open access under a CC BY -NC-SA 4.0 license (https://creativecommons.org/licenses/by -nc-sa/4.0/)
antibacterial activity and/or an indirect activity as antibiotic
resistance modifying compounds, which, combined with
antibiotics, increase their effectiveness (Haroun and Al -Kayali,
2016).
The use of plant extracts can be highly significant in therapeutic
treatments and there are many different medicinal plants used in
traditional medicine by the traditional herbalists to treat varieties
of human ailments (Aali et al., 2018). Numbers of different plants
have been used due to their antimicrobial activity as a result of
their active substances while others by a combination of their
common phytochemicals with antibiotics (Ahmed et al., 2010).
For this reason, the antibacterial activities of plant extract alone
and when it combined with different antibiotics have been
studied in many parts of the world by a number of researchers.
Farooqui et al . (2015) investigated synergistic antimicrobial
activity of Camellia sinensis and Juglans regia with nalidixic acid
against 350 Gram-positive and Gram-negative strains belonging
to 10 different bacterial species. While Liu et al. (2017) studied
synergistic antimicrobial effect of lipopeptides and tea
polyphenols against V. parahaemolyticus and their result showed
that the combination of lipopeptides and tea polyphenols
displayed strong synergistic antibacterial effect against V.
parahaemolyticus with a fractional inhibitory concentration
index of 0.19. In another study by Ennacerie et al . (2017) also
tested the antibacterial potency and evaluate the possible
synergistic effect between both aqueous and ethanolic extracts,
flower buds and fruits of Capparis spinosa , and antibiotics
against Klebsiella pneumoniae and Pseudomonas aeruginosa
and their results showed a synergistic effect with ICIF ranging
from 0.02 to 0.24. From the points of view, the present study
Vol.8,No.1,pp.7–11,March-2020
S.A. Shareef et al. / Science Journal of University of Zakho 8(1), 7-11, March-2020
8
conducted to investigate whether the combination of some
plant extracts with commonly used antibiotics has any
synergistic effects on some clinically isolated bacteria or not.
2. MATERIALS AND METHODS
2. 1. Plant materials and extraction
Henna (Lawsonia inermis) leaves, pomegranate (Punica
granatum) peel, Peanut (Arachis hypogaea) and Sesame
(Sesamum indicum) were selected as medicinal plants for
extraction.
The plant materials were processed by the methods described
previously by (Harborne, 1998). Briefly, pulverized plant
material (after cleaning and shade drying) was extracted
using normal hexane, methanol, and ethanol as solvents. To
remove the solvents from the extracts vacuum evaporator
was used to attain the crude extract of each fraction. The
extracts stored at -20 ºC and freshly dissolved in dimethyl
sulfoxide (DMSO, Merk Germany) before use.
2.1.1. Extraction of henna leaf: Fifty grams of henna leaves
were dried under shade, milled and extracted with n-hexane
by the use of Soxhlet extractor apparatus for about six hours.
Extraction was carried out the color of the solvent in the last
siphoning time became colorless. Then, the solvent
evaporated under reduced pressure by the use of a rotary
evaporator apparatus. The marc obtained from henna was
shade dried and re-extracted with methanol using the above
method.
2.1.2. Extraction of Pomegranate Peel: Fifty grams of
Pomegranate dry powder was mixed with ethanol, sonicated,
and filtered by Whatman paper no.1. The supernatant was
dried by rotary evaporator.
2.1.3. Volatile oil of sesame and peanut: One hundred
grams of both sesame and peanut weighted were put in 2000
ml rounded bottom capacity flask, separately. Both
Clevenger receiver and condenser were attached to the top of
the flask, 1000 ml of distilled water was added. The system
was heated to 100◦C for about 4h until the oil volume at the
upper part of the receiver fixed. The oil was pipetted and
dried over sodium sulfate anhydrous. Then, it was stored in a
dark container in the refrigerator till used (Harborne, 1998).
2.2. Preparing of oil concentrations
Each plants extract was dissolved in dimethyl sulfoxide
(DMSO) (50% of the final volume) and was then diluted with
Tryptic Soy Broth medium (TSB; Oxoid) to concentrations
(2, 4, 8, 16, 32, 64, 128) mg/ml.
2.3. Antimicrobials
Pure antimicrobial powders of Ciprofloxacin and
Erythromycin were selected as antimicrobials in this study.
The stock solution was prepared by dissolving the powder in
TSB in different concentrations (2, 4, 8, 16, 32, 64, 128)
mg/ml (Lalitha, 2004).
2.4. Bacterial isolates
Clinical isolates investigated in this study were;
Acinetobacter baumannii and Pseudomonas aeruginosa as
Gram-negative, Bacillus cereus and Staphylococcus aureus
as Gram-positive (The isolates were obtained from
Bacteriology laboratory of West Emergency Hospital in Erbil
City. Vitek II automated system (bioMérieux Marcy
l’´Etoile, France) (Vitek Systems Version: 06.01) was used
to identify the isolates.
2.5. Minimum inhibitory concentration (MIC) determination
Broth microdilution assay was performed to determine the
minimum inhibitory concentration for the galls extracts against
the identified isolates (Roberts et al., 2012). Ten µl of bacterial
cells (equilibrated to OD550 0.5) inoculated into 100µL TSB
containing a range of extracts or antimicrobials concentrations
beginning (1-128 mg ml-1) in the polystyrene microtiter plate
(MTP) wells. The MTPs have incubated overnight at 37 ºC. The
lowest concentration that did not show any obvious growth was
considered as minimum inhibitory concentration. To determine
the minimum bactericidal concentration, 100 µl from MTP wells
that did not show any obvious growth was streaked on sterile
plates of nutrient agar (NA; Oxoid). Nutrient agar (NA) plates
were incubated overnight at 37 ºC. Concentrations that have no
growth on NA plates were considered as minimum bactericidal
concentration (MBC). The level below the MICs was considered
as subinhibitory concentrations (SICs) which then used to
evaluate the synergistic effect of the extracts with antimicrobials.
Three replicates were considered on distinct occasions.
2.6. Checkerboard technique to determine the synergistic
antimicrobial activity
To investigate the effect of the combination of each extract with
the selected antimicrobials 150 μL of TSB medium containing a
mixture of SIC of each plant extract and each of the tested
antimicrobials were added to 96-well microtiter plates, TSB with
no extract and antimicrobials was used as a control. The wells
were inoculated with 10 μL of a bacterial suspension. All
experiments were achieved in triplicate, and the MTP was
incubated overnight at 37ºC. To determine the synergistic effect
of the extracts and the antimicrobials the broth in the wells were
sub-cultured on NA plates.
The combination between the extracts and the antibiotics were
evaluated by the checkerboard method as described by Petersen
et al (2006). The fractional inhibitory concentration (FIC) was
derived from the lowest concentrations of the extract in tryptic
soy broth tube after overnight incubation at 37 °C. FICs were
calculated using the following formula:
+
When antibiotic combined with a plant extract, synergy is
happened when FIC index ≤ 0.5, additivity when 5 < index FIC
≤ 1, indifference when 1< FIC index ≤ 4 and antagonism when
FIC index > 4 as described by Petersen et al. (2006). While
Kamatou et al (2006) defined synergy that happened when FIC
index<1.0, additivity when FIC index=1.0 and antagonism when
FIC index > 1.0. Hence, due to checkerboard assay, Olajuyigbe
and Afolayan (2012) indicated that synergy is determined when
FIC is less than or equal to 0.5 or is less than or equal to 1.
2.7. Statistical data analysis
All the data were analyzed by Statistical Package Social Science
(SPSS) version 21.0. The experimental results were expressed as
mean ± standard error of the mean (SEM). Groups were
compared by analysis of variance using Two-way ANOVA and
Dunnett's multiple comparisons test. Less than 0.05 of p-value
was regarded as statistically significant. Significance was defined
as p<0.05, p<0.01.
S.A. Shareef et al. / Science Journal of University of Zakho 8(1), 7-11, March-2020
9
3. RESULTS
The Minimum inhibitory concentration (MIC) of plant
extracts alone, and antibiotics alone were examined against
tested bacteria, as shown in Table (1), Figure (1).
Ciprofloxacin showed inhibited active on bacterial species at
(4 mg/ml) against all tested bacteria while erythromycin was
more active and inhibited tested bacteria at 1 mg/ml except
S. aureus (2 mg/ml).
The antibacterial activities of the plant extract varied in
relation to the tested organisms. Henna extract was the most
effective against all tested bacteria. The most active
concentration was 4 mg/ml concentration that inhibited the
growth of Staphylococcus aureus, Acinetobacter baumannii
and Bacillus cereus utterly, while Pseudomonas aeruginosa
was inhibited by 8 mg/ml concentration of henna extract.
Pomegranate also showed antibacterial activity with different
concentrations against all tested bacteria. Most effective
concentration was 4 mg/ml against P. aeruginosa, 16 mg/ml
against Acinetobacter while S. aureus and B. cereus were
inhibited at a minimum inhibitory concentration of 32 mg/ml.
Peanut showed similar effects to that of henna against P.
aeruginosa at MIC of 8 mg/ml and A. baumannii at MIC of
4 mg/ml, while it was less effective against B. cereus at MIC
of 32 mg/ml.
Table 1. Minimum inhibitory concentration (MIC) of plant extracts
alone and antibiotics alone against tested bacteria
Antibiotic and
Plant extract
MIC (mg/ml)
S.
aureus
B.
cereus
P.
aeruginosa
Henna
(Lawsonia inermis)
4
4
8
Pomegranate
(Punica granatum)
32
32
4
Peanut
(Arachis hypogaea)
64
32
8
Sesame
(Sesamum indicum)
128
64
64
Ciprofloxacin
4
4
4
Erythromycin
2
1
1
Figure 1. Minimum inhibitory concentration (MIC) of plant extracts
alone and antibiotics alone against tested
In vitro interaction between antimicrobials and tested plant
extracts by microdilution method showed a reduction of MIC
of the antimicrobials when combined with plant extracts. The
minimum inhibitory concentration of antibiotics in
combination with plant extracts against pathogenic bacteria
tested by microdilution method is shown in Table [2].
Table 2. Minimum inhibitory concentration of antibiotics in
combination with plant extracts against tested bacteria using the
microdilution method
Antibiotic and
Plant extract
combination
MIC (mg/ml)
S. aureus
B.
cereus
Cip + Sesame
2:64
N
Cip + Henna
N
2:2
Cip +
Pomegranate
N
N
2:8
Cip + Peanut
N
2:16
N
Ery + Sesame
1:64
0.5:32
N
Ery + Henna
N
N
N
Ery +
Pomegranate
N
N
N
Ery + Peanut
N
0.5:16
N
N= No effect
Antibacterial combination considered as synergism in ∑FIC ≤
1.0, indifference in 1.0 < ∑FIC ≤ 4 and antagonism in ∑FIC > 4
[Table 3]. According to the standard evaluation measures of
Kamatou et al. (2006) and Grytten et al. (1988). In combination
between ciprofloxacin and henna leaf extract, the MIC of
ciprofloxacin was reduced from 4 mg/ml to 2 mg/ml and showed
synergistic interaction against P. aeruginosa and Acinetobacter
(∑FIC is less than or equal to 1.0), while it was indifference (1.0
< ∑FIC ≤ 4) against B. cereus. The MIC of henna leaf extract was
decreased from 4, 8, 4 mg/ ml to 2, 4, 2 mg/ ml against B. cereus,
P. aeruginosa and Acinetobacter respectively. The combination
interaction between ciprofloxacin with pomegranate (against P.
aeruginosa and Acinetobacter) and peanut (against B. cereus)
showed a significant reduction of MIC for both antibiotic and
plant extract and the combination was classified as synergy
(∑FIC ≤ 1.0). Erythromycin and sesame activity against S.
aureus and B. cereus also were more effective when combined
together and reduced the MIC against these bacteria while no
synergistic effect of them was produced against the other tested
bacteria. No significant reduction of MIC occurred in
combination between erythromycin and both henna and
pomegranate, while there was a synergistic effect of the
combination of erythromycin and peanut and the MICs of both
of them decreased against B. cereus.
Table 3. Fractional inhibitory concentrations (FIC) of different
combination of the extracts and the antibiotics.
Antimicrobials
and Plant
extract
combination
Fractional Inhibitory Concentration
Tested
bacteria
FIC index
Remarks
Cip + Henna
B. cereus
1.25
Indifference
Cip + Henna
P. aeruginosa
0.5625
Synergy
Cip + Henna
A. baumannii
0.0625
Synergy
Cip + Peanut
B. cereus
0.12890625
Synergy
Cip+
Pomegranate
P. aeruginosa
1
Indifference
Cip+
Pomegranate
A. baumannii
0.25
Synergy
Cip + Sesame
S. aureus
0.3149414
Synergy
Ery + Peanut
B. cereus
0.0317383
Synergy
Ery + Sesame
S. aureus
0.015625
Synergy
Ery + Sesame
B. cereus
0.015625
Synergy
Cip= Ciprofloxacin, Ery = Erythromycin
S.A. Shareef et al. / Science Journal of University of Zakho 8(1), 7-11, March-2020
10
4. DISCUSSION
Many studies have been reported, assuring the antimicrobial
activities of an individual or combined extracts of medicinal
plants. In this study, henna extract was more effective against
all tested bacteria than the other plant extracts. Henna has
many traditional and commercial uses, the most common
being as a dye for hair, skin and fingernails because it
contains Lawsone (2-hydroxynaphthoquinone) which is one
of the component with (0.5-1.5%) responsible for dyeing. It
also contains tannic acid, mucilage, gallic acid and mannite
(Kelmanson et al., 2000). The antimicrobial effect of henna
may be according to numerous free hydroxyls which are able
to combine with the bacterial cell wall structures including
carbohydrates and proteins as suggested by Harborne and
Baxter (1995) and they attributed that to their attachment to
enzyme site rendering them inactive.
Results of the present study showed that pomegranate peel
extract was effective against tested bacteria. In particular,
among plants, Punica granatum used in traditional medicine,
is known for its pharmacological properties that have been
evaluated due to antiparasitic, antibacterial, antifungal,
antiproliferative, apoptotic, and anticancer effects (Jurenka,
2008). Literature data reported that extracts of Punica
granatum peel in different concentrations were effective
against different bacterial species such as S. aureus, E.
coli, Salmonella enterica, Shigella sonnei, Enterococcus
faecalis, and Bacillus subtilis (Pagliarulo et al., 2016;
Subramaniam et al, 2012; Rosas-Burgos et al, 2017 and Dey
et al, 2015). Pomegranate beverage contains several
compounds that are responsible for the antimicrobial activity,
depending on their abundance such as tannins which are
considered to be toxic to microorganisms (Viuda-Martos et
al., 2010). Their hydrophilic site cooperates with the polar
region of the bacterial cell membrane, while the hydrophobic
site is immersed in the non-polar region of the bacterial
membrane, this affects transporting of substances into the cell
(Cristani et al., 2007). Likewise, Naz et al. (2007) suggested
a phenolic toxicity through reactions with sulfhydryl groups
or through more non-specific interactions with proteins
leading to loss of function.
Sometimes the use of antibiotic alone does not give the
desired inhibitory effects, this can be defeated by a
combination of drugs which appears their synergistic effect,
and this is more significant than their effects alone (Kamatou
et al, 2006). Synergism is defined as a positive interaction
created when two agents are combined and together they
exert an inhibitory effect (on the targeted organisms) that is
greater than the sum of their individual effects (Levinson and
Jawetz, 2002). Consequently, mixing plant extracts with
antibiotics enhanced and synergized their effect and
decreased their MICs and this fact has clearly emerged in this
study. The synergistic effect could be related to the formation
of complex chemical products that can be greatly effective to
inhibit many species of microorganisms by preventing cell
wall to synthesize or may lead to lyses and finally, it dies
(Chanda and Rakholiya, 2011). There was a significant
synergistic effect of combination between both ciprofloxacin
and erythromycin with henna, sesame, pomegranate, and
peanut and the MIC of both of them was decreased against
tested bacteria and this could be referred to that these crude
extracts have many different phytochemicals which might
inhibit bacteria by different mechanisms (Duke et al., 2003).
This result reveals that plant extracts were potentiating the
effects of the ciprofloxacin and erythromycin. This double
attack of both agents on different target sites of the
bacteria could theoretically lead to either an additive or a
synergistic effect (Esimone et al. 2006). In a previous
similar study performed by Sato et al (2004), they were
combined between methanolic extract of pomegranate and
antibiotics and found that the antimicrobial activity of flavonoids
and polyphenols when they combined with antibiotics could alter
the bacterial resistant properties to be more effective. Cushnie et
al (2005) in different study indicated synergism between
antibiotics and flavonoids. Tsuchiya et al (1996) reported that
flavonoids disrupt bacterial cell membranes while Prasad et al
(2008) found that tannins precipitate bacterial protein. Yang et
al. (2005) and Aqil et al. (2005) reported in a previous in vitro
studies, significant decreasing in the MICs and synergistic effects
of the antibiotics when combined with number of plant extracts
against Staphylococcus aureus.
Ciprofloxacin is a second generation fluoroquinolone, interrupts
DNA replication by inhibiting both topoisomerase II (an enzyme
that reduces the amount of supercoiling of the DNA double-
stranded helix during the replication process) and IV thus
preventing cell division (Grohe et al, 1987). By the way, Liu et
al. (2011) stated that flavonoids are exist in many types of our
food such as vegetables, and fruits and they are able to combine
with fluoroquinolone antibiotics to exhibit an effective
antimicrobial agent.
5. CONCLUSION
Obtained results confirm the antibacterial activity of henna,
pomegranate, peanut and sesame extract and shows their
potential use as agents which enhance antibiotic activity.
Sometimes mixing plant extracts with different antimicrobials
enhanced and increase their antibacterial activity and the
antimicrobials that produce side effects can be used by reducing
dose concentration exploiting their synergy with the medicinal
plants. Mixing plant extracts with antimicrobials also increases
the spectrum of antibiotic, avoids the development of resistance
and decreases toxicity so exhibiting antibacterial activity better
than that estimated from each antibiotic alone. Further studies are
required to determine the specific substrates that have synergistic
effect and approved with in vivo studies.
REFERENCES
Ali M.H., Ismaiel N.J., AbdulMajid F.A. (2018). Antioxidant, and
Antimicrobial Activities of Phenolic and Flavonoid-Rich
Medicinal Plants (Fritillaria zagrica and Tulipa kurdica) Bulbs
Collected in Kurdistan Region of Iraq. Zanco Journal of Pure and
Applied Sciences, 30(5):1-16.
Abiramasundari P., Priya V., Jeyanthi G.P., Gayathri D.S. (2011).
Evaluation of the Antibacterial activity of Cocculus hirsutus.
HYGEIA - Journal for Drugs and Medicine, 3(2):26-31.
Agrawal P., Rai V., Singh R., B. (1996). Randomized, placebo-
controlled, single-blind trial of holy basil leaves in patients with
noninsulin-dependent diabetes mellitus. International Journal of
Clinical Pharmacology and Therapeutics, 34:406-9.
Ahmed A. A., Mawlood S.I., Ismaeel A.S. (2010). Role of solvent and
methods of extraction on the inhibitory effect of black seed
extracts on Pseudomonas aeruginosa. Zanco Journal of Pure and
Applied Sciences, 22, 3.
Ahmed, A. A. (2013). In vitro screening of Lactobacillus species from
homemade yoghurt for antagonistic effects against common
bacterial pathogens. Jordan Journal of Biological Sciences,
6(3):2011-16.
Aqil F., Khan M.S.A., Owais M. Ahmad I. (2005). Effect of certain
bioactive plant extracts on clinical isolates of -lactamase producing
methicillin resistant Staphylococcus aureus. Journal of Basic
Microbiology. 45:106-114.
Berge, A. C. and M. Wierup, M. (2012). Nutritional strategies to combat
Salmonella in mono-gastric food animal production. Animal, 6:
557-564.)
Chanda, S. and Rakholiya, K. (2011). Combination therapy: Synergism
between natural plant extracts and antibiotics against infectious
diseases. Science against microbial pathogens: communicating
current research and technological advances. Formatex, 1: 520-9.
Cristani M., D'Arrigo M., Mandalari G., Castelli F., Sarpietro M.G.,
Micieli D., Venuti V., Bisignano G., Saija A., Trombetta D.
S.A. Shareef et al. / Science Journal of University of Zakho 8(1), 7-11, March-2020
11
(2007). Interaction of four monoterpenes contained in
essential oils with model membranes: implications for their
antibacterial activity. Journal of Agricultural and Food
Chemistry, 25; 55(15):6300-8.
Cushnie T.P.T., Lamb A.J. (2005). Antimicrobial activity of
flavonoids. International Journal of Antimicrobial Agents.,
26: 34356.
Dey D., Ray R. and Hazra B. (2015). Antimicrobial activity of
pomegranate fruit constituents against drug-
resistant Mycobacterium tuberculosis and ß-lactamase
producing Klebsiella pneumonia (2015). Journal of
Pharmaceutical Biology, 53 (10): 147480.
DiMasi J.A., Grabowski H.G., Hansen R.W. (2016). Innovation in
the pharmaceutical industry: new estimates of R&D costs.
Journal of Health Economics, 47: 20-33.
Drobniewski F.A. (1993). Bacillus cereus and related species. Clin.
Microbiol. Rev. 6:324-338.
Duke J. A., JoBojeneschutz-Godwin M., DuCellier J., Duke P.A.K.
(2003). CRC, Handbook of Medical Plant. Boca Raton: CRC
Press, FL. 348.
Ennacerie F-Z., Filali F. R., Najia Moukrad, Ed-Dra A. (2017).
Antibacterial synergistic effect of extracts of the organs of
capparis spinosa and in combination with antibiotics.
International Journal of Advanced Research, 5(9), 1238-47.
Farooqui A., Khan A., Borghetto I., Kazmi S.U., Rubino S., Paglietti
B. (2015). Synergistic antimicrobial activity of Camellia
sinensis and Juglans regia against multidrug-resistant
bacteria. PLoS One, 10:e0118431.
Grohe K., Zeiler H.J., Metzger G. (1987). 7-amino-1-cyclopropyl-4-
oxo-1, 4-dihydro-quinoline and naphthyridine 3-carboxylic
acids and antibacterial agents containing these compounds.
US patent, 4: 670.
Grytten J., Scheie A.A., Giertsen E. (1988). Synergistic antibacterial
effects of copper and hexetidine against Streptococcus
sobrinus and Streptococcus sanguis. Acta Odontologica
Scandinavica, 46:181-3.
Harborne J.B. (1998). Phytochemical Methods A guide to modern
techniques of plant analysis. Third Ed: Chapman & Hall.
Harborne S.B and Baxter A. (1995). Phytochemical Dictionary. A
handbook of bioactive compounds from plants. Tylor and
Francis. London.
Haroun M. and Al-Kayali R.S. (2016). Synergistic effect of Thymbra
spicata L. extracts with antibiotics against multidrug-
resistant Staphylococcus aureus and Klebsiella
pneumoniae strains. Iranian Journal of Basic Medical
Sciences, 19:1193-200.
Kamatou G.P.P., Viljoen A.M., van Vuuren S.F., van Zyl R.L.
(2006). In vitro evidence of antimicrobial synergy between
Salvia chamelaeagnea and Leonotis leonurus. South African
Journal of Botany, 72:6346.
Kelmanson J.E., Jäger A.K., Van Staden J. (2000). Zulu medicinal
plants with antibacterial activity. Journal of
Ethnopharmacology, 69(3):241-6.
Kotiranta A., Lounatmaa K., Haapsalo M. (2000). Epidemiology and
pathogenesis of Bacillus cereus infections. Microbes
Infection, 2:189198.
Lalitha M.K. (2004). Manual on antimicrobial susceptibility testing.
Indian Association of Medical Microbiologists, 46.
Levinson W, Jawetz E. (2002). Medical microbiology and
immunology: Examination and board review. International.
7th ed., Lange Medical Books/McGraw-Hill, New York.
Liu G., Liang J., Wang X., Li Z. (2011). In vitro synergy of biochanin
A and ciprofloxacin against clinical isolates of
Staphylococuus aureus. Molecules, 16 (8):6656-66.
Liu, H.; Zhang, W.; Wu, Y.; Sun, L.; Wang, Y.; Liu, Y.; Zhang, X.;
Hong, P.; Ji, H. (2017). Synergistic antimicrobial effect and
mechanism of lipopeptides and tea polyphenols against Vibrio
parahaemolyticus. Food Sciences, 38: 14-19.
Naz, S., Siddiqi, R., Ahmad, S., Rasool, S.A., and Sayeed, S.A.J.
(2007). Antibacterial activity directed isolation of compounds
from Punica granatum. Journal of Food Science, 72 (9): 341-
5.
Olajuyigbe O. O., Afolayan a. J. (2012). Synergistic Interactions of
Methanolic Extract of Acacia mearnsii de wild with antibiotics
against bacteria of clinical relevance. International journal of
molecular sciences, 13(7): 891532.
Pagliarulo C., De vito V., Picariello G. (2016). Inhibitory effect of
pomegranate (Punica granatum) polyphenol extracts on the
bacterial growth and survival of clinical isolates of pathogenic
staphylococcus aureus and escherichia coli. Food chemistry, 190:
82431.
Petersen P.J., Labthavikul P., Jones C.H., Bradford P.A. Kamatou G.P.P.
(2006). In vitro antibacterial activities of tigecycline in
combination with other antimicrobial agents determined by
chequer board and time-kill kinetic analysis. Journal of
Antimicrobial Chemotherapy, 57:5736.
Prasad R.N., Viswanathan S., Devi J.R., Nayak V., Swetha V.C., Archana
B.R., Parathasarathy N., Rajkumar J. (2008). Preliminary
phytochemical screening and microbial activity of Samaea saman.
Journal of Medicinal Plant Research, 2:26870.
Roberts A.E., Maddocks S.E., Cooper R.A. (2012). Manuka honey is
bactericidal against Pseudomonas aeruginosa and results in
differential expression of oprF and algD. Microbiology, 158:3005-
13.
Rosas-Burgos E. C., Burgos-Hernández A., Noguera-Artiaga L. (2017).
Antimicrobial activity of pomegranate peel extracts as affected by
cultivar, Journal of the Science of Food and Agriculture, 97(3):
80210.
Rouveix B. (2007). Implications of multiple drug resistant efflux pumps
of pathogenic bacteria. Journal of Antimicrobial Chemotherapy,
5(96):12089.
Sato Y., Shibata H., Arai T., Yamamoto A., Okimura Y., Arakaki N.,
Higuti T. (2004). Variation in synergistic activity by flavone and
its related compounds on the increased susceptibility of various
strains of methicillin-resistant Staphylococcus aureus to β-lactam
antibiotics. International Journal of Antimicrobial Agents,
24:22633.
Subramaniam P., Dwivedi S., Uma E. and Girish Babu K. L. (2012).
Effect of pomegranate and aloe vera extract on streptococcus
mutans: An in vitro study, Dental Hypotheses, 3 (3): 99105.
Tsuchiya H., Sato M., Miyazaki T., Fujiwara S., Tanigaki S., Ohyama
M., Tanaka T., Iinuma M. (1996). Comparative study on the
antibacterial activity of phytochemical flavonones against
methicillin-resistant Staphylococcus aureus, Journal of
Ethnopharmacology, 50:2734.
Usha P.T.A., Jose S., Nisha A.R. (2010). Antimicrobial drug resistance -
a global concern. Veterinary World, 3:138-9.
Viuda-Martos M., Fernández-López J., Pérez-Álvarez J.A. (2010).
Pomegranate and its many functional components as related to
human health: a review. Comprehensive Reviews in Food Science
and Food Safety, 9:63554.
Yang Z.C., Wang B.C., Yang X.S., Wang Q., Ran L. (2005). The
synergistic activity of antibiotics combined with eight traditional
Chinese medicines against two different strains of Staphylococcus
aureus. Colloids and Surfaces B, Biointerfaces, 41:79-81.
... In this study, antimicrobials such as tetracycline and sulfonamide were tested as pure powders. Dissolving the powder in TSB at various concentrations (0.5, 2, 4, and 8 mg/ml) yielded the stock solution [14] . ...
... The wells were then filled with the various concentrations of plant extract (0.5, 2,4 and 8 mg/ml) (18 hours). After 24 hours of incubation, the area of inhibition (measured in mm) [14] . ...
... In addition, the combination may be helpful in the preclusion of the emergence of resistant bacteria and reducing the drug toxicity 27 . Various in vitro experiments have established the fact that a combination of plant extracts and antibiotics possess a synergistic effect, which results in a significant decrease in levels of minimum inhibitory concentration for the antibiotics 28,29 . ...
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... 19 Shareef et al studied the synergistic effect of methanol extract Lawsonia inermis, ethanol extract of Punica granatum, the volatile oil of Sesamum indicum, and Arachis hypogaea against Gram-positive and Gram-negative bacteria. 20 The interaction between the methanol extract of Ziziphus mucronata and antibiotics against bacteria enhanced the antibacterial activity. 21 One of the studies of Wess et al reported that a combination of four drugs (axitinib, erlotinib, dasatinib, and AZD4547) was significantly more potent for cancer treatment than monotherapies of all single drugs. ...
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