The antimicrobial activities of olive leaf extract against some pathogenic bacteria

Abstract

This study was designed to investigate the antibacterial activities of
olive leaf extract against five pathogenic bacteria (Bacillus cereu ,Salmonella typhimurium ATCC 14028 ,Pseudomonas aeruginosa ATCC 9027 , Staphylococcus aureus ATCC 25923 and Bacillus subtillus ATCC 6633).
The olive leaf extract , at a concentration of 300 ppm, produced the highest inhibitory potential on Salmonella typhimurium ATCC 14028 , Bacillus subtillus ATCC 6633 and Bacillus cereus with a zone of inhibition of 18.0 mm , 15.0 mm and 15.0 mm respectively, while increasing concentration extracts at 600ppm had the greatest activities in Salmonella typhimurium ATCC 14028 , Bacillus subtillus ATCC 6633 and Bacillus cereus with a zone of inhibition of 20.0 mm . These results therefore inferred the antibacterial efficacy of the olive leaf extracts.

Full text

Minia J. of Agric. Res. & Develop.
Vol. (34), No. 1, pp. 149-154, 2014
FACULTY OF AGRICULTUR THE ANTIMICROBIAL ACTIVITIES OF OLIVE LEAF
EXTRACT AGAINST SOME PATHOGENIC BACTERIA
Hassan, M.SH., M.M.Elsayed , A. M. Zaki , F.S.Hatour and Hanaa S. S. Gazwi
Agric. Bioch. Dept. Faculty of Agriculture , Minia University , Elminia Egypt Received: 7 Julay (2014) Accepted: 14 October (2014)
ABSTRACT
This study was designed to investigate the antibacterial activities of
olive leaf extract against five pathogenic bacteria (Bacillus cereu ,Salmonella
typhimurium ATCC 14028 ,Pseudomonas aeruginosa ATCC 9027 , Staphylococcus
aureus ATCC 25923 and Bacillus subtillus ATCC 6633).The olive leaf extract , at a
concentration of 300 ppm, produced the highest inhibitory potential on Salmonella
typhimurium ATCC 14028 , Bacillus subtillus ATCC 6633 and Bacillus cereus with a
zone of inhibition of 18.0 mm , 15.0 mm and 15.0 mm respectively, while increasing
concentration extracts at 600ppm had the greatest activities in Salmonella
typhimurium ATCC 14028 , Bacillus subtillus ATCC 6633 and Bacillus cereus with a
zone of inhibition of 20.0 mm . These results therefore inferred the antibacterial
efficacy of the olive leaf extracts.
Key words: Olive leaf extracts , antibacterial , pathogenic bacteria.
INTRODUCTION
Olive (Olea europaea) leaf has been
widely used in folk medicine for several
thousand of years within European
Mediterranean islands and countries (Gucci
et al., 1997). Historically, olive leaf was
used for the treatment of malaria and
associated fever (Benavente-Garcia et al.,
2000). Olive leaves extracts ( OLE ) are
rich in phenolic components (De Nino et
al., 1997), oleuropein being the most
prominent phenolic compound that may
reach concentrations of 60–90 mg g_1 of
dry matter (Ryan, et al., 2002).The major
physiological substances of olive leaf are
hydroxytyrosol, tyrosol, caffeic acid, p-
coumaric acid, vanillic acid, vanillin,
oleuropein, luteolin, diosmetin,
rutin,verbascoside, luteolin-7-glucoside,
apigenin-7-glucoside, and diosmetin-7-
glucoside (Bianco and Uccella, 2000;
Tasioula-Margari and Ologeri, 2001).
Several reports have been published on
olive leaf and presented the following: olive
leaf offered a capacity to lower blood
pressure and increase blood flow in the
coronary arteries (Khayyal et al.,2002). The
phenolic compounds extracted from olive
leaf possessed antimicrobial activity against
Helicobacter pylori, Campylobacter jejuni,
Staphylococcus

Hassan, et al.,2014
150
aureus (Sudjana et al., 2009). Pereira et al
.,(2007) reported that the antimicrobial
properties of phenolic compounds in olive
products refer to compounds obtained from
olive fruit,particularly hydroxytyrosol and
oleuropein.
Despite the many reports on olive leaf
and its phenolic compounds, the combined
effects of olive leaf phenolics in terms of
antimicrobial activities have not been
studied (Lee, and Lee 2010).
Pathogenic bacteria constitute a
major cause of morbidity and mortality in
humans. Sharma et al.,(2005) reported that
the emergence and spread of bacterial
resistance made the treatment of infectious
diseases more problematic. The
antimicrobial activity of a plant is highly
related to secondary substances that are
synthesized and produced by these plants
(Cowan 1999).
Secondary metabolites are substances of
low molecular weight, which were not the
products of the primary metabolic pathway
of the producing organism and at first
thought to be with no advantage to the
plant. Nowadays it is believed that they
have vital functions (Kant et al.,2010).The
utilization of plant extracts and
phytochemicals, with known antibacterial
characteristic, may be of immense
significance in therapeutic
treatments.Several studies have been
conducted in different countries to
substantiate such efficiency (Almagboul et
al.(1985) and Rakholiya and Chanda,
2012).
 This study has been aimed to
determine the antimicrobial activity of olive
leaf extracts against five pathogenic
bacteria (Bacillus cereu ,Salmonella
typhimurium ATCC 14028 ,Pseudomonas
aeruginosa ATCC 9027 , Staphylococcus
aureus ATCC 25923 and Bacillus
subtillus ATCC 6633).
MATERIAL AND METHODS
Olive Leaf Extract ( OLE ) Preparation Olive leaves were collected and put
in plastic bags. The plant material was then
dried at room temperature and powdered (20
mesh).Ground powdered leaves were
extracted as reported by Hassan et al. ( 2013 )
using ethanol (70% v/v) at 20% (w/v)
concentration. The mixture was mixed on
rotary shaker for three hours and filtered
through whatman no.4 , and then membrane
filter (0.45 um). To obtaine the solid residues
of the olive leaf extract , the extracts were
dried in rotary evaporator under lower
temperature.
Pathogenic indicators
The used bacterial indicators were,
Bacillus subtillus ATCC 6633 , Staphylococcus
aureus ATCC 25923, Pseudomonas
aeruginosa ATCC 9027, Salmonella
typhimurium ATCC 14028, Bacillus cereus.
These pathogens were kindly provided by the
staff members of The National Institute of
Oceanography and Fisheries (NIOF),
Alexandria branch.
Preparation of pathogenic bacterial
indicators:
The pathogenic bacteria
indicators were grown in nutrient broth and
incubated at 38 °C for 24 h. The cells were
centrifuged at 7000 rpm , and standardized to
OD 600 nm 0.1 and stored at 4 °C until ready
for use (Cwala et al., 2011).
Screening for antimicrobial activity:
The well-cut diffusion technique was
used to test the ability of the different
concentration from the crude extract to inhibit
the growth of indicator bacteria. Fifty
millimeters of nutrient agar medium
inoculated with indicator microorganism were
pored into plates. After solidifies, wells were
punched out using 0.5 cm cork

Hassan, et al.,2014
151
borer, and each of their bottoms was then
sealed with two drops of crude extract. One
hundred micro-liters of tested compounds
were transferred into each well. All plates
were incubated at 38 °C for 24 h, the
detection of clear inhibition zone around the
wells is an indication of antimicrobial
activities of the different isolates. (El-Masry et
al., 2002).
RESULTS AND DISCUSSION
Table ( 1 ) and Figures showed the
antibacterial activity of olive leaf extracts (at
300 and 600 ppm ) measured by inhibition
zone (mm) of the isolates against some
references of bacterial pathogens. In general
results indicated that olive leaf extracts
showed good inhibitory effects on pathogenic
bacteria.
Olive leaf extract (300ppm) showed good
antimicrobial abilities and the highest
inhibition of 15 mm against Bacillus subtillus
ATCC 6633 and against 12 mm
Staphylococcus aureus ATCC 25923, 12 mm
against Pseudomonas aeruginosa ATCC 9027,
18 mm against Salmonella typhimurium ATCC
14028, 15 mm against Bacillus cereus.
However , increasing of concentration olive
leaf extract (600 ppm ) showed a marked
increase in nhibition zone presented figures
(1).
Many studies confirm the positive role of
olive leaf extracts in inhibitory pathogenic
bacteria. Markin et al,(2003)
reported that water extract of olive leaf
with a concentration of 0.6% (w/v) killed
Staphylococcus. aureus in 3h exposure
and Bacillus. subtilis . On the other hand it
was inhibited only when the concentration was
increased to 20% (w/v) possibly due to spore
forming ability of this species . Pereira et al
.,(2007) revealed that the growth rates of S.
aureus was decreased while OLE
concentration increased .Sudjana et al, (2009)
studied the antibacterial activity of OLE with
a large variety of bacteria. In another study,
Korukluoglu et al.,(2010) investigated the
effect of the extraction solvent on the
antimicrobial efficiency of S. aureus.S.
thypimurium and they reported that solvent
type affected the phenolic distribution and
concentration in extracts, and antimicrobial
activity against tested bacteria.
Owen et al, (2003) added that phenolic
compounds within the OLE have shown
antimicrobial activities against several
microorganisms including
Staphylococcus.aureus, Bacillus. Cereus and
Salmonella. typhimurium .
In our study, the ethanolic olive
leaf extract of two concentrations showed
good antimicrobial abilities and highest
inhibition against pathogenic bacteria (
Bacillus cereu ,Salmonella typhimurium
ATCC 14028 Pseudomonas aeruginosa
ATCC 9027 and Staphylococcus aureus
ATCC 25923)
TABLE( 1 ) : Antibacterial efficiency of OLE at different concentrations (Inhibition zones in
millimeter) against some references bacterial pathogens:

Concentration (ppm)

600
300
Bacterial Pathogens
20
15
Bacillus cereu
20
1818
Salmonella typhimurium ATCC 14028
15
12
Pseudomonas aeruginosa ATCC 9027
15
12
Staphylococcus aureus ATCC 25923
20
15
Bacillus subtillus ATCC 6633


Hassan, et al.,2014
152



Figure 1):Antibacterial activity of OLE at different concentrations against various
bacterial pathogens.  


REFERENCES
Almagboul, A.Z.; Bashir, A.K.; Farouk, A.;
And Salih A. K . M .(1985) :
Antimicrobial activity of certain
Sudanese plants used in folkloric
medicine. Screening for antibacterial
activity. Fitoterapia 56, 331-337.
Benavente-Garcia, O .; Castillo, J.; Lorente,
J.; Ortuno, A. and Del Rio, J.A.,(2000):
Antioxidant activity of phenolics
extracted from Olea europaea L. leaves.
Food Chem. 68 , 457–462.
Bianco, A. and Uccella, N.,( 2000).
Biophenolic components of olives. Food
Res. Int. 33,475–485.
Cowan, M.M.(1999): Plant products as antimicrobial
agents. Clin.Microbiol. Rev., 12, 564-582.
Cwala, Z.; Lgbinosa, E. O. and Okoh, A. l. (2011):
Assessment of antibiotics
production potentials in four actinomycetes
isolated from aquatic environments of the
Eastern Cape Province of South Africa. Afr.
Pharm. Pharmacol. 5(2):118-124.
De Nino, A., Lombardo, N., Perri, E., Procopio, A.,
Raffaelli, A., and Sindona, G. (1997): Direct
identification of phenolic glucosides from
olive leaf extracts by atmospheric pressure
ionization tandem mass spectrometry.
Journal of Mass Spectrometry, 32, 533–541.
El-Masry, M. H.; Khalil, A.I.; Hassouna, M.S. and
Ibrahim, H.A.H . (2002): In situ and in vitro
suppressive effect of agricultural composts
and their water extracts on some
phytopathogenic fungi.World. J. Microbial.
Biotechno. 18:551-558.

Hassan, et al.,2014
153
Gucci, R., Lombardini, L. and Tattini, M. , (1997).
(1997):Analysis of leaf water relations in
leaves of two olive (Olea europaea)
cultivars differing in tolerance to salinity.
Tree Physiol. 17, 13–21.
Hassan, M.SH., M.M.Elsayed, A. M. Zaki ,
F.S.Hatour and Hanaa S. S.Gazwi
(2013) : The effects of olive leaf extracts (
OLEs) as antioxidants on changes
in canola oil during heating .J.Agric.Chem.
and Biotech.,
Mansoura.Univ,Vol.4 (10 ): 347-357.
Kant, R.U.; Pratibha, D.; Shoeb, A.
(2010):Screening of antibacterial activity of
six plant essential oils against pathogenic
bacterial strains. Asian J. Med. Sci. 2(3),
152-158.
Khayyal , M.T., El-Ghazaly , M.A., Abdallah, D.M.,
Nassar, N.N., Okpanyi, S. N. andeuter, M.
H., ( 2002): Blood pressure lowering
effect of an olive leaf extract
(Oleaeuropaea) in L-NAME induced
hypertension in rats. Arzneimittelforschung
52,797–802.
Korukluoglu. M; Sahan, Y; Yigit, A; Ozer, E.T and
Gucer. S (2010) : Antibacterial activity
and chemical constitutions of Olea
europaea L.leaf extracts ; Journal of ood
Processing and Preservation 34,383-396.
Lee, O.H and Lee , B.Y.(2010). Antioxidant and
antimicrobial activities of individual and
combined phenolics in Olea europaea leaf
extract. Bioresource Technology 101 .
3751–3754
Markin D, Duek L, Berdicevsky I. (2003) :
In vitro antimicrobial activity of
olive leaves. Mycoses;46:132-136.
Owen, R.W ; Haubner, R; Mier, W; Giacosa, A;
Hull, W.E; Spiegelhalder, B and Bartsch,
H. (2003): Olives and olive oil in cancer
prevention.. Food Chem. Toxicol; 41, 703-
717.
Pereira .A.P; Ferreira. I; Marcelino. F; Valentao. P;
Andrade. P. B; Seabra. R; Estevinho. L;
Bento. A. and Pereira. J.A.(2007): Phenolic
compounds and antimicrobial activity of
olive (Olea europaea L. Cv. Cobrançosa)
leaves. Molecules .12, 1153-1162.
Rakholiya K , Chanda S (2012) . In vitro
interaction of certain antimicrobial agents
in combination with plant extracts against
some pathogenic bacterial strains.
Asian Pac. J. Trop. Biomed. 2:876-880.
Ryan,D., Antolovich, M., Prenzler, P., Robards, K.,
and Lavee, S. (2002):
Biotransformations of phenolic compounds
in Olea europea L. Scientia
Horticulturae, 92, 147–176.
Sharma, R. ; Sharma, C.; Kapoor, B.
(2005):Antibacterial resistance: current
problems and possible solutions. Indian. J.
Med. Sci., .59(3),120--129.
Sudjana, A.N., D’Orazio, C., Ryan, V., Rasool, N.,
Ng, J., Islam, N., Riley, T.V.and
Hammer,K.A.,( 2009): Antimicrobial activity
of commercial Olea europaea (olive) leaf
extract. Int. J. Antimicrob. Agents 33, 461–
463.
Tasioula-margari, M. and Okogeri, O.
(2001):Isolation and Characterization of
Virgin Olive Oil Phenolic Compounds by
HPLC/UV and GC-MS. J .Food Sci.66,530-
534.
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