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The antibacterial activity of aqueous and 95% ethanol extracts of nettle leaf was tested against some Gram-negative and Gram-positive bacteria isolated from hospitalized patients by the agar well diffusion method. Staphylococcus aureus, Escherichia coli, Klebsiella spp., Bacillus Subtillus, Proteus spp. Salmonella spp. and Pseudomonas spp. were used. The results indicate that both extracts showed different antibacterial activities which were in favor of ethanolic extract because of more solubility of active ingredient in ethanol than in water. Staphylococcus aureus, Bacillus Subtillus, and Salmonella spp. showed the highest susceptibility to nettle extracts antibacterial effect, while E coli, Pseudomonas and Proteus were less susceptible. The only clear resistant bacteria isolate was Klebsiella spp.
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AL-Qadisiya Journal of Vet.Med.Sci. Vol./13 No./1 2014
1
Antibacterial effect of nettle (Urtica dioica)
N.A. Salih E.Dh. Arif D. J. Ali
Coll. of Vet. Med.Univ. of Sulaimani
Abstract
The antibacterial activity of aqueous and 95% ethanol extracts of nettle leaf were tested against
some Gram-negative and Gram-positive bacteria isolated from hospitalized patients by the agar well
diffusion method. Staphylococcus aureus, Escherichia coli, Klebsiella spp., Bacillus Subtillus, Proteus
spp. Salmonella spp. and Pseudomonas spp. were used. The results indicate that both extracts showed
different antibacterial activities which was in favor of ethanolic extract because of more solubility of
active ingredient in ethanol than in water. Staphylococcus aureus, Bacillus Subtillus and Salmonella
spp. showed the highest susceptibility to nettle extracts antibacterial effect, while E coli, Pseudomonas
and Proteus were less susceptible. The only clear resistant bacteria isolate was Klebsiella spp.
Key words: antibacterial, nettle, Urtica dioica, Gram negative bacteria, Gram positive bacteria.
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ةصخلا
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 
Introduction
Nettle (Urtica dioica) belongs to the family
Urticaceaeis recommended for complaints
associated with rheumatoid arthritis,
osteoarthritis and urinary tract infections,
allergies, Alzheimer’s, asthma, bladder
problems, bronchitis, cough, bursitis,
gingivitis, gout, hair growth and baldness,
hives, kidney stones, prostate enlargement,
sciatica, tendinitis (1,2). New antimicrobial
agents are needed to treat diseases in humans and
animals caused by drug resistant microorganisms.
Interest in plant-derived drugs has been
increasing, mainly due to the current widespread
belief that "green medicine" is safer and more
dependable than costly synthetic drugs (3,4).
Nettle is stated to possess antihaemorrhagic
and hypoglycemic properties. Traditionally, it
has been used for uterine hemorrhage,
cutaneous eruption, infantile and psychogenic
eczema, epistaxis, and melena and specifically
for nervous eczema (1, 2, 5). The German
Commission E approved internal use of nettle
leaf as irrigation therapy for inflammatory
disease of the lower urinary tract and
prevention of kidney gravel. The clinical
efficacy of many existing antibiotics is being
threatened by the emergence of multidrug-
resistant pathogens (3). Many infectious
diseases have been known to be treated with
herbal remedies throughout the history of
mankind. Natural products, either as pure
compounds or as standardized plant extracts,
provide unlimited opportunities for new drug
leads because of the unmatched availability of
chemical diversity. There is a continuous and
urgent need to discover new antimicrobial
AL-Qadisiya Journal of Vet.Med.Sci. Vol./13 No./1 2014
2
compounds with diverse chemical structures
and novel mechanisms of action for new
infectious diseases (6,7). Therefore,
researchers are increasingly turning their
attention to folk medicine, looking for new
leads to develop better drugs against microbial
infections (4, 1, 2, 8). So the present study
aimed to investigate the antibacterial effect of
nettle against some selected gram positive and
gram negative bacterial species.
Materials and methods
1. Samples: - one of the aerial parts which is
(leaves) of nettle was taken for the both extraction
procedures which obtained from Al-Kut city in
Iraq.
2. The microorganisms tested in this study were
isolated from hospitalized patients in the
Shoresh Hospital of Sulaimani city- Iraq.
Ethanolic extraction:
Plant were washed under tap water, and
then dried in room temperature in shade. The
dried plant was crushed by a laboratory
blender. Organic solvent extraction of nettle
was carried out by using ethanol (95% ethyl
alcohol) which is considered as a very
effective in extracting the active ingredients of
the plant according to method described by
(10, 11). This was done by using Soxhlet
apparatus, which consists of an electric heater
with a thermostat regulator upon which a
round bottom glass flask placed that fitted to
an extraction unit. The extracting unit contains
the solvent and cellulose (thumble) located
inside it that contains the dry plant powder. A
distiller unit is fitted on to the extraction unit.
For condensation of vapor solvent, 25 g. of
plant leaves powder was put inside the thumble
and 250 ml of 95% ethanol was put inside the
flask. The extraction was carried out for 24
hours by heating temperature that kept the
solvent at 50-60 Ċ. After that, the extract was
dried by using an electric oven at temperature
40-45 Ċ until dry extract was obtained. The
dry extract was placed in an incubator under
38-40 Ċ for complete dryness of the sample.
The dry ethanolic extract dissolved in
Dimethylsulphoxide (DMSO) to prepare
concentration of 30mg/ml that used for testing
its antibacterial activity.
Aqueous extraction:
Plants were washed under tap aqueous, and
then dried in room temperature in shade. The
dried plant was choped into small pieces and
then grinded by an electric grinder into
powder. Sixty grams from the grinded crude
powder was mixed with 100 ml distilled water.
Aqueous extract was carried out according to
(11,12) by using magnetic stirrer at 60C for 3
hours then filtrate was kept in incubator till
complete drying .The yield of dry powder was
15 gm. The dry powder dissolved in DMSO to
prepare concentration of 30mg/ml that used for
testing antibacterial activity.
Media preparation and antibacterial
activity:
The antimicrobial assay of aqueous and
ethanol extract of nettle was performed by agar
well diffusion method. The molten Mueller
Hinton agar was inoculated with 50 µl of the
tested microorganism (Cell suspension
containing 1 x 108 CFU/ml cells) according to
(McFarland standard 0.5).(6,7).For agar well
diffusion method, a well was prepared in the
plates with the help of a cork-borer (5mm).The
plates were incubated overnight at 37 C.
Microbial growth was determined by
measuring the diameter of zone of inhibition.
For each bacterial strain, the result was
obtained by observation of the zone of
inhibition (3, 9).
Bacterial isolate:
Clinical isolates of Staphylococcus aureus,
Escherichia coli, pseudomonas aeroginosa,
Proteus, Bacillus subtillus, Salmonella spp.
and Klebsiella spp. were used to test the effect
of nettle aqueous and ethanol extract to
demonstrate the antibacterial effect on these
microorganisms. Inhibition zone diameter of
5mm and above was taken as significant
susceptibility of each test micro organism to
the extract, we considered the range of
inhibition of (5-10mm)as slight and take one
plus(+), range of (10-15mm) as moderate and
take two plus (++), and range (=>15mm) as
high and take three plus (+++),while the
AL-Qadisiya Journal of Vet.Med.Sci. Vol./13 No./1 2014
3
negative result (-) refers to the absence of
inhibitory zone.
Results
Our objective was to evaluate the
antibacterial activity of nettle extract against
strains of bacteria isolated from sputum samples
of hospitalized patients (12, 13). The results
depicted in table (1) and also by figures (1, 2, 3, 4.
5. 6 and 7) indicating that the inhibition zone of
both extracts is so clear with some bacterial
isolate species. Seven different microbial
species were used to screen the possible
antimicrobial activity of both aqueous ant
ethanol extract of nettle. Nettle exhibited
antimicrobial activity against some of tested
microorganisms as shown in table (1), the
species used, Staphylococcus aureus is one of
the most common Gram-positive bacteria
causing food poisoning. Interestingly nettle
showed antibacterial activity against this
bacterium. As it is shown in table (1), other
generations of most bacteria was inhibited by
nettle are salmonella and pseudomonas. While
Escherichia coli, Proteus and Klebsiella are
resistant and show no antibacterial effect of
nettle.
Figure 1:- Effect of nettle aqueous extract 30 mg/ml (A) and ethanolic extract at 30 mg/ml(B),
against E. coli isolate growth.
Figure 2:- Effect of nettle aqueous extract 30 mg/ml (A) and ethanolic extract at 30 mg/ml(B),
against Salmonella growth.
Figure 3:- Effect of nettle aqueous extract 30 mg/ml (A) and ethanolic extract at 30 mg/ml(B),
against Proteus growth.
A
B
A
B
A
B
AL-Qadisiya Journal of Vet.Med.Sci. Vol./13 No./1 2014
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Figure 4:- Effect of nettle aqueous extract 30 mg/ml (A) and ethanolic extract at 30
mg/ml(B),against Klebsiella growth.
Figure 5:- Effect of nettle aqueous extract 30 mg/ml (A) and ethanolic extract at 30 mg/ml(B),
against Bacillus subtillus growth.
Figure 6:- Effect of nettle aqueous extract 30 mg/ml (A) and ethanolic extract at 30 mg/ml(B),
against Staphylococcus aureus growth.
Figure 7:- Effect of nettle aqueous extract 30 mg/ml (A) and ethanolic extract at 30 mg/ml(B),
against Pseudomonas growth.
A
B
A
B
A
A
B
AL-Qadisiya Journal of Vet.Med.Sci. Vol./13 No./1 2014
5
Inhibition zone diameter of 5mm and above
was taken as significant susceptibility of each
tested micro organism to the extract, we
considered the range of inhibition of (5-10mm)
as slight and take one plus (+), range of (10-
15mm) as moderate and take two plus(++), and
range (=>15mm) as high and take three
plus(+++).while the negative result (-) refers to
the absence of inhibitory zone.
Table (1) Antibacterial effect of aqueous
and ethanolic extract of nettle (Urtica dioica)
against some bacterial isolate
Microorganisms
Aqueous
Extract
(30mg/ml)
Ethanol
Extract
(30mg/ml)
E-Coli
_
+
Salmonella spp.
+++
+++
Proteus spp.
+
_
Klebsiella spp.
_
_
Bacillus subtillus
++
+++
Staphylococcus aureus
+
+++
Pseudomonas
aeruginosa
+
++
Discussion
Our results are in agreement with (8) and
(11) who refer that the extracted materials
from nettle have a powerful antibacterial
effect. Analysis of the chemical compounds of
nettle showed that nettle (Urtica dioica)
contained neophytadiene. Neophytadiene is
reported to be an antibacterial compound also
contain aromatic compounds including
carboxylic acids and esters were also.Reported
in this plant .Finally, fat acids including phtalic
acid, dibutyl ester, Bis (2 ethyl hexyl) maleat
and 1,2benzenedi carboxylic acid were
observed in this plant. These compounds are
reported to have anti putrefying and
antimicrobial effects. (16,22,23). Nettle have
other constituents which plays major role in
antibacterial effect such as alkaloids,
phenols, flavonoids, tannins and saponins in
these plants extract which have been claimed
to be responsible for their antimicrobial
activity (7,14).Alkaloid, it is antibacterial
activity may be due to its ability to react with
amino, carboxyl, sulfhydryl and hydroxyl
groups in bacterial protein as well as nucleic
acids, it highly reactive chemical compounds
that combine with proteins to give
intermolecular cross-links and intercalate with
DNA (15).Tanninic substances are another
constituents which are capable of precipitating
gelatin from solution, a property is known as
astringency. It has been reported to prevent the
development of microorganisms by
precipitating microbial protein and making
nutritional proteins unavailable for them
(9,16).Phenolic compound is mostly
hydrophobic, it has a hydroxyl group (-OH).
The importance of this group on antimicrobial
activity is well known, the site(s) and number
of hydroxyl groups on the phenol group are
thought to be related to their relative activity to
microorganisms (17,18).The high activity of
the phenolic components may be further
explained in terms of the alkyl substitution into
the phenolic nucleus, which is known to
enhance the antimicrobial activity of phenols.
The introduction of alkylation has been
proposed to alter the distribution ratio between
the aqueous and non-aqueous phases, including
bacterial phase, by reducing the surface tension
or altering the species selectivity. It was
suggested that plant products act via two main
mechanisms of action; the first is related to the
general hydrophobicity of plant products,
which facilitates their adhesion to the bacterial
surface inducing un-stabilization (9,14).The
second mechanism is the inactivation of
different molecules of the bacteria such as
enzymes or receptors by their adhesion to
specific sites (13, 18, and 19).Flavonoid
antibacterial activity is probably due to their
ability to complex with extracellular and
soluble proteins and to complex with bacterial
cell walls, more lipophilic flavonoids may also
disrupt microbial membranes.(20,21).The
differences in antimicrobial activity against
different bacterial isolate that noticed between
ethanolic and aqueous extract was in favor of
ethanolic extract may be because of the higher
solubility of some hydrophobic components
like phenols and flavonoids in ethanol than in
aqueous (20,21).Many researchers preferring
use of plant extract instead of antibiotics was
due to attenuation of pathogens virulence by
AL-Qadisiya Journal of Vet.Med.Sci. Vol./13 No./1 2014
6
plant extract as opposed to the direct killing of
pathogenic bacteria with antibiotic as a
strategy to combat infections is an interesting
concept. The idea that anti-pathogenic
molecules that prevents for instance the
production of toxins or abolish the ability of
bacteria to adapt to the host environment
would give a competitive advantage to the host
immune system to allow clearance of the
infectious organism. It is also anticipated that
such virulence attenuators would not affect
non-pathogenic bacteria communities or exert
a selective pressure for the development of
resistance as seen from the pressures exerted
by conventional antibiotics that targeted vital
bioprocesses in bacteria (9 and 19).
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... Contemporary research in this area has documented antihyperglycemic [16], diuretic [17], antifungal [18], and antibacterial [19] properties. Nettle extracts have been shown to treat arthritis [20] and breast cancer [21]. ...
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... The results of the statistical analysis also showed that E. coli, S. aureus and B. subtillus were the most sensitive with the plant extracts used in the experiment compared to the other isolates. Salih and Arif (2014) indicated the superiority of the nettle's alcoholic extract as an antibacterial on aqueous extract against some types of gram positive and negative bacteria which was attributed to the efficiency of alcohol in dissolving the hydrophobic compounds in the nettle plant represented by the phenols and flavonoids that were responsible for giving the inhibition properties against bacteria. These results agreed with those of Mirtaghi et al. (2016) w he re th ey con cl ude d th at the inhibitory effect of alcoholic nettle extracted at a concentration of 55 µg/disc against Pseudomonas and Klebsiella was due to its possession of carotenoids, terpenes, lignan, sterols and flavonoids. ...
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... Lhriga Leaves Decoction Oral 8 Urinary system problem Diabetes [6] Osteoporosis [30] Renal weakness, digestive system [1] Antibacterial [121] Antioxidant, antimicrobial, antiulcer, and analgesic [22] Evidence-Based Complementary and Alternative Medicine 11 Antioxidant activity [126] Antibacterial [127] 12 ...
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... demonstrated high susceptibility to nettle extracts, and they specify that both types of extracts exert antibacterial activities in different degrees. Ethanolic extracts have favorable effect compared to aqueous one due to the presence of more active ingredient in ethanol than in water 30 . ...
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... demonstrated high susceptibility to nettle extracts, and they specify that both types of extracts exert antibacterial activities in different degrees. Ethanolic extracts have favorable effect compared to aqueous one due to the presence of more active ingredient in ethanol than in water 30 . ...
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