Antibacterial Effect of Ethanolic and Methanolic Extracts of Plantago ovata
and Oliveria decumbens Endemic in Iran Against Some Pathogenic Bacteria
S.M. Seyyed Nejad
Plantago ovata (Plantaginaceae) and Oliveria decumbens (Umbliferae) are of important medicinal plants in Iran which have been used in traditional medicine. The aim of present study was to consider antibacterial properties of ethanolic and methanolic extracts of seed husk of Plantago ovata and aerial part of Oliveria decumbens. For this purpose, the 50 to 400 mg mL-1 concentration of these extracts were assayed against six Gram-negative and eight Gram-positive bacteria by disc diffusion method. Synthetic Antibiotic discs were used as control. Staphylococcus epidermidis and Staphylococcus aureus were the most sensitive species to the ethanolic and methanolic extracts of Plantago ovata while Pseudomonas aeruginosa was the most resistant to these extracts. Furthermore, Escherichia coli and Proteus mirabilis have shown resistance to ethanolic extract of this plant. Ethanolic extract of Oliveria decumbens was effective against all of tested bacteria and S. aureus was the most sensitive strain. In the case of the methanolic extract, Salmonella typhi, P. aeruginosa and P. mirabilis were more resistant than the others. The MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) values for Oliveria decumbens extracts against S. aureus were same (20 mg mL-1), whereas against Streptococcus pyogenes were different. Also, MIC for ethanolic extract of Plantago Ovata against S. aureus and Bordetella bronchiseptica were same (20 mg mL-1), whereas for methanolic extract were 20 and 10 mg mL-1, respectively. The MBC for these two bacteria werent found (>200 mg mL-1). On the basis of these results it can say that these plants have proper antibacterial effect and can be considered as a new source of antibiotic discovery and development for infectious disease treatment purposes.
to cite this article:
H. Motamedi, E. Darabpour, M. Gholipour and S.M. Seyyed Nejad, 2010. Antibacterial Effect of Ethanolic and Methanolic Extracts of Plantago ovata
and Oliveria decumbens Endemic in Iran Against Some Pathogenic Bacteria. International Journal of Pharmacology, 6: 117-122.
The medicinal plants have been used since ancient times. Evidence of using
these natural resources (Herbal remedies) in Iran goes back to the history itself
and there are lots of scientific documents in this area, Ibn-sina (Avicenna,
980-1037) has wrote many books on a wide range of topics but he is perhaps most
famous for his Laws of Medicines which contains sections on the formulation
of medicine, general medicine and other subjects that discuss the herbal medicines
in details (Lothfipour et al., 2008). In recent
years, antibiotic resistance has become a global concern and this problem is
more important especially in developing countries because infectious diseases
are still of important causes of morbidity and mortality among humans in these
countries. Plants readily synthesize substances for their defense against insects,
herbivores and microorganisms (Aboaba et al., 2006),
also plants maybe produce secondary antimicrobial metabolites as part of their
normal growth and development program or in response to stresses (Mirjana
et al., 2004). Plantago ovata Forssk. (Plantaginaceae)
and Oliveria decumbens Vent. (Umbliferae) are traditional medicinal
plants which have been used in Behbahan (Khuzestan, South West of Iran). Plantgo
ovata is a Winter annual plant that primarily inhabits desert regions of
the Northern hemisphere between the twenty-sixth and thirty- sixth latitudes
(Mayers and Liston, 2008). Plantago ovata is
commonly referred to Psyllium, indeed, Psylium is the husk from
the seed of P. ovata. Researches showed that this plant have hypocholesterolemic
(Salas-Salvado et al., 2007; Anderson
et al., 2000), anti-diarrhea (Washington et
al., 1998), anti-diabetic (Hannan et al.,
2006) and low anti-oxidant (Souri et al., 2008)
effects. Also, it has been reported that plantago ovata powder causes
allergic reaction when inhaled by occupationally exposed person (Bernedo
et al., 2008). Oliveria decumbens, a shrub commonly found
in South region of Iran, also has dispersed in South West of Anatolia, Syria
Oliveria decumbens is a relatively less explored plant. The limited
researches about therapeutic property of this plant are reported antibacterial
(Mahboubi et al., 2008) and antifungal (Mahboubi
and Feizabadi, 2008) activity of its essential oil, but about of its extracts
there isnt any reports. The aim of this study was to screen antibacterial
activity of ethanolic and methanolic extracts of O. decumbens aerial
parts and P. ovata seed husk against some clinical pathogens.
MATERIALS AND METHODS
Plant collection and identification: The plants used in this study were collected from hills around Behbahan (south east of Khuzestan, Iran) in May, 2008. The taxonomic identification of these plants was done by Herbarium in faculty of agriculture, Shahid Chamran University.
Plant extraction preparation: The seeds of P. ovata and aerial
parts of O. decumbens were shade dried at room temperature for ten days
and then were ground to a fine powder. One gram of powder was extracted by using
10 mL of alcohol (ethanol-or-methanol)-distilled water solution (8:2 v/v), centrifugation
(3000 rpm) for 15 min and collecting the supernatants. This process was repeated
for three times. Solvents then removed by evaporation (Seyyednejad
et al., 2001; Moazedi et al., 2007).
Bacterial strains: Fifteen bacterial species were used in this study. The Gram-positive species were Bacillus anthracis, Bacillus Pumilus, Bacillus cereus, Bacillus licheniformis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Streptococcus pyogenes and Corynebacterium renale. But S. pyogenes and C. renale were used only for O. decumbens and P. ovata, respectively. Gram-negative species were Pseudomonas aeruginosa, Bordetella bronchiseptica, Escherichia coli, Salmonella typhi, Proteus mirabilis and Klebsiella pneumoniae. These species were originally isolated from clinical specimens and identified by standard biochemical reactions.
Antibacterial susceptibility testing: The isolates were grown in Muller
Hinton Broth (MHB, Merck) medium at 37°C for 22 h. Final inoculum bacterial
number were adjusted to 108 cfu mL-1 with reference to
the Mc Farland turbidometry (Burt and Reinders, 2003;
Zuraini et al., 2007). A lawn culture was prepared
by pouring 0. 1 mL of bacterial suspension on Muller Hinton Agar (MHA Merck)
and dispersed by a sterile cotton swab and allowed to remain in contact for
1 min. Four concentrations of ethanolic and methanolic extracts (50, 100, 200,
400 mg mL-1) of both plants were prepared. The sterile filter paper
discs (6 mm diameter) (Cermelli et al., 2008;
Hsieh et al., 2001) were saturated through adding
50 μL of different concentrations of both extracts. Then, the discs were
placed on lawn cultures. The Petri dishes were subsequently incubated at 37°C
for 24 h and the inhibition zone around each disc was measured in mm. As positive
controls, discs containing different concentrations of seven antibiotics including
nafcillin 1 mcg, colistin 10 mcg, doxycycline 30 mcg, novobiocin 30 mcg, carbenicillin
100 mcg, methicillin 5 mcg and oxacillin 1 mcg were used. All these synthetic
antibiotics were produced by Difco. Discs impregnated with 80% of ethanol and
methanol was also included to test if they have inhibitory effect on the test
bacteria in this study.
MIC and MBC determination: MIC (Minimal Inhibitory Concentration) and
MBC (Minimal Bactericidal Concentration) of ethanolic and methanolic extracts
of these two plants were determined against two bacterial species (somewhat
important and more sensitive bacteria) S. aureus and Str. pyogenes
for O. decumbens and S. aureus and B. bronchiseptica for
P. ovata were determined. MIC was determined by macro broth dilution
assay method (Forbes et al., 1998). In the tube
dilution assay, standard bacterial suspension was added to tubes containing
1 mL MHB (Muller Hinton Broth) and different concentrations of extracts (5,
10, 20, 40, 80, 160, 200 mg mL-1). The tubes were incubated at 37°C
for 24 h. The first tube in the above series with no sign of visible growth
was reported as the MIC. MBC was determined by culturing one standard loop of
the tubes showing no apparent growth on MHA and subsequent incubation at 37°C
for 24 h. The least concentration that was inhibited no colony formation on
agar assumed as MBC for these extracts.
The results indicate that ethanolic and methanolic extracts of these plants
have antibacterial activities against both Gram-positive and Gram-negative bacteria.
Table 1 show that ethanolic extract of O. decumbens
was effective against all of the test bacteria even at one concentration. The
methanolic extract of this plant have antibacterial activity against the majority
of bacterial species, but S. typhi, P. mirabilis and K. pneumoniae
were resistant. However, S. aureus and B. bronchiseptica were
the most sensitive isolates to ethanolic and methanolic extracts, respectively.
Furthermore, inhibitory effect of both extracts of O. decumbens against
B. cereus observed only at 400 mg mL-1.
The antibacterial activities of both extracts from O. decumbens were
decreased in lower concentrations. Table 2 revealed that the
S. epidermidis and S. aureus were the most sensitive strains to
ethanolic and methanolic extracts of P. ovata, respectively. P. aeruginosa
was resistant to both extracts of this plant; also E. coli, L. monocytogenes
and P. mirabilis were resistant to ethanolic extract of P. ovata
even at highest concentration. Antibacterial activity of ethanolic and methanolic
extracts of P. ovata against all of sensitive isolates was decreased
at lower concentrations. Exceptionally, the effect of methanolic extract from
this plant against E. coli was increased with dilution of the extract.
Among tested Bacillus species, B. anthracis and B. licheniformis
had most sensitivity to O. decumbens and P. ovata, respectively.
zones (mm)* of antibiotic discs against some pathogenic bacteria
|*(6 mm) diameter disc, R: Resistant, -: Not used
In addition, results showed that the extracts of studied plants had good anti-Staphylococcocal
activity. The results of antibacterial activity of standard antibiotic discs
are shown in Table 3. All of the tested bacteria were resistant
to oxacillin and most of them presented resistance to colistin, nafcillin and
methicillin. MIC and MBC results are shown in Table 4. MIC
and MBC values for extracts of O. decumbens against S. aureus
were 20 mg mL-1. MICs for ethanolic and methanolic extracts of this
plant against S. pyogenes were 10 and 20 mg mL-1 and MBC values
were 20 and 40 mg mL-1, respectively. About P. ovata, MIC
values for both extracts against S. aureus were the same (20 mg mL-1).
The MICs for methanolic and ethanolic extract of this plant against B. bronchiseptica
were 10 and 20 mg mL-1, respectively. But MBCs for both extracts
of P. ovata against these two bacteria werent found (>200 mg
mL-1). Discs containing 80% ethanol and methanol did not have a zone
of inhibition probably due to the volatile nature of alcohol, so it was not
considered as a factor that might affect the results.
Nowadays, multiple drug resistant strains have been developed due to the indiscriminate
use of commercial antimicrobial drugs commonly used for infectious diseases
treatment. Unfortunately, bacteria have the genetic ability to transmit and
acquire resistance to drugs and chemicals (Nascimento et
al., 2000). Beyond the increasing prevalence of antibiotic resistance
among pathogenic bacteria, undesirable side effects of some synthetic antibiotics
add urgency to the search for new infection-fighting strategies, as well. Scientists
and pharmaceutical industries consider medicinal plants as a good choice, because
these natural resources have ordinary fewer side effects, are costless and effective
against broad spectrum of antibiotic resistant bacteria. In many parts of the
world, the extracts of medicinal plants are used for their antibacterial, antifungal
and antiviral properties (Hassawi and Kharma, 2006).
Plant species used in folk medicineare are potential for discovering extracts
with active biological compounds that have antibacterial activity. O. decumbens
and P. ovata are among the most important plants extensively used in
traditional medicine in South regions of Iran. Based on the results of the antibacterial
studies, shown in Table 1 and 2, the extracts
of both plants had antibacterial effect on both Gram-positive and Gram-negative
bacteria. Also, ethanolic extract of O. decumbens and methanolic extract
of P. ovata were the most effective extracts. Maximum activity of these
extracts frequently observed at the 200 and 400 mg mL-1 concentrations
so that at 400 mg mL-1, ethanolic and methanolic extracts of O.
decumbens had antibacterial activity against 100 and 86% of strains, respectively;
while at this concentration ethanolic and methanolic extracts of P. ovata
had inhibited 71 and 91% of isolates, respectively. Inhibitory activity of methanolic
extract of P. ovata against E. coli was increased along side with
decreasing concentrations; this may be due to the nature and number of available
bioactive compounds in the extract, cell membrane permeability or other factors.
In general, comparison of the inhibition zones diameter showed that extracts
of both plants are more effective against Gram-positive than Gram-negative bacteria.
This difference may be due to several possible reasons such as permeability
barrier provided by presence of cell wall with multilayer structure in Gram-negative
bacteria or the membrane accumulation mechanisms or presence of enzymes in periplasmic
space which are able to break down foreign molecules introduced from outside
(Parekh and Chanda, 2007; Abu-Shanab
et al., 2004; Holetz et al., 2002).
The diameter of inhibition zones around of the more active extracts in particular
ethanolic and methanolic extracts of O. decumbens and P. ovata,
respectively were comparable with standard antibiotics (Table
3). All of the Gram-positive and Gram-negative species were resistant to
oxacillin; and all of the tested bacteria except L. monocytogenes were
resistant to nafcillin. Furthermore, methicilin had no growth inhibition on
70% of tested isolates and more of the bacterial species were resistant to colistin.
S. aureus was resistant to methicillin and some of the other antibiotics,
while the extracts from both plants had noticeable activities against this bacterium.
This suggest new hopes for searching and discovering effective antibiotic against
MRSA (Methicillin Resistant Staphylococcus Aureus) strains which will
be a serious problem in future and can cause fatal infections. The MIC and MBC
(Table 4) for ethanolic and methanolic extracts of O. decumbens
were 20 mg mL-1, it is reported that for bactericidal antimicrobials
the MIC and MBC are often near or aquiline values (Reuben
et al., 2008) so we can say that extracts of O. decumbens
have bactericidal effect on S. aureus. Extracts from P. ovata
were unable to show bactericidal activity against S. aureus and B.
bronchiseptica even at 200 mg mL-1 (MBC>200 mg mL-1);
this result may be caused by high bacteriostatic effect of P. ovata extracts,
we can call these extracts as bacteriostatic agents which can inhibit bacterial
growth but generally do not kill them (Forbes et al.,
1998). Researches showed that the main components of the O. decumbens
essential oil are thymol (22%), carvacrol (22%) and p-cymene (19%); also carvacrol
is more effective against S. aureus than the others (Mahboubi
et al., 2007). Phytochemical investigations of Plantago species revealed
their high potential to produce a wide array of secondary bioactive metabolites,
i.e., iridoids, phenols, polysaccharides, sterols, alkaloids and cumarines that
have utilities as supplemented foods and as drugs to treat human diseases (Fons
et al., 2008). Moreover, considering the extracts of P. ovata
especially methanolic, it was more effective against Gram-positives while extracts
of O. decumbens especially ethanolic extract was more effective against
Gram-negative and Gram-positive bacteria, it can be concluded that perhaps these
two plants have two different mechanisms and target locations and can be used
to treat infection as synergism. In vitro assays may provide a guideline
to select the highly active plant extracts for subsequent isolation and identification
of potentially useful compounds, so isolation of bioactive constituents of active
extracts from studied plants can be a subject for next researches. Also, further
studies can evaluate in vivo efficacy of active extracts.
Finally, the extracts of both plants had antibacterial effect on both Gram-positive
and Gram-negative bacteria. Particularly, the extracts of studied plants had
good anti-staphylococcocal activity. Considering synergistic the effect of essential
oil of O. decumbens with vancomycin against S. aureus (Mahboubi
et al., 2007) and probable bactericidal activity of its extracts
on this bacterium, it seems that study of in vivo efficacy of O. decumbens
against S. aureus is important and suggested for further researches.
The authors wish to thank the vice chancellor for research of Shahid Chamran University, Ahvaz, Iran and Grant No. 672 for the research grant and financial support.
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