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Zahedan Journal of Research in Medical Sciences
Antibacterial Effect of Myrtus Communis Hydro-Alcoholic Extract on Pathogenic
Bacteria
Ali Taheri,*1 Amir Seyfan,2 Samira Jalalinezhad,3 Fatemeh Nasery4
1. Department of Fisheries (Seafood Sciences), Faculty of Marine Sciences, Chabahar Maritime and Marine Sciences University, Chabahar, Iran 2. Department of Fisheries, Faculty of Marine Sciences, Chabahar Maritime and Marine Sciences University, Chabahar, Iran 3. Medical Student, Zahedan Medicine University, Chabahar International Branch (Sina), Sistan and Balouchestan, Iran 4. Department of Agricultural Biotechnology, Iranian Veterinary Organization, Chabahar Office, Sistan and Balouchestan, Iran Article information
Abstract
Background: Today, due to the changes in the form of the resistance of pathogenic
bacteria, discovering new antimicrobial drugs is under study. So, the aim of this study is to evaluate the antimicrobial properties of the extract of the myrtle herb on some of Materials and Methods: Hydroalcoholic extract of the leaves of myrtle herb was
evaluated at 4 concentrations including 10-80 mg/ml on four strains of pathogenic bacteria using penetrative dissemination method together with the measuring diameter of the growth inhibition zone; then the results were compared to four conventional antibiotics. The minimum inhibitory and bactericidal concentrations were studied using macro dilution method. Results: Treatment by the concentration of 80 mg/ml extract of this herb showed the
Department of Fisheries (Seafood Sciences), Faculty of greatest effect on the bacterium Staphylococcus aureus and Vibrio cholera serotype Ogawa which had a significant difference with all other treatments and standard antibiotics (p< 0.05). The extract showed no effect on the bacterium Pseudomonas aeruginosa and just concentration of 80 mg/ml showed a little effect on E. coli and other antibiotics had no significant effect except tetracycline which has little effect on this strain. Minimum inhibitory concentration was 0.2 mg/ml for bacterium Staphylococcus aureus (S. aureus)
and the maximum for E.coli by 8 mg/ml.
Conclusion: This study showed that under study bacteria were more resistant to the
antibiotics and the extract of Myrtus communis leaves showed greatest antibacterial effect
against S. aureus and V. cholerae cerotype Ogawa.
Copyright 2013 Zahedan University of Medical Sciences. All rights reserved.
Introduction
oday, environmental problems and high prices of Khorasan River Valley, Sarab, Guilan-e-gharb, Kerman, some antibiotics have reinforced a tendency to Maharlu in Shiraz, Neyriz, Fasa, Mamasani and Bandar replace them with less harmful substances. Among Abbas [5]. The dried leaves of this herb contain the various materials to replace antibiotics, products of terpineolene, cineol, linalool, terpineole, linalyl acetate, plant origin have recently gotten a special place [1, 2]. tannins and flavonoids compounds and there are Herbal medicines have been the only source of pain numerous reports about the anti-parasitic and anti- treatment during the centuries and now with the infective properties of the extract of this herb [6- 10]. advancement of sciences and development in the Also its anti-virus effect has led to the production of anti- application of synthetic drugs, medicinal plants are still used in large-scale [3]. This issue is very important for the Today, the anti-parasitic and antimicrobial drug application in drug therapy or side applications such as resistance is a major problem in the world which is due to the uncontrolled use of antimicrobial drugs. This However, due to the climatic variation and the vast area resistance is so important that the theme of 2011 World of Iran, wide spectrum of medicinal plants are found there Health Organization was "Resistance to antimicrobial which are the basis of Iran's traditional medicine [4-6]. drugs is a global threat". This resistance can be Thus, in recent years, extensive researches have been transmitted by microbes from one generation to another conducted to evaluate the antimicrobial effect of the generation and even from a microbial species to another essential oils and extracts which shows the strength and species through the creation of an antibiotic-resistant gene ability of these compounds to inhibit the growth of a wide by them and ultimately the high levels of infection range of pathogenic microorganisms [7]. Myrtle (Myrtus remained stable despite the administration of antibiotics. communis) of the Mytaseae family is a shrub, evergreen Until recently, several studies have been conducted on the and aromatic herb with numerous stems and branches. antimicrobial properties of the leaf and stem extracts of This herb is found in the white Lab of Bakhtiari valley, myrtle against pathogenic bacteria and good results have Zahedan J Res Med Sci 2013 June; 15(6): 19-24. been obtained about its effects on staphylococcus aureus Concentration of bacteria became equal to the [12-4], E. coli [15-16], Lactobacillus plantarum [16], McFarland standard tube No. 0.5 (108×1.5) after 24 h Bacillus cereus [12], Listeria monocytogenes and incubation in the logarithmic phase of growth, using a Pseudomonas aeruginosa, [17-18], Klebsiella and spectrophotometer. This suspension was considered as a Shigella [17], though its antimicrobial effect against some reserve and diluted in a similar medium based on 1:100 strains of bacteria such as E. coli has been rejected in ratio while taking on the same day (6 10×5/1) [23]. Susceptibility to the nosocomial microorganisms to the Some gram-negative bacteria such as E. coli and myrtle extract was conducted using penetrative Pseudomonas aeruginosa and some gram-positive dissemination method. Bacterial suspension was seeped bacteria such as Staphylococcus aureus have specific on the medium using 20 ml sampler and was spread on characteristics as far as bacterial infections are concerned the medium with a sterile cotton swap; then it was dried and they are removed or isolated from the most of the for 5 minutes and incubated for 15 minutes in order to clinical samples referred to the diagnostic laboratories equilibrate the moisture inside the plates. [19, 20]. On the other hand, some reports on the cases of Sterile crude discs with 6 mm diameter were placed on being infected with cholera by Vibrio cholerae which is a the surface of medium and 15 microliters of the myrtle gram-negative bacterium are annually published and solution was seeped in a specified concentration on the being infected with this bacterium has been observed in discs. Standard discs of streptomycin 10, gentamicin 10 the southern districts of the country due to the and erythromycin 15 and tetracycline 30, produced by consumption of contaminated raw or semi raw fishes and vegetables irrigated with sewage. Accordingly, given the Plates were incubated for 24 hours at 37°C and bacterial importance of medicinal plants in the traditional medicine inhibition zone was measured and recorded using Vernier and very few side effects of these medicines on human calipers. Experiments were conducted with three and also due to the changes in the form of the resistance replications. The minimum growth inhibitory of pathogenic bacteria which requires monitoring of concentration and Minimum Lethal Concentration (MLC) antibacterial effects substances in periodic intervals, the of antimicrobials were determined using the tube dilution antimicrobial effects of hydroalcoholic extract of Myrtus method [24, 25]. A series of nine test tubes were used for communis leaves against the above-mentioned pathogenic each concentration and each bacterium. There were 7 bacteria have been evaluated in this study. tubes for different dilutions, a positive control tube and a negative control tube. 9 ml nutrient broth was added to Materials and Methods
the test tubes and then was sterilized. 1000 microliters of the diluted extract was added to the first tube and after This study was conducted in 2011 at the Laboratory of being homogenized, 1000 microliters of the homogenized Microbiology of Chabahar University of Maritime and fluid was added to the second tube and this operation was Marine Sciences. Extraction was carried out with slight continued and 1000 microliters of homogenized solution variations based on the study conducted by Sadeghi [21]. (from 7th tube) was discarded. 10 microliters of the Accordingly, the leaves of myrtle were collected in spring bacterial suspension was added to all tubes except the from Zaranjan located in the district of Fasa and they negative control tube based on McFarland tube 1. were dried in the shade after washing. 600 grams of the All tubes were incubated for 24 h at 37 C and then the ground leaf powder was mixed with 500 ml of distilled tubes were evaluated in terms of turbidity due to the water and ethyl alcohol 96 at 1:1 ratio and was kept in a bacterial growth. The last tube in which no turbidity was dark place for 48 hours. The contents of the Erlenmeyer seen was considered as the minimum growth inhibitory flasks were stirred for 25 minutes once every 16 hours. concentration. The solutions inside all tubes with no After 48 hours, the contents of the Erlenmeyer flask were growth turbidity were cultured using pour plate method smoothed by the filter paper and smoothed fluid was for determining minimum bactericidal concentrations of extracted in the rotary evaporation apparatus under the myrtle extract and the final concentration of the vacuum and 50̊C temperature. The concentrated extract extract which was able to kill 99.9% of the live bacteria, was poured in sterile Petri dish and dried in the oven with 40̊C. Dried powders were collected and were prepared at concentration of the microorganisms [26, 27]. concentrations of 10, 20, 40 and 80 mg using sterile Statistical analysis: Experiments were performed in
distilled water. Staphylococcus aureus, Pseudomonas three replications and Graphpad-Prism 7.00 software was aeruginosa, Vibrio cholerae serotype Ogawa and E. coli used for the data analysis under analysis of variance test; Bacteria were prepared from the samples of patients and Duncan test was used to compare treatments at 95% referred to Imam Ali hospital of Chabahar city after doing specialized microbiologic tests for confirming the presence of microorganisms. Cultures were purred and stored at refrigeration temperature until the start of the study [22]. One day before experiments, a small portion Table 1 shows the results of penetrating emissions test of the mother culture was added to the Hinton broth of the myrtle extract with different concentrations on under-study microorganisms. Accordingly, the concentration of 80 mg/ml of myrtle extract showed the Antibacterial effect of myrtus communis Taheri A et al. greatest effect on the Staphylococcus aureus bacterium Table 2. Comparison of the minimum growth inhibitory concentration
and no significant difference was seen with all other and minimum bactericidal concentrations of the myrtle leaf extract on microorganisms treatments and control antibiotics and showed a higher efficacy (p<0.05). Lower concentrations also showed a significant difference with each other and all of them had significant differences with antibiotics except the concentration of 10 mg/ml that had no significant difference with gentamicin (p<0.05). Erythromycin and streptomycin did not show any effect on bacteria. Myrtle extract, at any concentration, did not show any effect on the bacterium Pseudomonas aeruginosa and the highest Discussion
effect of control group of antibiotics was observed in gentamicin which had a significant difference with In the present study, hydroalcoholic extract of the myrtle leaves showed the greatest effect on Staphylococcus The study conducted on the effects of myrtle extract on aureus which had significant difference with the standard E. coli bacteria also showed that only the concentration of antibiotics. The positive effect of the myrtle extract on 80 mg/ml has the effect on this strain of bacteria and Vibrio cholerae serotype Ogawa was well. In this study except for the very light effect of tetracycline on this on these two strains of bacteria, concentration of the used strain, other control antibiotics did not have any effect. extract also caused a significant difference in the results. Concentration of 80 mg/ml showed the highest effect on Myrtle extract showed a very little effect on E. coli only the Vibrio chlorea Bacterium which had a significant at the highest concentration but Pseudomonas aeruginosa, difference with all other treatments (p<0.05). was completely resistant to the extract. Lower concentrations also showed a positive effect, all For centuries in Iran, several herbs have been used to of them had a significant difference with each other treat diseases. Different generations' trust in the treatment (p<0.05). Tetracycline was the only antibiotic effective on with the traditional medicine demonstrates the positive this bacteria which had a significant difference with other impact of this type of treatment. On the other hand, the concentrations except that concentrations of 10 and 20 increasing resistance of pathogenic bacteria and mg/ml (p<0.05). Results of minimum inhibitory continuous change of the resistance form of these concentration of growth and minimum bactericidal microorganisms have led to the major challenges in the concentrations after 24 h culture were shown in table 2. use of common antibiotic drugs which requires the search According to the results of this table, myrtle extract had for the new compounds with anti-bacterial properties. no effect on the bacterium Pseudomonas aeruginosa. Myrtle is an aromatic and medicinal herb for which Minimum inhibitory concentration belonged to the antibacterial, antifungal, antiviral, antioxidant and anti- bacterium Staphylococcus aureus and the highest rate mutagenicity properties have been reported [28-32]. belonged to the E. coli. Similar results were obtained Positive properties of the myrtle extract on hospital about the minimum bactericidal concentration. bacteria have been reported. In a study, effects of several plants, including myrtle against Streptococcus Table 1. Comparison of the inhibition zone diameter (mm) of myrtle
pneumoniae, Haemophilus influenzae and Moraxella extracts and standard antibiotics on under study microorganisms catarrhalis isolated from the patients in hospitals were evaluated and the results showed that the myrtle extract creates 30, 50 and 22 mm growth inhibition zone in the diffusion penetration test; therefore the use of myrtle extract for the treatment of sinusitis and bronchitis was The positive effect of the myrtle extract on S. aureus in this study was consistent with the results obtained by Salvagnini. These researchers were studied the effect of the oil and ethanolic extract of myrtle on several strains of bacteria and reported that the ethanolic extract of myrtle has a positive effect on S. aureus with 12 mm inhibition zone. But the oil has shown a greater effect [29]. Alem also reported the antibacterial effects of the myrtle extract on S.aureus isolated from human samples with 0.5 mg/ml of the minimum inhibitory concentration [14]. Ghlamhsynyan Najjar et al. who had observed a very positive effect of the chloroform, ethyl acetate and * There was no inhibition zone; treatment of any columns indicated methanol extracts of the myrtle leaves on S. aureus significant difference with dissimilar letters acknowledged that antimicrobial effects of myrtle extract on this bacterial strain is partly related to the stimulation of free radicals and the concentration of the extract has an Zahedan J Res Med Sci 2013 June; 15(6): 19-24. effect on antibacterial activity [34]. In the study of control the bacteria [36- 38]. Due to this reason, a gram-positive bacterium is more susceptible to the extract than gram- concentrations of the myrtle extract on Pseudomonas No report on the effects of myrtle extract against the Fewer efficacies of these antibiotics can confirm the bacteria causing cholera (i.e. Vibrio cholera) has been resistance of the mentioned strains of bacteria and on the reported yet. The results of this study show the positive other hand, various studies have shown the effects of effect of the hydroalcoholic extract of the myrtle on this myrtle extract on P. aeruginosa [6, 35]. strain of bacteria; serotype Ogawa which is much better For example Alem reported the effect of myrtle extract than that of tetracycline which is the only antibiotic on Pseudomonas aeruginosa isolated from human cases effective on this bacterium. In a study conducted by with a minimum inhibitory concentration of 120 mg/l Rahbar et al. Vibrio cholerae serotype Inaba and Ogawa [14]. But the myrtle extract used in this study had no were sensitive to Tetra Doxycycline, erythromycin and effect on bacteria which could be due to the mutations in ampicillin [39]. It can be said that the less efficacy of the bacterial strains which are still sensitive to antibiotics antibiotics in this study can be due to the resistance of this but are resistant to the myrtle extract, although differences strain of pathogenic bacteria. Several studies have in the extracted essential oil and even used concentrations reported the antibacterial activity of essential oil extracted could be another reason for the lack of conformity with from the herb. In recent years, many of these researchers the present findings. In a study conducted by Amensour, came to this understanding that the presence of phenolic leaf and stem extract of myrtle was examined on 15 compounds in the leaves of myrtle herb is due to the strains of bacteria which cause food borne diseases. They presence of Flavenols (Quercetin glycosides and reported that the methanol and ethanol extracts of the myricetin) and derivatives of Galoyl including Galoyl myrtle leaves and branches have respectively glycosides, Alajytanyn, the Galvyl–quinic acid [40] and antimicrobial properties on Listeria monocytogenes, they attributes the antibacterial activity of these Pseudomonas aeruginosa and Staphylococcus aureus and compounds to the presence of polyphenols [41]. the diameters of the inhibition zone of ethanol extract in Many studies have shown that the mechanism of this these bacteria are respectively 30, 23 and 37 mm and effect is due to the influence on the cell wall and reported minimum bactericidal concentration for Staphylococcus that the cell wall and cell membrane are affected and their aureus was reported less than 0.075 mg/ml. Also the permeability is changed and lead to the release of highest antibacterial activity was against gram-positive intracellular contents which can be accompanied with the bacteria [18]. Comparison of the results of this study disrupt in the membrane function such as electron shows that the extract has no effect on Pseudomonas transfer, enzyme activity or nutrient absorption [18]. aeruginosa and the inhibition zone of Staphylococcus In sum, it can be said that the results of this study aureus was about 20 mm in the best condition and the indicate the antibacterial effect of hydroalcoholic extract minimum bactericidal concentration for Staphylococcus of the leaves of myrtle herb on some pathogenic bacteria, especially Staphylococcus aureus and Vibrio chloreae The comparison shows the difference between which can be an alternative for the treatment of diseases pathogenic bacteria and food born disease agents and may resulted from these bacterial strains that the resistance be another proof for the resistance of bacteria studied in form of which has been changed as an herbal antibiotic this study. In the studies conducted on the effects of with side effects fewer than conventional antibiotics. Also extracts on E. coli, most of the reports show that the working on other extraction materials and methods such myrtle extract has no effect on these bacterial strains [18, as extracting by methanol and ethyl acetate may indicate 29]. In a study conducted in Iran in the past, the positive different functions. Also clinical confirmation and effect of alcoholic extract of the myrtle herb on E. coli pharmacological standardization is required before their had been reported. Also according to the report of Ghasemi Pirbalouti et al. although the methanol extract showed no effect on the diffusion penetrating activity, but Acknowledgements
the minimum lethal concentration for E. coli was 10 mg/ml [6, 35]. Comparison of the results of this study We sincerely appreciate the staff of Imam Ali Hospital revealed that myrtle extract, even in the highest and Chabahar Veterinary Office for their cooperation with concentration, has little effect on the diffusion penetration us. This study has been conducted in the framework of of E. coli and minimum lethal concentration was Mr. A. Seyfan's research project of Fisheries Engineering determined as 40 mg/ml. The resistance of this strain of at the University of Maritime and Marine Sciences of pathogenic bacteria could be a reason for this finding. It Chabahar. Therefore, we appreciate the authorities of this has been generally reported that the gram-negative university for the provision of the laboratory facilities for bacteria are more resistant to the extracts and essential oils of this herbs, because hydrophilic structure of the cell walls of the gram-positive bacteria is mainly composed of Authors’ Contributions
penetration of hydrophobic oil and also prevents All authors had equal role in design, work, statistical accumulation of its compounds in the cell membrane of Antibacterial effect of myrtus communis Taheri A et al. Conflict of Interest
The authors declare no conflict of interest. Funding/Support
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