ANTIMICROBIAL ACTIVITY OF SOME IRANIAN MEDICINAL PLANTS

The major aim of this study was to determine the antimicrobial activity of the extracts of eight plant species which are endemic in Iran. The antimicrobial activities of the extracts of eight Iranian traditional plants, including Hypericum scabrum, Myrtus communis, Pistachia atlantica, Arnebia euchroma, Salvia hydrangea, Satureja bachtiarica, Thymus daenensis and Kelussia odoratissima, were investigated against Escherichia coli O157:H7, Bacillus cereus, Listeria monocytogenes and Candida albicans by agar disc diffusion and serial dilution assays. Most of the extracts showed a relatively high antimicrobial activity against all the tested bacteria and fungi. Of the plants studied, the most active extracts were those obtained from the essential oils of M. communis and T. daenensis. The MIC values for active extract and essential oil ranged between 0.039 and 10 mg/ml. It can be said that the extract and essential oil of some medicinal plants could be used as natural antimicrobial agents in food preservation.


INTRODUCTION
Food spoilage is one of the most important issues facing the food industry.In fact, food-borne illnesses are a global problem, even in developing and developed countries.Food spoilage or deterioration is predominantly caused by the growth of microorganisms.Many pathogenic microorganisms, including Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Campylobacter jejuni, Candida spp., Zygosaccharomyces spp., Fusarium spp., Aspergillus spp., Rhizopus spp., Penicillium spp., and Salmonella spp.have been identified as the causal agents of food-borne diseases or food spoilage (Betts et al., 1999;Solomakos et al., 2008).
Listeria monocytogenes is a Gram-positive bacterium responsible for the severe food-borne illness, listeriosis.This disease is primarily transmitted through various foods, fish, dairy products, cured or processed meat, egg, poultry, seafood, salad, fruits and vegetables (Garcia et al., 2004).Among severe infections, listeriosis has been associated with a mortality rate as high as 30-40% (Datta, 2003).
Bacillus cereus is a species of Gram-positive bacteria inhabiting numerous environments including soil, plant materials and many foods.Due to their spore-forming ability they can pose problems in the food industry (Andersson et al., 1995).In addition, the production of toxins in foods (emetic) or in the human intestine can lead to emesis or diarrhea, respectively.At least three enterotoxins are involved in the pathogenesis of B. cereus diarrheal gastroenteritis (Granum and Lund, 1997).
E. coli O157:H7 is recognized as an important cause of food-borne disease.This pathogen is a Gram-negative, facultative anaerobe bacterium, with a low infection dose of 50-5 organisms (Betts, 2000).According to reports, less than 1% of these food-borne diseases were due to E. coli.
Candida albicans, which exists as a commensal organism in the mucocutaneous cavities of the skin, vagina and intestine in humans, can cause infections under altered physiological and pathological conditions, such as infancy, pregnancy, diabetes, prolonged broad-spectrum antibiotic treatment, steroidal chemotherapy and AIDS (Friedman et al., 2000;Kennedy et al., 2000).
Antibiotic resistance has become a global concern.In recent years there is increasing incidence of multiple resistance in human pathogenic microorganisms, largely due to the indiscriminate use of commercial antimicrobial drugs commonly employed in the treatment of infectious diseases (Westh et al., 2004).This has forced scientist to search for new antimicrobial substances from various sources like medicinal plants.Search for new antibacterial agents should be continued by the screening of many plant families (Parekh and Chanda, 2007).

Plant material
An ethnobotanical survey was conducted in Chaharmahal va Bakhtiari Province, South-West of Iran.The survey was conducted by interviewing traditional healers in each locality using the local language.Each interview followed a semistructured questionnaire designed to obtain the following information: scientific and local plant names; habit; plant parts used; uses/ailments treated.The plants were collected from the mountain areas of Zagross, Chaharmahal va Bakhtiari district, during May -September, 2008.Their identity was confirmed and voucher specimens were deposited at the Research Centre of Medicinal and Aromatic Plants, Islamic Azad University, Shahrekord Branch, Iran.

Extract preparation
Dried plant materials were powdered (200 g) and subjected to hydro-distillation (in 2 000 ml distilled water) for 4 h, using a Clevenger-type apparatus according to the method recommended in British pharmacopoeia (British Pharmacopoeia, 1988).The leaves, roots and flowers of some of the plants, shade dried and ground into a powder (100 g), macerated in 200 ml of 80% ethanol, filtered and dried at 35°C under a rotary vacuum (Model Zirbus 302®, Italy).The extract samples were stored in universal bottles and refrigerated at 4°C prior to use.

Bacterial and fungal strains
The two Gram-positive (L.monocytogenes and B. cereus) and one Gram-negative (E. coli O157:H7) bacterial strains and one fungal strain (C.albicans) were all clinical isolates obtained from the Food Microbiology Laboratory, Veterinary Medicine Faculty, Islamic Azad University of Shahrekord Branch, Iran.They were identified using conventional morphological as well as biochemical tests.Stock cultures of the bacteria were kept in 20% glycerol PBS (phosphate buffered saline) at -70°C.Active cultures were generated by inoculating 100 μl of the thawed microbial stock suspensions into 5 ml of nutrient broth (Merck, Germany), followed by overnight incubation at 37°C.The yeast was cultured overnight at 35°C in Sabouraud dextrose agar (SDB) (Merck, Germany).Each microorganism was suspended in sterile saline and diluted at ca. 10 7 colony-forming unit (CFU/ml).

Antimicrobial test Disc diffusion assay
The disc diffusion method of Iennette (1985) was used with some modification to determine the rate of growth inhibition of bacteria and fungi by the examined plant extracts and essential oils.BHI agar and Sabouraud dextrose agar (Merck, Germany) were used to prepare the culture medium and autoclaved at 121°C for 15 min.Plates (8-cm diameter) were prepared with 10 ml agar inoculated with 1 ml of each microbial suspension.The extracts were dissolved in dimethyl sulfoxide (DMSO, 20 μl) before testing for antimicrobial activity.Sterile paper discs (6 mm in diameter) were impregnated with 60 μl of dilutions of known extract concentrations (100 μg/disc) and incubated at 37°C for 48 h.The plates contained L. monocytogenes incubated at 4° C for 48 h.A disc (6 mm diameter) of erythromycin (10 μg) was used as a positive control.Amphotericin B (5 mg/ml) was dissolved in DMSO and served as a positive control.Microbial growth inhibition was determined as the diameter of the inhibition zones around the discs (mm).The growth inhibition diameter was the average of three measurements, taken at three different directions.All tests were performed in triplicate.

Serial dilution
The minimal inhibitory concentration (MIC) value was determined using serial dilution assay.The MIC was defined as the lowest concentration of the compound to inhibit the growth of 50% of microorganisms.All extracts were initially tested at 10 000 μg/ml and serially diluted to 39 μg/ml.Each tube was inoculated with 5 ml of microbial suspension at a density of 10 7 CFU/ml and incubated at 37°C for 48 h.The plates contained L. mono-cytogenes incubated at 4°C for 48 h.The growth of microorganisms was observed as turbidity determined by the measure of optical density at 600 nm (Eppendorf spectrophotometer, AG, Germany).Erythromycin and amphotericin B were included as positive controls in each assay.Extract-free solution was used as a negative control.Control tubes were incubated under the same condition.All assays were carried out in triplicate.The inhibition demonstrated by the extracts is expressed by the following equation (Zampini et al., 2005): where OD c is the OD 600 for the negative control (containing no extract) and OD t is the OD 600 for the sample treated with the antimicrobial compounds.

Ethnobotanical survey
The results of the survey are presented in Table 1.

Antibacterial test
The growth inhibition value of extracts and essential oils on microbial strains are shown in table 2. The extracts from the different plant species studied showed antimicrobial activities, with the diameters of the inhibition zone ranging from 7 to 30 mm.There were significant differences (P ≤ 0.05) in the antimicrobial activities of the plant extracts.Among the plants tested, the essential oils of M. communis and T. daenensis showed the best antimicrobial activity.These were followed by the ethanol extracts of H. scabrum and P. atlantica, the essential oil of S. hydrangea and erythromycin and amphotericin B which exhibited strong inhibitory activities (Table 2).The results showed that most of the extracts and essential oils could effectively inhibit the growth of B. cereus.
Among the plants that were tested, only erythromycin and the ethanol extract of P. atlantica and H. scabrum could effectively inhibit the growth of E. coli O157:H7; erythromycin and the essential oils of M. communis and T. daenensis could effectively inhibit the growth of L. monocytogenes.Interestingly, the essential oils of T. daenensis, M. communis, S. hydrangea and S. bachtiarica showed promising inhibitory activity against C. albicans (Table 2).
Subsequent experiments were conducted to determine the minimal inhibitory concentration of all selected plant extracts and essential oils.The results are presented in Tables 3 and 4.Among the plants tested, T. daenensis, S. hydrangea and S. bachtiarica showed the best antimicrobial activities (Table 3).Also, the essential oils of M. communis and K. odoratissima and the ethanol extract of T. daenensis, M. communis, S. hydrangea and H. scabrum showed promising antimicrobial activities against L. monocytogenes, B. cereus and C. albicans (Table 3).The MIC (>50% growth inhibition) for the extracts and essential oils of the medicinal plants is presented in Table 4.The MIC values for active extracts and essential oils ranged between 0.039 and 10 mg/ml.The results obtained appeared to confirm the antimicrobial potential of the plants investigated.The essential oil of T. daenensis showed the best MIC value and activity against three bacteria strains and yeast used.

DISCUSSION AND CONCLUSION
The presented results show that Gram-positive bacteria (L.monocytogenes and B. cereus) were more sensitive than Gram-negative bacteria (E. coli O157:H7) (Table 2-4).The antimicrobial activities of the extracts and essential oils of the plants varied in relation to the test organisms.The most active was a 10 mg/ml concentration that inhibited completely the growth of all Gram-positive bacteria and yeast.Cos et al., 2006, reported that Gramnegative bacteria are generally more resistant compared to the Gram-positive ones.Also, Shan et al., 2007  cereus) were generally more sensitive to the tested extracts than Gram-negative (Escherichia coli and Salmonella anatum).S. aureus was the most sensitive, while E. coli was the most resistant.In the serial dilution assay C. albicans was more sensitive than the bacterial strains.
A possible explanation for these observations may lie in the significant differences in the outer layers of Gram-negative and Gram-positive bacteria.Gram-negative bacteria possess an outer membrane and a unique periplasmic space not found in Gram-positive bacteria (Duffy and Power, 2001).The resistance of Gram-negative bacteria towards antibacterial substances is related to the hydrophilic surface of their outer membrane which is rich in lipopolysaccharide molecules, presenting a barrier to the penetration of numerous antibiotic molecules.It is also associated with the enzymes in the periplasmic space, which are capable of breaking down the molecules introduced from outside (Nikaido, 1994;Gao et al., 1999).Grampositive bacteria do not possess this type of outer membrane and cell wall structure.Antibacterial substances can easily destroy the bacterial cell wall and cytoplasmic membrane and result in a leakage of the cytoplasm and its coagulation (Kalemba and Kunicka, 2003).
The strongest activity (MIC 0.039 mg/ml) was shown by the essential oils of T. daenensis and S. bachtiarica against B. cereus and C. albicans, and the essential oils of M. communis, S. hydrangea against L. monocytogenes and C. albicans.In this study, the extracts and essential oils were significantly more active against Gram-positive bacteria and yeast (MICs ranging from 0.039 to 10 mg/ml) than against Gram-negative bacteria (MICs > 2.5 mg/ml).Good activity was also observed in the essential oils of T. daenensis and S. bachtiarica, which inhibited microbial growth from 0.039 to 2.5 mg/ml.Other extracts showed only slight inhibition of the tested microorganisms.
Some studies claim that the phenolic compounds present in spices and herbs might also play a major role in their antimicrobial effects (Hara-Kudo et al., 2004).There has been no large scale, systematic investigation of the relationship between bacterial inhibition and the total phenolic content of spices and herbs.Previous studies (Shan et al., 2005) showed that a highly positive linear relationship exists between antioxidant activity and total phenolic content in some spices and herbs.Many herb and spice extracts, for example T. daenensis and S. bachtiarica, contained high levels of phenolics and exhibited antibacterial activity against food-borne pathogens.Previous studies (Rasooli et al., 2006) on the antimicrobial activity of the essential oils of some Thymus spp., most of them possessing large quantities of phenolic monoterpenes, have shown activity against viruses, bacteria, food-derived microbial strains and fungi.
Pervious works (Ghasemi Pirbalouti et al., 2009) showed that the essential oils of T. daenensis and Thymus spp.(Elam ecotype) flowers exhibited antibacterial activities against L. monocytogenes from chicken meat.In a previous study, the minimum inhibitory concentration (MIC > 50 % growth inhibition) against L. monocytogenes for T. daenensis and Thymus spp.were 0.700 and 1.7 mg/ml, respectively.Fazeli et al., 2007 studied the antimicrobial effects of two medicinal plants (Rhus coriaria L. and Zataria multiflora Boiss.)used in Iranian traditional medicine which were investigated against some pathogenic food-borne bacteria.The minimum inhibitory concentrations of Rhus coriaria and Zataria multiflora were determined against several strains of Gram-positive and Gram-negative bacteria.They have reported that Bacillus cereus was found to be the most sensitive bacteria to Rhus coriaria, showing a MIC of 0.05%., while S. aureus and Proteus vulgaris ranked next with 0.10% followed by Shigella flexneri, E. coli and Salmonella typhi with a MIC of 0.20%.According to a report (Rasooli et al., 2006) the extracts and essential oils of Thymus erioealyx and Thymus porlock inhibited the growth of L. monocytogenes.The essential oils and extracts of some aromatic plants (e.g. the mint family, Lamiaceae) with a higher percentage of cavracrol and thymol have a higher efficacy against strain bacterial (Rasooli et al., 2006).
The result of a study (Ghasemi Pirbalouti et al., 2008) showed that the extracts and essential oils of S. bachtiarica, Scrophularia deserti and Zizyphus spina-christi inhibited the growth of C. albicans.Ghasemi Pirbalouti et al., 2009 reported that the essential oils of T. daenensis and Thymus khuzestanicum (MIC > 50% = 0.63 μl ml -1 and MLC > 99.9% = 22 μl ml -1 ) and the ethanol extract of Mentha longifolia showed higher of inhibition against Saprolegnia parasitica than the other extracts.Kokoska et al. (2002) reported that the ethanol extract of Salvia officinalis had strong antimicrobial activity against B. cereus, E. coli, and S. aureus.
According to the findings of this study, most of the extracts showed relatively high antimicrobial activity against all the tested bacteria and fungi.The essential oils of S. bachtiarica and T. daenensis leaves and flowers had antimicrobial activities.The present study suggests that the essential oil of these plants is a potential source of natural antibacterial agents.After this screening experiment, further work should be performed to describe the antimicrobial activities in more detail as well as their activity in vivo.Also phytochemical studies will be necessary to isolate the active constituents and evaluate the antibacterial activities against a wide range of bacteria population.