In vitro antibacterial activity and phytochemical analysis of some medicinal plants

Essential oils of medicinal plants have been used traditionally against pathogenic bacteria that caused infectious disease in human and microbial spoilage of food and have been used safely in herbal medicine as antibacterial compounds. In the present study, the antibacterial activities of the oils were evaluated against human and animals pathogenic bacteria. In this assay, the selective plants reported ethnobotanical uses traditionally and also were referenced in some herbal medicine text. The essential oil of Stachys pubescens, Mentha piperita, Clinopodium vulgare and Satureja hortensis were prepared by hydrodistillation and were analyzed by gas chromatography/mass spectrometry (GC/MS). The number 23, 22, 21 and 21 components were identified in S. pubescens, S. hortensis, M. piperita and C. vulgare, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of oils were determined with broth microdilution and agar diffusion method on bacterial strains. Results from the antibacterial testing indicated that S. pubescens, M. piperita and C. vulgare essential oils showed high activities and inhibited the growth of all the selected bacteria. While the essential oil of S. hortensis displayed the moderate potential activity. Our finding supported the notion that plant essential oils composition or total extract may have a role as pharmaceuticals and preservatives effects as safely and effective drugs with low resistance against microorganisms. Therefore, these essential oils could be used for management of these pathogens as a potential source of sustainable eco-friendly botanical bactericides.


INTRODUCTION
Herbal medicine has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural resources as a good choice, because these natural resources have ordinarily fewer side effects (Zargari, 1996).The medicinal plants have been proved effective in the treatment of infectious diseases and simultaneously decrease many of the side effects (Iwu et al., 1999).Also, they are costless and effective against a broad spectrum of antibiotic resistant microorganisms and they have very potent natural biologically active agents (Nychas et al., 2003).In many parts of the world, the extracts and essential oil of medicinal plants with active biological compounds are used for their antimicrobial and antiviral properties (Hassawi and Kharma, 2006) that have been used in folk medicine.The increasing occurrence of antimicrobial resistance represents a worldwide major concern for both human and veterinary medicine (Lorian, 1996).For this reason, there is a growing interest in the antimicrobial screening of extracts and essential oils from plants in order to discover new antimicrobial agents.Nowadays, about 25% of the drugs prescribed worldwide come from plants and 252 of them are considered as basic and essential by the World Health Organization (WHO).The WHO considers phytotherapy in its health programs and suggests basic procedures for the validation of drugs in developing countries.Infectious diseases are the second leading cause of death worldwide (Fazly-Bazzaz et al., 2005).From the time of the ancient Iranian, the plants were considered to protect against diseases.Iran has a very honorable past in traditional medicine, which goes back to the time of Babylonian, Assyrian civilization.One of the most significant ancient heritages is sophisticated experience of people who have tried over millennia to find useful plants for health improvement, with each generation adding its own experience to this tradition (Naghibi et al., 2005).Based on literature search, 18% of the plant species are used for medicinal purposes in Iran.Treatment of infections continues to be a problem in modern time, because of side effects of some drugs and growing resistance to antimicrobial agents.To investigate for novel, safer and more potent antimicrobials is a pressing need.Herbal medicines have received much attention as a source of new antibacterial with low side effect and significant activity (Fazly-Bazzaz et al., 2005).In the present study, the biological activities of four plants: Stachys pubescens, Mentha piperita, Satureja hortensis and Clinopodium vulgare were evaluated.Nowadays, there is a considerable research interest towards the compositional analysis of essential oil and extract.It has been reported that essential oil yield and their components in plants is related to genetic (Mohammed and Al-Bayati, 2009), climate, elevation, topography (Pourohit and Vyas, 2004;Rahimmalek et al., 2009a) and genotype (G), growing conditions (E) and their interaction (G × E) (Basu et al., 2009;Shafie et al., 2009).Previous studies have shown that these selected plant species have potential medicinal activity (Iscan et al., 2002;Mimica-Dukić et al., 2003;Saeed and Tariq, 2005;Andoğan et al., 2002;Mathur et al., 2011;Hammer et al., 1999;Adam et al., 1998;Sahin et al., 2003;Gulluce et al., 2003;Razzaghi-Abyaneh et al., 2008;Adinguzel et al., 2007;Boyraz and Özcan, 2006;Dikbas et al., 2009;Azaz et al., 2005;Chorianopoulos et al., 2004;Mihajilov-Krstev et al., 2010;Mihajilov-Krstev et al., 2009;Karami-Osboo et al., 2010).The antibacterial activity of essential oil of S. pubescens and C. vulgare has never been evaluated and was carried out for the first time in this study.The widespread use of antibiotics both inside and outside medicine is playing a significant role in the emergence of bacterial resistant (Goossens et al., 2005).Although, there were low levels of preexisting anti-biotic resistant bacteria before the widespread use of antibiotics; evolutionary pressure from their use has played a role in the development of multidrug resistance varieties and the spread of resistance between bacterial species (Hawkey and Jones, 2009).Biological cost or metabolic price is a measure of the increased energy metabolism required to achieve a function.Drug resistance has a high metabolic price (Steven and Timothy, 2010) in pathogens for which this concept is relevant (Wichelhaus et al., 2002).Although several strategies have been proposed to overcome and control this situation.However, a clear solution has not yet been elucidating due to the antibiotic resistance, consequences, and side effects of antimicrobial drugs.Many plants are used in Iran in the form of oils and crude extracts, infusion or plaster to treat common infections without any scientific evidence of efficacy.Pharmacological studies carried out on essential oils of some aromatic plants' species that were obtained in central regions of Iran, have shown antimicribial activity which is coherent with the use of these plants in folk medicine.It is interesting to determine whether their traditional uses are supported by actual pharmacological effects or merely based on folklore.In the present study, three medicinal plants were selected which are widely used in the folk medicine in our region.All the plants have been used in the treatment of infectious diseases with different geographical area (Rechinger, 1982a, b;Chevallier, 1996).The aim of this study was to evaluate the antibacterial potential of the essential oils derived from S. pubescens, M. piperita, S. hortensis and C. vulgare that grows in the wild in the central part of Iran against standard strains.The selected strains: Staphylococcus aureus (PTCC:1431), Listeria monocytogenes (PTCC:1163), Streptococcus pneumoniae (PTCC:1240), Pseudomonas aeruginosa (PTCC:1430), Klebsiella pneumoniae (PTCC:1053), Escherichia coli (PTCC:1329) and Salmonella typhi (PTCC:1609) were purchased from Iranian Research Organization for Science and Technology (IROST).The antimicrobial potential was performed by disc diffusion (DD) and broth microdilution method (BMD) to determine the minimum inhibitory concentration (MICs) and maximum bactericidal concentration (MBCs).

Collection of plant and essential oil extraction
The plants were collected from their wild habitat in Semnan city in the central part of Iran between April and June, 2011 which are shown as geographical and environmental conditions in Air-drying of plant material was performed in a shady place at room temperature for 4 days.Ground and dried leaves of plants (100 g) were subjected to hydro-distillation for 3 h, using a Clevenger-type apparatus.The distillated oils were dried over anhydrous sodium sulfate and stored in tightly closed dark vials at 4°C until analysis.

Gas chromatography/mass spectrometry (GC/MS) analysis
The essential oils were analyzed on an Agilent Technologies 7890A GC system coupled to a 5975C VLMSD mass spectrometer with an injector 7683B series device.A fused silica capillary column DB-5 (30 μm, 0.25 mm i.d, film thickness 0.25 μm) and a flame ionization detector (FID) was used for the separation.Helium was used as a carrier gas at a flow rate of 1 ml/min.The oven temperature was programmed at 60°C (4 min), and then rising to 300°C at 4°C min -1 .The injector and detector temperature were kept at 250 and 300°C, respectively.The mass spectrometer was operated in electronimpact ionization (EI) mode with 70 eV energy with MS transfer line at temperature of 300°C was used.Ion source and interface temperatures were 200 and 250°C, respectively.The split ratio was 1:50.The percentage compositions were obtained from electronic integration measurements using flame ionization detector (FID), set at 250°C.The column was programmed as follows: 60°C for 2 min and then increased by 3°C min -1 up to 300°C.Volume of injected samples was 0.5 µl.Identification of components was based on the comparison of retention times (RT) and the computer mass spectra libraries using Wiley 275 GC/MS Library (Wiley, New York), those found in the literature (Adams, 2001;McLafferty, 1993) and the mass spectrometry data bank (NIST).The percentage composition of the essential oil was computed by the normalization method from the GC peak areas measurements (Table 4).

Preparation of inoculums
Bacterial strains were prepared by suspending one isolated colony from MH agar plates in 5 ml of MH broth and overnight broth cultures.The suspensions were adjusted in 0.5 McFarland standard turbidity to obtain final inoculums of 5 × 10 5 to 5 × 10 6 CFU/ml after 24 h of growth at 37°C and confirmed using a spectrophotometer.The essential oils were dissolved in dimethyl sulfoxide (DMSO, 25 mg/ml) and diluted to MH broth for antibacterial tested.All strains were tested by BMD and disk diffusion (DD) techniques according to the National Committee for Clinical Laboratory Standards (NCCLS, 2003a, b).

Serial dilution method
MICs and MBCs of essential oils were determined by using BMD method as described by NCCLS (2003a) in flat-bottomed 96-well clear plastic tissue-cultured plates (Greiner, 650161).The MIC was assayed using two-fold BMD method in MH Broth in 96-well plates.
Plates contained two-fold dilutions of antibacterial agents at the concentration ranges: 0.5 to 64 μg/ml (25%, v/v).These dilutions were used to dispense 100 µl into each of the sterile 96-wells and an equal volume of bacterial inoculums was added to each well on the microtiter plate.After incubation for 24 h at 37 °C, the microdilution trays were checked with unaided eyes to detect the growth inhibition of the bacteria, and then the MICs were determined with spectrophotometer.The MIC was defined as the lowest concentration of the essential oil at which the microorganism does not demonstrate visible growth.The final concentration of DMSO in the assays did not interfere with the bacterial proliferation which is used as a control.Negative controls were prepared with noninoculated medium with oils, and one non-inoculated well, without antimicrobial agents, was also included to ensure medium sterility.
The commercial antimicrobials Ciprofloxacin (Sigma) and Gentamicin (Merk) were included as positive controls.One inoculated well was included to allow control of the broth suitability for organism growth.To determine the MBCs, the suspensions (20 µl) were taken from each well without visible growth and inoculated in MH agar for 24 h at 37°C.The MBC was defined as the lowest concentration of the essential oil at which incubated microorganisms are completely killed.Tests were performed in triplicate for each test concentration (P > 0.05).

Disc diffusion method
Agar diffusion method was carried out for the assessment of the essences antibacterial activity as recommended by NCCLS (2003b).The potential activity of oils were confirmed by the inhibitory effect on bacterial growth as reflected by the inhibition zone (IZ) compared to known standard antibiotics.Essential oils were diluted in DMSO to different concentrations (0.5, 1, 2, 4, 8, 16, 32 and 64 µg/ml).50 µl of standardized inoculums according to 0.5 McFarland turbidity standard solutions (10 5 to 10 6 CFU/ml) of the selected strains were spread onto the surface of Mueller Hinton (MH) agar and kept for 2 h at 4°C for absorption.Sterilized paper discs (Whatman, 6 mm diameter) containing approximately 20 µl of the essential oils were impregnated with different amount of essential oils.The prepared discs of the oils and standard antibiotics were placed on the surface of MH agar media.The inoculated plates were incubated at 37°C for 24 h and the resulting  zone of inhibition (diameter) was measured in millimeters by comparing the different concentrations of oils and the standard antibiotics.The MIC was defined as the lowest concentration, resulting in a clear zone of growth inhibition around the disc after incubation period.Gentamicine (Merk) and Ciprofloxacin (Sigma) discs were applied over the test plates as a positive control.Negative controls were prepared using the solvent to dissolve the essential oil solution.All experiments were performed in triplicate.

Statistical analysis
Comparison of data was performed using the one way analysis of variance (ANOVA) or the unpaired Student's t-test and is presented as mean ± standard deviation.Comparison of MIC and MBC values, tests were made in triplicate for quantification.Values of p < 0.05 were considered significant.

RESULTS
All essential oils showed effective antibacterial activities on the selected pathogenic bacteria.Antibacterial activities of essential oils were investigated by broth microdilution and the disc diffusion method.The MICs and MBCs and diameter of inhibition zone (DD) of the selected oils on the bacteria are shown in Tables 2 and 3.
The results showed that essential oil of the plants were active against all the pathogenic bacteria species with different degree in the following range of concentrations: essential oil of S. pubescens and M. piperita had the best antibacterial effect and its MIC value was between 0.5 and 8 μg/ml.C. vulgare is the second degree with MIC values between 0.5 and 16 μg/ml.Otherwise, S. hortensis had a lowest antibacterial effect comparison to the earlier essences and its MIC value was 1 to 32 μg/ml.Ciprofloxacin and Gentamicin used as positive control as well as DMSO as a negative control which did not show any inhibition against the pathogens bacteria.MIC range of standard antibiotics "Ciprofloxacin and Gentamycin" were 0.5 to 1 μg/ml and 0.5 to 2 μg/ml, respectively.Even at low concentrations, the plant's species showed antibacterial activity more or nearly equal to the commercial bactericidal agents.All of the oils had the best inhibitory activities against S. Pneumonia, S. aureus and P. aeruginosa.The weakest activity was observed against L. monocytogenes and K. pneumoniae with the highest MIC and MBC, and K. pneumoniae was resistance against S. hortensis.The results of the chemical analyses using GC/MS of the essential oils were listed in Table 4. Number of indentified constituents

DISCUSSION
This study was attempted to purify the selected plant's oils that were native in our region in order to identify their essential oils as antibacterial properties.In addition, components of plants were determined and the result was compared with other studies.This is due to several reasons, namely, conventional medicine can have side effects, high coast, abusive or incorrect usage of synthetic drugs result in complications, and the large percentage of the world's population do not have access to conventional pharmacological treatment.The best antibacterial activities were seen in M. piperita, S. pubescens and C. vulgare, while S. hortensis displayed a moderate response against bacterial species.In comparison to the standard drugs, these data showed M. piperita and S. pubescens had the highest activity; C. vulgare had the lower activity but with the lowest different, while the different properties of S. hortensis was more.The results confirmed the antibacterial potency of these plants.In other studies concerning the antimicrobial activity of these plants, inhibition effects of M. piperita on some microorganisms such as S. paratyphi, Proteus mirabilis, Proteus vulgaris, Streptococcus mutans, Streptococcus faecalis, Streptococcus pyogenes, Lactobacillus acidophilus, Bacillus subtilis, Enterobacter aerogenes, Shigella dysenteriae and Yersinia enterocolitica growth was studied and this plant showed the highest antimicrobial activity (Iscan et al., 2002;Mimica-Dukić et al., 2003;Saeed and Tariq, 2005;Mathur et al., 2011;Andogan et al., 2002).In previous studies, S. hortensis showed antimicrobial activity against some of the standard and clinical microorganisms (Iscan et al., 2002;Sahin et al., 2003;Gulluce et al., 2003;Adinguzel et al., 2007;Boyraz and Özcan, 2006;Azaz et al., 2005;Chorianopoulos et al., 2004;Mihajilov-Krstev et al., 2010;Mihajilov-Krstev et al., 2009;Karami-Osboo et al., 2010;Baser et al., 2004).Other study concerning the extract of C. vulgare, inhibited the growth of some bacterial species with different degrees (Opalchenova and Obreshkova, 1999).Our result with other studies confirmed that a variety of bacterial species are affected by essential oil of selected plants, especially about the essential oils of S. pubescens and C. vulgare evaluated for the first time.The aforementioned finding supports their traditional usage of these oils as antibiotic and antiseptic (Riley, 2005).Briefly, the results of this study showed that the essential oils of these plants have a very broad spectrum of antibacterial activities with notable MICs and MBCs, which are near or lower than dose synthetic drugs.These plants could safely be used as organic preservatives to replace synthetic antibiotics in the prevention and cure of some human and animal infectious disease as well as food industrial preservatives.However, it is necessary to determine the toxicity of the active constituents, their side effects and pharmaco-kinetic properties.In comparison to the other studies, the composition of the plants showed little difference.Similar to our result, previous studies on M. piperita showed that the main components of the oils and extracts were menthol, menthofuran, menthone and menthyl acetate (Iscan et al., 2002;Maffei et al., 1999;Soković et al., 2009;Rohloff, 1999).There are three numbers of these components in our main components, but in the other study in Iran, the main components were very different: α-terpinene, isomenthone, trans-carveol, pipertitinone oxide (Rasooli et al., 2006).Menthone and menthol has been reported to be responsible for the antimicrobial activity of M. piperita (Gupta and Saxena, 2010;Bassolé et al., 2010;Kizil et al., 2010).But, the antibacterial activity of menthofuran has not been deter-mined.Since M. piperita showed the highest antibacterial activities, then it was suggested the menthol, menthofuran or menthone alone or mix together (synergic effect) play a major antibacterial role.Since, each of menthol, menthofuran or menthone alone do not have considerable amount in the total extract; therefore, its antibacterial activity also belongs to the mixture of the whole components.With attention to other, its components were less than 8%.But, a point of consideration is the study that showed the good antimicrobial activity of M. piperita, while there are any these three components in their chemical composition (Rasooli et al., 2006).Concerning the S. pubescens, there are a few studies which determined the chemical composition.Many studies have not been conducted so far; Iranian researcher reported (Z)-β-Ocimene, Germacrene D and Bicyclogermacrene as the main components (Baher and Mirza, 2006).Previous studies related to the chemical composition of S. hortensis, thymol, Carvacrol, γ-terpinene and p-cymene reported as main components which are similar to our result (Gulluce et al., 2003;Adinguzel et al., 2007;Azaz et al., 2005;Sefidkon et al., 2006).Since, in these studies, S. hortensis display the high antimicrobial activity, therefore, it is concluded that the main components like thymol and carvacrol have an antimicrobial activity.But, the important point in these studies confirmed the antimicrobial activity of S. hortensis (Gulluce et al., 2003;Adinguzel et al., 2007;Mihajilov-Krstev et al., 2009) which is the lack of thymol or carvacrol as part of the main compounds, and the amount of these components was inconsiderable against whole extract.Therefore, it can be said that the total extract of S. hortensis has better antimicrobial activity than pure components, because in this status, there is synergistic effect between components as was observed in this study.Concerning the C. vulgare, in the previous study, the constituents of sabinene, germacrene D, E-caryophyllene, (Z)-β-ocimene and γ-terpinene was reported as the main components (Nurzyn´ska-Wierdak, 2009).The main components of their plants were similar to our results, but in the other study the main components were different to our result, in that the study of germacrene-D, b-caryophyllene and bcaryophyllene oxide was reported as the main components (Kökdil, 1998).In the present study, the similarity of composition to other studies is low.Nonetheless, all of them have a good antimicrobial activity and this subject describes that the special compound cannot have the major antimicrobial potency and the antibacterial of this plant yielded the mixture of their components.Although, these plants are in the same family, but their main component is different, and this variety in biological activity is related to their composition.The two Gram positive: S. aureus, S. pneumonia and two Gram negative: P. aeruginosa, E. coli bacteria showed high sensitivity against the essential oils with the lowest MICs and MBCs.Some studies reported that these plants inhibited both Gram positive and Gram negative bacteria (Dikbas et al., 2006;Hoferl et al., 2009).It was suggested that these differences in components could be due to the variety of the ecotype system reported by other scientists and references (Asbaghian et al., 2011).Since the essential oils are complex mixtures of several compounds, it is difficult to attribute their biological activity to a particular constituent.Usually, major compounds are the ones responsible for the antimicrobial activity of the essential oils.However, some studies showed that minor components may have a crucial role in the biological activity of the oils (Koroch et al., 2007).Further studies are needed to determine the antibacterial activities of the bioactive compounds responsible for the observed potential value.Natural plant-derived bactericidal may be a source of a new alternative active compounds.In attention to, in the present study, most isolates showed a less difference concentrations of essential oils between bacteriostatic and bactericidal values.Suggesting that the essential oils of the selected plants could be a possible source to obtain new and effective herbal medicines to treat infections caused by multi-drug resistant strains of microorganisms and also in the search for novel antibacterial agents with the potential application of some major or minor constituents alone, mixture of the extract or in combination with antibiotics for the treatment and prevention of pathologies associated with multi resistant bacteria.However, the mechanism of inhibitory effects of these plant's oils against infectious bacteria is still unclear.Further investigations regarding the in vitro and in vivo should be conducted in order to clear mechanisms pathway and develop such products.

Table 1 .
Plants were identified by experts of the University of Applied Science and Technology (UAST) Education Center in Semnan branch.The leaves of S. pubescens, M. piperita, S. hortensis and C. vulgare in full flowering stage were collected to determine antibacterial activity.A voucher specimen for each plant has been deposited in the herbarium of Medicinal Plants Research, UAST.

Table 1 .
Geographical and environmental conditions.

Table 2 .
MIC and MBC (μg/ml)values for different essential oils of plants.

Table 3 .
Antibacterial activity screening of antibacterial agents by zone of inhibition (mm diameter) in disc diffusion method.

Table 4 .
Chemical analyses of essential oils.