MATERIALS AND METHODS

Collection and preparation of samples: Samples were collected from Cairo University Farm, Cairo, Egypt. Apparently healthy plants were collected, washed thoroughly in tap water and dried at dark room temperature for 15 days. The leaves was powdered and extracted by hydro-distillation following the previous published procedures with N. Sativa seeds oil extract slight modification as standardized in Department of Medicinal Plant, NRC, Egypt by Dr. S. Mahfouz (Eweis and Gad, 2011; Eweis et al., 2012).

Identification of its constituents: Conditions of essential oil on GC Instrument Gies-agilent technolo.6890 n network GC system USA. Oven initial temp. 70°C, initial time,1 min, rate c min^-1 4, final temp. 190°C, final time 13 min. intel temp. 250°C, detector temp. 280°C (FID) flame ionization detector flow 2 mL min^-1, carrier gas n230 mL min^-1, H2, 30 mL min^-1, air, 300 mL min^-1, capillary column HP-5 (5% phenyl methy siloxane) length 30 m, diameter 320 μm film thickness, 0.25 μm NRC-Dokki-Egypt.

Test organisms: Identified pure culture of test organisms representing different species were obtained by sub-culture from glycerol stock cultures from the Department of Microbiology and Immunology Department, Faculty of Pharmacy, Al-Azhar University. The bacteria studied, standard strains and clinical isolates, included three Gram-positive: Bacillus cereus, Staphylococcus epedrmidis and Staphylococcus aureus (ATCC 6538), six Gram-negative: Escherichia coli ATCC (8739), Salmonella Typhi, Klebsiella pneumonia, Psedumonas aeruginosa (ATCC 27853), Proteus mirabilis and Shigella flexneri. The clinical isolates were resistant to several antibiotics. In addition, two fungi, one yeast like and one filamentous, were used in this assay Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404), respectively. The bacteria were stored by culturing on nutrient agar slants; however fungi were cultured on Sabouraud dextrose agar.

Assay for antimicrobial activity: The antimicrobial activities of the extracted essential oils were systematically performed against 11 different bacterial and fungal species by agar cup plate diffusion method based on the methodology used by Bauer et al. (1966) with some modifications. The sterile Müeller-Hinton agar (Oxoid Ltd, UK) (for bacteria) or Sabouraud dextrose agar (for fungi) was poured into sterile Petri plates aseptically and allowed to solidify at room temperature. Both media were poured into Petri plates on a horizontal surface to give a uniform depth of approximately. All Petri plates were flooded with 400 μL mL^-1 of the microbial suspension in sterilized saline equivalent to McFarland 0.5 standard solution (1.5x10⁸ CFU mL^-1) and uniformly distributed by sterile glass rod, then allowed to dry for 20 min with lid in place. A loop full of fresh bacterium suspended in sterilized distilled water and its optical density adjusted to OD = 0.13 using spectrophotometer at 600 nm wavelength. In each plate, 6 wells of 6 mm diameter were made with a sterile borer and the agar plugs were taken out carefully so as not disturb the surrounding medium. In addition, a disc of standard drug ciprofloxacin (5 μg disc^-1) served as reference standard for bacteria and nystatin (30 μg disc^-1) for fungi. Precisely 100 μL of the test solution was added to the cups aseptically and labeled accordingly as well as DMSO. Test activities of Menthe piperita, Pelargonium graveolens (Geranium) and Nigella sativa essential oils were carried out by agar diffusion technique, in particular, cup plate method. The antibacterial solutions are the crude oil and different dilutions (1:1, 1:5, 1:10 and 1:20) in Dimethyl sulfoxide (DMSO). After holding the plates at room temperature for 1 h to allow diffusion of test samples into the agar, the plates were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. The diameter of the clear zone of inhibition surrounding each well was measured twice at right angles and the average of the two readings was recorded to the nearest mm. The inhibition effects were compared with that of standard drug ciprofloxacin (5 μg disc^-1) for bacteria and nystatin (30 μg disc^-1) for fungi. Negative controls were set up with equivalent quantities of DMSO. All the tests were performed in triplicate. Values are presented as means of three parallel measurements. The zone of inhibition above 7 mm in diameter was taken as positive result.

RESULTS

The chemical compositions of the essential oils of Nigella sativa oil, Pelargonium graveolens (Geranium oil) and Menthe piperita oil were characterized using GC analyses, listed in Table 1, 2 and 3, respectively, according to their elution order.

Table 1:	Main chemical constituents of Nigella sativa essential oil

Table 2:	The main chemical constituents of Pelargonium graveolens (Geranium) oil

Table 3:	The main chemical constituents of Mentha piperitta essential oil

Table 4:	Antimicrobial activity profile of the extracted essential oils by agar well diffusion method
1zone size, mm; oil 1, stands for Menthe piperitta oil; oil 2, stands for Pelargonium graveolens (geranium oil); oil 3, stands for Nigella sativa oil, a, crude oil; b, c, d, e, 1:1, 1:5, 1:10, 1:20 dilutions in DMSO, respectively; 1 average diameter in mm of three measures in inhibition zone; “-” indicates no inhibition

In addition, the antimicrobial activity, compared with standard drug ciprofloxacin (5 μg disc^-1), was assessed against three Gram-positive bacteria, six Gram-negative bacteria. In addition, the antifungal activity of the oils, compared with standard drug nystatin (30 μg disc^-1) was assessed against two fungi. The zone of inhibition above 7 mm in diameter was considered as positive result. Presence or absence of growth inhibition zones and zones diameter produced by the three essential oils are summarized in Table 4. Generally, the results revealed that the selected essential oils showed good antibacterial and antifungal activities against the specific organisms tested with varying magnitudes. Most of the tested microorganisms were sensitive to the three essential oils, especially Gram-positive bacteria and fungi; however, some Gram-negative bacteria were sensitive to one or two of the tested oils. The three essential oils showed strong antimicrobial activity against C. albicans and A. niger when compared to standard drug nystatin (30 μg disc^-1). In addition, the size of zone of inhibition indicates increasing in the antimicrobial activity with increasing in concentration of oils as evident by the size of zone of inhibition of crude oil compared with the diluted ones in DMSO. The Pelargonium graveolens (Geranium oil) showed maximum activity against tested organisms compared with the other two. There was no inhibition of growth with the negative control DMSO.

DISCUSSION

Infectious diseases, also called as communicable diseases, are disorders caused by pathogenic microorganisms like bacteria, viruses and fungi, however the highest percentage are commonly caused by bacteria (Khosravi et al., 2007; Mallam et al., 2012). These illnesses were found to be the most significant cause of morbidity and mortality in developing countries, especially in immune compromised patients. Importantly, many of these infectious microorganisms are resistant to synthetic drugs, in addition to the side effects of synthetic antibiotics, thus medicinal plants are gaining attractiveness over these drugs as an alternative therapy (Al-Bari et al., 2006). Indeed, the results of different studies have provided evidences that some medicinal plants might be potential sources of new antimicrobial and/or antifungal agents (Kone et al., 2004; Mallam et al., 2012). Above all, the antimicrobial properties of the essential oils and their components are established and exploited in such diverse commercial products as antiseptics and preservatives (Burt, 2004). Thus it is required to investigate those plants which have been used in traditional medicine to improve the quality of healthcare (Prabuseenivasan et al., 2006).

The chemical compositions of the essential oils of Nigella sativa oil, Pelargonium graveolens (Geranium oil) and Menthe piperita oil, characterized using GC- analyses, showed variations in comparison to compositions of oils collected from other countries. These differences may be attributed to climate conditions and types and methods of distillation (Ghannadi et al., 2012).

The in vitro antimicrobial activity of the three essential oils was carried out in this study by cup plate diffusion method which is mostly used for investigation of antimicrobial activity of essential oils. There is no a standard methodology to evaluate the inhibitory activity of plant extracts, thus most of the methods are based on the ones used to evaluate the resistance and/or susceptibility to antibiotics (Calvo et al., 2012). However, since the outcome of this test can be affected by factors such as the method used to extract the essential oil from plant material, the volume of inoculum, growth phase, culture medium used, pH of the media and incubation time and temperature, comparison of published data is complicated (Friedman et al., 2002; Burt, 2004).

In vitro studies in this study showed that the essential oils of Nigella sativa, Menthe piperita and Pelargonium graveolens inhibited both the fungal and bacterial growth although their effectiveness varied. This is consistent with the finding that antimicrobial activity of many essential oils were previously investigated and classified as strong, medium or weak (Zaika, 1988). In addition, it was found that the three essential oils exhibited better inhibitory effects and/or greater zone of inhibition to S. aureus, S. epidermidis and B. cereus species (Gram-positive) in comparison to Gram-negative ones. This finding is consistent with Al-Bayati (2008) and Haloci et al. (2012) findings that Gram-positive bacteria are more susceptible to essential oils than Gram-negative bacteria. That could be explained that tolerance of Gram negative bacteria to essential oils is due to the presence of a hydrophilic outer membrane that obstruct the penetration of hydrophobic essential oils into target cell membrane (Al-Bayati, 2008). The essential oil of Menthe piperita showed an inhibitory effect on the growth of S. aureus, S. epidermidis, B. cereus, S. typhi and P. mirabilis. The essential oil of Pelargonium graveolens inhibited the growth of S. aureus, S. epidermidis, B. cereus, S. typhi, K. pneumonia, E. coli, P. aeruginosa and S. flexneri. The essential oil of Nigella sativa showed growth inhibition of S. aureus, S. epidermidis, B. cereus, S. typhi, K. pneumonia, E. coli and P. mirabilis. In addition, the three essential oils showed good inhibition of the growth of the two tested fungi C. albicans and A. niger. In addition to the crude oil, four different dilutions 1:1, 1:5,1:10,1:120 were used against the tested microorganisms. Among them the crude oil and 1:1 dilution showed more inhibition to the growth of all organisms; indicates increasing in the antimicrobial activity with increasing in concentrations of oil constituents. This finding is consistent with results of Ara et al. (2005) study.

CONCLUSION

In the present study we report the main chemical compositions of the Egyptian essential oils of Nigella sativa, Menthe piperita and Pelargonium graveolens. In addition, this study concluded that the essential oils of these traditional medicinal plants have antimicrobial activity against different species of pathogenic microorganisms. Thus, our results are a preliminary scientific validation for the use of these essential oils for antimicrobial activity against human pathogens. It is essential that research should continue to isolate and purify the active components of these oils and testing in experimental animals is warranted.