Evaluation of Antibacterial Activity of Essential Oil of Ziziphora clinopodioides and Achillea wilhelmsii on Antibiotic-resistant Strains of Staphylococcus aureus

Background: The use of drugs to treat diseases led to the emergence of resistant strains of bacteria. Bacterial resistance to antibiotics is the most common problems in medical science. This study was aimed to evaluate the antimicrobial effects of essential oil of Ziziphora clinopodioides and Achillea wilhelmsii on against resistant clinical strains of Staphylococcus aureus. Methods: After collecting plants and validate its scientific name by botanists of Agricultural Organization and after drying in the shade, essential oil of Z. clinopodioides and A. wilhelmsii, extracted with steam distillation method by Clevenger and antimicrobial effects of essential oil by well diffusion on bacteria mentioned above were interpreted. The amount of essential oil were injected to gas chromatography linked to mass spectrometry (GC/Ms), and the amount and type compounds of the essential oils were identified. Results: The results showed that the extracted essence essential oil from the Z. clinopodioides and A. wilhelmsii has a bactericidal effect. In the obtained results by GC/Ms chromatography, in Z. clinopodioides and A. wilhelmsii essential oils, 22 and 18 compounds were identified, respectively. Conclusion: Our results showed Z. clinopodioides and A. wilhelmsii have considered in herbals groups with antibacterial properties and after evaluating their effects in-vivo condition and to identify the active ingredients, as an alternative to synthetic drugs that commonly used to treat infections are used.


INTRODUCTION
One of the most important health challenges is the treatment of infectious diseases due to their much higher prevalence and spread.Since the discovery of penicillin in the late 40's, and widely use of it, new antibiotics have been continuously presented to treat infections.The result was the increased use of clinical or synthetic antibiotics to heal clinical infectious disease.The indiscriminate use of antimicrobial drugs caused the increased drug resistance against different antibiotics in most bacteria (1)(2)(3)(4)(5)(6).It has been one of the reasons of the increasing use of plants as natural, safe, accessible and affordable materials in treating bacterial infections in comparison with synthetic antibiotics.Also, the consumption of herbal medicines is more acceptable than chemical drugs (7)(8)(9).Moreover, that is why a new wave of expensive global studies has raised, and the antibacterial effects of dif-Thirty-two components constituting 97.1 % of the essential oil were identified (13).Achillea wilhelmsii C.Koch is one of the most famous medicinal plants that about 85 species of it have been identified that 7 of them are exclusively found in Iran.Native Achillea species has a relatively wide distribution in different parts of the country and are commonly grown in fields, roadsides and mountainous areas (14).New flowers of Achillea are rich in chemicals and used to treat respiratory problems as anti-allergic, anti-congestion and mucus.Essential oil of them contains chamazulene, cineol and borneol which have shown anti-inflammatory, antispasmodic, anti-dandruff properties, hair growth stimulation, and topical healing of skin (14)(15).Aljancic et al. (1999) in their study showed the significant inhibitory effect of A. wilhelmsii on Candida albicans and Bacillus subtilis.According to them, in addition to the inhibitory effect of two above microbes, the flavonoids of A. wilhelmsii essential oil also the growth of Aspergillus Niger effectively (16).
In the study by Sokmen et al. (2003), the antimicrobial effect of the essential oil, aqueous and methanol extracts of A. sintenisii on 12 bacteria and two yeast species were examined comparatively (17).The current study intends to investigate the chemical compounds and anti-bacterial effects of Z. clinopodioides and A. wilhelmsii on Staphylococcus aureus resistant to the antibiotic.

Plant sampling and investigation of its anti-microbial properties
Plant samples were collected from the nature areas of Eyshabad village, Marand city, and East-Azerbaijan province in two stages in late May to early June.The samples were cleaned after collection and transmission, and also, they were dried in a large and proper space under the conditions away from direct sunlight.After they had been dried completely, their aerial parts (stems and leaves) were separated from the roots and prepared to be grinded.Then, the essential oil of their aerial parts was extracted for 5 hours by a Clevenger-type apparatus.After washing with sodium sulfate and dissolving in n-hexane, their essential oil was used to investigate antibacterial properties.The bacteria employed in this study was S. aureus isolated from patients.Initially, a suspension equivalent to standard McFarland 0.5 turbidity was prepared from each bacterium.After spreading the bacterial suspension on Müller-Hinton agar media, wells of 5 mm in diameter were immediately made.About 50 µL and 100 µL of essential oils were added aseptically in wells.The same amount of n-hexane solvent (as con-trol) was added to plates and then were incubated for 24 hours at 37°C (18-19).

Bacterial isolates and their resistance to antibiotics
Thirty samples used in this study were selected from the clinical samples of Marand Razi hospital and cultured on mannitol salt agar and blood agar media.

Essential oil analysis by gas chromatography-mass spectrometry (GC/Ms)
The essential oil sample was injected in GC/Ms (type: Agilent GC (USA)) after finding thermal planning system.The compounds were identified by the database of the device and comparing their mass spectra.SPSS V.19 software performed variance analysis of data, and Duncan's multiple range test at the confidence level of 99% was used for the mean.

Bacterial responses to antibiotics
In-vitro antibacterial activities of some antibiotics against obtained isolates were evaluated.The results revealed that in the presence of antibiotics, each bacterial isolate responded somewhat similarly, however, there were differences among the isolates in terms of antimicrobial resistance or antimicrobial sensitivity.Some differences were also observed in inhibition mechanisms of antibiotics, as the antibacterial effect of penicillin and erythromycin was much less than other antibacterial drugs (Table 1).

Antimicrobial activity of essential oils
Because of various geographical and climatic conditions in Iran, there is a rich and varied source of plant species in it.Some of these herbal plants have medicinal properties The n-hexane solvent was used as control sample that showed no inhibitory effect.Given the observed clear relationship between the plant derivate (essential oils) and their antimicrobial activity, it can be said that they can be used as new biologically active substances against some virulent infectious agents.

Chemical compounds obtained from the essential oils of Z. clinopodioides and A. wilhelmsii
Chemical compounds obtained from the essential oils of the aerial part of Z. clinopodioides and A. wilhelmsii were investigated in-vitro.The essential oils were extracted by steam distillation with Clevenger and injected to GC/Ms after purification (removing from the plant sweat), and the amount and type of the essential oil were determined.Totally, 22 compounds of Z. clinopodioides essential oil, including 95.4% of compounds, and 18 compounds of A. wilhelmsii essential oils, including 91.31% of compounds, were identified.Table3 shows the ingredients of each essential oil.

DISCUSSION
According to increased bacterial resistance to a variety of antibiotics, it has been tried to access and use the compounds of plants in the treatment of various diseases.From thousands of years ago, plants have played a critical role in maintaining health and improving the life quality of human beings.Medicinal herbs have useful properties that their antibacterial, anti-parasitic, antifungal and antioxidant properties can be noted (20).Chalibian et al. ( 2003) have proved that A. wilhelmsii has an antimicrobial effect on S. aureus that the results of this study are consistent with it (21).Ghaderi et al. (2012) shown that A. wilhelmsii had inhibitory effect on Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus and used GC/MS to separate and identify the compounds of essential oil that the results of the chemical compounds of the current study are consistent with the results of their study (22).In the study by Aljancic et al., it has been found that A. wilhelmsii prevents the growth of Bacillus subtilis, C. albicans and A. niger in in-vivo conditions.They have identified the flavonoids extracted from the essential oils as a factor of antibacterial effects (23).
Kermanshah et al. have studied the antibacterial effect of the hydroalcoholic extracts of common sage (Salvia officinalis) and A. wilhelmsii on cariogenic microorganisms and concluded that both essential oils have an inhibitory effect on the growth of Streptococcus mutans, Lactobacillus rhamnosus and Actinomyces viscous (24).Terpenes can damage the cell membrane and penetrate the lipid structure of the cellular wall of bacteria that this leads to protein denaturation and the collapse of the cell structure and leakage of cytoplasm and finally cell death (26).Previous studies indicated that the extracts of Z. persica and Z. clinopodioides could prevent the growth of broad range of gram-positive and gram-negative bacteria (27,28).

CONCLUSION
In general, in-vitro investigations showed that of the essential oils extracted from Z. clinopodioides and A. wilhelmsii have antimicrobial activity against the S. aureus resistant to antibiotics that would be considered as an alternative option in producing herbal medicines against the Staphylococcus aureus bacterium with minimal side effects.Since that they are non-toxic and natural components after performing further investigation on experimental animals.
Khanaki et al. (2010) have identified the chemical compounds of Z. clinopodioides by GC/MS.The results of the current study are consistent with their results (25).Oussalah et al. (2006) have identified the main compounds of Z. clinopodioides essential oil.Pulegone is a ketone and of Monoterpene.

Table 1 .
Antibiotic susceptibility pattern of S. aureus isolates (%) In our study, the antibacterial effects of essential oils were as follows: essential oils of Z. clinopodioides and A. wilhelmsii could inhibit S. aureus growth.The antibacterial effect of Z. clinopodioides was more than the one of A. wilhelmsii.The results of the antimicrobial activity of Z. clinopodioides and A. wilhelmsii are represented in Table2.
R: Resistant; S: Sensitive; I: Intermediate such as antibacterial activity.

Table 3 .
Chemical compounds obtained from the essential oils of Z. clinopodioides and A. wilhelmsii

Table 2 .
Antimicrobial effect of essential oils of Z. clinopodioides and A. wilhelmsii on S. aureus resistant to antibiotics (mean±SD)