Antimicrobial effects of yarrow ( Achillea millefolium ) essential oils against Staphylococcus species

Staphylococcus aureus, a gram positive, non-motile, catalase and coagulase positive, facultative anaerobe coccus is a common type of bacteria that normally lives on the skin and nasal passages of healthy people. The genus Achillea (Asteraceae) was named after the mythological Greek warrior Achilles who used Achillea species for healing wounded-soldiers during the Trojan war. The genus Achillea comprises of ~85 species, most of which are endemic to Europe and the Middle East. The aim of this study was to evaluate antimicrobial effects of yarrow (Achillea millefolium) essential oils against Staphylococcus spp., fourteen clinical isolates of Staphylococcus cultured from patients. The disc diffusion method was used for determination of antimicrobial activity of essential oil. Results showed that this inhibitory effect is dose-dependent to wit, by increasing the concentration of the extract in the culture media; reduction in growth was obviously revealed. In conclusion, it can be stated that yarrow essential oils have inhibitory effect against Staphylococcus spp.


INTRODUCTION
Plant substances play a major role in primary health care as therapeutic remedies in many developing countries until today (Zakaria, 1991).Medicinal herbs contain physiologically active principles that over the years have been exploited in traditional medicine for the treatment of various ailments, as they contain anti-microbial properties (Kelmanson et al., 2000;Srinivasan et al., 2001).The antifungal and antibacterial activity exhibited by extract and essential oil of medicinal plants has been demonstrated by several researchers (Delgado et al., 2004;Nasar-Abbas and Kadir Halkman, 2004;Ozcan and Erkmen, 2001;Fazeli et al., 2007) but unfortunately, there are few data related to the antimicrobial activity of extracts obtained from different medicinal plants in *Corresponding author.E-mail: Dr.e.issabeagloo@gmail.com.Tell: +989144079927.

Chaharmahal va Bakhtiari (Iran).
Staphylococcus aureus, a gram positive, non-motile, catalase and coagulase positive, facultative anaerobe coccus is a common type of bacteria that normally lives on the skin and nasal passages of healthy people.When it enters the body through a cut or other medical devices, it can cause local or serious infections (Franklin, 1998).Methicillin resistant S. aureus (MRSA) has become one of the major causes of nosocomial and community pathogens causing significant morbidity and mortality because there are multi drug resistant pathogens that are resistant to all penicillins, so the option antibiotics for treatment of MRSA infections are limited to antibiotics such as vancomycin, tigecycline, lincozolid and mupirocin (Simor et al., 2007).The patterns of antimicrobial susceptibility of S. aureus have been changed worldwide and it has been reported increasingly to be less effective.
Development of mupirocin (dos Santos et al., 2007) and vancomycin (Appelbaum, 2006) (Cheers, 1999).The genus Achillea comprises of ~85 species, most of which are endemic to Europe and the Middle East.Turkish flora possesses 42 Achillea species and 23 of them are endemic (Duman and Achillea, 2000).These species have some interesting properties and are used in cosmetics, fragrances and agriculture, for example, plant protection (Senatore et al., 2005).Some Achillea species have been known to be ethnopharmacologically used in folk remedies for various purposes such as hemorrhoid and wound healing (Baytop, 1999).Herbal teas prepared from some Achillea species are very often used in folk medicine as diuretic, for abdominal pain, against diarrhea, flatulence and emmenagogue, moreover for wound healing purposes (Fujita et al., 1995;Honda et al., 1996;Yesilada et al., 1993).A. biebersteinii is locally named yarrow, and other species widely used as a folk remedy to treat abdominal pain, wounds and stomachache as well (Sezik et al., 2001;Baytop, 1997).
The aim of this study was to evaluate antimicrobial effects of yarrow (A.millefolium) essential oils against Staphylococcus spp.

Extract preparation
The aerial parts of A. millefolium were air-dried and then ground into fine powder using grinder and stored at room temperature.Dried plant materials were powdered (200 g) and subjected to hydro-distillation (in 2000 ml distilled water) for 4 h, using a Clevenger-type apparatus according to the method recommended in British Pharmacopoeia (1988).The essential oil samples were stored in universal bottles and refrigerated at 4°C prior to use.

cultures and preparation of yarrow extracts
Fourteen clinical isolates of Staphylococcus cultured from patients and S. aureus ATCC 25923 were used in all experiments.Methicillin resistant S. aureus directed was detected on CHROMagarTM MRSA (CHROMagar Paris, France).Bacterial suspensions were made in Brain Heart Infusion (BHI) broth to concentration of approximately 108 CFU/ml using standard routine spectrophometrical method.Subsequent dilutions were prepared from the above suspensions, which were then used in the tests.

Disc diffusion method
The disc diffusion method was used for determination of antimicrobial activity of essential oil.Briefly, using a sterile cotton swab, above microbial suspensions was spread on the Mueller Hinton Agar (MHA) plates.Sterile paper discs (6 mm in diameter) were impregnated with 10, 15, 20 of each oil and were placed on the inoculated plates.After remaining at 4°C for 2 h, plates were incubated for 24 h at 37°C.The diameters of the inhibition zones were measured in millimeters.All tests were performed in triplicate (NCCLS, 2009).
Chemical composition of A. millefolium L. essential oil is shown in Table 1 (Bimbiraite et al., 2008).
Biochemical test such as catalase, oxidase, coagulase and OF was carried out to proven the genera and data are  shown in the Table 2. Inhibitory effect of yarrow extract was determined by different concentrations of this herbal extract and results showed that this inhibitory effect is dose-dependent, to wit, by increasing the concentration of the extract in the culture media, reduction in growth was obviously revealed (Table 3).

DISCUSSION
Essential oils of higher plants have been evaluated against pathogenic microorganisms (bacteria, fungi and yeast) by many other workers (Pandey et al., 2010;Rasooli and Rezaei, 2002;Bussmann and Glenn, 2011) and the results support the findings of the present investigation.An important characteristic of essential oils and their components is their hydrophobicity, which enables them to partition in the lipids of bacterial cell membrane and mitochondria, disturbing the structures and rendering them more permeable as a result of which leakage of ions and other cell contents can then occur (Pirbalouti et al., 2011).Although, a certain amount of leakage from bacterial cells may be tolerable without loss of viability, extensive cell contents loss or the exit of critical molecules and ions will lead to death.It has also been reported that gram-negative strains are less susceptible to essential oils due to the presence of an outer membrane surrounding the cell wall which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering (Fredj et al., 2007).
The results obtained in the present study revealed that essential oil exhibited variable levels of antibacterial activity against all tested bacterial strains.According to the literature data (Candan et al., 2003;Mimica-Dukic et al., 2004), gram-positive bacteria seemed to be more sensitive to the examined essential oil than gramnegative bacteria.Furthermore, the essential oil obtained from A. collina s.l. in the most of the cases exhibited stronger antibacterial activity than A. pannonica oil (in some of tested S. aureus and Streptococcus strains).This could be due to the presence of high ratio of chamazulene in the essential oil.
On the other hand, stronger bacteriostatic activity of A. pannonica was observed on Streptococcus hyicus and one strain of Str.agalactiae in comparison to A. collina oil.This could be explained by notable amounts of 1,8cineole (40.40%), camphor (11.10%) and borneol (3.22%) in the essential oil.All of the three substances are confirmed as strong antimicrobials on a different range of bacteria (Candan et al., 2003;Tabanca et al., 2001;Tzakou et al., 2001).
Gram-positive bacteria are known to be more susceptible to essential oils than gram-negative bacteria (Inouye et al., 2001).P. aeruginosa was least susceptible to the essential oils.The weak antibacterial activity against gram-negative bacteria was ascribed to the presence of their cell wall, lip polysaccharide (Nostro et al., 2000).B. subtilis was the most susceptible micro-organism to the rosemary essential oil.Concerning the activity of pure active compounds, the most susceptible bacteria to thymol was B. subtilis (23.0 mm) and the most resistant was P. aeruginosa (11.5 mm).

Conclusion
From the obtained results, it is obvious that the chemical composition of the essential oil has important impact on both antioxidant and antimicrobial effects of A. millefolium obtained from different biological sources.The presence of chamazulene increases the antibacterial activity, whereas the antioxidant and scavenging effects of essential oil are related to some other substances such as camphor and borneol.

Table 1 .
Chemical composition of A. millefolium L. essential oil.

Table 2 .
Results obtained from different biochemical tests.

Table 3 .
Anti-staphylococcal activity of yarrow essential oils by disc diffusion method.