Antibacterial Activity of Water and Alcoholic Crude Extract of Flower Achillea millefolium

The aim of this study was to determine the antimicrobial effect of yarrow and it is flower extract on certain microorganisms. The ethanolic (alcoholic) extract of the flower and aqueous (water) extract was tested against: Pseudomonasa aeruginosa, Salmonella enterica enterica (Typhimurium), Shigella flexneri, Micrococcus luteus, Staphylococcus aureus and Enterococcus faecalis. Using the diffusion test, the alcoholic and aqueous flower extract at 200 mg /ml possessed abroad spectrum of antimicrobial activity against all tested microorganisms at different conc., alcoholic extract had the greatest inhibitory effect (diameter of inhibition zone) against Pseudo. aeruginosa 30 mm, Staph. aureus 24 mm and Micro. lutues 23 mm, While the aqueous extract slightly inhibited Pseudomonasa aeruginosa and Micrococcus luteus 12mm and 10 mm respectively compared with standard antibiotic Ciprofloxacin 26 mm, 22mm and 22mm but Entero. faecalis, S. enterica enterica (Typhimurium) and Shig. flexneri show high resistance for both extract 8 mm, 0 mm, 9 mm, 12 mm and 8 mm, 10 mm but sensitivity for Ciprofloxacin 23 mm, 30 mm and 31 mm. Minimum inhibitory concentration (MIC) were determined for alcoholic extract against sensitivite microorganisms: Pseudo. aeruginosa 50 mg/ml, Staph. aureus 100 mg/ ml and Micro. luteus 100mg/ ml. It could be concluded that the alcoholic extract of yarrow flower have considerable antimicrobial effect on some pathogenic which cause infection.


INTRODUCTION
Plants, a source of medicinal compounds have continued to play a dominant role in maintenance of human health since ancient times.The World Health Organization estimates that plant extracts or their active constituents were used as folk medicine in traditional therapies of 80% of the world population (Anonynous, 1993).Over 50% of all modern clinical drugs are of natural origin (Baker et al., 1995).Iraqi People have traditionally used a number of plants species for treatment of infections disease and various ailments (Najla and Awaz, 2009).
The treatment of infectious diseases with antimicrobial agents continues to present problems in modern day, medical studies showed a significant increase in the incidence of side effects and the resistance that pathogenic microorganisms build against several antibiotic (Kunin, 1993;Finch, 1998;Iwu, 1999;Nascimento, 2000).However attention has been paid to plant and biologically active compounds isolated from them used in herbal medicine such as alkaloids, flavonoids, isoflavonoids, tannins,glycosides and phenolic compounds (Evans et al., 2002;Romero, 2005).
Achillea Millefolium L. (Yarrow) is flowering plant in the family Achillea Asteraceae grows in Europe, North America and Asia.a number of species were used as garden omamentals.The aerial parts of yarrow have along history of traditional herb medicine in veterinary medicine (Ludwing, 1996;Hirti, 2000).Preparation in the form of decoctions or fresh juices have applied against anorexia, stomach, cramps, flatulence, gastritis, enteritis, internal and external bleeding (bloody cough, nosebleed, and menstrual bleeding, bloody urine), wounds, sores, skin rash as well as dog and snake bite sites.Its name is derived from Achilles (means a mythical character), who carried it with his army to treat the wounds of his fellow soldiers during Trojan war, its specific name means (a thousand leaves) and refers to its shape like bird feather.This medicinal utillsation is also reflected in some of its common name: wound wort A, nosebleed, blood wort, sanguinar, herb milltaris and knights milfoil (Chandler et al., 1982).
The genus Achillea consists of 140 perennial herbs native to the northern hemispheres.Yarrow (fig. 1) aerial parts of the Achillea which has the widest range of herb applications in the world, it contains a volatile oil rich in sesquiterpene lactones and alkaloids (Muller et al., 1994).In vitro studies yarrow flower extract showed its antimicrobial activity to be more effective against certain microorganisms than yarrow leaves and stems (Chandler et al., 1982;Bisset, 1994;Pattnak, 1997;Nemeth and Bernath, 2008).
Fig. 1: Achillea Millefolium (aerial parts) (Chandler et al., 1982) Many plants in Mosul city are familiar and widely used medicinal applications or have the potential to cure some diseases.Yet, these plant do not receive enough assessment therefore this study was conducted to determine the antibacterial activity of yarrow flower against some gram positive and gram negative bacteria.

MATERIAL AND METHOD
Plant: Dried yarrow flower were collected from Mosul herbal herbarium and identified according to the relevant literature (The complete medicinal herb) by herbalist in College of Agriculture, Mosul University (Penlope, 1999).
Extract preparation: 10 gm of dried powder of yarrow flower was suspended in 150 ml of distilled water or absolute ethanol stirred on magnetic stirrer without heating until mixing well.then the mixture was left in refrigerator at 4C o for 24hr after that both infusions were filtered through many layers of gauze and by using Whatman No.1 filter paper, after drying the two samples of dried extract weighted and stored at 4 C o in dark bottles with aluminum foil to prevent negative effect of light (Al -Joboory and Al -Rawi,1994).Two sample stock solutions were prepared by suspending 1 gm of dried extract in 5 ml of distilled water or dimethyl sulfoxide for aqueous and alcoholic extract respectively 200 mg/ ml, then aqueous stock solution was sterilized by membrane filter o.22µ but alcoholic stock solution was sterilized by pasteurization at 62 C o for 10 min.(Al-Neman ,1998).Then tested against all microorganisms, compared with standard antibiotic Ciprofloxacin 5µg/ml, the filter paper disks were impregnated into extract and antibiotic solution (Collee et al., 1996).
Preparation of microbial cultures: The six bacteria were inoculated on nutrient broth incubated for 24hr., bacteria were inoculated in sterile Mueller Hinton agar (10 8 CFU /ml).Discs were applied on the surface agar medium by pressing slightly Petri dishes and then incubated at 35C o for 24hr.at the end of period, inhibition zones were measured in millimeters (Collins and Lyne, 1987).
Determination of MIC: the MIC for alcoholic extract showed highest antimicrobial effect, Dilution were prepared at concentration 150mg/ml, 100mg/ml, 50mg/ml and 25mg/ml for sensitive microorganisms (Atlas et al., 1995).

RESULTS AND DISCUSSION
In vitro antimicrobial activity of the aqueous and alcoholic extracts of yarrow flower 200 mg/ml was tested against three species of Gram -negative bacteria (Pseudo.aeruginosa, S. enterica enterica (Typhimurium) and Shig.flexneri) and three Grampositive species (Micro.luteus, Staph.aureus and Entero.faecalis) compared with standard antibiotic ciprofloxacin 5µg/ml was shown in Table .1 and Fig. 2. The alcoholic and aqueous flower extract of yarrow possessed a board spectrum of antimicrobial activity using the Kirby -Bauer disk diffusion test against all tested microorganisms at different raties, alcoholic extract had the greatest inhibitory effect (diameter of zone of inhibition) against Pseudo.aeruginosa 30 mm (1), Staph.aureus 24mm (2), and Micro.lutues 23 mm (3), while the aqueous extract slightly inhibited Pseudomonasa aeruginosa and Micrococcus luteus 12 mm and 10 mm respectively compared with standard antibiotic Ciprifloxcain 5 µg/ml: 26 mm, 22 mm and 22 mm but Entero.faecalis, S. enterica enterica (Typhimurium) and Shig.flexneri show high resistance for both alcoholic and aqueous extracts 8mm, 9mm, 8mm, 0mm , 12mm, 10mm compared to the sensitivity for ciprofloxacin : 23m, 30mm and 31mm, the results also showed that the most sensitive organism to both extracts was Pseudo.aeruginosa and the least sensitive organisme Entero.faecalis, then according to the results of dilution of alcoholic extract, the minimum inhibitory concentrations (MIC) sensitive microorganisms : Pseudo.aeruginosa (MIC=50 mg/ml), Staph.aureus (MIC=100 mg/ml) and Micro.luteus (MIC=100mg/ml).

Alcoholic aqueous
The result of this study agreed with recent investigation which have also pointed out to the notable effect of extracts of aerial parts of yarrow as it showed a board spectrum antimicrobial activities against Staph.aureus, E. coli, K. pneumoniae, A. niger and C. albicans (Candan et al., 2003 ;Skoeibusic et al., 2004;Radulovic, 2005).
It could be concluded that the alcoholic extract of yarrow flower have considerable antibacterial effect on some pathogenic microorganisms which cause infections.Therefore, befor application of these extracts as antimicrobial agents it must be evaluated in vivo and clinically.

CONCLUSION
On the light of these finding and other reports in this field, it could be concluded that: Yarrow flower extract have potential antibacterial effect against certain microorganisms which cause infections.
Possible benefits from other herbs with antibacterial activity.Further large -scale, well -designed clinical trails are required to prove more conclusive proof of their efficacy, the problem of microbial resistance is growing and the out look for use antimicrobial drugs in future is still uncertain, therefore, action must be taken to reduce this problem, control the use of antibiotic, develop research to better understand the genetic mechanisms of resistance and to continue studies to develop new drugs, either synthetic or natural.The aim is to offer appropriate and efficient antibacterial drugs to patient.
Fig. 2: Alcoholic and aqueous extract comparison with Ciprofloxacin.

Table 1 :
Antibacterial activity of yarrow flower extract.