Antibacterial Effects of Camellia sinensis and Achillea millefolium on Several Antibiotic-resistant Bacteria

out collaboration between all Authors AA and SAM designed the study and wrote the first draft of the manuscript. Author AA performed the statistical analysis. Author MRK managed the literature searches. Author FK prepared the plant and author DS prepared the bacteria. the ABSTRACT Background and Objectives: Infectious diseases are among the most known important causes of morbidity and mortality in the world. The ongoing explosion antibiotic-resistant bacteria and side effects of medications, application of assistive methods including the use of medicinal plants for treatment have become particularly important. Materials and Methods: In this study, the ethanol extract of green tea ( Camellia sinensis ) and yarrow flower ( Achillea millefolium ) were prepared by maceration method and its antibacterial effect on four clinical strains of antibiotic resistant bacteria including Klebsiella pneumoniae, Acinetobacter, Escherichia coli and Pseudomonas aeruginosa were evaluated with microtiter plate method for determining the MIC (Minimum Inhibitory Concentration). Results: The MICs of green tea extract for mg/ml for Klebsiella pneumoniae, Pseudomonas aeruginosa Achillea millefolium extract for Acinetobacter baumannii against Klebsiella pneumoniae and Conclusion: Considering the antimicrobial effects of green tea and Achillea on four strains of resistant bacteria, the extract of this plant can be used in control and treatment of infections caused by these infectious agents.


INTRODUCTION
Emerging infectious diseases imposes a significant financial burden on global economies and health [1]. Increasing resistance to common antibiotics due to irregular use of them, threated the public health. To prevent this threat, identification and use of new antibacterial compounds is required [2]. Natural products derived from medicinal plants as major of biologically active compounds have been important candidates for production of new chemicals in the pharmacy. There are about 500,000 plant species around the world that only one percent of their phytochemicals have been evaluated, and there is a great potential for the discovery of novel bioactive compounds Recently, the use of herbal medicines are widely used because of low side effects, low cost, easy access and compatible with human nature The plants have been used in traditional medicine for thousands of years and even with the advances of modern medicine, products derived from medicinal plants have become the basis for expansion of new chemical drugs in pharmacy [5].
Green tea (Camellia sinensis ) is one of the most consumed beverage in the world especially Iran [6], increase in the use of green tea is day by day due to its wide range of disease treating potential. Green tea contains protein, amino acids, carbohydrates minerals, trace elements ,lipids , vitamins B, C and E, caffeine, theophylline, poly phenolic compounds Green tea has showed antibacteri alactivity against a many strain of bacteria, both Gram positive and Gram negative bacteria showed antiviral effect [7].

2
The MICs of green tea extract for Acinetobacter baumannii was 15.6 mg/ml and 31.25 Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli. The MIC of Acinetobacter baumannii and Escherichia coli was 125 mg/ml and and Pseudomonas aeruginosa was 250 mg/ml. Considering the antimicrobial effects of green tea and Achillea on four strains of resistant bacteria, the extract of this plant can be used in control and treatment of infections caused Achillea millefolium; minimum inhibitory concentration; Emerging infectious diseases imposes a significant financial burden on global economies Increasing resistance to common antibiotics due to irregular use of them, has public health. To prevent this threat, identification and use of new antibacterial . Natural products derived from medicinal plants as major sources of biologically active compounds have been important candidates for production of new chemicals in the pharmacy. There are about 500,000 plant species around the world that only one percent of their phytochemicals have been a great potential for the discovery of novel bioactive compounds [3]. Recently, the use of herbal medicines are widely used because of low side effects, low cost, easy access and compatible with human nature [4]. The plants have been used in traditional edicine for thousands of years and even with the advances of modern medicine, products derived from medicinal plants have become the basis for expansion of new chemical drugs in ) is one of the most consumed beverage in the world especially Iran , increase in the use of green tea is day by day due to its wide range of disease treating potential. Green tea contains protein, amino acids, carbohydrates minerals, trace elements lipids , vitamins B, C and E, caffeine, theophylline, poly phenolic compounds [7]. Green tea has showed antibacteri alactivity against a many strain of bacteria, both Gram positive and Gram negative bacteria [8] and L. belonging to the Family that grows in Asia, Europe, North [9]. Achillea species contains Proazulenes, essential oils, flavonoids, coumarins, terpenoids, sterols, lignans, amino acids and alkamides species have several effects such as antibacterial, anti-inflammatory, antitumor, anti cirrhosis, anti fever, and useful for curing insomnia,stopping the bleeding and for wound healing [9,11].

Yarrow
The aim of this study is evaluation of antibacterial effects of ethanol extract of millefolium and green tea on several strains of antibiotic-resistant human pathogenic bacteria in the laboratory condition.
; Article no.EJMP.33395 was 15.6 mg/ml and 31. 25 . The MIC of was 125 mg/ml and Considering the antimicrobial effects of green tea and Achillea on four strains of resistant bacteria, the extract of this plant can be used in control and treatment of infections caused minimum inhibitory concentration; antibioticflavonoids, coumarins, terpenoids, sterols, lignans, amino acids and alkamides [10]. Achillea species have several effects such as inflammatory, antitumor, anti cirrhosis, anti fever, and useful for curing insomnia,stopping the bleeding and for wound The aim of this study is evaluation of effects of ethanol extract ofAchillea and green tea on several strains of resistant human pathogenic bacteria in

Plant Preparation and Extraction
Yarrow plants in late June, its flowering season, were collected from the region of Sardasht, Lordegan city located in the province of Chahar Mahal and Bakhtiari. The plant samples were evaluated and registered as Achillea wilhelmsii with herbarium number 19706 by Leila Ghaem Maghami at University of Isfahan.
Flowers were isolated and dried under appropriate conditions, away from light and moisture. Then for optimized extraction of flowers, they were powdered with electric mill.
Green tea was purchased from medicinal plants market of Isfahan.
The dried powder was mixed with 50% ethanol solution. After 72-48 hours it was filtered through Buchner funnel. Then extract was placed in the rotary evaporator (Heidolph Company, Germany) in order to separate the solvent from the extract [12]. Then, they were put in the oven at 40°C to dry completely.

Bacterial Isolates and Media Preparation
Four antibiotic resistant isolates of bacteria including Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa were isolated from patients who referred to Al-Zahra hospital, Isfahan, IRAN.

Determination of Minimum Inhibitory Concentration (MIC)
The extracts were tested against pathogens by a micro-dilution broth method for determine of minimum inhibitory concentration (MIC). bacteria fresh culture was diluted at tryptose soy broth (TSB; Quelab, Montreal, Canada), the turbidity equal to 0.5 McFarland was prepared and diluted at a ratio of 1 to 100 to obtain turbidity equal to 1×10 6 . Different dilutions (Serial dilutions) extracts were prepared in broth media, .Then 100 µl of different dilutions of extract containing 100 µl of bacterial suspension were poured in 96-well polystyrene plates [13]. Also, wells containing 200 µl of broth media and wells containing broth media and bacteria, were considered as negative and positive controls, respectively. Some wells containing 100 µl media and 100 µl from each dilution were considered as turbidity controls. Three replications were considered for each bacteria. Then, the plate surfaces were covered and incubated in incubator for 24 h at 37°C. The growth of bacteria was measured at 630 nm using a micro-plate reader (AWARENESS, Technology INC, Atat fax 2100).

Statistic Analysis
The data were statistically analyzed (One way ANOVA and Tukey's post hoc tests) on Graf Pad Prism 5 software (Graph Pad Software Inc., CA, USA). A P-value of less than0.05 was considered statistically significant.

RESULTS
Results of tests for evaluation of sensitivity of bacteria to several antibiotics are shown in Table  1 indicated that this bacteria have resistant to many of examined antibiotics.
In this study, the minimum inhibitory concentrations of green tea and yarrow flower extracts against four strains of antibiotics resistant pathogenic bacteria were investigated and the results are shown in Table 2.
MIC of green tea for Acinetobacter baumannii was 15.6 mg/mL which was significantly less than that for the other three bacteria (P<0.05). and for Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa was 31.25 mg/mL In the case of yarrow flowers, the highest MIC, 250 mg/ml, was related to Klebsiella pneumoniae and Pseudomonas aeruginosa, which was significantly (P <0/05) higher than two other bacteria. The MIC of green tea on four bacterial strains was less than that of Achila millefolium (P <0/05), indicating its higher antibacterial activity.
The inhibitory effects of different concentrations of green tea and yarrow flower (Achila millefolium) extracts on the growth of the tested bacteria are shown in Fig. 1. In 500-125 mg/mL concentrations of yarrow flower, the growth of Escherichia coli and Acinetobacter baumannii decreased >90% and in 500-250 mg/mL of yarrow flower, the growth of Klebsiella pneumoniae and Pseudomonas aeruginosa decreased >90%. In addition, in 500-31.25 mg/mL concentrations of Green tea the growth of Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosain its 500-15.6 mg/mL concentration, the growth of S. sanguis decreased >90%.

DISCUSSION
Recently, there has been a growing interest in the use of herbal medicine in developed and developing countries [14]. plant medicine has been considered to treat different diseases, especially infectious ones. Today, there is a universal tendency toward utilizing plant drugs instead of chemical drugs. Wide acceptance of the plant drugs in the field of Medical sciences has been observed due to less side effects, as well as appropriate prices [15].
Green tea is the second oldest and largest most consumed drinks in the world [7]. Recent studies suggest that green tea help to reduction in the risk of cardiovascular disease and cancer, help to oral health and have anti-hypertensive effect, body weight control, antibacterial and antivirasic activity, UV protection [16].
Achillea millefolium L. is belongs to the Asteraceae family that is medicinal plant and used in folk medicine against gastrointestinal disorders, lack of appetite [17].
In this study, antimicrobial activity of green tea and yarrow flower on four strains of antibiotic resistant bacteria including Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa was evaluated in vitro. The results showed that green tea and yarrow flower (Achillea millefolium) extracts has antibacterial effects these bacteria.
There are several study regarding to antimicrobial activity of Green tea and yarrow: In our study, it was showed that effect of green tea on growth bacteria was significantly higher than that of yarrow flower (P<0.05). The MIC of green tea for Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa were less than those of yarrow flower (P<0.05).However, further investigations are needed to determine cause of the findings.

CONCLUSION
Due to the positive effects of two Herbal extracts on hospital bacteria resistant to several common antibiotics, the plants can be used in different pharmaceutical formulation for the prevention and control of infectious diseases associated with these bacteria. It is recommended that additional studies be conducted to establish the optimal effect of the above extracts.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.