In vitro activity of heather [Calluna vulgaris (L.) Hull] extracts on selected urinary tract pathogens

Calluna vulgaris L. Hull (Ericaceae) has been used for treatment of urinary tract infections in traditional medicine. In this study we analyzed in vitro antibacterial activity of the plant extracts on diff erent strains of Escherichia coli, Enterococcus faecalis and Proteus vulgaris, as well as the concentrations of total phenols and fl avonoids in the extracts. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. Th e concentrations of total phenols were examined by using Folin-Ciocalteu reagent and ranged between 67.55 to 142.46 mg GAE/g. Th e concentrations of fl avonoids in extracts were determined using spectrophotometric method with aluminum chloride and the values ranged from 42.11 to 63.68 mg RUE/g. Th e aqueous extract of C. vulgaris showed a signifi cant antibacterial activity. Th e values of MIC were in the range from 2.5 mg/ml to 20 mg/ml for this extract. Proteus vulgaris strains were found to be the most sensitive. Th e results obtained suggest that all tested extracts of C. vulgaris inhibit the growth of human pathogens, especially the aqueous extract.


INTRODUCTION
Heather, (Calluna vulgaris L. Hull, fam. Ericaceae) is an evergreen shrub. C. vulgaris can be found in most parts of Europe and Northern America from lowland up to alpine regions. Plant is traditionally used to treat urinary tract infection and infl ammatory disorders [1].
C. vulgaris has been used in ethnopharmacology as an antiseptic, antibacterial, cholagogue, diuretic, expectorant, antirheumatic and anti-inflammatory agent [2]. Several studies revealed antioxidant [3][4][5], antitumor and anti-inflammatory effects of C. vulgaris extract [6]. Heather leaves represent a very promising source of triterpenoids [7]. Numerous triterpenoids, including ursolic and oleanolic acid, possess antitumor and anti-inflammatory properties [8]. A new property of ursolic acid has been described in an acetone-extract of heather which could help explain the anti-inflammatory characteristics of this plant [9]. Previous research showed the activity C. vulgaris in the treatment of inflammatory diseases and wounds in the Swedish traditional medicine [10]. A large number of plant species are used in Danish folk medicine for treatment of depression and anxiety. One of the three most active extracts was the aqueous extract of aerial parts of C. vulgaris for antidepressive treatment [11]. The content of quercetin in C. vulgaris might explain the reported nerve calming effect of the plant [12].
A review of relevant literature revealed little data on antibacterial activity of diff erent extracts of C. vulgaris on the most frequent urinary tract pathogens. Th e aim of this study was to investigate in vitro antibacterial activity of aqueous, ethanol and ethyl acetate extract from leaves and fl owers of this plant. Th e second aim was to determine a total phenol and fl avonoid content in the extracts using spectrophotometric methods.

Plant material
During summer of 2010, leaves and fl owers of C. vulgaris were collected from natural populations on Borja Mountain in the region of Teslić municipality in Bosnia and Herzegovina. Plants identifi ed were confi rmed and voucher specimen was deposited at the Herbarium of the Department of Biology and Ecology, Faculty of Science, University of Kragujevac. Th e collected plant material was air dried in darkness at ambient temperature (20°C). Th e dried plant material was cut up and stored in paper bags until performing analysis.

Preparation of plant extracts
Prepared plant material (10g) was transferred to dark-colored fl asks with 200 ml of solvent (water, ethanol, ethyl acetate) and stored at room temperature. After 24 h, infusions were fi ltered through Whatman No. 1 fi lter paper and residue was re-extracted with equal volume of solvents. After 48 hours, the process was repeated. Combined supernatants were evaporated to dryness under vacuum at 40 °C using Rotary evaporator. Aqueous extract of herb was prepared at 80°C. Th e obtained extracts were kept in sterile sample tubes and stored at -20 ºC.
Determination of total phenolic content Th e C. vulgaris extracts were analyzed by spectrophotometry for total phenolics according to Folin-Ciocalteu procedure [13]. Th e reaction mixture was prepared by mixing 0.2 ml of methanolic solution of extract (1 mg/ml) and 1.5 ml of 110 Folin-Ciocalteu reagent dissolved in water. Th e mixture was allowed to equilibrate for 5 min and then mixed with 1.5 ml 6 NaCO 3 solution. After incubation for 90 minutes at room temperature in darkness, the absorbance of the mixture was read at 725 nm against a blank using spectrophotometer. Th e blank was prepared with methanol instead of extract solution. Th e samples were prepared in triplicate and the mean value of absorbance was obtained. Th e same procedure was repeated for gallic acid, which was used for calibration of standard curve. Total phenol content is reported as gallic acid equivalents by reference to linear equation of the standard curve (y = 0.008x + 0.0077, R² = 0.998). Th en the total phenolic content was expressed as gallic acid equivalents in miligrams per gram of extract (mg GAE/g of extract).

Determination of fl avonoid concentration
Th e concentrations of fl avonoids were determined using spectrophotometric method with aluminum chloride [14]. Th e sample contained 1 ml of methanolic solution of the extract in the concentration of 1 mg/ml and 1 ml of 2 AlCl 3 solution dissolved in methanol. Th e mixture was vigorously shaken, and after 10 minutes of incubation at room temperature, the absorbance versus a prepared blank was read at 430 nm using spectrophotometer. Th e samples were prepared in triplicate and the mean value of absorbance was obtained. Rutin was used as a standard for calibration of standard curve.

Macrodilution method
Th e minimum inhibitory concentration (MIC) of the extracts had been determined using the tube dilution method through the series of dilutions [16]. In the test tubes fi lled with the Mueller Hinton broth, the solution of the extracts is added and the series of double dilutes was made. In each of the test tubes 100μl of the suspension of the tested bacteria was added. Th e mixture was incubated for 24 hours at the temperature of 37 ºC. Th e same method was used to identify the MIC value for amoxicillin. Th ese values have been determined by inoculating the Mueller Hinton agar with the test tube content. Amoxicillin was used as a positive control. Whereas the extracts were dissolved in 10 DMSO, solvent control test was performed to study the eff ects of 10 DMSO on the growth of bacterial strains. It was observed that 10 DMSO did not inhibit the growth of bacteria.

Statistical analysis
Data are presented as means ± standard deviations where appropriate. All statistical analyses were performed using SPSS package (IBM Corp. Chicago, USA).

Total phenolic content and fl avonoid concentrations
Th e results of total phenolic content and fl avonoid concentrations in tested extracts from C. vulgaris are presented in Table 1. Th e total phenolic content was expressed as gallic acid equivalents. Th e aqueous extract had the highest phenolic content with 142.46 mg of GAE/g of extract. Th e ethanol extract was richer in phenolic active compounds than ethyl acetate extract. Th e content of fl avonoids was expressed as rutin equivalent. Total fl avonoid content in plant extracts ranged between 42.11 to 63.68 mg RUE/g of extract. High concentration of fl avonoids was measured in ethyl acetate extract from C. vulgaris.

Antibacterial activity
In vitro antibacterial activities of aqueous, ethanol and ethyl acetate extracts of C. vulgaris leaves and fl owers were studied on strains of Gram-positive and Gram-negative bacteria. Th e results of antibacterial activities of extracts against 30 strains of pathogenic bacteria are presented in Table 2. Antibacterial activity of tested extracts was evaluated by determining MICs and MBCs values. All tested extracts from C. vulgaris inhibited urinary pathogens extracted from urine samples. Th e strongest antibacterial activity was detected on P. vulgaris while the activities on E. coli and E. faecalis strains were similar. Aqueous extract showed the strongest eff ect against all tested bacterial strains. In general, the tested extracts demonstrated selective antibacterial activity and the activity depended both on the species of bacteria and on the type of extract. Ethyl acetate extract showed excellent activity on strains of P. vulgaris.

DISCUSSION
Previous research has indicated that phenolic compounds have antibacterial properties [17].
Phenolics belong to the group of secondary plant compounds and have various bioactivities such as antioxidat [18], anti-ulcer [19], and anti-infl ammatory [20]. Th e interest in possible health benefi ts of fl avonoids has increased due to their powerful antimicrobial activities [21].
Extract of C. vulgaris was found eff ective against M. tuberculosis bacillus [21] and the results of this study provide scientifi c basis for the traditional use of this plant in the treatment of tuberculosis [22].
Huttunen et al. [23] investigated antimicrobial activity of fi ve Finnish honey products against important human pathogens Streptococcus pneumoniae, S. pyogenes and Staphylococcus aureus. In this research, heather (C. vulgaris) honey showed signifi cant antimicrobial activity against all tested pathogens. Heather honey was tested against Staphylococcus aureus and C. vulgaris was shown it could be a source that could provide honey with high antibacterial activity [24].
Our previous studies have shown that species belonging to the family Ericaceae are rich in phenolics [25,26]. Th e antimicrobial activity of phenolic compounds from heather is also signifi cant. Antibacterial eff ects of diff erent extracts of C. vulgaris showed that phenolic compounds and fl avonoids were responsible for the growth inhibition of bacterial strains.
Th is study indicated that extracts of C. vulgaris have antibacterial activity and may have potential medical use. Studies of medicinal plants are important sources of knowledge for development of less harmful and more eff ective antimicrobial agents for treatment of urinary infections.

CONCLUSIONS
Th e results of this research suggest that aqueous, ethanol and ethyl acetate extract from C. vulgaris, tested in vitro, show great potential as a natural antibacterial agents. Th us, they may be useful in the treatment of infectious diseases caused by urinary tract pathogens. Aqueous extract may have a signifi cant eff ect on the prevention of the infections of urinary tract. Th e levels of the total phenolic content indicated that C. vulgaris extracts are rich source of the phenolic compounds. ¹Minimum inhibitory concentration (MIC) and ²minimum bactericidal concentration (MBC) values are given as mg/ml for plant extracts and μg/ml for antibiotic.