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

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 inflammatory 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 [3-5], 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 different extracts of C. vulgaris on the most frequent urinary tract pathogens. The aim of this study was to investigate in vitro antibacterial activity of aqueous, ethanol and ethyl acetate extract from leaves and flowers of this plant. The second aim was to determine a total phenol and flavonoid content in the extracts using spectrophotometric methods.

MATERIALS AND METHODS

Chemicals

Gallic acid, rutin hydrate and aluminium chloride hexahydrate (Al[Cl.sub.3]) were purchased from Acros Organics, New Jersey, USA. Organic solvents and sodium hydrogen carbonate were purchased from "Zorka pharma" Sabac, Serbia. Chlorogenic acid, Folin-Ciocalteu phenol reagent and 2,2-diphenyl-i-picrylhydrazyl (DPPH) were obtained from Sigma Chemicals Co, St Louis, MO, USA. Sodium carbonate ([Na.sub.2]C[O.sub.3]) was obtained from MP-Hemija, Belgrade, Serbia. Nutrient liquid medium, a Mueller-Hinton broth was purchased from Liofilchem, Italy. An antibiotic, amoxicillin, was purchased from Panfarma, Belgrade, Serbia.

Plant material

During summer of 2010, leaves and flowers of C. vulgaris were collected from natural populations on Borja Mountain in the region of Teslic municipality in Bosnia and Herzegovina. Plants identified were confirmed and voucher specimen was deposited at the Herbarium of the Department of Biology and Ecology, Faculty of Science, University of Kragujevac. The collected plant material was air dried in darkness at ambient temperature (20[degrees]C). The 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 flasks with 200 ml of solvent (water, ethanol, ethyl acetate) and stored at room temperature. After 24 h, infusions were filtered through Whatman No. 1 filter 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[degrees]C using Rotary evaporator. Aqueous extract of herb was prepared at 80[degrees]C. The obtained extracts were kept in sterile sample tubes and stored at -20[degrees]C.

Determination of total phenolic content

The C. vulgaris extracts were analyzed by spectrophotometry for total phenolics according to Folin-Ciocalteu procedure [13]. The 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. The mixture was allowed to equilibrate for 5 min and then mixed with 1.5 ml 6% NaC[O.sub.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. The blank was prepared with methanol instead of extract solution. The samples were prepared in triplicate and the mean value of absorbance was obtained. The 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, [R.sup.2] = 0.998). Then the total phenolic content was expressed as gallic acid equivalents in miligrams per gram of extract (mg GAE/g of extract).

Determination of flavonoid concentration

The concentrations of flavonoids were determined using spectrophotometric method with aluminum chloride [14]. The sample contained 1 ml of methanolic solution of the extract in the concentration of 1 mg/ml and 1 ml of 2% Al[Cl.sub.3] solution dissolved in methanol. The 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. The samples were prepared in triplicate and the mean value of absorbance was obtained. Rutin was used as a standard for calibration of standard curve. The concentrations of flavonoids were calculated from the linear equation of standard curve (y = 0.021x + 0.040, [R.sup.2] = 0.999). Then the concentrations of flavonoids were expressed as milligram of rutin equivalent per gram of extract (mg of RUE/g of extract).

Tested bacterial strains

Antibacterial activity of aqueous, ethanol and ethyl acetate extract from C. vulgaris was tested against urinary pathogens, extracted from urine samples, including ten different Escherichia coli strains ([Mf-Ec.sub.1], [Mf-Ec.sub.2], [Mf-Ec.sub.3], [Mf-Ec.sub.4], [Mf-Ec.sub.5], [Mf-Ec.sub.6], [Mf-Ec.sub.7], [Mf-Ec.sub.8], [Mf-Ec.sub.9], [Mf-Ec.sub.10]), ten Enterococcus faecalis strains ([Mf-Ef.sub.1], [Mf-Ef.sub.2], [Mf-Ef.sub.3], [Mf-Ec.sub.4], [Mf-Ef.sub.5], [Mf-Ef.sub.6], [Mf-Ef.sub.7], [Mf-Ef.sub.8], [Mf-Ef.sub.9], [Mf-Ef.sub.10]) and ten different strains of Proteus vulgaris ([Mf-Pv.sub.1], [Mf-Pv.sub.2], [Mf-Pv.sub.3], [Mf-Pv.sub.4], [Mf-Pv.sub.5], [Mf-Pv.sub.6], [Mf-Pv.sub.7], [Mf-Pv.sub.8], [Mf-Pv.sub.9], [Mf-Pv.sub.10]).

The Escherichia coli strains and Proteus vulgaris strains represented Gram-negative bacteria. Bacterial strains of Enterococcus faecalis were Gram-positive. These pathogens are the most frequent cause of urinary tract infections [15]. All clinical isolates were a generous gift from the Institute of Public Health, Banja Luka.

Suspension preparation

The original density of the bacterial suspension was 0.5 Mc Farland after which the additional dilution in saline at the proportion of 110 is made. The final concentration of the bacteria in the test tubes was 106 colony forming units (CFU)/ml.

Macrodilution method

The 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 filled 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 100 [micro]l of the suspension of the tested bacteria was added. The mixture was incubated for 24 hours at the temperature of 37[degrees]C. The same method was used to identify the MIC value for amoxicillin. These 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 effects 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 [+ or -] standard deviations where appropriate. All statistical analyses were performed using SPSS package (IBM Corp. Chicago, USA).

RESULTS

Total phenolic content and flavonoid concentrations

The results of total phenolic content and flavonoid concentrations in tested extracts from C. vulgaris are presented in Table 1. The total phenolic content was expressed as gallic acid equivalents. The aqueous extract had the highest phenolic content with 142.46 mg of GAE/g of extract. The ethanol extract was richer in phenolic active compounds than ethyl acetate extract. The content of flavonoids was expressed as rutin equivalent. Total flavonoid content in plant extracts ranged between 42.11 to 63.68 mg RUE/g of extract. High concentration of flavonoids 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 flowers were studied on strains of Gram-positive and Gram-negative bacteria. The 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.

The 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 effect 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-inflammatory [20]. The interest in possible health benefits of flavonoids has increased due to their powerful antimicrobial activities [21].

Extract of C. vulgaris was found effective against M. tuberculosis bacillus [21] and the results of this study provide scientific basis for the traditional use of this plant in the treatment of tuberculosis [22].

Huttunen et al. [23] investigated antimicrobial activity of five Finnish honey products against important human pathogens Streptococcus pneumoniae, S. pyogenes and Staphylococcus aureus. In this research, heather (C vulgaris) honey showed significant 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]. The anti-microbial activity of phenolic compounds from heather is also significant. Antibacterial effects of different extracts of C. vulgaris showed that phenolic compounds and flavonoids were responsible for the growth inhibition of bacterial strains.

This 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 effective antimicrobial agents for treatment of urinary infections.

CONCLUSIONS

The 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. Thus, they may be useful in the treatment of infectious diseases caused by urinary tract pathogens. Aqueous extract may have a significant effect on the prevention of the infections of urinary tract. The levels of the total phenolic content indicated that C. vulgaris extracts are rich source of the phenolic compounds.

ACKNOWLEDGEMENTS

This study was supported by the Ministry of Education and Science of the Republic of Serbia, grants No. OI173032 and No. 41010.

DECLARATION OF INTEREST

The authors declare that they have no competing interests.

REFERENCES

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[25] Vucic DM, Petkovic MR, Rodic-Grabovac BB, Stefanovic OD, Vasic SM, Comic LjR. Phenolic content, antibacterial and anti-oxidant activities of Erica herbacea L. Acta Pol Pharm 2013; 7(45)5130-5136.

[26] Vucic DM, Petkovic MR, Rodic-Grabovac BB, Stefanovic OD, Vasic SM, Comic LjR. Antibacterial and antioxidant activities of bilberry (Vaccinium myrtillus L.) in vitro. Afr J Microbiol Res 2013; 7(45)5130-5136.

Dragana M. Vucic (1) *, Miroslav R. Petkovic (1), Branka B. Rodic-Grabovac (2), Olgica D. Stefanovic (3), Sava M. Vasic (3), Ljiljana R. Comic (3)

(1) Department of Microbiology, Medical Faculty, University of Banja Luka, Banja Luka, Bosnia and Herzegovina. (2) Department of Organic Chemistry, Faculty of Technology, University of Banja Luka, Banja Luka, Bosnia and Herzegovina. (3) Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Kragujevac, Serbia

* Corresponding author: Dragana Vucic, Koste Jarica 34, 78 000 Banja Luka, Bosnia and Herzegovina, Tel: +387 65 456478, +387 51 437245, E-mail draganavucicbl@gmail.com

Submitted: 27 January 2014 / Accepted: 10 June 2014
TABLE 1. Total phenolic contents and concentrations of
flavonoids in leaves and flowers of C vulgaris extracts

Type of         Total phenolic content1   Flavonoid concentration1
extract          (mg GAE/g of extract)     (mg RUE/g of extract)

Water            142.46 [+ or -] 0.50       42.11 [+ or -] 0.32
Ethanol           81.86 [+ or -] 0.95       43.03 [+ or -] 0.11
Ethyl acetate     67.55 [+ or -] 0.38       63.68 [+ or -] 0.19

(1) Values represent mean [+ or -] standard deviation.

TABLE 2. Antibacterial activities of aqueous, ethanol
and ethyl acetate extracts from leaves and flowers of
C. vulgaris against tested strains of bacteria.

Species                   Aqueous extract     Ethanol
                                              extract

                         MIC (1)   MBC (2)   MIC   MBC

E. coli Mf-Ec1             20        40      20    40
E. coli Mf-Ec2             20        40      20    40
E. coli Mf-Ec3             10        20      20    40
E. coli Mf-Ec4             20        40      20    40
E. coli Mf-Ec5             10        20      20    40
E. coli Mf-Ec6             10        20      20    40
E. coli Mf-Ec7             10        20      20    40
E. coli Mf-Ec8             10        20      20    40
E. coli Mf-Ec9             10        20      20    40
E. coli Mf-Ec10            10        20      20    40
E. faecalis Mf-Ef1         20        40      20    >40
E. faecalis Mf-Ef2         10        20      20    >40
E. faecalis Mf-Ef3         10        20      20    >40
E. faecalis Mf-Ef4         10        20      20    >40
E. faecalis Mf-Ef5         20        40      20    >40
E. faecalis Mf-Ef6         20        40      20    >40
E. faecalis Mf-Ef7         10        20      20    >40
E. faecalis Mf-Ef8         10        20      20    >40
E. faecalis Mf-Ef9         10        20      20    >40
E. faecalis Mf-Ef10        10        20      20    >40
P. vulgaris Mf-Ef1         2.5        5      10    40
P. vulgaris Mf-Ef2         2.5        5      10    40
P. vulgaris Mf-Ef3         2.5        5      10    40
P. vulgaris Mf-Ef4         2.5        5      10    40
P. vulgaris Mf-Ef5         2.5        5      10    40
P. vulgaris Mf-Ef6         2.5        5      10    40
P. vulgaris Mf-Ef7         2.5        5      10    40
P. vulgaris Mf-Ef8         2.5        5      10    40
P. vulgaris Mf-Ef9         2.5        5      10    40
P. v ulga ris Mf-Ef10      2.5        5      10    40

Species                    Ethyl      Amoxicillin
                          acetate
                          extract

                         MIC   MBC    MIC     MBC

E. coli Mf-Ec1           40    >40   1000    2000
E. coli Mf-Ec2           40    >40   4000    8000
E. coli Mf-Ec3           40    >40     4       8
E. coli Mf-Ec4           40    >40     2       4
E. coli Mf-Ec5           40    >40   2000    4000
E. coli Mf-Ec6           40    >40   2000    4000
E. coli Mf-Ec7           40    >40     4       4
E. coli Mf-Ec8           40    >40   1000    2000
E. coli Mf-Ec9           40    >40   4000    8000
E. coli Mf-Ec10          40    >40   1000    2000
E. faecalis Mf-Ef1       40    >40   0.977    125
E. faecalis Mf-Ef2       40    >40   0.488   >125
E. faecalis Mf-Ef3       40    >40   0.488   >125
E. faecalis Mf-Ef4       40    >40   0.488   >125
E. faecalis Mf-Ef5       40    >40   0.488   >125
E. faecalis Mf-Ef6       40    >40   0.488   >125
E. faecalis Mf-Ef7       40    >40   0.977   >125
E. faecalis Mf-Ef8       40    >40   0.488    125
E. faecalis Mf-Ef9       40    >40   0.488   >125
E. faecalis Mf-Ef10      40    >40   0.488   >125
P. vulgaris Mf-Ef1        5    10    >4000   >4000
P. vulgaris Mf-Ef2        5    20    >4000   >4000
P. vulgaris Mf-Ef3        5    10    >4000   >4000
P. vulgaris Mf-Ef4        5    10    0.977   7.812
P. vulgaris Mf-Ef5        5    20    >4000   >4000
P. vulgaris Mf-Ef6        5    20    0.977   7.812
P. vulgaris Mf-Ef7        5    10    >4000   >4000
P. vulgaris Mf-Ef8        5    20    2000    >4000
P. vulgaris Mf-Ef9        5    20    >4000   >4000
P. v ulga ris Mf-Ef10     5    20    >4000   >4000

(1) Minimum inhibitory concentration (MIC) and
(2) minimum bactericidal concentration (MBC)
values are given as mg/ml for plant extracts
and [micro]g/ml for antibiotic.
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Author:Vucic, Dragana M.; Petkovic, Miroslav R.; Rodic-Grabovac, Branka B.; Stefanovic, Olgica D.; Vasic, S
Publication:Bosnian Journal of Basic Medical Sciences
Article Type:Report
Geographic Code:4EXBO
Date:Nov 1, 2014
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