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Antimicrobial activity of selected plant species of Genera arbutus l., Bruckenthalia rchb., Calluna salisb. and Erica l. (Ericaceae)

Antimicrobial activity of selected plant species of Genera arbutus l., Bruckenthalia rchb.,... ACTA FACULTATIS MEDICAE NAISSENSIS DOI: 10.2478/afmnai-2014-0009 UDC: 633.886 Scientific Journal of the Faculty of Medicine in Nis 2014;31(1):81-85 Original article Dragana Pavlovi1, Branislava Lakusi2, Dusanka Kiti1, Milica Milutinovi1, Milica Kosti1, Bojana Miladinovi1, Nada Kovacevi3 University of Nis, Faculty of Medicine, Department of Pharmacy, Serbia University of Belgrade, Faculty of Pharmacy, Institute of Botany, Serbia 3 University of Belgrade, Faculty of Pharmacy, Institute of Pharmacognosy, Serbia SUMMARY Uvae ursi folium (Arctostaphylos uva ursi, Ericaceae) is the best known and most widely used herbal urinary antiseptic. In traditional medicine, other Ericaceae species are also used for the treatment of urinary tract infections. The present study investigates antimicrobial activity of five species of Ericaceae family native to the Balkan Peninsula: Arbutus unedo, Bruckenthalia spiculifolia, Calluna vulgaris, Erica arborea and Erica carnea. Ethanolic extracts were tested against 10 different gram-positive and gram-negative bacteria by the disc diffusion technique, where standard tetracycline, streptomycin and penicillin discs and discs containing crystal violet (1 mg/ml) and solvent (70.0% v/v ethanol) were used as controls. The most prominent antibacterial effect was achieved on Staphylococcus aureus with extracts of Calluna vulgaris and Erica carnea. Tested samples showed no activity against the gram-negative strains Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Inhibitory effects on the growth of gram-positive bacteria were more potent. The exception is Arbutus unedo ethanol extract which exhibited certain activity against a laboratory strain of wild Escherichia coli. Antimicrobial activity of the ethanolic extracts against 10 tested strains of bacteria in disc diffusion assay was generally weak, even for sample in which HPLC determination confirmed the presence of arbutin (secondary metabolite responsible for most of the antibacterial activity of Uvaeursi folium). Key words: Ericaceae, antimicrobial activity, disc diffusion, arbutin Corresponding author: Dusanka Kiti · phone: 063 104 50 43 · e-mail: duska@medfak.ni.ac.rs · 81 ACTA FACULTATIS MEDICAE NAISSENSIS, 2014, Vol 31, No 1 INTRODUCTION Arctostaphylos uva ursi, the famous member of the Ericaceae family, is an ancient astringent and urinary antiseptic. Therapeutic indication for bearberry leaf, Uvae ursi folium, is uncomplicated infections of the lower urinary tract such as cystitis, when antibiotic treatment is not considered essential (1). The antimicrobial effect of Uvae ursi folium is associated with the aglycone hydroquinone released from arbutin or arbutin waste products in the alkaline urine (2). In traditional medicine, other Ericaceae species are also used for the treatment of urinary tract infections (3-5). Our research involved five species from Ericaceae family that grow wild in Serbia and Montenegro, in addition to Arctostaphylos uva ursi: Arbutus unedo L., Bruckentalia spiculifolia (Salisb.) Reichenb., Calluna vulgaris Salisb., Erica arborea L. and Erica carnea L. Despite the fact that these species are often used in popular medicine in their region of origin, only few of these selected species have been partly investigated. on) and NCIMB (National Collection of Industrial Bacteria, Aberdeen). Gram-positive bacteria: Staphylococcus aureus (ATCC29213), Staphylococcus epidermidis (ATCC 12228), Micrococcus flavus (ATCC10240), Bacillus subtilis (ATCC10707), Sarcinalutea (ATCC9391). Gram-negative bacteria: Pseudomonas aeruginosa (ATCC9027), Klebsiella pneumoniae (NCIB9111) and Escherichia coli (E. coli) (ATCC25922). Two laboratory strains of E. coli were also included in the study: SY252 (strain with weakened wall) and LPCA (the wild laboratory strain). The bacterial inoculates were made up from overnight broth cultures. Suspensions with microorganisms were adjusted to 0.5 McFarland standard turbidity according to consensus standard of the National Committee for Clinical Laboratory Standards (10). Suspensions were spread on Muller-Hinton agar (MHA, Torlak) in sterilized Petri dished (90 mm in diameter). Experimental Procedure The antimicrobial study was carried out by disc diffusion technique (11). All tested extracts were weighed under aseptic conditions in sterile volumetric flasks, and dissolved with 70.0% sterile ethanol to obtain different extract concentrations (concentrations 1, 2, 3 and 4 contained 40.0, 20.0, 10.0 and 5.0% of crude extract, respectively). Standard tetracycline, streptomycin and penicillin discs and discs containing crystal violet (1 mg/ml) and solvent (70.0% v/v ethanol) were used as controls. All probes were applied in 20 l volumen per disk. According to this method, the antimicrobial potency of the test samples was measured by determining the diameter of the zones of inhibition in millimetres. All determinations were made in duplicate. MATERIAL AND METHODS Plant material Plant materials were collected from wild growing species of Ericaceae family: Arbutus unedo (Lustica, Montenegro) HFF (Herbarijum Farmaceutskog Fakulteta Univerziteta u Beogradu - Herbarium collection of the Faculty of Pharmacy, University of Belgrade) No. 1173; Bruckentalia spiculifolia (Kopaonik - Panciev vrh, Serbia) HFF No. 1217; Calluna vulgaris (Loznica - Gucevo, Serbia) HFF No. 1272; Erica arborea (Lustica, Montenegro) HFF No. 1430; Ericacarnea (syn.: Erica herbacea L., Erica saxatilis Salisb.) (Mokra Gora, Serbia) HFF No. 1431. Authenticated voucher herbarium specimens have been deposited in the Herbarium collection of the Faculty of Pharmacy, University of Belgrade (HFF). Determination was done according to Flora of SR Srbija (6), Flora Europea (7) and Anatomy of the Dicotyledons (8). RESULTS The antimicrobial activity of tested samples against selected microorganisms is showed in Table 1. Diameter of sterile paper disk was 13 mm. Thus, diameters of the inhibition zones larger than 13 mm were presented as positive results. Applied extract concentrations (40.0, 20.0, 10.0 and 5.0% of crude extract) were labelled as concentrations 1, 2, 3 and 4, respectively. As referent antimicrobial drugs, tetracycline, streptomycin and penicillin alongside discs containing crystal violet exhibited obviously higher antimicrobial activity than tested ethanolic extracts of selected Ericaceae species. Extract solvent, ethanol, did not affect any of tested bacterial strains. Extraction procedure Plant material, dried leaves of selected Ericaceae species, was reduced to a fine powder and extracted with ethanol (70.0%, v/v) by percolation, as described in European Pharmacopeia 6.0 (9). Ethanolic extracts of A. unedo (AE), B. spiculifolia (BE), C. vulgaris (VE), E. arborea (EE) and E. carnea (CE) were obtained after evaporation to the dryness in vacuo below 40ºC and extraction yields were 45.05, 32.35, 33.84, 38.98 and 38.61% (w/w), respectively. Test Organisms Both gram-positive and gram-negative bacterial strains used for the experiment were collected as pure cultures from ATCC (American Type of Culture Collecti82 Dragana Pavlovi et al. Table 1. Results of antimicrobial assay showed as diameters of the of inhibitory zones in millimetres Ethanol 70% v/v / / / / / / 13.5 / / / Crystal violet Microorganism AE BE VE 1=14.5 2=14 3=13.5 / / 1,2=14 3=13.5 / / / / / / EE CE 1=16 2=15 3=14 / / 1-4=14 1=13.5 / / / / / Antibiotic Staphylococcus aureus Staphylococcus epidermidis Micrococcusflavus Bacillus subtilis Sarcinalutea Pseudomonas aeruginosa Klebsiella pneumoniae E. coli E. coli SY252 E. coli LPCA / 1,2=14.5 3=14 1=17 2,3=16 4=15 1=14.5 2,3=14 1,2=14.5 3=14 / / / / 1=15 2=14 3=13.5 1=14 2,3=13.5 / 1=14.5 2=14 1,2,3=14 1=15 2=14.5 / / / / / / / 1,2=13.5 1=14 1,2=13.5 / / / / / 17 21 22 20 20 / 16 / 14 21 DISCUSSION Ethanolic extracts of A. unedo, B. spiculifolia, C. vulgaris, E. arborea and E. carnea were compared between each other and with A. uva ursi, species of this family well known for its antimicrobial properties. The disc diffusion method was used to determine the inhibition zones of the five ethanolic extracts from different Ericaceae species. The five gram-positive and five gram-negative bacterial strains have been used. In general, phenolics are the predominant active chemicals in plants, with gram-positive bacteria being the most sensible germs (12). Our results for AE, BE, VE, EE and CE antimicrobial activity string along these findings. According to the results in Table 1, different ethanolic extracts showed certain antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus flavus, Bacillus subtillis and Sarcinalutea. Antimicrobial activity against the Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli was not observed. The only exception is mild impact of A. unedo extract (AE) on E. coli LPCA (the wild laboratory strain of E. coli). The most prominent antimicrobial effect was achieved on Micrococcus flavus with AE. Inhibition zones observed for concentration 1 (17 mm) were slightly larger than those observed for concentrations 2 and 3 (16 mm) and 4 (15 mm) of A. unedo ethanolic extracts. The antimicrobial activity of A. unedo root extract against E. coli and Staphylococcus aureus (13) and antiprotosoal activity of A. unedo leaf extract against Leishmania tropica (14) and Trichomonas vaginalis (15) has already been reported. According to Kumarasamy et al. (2002) methanolic extract of C. vulgaris seed showed bacteriostatic activity against the gram-positive strains Staphylococcus aureus and Staphylococcus hominis (with minimal inhibitory concentration 0,1 g/ml) (16). Our results for VE indicate very mild impact on Staphylococcus aureus and Bacillus subtilis. The 30.0% ethanol extract of Uvaeursi folium inhibited the growth in vitro of Bacillus subtilis, E. coli, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens and Staphylococcus aureus. Ethanol extract were also active in vitro against E. coli, Proteus vulgaris, Staphylococcusfaecalis and Enterobacter aerogenes. However, 95.0% ethanol extract had no antibacterial activity (17). Arbutin is generally considered to be respon83 ACTA FACULTATIS MEDICAE NAISSENSIS, 2014, Vol 31, No 1 sible for the antibacterial activity of the Uvae ursi folium extract. Preliminary chemical analysis showed the presence of hydroquinone and phenil propanoid derivates, flavonoids and tannins in A. unedo, B. spiculifolia, C. vulgaris, E. arborea and E. carnea. Our previous results, obtained using the HPLC method, also confirmed the presence of arbutin only in leaves of Arbutus unedo (1.21 ±0.03%) and absence of hydroquinone in all tested samples (18). The amount of arbutin in dry A. uva ursi leaves ranges from 5.0 to 15.0% (17), while according to the monograph of European Pharmacopoeia 6.0 (9), dried drug Uvae ursi folium should contain minimum 7.0% of anhydrous arbutin. Thus, the presence of arbutin in AE could be, at least partly, responsible for measured antibacterial activity. However, in this preliminary screening, it was observed that antimicrobial activity of all tested samples was generally weak. CONCLUSION Based on the results of this in vitro tests on antimicrobial activity of leaf ethanolic extracts of Arbutus unedo, Bruckentalia spiculifolia, Calluna vulgaris, Erica arborea and Erica carnea, and bearing in mind that only leaves of Arbutus unedo contain arbutin (although in significantly smaller quantity than Uvae ursi folium), the traditional use of leaf of this species in the treatment of urinary tract infections cannot be considered plausible. Acknowledgement This research was supported by the Ministry of Education and Science of the Republic of Serbia (Grants No.III 41018 and III 46013). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Medicae Naissensis de Gruyter

Antimicrobial activity of selected plant species of Genera arbutus l., Bruckenthalia rchb., Calluna salisb. and Erica l. (Ericaceae)

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de Gruyter
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2217-2521
DOI
10.2478/afmnai-2014-0009
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Abstract

ACTA FACULTATIS MEDICAE NAISSENSIS DOI: 10.2478/afmnai-2014-0009 UDC: 633.886 Scientific Journal of the Faculty of Medicine in Nis 2014;31(1):81-85 Original article Dragana Pavlovi1, Branislava Lakusi2, Dusanka Kiti1, Milica Milutinovi1, Milica Kosti1, Bojana Miladinovi1, Nada Kovacevi3 University of Nis, Faculty of Medicine, Department of Pharmacy, Serbia University of Belgrade, Faculty of Pharmacy, Institute of Botany, Serbia 3 University of Belgrade, Faculty of Pharmacy, Institute of Pharmacognosy, Serbia SUMMARY Uvae ursi folium (Arctostaphylos uva ursi, Ericaceae) is the best known and most widely used herbal urinary antiseptic. In traditional medicine, other Ericaceae species are also used for the treatment of urinary tract infections. The present study investigates antimicrobial activity of five species of Ericaceae family native to the Balkan Peninsula: Arbutus unedo, Bruckenthalia spiculifolia, Calluna vulgaris, Erica arborea and Erica carnea. Ethanolic extracts were tested against 10 different gram-positive and gram-negative bacteria by the disc diffusion technique, where standard tetracycline, streptomycin and penicillin discs and discs containing crystal violet (1 mg/ml) and solvent (70.0% v/v ethanol) were used as controls. The most prominent antibacterial effect was achieved on Staphylococcus aureus with extracts of Calluna vulgaris and Erica carnea. Tested samples showed no activity against the gram-negative strains Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Inhibitory effects on the growth of gram-positive bacteria were more potent. The exception is Arbutus unedo ethanol extract which exhibited certain activity against a laboratory strain of wild Escherichia coli. Antimicrobial activity of the ethanolic extracts against 10 tested strains of bacteria in disc diffusion assay was generally weak, even for sample in which HPLC determination confirmed the presence of arbutin (secondary metabolite responsible for most of the antibacterial activity of Uvaeursi folium). Key words: Ericaceae, antimicrobial activity, disc diffusion, arbutin Corresponding author: Dusanka Kiti · phone: 063 104 50 43 · e-mail: duska@medfak.ni.ac.rs · 81 ACTA FACULTATIS MEDICAE NAISSENSIS, 2014, Vol 31, No 1 INTRODUCTION Arctostaphylos uva ursi, the famous member of the Ericaceae family, is an ancient astringent and urinary antiseptic. Therapeutic indication for bearberry leaf, Uvae ursi folium, is uncomplicated infections of the lower urinary tract such as cystitis, when antibiotic treatment is not considered essential (1). The antimicrobial effect of Uvae ursi folium is associated with the aglycone hydroquinone released from arbutin or arbutin waste products in the alkaline urine (2). In traditional medicine, other Ericaceae species are also used for the treatment of urinary tract infections (3-5). Our research involved five species from Ericaceae family that grow wild in Serbia and Montenegro, in addition to Arctostaphylos uva ursi: Arbutus unedo L., Bruckentalia spiculifolia (Salisb.) Reichenb., Calluna vulgaris Salisb., Erica arborea L. and Erica carnea L. Despite the fact that these species are often used in popular medicine in their region of origin, only few of these selected species have been partly investigated. on) and NCIMB (National Collection of Industrial Bacteria, Aberdeen). Gram-positive bacteria: Staphylococcus aureus (ATCC29213), Staphylococcus epidermidis (ATCC 12228), Micrococcus flavus (ATCC10240), Bacillus subtilis (ATCC10707), Sarcinalutea (ATCC9391). Gram-negative bacteria: Pseudomonas aeruginosa (ATCC9027), Klebsiella pneumoniae (NCIB9111) and Escherichia coli (E. coli) (ATCC25922). Two laboratory strains of E. coli were also included in the study: SY252 (strain with weakened wall) and LPCA (the wild laboratory strain). The bacterial inoculates were made up from overnight broth cultures. Suspensions with microorganisms were adjusted to 0.5 McFarland standard turbidity according to consensus standard of the National Committee for Clinical Laboratory Standards (10). Suspensions were spread on Muller-Hinton agar (MHA, Torlak) in sterilized Petri dished (90 mm in diameter). Experimental Procedure The antimicrobial study was carried out by disc diffusion technique (11). All tested extracts were weighed under aseptic conditions in sterile volumetric flasks, and dissolved with 70.0% sterile ethanol to obtain different extract concentrations (concentrations 1, 2, 3 and 4 contained 40.0, 20.0, 10.0 and 5.0% of crude extract, respectively). Standard tetracycline, streptomycin and penicillin discs and discs containing crystal violet (1 mg/ml) and solvent (70.0% v/v ethanol) were used as controls. All probes were applied in 20 l volumen per disk. According to this method, the antimicrobial potency of the test samples was measured by determining the diameter of the zones of inhibition in millimetres. All determinations were made in duplicate. MATERIAL AND METHODS Plant material Plant materials were collected from wild growing species of Ericaceae family: Arbutus unedo (Lustica, Montenegro) HFF (Herbarijum Farmaceutskog Fakulteta Univerziteta u Beogradu - Herbarium collection of the Faculty of Pharmacy, University of Belgrade) No. 1173; Bruckentalia spiculifolia (Kopaonik - Panciev vrh, Serbia) HFF No. 1217; Calluna vulgaris (Loznica - Gucevo, Serbia) HFF No. 1272; Erica arborea (Lustica, Montenegro) HFF No. 1430; Ericacarnea (syn.: Erica herbacea L., Erica saxatilis Salisb.) (Mokra Gora, Serbia) HFF No. 1431. Authenticated voucher herbarium specimens have been deposited in the Herbarium collection of the Faculty of Pharmacy, University of Belgrade (HFF). Determination was done according to Flora of SR Srbija (6), Flora Europea (7) and Anatomy of the Dicotyledons (8). RESULTS The antimicrobial activity of tested samples against selected microorganisms is showed in Table 1. Diameter of sterile paper disk was 13 mm. Thus, diameters of the inhibition zones larger than 13 mm were presented as positive results. Applied extract concentrations (40.0, 20.0, 10.0 and 5.0% of crude extract) were labelled as concentrations 1, 2, 3 and 4, respectively. As referent antimicrobial drugs, tetracycline, streptomycin and penicillin alongside discs containing crystal violet exhibited obviously higher antimicrobial activity than tested ethanolic extracts of selected Ericaceae species. Extract solvent, ethanol, did not affect any of tested bacterial strains. Extraction procedure Plant material, dried leaves of selected Ericaceae species, was reduced to a fine powder and extracted with ethanol (70.0%, v/v) by percolation, as described in European Pharmacopeia 6.0 (9). Ethanolic extracts of A. unedo (AE), B. spiculifolia (BE), C. vulgaris (VE), E. arborea (EE) and E. carnea (CE) were obtained after evaporation to the dryness in vacuo below 40ºC and extraction yields were 45.05, 32.35, 33.84, 38.98 and 38.61% (w/w), respectively. Test Organisms Both gram-positive and gram-negative bacterial strains used for the experiment were collected as pure cultures from ATCC (American Type of Culture Collecti82 Dragana Pavlovi et al. Table 1. Results of antimicrobial assay showed as diameters of the of inhibitory zones in millimetres Ethanol 70% v/v / / / / / / 13.5 / / / Crystal violet Microorganism AE BE VE 1=14.5 2=14 3=13.5 / / 1,2=14 3=13.5 / / / / / / EE CE 1=16 2=15 3=14 / / 1-4=14 1=13.5 / / / / / Antibiotic Staphylococcus aureus Staphylococcus epidermidis Micrococcusflavus Bacillus subtilis Sarcinalutea Pseudomonas aeruginosa Klebsiella pneumoniae E. coli E. coli SY252 E. coli LPCA / 1,2=14.5 3=14 1=17 2,3=16 4=15 1=14.5 2,3=14 1,2=14.5 3=14 / / / / 1=15 2=14 3=13.5 1=14 2,3=13.5 / 1=14.5 2=14 1,2,3=14 1=15 2=14.5 / / / / / / / 1,2=13.5 1=14 1,2=13.5 / / / / / 17 21 22 20 20 / 16 / 14 21 DISCUSSION Ethanolic extracts of A. unedo, B. spiculifolia, C. vulgaris, E. arborea and E. carnea were compared between each other and with A. uva ursi, species of this family well known for its antimicrobial properties. The disc diffusion method was used to determine the inhibition zones of the five ethanolic extracts from different Ericaceae species. The five gram-positive and five gram-negative bacterial strains have been used. In general, phenolics are the predominant active chemicals in plants, with gram-positive bacteria being the most sensible germs (12). Our results for AE, BE, VE, EE and CE antimicrobial activity string along these findings. According to the results in Table 1, different ethanolic extracts showed certain antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus flavus, Bacillus subtillis and Sarcinalutea. Antimicrobial activity against the Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli was not observed. The only exception is mild impact of A. unedo extract (AE) on E. coli LPCA (the wild laboratory strain of E. coli). The most prominent antimicrobial effect was achieved on Micrococcus flavus with AE. Inhibition zones observed for concentration 1 (17 mm) were slightly larger than those observed for concentrations 2 and 3 (16 mm) and 4 (15 mm) of A. unedo ethanolic extracts. The antimicrobial activity of A. unedo root extract against E. coli and Staphylococcus aureus (13) and antiprotosoal activity of A. unedo leaf extract against Leishmania tropica (14) and Trichomonas vaginalis (15) has already been reported. According to Kumarasamy et al. (2002) methanolic extract of C. vulgaris seed showed bacteriostatic activity against the gram-positive strains Staphylococcus aureus and Staphylococcus hominis (with minimal inhibitory concentration 0,1 g/ml) (16). Our results for VE indicate very mild impact on Staphylococcus aureus and Bacillus subtilis. The 30.0% ethanol extract of Uvaeursi folium inhibited the growth in vitro of Bacillus subtilis, E. coli, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens and Staphylococcus aureus. Ethanol extract were also active in vitro against E. coli, Proteus vulgaris, Staphylococcusfaecalis and Enterobacter aerogenes. However, 95.0% ethanol extract had no antibacterial activity (17). Arbutin is generally considered to be respon83 ACTA FACULTATIS MEDICAE NAISSENSIS, 2014, Vol 31, No 1 sible for the antibacterial activity of the Uvae ursi folium extract. Preliminary chemical analysis showed the presence of hydroquinone and phenil propanoid derivates, flavonoids and tannins in A. unedo, B. spiculifolia, C. vulgaris, E. arborea and E. carnea. Our previous results, obtained using the HPLC method, also confirmed the presence of arbutin only in leaves of Arbutus unedo (1.21 ±0.03%) and absence of hydroquinone in all tested samples (18). The amount of arbutin in dry A. uva ursi leaves ranges from 5.0 to 15.0% (17), while according to the monograph of European Pharmacopoeia 6.0 (9), dried drug Uvae ursi folium should contain minimum 7.0% of anhydrous arbutin. Thus, the presence of arbutin in AE could be, at least partly, responsible for measured antibacterial activity. However, in this preliminary screening, it was observed that antimicrobial activity of all tested samples was generally weak. CONCLUSION Based on the results of this in vitro tests on antimicrobial activity of leaf ethanolic extracts of Arbutus unedo, Bruckentalia spiculifolia, Calluna vulgaris, Erica arborea and Erica carnea, and bearing in mind that only leaves of Arbutus unedo contain arbutin (although in significantly smaller quantity than Uvae ursi folium), the traditional use of leaf of this species in the treatment of urinary tract infections cannot be considered plausible. Acknowledgement This research was supported by the Ministry of Education and Science of the Republic of Serbia (Grants No.III 41018 and III 46013).

Journal

Acta Facultatis Medicae Naissensis – de Gruyter

Published: Mar 1, 2014

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