FEMS Microbiology Letters 240 (2004) 111–116 www.fems-microbiology.org
Galangin expresses bactericidal activity against multiple-resistant bacteria: MRSA, Enterococcus spp. and Pseudomonas aeruginosa Stjepan Pepeljnjak, Ivan Kosalec
*
Institute of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Schrottova 39/I, HR-10 000 Zagreb, Croatia Received 28 May 2004; received in revised form 1 September 2004; accepted 16 September 2004 First published online 28 September 2004 Edited by H.B. Deising
Abstract The antimicrobial activity of three propolis ethanol extracts (EEP) was examined for various Gram-negative and Gram-positive bacterial species, including multiple-resistant Staphylococcus aureus, Enterococcus spp. and Pseudomonas aeruginosa strains. EEP had a good bactericidal activity against Gram-positive species, and all multiple-resistant bacterial strains tested were sensitive to EEP. Minimal inhibitory concentrations (MICs) were lower in samples of higher flavonoid content (from 0.65 to 7.81 mg mL 1), indicating the influence of the concentration of some potent bactericidal compound(s) in propolis or synergism among some bactericidal compounds. Antimicrobial-guided separation of flavonoid aglycones (bioassay in situ on thin-layer chromatogram) showed that galangin (3,5,7-trihydroxyflavone) is one compound in EEP with bactericidal activity. Galangin was isolated by preparative chromatography. After determining the quantity present, the MIC against multiple-resistant bacteria was determined. The MIC of galangin against multiple-resistant bacterial strains was significantly lower (from 0.16 to 0.44 mg mL 1, p < 0.05) than that of EEP. The bactericidal activity of galangin against P. aeruginosa strains was present at 0.17 ± 0.05 mg mL 1. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Galangin; 3,5,7-Trihydroxyflavone; Flavonoids; Bacteria; Resistance; Staphylococcus aureus; Enterococcus spp.; Pseudomonas aeruginosa
1. Introduction The resistance of bacteria and other microorganisms on antimicrobial agents has become a wide-spread medical problem especially as nosocomial pathogens. Treatment options for these infections are often limited, especially in debilitated and immunocompromised patients. Among nosocomial pathogens, methicillin- and vancomycin-resistant Staphylococcus aureus strains, multiple-resistant Enterococcus spp. and Pseudomonas aeruginosa as well as members of the Enterobacteriaceae *
Corresponding author. Tel./fax: +385 1 46 36 371. E-mail address:
[email protected] (I. Kosalec).
family (resistant on five or more antibiotics) are the most common bacterial isolates [1–3]. One of the untapped reservoirs of the new biologically active molecules are plants [4,5]. Large classes of polyphenols, which are wide-spread in plants, are flavonoids, and these classes of compounds possess antioxidative, antiinflammatory, antiviral and antimicrobial, antiproliferative, anticarcinogenic, analgesic, spasmolytic and hepatoprotective characteristics [6–9]. The antimicrobial activity of flavonoids is very interesting for the research of the new compounds against multiple-resistant bacterial strains [10]. Therefore, we have examined the antimicrobial activity of different propolis samples as they are natural substances with a very high
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.09.018
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concentration of flavonoids. Propolis is a resinous substance collected by honeybees (Apis mellifica L.) and in folk medicine propolis has been used as an antiseptic [11]. The chemical composition, colour and smell differ greatly due to variations in geographical and botanical origin [11,12]. The biological activities of propolis depend mainly on the presence of flavonoids or phenolic and benzoic acids and their esters. Among the many biological activities of propolis extracts, antimicrobial effects have been widely reported, especially against Gram-positive bacterial species and yeasts [13–16]. Ethanolic extracts of propolis have been found to be effective against protozoa, Toxoplasma gondii and Trichomonas vaginalis [13]. In this study, we have analysed the spectrum of antimicrobial activity of ethanolic extracts of propolis samples on various bacterial and fungal species. Antimicrobial activity against clinical isolates of the multiple-resistant bacterial strains of S. aureus species, Enterococcus spp. and on P. aeruginosa strains, was measured. In order to elucidate which compounds in the complex mixture of propolis extracts have bactericidal activity against multiple-resistant bacteria, we fractioned the extract. The bactericidal compound was isolated with preparative chromatography. The re-isolated compound was tested on multiple-resistant bacterial species, and MICs were compared.
2. Materials and methods 2.1. Propolis samples Three samples of raw propolis collected by honeybees (Apis mellifica L.) were obtained from Croatian bee keepers in autumn 2001. Sample number 5587 was collected around the city of Zagreb (continental part of Croatia) and samples 5582 and 5581 were from the hives around the city of Imotski (Mediterranean part of Croatia). Prior to the analysis, the propolis samples were kept at room temperature in the dark. Crude propolis was ground into powder and dissolved in 80% ethanol (Kemika, Croatia) at 37 C for 48 h by shaking. After dissolving, ethanolic extracts of propolis (EEP) were filtered through Whatman No. 1 filter paper. EEPs were dissolved with 80% ethanol to a final concentration of 0.25 g mL 1. 2.2. Thin-layer and preparative chromatography The thin-layer chromatography (TLC) was performed by the Kosalec method [11] using pre-coated silica gel plates (10 · 20 cm, 0.25 cm thick) with a fluorescent indicator (Macherey-Nagael, Germany) as the stationary phase and toluene:ethyl-acetate:formic acid = 5:4:1 (v/v/v) as the mobile phase. Spots were vis-
ualized by UV irradiation at 366 nm after spraying with 1% (w/v) methanolic solution of diphenylboric acid aminoethyl ester (Sigma, Germany) followed by 5% (v/v) ethanolic solution of polyethylene glycol 4000 (Sigma, Germany). EEPs were mixed 1 + 4 with 80% ethanol and 5 lL was applied on the plates with a microsyringe (Hamilton, Switzerland). TLC plates were run in duplicate and one set was used as the reference chromatogram (for detecting flavonoid aglycones). The other set was used for bioautography. As reference substances, 0.05% (w/v) solution in 80% ethanol of flavonoid aglycones were used: naringenin, chrysin, quercetin, galangin and caffeic acid (all from Sigma–Aldrich, USA). Silica gel H (Kemika, Croatia) plates were used for preparative chromatography (20 · 20 cm, 1 mm thick). The TLC plates were developed, then air dried and specific compounds were isolated by scraping the silica gel. 2.3. Analysis of flavonoids from propolis – spectrometric and liquid chromatography methods Total flavonoids in raw propolis were quantitatively analyzed by two colorimetric methods described by Chang et al. [17], and the concentration of galagin was determined by Kosalec method [11]. Quercetine was used as a standard for the calculation of flavones and flavonols concentration, and naringenin for the calculation of flavanones concentration in EEP. Absorbances were measured using PU 8626 UV/VIS model of spectrophotometer (Philips, The Netherlands) at 415 nm for the flavones and flavonols, and at 495 for the flavanones. Isolation and measurement of galangin was performed by HPLC by the previously published method [11]. Galangin in the EEP was identified with HPLC by comparison with commercially available galangin dissolved in ethanol (96%, v/v) and its retention time and UV spectral data. 2.4. Microorganisms Microorganisms used in this research for the determination of spectrum of antimicrobial activity are displayed in Table 1. Multiple-resistant bacterial strains used in our research were obtained from swab specimens such as wounds (decubitus), catheter tips (central venous), or from broncho-alveolar lavage and from bloodor urine-culture. Susceptibility testing (disk diffusion tests on Mu¨ller–Hinton agar) was performed on all bacterial strains after isolation and identification using NCCLS recommendations [18] with BBL disks. Susceptibility of isolates was as follows: 20 clinical isolates of S. aureus: ten strains were methycillin-resistant (MRSA, also resistant to oxacilline, azytromicine, clindamycine, gentamicin, ciprofloxacin, netilmicine) and ten methycillin-susceptible (MSSA); 17 clinical isolates of Enterococcus spp. All enterococci strains were resistant
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Table 1 Spectrum of bactericidal activity of EEP Microorganism
Sample code
Control (80% ethanol)
Inhibition zones (mm)
Bacillus subtilis NCTC 8236 B. cereus ATCC 11778 Micrococcus lufeus ATCC 9341 Staphylococcus aureus ATCC 6538 P S. aureus ATCC 25923 Enterococcus faecalis ATCC 29212 Listeria monocytogenes MFBF 11 Salmonella enteritidis MFBF Proteus mirabilis MFBF 1812 Serratia marcescens MFBF 2 Pseudomonas aeruginosa ATCC 27853 Klebsiella oxytoca MFBF 1 Yersinia enterocolitica 0:3 MFBF 3 Escherichia coli 0157:A2 MFBF
5587a
5582a
5581a
19 21 13 19 17 7 21 0 0 0 17 0 20 8
13 12 8 11 10 8 9 0 0 0 0 0 0 0
21 23 15 19 18 15 21 0 0 0 11 0 17 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
Abbreviations: ATCC, American Type Culture Collection; Rockville, USA; NCTC, National Collection of Type Cultures; London, Great Britain; MFBF, number of strains from collection of microorganisms from the Institute of Microbiology, Faculty of Pharmacy and Biochemistry University of Zagreb, Zagreb, Croatia. a Statistical differences of antimicrobial activity were noticed in comparison of samples 5587 and 5581 with sample 5582 (p < 0.05).
to tetracycline, gentamycin, erythromycin, ciprofloxacin, norfloxacin, azitromycin, oxacillin, ceftazidime and ceftriaxone; 10 clinical isolates of P. aeruginosa were resistant to: gentamicin, amikacin, norfloxacin, ciprofloxacin, impinem, netilmicin, cefuroxim and nitrofurantoin.
ber of colonia after removing the loop with 10 lL of each dilution on tryptic-soy agar and incubation at 37 C for 18 h. MBC was defined as the lowest concentration of EEP that allows no growth of bacteria. 2.6. Bioautography – bioassay in situ
2.5. Diffusion and dilution methods Inoculum was prepared with fresh cultures of bacterial strains, cultured on tryptic-soy agar (Merck, Germany) for 18 h at 37 C with physiological saline with 3 · 106 cells mL 1. Density of inoculum was compared with MacFarlandÕs standard solution of BaSO4 (0.1 mL of 1% BaCl2 + 9.9 mL 1% H2SO4). Yeasts were cultivated on Sabouraud 2%-dextrose agar (Biolife, Italy) with the addition of chloramphenicol (50 mg L 1) for 5 days at 25 C. One mL of inoculum was mixed with 22 mL of Mu¨ller–Hinton agar (Merck, Germany) for bacterial strains. After cooling the inoculated agars at room temperature for 25 min, holes (d = 6 mm) were made with stainless steel cylinders. Four holes were made per plate. 40 lL of EEPs was dropped into holes and 40 lL of 80% ethanol as control. In order to accelerate the diffusion of EEP into agar, plates were incubated at +4 C for 1 h and then were incubated at +37 C for 18 h under aerobic conditions. The diameter of the inhibition zone around each hole was measured and recorded. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the broth twofold macro-dilution method in Mu¨ller–Hinton broth (Merck, Germany). MIC was defined as the lowest concentration of EEP that allows no more than 20% growth of the bacteria which is seen as a decreased num-
TLC developed plates were air dried for 3 days at room temperature in the dark. After drying, plates were placed in a glass chamber with a cover and sprayed with sterile physiological saline. Inoculum of investigated microorganisms, containing 106 CFU mL 1 in molted Mu¨ller–Hinton agar, was distributed over the plates with thickness 2 mm. After solidification of the medium for maximum 25 min, TLC plates were incubated for 18 h at 37 ± 1 C. Bioassay plates were sprayed with 1% (w/v) solution of 2,3,5-triphenyltetrazolium chloride (Sigma, Germany) in sterile distilled water and incubated at 37 C for 10 min. Inhibition zones were seen as clear spots around the developed flavonoids with antibacterial activity against the red background. 2.7. Statistical analysis The data from diffusion and dilution method were compared and the non-parametric Kruskal–Wallis test was used (GraphPad Prism software, GraphPad Software, USA). The chosen level of significance was p < 0.05.
3. Results and discussion There are many papers which describe the antimicrobial activity of propolis ethanolic extracts. In general,
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EEP has antimicrobial activity against Gram-positive bacteria but lower activity against Gram-negative bacteria and good activity against yeasts [13–15]. It has been suggested that the antimicrobial activity of propolis depends on synergism among some flavonoids, phenolic acids and other compounds in propolis [16,19]. The mode of antimicrobial activity of propolis compounds is not clear, but possible explanations are: inhibition of bacterial motility, affection of membrane potential, inhibition of RNA-polymerase, disorganization of cytoplasm, cytoplasmic membrane and cell wall, and inhibition of protein synthesis [20,21]. We compared three samples of raw propolis collected from Croatia and examined the antimicrobial activities of their ethanolic extracts (Table 1). Propolis samples from the continental part of Croatia (sample 5587) and one from the Mediterranean part of Croatia (sample 5581) inhibit the growth of all tested Gram-positive bacteria with the inhibition zones from 7 to 21 mm (sample 5587) and between 15 and 23 mm (sample 5581), respectively. Another sample (5582) from the Mediterranean part of Croatia caused smaller inhibition zones (8–13 mm). Only P. aeruginosa and Yersinia enterocolitica, as Gram-negative bacterial species, were sensitive to EEP samples 5587 and 5581 and E. coli O157:A2 was sensitive to EEP sample 5587. Our data suggest better activity of EEP on Gram-positive bacterial species, which is in agreement with previously published results. The results of the bactericidal activity of EEP encouraged us to test its activity against nosocomial isolates of multiple-resistant bacteria. All tested bacterial isolates from nosocomial infections (methicillin-resistant S. aureus, multiple-resistant enterococci and P. aeruginosa strains) showed sensitivity to the three EEPs with the inhibition zones in diffusion method (Table 2). The largest inhibition zones were noticed against MRSA/MSSA strains followed by P. aeruginosa and Enterococcus spp. strains. In comparison of bactericidal activity by the diffusion method, there were no differences between MRSA and MSSA strains. The MIC and MBC are presented in Table 3. Samples of EEP 5587 and 5581 have lower MIC values than sample
5582 for all tested resistant bacterial strains. Lowest MICs were noticed against MRSA and MSSA and highest against P. aeruginosa strains. The results of bactericidal activity, both in diffusion and dilution methods, indicate higher concentrations of bactericidal compounds in two samples (5587 and 5581) than in sample 5582. Therefore, we conducted bioactivity-guided (bioassay in situ) separation of compound(s) from propolis ethanolic extract with bactericidal activity. The results of bioassay showed that the biggest zone of bactericidal activity was around flavonoid galangin, which was confirmed with purchased galangin. After the determination of one of bactericidal compounds in EEP we used preparative chromatography to isolate galangin. The purity of the extracted galangin was tested by comparing its UV spectra with commercially available galangin. Ethanolic solution of isolated galangin was used for the determination of the inhibition zones and MICs and MBCs on multiple-resistant bacterial strains. The results are presented in Tables 4 and 5. Galangin has a lower inhibition zone than ethanolic extract from which it was extracted, but MIC and MBC values were significantly lower (Fig. 1). Quantitative analysis of flavonoids in propolis samples shows differences in concentrations within particular groups of flavonoids (Table 6). The concentration of the bactericidal flavonoid galangin also varies in samples of EEP in a range from 0.002 to 0.305 mg mL 1. The highest concentration of galangin in sample 5581 could explain the lowest MIC and MBC values in comparison with other two samples. EEP sample 5581 has fungicidal activity against all yeasts and dermatophytes tested, and future research has to determine compound(s) with fungicidal activity in EEP. It is interesting that the propolis samples and their ethanolic extracts which were investigated here have antimicrobial activity on P. aeruginosa in dilution method. Nieva Moreno et al. [22] analyzed antimicrobial activity of ethanolic extracts of propolis samples from four localities in Argentina, but they did not find any activities against Gram-negative bacterial species
Table 2 Inhibition zones of three EEPs on resistant bacterial strains by diffusion method EEP samples
Control (80% ethanol)
ZIa (mean ± SD) 5587b MRSA (n = 10) MSSA (n = 10) Enterococcus spp. (n = 17) Pseudomonas aeruginosa (n = 10) a b c
5582b c
18.6 ± 2.9 22.0 ± 5.4c 12.3 ± 2.9 15.0 ± 3.2
5581b c
11.7 ± 2.5 12.6 ± 5.6c 9.3 ± 3.1 9.4 ± 1.9
20.6 ± 5.7c 22.1 ± 6.8c 11.5 ± 2.5 12.5 ± 2.5
0 0 0 0
Measured as diameter of inhibition zones in mm. Inhibition zones of all EEP samples are significantly different (p < 0.05) from control. Comparison of inhibition zones between MRSA and MSSA strains of same sample were not significantly different (p > 0.05).
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Table 3 Minimal inhibitory concentrations and minimal bactericidal concentrations of EEPs on resistant bacteria EEP sample Concentration (mean ± SD; mg/mL) 5587
5582 a
MRSA (n = 10) MSSA (n = 10) Enterococcus spp. (n = 17) Pseudomonas aeruginosa (n = 10) a b c
b
MIC
MBC
MIC
MBC
MIC
MBC
1.06 ± 0.57c 0.89 ± 0.71c 9.09 ± 5.32 7.63 ± 3.32
2.00 ± 1.14c 1.73 ± 1.45c 16.24 ± 10.18 10.65 ± 3.94
4.98 ± 2.70c 5.68 ± 2.04c 8.54 ± 2.83 14.16 ± 5
9.37 ± 5.58c 10.65 ± 3.94c 17.71 ± 5.50 23.44 ± 8.56
1.19 ± 1.06c 0.65 ± 0.31c 2.33 ± 1.83 7.81 ± 4.19
2.37 ± 2.14c 1.38 ± 0.57c 4.67 ± 3.66 12.78 ± 7.22
Minimal inhibitory concentration. Minimal bactericidal concentration. Comparison of MICs and MBCs between MRSA and MSSA strains of same sample were not significantly different (p > 0.05).
Table 4 Inhibition zones of galangin on resistant bacteria
10 9
Inhibition zones (mm) (mean ± SD)
Control (80% ethanol)
10.9 ± 1.4 9.8 ± 1.0 6.4 ± 4.6
0 0 0
Table 5 Minimal inhibitory and bactericidal concentrations of galangin on resistant Galangin concentration (mg/mL) (mean ± SD) a
MIC MRSA (n = 10) Enterococcus spp. (n = 17) Pseudomonas aeruginosa (n = 10) b c
b
MBC
Control (80% ethanol) MIC/MBC c
0.16 ± 0.03 0.24 ± 0.05
0.27 ± 0.06 0.44 ± 0.13
ND ND
0.17 ± 0.05
0.23 ± 0.08
ND
Minimal inhibitory concentration. Minimal bactericidal concentration. ND, not detected.
Table 6 The flavonoid and galangin contents of three raw propolis samples Sample code
5587 5582 5581 a b c
Flavonoid content (%) Flavones and flavonolsa,b
Flavanonesa,b
Galanginc
2.1 ± 0.1 1.3 ± 0.3 2.3 ± 0.2
19.6 ± 1.0 3.9 ± 0.3 17.9 ± 0.1
0.136 0.002 0.305
Results were presented as mean ± SD (n = 3). Data obtained by spectrophotometric analysis. Data obtained by HPLC analysis.
(E. coli, Klebsiella pneumoniae, Serratia marcescens, Acinetobacter spp., P. aeruginosa and Stenotrophomonas maltophilia), which is different from our experience because we found this activity against E. coli in EEP sample 5587 and against Y. enterocolitica in samples 5587 and 5581.
concentration (mg/mL)
MRSA (n = 10) Enterococcus spp. (n = 17) Pseudomonas aeruginosa (n = 10)
a
5581
8
propolis
galangin
7 6 5 4 3 2 1 0 MRSA
Enterococcus spp.
P. aeruginosa
Fig. 1. Comparison of minimal inhibitory concentrations between propolis (sample 5581 from Mediterranean part of Croatia) and galangin (isolated from the same sample of propolis) mean ± SD, *p < 0.05, **p < 0.01).
In our research we have found bactericidal activity of two EEP (5587 and 5581) against P. aeruginosa by diffusion method, and against clinical isolates of P. aeruginosa strains. Differences between activities were probably due to different concentrations of some bactericidal compounds in extracts. One of the flavonoid compounds expressing bactericidal activity was galangin (3,5,7-trihidroxyflavone), and we found its activity both against S. aureus, Enterococcus spp. and P. aeruginosa strains. Our future research will be on the antimicrobial activity of lipophilic compounds and its determination from EEP against yeasts, as well as defining the additional functional groups in the galangin molecule as a possible improvement of its bactericidal activity. Acknowledgements We thank to Asja Smolcˇic´-Bubalo and Marina Bakmaz for analysis of propolis samples.
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