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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis a
b
Elżbieta Studzińska-Sroka , Elżbieta Hołderna-Kędzia , c
a
d
Agnieszka Galanty , Wiesława Bylka , Karol Kacprzak & Karolina Ćwiklińska
e
a
Department of Pharmacognosy, Poznan University of Medical Sciences, Święcickiego 4, 60-781 Poznań, Poland b
Click for updates
Institute of Natural Fibres and Medicinal Plants, Libelta 27, 61-707 Poznań, Poland c
Department of Pharmacognosy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland d
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznań, Poland e
Department of Laboratory Diagnostics, Josef Strus Multidisciplinary Municipal Hospital, Szwajcarska 3, 61-285 Poznań, Poland Published online: 03 Feb 2015.
To cite this article: Elżbieta Studzińska-Sroka, Elżbieta Hołderna-Kędzia, Agnieszka Galanty, Wiesława Bylka, Karol Kacprzak & Karolina Ćwiklińska (2015): In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1005616 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1005616
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1005616
SHORT COMMUNICATION In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis Elz˙bieta Studzin´ska-Srokaa*, Elz˙bieta Hołderna-Ke˛dziab, Agnieszka Galantyc, ´ wiklin´skae Wiesława Bylkaa, Karol Kacprzakd and Karolina C Department of Pharmacognosy, Poznan University of Medical Sciences, S´wie˛cickiego 4, 60-781 Poznan´, Poland; bInstitute of Natural Fibres and Medicinal Plants, Libelta 27, 61-707 Poznan´, Poland; c Department of Pharmacognosy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland; dFaculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´, Poland; e Department of Laboratory Diagnostics, Josef Strus Multidisciplinary Municipal Hospital, Szwajcarska 3, 61-285 Poznan´, Poland (Received 22 July 2014; final version received 4 January 2015)
DIETHYL ETHER
CH3 O H3 C
OH
20
S.aureus
COOH
O OHC
OH CH3
HEPTANE
ACETONE
H3C
OH H3C
HO
O
MICµg/mL
H 3CO
MRSA
16 12 8
O
4 CH3
0
no U sn l i am c a ci at d i c n am A e ac ce (M id ph to R en ne S A ic A (M 1 ol ce R ,3 to S ,5 ne A ,7 , (M 2,4 9) , R 6 S ,8 A ) 10 ) or
A
ce
to
S
hl
)(-
qu
ha
C
er th
to
.e
ce
et
A
M
ta
ne
CH3
ne
OH
ep
METHANOL
O
D
O
H
Downloaded by [Selcuk Universitesi] at 11:18 10 February 2015
a
Heptane (Hep), diethyl ether (Et2O), acetone (Me2CO) and methanolic (MeOH) extracts, as well as (2)-usnic acid and squamatic acid, were obtained from thallus of Cladonia uncialis (Cladoniaceae). The antimicrobial activities of these extracts, (2 )usnic acid and squamatic acid, were tested against reference strains: Staphylococcus aureus, Escherichia coli and Candida albicans. In addition, Me2CO extract was analysed against 10 strains of Methicillin-resistant S. aureus (MRSA) isolated from patients. All extracts exerted antibacterial activity against the reference strain S. aureus, comparably to chloramphenicol [minimum inhibitory concentration (MIC) ¼ 5.0 mg/mL]. The Me2CO extract exhibited the strongest activity against S. aureus (MIC ¼ 0.5 mg/mL), higher than ( 2 )-usnic acid, whereas squamatic acid proved inactive. The Me2CO extract showed potent antimicrobial activity against MRSA (MIC 2.5– 7.5 mg/mL). Also no activity of C. uncialis extracts against E. coli and C. albicans was observed. Keywords: Cladonia uncialis; extracts; usnic acid; squamatic acid; antimicrobial activity
1. Introduction Lichens are widespread symbiotic organisms consisting of fungal partners, mycobionts and one or more photosynthetic partners, the photobionts, are green algae or cyanobacteria. The specific
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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E. Studzin´ska-Sroka et al.
secondary metabolites of lichens exhibit wide a spectrum of biological activity such as antimicrobial, antiviral, antiprotozoal, antitumour, anti-inflammatory, analgesic, antipyretic, antiproliferative, antioxidant and antiherbivoral (Mu¨ller 2001). Cladonia uncialis (L.) Weber ex F.H. Wigg. belongs to the order Lecanorales and to the family Cladoniaceae. The thallus of this lichen is yellow-green or greenish, often brownish towards the pointed apices. C. uncialis frequently grows on soil among mosses, in well-lit and typically, dry places. It is common in coniferous forests, on heathland and sand dunes in Europe, North America and Asia (Purvis et al. 1992). C. uncialis produces dibenzofurans such as (2)-usnic acid and depsides: squamatic acid and thamnolic acid (Culberson 1969; Purvis et al. 1992; BeGora & Fahlset 2001). The bacteria (Gram-positive and Gram-negative) and fungi are the agents responsible for numerous human diseases. The serious threat is the antibiotic-resistant strains of bacteria, e.g. Methicillin-resistant Staphylococcus aureus (MRSA) which is a frequent cause of nosocomial infection leading to a wide range of diseases: endocarditis, osteomyelitis, toxic shock syndrome, pneumonia, food poisoning and carbuncles (Chambers 2003). It is known that lichen and compounds isolated from them were found active against the various classes and species of the microorganisms. While a number of reports concern the antimicrobial effects of usnic acid, there are no data available on the biological activity including the antimicrobial properties of crude extracts from C. uncialis and squamatic acid (Mu¨ller 2001; Ingo´lfsdo´ttir 2002). The aim of our study was to investigate the antimicrobial activity by the broth microdilution method of heptane (Hep), diethyl ether (Et2O), acetone (Me2CO) and methanolic (MeOH) extracts, as well as isolated (2)-usnic and squamatic acids obtained from C. uncialis against three reference strains: S. aureus, Escherichia coli and Candida albicans. The Me2CO extract has also been tested against MRSA isolated from patients. In addition the acetone extract was examined by LC –MS and GC – MS analyses. Moreover, the content of (2)-usnic acid in the extracts from C. uncialis was studied using the HPLC method to explain whether and to what extent the dibenzofuran could be responsible for the biological properties of the extracts. 2. Results and discussion 2.1. Phytochemical analysis C. uncialis (32.6 g) was extracted with heptane and then with acetone, successively. Extracts were obtained in a similar quantity (280 and 272 mg, respectively). (2)-Usnic acid (92.3 mg) and squamatic acid (90.3 mg) (Supplementary Figure S1 – online only) were isolated from the Hep and Me2CO extract, respectively, and identified by the LC – MS (Supplementary Figure S2 – online only) and TLC analyses as well as spectroscopic methods (UV, 1H NMR, 13C NMR and ESI-MS). The optical rotation of (2)-usnic acid was also determined confirming its high optical purity. For antimicrobial assays, heptane (Hep; 33.7 mg), diethyl ether (Et2O; 38.05 mg), acetone (Me2CO; 69.8 mg) and methanolic (MeOH; 270.6 mg) extracts were received from 5 g of the thalli of C. uncialis using the ultrasonic bath. The TLC analysis of these extracts showed that (2)-usnic acid was present in all extracts, mainly in the Hep extract. Squamatic acid can be conveniently extracted with diethyl ether and acetone, while the Hep extract did not contain this compound, and the MeOH extract did contain only a small amount. The absolute quantification of (2)-usnic acid was determined by HPLC. Heptane and diethyl ether were the most effective solvents for (2)-usnic acid extraction, with the content of 0.633 ^ 0.03 and 0.437 ^ 0.01 mg/ mL, respectively. The concentration of (2)-usnic acid was about two-and-a-half times lower (0.284 ^ 0.03 mg/mL) in the Me2CO and three times lower in the MeOH extract (0.190 ^ 0.01 mg/mL), in comparison with the Hep extract. Acetone extract was also analysed by the LC –MS method on reversed phase within 60 min analysis under increasing gradient of
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Table 1. Compounds identified in the acetone extract of C. uncialis using GC-MS.
No. 1 2 3 4
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5 6 7
Name 3-Methoxy-5-methylphenol (syn. orcinol monomethyl ether) 2,5-Dimethyl-1,4-benzenediol 2,4-Dihydroxy-6-methylbenzaldehyde (syn. o-orsellinaldehyde) Methyl 2,4-dihydroxy-3,6-dimethylbenzoate (syn. atraric acid) 5-Pentyl-1,3-benzenediol (syn. olivetol) Ethanone, 1-(4,6-dihydroxy-2,3,5-trimethyl7-benzofuranyl) (syn. usnetol) (9bS)-2,6-Diacetyl-3,7,9-trihydroxy-8,9bdimethyldibenzofuran-1-one (syn. usnic acid)
Retention Peak area Match Molecular Molecular time (%) factor mass (g/mol) formula (min) 10.160
26.60
878
138
C8H10O2
11.484 13.154
43.41 3.66
880 834
138 152
C8H10O2 C8H8O3
14.942
1.67
824
196
C10H12O4
15.462 18.494
4.98 1.12
914 808
180 234
C11H16O2 C13H14O4
24.103
5.59
922
344
C18H16O7
MeOH (up to 100%). All the tested detection modes (UV, negative and positive ions) proved the presence of both (2)-usnic and squamatic acids as well as some predominantly low-molecular yet undefined substances (Supplementary Figure S2 – online only). The GC – MS analysis provided the composition of the volatiles of acetone extract from C. uncialis (Table 1). The compounds were identified with match factor . 800. Taking into account the peak intensities, it can be established that among the seven compounds identified using GC – MS in the acetone extract, the main compounds were 2,5-dimethyl-1,4-benzenediol (43.41%) and orcinol monomethyl ether (26.60%). Usnic acid, olivetol, o-orsellinaldehyde, atraric acid and usnetol were also presented but in smaller quantities (Table 1). It is probable that the volatility of other compounds of the acetone extract was not sufficient for GC –MS analysis. The volatiles of the acetone extract of C. uncialis identified in this study were also described in other lichens (e.g. orcinol monomethyl ether, olivetol or atraric acid in Hypogymnia physodes or Evernia prunastri) (Stojanovic´ et al. 2011). 2.2. Antimicrobial assay The potency of the antimicrobial effect depended on the type of the extracts of C. uncialis and the tested microorganism. All the extracts tested showed strong antimicrobial activity against the reference strain of the Gram-positive bacteria S. aureus. The strongest activity against S. aureus (10 times higher than chloramphenicol) was observed for the Me2CO extract (MIC ¼ 0.5 mg/ mL). The antibacterial activity of the Et2O, Hep and MeOH extracts was two times higher, equal or two times lower (MIC ¼ 2.5, 5.0 and 10.0 mg/mL, respectively) than the reference compound. The activity of the isolated (2)-usnic acid was five times weaker than the activity of the examined Me2CO extract (MIC ¼ 2.5 mg/mL), whereas the squamatic acid was characterised by weak antibacterial activity (MIC ¼ 1250.0 mg/mL). Extracts from C. uncialis were inactive against the Gram-negative E. coli strain (MIC , 1000 mg/mL in comparison with chloramphenicol MIC ¼ 100 mg/mL). C. albicans was susceptible to lower concentration of the tested extract than E. coli bacteria; however, this action was lower than the reference amphotericin B (MIC 250 – 750 mg/mL and MIC ¼ 1 mg/mL, respectively) (Table 2). In the same manner, the activity of the MeOH extract against yeast C. albicans was higher than that of the Hep, Et2O and Me2CO extracts. Our results were compatible with those published in the literature, according to which lichenic compounds and extracts are potent active against Grampositive than Gram-negative bacteria and yeast (Mu¨ller 2001; Ingo´lfsdo´ttir 2002; Yilmaz et al.
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Table 2. MICs of the analysed material against tested micro-organisms. MIC (mg/mL)
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Tested material C. uncialis extracts Heptane Diethyl ether Acetone Methanolic Compounds (2)-Usnic acid Squamatic acid Antibiotics Chloramphenicol Amphothericin B
S. aureusa
E. coli
b
C. albicansc
5 2.5 0.5 10
1000 1000 1000 1000
750 750 750 250
2.5 1250
– –
– –
5 –d
100 –
– 1
Note: Values given are the mean of three replicates (n ¼ 3). a Gram-positive: S. aureus – S. aureus ATCC 6538 P. b Gram-negative: E. coli – E. coli PZH 26B6. c fungi: C. albicans – C. albicans PCM 1409 PZH. d –, not tested.
2005). The Me2CO extract with strong activity against standard S. aureus led us to investigate the antimicrobial activity against 10 strains of MRSA, isolated from various types of wounds and from bronchial secretions from patients (Table 3). The results indicated that the Me2CO extract also showed potent activity against MRSA (Table 3). The activity of Me2CO extract was different depending on the examined MRSA strain. The strongest action of Me2CO extract was noticed on No. 10 S. aureus strain (MIC ¼ 2.5 mg/mL). This was higher than the activity of chloramphenicol (MIC ¼ 5.0 mg/mL against the reference strain). The antibacterial activity of the Me2CO extract, demonstrated against other clinical strains, was MIC 5.0– 7.5 mg/mL. The lower content of (2)-usnic acid in the Me2CO extract than that of the Hep extract, which was characterised by weaker activity against S. aureus, indicated that the antimicrobial activity could be due to the synergism between (2)-usnic and squamatic acids and the presence Table 3. MRSA isolated from patients used in the experiment and MICs of the acetone extract of C. uncialis against MRSA. Antibiotica resistant profile No.
Clinical source of MRSA
Resistant
Sensitive
1 2 3 4 5 6 7 8 9 10
Burn wound Burn wound Traumatic wound Internal wound Traumatic wound Decubitus wound Burn wound Burn wound Bronchial secretion Bronchial secretion
Cpx, – Cpx, Cpx, Cpx, Cpx, Cpx, Cpx, Cpx, Cpx,
R, Ti, V, Tei, L R, Ti, V, Tei, L R, V, Tei, C R, V, Tei, C R, V, Tei, C V, Tei, C R, V, Tei, C R, V, Tei, C Cl, R, Tei, D R, V, Tei
Cl, E, G Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, E, Tri/S, G Cl, E, Tri/S,
D, G G D, G D, C D, G D, G D, G
MIC (mg/mL) of acetone extract 5.0 7.5 5.0 7.5 5.0 7.5 5.0 7.5 5.0 2.5
Note: Values given are the mean of three replicates (n ¼ 3). a Cpx, Ciprofloxacin; Cl, Clindamycin; E, Erythromycin; R, Rifampicin; Ti, Tigecycline; Tri/S, Trimethoprim/ Sulphamethoxazole; V, Vancomycin; Tei, Teicoplanine; L, Linezolide; D, Doxycycline; G, Gentamycin; C, Chloramphenicol; ‘ –’ not detected.
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of others components. The isolation showed that the Me2CO extract contained considerable amounts of squamatic acid, but the compound did not show the antibacterial activity against the tested strains of microorganisms. However, the LC – MS and GC –MS analyses show that besides usnic and squamatic acids, the Me2CO extract contains the secondary metabolites of low molecular weight (Table 1), the presence of which could show the antimicrobial activity (Marante et al. 2003). There is evidence of synergistic interactions between individual components of the same extract also among extracts of different plants (Williamson 2001). Interaction of usnic acid in combination with some antibiotics, against Methicillin-resistant clinical strains of S. aureus isolates, was observed (Segatore et al. 2012). Positive interactions of plant extracts and extracts from lichens with synthetic antibiotics (ampicillin or tetracycline) were also documented (Agboke et al. 2011; Agboke & Simone 2011). 3. Conclusion In summary, the results show varying effects of the antimicrobial activity of Hep, Et2O, Me2CO and MeOH extracts of C. uncialis. Significant activity was exhibited by the Me2CO extract against S. aureus, potent activity was also shown against MRSA isolated from patients, close to that of chloramphenicol against the reference strain. The activity of the Me2CO extract against S. aureus was greater than that of the isolated (2)-usnic acid, whereas squamatic acid present in this extract proved to be inactive. The content of (2)-usnic acid in the most active Me2CO extract was lower than in the less active Hep extract, which indicated that the antimicrobial activity was associated with the presence of other components or a synergic action between compounds of acetone extract. In our opinion, the Me2CO extract can be considered as an active antimicrobial material, for example in the treatment of bacterial infections involving MRSA. This requires further studies including the cytotoxicity tests. Supplementary material Experimental details relating to this paper are available online, alongside Figures S1 and S2. References Agboke A, Jackson C, Adedokun M, Momoh MA. 2011. In vitro evaluation of the interaction between methanol extract of lichen (Ramalina farinacea) and tetracycline against clinical isolates of Staphylococcus aureus. Afr J Biotechnol. 10:2314–2318. Agboke AA, Simone CO. 2011. Antimicrobial evaluation of the interaction between methanol extract of the lichen, Ramalina farinace (Ramalinacea) and ampicillin against clinical isolates of Staphylococcus aureus. J Med Plant Res. 5:644–648. BeGora MD, Fahselt D. 2001. Usnic acid and atranorin concentrations in lichens in relation to bands of UV irradiance. Bryologist. 104:134–140, doi:10.1639/0007-2745(2001)104[0134:UAAACI]2.0.CO;2. Chambers HF. 2003. Tracking the spread of CMRSA. APUA Newsletter. 21:1–5. Culberson CF. 1969. Chemical and botanical guide to lichen products. Koenigstein: Carolina Press. The University of North. Reprint 1979 by Otto Koeltz Science Publishers. Ingo´lfsdo´ttir K. 2002. Usnic acid. Phytochemistry. 61:729–736. Marante FJT, Castellano AG, Rosas FE, Aguiar JQ, Barrera JJB. 2003. Identification and quantitation of allelochemicals from the lichen Lethariella canariensis: phytotoxicity and antioxidative activity. Chem Ecol. 29:2049– 2071, doi:10.1023/A:1025682318001. Mu¨ller K. 2001. Pharmaceutically relevant metabolites from lichens. Appl Microbiol Biotechnol. 56:9–16. Purvis OW, Coppins BJ, Hawksworth DL, James PW, Moore DM. 1992. The lichen flora of Great Britain and Irleand. London: Natural History Publications. Segatore B, Bellio P, Setacci D, Brisdelli F, Piovano M, Garbarino JA, Nicoletti M, Amicosante G, Perilli M, Celenza G. 2012. In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant staphylococcus aureus clinical isolates determined by FICI and DE model methods. Phytomedicine. 19:341–347, doi:10.1016/j.phymed.2011.10.012.
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Stojanovic´ IZˇ, Radulovic´ NS, Mitrovic´ TLj, Stamenkovic´ SM, Stojanovic´ GS. 2011. Volatile constituents of selected Parmeliaceae lichens. J Serb Chem Soc. 76:987–994, doi:10.2298/JSC101004087S. Yilmaz M, Tay T, Kivanc M, Tu¨rk H, Tu¨rk AO. 2005. The antimicrobial activity of extracts of the lichen Hypogymnia tubulosa and its 3-hydroxyphysodic acid constituent. Z Naturforsch C. 60:35–38. Williamson EM. 2001. Synergy and other interactions in phytomedicines. Phytomedicine. 8:401–409, doi:10.1078/ 0944-7113-00060.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis a
b
Elżbieta Studzińska-Sroka , Elżbieta Hołderna-Kędzia , c
a
d
Agnieszka Galanty , Wiesława Bylka , Karol Kacprzak & Karolina Ćwiklińska
e
a
Department of Pharmacognosy, Poznan University of Medical Sciences, Święcickiego 4, 60-781 Poznań, Poland b
Click for updates
Institute of Natural Fibres and Medicinal Plants, Libelta 27, 61-707 Poznań, Poland c
Department of Pharmacognosy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland d
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznań, Poland e
Department of Laboratory Diagnostics, Josef Strus Multidisciplinary Municipal Hospital, Szwajcarska 3, 61-285 Poznań, Poland Published online: 03 Feb 2015.
To cite this article: Elżbieta Studzińska-Sroka, Elżbieta Hołderna-Kędzia, Agnieszka Galanty, Wiesława Bylka, Karol Kacprzak & Karolina Ćwiklińska (2015): In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1005616 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1005616
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Selcuk Universitesi] at 11:18 10 February 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1005616
SHORT COMMUNICATION In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis Elz˙bieta Studzin´ska-Srokaa*, Elz˙bieta Hołderna-Ke˛dziab, Agnieszka Galantyc, ´ wiklin´skae Wiesława Bylkaa, Karol Kacprzakd and Karolina C Department of Pharmacognosy, Poznan University of Medical Sciences, S´wie˛cickiego 4, 60-781 Poznan´, Poland; bInstitute of Natural Fibres and Medicinal Plants, Libelta 27, 61-707 Poznan´, Poland; c Department of Pharmacognosy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland; dFaculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´, Poland; e Department of Laboratory Diagnostics, Josef Strus Multidisciplinary Municipal Hospital, Szwajcarska 3, 61-285 Poznan´, Poland (Received 22 July 2014; final version received 4 January 2015)
DIETHYL ETHER
CH3 O H3 C
OH
20
S.aureus
COOH
O OHC
OH CH3
HEPTANE
ACETONE
H3C
OH H3C
HO
O
MICµg/mL
H 3CO
MRSA
16 12 8
O
4 CH3
0
no U sn l i am c a ci at d i c n am A e ac ce (M id ph to R en ne S A ic A (M 1 ol ce R ,3 to S ,5 ne A ,7 , (M 2,4 9) , R 6 S ,8 A ) 10 ) or
A
ce
to
S
hl
)(-
qu
ha
C
er th
to
.e
ce
et
A
M
ta
ne
CH3
ne
OH
ep
METHANOL
O
D
O
H
Downloaded by [Selcuk Universitesi] at 11:18 10 February 2015
a
Heptane (Hep), diethyl ether (Et2O), acetone (Me2CO) and methanolic (MeOH) extracts, as well as (2)-usnic acid and squamatic acid, were obtained from thallus of Cladonia uncialis (Cladoniaceae). The antimicrobial activities of these extracts, (2 )usnic acid and squamatic acid, were tested against reference strains: Staphylococcus aureus, Escherichia coli and Candida albicans. In addition, Me2CO extract was analysed against 10 strains of Methicillin-resistant S. aureus (MRSA) isolated from patients. All extracts exerted antibacterial activity against the reference strain S. aureus, comparably to chloramphenicol [minimum inhibitory concentration (MIC) ¼ 5.0 mg/mL]. The Me2CO extract exhibited the strongest activity against S. aureus (MIC ¼ 0.5 mg/mL), higher than ( 2 )-usnic acid, whereas squamatic acid proved inactive. The Me2CO extract showed potent antimicrobial activity against MRSA (MIC 2.5– 7.5 mg/mL). Also no activity of C. uncialis extracts against E. coli and C. albicans was observed. Keywords: Cladonia uncialis; extracts; usnic acid; squamatic acid; antimicrobial activity
1. Introduction Lichens are widespread symbiotic organisms consisting of fungal partners, mycobionts and one or more photosynthetic partners, the photobionts, are green algae or cyanobacteria. The specific
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Downloaded by [Selcuk Universitesi] at 11:18 10 February 2015
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E. Studzin´ska-Sroka et al.
secondary metabolites of lichens exhibit wide a spectrum of biological activity such as antimicrobial, antiviral, antiprotozoal, antitumour, anti-inflammatory, analgesic, antipyretic, antiproliferative, antioxidant and antiherbivoral (Mu¨ller 2001). Cladonia uncialis (L.) Weber ex F.H. Wigg. belongs to the order Lecanorales and to the family Cladoniaceae. The thallus of this lichen is yellow-green or greenish, often brownish towards the pointed apices. C. uncialis frequently grows on soil among mosses, in well-lit and typically, dry places. It is common in coniferous forests, on heathland and sand dunes in Europe, North America and Asia (Purvis et al. 1992). C. uncialis produces dibenzofurans such as (2)-usnic acid and depsides: squamatic acid and thamnolic acid (Culberson 1969; Purvis et al. 1992; BeGora & Fahlset 2001). The bacteria (Gram-positive and Gram-negative) and fungi are the agents responsible for numerous human diseases. The serious threat is the antibiotic-resistant strains of bacteria, e.g. Methicillin-resistant Staphylococcus aureus (MRSA) which is a frequent cause of nosocomial infection leading to a wide range of diseases: endocarditis, osteomyelitis, toxic shock syndrome, pneumonia, food poisoning and carbuncles (Chambers 2003). It is known that lichen and compounds isolated from them were found active against the various classes and species of the microorganisms. While a number of reports concern the antimicrobial effects of usnic acid, there are no data available on the biological activity including the antimicrobial properties of crude extracts from C. uncialis and squamatic acid (Mu¨ller 2001; Ingo´lfsdo´ttir 2002). The aim of our study was to investigate the antimicrobial activity by the broth microdilution method of heptane (Hep), diethyl ether (Et2O), acetone (Me2CO) and methanolic (MeOH) extracts, as well as isolated (2)-usnic and squamatic acids obtained from C. uncialis against three reference strains: S. aureus, Escherichia coli and Candida albicans. The Me2CO extract has also been tested against MRSA isolated from patients. In addition the acetone extract was examined by LC –MS and GC – MS analyses. Moreover, the content of (2)-usnic acid in the extracts from C. uncialis was studied using the HPLC method to explain whether and to what extent the dibenzofuran could be responsible for the biological properties of the extracts. 2. Results and discussion 2.1. Phytochemical analysis C. uncialis (32.6 g) was extracted with heptane and then with acetone, successively. Extracts were obtained in a similar quantity (280 and 272 mg, respectively). (2)-Usnic acid (92.3 mg) and squamatic acid (90.3 mg) (Supplementary Figure S1 – online only) were isolated from the Hep and Me2CO extract, respectively, and identified by the LC – MS (Supplementary Figure S2 – online only) and TLC analyses as well as spectroscopic methods (UV, 1H NMR, 13C NMR and ESI-MS). The optical rotation of (2)-usnic acid was also determined confirming its high optical purity. For antimicrobial assays, heptane (Hep; 33.7 mg), diethyl ether (Et2O; 38.05 mg), acetone (Me2CO; 69.8 mg) and methanolic (MeOH; 270.6 mg) extracts were received from 5 g of the thalli of C. uncialis using the ultrasonic bath. The TLC analysis of these extracts showed that (2)-usnic acid was present in all extracts, mainly in the Hep extract. Squamatic acid can be conveniently extracted with diethyl ether and acetone, while the Hep extract did not contain this compound, and the MeOH extract did contain only a small amount. The absolute quantification of (2)-usnic acid was determined by HPLC. Heptane and diethyl ether were the most effective solvents for (2)-usnic acid extraction, with the content of 0.633 ^ 0.03 and 0.437 ^ 0.01 mg/ mL, respectively. The concentration of (2)-usnic acid was about two-and-a-half times lower (0.284 ^ 0.03 mg/mL) in the Me2CO and three times lower in the MeOH extract (0.190 ^ 0.01 mg/mL), in comparison with the Hep extract. Acetone extract was also analysed by the LC –MS method on reversed phase within 60 min analysis under increasing gradient of
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Table 1. Compounds identified in the acetone extract of C. uncialis using GC-MS.
No. 1 2 3 4
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5 6 7
Name 3-Methoxy-5-methylphenol (syn. orcinol monomethyl ether) 2,5-Dimethyl-1,4-benzenediol 2,4-Dihydroxy-6-methylbenzaldehyde (syn. o-orsellinaldehyde) Methyl 2,4-dihydroxy-3,6-dimethylbenzoate (syn. atraric acid) 5-Pentyl-1,3-benzenediol (syn. olivetol) Ethanone, 1-(4,6-dihydroxy-2,3,5-trimethyl7-benzofuranyl) (syn. usnetol) (9bS)-2,6-Diacetyl-3,7,9-trihydroxy-8,9bdimethyldibenzofuran-1-one (syn. usnic acid)
Retention Peak area Match Molecular Molecular time (%) factor mass (g/mol) formula (min) 10.160
26.60
878
138
C8H10O2
11.484 13.154
43.41 3.66
880 834
138 152
C8H10O2 C8H8O3
14.942
1.67
824
196
C10H12O4
15.462 18.494
4.98 1.12
914 808
180 234
C11H16O2 C13H14O4
24.103
5.59
922
344
C18H16O7
MeOH (up to 100%). All the tested detection modes (UV, negative and positive ions) proved the presence of both (2)-usnic and squamatic acids as well as some predominantly low-molecular yet undefined substances (Supplementary Figure S2 – online only). The GC – MS analysis provided the composition of the volatiles of acetone extract from C. uncialis (Table 1). The compounds were identified with match factor . 800. Taking into account the peak intensities, it can be established that among the seven compounds identified using GC – MS in the acetone extract, the main compounds were 2,5-dimethyl-1,4-benzenediol (43.41%) and orcinol monomethyl ether (26.60%). Usnic acid, olivetol, o-orsellinaldehyde, atraric acid and usnetol were also presented but in smaller quantities (Table 1). It is probable that the volatility of other compounds of the acetone extract was not sufficient for GC –MS analysis. The volatiles of the acetone extract of C. uncialis identified in this study were also described in other lichens (e.g. orcinol monomethyl ether, olivetol or atraric acid in Hypogymnia physodes or Evernia prunastri) (Stojanovic´ et al. 2011). 2.2. Antimicrobial assay The potency of the antimicrobial effect depended on the type of the extracts of C. uncialis and the tested microorganism. All the extracts tested showed strong antimicrobial activity against the reference strain of the Gram-positive bacteria S. aureus. The strongest activity against S. aureus (10 times higher than chloramphenicol) was observed for the Me2CO extract (MIC ¼ 0.5 mg/ mL). The antibacterial activity of the Et2O, Hep and MeOH extracts was two times higher, equal or two times lower (MIC ¼ 2.5, 5.0 and 10.0 mg/mL, respectively) than the reference compound. The activity of the isolated (2)-usnic acid was five times weaker than the activity of the examined Me2CO extract (MIC ¼ 2.5 mg/mL), whereas the squamatic acid was characterised by weak antibacterial activity (MIC ¼ 1250.0 mg/mL). Extracts from C. uncialis were inactive against the Gram-negative E. coli strain (MIC , 1000 mg/mL in comparison with chloramphenicol MIC ¼ 100 mg/mL). C. albicans was susceptible to lower concentration of the tested extract than E. coli bacteria; however, this action was lower than the reference amphotericin B (MIC 250 – 750 mg/mL and MIC ¼ 1 mg/mL, respectively) (Table 2). In the same manner, the activity of the MeOH extract against yeast C. albicans was higher than that of the Hep, Et2O and Me2CO extracts. Our results were compatible with those published in the literature, according to which lichenic compounds and extracts are potent active against Grampositive than Gram-negative bacteria and yeast (Mu¨ller 2001; Ingo´lfsdo´ttir 2002; Yilmaz et al.
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Table 2. MICs of the analysed material against tested micro-organisms. MIC (mg/mL)
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Tested material C. uncialis extracts Heptane Diethyl ether Acetone Methanolic Compounds (2)-Usnic acid Squamatic acid Antibiotics Chloramphenicol Amphothericin B
S. aureusa
E. coli
b
C. albicansc
5 2.5 0.5 10
1000 1000 1000 1000
750 750 750 250
2.5 1250
– –
– –
5 –d
100 –
– 1
Note: Values given are the mean of three replicates (n ¼ 3). a Gram-positive: S. aureus – S. aureus ATCC 6538 P. b Gram-negative: E. coli – E. coli PZH 26B6. c fungi: C. albicans – C. albicans PCM 1409 PZH. d –, not tested.
2005). The Me2CO extract with strong activity against standard S. aureus led us to investigate the antimicrobial activity against 10 strains of MRSA, isolated from various types of wounds and from bronchial secretions from patients (Table 3). The results indicated that the Me2CO extract also showed potent activity against MRSA (Table 3). The activity of Me2CO extract was different depending on the examined MRSA strain. The strongest action of Me2CO extract was noticed on No. 10 S. aureus strain (MIC ¼ 2.5 mg/mL). This was higher than the activity of chloramphenicol (MIC ¼ 5.0 mg/mL against the reference strain). The antibacterial activity of the Me2CO extract, demonstrated against other clinical strains, was MIC 5.0– 7.5 mg/mL. The lower content of (2)-usnic acid in the Me2CO extract than that of the Hep extract, which was characterised by weaker activity against S. aureus, indicated that the antimicrobial activity could be due to the synergism between (2)-usnic and squamatic acids and the presence Table 3. MRSA isolated from patients used in the experiment and MICs of the acetone extract of C. uncialis against MRSA. Antibiotica resistant profile No.
Clinical source of MRSA
Resistant
Sensitive
1 2 3 4 5 6 7 8 9 10
Burn wound Burn wound Traumatic wound Internal wound Traumatic wound Decubitus wound Burn wound Burn wound Bronchial secretion Bronchial secretion
Cpx, – Cpx, Cpx, Cpx, Cpx, Cpx, Cpx, Cpx, Cpx,
R, Ti, V, Tei, L R, Ti, V, Tei, L R, V, Tei, C R, V, Tei, C R, V, Tei, C V, Tei, C R, V, Tei, C R, V, Tei, C Cl, R, Tei, D R, V, Tei
Cl, E, G Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, Cl, E, Tri/S, E, Tri/S, G Cl, E, Tri/S,
D, G G D, G D, C D, G D, G D, G
MIC (mg/mL) of acetone extract 5.0 7.5 5.0 7.5 5.0 7.5 5.0 7.5 5.0 2.5
Note: Values given are the mean of three replicates (n ¼ 3). a Cpx, Ciprofloxacin; Cl, Clindamycin; E, Erythromycin; R, Rifampicin; Ti, Tigecycline; Tri/S, Trimethoprim/ Sulphamethoxazole; V, Vancomycin; Tei, Teicoplanine; L, Linezolide; D, Doxycycline; G, Gentamycin; C, Chloramphenicol; ‘ –’ not detected.
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of others components. The isolation showed that the Me2CO extract contained considerable amounts of squamatic acid, but the compound did not show the antibacterial activity against the tested strains of microorganisms. However, the LC – MS and GC –MS analyses show that besides usnic and squamatic acids, the Me2CO extract contains the secondary metabolites of low molecular weight (Table 1), the presence of which could show the antimicrobial activity (Marante et al. 2003). There is evidence of synergistic interactions between individual components of the same extract also among extracts of different plants (Williamson 2001). Interaction of usnic acid in combination with some antibiotics, against Methicillin-resistant clinical strains of S. aureus isolates, was observed (Segatore et al. 2012). Positive interactions of plant extracts and extracts from lichens with synthetic antibiotics (ampicillin or tetracycline) were also documented (Agboke et al. 2011; Agboke & Simone 2011). 3. Conclusion In summary, the results show varying effects of the antimicrobial activity of Hep, Et2O, Me2CO and MeOH extracts of C. uncialis. Significant activity was exhibited by the Me2CO extract against S. aureus, potent activity was also shown against MRSA isolated from patients, close to that of chloramphenicol against the reference strain. The activity of the Me2CO extract against S. aureus was greater than that of the isolated (2)-usnic acid, whereas squamatic acid present in this extract proved to be inactive. The content of (2)-usnic acid in the most active Me2CO extract was lower than in the less active Hep extract, which indicated that the antimicrobial activity was associated with the presence of other components or a synergic action between compounds of acetone extract. In our opinion, the Me2CO extract can be considered as an active antimicrobial material, for example in the treatment of bacterial infections involving MRSA. This requires further studies including the cytotoxicity tests. Supplementary material Experimental details relating to this paper are available online, alongside Figures S1 and S2. References Agboke A, Jackson C, Adedokun M, Momoh MA. 2011. In vitro evaluation of the interaction between methanol extract of lichen (Ramalina farinacea) and tetracycline against clinical isolates of Staphylococcus aureus. Afr J Biotechnol. 10:2314–2318. Agboke AA, Simone CO. 2011. Antimicrobial evaluation of the interaction between methanol extract of the lichen, Ramalina farinace (Ramalinacea) and ampicillin against clinical isolates of Staphylococcus aureus. J Med Plant Res. 5:644–648. BeGora MD, Fahselt D. 2001. Usnic acid and atranorin concentrations in lichens in relation to bands of UV irradiance. Bryologist. 104:134–140, doi:10.1639/0007-2745(2001)104[0134:UAAACI]2.0.CO;2. Chambers HF. 2003. Tracking the spread of CMRSA. APUA Newsletter. 21:1–5. Culberson CF. 1969. Chemical and botanical guide to lichen products. Koenigstein: Carolina Press. The University of North. Reprint 1979 by Otto Koeltz Science Publishers. Ingo´lfsdo´ttir K. 2002. Usnic acid. Phytochemistry. 61:729–736. Marante FJT, Castellano AG, Rosas FE, Aguiar JQ, Barrera JJB. 2003. Identification and quantitation of allelochemicals from the lichen Lethariella canariensis: phytotoxicity and antioxidative activity. Chem Ecol. 29:2049– 2071, doi:10.1023/A:1025682318001. Mu¨ller K. 2001. Pharmaceutically relevant metabolites from lichens. Appl Microbiol Biotechnol. 56:9–16. Purvis OW, Coppins BJ, Hawksworth DL, James PW, Moore DM. 1992. The lichen flora of Great Britain and Irleand. London: Natural History Publications. Segatore B, Bellio P, Setacci D, Brisdelli F, Piovano M, Garbarino JA, Nicoletti M, Amicosante G, Perilli M, Celenza G. 2012. In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant staphylococcus aureus clinical isolates determined by FICI and DE model methods. Phytomedicine. 19:341–347, doi:10.1016/j.phymed.2011.10.012.
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Stojanovic´ IZˇ, Radulovic´ NS, Mitrovic´ TLj, Stamenkovic´ SM, Stojanovic´ GS. 2011. Volatile constituents of selected Parmeliaceae lichens. J Serb Chem Soc. 76:987–994, doi:10.2298/JSC101004087S. Yilmaz M, Tay T, Kivanc M, Tu¨rk H, Tu¨rk AO. 2005. The antimicrobial activity of extracts of the lichen Hypogymnia tubulosa and its 3-hydroxyphysodic acid constituent. Z Naturforsch C. 60:35–38. Williamson EM. 2001. Synergy and other interactions in phytomedicines. Phytomedicine. 8:401–409, doi:10.1078/ 0944-7113-00060.
