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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
Helichrysum arenarium subsp. arenarium: phenolic composition and antibacterial activity against lower respiratory tract pathogens a
b
a
Adina C. Gradinaru , Mihaela Silion , Adriana Trifan , Anca Miron
a
a
& Ana C. Aprotosoaie a
Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa-Iasi, Universitatii Str. 16, 700115 Iasi, Romania b
Institute of Macromolecular Chemistry Petru Poni, Grigore Ghica Voda Al. 41A, 700487 Iasi, Romania Published online: 16 Jun 2014.
To cite this article: Adina C. Gradinaru, Mihaela Silion, Adriana Trifan, Anca Miron & Ana C. Aprotosoaie (2014) Helichrysum arenarium subsp. arenarium: phenolic composition and antibacterial activity against lower respiratory tract pathogens, Natural Product Research: Formerly Natural Product Letters, 28:22, 2076-2080, DOI: 10.1080/14786419.2014.924931 To link to this article: http://dx.doi.org/10.1080/14786419.2014.924931
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. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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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, 2014 Vol. 28, No. 22, 2076–2080, http://dx.doi.org/10.1080/14786419.2014.924931
SHORT COMMUNICATION Helichrysum arenarium subsp. arenarium: phenolic composition and antibacterial activity against lower respiratory tract pathogens Adina C. Gradinarua, Mihaela Silionb, Adriana Trifana, Anca Mirona* and Ana C. Aprotosoaiea a
Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa-Iasi, Universitatii Str. 16, 700115 Iasi, Romania; bInstitute of Macromolecular Chemistry Petru Poni, Grigore Ghica Voda Al. 41A, 700487 Iasi, Romania
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(Received 23 December 2013; final version received 13 May 2014) The aim of this study was to investigate the phenolic content and antibacterial activity of the methanol extract from Helichrysum arenarium (L.) Moench subsp. arenarium inflorescences against lower respiratory tract pathogens (standard strains and clinical isolates). The extract was characterised by a total phenolic content of 160.17 mg/g. Several caffeic acid conjugates (chlorogenic acid and dicaffeoylquinic acids) and flavonoids (apigenin, naringenin, apigenin-7-O-glucoside and naringenin-O-hexosides) were identified as major constituents by HPLC-DAD-ESI-MS. Staphylococcus aureus ATCC 25923 was more susceptible to Helichrysum extract than Streptococcus pneumoniae ATCC 49619 (minimum inhibitory concentration [MIC] ¼ 0.62 and 1.25 mg/mL, respectively). The extract exhibited similar antibacterial effects against methicillin-resistant S. aureus and penicillin-resistant S. pneumoniae clinical isolates (MIC ¼ 2.5 mg/mL) displaying a higher activity against ampicillin-resistant Moraxella catarrhalis isolate (MIC ¼ 0.15 mg/mL). The combination with ciprofloxacin exhibited additivity against both standard strains (fractional inhibitory concentration [FIC] index ¼ 0.75 and 0.73) and S. aureus isolates (FIC index ¼ 0.62) and synergy against S. pneumoniae isolates (FIC index ¼ 0.5). Keywords: HPLC-DAD-ESI-MS; ciprofloxacin; clinical isolates; penicillin-resistant Streptococcus pneumoniae; synergistic effects
1. Introduction The genus Helichrysum Mill. (Asteraceae) consists of more than 500 species widespread throughout the world (Wichtl 2004; Gouveia & Castilho 2012). Many Helichrysums have been used in the traditional medicine to cure various ailments such as infections of the skin, gallbladder, respiratory and digestive tracts disorders and kidney stones (Lourens et al. 2008, 2011; Su¨zgec -Selc uk & Birtekso¨z 2011; Tirillini et al. 2013). Moreover, in South Africa, several species are used to treat tuberculosis and related symptoms (Lourens et al. 2008). In recent years, extracts from different parts of Helichrysum species have been reported to exhibit a broadspectrum antimicrobial activity against Gram-positive bacteria (Enterococcus faecalis, Bacillus cereus, Bacillus subtilis, Bacillus brevis, Staphylococcus aureus), Gram-negative bacteria (Klebsiella pneumoniae, Yersinia enterocolitica, Pseudomonas aeruginosa, Escherichia coli) and yeasts (Cryptococcus neoformans, Candida albicans) (Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). Two subspecies of Helichrysum arenarium (L.) Moench (yellow everlasting) are very common and widespread in Romania: arenarium and ponticum; the latter grows only on the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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western coast of the Black Sea (Ciocarlan 2000). Although many Helichrysum species have been intensively studied, a literature survey showed only few investigations on Turkish H. arenarium subspecies (erzincanicum, rubicundum, aucheri) and H. arenarium (subspecies not mentioned) collected from different parts of Poland (Sroka et al. 2004; Bryksa-Godzisz et al. 2006; Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). Due to the increasing prevalence of resistance to antibiotic treatment and severe side effects of many antibiotics, the identification of antimicrobials to improve the efficacy and safety of antibiotics is an important area of research (Kyaw et al. 2012). In this respect, this work aimed to investigate the antibacterial efficacy of a methanol extract from H. arenarium subsp. arenarium inflorescences, alone and in combination with ciprofloxacin, against antibiotic-resistant clinical isolates of S. aureus, Streptococcus pneumoniae and Moraxella catarrhalis; in addition, the antibacterial effects against standard strains (S. aureus ATCC 25923, S. pneumoniae ATCC 49619), the phenolic content and profile of the extract were also studied. 2. Results and discussion 2.1. Phenolic content and profile The total phenolic content of Helichrysum extract, as estimated by the Folin – Ciocalteu assay, was 160.17 ^ 0.96 mg gallic acid equivalents (GAE) per g extract. The phenolic profile of the extract was investigated by HPLC-DAD-ESI-MS (Figure S1). Chlorogenic and caffeic acids, apigenin-7-O-glucoside, naringenin, apigenin and kaempferol were undoubtedly identified by comparing their UV and MS spectra with those of authentic standards analysed in the same experimental conditions. Quinic and two dicaffeoylquinic acids together with three naringeninO-hexosides were tentatively identified on the basis of the UV and MS spectral data reported in the literature (Table S1) (Kammerer et al. 2005; Gouveia & Castilho 2010, 2012). Except quinic and dicaffeoylquinic acids, all other compounds have been previously reported in H. arenarium flowers collected from other sites or belonging to other subspecies. Total phenolic contents of 115.76, 125.57 and 71.81 mg GAE per g were determined in the methanol extracts of dried aerial parts of H. arenarium (L.) Moench subsp. aucheri, subsp. erzincanicum and subsp. rubicundum, respectively; all three subspecies belong to Turkish flora. Several phenolic acids (chlorogenic, caffeic and p-coumaric acids), flavonoids (apigenin, apigenin-7-glucoside, luteolin and naringenin) and resveratrol were identified in the same extracts (Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). Numerous other polyphenols (kaempferol, quercetin and their derivatives, naringenin glucosides, isosalipurposide, ferulic, syringic, p-hydroxybenzoic and protocatechuic acids) have been reported in the methanol extracts of inflorescences of H. arenarium (L.) Moench (subspecies not specified) from Poland (Sroka et al. 2004; Bryksa-Godzisz et al. 2006). 2.2. Antimicrobial activity There are literature reports on the antimicrobial activity of different Helichrysum species, including several H. arenarium subspecies (erzincanicum, rubicundum, aucheri), against Grampositive and Gram-negative bacteria and yeasts (Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). These studies were performed on standard control strains which are known to have a lower virulence in comparison to clinical isolates (Fux et al. 2005). In this respect, assessment of antimicrobial activity of Helichrysum species against clinical isolates is an important approach for predicting the therapeutic outcome in patients with infections. Initially, this study evaluated the antimicrobial activity of Helichrysum extract against clinical isolates of antibiotic-resistant bacteria responsible for severe lower respiratory tract infections (methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae and
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ampicillin-resistant M. catarrhalis). In the agar well diffusion assay, the extract (1 mg/well) exhibited high activity against all tested clinical isolates with inhibition zones larger than 14 mm (Table S2). According to the minimum inhibitory concentration (MIC) values, determined as described by Basile et al. (2006), S. aureus and S. pneumoniae isolates displayed a similar susceptibility to Helichrysum extract (MIC ¼ 2.5 mg/mL) while M. catarrhalis isolate was more sensitive (MIC ¼ 0.15 mg/mL) (Table S2). The antibacterial activity against standard strains was also tested. According to the MIC values, both S. aureus ATCC 25923 (MIC ¼ 0.62 mg/mL) and S. pneumoniae ATCC 49619 (MIC ¼ 1.25 mg/mL) displayed a higher susceptibility to Helichrysum extract in comparison to the clinical isolates (Table S2). Polyphenols are undoubtedly involved in the antibacterial activity of Helichrysum extract. In general, polyphenols damage bacterial membrane causing a considerable increase in membrane permeability, inhibit extracellular bacterial enzymes and alter bacterial metabolism (Daglia 2012). Caffeic acid derivatives and flavonoids identified by HPLC-DAD-ESI-MS in Helichrysum extract cannot justify the significant activity against M. catarrhalis isolate, a Gramnegative bacteria. The high efficacy against M. catarrhalis might be not only due to other constituents, but also due to possible interactions between constituents. Although extracts from different Helichrysum species were reported to be active against Gram-negative bacteria (Lourens et al. 2008), there is little information on the bioactive constituents. Two chalcone derivatives, two acylphloroglucinols, 3-methoxyquercetin and several diterpenes isolated from Helichrysum gymnocomum and Helichrysum tenax, respectively, were tested regarding their ability to inhibit the growth of Gram-negative bacteria (K. pneumoniae, P. aeruginosa and E. coli). They exhibited antibacterial activity with MIC values ranging from 41.5 to 625 mg/mL (Drewes et al. 2006; Drewes & Van Vuuren 2008). 2.3. Antimicrobial activity in combination with ciprofloxacin Ciprofloxacin has a broad spectrum of activity including most of the pathogens responsible for lower respiratory tract infections, being more active against Gram-negative bacteria (Arpi et al. 1987). Identification of antimicrobial agents having the ability to reduce the minimum efficient dose of ciprofloxacin and consequently, its side effects, is of great interest. According to fractional inhibitory concentration (FIC) index evaluation described by Kyaw et al. (2012), the combination Helichrysum extract – ciprofloxacin showed a positive interaction (additivity) on standard strains (FIC index ¼ 0.75 on S. aureus ATCC 25923, FIC index ¼ 0.73 on S. pneumoniae ATCC 49619). Positive interactions were also found on Gram-positive clinical isolates: synergy (FIC index ¼ 0.5) on penicillin-resistant S. pneumoniae isolates and additivity (FIC index ¼ 0.62) on methicillin-resistant S. aureus isolates. The combination showed no interaction – indifference (FIC index ¼ 1.2) on Gram-negative M. catarrhalis isolate (Table S3). In the combination Helichrysum extract – ciprofloxacin, the MIC of ciprofloxacin decreased considerably by 4 and 8 times depending on the clinical isolate (S. pneumoniae and S. aureus, respectively). It is obvious that the antimicrobial constituents in Helichrysum extract target the same sites as ciprofloxacin but also different sites leading to additive and synergistic effects. Although the antimicrobial effects of different Helichrysum species have been investigated, neither the efficacy against clinical isolates nor possible interactions with antibiotics have been reported. To the best of our knowledge, this is the first study on the antibacterial efficacy of a Helichrysum extract, alone and in combination with an antibiotic, against clinical isolates of antibiotic-resistant bacteria. It is worthy to note that in recent years, the prevalence of penicillin-resistant S. pneumoniae strains has dramatically increased worldwide, these strains being involved in severe pulmonary infections. Due to poor activity against S. pneumoniae, the use of ciprofloxacin in the treatment
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of community-acquired pneumonia is considerably limited (Woodhead et al. 2011). The results of our study suggest a possible use of the methanol extract from H. arenarium subsp. arenarium inflorescences to increase the antibacterial activity of ciprofloxacin against penicillin-resistant S. pneumoniae. In addition, isolation of bioactive constituents might be of interest. 3. Conclusions HPLC-DAD-ESI-MS led to the identification of 12 constituents in the methanol extract from H. arenarium subsp. arenarium inflorescences: quinic and caffeic acids, caffeic acid derivatives, naringenin, apigenin and their glycosides, kaempferol. The extract exhibited antibacterial activity against methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae and ampicillin-resistant M. catarrhalis clinical isolates. Helichrysum extract reduced the effective dose of ciprofloxacin on S. aureus and S. pneumoniae isolates and showed synergistic activity in combination with ciprofloxacin against the latter. This result encourages further studies to evaluate the effects of the combination against other bacterial strains and to assess the in vivo significance of the antibacterial activity. Supplementary material Experimental details relating to this article are available online, alongside Figure S1 and Tables S1, S2 and S3. References ¨ . 2010. Phenolic compounds, antioxidant and antimicrobial properties of Albayrak S, Aksoy A, Sag˘dic O, Budak U Helichrysum species collected from Eastern Anatolia, Turkey. Turk J Biol. 34:463– 473. Albayrak S, Aksoy A, Sagdic O, Hamzaoglu E. 2010. Compositions, antioxidant and antimicrobial activities of Helichrysum (Asteraceae) species collected from Turkey. Food Chem. 119:114–122. Arpi M, Gahrn-Hansen B, Søgaard P, Bentzon MW. 1987. Comparative in vitro activities of pefloxacin, ofloxacin, enoxacin and ciprofloxacin against 256 clinical isolates. Acta Pathol Microbiol Immunol Scand B Microbiol. 95:141–146. Basile A, Senatore F, Gargano R, Sorbo S, Del Pezzo M, Lavitola A, Ritieni A, Bruno M, Spatuzzi D, Rigano D, Vuotto ML. 2006. Antibacterial and antioxidant activities in Sideritis italica (Miller) Greuter et Burdet essential oils. J Ethnopharmacol. 107:240–248. Bryksa-Godzisz M, We˛glarz Z, Przybył J. 2006. Phenolic compounds in yellow everlasting (Helichrysum arenarium (L.) Moench) growing wild in the middle part of the Bug river valley. Herba Pol. 52:26–31. Ciocarlan V. 2000. Flora ilustrata a Romaniei, Pteridophyta et Spermatophyta: fam. Asteraceae (Compositae). Bucuresti: Ed. Ceres. Daglia M. 2012. Polyphenols as antimicrobial agents. Curr Opin Biotechnol. 23:174–181. Drewes SE, Mudau KE, Van Vuuren SF, Viljoen AM. 2006. Antimicrobial monomeric and dimeric diterpenes from the leaves of Helichrysum tenax var tenax. Phytochemistry. 67:716–722. Drewes SE, Van Vuuren SF. 2008. Antimicrobial acylphloroglucinols and dibenzyloxy flavonoids from the flowers of Helichrysum gymnocomum. Phytochemistry. 69:1745 –1749. Fux CA, Shirtliff M, Stoodley P, Costerton JW. 2005. Can laboratory reference strains mirror ‘real-world’ pathogenesis? Trends Microbiol. 13:58–63. Gouveia S, Castilho PC. 2010. Characterization of phenolic compounds in Helichrysum melaleucum by highperformance liquid chromatography with on-line ultraviolet and mass spectrometry detection. Rapid Commun Mass Spectrom. 24:1851–1868. Gouveia S, Castilho PC. 2012. Helichrysum monizii Lowe: phenolic composition and antioxidant potential. Phytochem Anal. 23:72–83. Kammerer B, Kahlich R, Biegert C, Gleiter CH, Heide L. 2005. HPLC-MS/MS analysis of willow bark extracts contained in pharmaceutical preparations. Phytochem Anal. 16:470–478. Kyaw BM, Arora S, Lim CS. 2012. Bactericidal antibiotic –phytochemical combinations against methicillin resistant Staphylococcus aureus. Braz J Microbiol. 43:938– 945. Lourens ACU, Van Vuuren SF, Viljoen AM, Davids H, Van Heerden FR. 2011. Antimicrobial activity and in vitro cytotoxicity of selected South African Helichrysum species. S Afr J Bot. 77:229–235.
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Lourens ACU, Viljoen AM, Van Heerden FR. 2008. South African Helichrysum species: a review of the traditional uses, biological activity and phytochemistry. J Ethnopharmacol. 119:630–652. Sroka Z, Kuta I, Cisowski W, Drys´ A. 2004. Antiradical activity of hydrolyzed and non-hydrolyzed extracts from Helichrysi inflorescentia and its phenolic contents. Z Naturforsch C. 59:363–367. Su¨zgec -Selc uk S, Birtekso¨z AS. 2011. Flavonoids of Helichrysum chasmolycicum and its antioxidant and antimicrobial activities. S Afr J Bot. 77:170–174. Tirillini B, Menghini L, Leporini L, Scanu N, Marino S, Pintore G. 2013. Antioxidant activity of methanol extract of Helichrysum foetidum Moench. Nat Prod Res. 27:1484– 1487. Wichtl M, ed. 2004. Herbal drugs and phytopharmaceuticals: a handbook for practice on a scientific basis. 3rd ed. Stuttgart: Medpharm GmbH Scientific Publishers. Woodhead M, Blasi F, Ewig S, Garau J, Huchon G, Ieven M, Ortqvist A, Schaberg T, Torres A, van der Heijden G, et al. 2011. Guidelines for the management of adult lower respiratory tract infections – summary. Clin Microbiol Infect. 17:1–24.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Helichrysum arenarium subsp. arenarium: phenolic composition and antibacterial activity against lower respiratory tract pathogens a
b
a
Adina C. Gradinaru , Mihaela Silion , Adriana Trifan , Anca Miron
a
a
& Ana C. Aprotosoaie a
Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa-Iasi, Universitatii Str. 16, 700115 Iasi, Romania b
Institute of Macromolecular Chemistry Petru Poni, Grigore Ghica Voda Al. 41A, 700487 Iasi, Romania Published online: 16 Jun 2014.
To cite this article: Adina C. Gradinaru, Mihaela Silion, Adriana Trifan, Anca Miron & Ana C. Aprotosoaie (2014) Helichrysum arenarium subsp. arenarium: phenolic composition and antibacterial activity against lower respiratory tract pathogens, Natural Product Research: Formerly Natural Product Letters, 28:22, 2076-2080, DOI: 10.1080/14786419.2014.924931 To link to this article: http://dx.doi.org/10.1080/14786419.2014.924931
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. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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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, 2014 Vol. 28, No. 22, 2076–2080, http://dx.doi.org/10.1080/14786419.2014.924931
SHORT COMMUNICATION Helichrysum arenarium subsp. arenarium: phenolic composition and antibacterial activity against lower respiratory tract pathogens Adina C. Gradinarua, Mihaela Silionb, Adriana Trifana, Anca Mirona* and Ana C. Aprotosoaiea a
Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa-Iasi, Universitatii Str. 16, 700115 Iasi, Romania; bInstitute of Macromolecular Chemistry Petru Poni, Grigore Ghica Voda Al. 41A, 700487 Iasi, Romania
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(Received 23 December 2013; final version received 13 May 2014) The aim of this study was to investigate the phenolic content and antibacterial activity of the methanol extract from Helichrysum arenarium (L.) Moench subsp. arenarium inflorescences against lower respiratory tract pathogens (standard strains and clinical isolates). The extract was characterised by a total phenolic content of 160.17 mg/g. Several caffeic acid conjugates (chlorogenic acid and dicaffeoylquinic acids) and flavonoids (apigenin, naringenin, apigenin-7-O-glucoside and naringenin-O-hexosides) were identified as major constituents by HPLC-DAD-ESI-MS. Staphylococcus aureus ATCC 25923 was more susceptible to Helichrysum extract than Streptococcus pneumoniae ATCC 49619 (minimum inhibitory concentration [MIC] ¼ 0.62 and 1.25 mg/mL, respectively). The extract exhibited similar antibacterial effects against methicillin-resistant S. aureus and penicillin-resistant S. pneumoniae clinical isolates (MIC ¼ 2.5 mg/mL) displaying a higher activity against ampicillin-resistant Moraxella catarrhalis isolate (MIC ¼ 0.15 mg/mL). The combination with ciprofloxacin exhibited additivity against both standard strains (fractional inhibitory concentration [FIC] index ¼ 0.75 and 0.73) and S. aureus isolates (FIC index ¼ 0.62) and synergy against S. pneumoniae isolates (FIC index ¼ 0.5). Keywords: HPLC-DAD-ESI-MS; ciprofloxacin; clinical isolates; penicillin-resistant Streptococcus pneumoniae; synergistic effects
1. Introduction The genus Helichrysum Mill. (Asteraceae) consists of more than 500 species widespread throughout the world (Wichtl 2004; Gouveia & Castilho 2012). Many Helichrysums have been used in the traditional medicine to cure various ailments such as infections of the skin, gallbladder, respiratory and digestive tracts disorders and kidney stones (Lourens et al. 2008, 2011; Su¨zgec -Selc uk & Birtekso¨z 2011; Tirillini et al. 2013). Moreover, in South Africa, several species are used to treat tuberculosis and related symptoms (Lourens et al. 2008). In recent years, extracts from different parts of Helichrysum species have been reported to exhibit a broadspectrum antimicrobial activity against Gram-positive bacteria (Enterococcus faecalis, Bacillus cereus, Bacillus subtilis, Bacillus brevis, Staphylococcus aureus), Gram-negative bacteria (Klebsiella pneumoniae, Yersinia enterocolitica, Pseudomonas aeruginosa, Escherichia coli) and yeasts (Cryptococcus neoformans, Candida albicans) (Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). Two subspecies of Helichrysum arenarium (L.) Moench (yellow everlasting) are very common and widespread in Romania: arenarium and ponticum; the latter grows only on the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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western coast of the Black Sea (Ciocarlan 2000). Although many Helichrysum species have been intensively studied, a literature survey showed only few investigations on Turkish H. arenarium subspecies (erzincanicum, rubicundum, aucheri) and H. arenarium (subspecies not mentioned) collected from different parts of Poland (Sroka et al. 2004; Bryksa-Godzisz et al. 2006; Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). Due to the increasing prevalence of resistance to antibiotic treatment and severe side effects of many antibiotics, the identification of antimicrobials to improve the efficacy and safety of antibiotics is an important area of research (Kyaw et al. 2012). In this respect, this work aimed to investigate the antibacterial efficacy of a methanol extract from H. arenarium subsp. arenarium inflorescences, alone and in combination with ciprofloxacin, against antibiotic-resistant clinical isolates of S. aureus, Streptococcus pneumoniae and Moraxella catarrhalis; in addition, the antibacterial effects against standard strains (S. aureus ATCC 25923, S. pneumoniae ATCC 49619), the phenolic content and profile of the extract were also studied. 2. Results and discussion 2.1. Phenolic content and profile The total phenolic content of Helichrysum extract, as estimated by the Folin – Ciocalteu assay, was 160.17 ^ 0.96 mg gallic acid equivalents (GAE) per g extract. The phenolic profile of the extract was investigated by HPLC-DAD-ESI-MS (Figure S1). Chlorogenic and caffeic acids, apigenin-7-O-glucoside, naringenin, apigenin and kaempferol were undoubtedly identified by comparing their UV and MS spectra with those of authentic standards analysed in the same experimental conditions. Quinic and two dicaffeoylquinic acids together with three naringeninO-hexosides were tentatively identified on the basis of the UV and MS spectral data reported in the literature (Table S1) (Kammerer et al. 2005; Gouveia & Castilho 2010, 2012). Except quinic and dicaffeoylquinic acids, all other compounds have been previously reported in H. arenarium flowers collected from other sites or belonging to other subspecies. Total phenolic contents of 115.76, 125.57 and 71.81 mg GAE per g were determined in the methanol extracts of dried aerial parts of H. arenarium (L.) Moench subsp. aucheri, subsp. erzincanicum and subsp. rubicundum, respectively; all three subspecies belong to Turkish flora. Several phenolic acids (chlorogenic, caffeic and p-coumaric acids), flavonoids (apigenin, apigenin-7-glucoside, luteolin and naringenin) and resveratrol were identified in the same extracts (Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). Numerous other polyphenols (kaempferol, quercetin and their derivatives, naringenin glucosides, isosalipurposide, ferulic, syringic, p-hydroxybenzoic and protocatechuic acids) have been reported in the methanol extracts of inflorescences of H. arenarium (L.) Moench (subspecies not specified) from Poland (Sroka et al. 2004; Bryksa-Godzisz et al. 2006). 2.2. Antimicrobial activity There are literature reports on the antimicrobial activity of different Helichrysum species, including several H. arenarium subspecies (erzincanicum, rubicundum, aucheri), against Grampositive and Gram-negative bacteria and yeasts (Albayrak, Aksoy, Sag˘dic & Budak 2010; Albayrak, Aksoy, Sagdic & Hamzaoglu 2010). These studies were performed on standard control strains which are known to have a lower virulence in comparison to clinical isolates (Fux et al. 2005). In this respect, assessment of antimicrobial activity of Helichrysum species against clinical isolates is an important approach for predicting the therapeutic outcome in patients with infections. Initially, this study evaluated the antimicrobial activity of Helichrysum extract against clinical isolates of antibiotic-resistant bacteria responsible for severe lower respiratory tract infections (methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae and
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ampicillin-resistant M. catarrhalis). In the agar well diffusion assay, the extract (1 mg/well) exhibited high activity against all tested clinical isolates with inhibition zones larger than 14 mm (Table S2). According to the minimum inhibitory concentration (MIC) values, determined as described by Basile et al. (2006), S. aureus and S. pneumoniae isolates displayed a similar susceptibility to Helichrysum extract (MIC ¼ 2.5 mg/mL) while M. catarrhalis isolate was more sensitive (MIC ¼ 0.15 mg/mL) (Table S2). The antibacterial activity against standard strains was also tested. According to the MIC values, both S. aureus ATCC 25923 (MIC ¼ 0.62 mg/mL) and S. pneumoniae ATCC 49619 (MIC ¼ 1.25 mg/mL) displayed a higher susceptibility to Helichrysum extract in comparison to the clinical isolates (Table S2). Polyphenols are undoubtedly involved in the antibacterial activity of Helichrysum extract. In general, polyphenols damage bacterial membrane causing a considerable increase in membrane permeability, inhibit extracellular bacterial enzymes and alter bacterial metabolism (Daglia 2012). Caffeic acid derivatives and flavonoids identified by HPLC-DAD-ESI-MS in Helichrysum extract cannot justify the significant activity against M. catarrhalis isolate, a Gramnegative bacteria. The high efficacy against M. catarrhalis might be not only due to other constituents, but also due to possible interactions between constituents. Although extracts from different Helichrysum species were reported to be active against Gram-negative bacteria (Lourens et al. 2008), there is little information on the bioactive constituents. Two chalcone derivatives, two acylphloroglucinols, 3-methoxyquercetin and several diterpenes isolated from Helichrysum gymnocomum and Helichrysum tenax, respectively, were tested regarding their ability to inhibit the growth of Gram-negative bacteria (K. pneumoniae, P. aeruginosa and E. coli). They exhibited antibacterial activity with MIC values ranging from 41.5 to 625 mg/mL (Drewes et al. 2006; Drewes & Van Vuuren 2008). 2.3. Antimicrobial activity in combination with ciprofloxacin Ciprofloxacin has a broad spectrum of activity including most of the pathogens responsible for lower respiratory tract infections, being more active against Gram-negative bacteria (Arpi et al. 1987). Identification of antimicrobial agents having the ability to reduce the minimum efficient dose of ciprofloxacin and consequently, its side effects, is of great interest. According to fractional inhibitory concentration (FIC) index evaluation described by Kyaw et al. (2012), the combination Helichrysum extract – ciprofloxacin showed a positive interaction (additivity) on standard strains (FIC index ¼ 0.75 on S. aureus ATCC 25923, FIC index ¼ 0.73 on S. pneumoniae ATCC 49619). Positive interactions were also found on Gram-positive clinical isolates: synergy (FIC index ¼ 0.5) on penicillin-resistant S. pneumoniae isolates and additivity (FIC index ¼ 0.62) on methicillin-resistant S. aureus isolates. The combination showed no interaction – indifference (FIC index ¼ 1.2) on Gram-negative M. catarrhalis isolate (Table S3). In the combination Helichrysum extract – ciprofloxacin, the MIC of ciprofloxacin decreased considerably by 4 and 8 times depending on the clinical isolate (S. pneumoniae and S. aureus, respectively). It is obvious that the antimicrobial constituents in Helichrysum extract target the same sites as ciprofloxacin but also different sites leading to additive and synergistic effects. Although the antimicrobial effects of different Helichrysum species have been investigated, neither the efficacy against clinical isolates nor possible interactions with antibiotics have been reported. To the best of our knowledge, this is the first study on the antibacterial efficacy of a Helichrysum extract, alone and in combination with an antibiotic, against clinical isolates of antibiotic-resistant bacteria. It is worthy to note that in recent years, the prevalence of penicillin-resistant S. pneumoniae strains has dramatically increased worldwide, these strains being involved in severe pulmonary infections. Due to poor activity against S. pneumoniae, the use of ciprofloxacin in the treatment
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of community-acquired pneumonia is considerably limited (Woodhead et al. 2011). The results of our study suggest a possible use of the methanol extract from H. arenarium subsp. arenarium inflorescences to increase the antibacterial activity of ciprofloxacin against penicillin-resistant S. pneumoniae. In addition, isolation of bioactive constituents might be of interest. 3. Conclusions HPLC-DAD-ESI-MS led to the identification of 12 constituents in the methanol extract from H. arenarium subsp. arenarium inflorescences: quinic and caffeic acids, caffeic acid derivatives, naringenin, apigenin and their glycosides, kaempferol. The extract exhibited antibacterial activity against methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae and ampicillin-resistant M. catarrhalis clinical isolates. Helichrysum extract reduced the effective dose of ciprofloxacin on S. aureus and S. pneumoniae isolates and showed synergistic activity in combination with ciprofloxacin against the latter. This result encourages further studies to evaluate the effects of the combination against other bacterial strains and to assess the in vivo significance of the antibacterial activity. Supplementary material Experimental details relating to this article are available online, alongside Figure S1 and Tables S1, S2 and S3. References ¨ . 2010. Phenolic compounds, antioxidant and antimicrobial properties of Albayrak S, Aksoy A, Sag˘dic O, Budak U Helichrysum species collected from Eastern Anatolia, Turkey. Turk J Biol. 34:463– 473. Albayrak S, Aksoy A, Sagdic O, Hamzaoglu E. 2010. Compositions, antioxidant and antimicrobial activities of Helichrysum (Asteraceae) species collected from Turkey. Food Chem. 119:114–122. Arpi M, Gahrn-Hansen B, Søgaard P, Bentzon MW. 1987. Comparative in vitro activities of pefloxacin, ofloxacin, enoxacin and ciprofloxacin against 256 clinical isolates. Acta Pathol Microbiol Immunol Scand B Microbiol. 95:141–146. Basile A, Senatore F, Gargano R, Sorbo S, Del Pezzo M, Lavitola A, Ritieni A, Bruno M, Spatuzzi D, Rigano D, Vuotto ML. 2006. Antibacterial and antioxidant activities in Sideritis italica (Miller) Greuter et Burdet essential oils. J Ethnopharmacol. 107:240–248. Bryksa-Godzisz M, We˛glarz Z, Przybył J. 2006. Phenolic compounds in yellow everlasting (Helichrysum arenarium (L.) Moench) growing wild in the middle part of the Bug river valley. Herba Pol. 52:26–31. Ciocarlan V. 2000. Flora ilustrata a Romaniei, Pteridophyta et Spermatophyta: fam. Asteraceae (Compositae). Bucuresti: Ed. Ceres. Daglia M. 2012. Polyphenols as antimicrobial agents. Curr Opin Biotechnol. 23:174–181. Drewes SE, Mudau KE, Van Vuuren SF, Viljoen AM. 2006. Antimicrobial monomeric and dimeric diterpenes from the leaves of Helichrysum tenax var tenax. Phytochemistry. 67:716–722. Drewes SE, Van Vuuren SF. 2008. Antimicrobial acylphloroglucinols and dibenzyloxy flavonoids from the flowers of Helichrysum gymnocomum. Phytochemistry. 69:1745 –1749. Fux CA, Shirtliff M, Stoodley P, Costerton JW. 2005. Can laboratory reference strains mirror ‘real-world’ pathogenesis? Trends Microbiol. 13:58–63. Gouveia S, Castilho PC. 2010. Characterization of phenolic compounds in Helichrysum melaleucum by highperformance liquid chromatography with on-line ultraviolet and mass spectrometry detection. Rapid Commun Mass Spectrom. 24:1851–1868. Gouveia S, Castilho PC. 2012. Helichrysum monizii Lowe: phenolic composition and antioxidant potential. Phytochem Anal. 23:72–83. Kammerer B, Kahlich R, Biegert C, Gleiter CH, Heide L. 2005. HPLC-MS/MS analysis of willow bark extracts contained in pharmaceutical preparations. Phytochem Anal. 16:470–478. Kyaw BM, Arora S, Lim CS. 2012. Bactericidal antibiotic –phytochemical combinations against methicillin resistant Staphylococcus aureus. Braz J Microbiol. 43:938– 945. Lourens ACU, Van Vuuren SF, Viljoen AM, Davids H, Van Heerden FR. 2011. Antimicrobial activity and in vitro cytotoxicity of selected South African Helichrysum species. S Afr J Bot. 77:229–235.
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Lourens ACU, Viljoen AM, Van Heerden FR. 2008. South African Helichrysum species: a review of the traditional uses, biological activity and phytochemistry. J Ethnopharmacol. 119:630–652. Sroka Z, Kuta I, Cisowski W, Drys´ A. 2004. Antiradical activity of hydrolyzed and non-hydrolyzed extracts from Helichrysi inflorescentia and its phenolic contents. Z Naturforsch C. 59:363–367. Su¨zgec -Selc uk S, Birtekso¨z AS. 2011. Flavonoids of Helichrysum chasmolycicum and its antioxidant and antimicrobial activities. S Afr J Bot. 77:170–174. Tirillini B, Menghini L, Leporini L, Scanu N, Marino S, Pintore G. 2013. Antioxidant activity of methanol extract of Helichrysum foetidum Moench. Nat Prod Res. 27:1484– 1487. Wichtl M, ed. 2004. Herbal drugs and phytopharmaceuticals: a handbook for practice on a scientific basis. 3rd ed. Stuttgart: Medpharm GmbH Scientific Publishers. Woodhead M, Blasi F, Ewig S, Garau J, Huchon G, Ieven M, Ortqvist A, Schaberg T, Torres A, van der Heijden G, et al. 2011. Guidelines for the management of adult lower respiratory tract infections – summary. Clin Microbiol Infect. 17:1–24.
