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1 This article was downloaded by: [Univ Studi Della Calabria] On: 18 June 2012, At: 03:20 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: Antimicrobial and antioxidant properties of Betula aetnensis Rafin. (Betulaceae) leaves extract Rosaria Acquaviva a, Francesco Menichini b, Salvatore Ragusa c, Carlo Genovese d, Andrea Amodeo d, Rosa Tundis b, Monica R. Loizzo b & Liliana Iauk d a Department of Pharmaceutical Sciences, Biochemistry Section, University of Catania, Viale A. Doria 6, Catania, Italy b Department of Pharmaceutical Sciences, University of Calabria, via P. Bucci, Arcavacata di Rende (CS), Italy c Department of Pharmacobiological Sciences, University Magna Graecia of Catanzaro, Viale Europa-Località Germaneto, Catanzaro, Italy d Department of Bio-medical Sciences, University of Catania, via Androne 81, Catania, Italy Available online: 18 Jun 2012 To cite this article: Rosaria Acquaviva, Francesco Menichini, Salvatore Ragusa, Carlo Genovese, Andrea Amodeo, Rosa Tundis, Monica R. Loizzo & Liliana Iauk (2012): Antimicrobial and antioxidant properties of Betula aetnensis Rafin. (Betulaceae) leaves extract, Natural Product Research: Formerly Natural Product Letters, DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use:

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3 Natural Product Research 2012, 1 5, ifirst SHORT COMMUNICATION Antimicrobial and antioxidant properties of Betula aetnensis Rafin. (Betulaceae) leaves extract Rosaria Acquaviva a, Francesco Menichini b, Salvatore Ragusa c, Carlo Genovese d, Andrea Amodeo d, Rosa Tundis b *, Monica R. Loizzo b and Liliana Iauk d a Department of Pharmaceutical Sciences, Biochemistry Section, University of Catania, Viale A. Doria 6, Catania, Italy; b Department of Pharmaceutical Sciences, University of Calabria, via P. Bucci, Arcavacata di Rende (CS), Italy; c Department of Pharmacobiological Sciences, University Magna Graecia of Catanzaro, Viale Europa-Localita` Germaneto, Catanzaro, Italy; d Department of Bio-medical Sciences, University of Catania, via Androne 81, Catania, Italy (Received 1 December 2011; final version received 2 May 2012) This study aimed to evaluate the antimicrobial effects, the radical scavenging activity (by DPPH and ABTS tests) and the antioxidant capacity (by -carotene bleaching test) of Betula aetnensis leaves extract. The antimicrobial activity was tested against 14 Gram-positive clinical strains, 2 ATCC Gram-positive strains, 10 Gram-negative clinical strains and 4 Gram-negative ATCC strains. Streptococcus pyogenes Ery-S and Ery-R1 were the most sensitive. Betula aetnensis was considerably active against three bacterial strains, namely Haemophilus influenzae ATCC 49247, Amp-R1 and Moraxella catarrhalis ATCC Standard ATCC strains of Gram-positive bacteria were more sensitive than Gram-negative. Betula aetnensis showed also an interesting reducing power with TEAC values of 9.7 and a good inhibition of linoleic acid oxidation with an IC 50 value of 22.0 mgml 1 after 30 min of incubation. The total phenol and flavonoid contents were determined with the purpose to evaluate the relationship with the observed bioactivities. Keywords: Betula aetnensis; antimicrobial effects; Haemophilus influenzae; Streptococcus pyogenes Ery-S and Ery-R1; antioxidant activity 1. Introduction Numerous studies demonstrated that medicinal plants are sources of nutrient and nonnutrient compounds, many of which display antioxidant and antimicrobial properties which can protect the human body against both cellular oxidation reactions and pathogens. Thus, it is important to characterise the different types of medicinal plants for their antioxidant and antimicrobial potentials (Dallenbach-To lke, Nyiredy, Gross, & Sticher, 1986). Currently, of the one-quarter to one-half of all pharmaceuticals dispensed in the United States having higher-plant origins, very few are intended for use as antimicrobials, since we have relied on bacterial and fungal sources for these activities. Since the advent *Corresponding author. tundis@unical.it ISSN print/issn online ß 2012 Taylor & Francis

4 2 R. Acquaviva et al. of antibiotics in the 1950s, the use of plant derivatives as antimicrobials has been virtually non-existent. Plants have an almost limitless ability to synthesise aromatic substances, most of which are phenols or their oxygen-substituted derivatives. In many cases, these substances serve as plant defence mechanisms against predation by microorganisms, insects and herbivores. In this context are included polyphenolic compounds. The site(s) and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms, with evidence that increased hydroxylation results in increased toxicity. In addition, some authors have found that more highly oxidised phenols are more inhibitory. The mechanisms thought to be responsible for phenolic toxicity to microorganisms include enzyme inhibition by the oxidised compounds, possibly through reaction with sulphhydryl groups or through more non-specific interactions with the proteins. Betula aetnensis Rafin. (Birch Etna) is a medium-sized deciduous tree, typically reaching 5 20 m tall, which belongs to the family Betulaceae. It grows on the eastern slope of Etna, at an altitude between 1200 and 2000 m (Rapp & Pashinskii, 1999). Many Betula species are used in folk medicine to treat skin diseases, especially eczema, infections, inflammations, rheumatism and urinary disorders (Kumaraswamy, Kavitha, & Satish, 2008; Pignatti, 1997). Some species, like Betula utilis, Betula pendula and Betula papyrifera were evaluated for their antibacterial activity (Cowan, 1999; Dallenbach-To lke et al., 1986; Saric et al., 2009). Betula platyphylla var. japonica showed antioxidant activity, had protective effects against hydrogen peroxide and induced apoptotic cell death in human promyelocytic leukaemia cells (Ju, Lee, Hwang, & Kim, 2004). Nearly, all species contain flavonoids, tannins, saponins and sterols as the most characteristic metabolites. Essential oils were also obtained from several Betula species, namely B. pendula, Betula browicziana, Betula litwinowii, Betula recurvata and Betula medwediewii (Bas er & Demirci, 2007; Kumaraswamy et al., 2008; Pignatti, 1997). Therefore, given the interesting biological properties reported for Betula species, the objective of this study is to investigate for the first time the in vitro antibacterial activity of B. aetnensis leaves extract against common clinical isolates, mainly those associated with respiratory infections. Furthermore, being commonly accepted that reactive oxygen species play an important role in the pathogenesis of various diseases, the antioxidant activity (assessed by DPPH assay, ABTS assay and -carotene bleaching test) was evaluated and was related to the total phenol and flavonoid contents. 2. Results and discussion In this study B. aetnensis leaves extract was evaluated for its antimicrobial and antioxidant effects. It was tested against 14 Gram-positive clinical strains and 2 ATCC Gram-positive strains with MIC in the range mgml 1 and its antimicrobial activity compared to that of five reference antibiotics: erythromycin, methicillin, penicillin G, ampicillin and ciprofloxacin (CLSI, 2008). As presented in Table 1, Streptococcus pneumoniae ATCC 49619, Pen-R2, Ery-R1, Ery-R2, Pen-R Ery-R2 and Streptococcus pyogenes Ery-S, Ery-R1 were the most sensitive (MIC 225 mgml 1 ) while Staphylococcus aureus Met-R1 and Met-R2 were the least sensitive (MIC 900 mgml 1 ). The antibacterial activity was also evaluated on 10 Gramnegative clinical strains and 4 Gram-negative ATCC strains with a variable degree of susceptibility. It was considerable only against three bacterial strains: Haemophilus influenzae ATCC 49247, Amp-R1 and Moraxella catarrhalis ATCC (MIC 900 mgml 1 ). Haemophilus influenzae Amp-R2 was less susceptible (MIC 1800 mgml 1 );

5 Natural Product Research 3 Table 1. Antibacterial activity (MIC (mgml 1 )) of B. aetnensis extract on Gram-positive and Gram-negative strains. Betula aetnensis Amp Cip Ery Met Pen Gram-positive strains Staphylococcus aureus ATCC Staphylococcus aureus Met-R Staphylococcus aureus Met-R Staphylococcus epidermidis Met-S Staphylococcus epidermidis Met-R Staphylococcus epidermidis Met-R Streptococcus pneumoniae ATCC Streptococcus pneumoniae Pen-R Streptococcus pneumoniae Pen-R Streptococcus pneumoniae Ery-R Streptococcus pneumoniae Ery-R Streptococcus pneumoniae Pen-R Ery-R Streptococcus pneumoniae Pen-R Ery-R Streptococcus pyogenes Ery-S Streptococcus pyogenes Ery-R Streptococcus pyogenes Ery-R Gram-negative strains Haemophilus influenzae ATCC Haemophilus influenzae Amp-R Haemophilus influenzae Amp-R Moraxella catarrhalis ATCC Escherichia coli ATCC Escherichia coli Amp-R Escherichia coli Amp-R Escherichia coli Cip-R Escherichia coli Cip-R Escherichia coli Amp-R Cip-R Escherichia coli Amp-R Cip-R Pseudomonas aeruginosa ATCC Pseudomonas aeruginosa multi-r Pseudomonas aeruginosa multi-r Notes: Ery, erythromycin; Met, methicillin; Pen, penicillin G; Amp, ampicillin; and Cip, ciprofloxacin. Escherichia coli and Pseudomonas aeruginosa strains were resistant to all extract dilutions (MIC mgml 1 ). Our data revealed that standard ATCC strains of Gram-positive bacteria were more sensitive than Gram-negative. Since B. aetnensis extract showed a good content of both polyphenols and flavonoids, it is possible to ascribe the bioactivity to these classes of natural products (Dallenbach- To lke et al., 1986; Taguri, Tanaka, & Kouno, 2004). The antibacterial activity of several Betula species was previously reported. Betula utilis bark petroleum ether, chloroform, methanol, ethanol and water extracts were tested against 14 important bacterial strains by agar well diffusion method (Dallenbach-To lke et al., 1986). The methanol extract showed a significant activity against all tested bacteria, followed by ethanol and aqueous extract. In particular, B. utilis exhibited an interesting inhibitory activity against Shigella boydii, E. coli, P. aeruginosa, Salmonella paratyphi A and Shigella sonnei. The antibacterial activity of B. pendula ethanol extract against Bacillus cereus,

6 4 R. Acquaviva et al. Table 2. Total phenol and flavonoid contents and antioxidant activity of B. aetnensis extract. Total phenols a Total flavonoids b DPPH test c ABTS test d -Carotene bleaching test c 30 min 60 min Betula aetnensis ** ** ** ** Ascorbic acid BHT (þ)-catechin Propyl gallate Notes: Data are expressed as mean SD (n ¼ 3). a mg Chlorogenic acid equivalents per gram extract; b mg quercetin equivalents per gram extract; c IC 50 values expressed as mgml 1 ; and d TEAC value. Differences within and between groups were evaluated by one-way analysis of variance test followed by a multi-comparison Dunnett s test (**p ) compared with the positive control. Figure 1. Concentration response effects of B. aetnensis extract using -carotene linoleate system after 30 and 60 min of incubation. Data are given as means SD (n ¼ 3). Enterococcus faecalis, Salmonella choleraesuis, S. aureus, Proteus mirabilis and E. coli was reported (Saric et al., 2009). Betula pendula showed the highest antibacterial potential against B. cereus (MIC of 10 mg ml 1 ). This extract exhibited moderate antimicrobial activity towards the other investigated bacteria, except for E. faecalis and S. choleraesuis. Betula papyrifera flowers, leaves and bark extracts were previously investigated against S. aureus, E. coli, P. aeruginosa and Candida albicans (Cowan, 1999). Betula papyrifera leaves extract was most effective against S. aureus. The radical scavenging activity of B. aetnensis was tested through different in vitro methods such as DPPH and ABTS assays. A concentration response relationship was observed. Results are summarised in Table 2. Betula aetnensis extract showed a higher radical scavenging activity of the positive standard (þ)-catechin with IC 50 values of 31.0 and 35.0 mgml 1, respectively. A reducing power with a TEAC value of 9.7 compared to the positive control ascorbic acid (1.7) was observed in the ABTS test. Moreover, the antioxidant activity of B. aetnensis was studied using the -carotene bleaching method based on oxidative decomposition of -carotene in the presence of linoleic acid.

7 Natural Product Research 5 Betula aetnensis showed an IC 50 value of 22.0 mgml 1 after 30 min of incubation (Figure 1). After 60 min incubation, an increase in the IC 50 value was observed, indicating a decrease in the antioxidant activity of the extract over reaction time (IC 50 value of 27.5 mgml 1 ). The positive control propyl gallate showed IC 50 values of 1.0 mgml 1 after both 30 and 60 min incubation. The antiradical scavenging and antioxidant activities of B. aetnensis could be related to the level of phenolic components. In fact, the extract showed a total phenol content of mg chlorogenic acid equivalents per gram extract and a total flavonoid content of mg quercetin equivalents per gram extract. It is well-known that phenolic compounds contribute to modify plants colour, taste, aroma and flavour and also to provide health beneficial effects. They have also a role in plant defence mechanisms in order to survive and prevent molecular damage and damage by microorganisms, insects and herbivores (Vaya, Belinky, & Aviram, 1997). 3. Conclusions The results obtained in this study demonstrate the antioxidant properties and a strong bioactivity of B. aetnensis polyphenolic extract against S. pneumoniae. This bacteria is responsible of serious respiratory infections such as acute otitis media, sinusitis and pneumonia. Streptococcus pneumoniae can evade host defences in normal and impaired hosts and spread to the upper or lower respiratory tract, provoking correlate infections or it may invade the bloodstream, causing invasive diseases. Besides, it is constantly being studied for the frequency with which resistance occurs against the most common antibiotics. Therefore, polyphenolic compounds present in B. aetnensis could be useful in the treatment of infections caused by this bacterial strain. Supplementary material Experimental details relating to this article are available online. References Bas er, K.H.C., & Demirci, B. (2007). Studies on Betula essential oils. Arkivoc, 7, Cowan, M.M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12, Clinical and Laboratory Standards Institute (CLSI). (2008). Performance standards for antimicrobial susceptibility testing, 18th Informational Supplement, CLSI Document M100-S18, Wayne, PA. Dallenbach-To lke, K., Nyiredy, S., Gross, G.A., & Sticher, O. (1986). Flavonoid glycosides from Betula pubescens and Betula pendula. Journal of Natural Products, 49, Ju, E.M., Lee, S.E., Hwang, H.J., & Kim, J.H. (2004). Antioxidant and anticancer activity of extract from Betula platyphylla var. japonica. Life Sciences, 74, Kumaraswamy, M.V., Kavitha, H.U., & Satish, S. (2008). Antibacterial evaluation and phytochemical analysis of Betula utilis D. Don against some human pathogenic bacteria. World Journal of Agricultural Science, 4, Pignatti, S. (1997). Flora Europaea. Bologna: Edagricole. Rapp, O., & Pashinskii, V. (1999). Comparative study of pharmacological activity of birch bark ethanol extracts. Pharmaceutical Chemistry Journal, 33(1), 1 3. Saric, C.L., Cabarkapa, S.I., Beljkasˇ, M.B., Misˇan, C.A., Sakac, B.M., & Plavsˇic, V.D. (2009). Antimicrobial activity of plant extracts from Serbia. Food Processing, Quality and Safety, 1 2, 1 5. Taguri, T., Tanaka, T., & Kouno, I. (2004). Antimicrobial activity of 10 different plant polyphenols against bacteria causing food-borne disease. Biological and Pharmaceutical Bulletin, 27, Vaya, J., Belinky, P.A., & Aviram, M. (1997). Antioxidant constituent from licorice roots. Free Radical Biology and Medicine, 23,