Phenolic composition and in vitro activity of the Brazilian fruit tree Caryocar coriaceum Wittm.

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European Journal of Integrative Medicine 5 (2013) 178–183

Original article

Phenolic composition and in vitro activity of the Brazilian fruit tree Caryocar coriaceum Wittm. Mariana K.A. Araruna a , Karla K.A. Santos b , José G.M. da Costa c , Henrique D.M. Coutinho b,∗ , Aline A. Boligon d , Sílvio T. Stefanello d , Margareth L. Athayde d , Rogerio A. Saraiva d , João Batista T. da Rocha d , Marta R. Kerntopf a , Irwin R.A. de Menezes a a

Laboratory of Pharmacology and Molecular Chemistry (LFQM), Chemical-Biology Department, Regional University of Cariri – URCA, Cel. Antonio Luis 1161, Crato, CE, Brazil b Laboratory of Microbiology and Molecular Biology (LMBM), Chemical-Biology Department, Regional University of Cariri – URCA, Cel. Antonio Luis 1161, Crato, CE, Brazil c Laboratory of Natural Products Research (LPPN), Chemical-Biology Department, Regional University of Cariri – URCA, Cel. Antonio Luis 1161, Crato, CE, Brazil d Universidade Federal de Santa Maria – UFSM, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil Received 18 May 2012; received in revised form 23 November 2012; accepted 24 November 2012

Abstract Introduction: Caryocar coriaceum Wittm. (Caryocaraceae) is a plant found in the region of Araripe (Ceará State, Brazil) and is widely used in folk medicine. The present work aimed to evaluate the chemical composition and synergistic–antibiotic activity of a hydroethanol leaf extract (LECC) and a methanol fraction (LECC-MF) of C. coriaceum. Methodology: Qualitative phytochemical analysis of both extracts was performed and the quantification of phenolic and flavonoid compounds was performed by HPLC-DAD. The antimicrobial activity of a hydroethanol leaf extract of C. coriaceum (LECC) and a methanol fraction (LECC-MF) were analyzed by the microdilution test using standard and clinical multiresistant bacterial and fungal strains (Staphylococus aureus ATCC25923, Escherichia coli ATCC10536, Pseudomonas aeruginosa ATCC15442, Klebsiella pneumonae ATCC4362, E. coli 27, S. aureus 358, Candida albicans ICB12 and Candida krusei ATCC6258). The synergism assay was performed with aminoglycosides (as amikacin, Kanamycin, gentamicin and neomycin) and antifungal drugs (as metronidazole, amphotericin B, nystatin, and benzoylmetronidazole). Results: The phytochemical analysis revealed the presence of pyrogallic and hydrolisable tannins, phenolic compounds, flavonoids and other phytocompounds. HPLC analysis showed a variety of compounds in both of the extracts, including gallic acid, chlorogenic acid, caffeic acid, rutine and quercetine. Both the hydroethanol extract and methanol fraction showed no clinically relevant antimicrobial effect against the tested microorganisms. However, a synergistic activity was demonstrated by both natural products against E. coli 27 and S. aureus 358. Conclusion: These findings suggest that LECC and LECC-MF could be used as an important source of compounds with the potential to reduce the bacterial resistance to aminoglycosides. © 2013 Elsevier GmbH. All rights reserved. Keywords: Synergism; Antimicrobial activity; Aminoglycoside modulatory activity; Caryocar coriaceum

Introduction The faith of many communities in the healing power of the plants, has been based in cultural and more recently in empirical

∗ Corresponding author at: Departamento de Química Biológica, Universidade Regional do Cariri – URCA, Crato-CE, Rua Cel. Antonio Luis 1161, Pimenta 63105-000, Brazil. Tel.: +55 88 31021212; fax: +55 88 31021291. E-mail address: [email protected] (H.D.M. Coutinho).

1876-3820/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.eujim.2012.11.007

knowledge of several researchers in the search of bioactive drugs since remote times [1–3]. Despite folk observations about the healing properties of natural products [4], the chemical composition of many of these plants have not been studied and there is a lack of evidence based on their potential therapeutic properties [5]. The uncontrolled use of antimicrobial drugs selected microbial populations with resistance to these drugs has increased the resistance of these microorganisms [6]. Such problems limit the use of these drugs and their treatment of many infectious

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diseases. The study of new promising substances to combat multidrug resistance is therefore critical. The development of new drugs and the evaluation of natural products has been the object of several studies [7,8]. Natural products can be interesting alternatives to combat the microbial resistance due their complex chemical composition [9,10]. Several microorganisms including fungi and bacteria are the causal agents of pathologies. The worldwide spread of antibiotic-resistant bacteria, such as the methicillin-resistant Staphylococcus aureus (MRSA) has stimulated the search by antimicrobial compounds from natural sources [9]. Candida infections are reported as the most common diseases of female urinary genital tract. Candidiasis is the most common mycosis infection, being caused mainly by Candida albicans, although Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea and Candida tropicallis have been also reported as etiological agents [11]. Combinations of drugs may result in an antagonistic or synergistic effect of the drug or a decrease in side effects [12]. Costa and coworkers [13] showed an antibacterial modulatory activity of Lantana camara, observing the synergistic effect between essential oil and aminoglycosides. Aminoglycosides are inhibitors of bacterial protein synthesis, and bacterial resistance against aminoglycosides is a serious public health problem [14,15]. Caryocar coriaceum Wittm. (Caryocaraceae) is known in Brazil by “pequizeiro”, is found in the south of Ceara state [16,17]. Its leaves are used in the traditional medicine used against viral infections and other infectious diseases [2]. The extract of leaves of Caryocar brasilienses have demonstrated an antimicrobial activity, inhibiting the growth of pathogenic bacteria [18]. This research investigated the chemical composition and identified the main phenolic compounds of the hydroethanol extract of leaves of C. coriaceum Wittm. (LECC) and the methanol fraction (LECC-FM). The antimicrobial and modulatory activities of these natural products in association with antibiotics and antifungals were also evaluated in this work. Materials and methods Plant material C. coriaceum Wittm. leaves were collected in Araripe plateau, Crato, Ceará State, Northeastern Region of Brazil (S7◦ 21 53.1 ; W39◦ 28 42.6 ) in April/2009. The identification of the voucher specimen was performed by Prof. Dr. Lígia Queiroz Matias and a voucher specimen was deposited in the Herbarium Prisco Bezerra of the Universidade Federal do Ceará (#No. 44523). Chemicals and solutions Only chemicals of analytical grade were used. Dimethylsulfoxide (DMSO), methanol, ethanol, acetic acid, gallic acid and chlorogenic acid were purchased from Merck (Darmstadt, Germany). Resazurin, quercetin, rutin, gallic and chlorogenic acids were acquired from Sigma Chemical Co. (St. Louis, MO, USA). The following aminoglycosides: amikacin, kanamycin,

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gentamicin and neomycin and the antifungals metronidazole, amphotericin B, nystatin, benzoylmetronidazole were purchased from GSM pharmaceutical (Brazil). All natural products were solubilized in DMSO at following ratios: 200 mg of the sample was solubilized using 1 mL of DMSO (f.c. 200 mg/mL). This sample was diluted to obtain a solution of 10 mg/mL that was diluted again to a final test concentration of 1024 ␮g/mL. This stock solution was be used in the microdilution procedure, with serial dilutions leading to final concentrations varying from 512 to 8 ␮g/mL.

Preparation of the extracts 500 g of leaves was washed, dried and extracted by maceration using a mixture of water:ethanol (1:1) for a period of 72 h at room temperature. The hydroethanol leaf extract of C. coriaceum was concentrated under low pressure at 50 ◦ C and dried by liophylization (LECC). The extract was partitioned with methanol, filtered and the solvent was evaporated in a vacuum rotator (LECC-MF). The extract and fraction were stored in ambar glass at 10 ◦ C to further analysis.

Phytochemical characterization LECC and LECC-MF were subjected to several reactions to detect qualitatively the presence of secondary metabolites such as phenolic compounds, flavonoids, tannins and alkaloids, using the methods described by Matos [19] and Simões et al. [20].

Quantification of phenolic and flavonoid compounds by HPLC-DAD Reverse phase chromatographic analyses were carried out under gradient conditions using C18 column (4.6 mm × 250 mm) packed with 5 ␮m diameter particles; the mobile phase was water containing 2% acetic acid (A) and methanol (B), and the composition gradient was: 5% (B) for 2 min; 25% (B) until 10 min; 40, 50, 60, 70 and 80% (B) every 10 min. All the samples and mobile phase were filtered through 0.45 ␮m membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.05–0.25 mg/mL for quercetin and rutin, and 0.02–0.2 mg/mL for gallic, chlorogenic and caffeic acids. Quantification was carried out by integration of the peaks using the external standard method, at 257 nm for gallic acid, 325 nm for chlorogenic and caffeic acids, and 365 for quercetin and rutin. The flow rate was 0.8 mL/min and the injection volume was 40 ␮L. The chromatography peaks were confirmed by comparing their retention time and Diode-Array-UV spectra with those of the reference standards. All chromatography operations were carried out at ambient temperature and in triplicate.

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Strains The following standard bacterial strains S. aureus ATCC25923, Escherichia coli ATCC10536, Pseudomonas aeruginosa ATCC15442 and Klebsiella pneumonae ATCC4362; the multi-resistant clinical strains E. coli 27 (EC27) and S. aureus 358 (SA358) (resistance profile is showed in Table 4); and standard fungal strains C. albicans ICB12 and C. krusei ATCC6258, obtained from the collection of microorganisms of the Laboratory of Mycology, Universidade Federal da Paraíba, Brazil were used in this study. All strains were maintained on heart infusion agar slants (HIA, Difco Laboratories Ltd.) and prior to assay, the cells were grown overnight at 37 ◦ C in brain heart infusion (BHI, Difco Laboratories Ltd.). Antimicrobial activity of C. coriaceum extracts The minimum inhibitory concentrations (MIC) of the leaf extract of C. coriaceum (LECC) and methanol fraction (LECCMF), aminoglycosides and antifungals were determined in BHI by a microdilution assay using suspensions of 105 CFU/mL and a drug concentration ranging from 512 to 8 ␮g/mL (twofold serial dilutions) [21]. Each sample was inoculated with 100 ␮L of microorganism suspension; and the incubation was performed at 37 ◦ C. After incubation for 24 h, 25 ␮L of resazurin (0.01%) was added to each well with bacterial growth and incubated 1 h at 37 ◦ C. The MIC for both bacteria and fungi was defined as the lowest drug concentration that inhibited the bacterial growth. The verification of fungal growth was using the visual detection of turbidity enhancement. In order to confirm the inhibition of bacterium growth, a colour-change of resazurin from blue to pink in each sample were also evaluated. All the experiments were performed in triplicate. Modulation of antibiotic or antifungal activity by direct contact For the evaluation of the modulatory activity of antibiotics or antifungals, the MIC of the antibiotics and antifungals were determined in the absence and presence of the extract and methanolic fraction in a sub-inhibitory concentration (MIC/8). The bacterial clinical isolates EC27 and SA358, and fungal strains C. albicans ICB12 and C. krusei ATCC6258 were assayed with four different aminoglycosides and antifungals at concentrations of 1024–1 ␮g/mL. All plates were incubated aerobically for 24 h at 37 ◦ C. The growth observation used the same methodology cited on the MIC assay. All experiments were performed in triplicate. Results Phytochemical characterization Phytochemical screening is showed in Table 1. This analysis revealed several secondary metabolites including pyrogallic and hydrolisable tannins, phenolic compounds, flavonoids (flavones,

Table 1 Phytochemical characterization of Leaf extract of Caryocar coriaceum (LECC) and methanol fraction (LECC-MF). Secondary metabolite

LECC

LECC-MF

Phenolic compounds Flavonoids Flavones Flavonols Xantones Chalcones and aurones Flavononols Antocianines Antocianidines Leucoantocianidines Catequines Flavonones Pyrrogallic tannins Hydrolysable tannins Alkaloids

+

+

+ + + − − − − − − − + − −

+ + + − + − − − − − − + −

Note: (+): presence; (−): absence.

flavonols and xanthones) and phenolic compounds. In addition, alkaloids were not detected in either natural products. HPLC analysis The HPLC profile of LECC and LECC-MF is shown in Fig. 1. The samples of C. brasiliensis demonstrated gallic acid (retention time-tR 12.63 min, peak 1), chlorogenic acid (tR = 21.97 min, peak 2), caffeic acid (tR = 28.27 min, peak 3), rutin (tR = 37.83 min, peak 4) and quercetin (tR = 46.42 min, peak 5). The quantification of quercetin, rutin, gallic acid, caffeic acid and chlorogenic acid by HPLC-DAD was based on reference standard calibration curves (Table 2). Calibration curve for gallic acid: Y = 25681x − 1536.2 (r = 0.9971); chlorogenic acid: Y = 27235x − 1604.6 (r = 0.9799); caffeic acid: Y = 23674x − 1288.4 (r = 0.9993); rutin: Y = 29767x − 1258.7 (r = 0.9989) and quercetin: Y = 28077x − 1741.5 (r = 0.9965). Antimicrobial activity Both LECC and LECC-MF demonstrated a MIC ≥ 1024 ␮g/mL against all bacterial and fungal strains assayed, demonstrating no clinically relevant antimicrobial activity. However, the extract and fraction showed an antibiotic modulatory effect when combined with the used aminoglycosides (Table 3). The association of amikacin or gentamicin with Table 2 Phenolic and flavonoid composition of LECC and LECC-MF extracts by HPLC analysis. Results are expressed as media (M) and standard deviation (SD) = (M ± SD) of three determinations. Compounds

LECC (mg/g)

Gallic acid Chlorogenic acid Caffeic acid Rutin Quercetin

19.0 31.4 18.7 68.1 26.9

± ± ± ± ±

0.15 0.26 1.02 0.04 0.17

LECC-MF (mg/g) 11.2 56.8 7.5 32.5 18.5

± ± ± ± ±

0.34 0.16 0.09 0.05 0.03

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Fig. 1. High performance liquid chromatography phenolics and flavonoids profile of LECC (a) and LECC-MF (b). Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), rutin (peak 4) and quercetin (peak 5). Table 3 Modulatory activity of leaf extract (LECC) and methanol fractions (LECC-MF) of Caryocar coriaceum associated with aminoglycoside. S. aureus 358 AMI

E. coli 27 KAN

Modulatory activity of natural products (MIC/8 = 128 ␮g/mL) 625 312.5 Positive control LECC 312.5 1250 50% −300a % Increase 78.125 1250 LECC-MF % Increase 87.5% −300a

GEN

NEO

AMI

KAN

GEN

NEO

19.53 4.88 75% 19.53 0

312.5 312.5 0 312.5 0

2500 2500 0 312.5 87.5%

2500 2500 0 2500 0

19.53 19.53 0 312.25 −1498.8a

625 156.25 75% 156.25 75%

Positive control: only antibiotic effect; %: increase of percentual inhibition. a Negative value = antagonism.

leaf extract of C. coriaceum (LECC) showed a synergistic effect against the MRSA strain S. aureus 358, with 50 and 75% of antibiotic activity enhancement. Similar results were observed when LECC was combined with neomycin against E. coli 27 (75%). Synergism was observed when the methanol fraction (LECC-MF) was combined with amikacin against S. aureus 358 and E. coli 27 (87.5% for both strains) and with neomycin against E. coli 27 (75%) (Table 4). Antagonistic effects were observed against SA358 and EC27 when LECC and LECC-MF were combined with kanamycin and gentamicin respectively. No modulatory effect was observed when antifungals and natural products were combined. Discussion Extracts from plants are complex mixtures due to the presence of several secondary metabolites [22]. The use of natural products as antimicrobial agents presents a low risk of increasing the drug resistance, difficulties in changing the adaptative

microbial response [23]. So, natural products can be important drugs due their direct action against the microorganism. However, these same drugs can influence the antibiotic effect, increasing or reversing the antibiotic activity and the antibiotic resistance [3,9]. In this study, the antimicrobial effect for the extract and fraction showed a MIC ≥ 1024 ␮g/mL against all bacterial and fungal strains tested. Aligiannis et al. [24] proposed a classification for plant materials based on MIC: strong inhibitors – MIC ≥ 0.5 ␮g/mL; moderate inhibitors – MIC between 0.6 and 1.5 ␮g/mL; weak inhibitors – MIC ≤ 1.6 ␮g/mL. So, the results demonstrated that LECC and LECC-MF showed moderate antibacterial effect. The phytochemical characterization (Table 1) of LECC and LECC-MF is in accordance with data from literature regarding the chemical compounds in the family Caryocaraceae. The presence of tannin and flavonoids is also observed in leaf extracts from C. brasilienses [25]. In the same form, tannins and flavonoids are important biologically active secondary

Table 4 Resistance profile to antibiotics of clinical isolated bacterial strains used. Clinical isolated bacterial strain

Origin

Resistance profile

E. coli EC27 S. aureus SA358

Surgical wound Surgical wound

Azt, Amp, Ami, Amox, Ca, Cfc, Cf, Caz, Cip, Clo, Im, Kan, Szt, Tet, Tob Oxa, Gen, Tob, Ami, Can, Neo, Paro, But, Sis, Net

Azt, aztreonam; Amp, ampicillin; Ami, amikacin; Amox, amoxicillin; Ca, cefadroxil; Cfc, cefaclor; Cf, cephalothin; Caz, ceftazidime; Cip, ciprofloxacin; Clo, chloramphenicol; Im, imipenem; Kan, kanamycin; Szt, sulphametrim; Tet, tetracycline; Tob, tobramycin; Oxa, oxacillin; Gen, gentamicin; Neo, neomycin; Paro, paromomycin; But, butirosin; Sis, sisomicin; Net, netilmicin.

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metabolites. The tannins present a redox potential and can form complexes with proteins or metals as iron (Fe+3). Due to these actions, some tannins could inhibit directly microbial processes [26,27]. Tannins can affect bacterial growth by several mechanisms: inhibiting extracellular enzymes; chelating substrates required for the microbial growth or affecting the oxidative metabolism [26,28–32]. Due to these mechanisms, Hatano et al. [33] showed that hydrolysable tannins as theasinensin A, (−)epigallocatechin gallate and proanthocyanidins from fruits of Zizyphus jujuba var. suppressed the antibiotic resistance of MRSA strains. Khare and coworkers [34] showed that rutin exhibited strong activity against Bacillus cereus, P. aeruginosa and K. pneumoniae with MIC values of 0.03 ␮g/mL. The work of Fattouch et al. [35] on the antimicrobial activity of Cydonia oblonga and phenolic compounds as chlorogenic acid, rutin and quercetin demonstrated a high antimicrobial activity against S. aureus and P. aeruginosa, these strains being susceptible to polyphenols than E. coli and C. albicans. Pure phenolic compounds showed a low antibacterial activity, indicating that polyphenols, especially the chlorogenic acid, may act synergistically with other compounds from the extracts to exhibit the total antimicrobial activity, as demonstrated by the modulatory results of the methanol fraction in our work. This result is also accordance with the low antifungal activity demonstrated against C. albicans and C. krusei. The flavonoids constitute an important class of polyphenols and a large group of secondary metabolites with biological properties used for the maintenance of human health [36]. Flavonoids have attracted a great interest due to in vitro and in vivo studies demonstrating their potential as antioxidants, free radical scavengers [37] and metal chelators, including anti-inflammatory [8,38], antiallergic, anticarcinogenic, antihypertensive, antiarthritic and anti-microbial effects [31]. de Nadra and coworkers [39] showed the antimicrobial effect of seven pure phenolic compounds: four phenolic acids: gallic, vanillic, protocatechuic and caffeic; and three flavonoids: rutin, catechin and quercetin against Serratia marcescens, Proteus mirabilis, E. coli, K. pneumoniae and Flavobacterium sp. This study demonstrated that quercetin was the only phenolic compound with an antibacterial effect against all bacteria assayed. In the LECC and LECC-MF, rutin and quercetin were the compounds present in the chemical composition of the plant. However, the presence of chlorogenic acid in high concentration in the fraction could explain the effect of these products. Conclusions This work has contributed to the knowledge about the antimicrobial properties of extracts and fractions of C. coriaceum Wittm. against the bacteria that affect human health. Our studies demonstrated a modulatory activity of aminoglycosides, probably due to the interactions between the polyphenols with these natural products. These results may provide a new tool to combat against the growth of the antibiotic resistance, but indicate the necessity of more research on with the main compounds to determine the mechanism of action.

Conflict of interest The authors do not have any conflicts of interest to disclose. Acknowledgments This work was supported by CAPES, CNPq, FUNCAP (Process BPI-0031-00107.01.00/10) and BNB/ETENE, Brazilian government agency, for the financial support. References [1] Maciel MAM, Pinto AC, Veiga Junior VF. Plantas medicinais: a necessidade de estudos multidisciplinares. Quimica Nova 2002;25:429–38. [2] Agra MF, Freitas PF, Barbosa-Filho JM. Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. The Revista Brasileira de Farmacognosia 2007;17:114–40. [3] Oliveira DR, Brito-Junior FE, Bento EN, Matias EF, Sousa AC, Costa JG, et al. Antibacterial and modulatory effect of Stryphnodendron rotundifolium. Pharmaceutical Biology 2011;49:1265–70. [4] Alviano DS, Alviano CS. Plant extracts: search for new alternatives to treat microbial diseases. Current Pharmaceutical Biotechnology 2009;10:106–21. [5] Brandão MGL, Zanetti NNS, Oliveira P, Grael CFF, Santos ACP, MonteMór RLM. Brazilian medicinal plants described by 19th century European naturalists and in the Official Pharmacopoeia. Journal of Ethnopharmacology 2008;120:141–8. [6] Varaldo PE. Antimicrobial resistance and susceptibility testing: an evergreen topic. Journal of Antimicrobial Chemotherapy 2002;50:1–4. [7] Keith CT, Borisy AA, Stockwell BR. Multicomponent therapeutics for networked systems. Nature Reviews Drug Discovery 2005;4:71–8. [8] Cazarolli LH, Zanatta L, Alberton EH, Figueiredo MS, Folador P, Damazio RG, et al. Flavonoids: prospective drug candidates. Mini Reviews in Medicinal Chemistry 2008;8:1429–40. [9] Coutinho HDM, Costa JG, Lima EO, Falcão-Silva VS, Siqueira-Júnior JP. Enhancement of the antibiotic activity against a multiresistant Escherichia coli by Mentha arvensis L. and chlorpromazine. Chemotherapy 2008;54:328–30. [10] Ameenah G-F. Medicinal plants: traditions of yesterday and drugs of tomorrow. Molecular Aspects of Medicine 2006;27:1–93. [11] Sobel JD, Fisher JF, Kauffman CA, Newman CA. Candida urinary tract infections––epidemiology. Clinical Infectious Diseases 2011;52(Suppl. 6):S433–6. [12] Izzo AA, Ernst EE. Interactions between herbal medicines and prescribed drugs: an updated systematic review. Drugs 2009;69:1777–98. [13] Sousa EO, Silva NF, Rodrigues FF, Campos AR, Lima SG, Costa JG. Chemical composition and resistance-modifying effect of the essential oil of Lantana camara linn. Pharmacognosy Magazine 2010;6:79–82. [14] Jana S, Deb J. Molecular understanding of aminoglycoside action and resistance. Applied Microbiology and Biotechnology 2006;70:140–50. [15] Newman DJ, Cragg GM, Snader KM. Natural products as sources of new drugs over the period 1981–2002. Journal of Natural Products 2003;66:1022–37. [16] Costa IR, Araújo FS, Lima-Verde LW. Flora e aspectos auto-ecológicos de um encrave de cerrado na chapada do Araripe, Nordeste do Brasil. Acta Botanica Brasilica 2004;18:759–70. [17] Oliveira MEB, Guerra NB, Maia AHN, Alves RE, Matos NMS, Sampaio FGM, et al. Chemical and physical–chemical characteristics in pequi from the Chapada do Araripe, Ceará, Brazil. Revista Brasileira de Fruticultura 2010;32:114–25. [18] Paula-Ju WD, Rocha FH, Donatti L, Fadel-Picheth CMT, Weffort-Santos AM. Leishmanicidal, antibacterial, and antioxidant activities of Caryocar brasiliense Cambess leaves hydroethanolic extract. Revista Brasileira de Farmacognosia 2006;16:625–30.

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