Journal of Ethnopharmacology 156 (2014) 88–96

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Journal of Ethnopharmacology

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Research Paper Quantification of polyphenols and evaluation of antimicrobial, analgesic and anti-inflammatory activities of aqueous and acetone–water extracts of Libidibia ferrea, rigida and Psidium guajava

Aurigena Antunes de Araújo a,n, Luiz Alberto Lira Soares b, Magda Rhayanny Assunção Ferreira b, Manoel André de Souza Neto c, Giselle Ribeiro da Silva c, Raimundo Fernandes de Araújo Jr.d, Gerlane Coelho Bernardo Guerra e, Maria Celeste Nunes de Melo f a Postgraduate program in Public Health, Postgraduate program in Pharmaceutical Science, Department of Biophysics and Pharmacology, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Av. Senador Salgado Filho, Campus Universitário, Lagoa Nova, 59072-970, Natal, RN, Brazil b Postgraduate program in Pharmaceutical Science/Department of Pharmacy, Universidade Federal de Pernambuco, Av. Artur de Sá, Cidade Universitária, 50740-520, Recife, PE, Brazil c Postgraduate program in Pharmaceutical Science, Universidade Federal do Rio Grande do Norte, Rua Gen. Gustavo Cordeiro de Farias, 59010-180, Natal, RN, Brazil d Postgraduate program in Functional and Structural Biology, Postgraduate program in Health Science, Department of Morphology, Universidade Federal do Rio Grande do Norte, Av. Senador Salgado Filho, Campus Universitário, 59072-970, Natal, RN, Brazil e Department of Biophysics and Pharmacology, Universidade Federal do Rio Grande do Norte, Av. Senador Salgado Filho, Campus Universitário, 59072-970, Natal, RN, Brazil f Postgraduate program in Parasitary Biology, Postgraduate program in Biology Science, Department of Microbiology and Parasitology, Universidade Federal do Rio Grande do Norte, Av. Senador Salgado Filho, Campus Universitário, 59072-970, Natal, RN, Brazil article info abstract

Article history: Ethnopharmacological relevance: Vast numbers of species from northeastern Brazil have not yet Received 22 January 2014 been phytochemically or biologically evaluated. Received in revised form Aim of the study: The goal of this work was to obtain, characterize and show the antimicrobial, analgesic 5 June 2014 and anti-inflammatory activities of aqueous and acetone–water extracts of Libidibia ferrea, Parapiptadenia Accepted 9 July 2014 rigida and Psidium guajava. Available online 12 August 2014 Materials and methods: The plant material (100 g) was dried, and the crude extracts were obtained by Keywords: using turbo-extraction (10%; w/v) with water or acetone:water (7:3, v/v) as the extraction solvent. High- Libidibia ferrea performance liquid chromatography (HPLC) methods were used to screen the crude extracts for Parapiptadenia rigida hydrolysable (gallic acid) and condensed tannins (catechins). The antibacterial activity was Psidium guajava evaluated by agar-diffusion and microdilution methods against Gram-positive strains (Staphylococcus Polyphenols aureus ATCC 25923, Staphylococcus epidermidis INCQS 00016, Enterococcus faecalis ATCC 29212 and a Antimicrobial Antiinflammatory clinical isolate of methicillin-resistant Staphylococcus aureus) as well as Gram-negative strains (Escher- ichia coli ATCC 25922, Salmonella enteritidis INCQS 00258, Shigella flexneri and Klebsiella pneumoniae). To evaluate the anti-inflammatory activity, a leukocyte migration model was used. Analgesic activity was determined by the hot plate test and the acetic acid-induced abdominal writhing test. Data were analyzed by analysis of variance (ANOVA) at a significance level of 5%. Results: Parapiptadenia rigida presented the highest amount of total polyphenols (35.8270.20%), while the greatest catechin content was found in the acetone–water extract of Psidium guajava (EAWPg;

Abbreviations: HPLC, High-performance liquid chromatography; g, grams; w/v, weight/volume; INCQS, Instituto Nacional de Controle de Qualidade em Saúde (National Institute of Quality Control in Health); ATCC, American Type Culture Collection; MRSA, methicillin-resistant Staphylococcus aureus; ANOVA, analysis of variance; EAWPg, acetone–water extract of Psidium guajava; EAWLf, acetone–water extract of Libidibia ferrea; EAWPr, acetone–water extract of Parapiptadenia rigida; EAPg, aqueous extract of Psidium guajava; EALf, aqueous extract of Libidibia ferrea; EAPr, aqueous extract of Parapiptadenia rigida; MIC, minimum inhibitory concentration; UFRN, Federal University of Rio Grande do Norte; IPA, Agronomy Institute of Pernambuco; UFPE, Federal University of Pernambuco; TLC, Thin layer chromatography; TPC, total polyphenolic content; DMSO, dimethyl sulfoxide; M-H, Muller-Hinton n Corresponding author. Tel.: þ 55 84 3215 3419. E-mail address: [email protected] (A.A. de Araújo). http://dx.doi.org/10.1016/j.jep.2014.07.031 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved. A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96 89

1.04 μg/g). The largest amounts of catechins were found in the aqueous extract of Libidibia ferrea (EALf; 1.07 μg/g) and the acetone–water extract of Parapiptadenia rigida (EAWPr; 1.0 μg/g). All extracts showed activity against Gram-positive bacteria. The aqueous and acetone–water extracts of Psidium guajava showed the greatest inhibition zones in the agar diffusion tests. In the evaluation of the minimum inhibitory concentration (MIC), the most susceptible Gram-positive bacterium was Staphylococcus epidermidis and the most susceptible Gram-negative bacterium was Shigella flexneri. EAPg and EAWPg showed the greatest MIC values. All extracts were significant inhibitors of leukocyte migration (po0.05). Using the writhing test, significant analgesic activity was found for EAPr (50 mg/kg), EAWPr (100 mg/kg and 200 mg/kg) and EAWPg (50 mg/kg) (po0.05). Conclusions: Thus, the appropriate extraction procedure preserves the chemical components such as gallic acid and catechin, and showed antimicrobial, anti-inflammatory and analgesic properties. & 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction extracts of Libidibia ferrea, Parapiptadenia rigida and Psidium guajava. Brazil is one of the countries with the greatest diversity of with different biomes, and these plants may have thera- peutic potential. The semi-arid Caatinga biome that exists in 2. Material and methods northeastern Brazil has been little studied (de Albuquerque et al., 2007). Therefore, it is vital to study the utilitarian plant species 2.1. Plants from semi-arid regions of northeastern Brazil because new eco- nomic alternatives can ensure the livelihood and maintenance of Samples of Libidibia ferrea bark, Parapiptadenia rigida bark and these species. Among the species popularly used in the northern Psidium guajava leaves were collected in the states of Pernambuco, and semi-arid northeastern regions of Brazil are Libidibia ferrea, Paraíba and Rio Grande do Norte, respectively. Voucher specimens Parapiptadenia rigida and members of the genus Psidium (Sampaio were deposited at the Agronomy Institute of Pernambuco (IPA), et al., 2009; Costa et al., 2012; De Souza et al., 2012). the Department of Botany at the Federal University of Pernambuco Libidibia ferrea (Mart.) L. P. Queiroz belongs to the Caesalpina- (UFPE) and the Department of Botany at the Federal University of ceae family and, as a medicinal plant, its use has been reported in Rio Grande do Norte (UFRN), under numbers 88145, 83115 and the treatment of virus, inflammation and diarrhea (Pereira et al., 8214, respectively. The plant names have been verified with www. 2012; de Medeiros et al., 2013; Lopes et al., 2013). Its popular name theplantlist.org. is ironwood, and it is composed of the following phenolic compounds: condensed tannins, catechins and hydrolysable tan- nins such as gallic acid and ellagic acid (Frasson et al., 2003; 2.1.1. Extract collection Vasconcelos et al., 2011). The plant material (100 g) was dried under forced-air circula- Parapiptadenia rigida (Benth.) Brenan belongs to the tion at 4572 1C until its weight stabilised. The crude extract was family and is popularly known as red angico. As a medicinal plant, obtained by turbo-extraction (10%; w/v) using water or acetone: fl the bark is used in the treatment of in ammation, gastrointestinal water (7:3, v/v) as the extraction solvent for a period of 20 min. disorders, tumors and chronic diseases (Souza et al., 2004). The Next, the crude extracts were filtered, concentrated under reduced bark has phenolic compounds such as tannins, catechin derivatives pressure using a rotavapor and then lyophilized. and proanthocyanidins (Schmidt et al., 2011). Psidium guajava L. belongs to the Myrtaceae family and is popularly known as guava. It is present in all tropical and 2.1.2. Phytochemical analysis: thin layer chromatography (TLC) subtropical regions of the world, including Latin America, Europe, analysis Asia and Africa (Gutierrez et al., 2008). In northeastern Brazil, its The methods described by Wagner and Bladt (1996) were used leaves are used to treat dysentery, digestive problems, inflamma- to screen the crude extracts for hydrolysable tannins (gallic acid) tion, gingivitis and throat afflictions (de Albuquerque et al., 2007). and condensed tannins (catechins). The phytochemical profile was fl s Its bark and leaves have tannins, avonoids, saponins and alka- determined by using TLC on 60-F254 silica gel plates (Merck ) with loids (Tona et al., 2000). The biological characteristics of Psidium the appropriate mobile phase, reagents and standards. guajava include antidiarrheal, amoebicidal, spasmolytic, antibac- terial and antifungal activities (Birdi et al., 2010; Rahim et al., 2010). 2.1.3. Estimation of total polyphenolic content (TPC) In this context, most of the plant compounds that have been The methods described by the European Pharmacopoeia found to be medicinally useful and interesting tend to be second- (Pharmacopoeia, 2005) and the Brazilian Pharmacopeia (2010) ary metabolites. The choice of extraction solvent strongly affects for the determination of TPC were used as guidelines for the the yield and the number of metabolites obtained. Different dilution of the crude extracts and standard (pyrogallol). Solutions solvent systems have been used to extract secondary metabolites of the standard (500 μg/mL) and crude extracts (1000 μg/mL) from plant materials because their extraction efficacy depends on were prepared in distilled water. Each solution (at the appropriate their chemical nature. For example, ethyl alcohol extracts a large dilution) was transferred to a 25-mL flask containing distilled number of flavonoids; while aqueous mixtures of ethanol or water (10 mL) and the Folin–Ciocalteu reagent (1 mL); the volume acetone have been used to extract polyphenols from plant material was completed with 10.75% anhydrous sodium carbonate (w/v). (Garcia-Salas et al., 2010). The samples were scanned (400–900 nm) in an ultraviolet/visible Thus, the aim of this work was to obtain and quantitate the spectrophotometer (ThermoScientifics) at 30 min after the addi- polyphenol content and verify the antimicrobial, analgesic tion of the last reagent. Distilled water was used as the blank. Tests and anti-inflammatory activities of aqueous and acetone–water were carried out in triplicate. 90 A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96

2.1.4. High-performance liquid chromatography (HPLC) analysis 200 μL of extract mixed with M-H broth was added to the first The main phytochemical markers (gallic acid and catechin) wells of each row. Then, a serial two-fold dilution was performed were quantified by HPLC analysis using a ThermoScientific system until the eighth well of each row. Lastly, 100 μL of bacterial (Ultimate 3000) equipped with a photodiode array detector 3000 inoculum diluted in M-H broth was added to the wells, resulting (RS), Binary Pump HPG-3 00RS and ACC-3000 autosampler. The in concentrations ranging from 10 to 0.078 mg/mL (Libidibia ferrea chromatographic separation was performed using a C18 column and Parapiptadenia rigida) and from 5 to 0.039 mg/mL (Psidium with 250 mm 4 mm i.d., 5 mm (Dionexs), protected by a pre- guajava). The plates were incubated aerobically at 35 1C for 24 h. column packet of the same material (Phenomenexs). Chromeleon The ninth through 12th wells were reserved as viability controls 6.0 (Dionexs) software was used for data acquisition and proces- (with and without DMSO) and sterility controls (with and without sing. A gradient elution was performed by varying the proportion plant extracts). As reference antibiotics, cephalothin, gentamicin of solvent B (methanol with 0.05%, v/v, trifluoracetic acid) to and vancomycin (Sigma, St. Louis, MO, USA) were employed. The solvent A (water with 0.05%, v/v, trifluoracetic acid) at a flow rate MIC was considered as the lowest concentration of the extract that of 0.8 mL/min, according to the following gradient program: 10– prevented visible growth or changed the color of phenol red from 25% B (10 min), 25–40% B (10 min), 40–75% B (5 min) and 75–10% red to yellow due to the formation of acidic metabolites corre- B (5 min). The crude extracts of Libidibia ferrea, Parapiptadenia sponding to microbial growth. Tests were carried out in duplicate. rigida and Psidium guajava as well as the standards were dissolved in water and filtered through a 0.45-mm membrane (Macherey- s 2.2.4. Evaluation of synergism between plant extracts and Nagel , Germany) prior to sample injection (20 mL). The peaks of antimicrobials each marker substance in the crude extract were initially identi- The synergism test was executed according to the agar-well fied by comparing the retention times and UV spectra. After that, diffusion assay protocol (Valgas et al., 2007), except that the the peaks were confirmed by spiking the sample with a small acetone–water extracts were diluted in M-H agar (HiMedias, amount of the standards. Mumbai, India) at a concentration equal to half the MIC (Probst, 2012). The synergistic effect was evaluated for the acetone–water 2.2. In vitro studies: microbiological analyses extracts and the antimicrobials oxacillin (1 mg; DMEs) and peni- cillin (10 U; DMEs) against the MRSA clinical isolate. The anti- 2.2.1. Bacterial strains microbial discs were added in duplicate on the inoculated agar Antibacterial activity was evaluated against the following surface and, after incubation for 24 h, the inhibition zones were microorganisms: Staphylococcus aureus ATCC 25923, Staphylococ- recorded. A plate that only contained the reference antimicrobials cus epidermidis INCQS 00016, Enterococcus faecalis ATCC 29212, was reserved as a control. Tests were carried out in duplicate. Escherichia coli ATCC 25922, Salmonella enteritidis INCQS 00258, and against the clinical isolates of Shigella flexneri, Klebisiella pneumoniae and a methicillin-resistant Staphylococcus aureus 2.2.5. Evaluation of synergism of plant extract combinations (MRSA) strain, which is a representative of the Brazilian epidemic The synergism test was executed according the agar-well diffu- clone (Teixeira et al., 1995). All strains were used for the diffusion sion technique (Valgas et al., 2007), except that the aqueous and – and microdilution tests, and only the MRSA strain was used for the acetone water extracts were diluted in M-H agar at a concentration synergism assay. Bacterial suspensions were standardized to a equal to half the MIC (Probst, 2012).Thesynergismofthecombina- þ – þ – turbidity equivalent to 0.5 on the McFarland scale, according to the tion of extracts (aqueous aqueous/acetone water acetone water) Clinical and Laboratory Standards Institute M07-A9 document was evaluated by placing the extracts into the agar and in the well. As – (CLSI, 2012). controls, aqueous and acetone water extracts were added to the wells of plates with pure agar. The plates were incubated at 35 1Cfor 24 h. A synergistic effect was considered when the mean inhibition 2.2.2. Agar-well diffusion assay zone was Z2mm(Oliveira et al., 2006). The agar-well diffusion assay was executed according to pre- viously described methods (Valgas et al., 2007). A standardized bacterial inoculum was uniformly spread on a Mueller-Hinton agar 2.3. In vivo studies: anti-inflammatory and analgesic activities (MH agar, HiMedias, Mumbai, India) surface using sterile cotton swabs. Immediately, wells of approximately 6 mm in diameter 2.3.1. Animals were made on the agar surface using a sterile metallic cylinder. Male and female Swiss mice weighing 25–35 g and male Wistar Next, 80 μL of each plant extract dissolved in dimethyl sulfoxide rats weighing 240–290 g were obtained from the vivarium of the (DMSO; 62.5 mg/mL) was added into the wells, and the plates Department of Biophysics and Pharmacology, Federal University of were incubated at 35 1C for 24 h. At the end of the incubation, the Rio Grande Norte. All animals were housed in an animal room diameters of the inhibition zones formed around the wells were under standard laboratory conditions of 22þ2 1C and a 12 h light/ measured. A cloranfenicol (30 μg) antibiotic disc (DMEs, São 12 h dark cycle, and fed with pelleted food and water ad libitum. Paulo, Brazil) and a well containing only solvent were used as They were acclimatized for seven days before the experiments controls. All tests were done in triplicate. were started and fasted for 12 h prior to the experiments. The welfare of the animals and the experimental procedures were in strict accordance with the Ethics Committee on Animal Use/UFRN 2.2.3. Minimum inhibitory concentration (MIC) approved protocol (N1 025/2013). The MIC determination was performed by the broth microdilu- tion method according to the CLSI M07-A9 guidelines (CLSI, 2012) and Mbosso et al. (2010). Initially, aqueous and acetone–water 2.3.2. Drug administration extracts were dissolved in 50% DMSO in order to make stock Before administration to determine their effects, the aqueous solutions of 100 mg/mL (Libidibia ferrea and Parapiptadenia rigida) extracts and the acetone–water extracts were dissolved in DMSO and 50 mg/mL (Psidium guajava). Afterwards, 100 μL of Muller- to make solutions of 50, 100 and 200 mg/kg. DMSO (VETEC Hinton (M-H) broth (HiMedias, Mumbai, India), supplemented Química, Rio De Janeiro, Brazil), indomethacin (INDOCID 25 mg with glucose (1%, w/v) and phenol red (0.0025%, w/v), was added Aspen Pharma) and morphine sulfate (Dimorf 10 mg/mL in 1 mL of to the second through eighth wells of each row. A volume of Cristálias São Paulo, Brazil). A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96 91

2.3.3. Mouse carrageenan peritonitis (anti-inflammatory activity) Table 1 The method of Ribeiro et al. (1997) was used for this test. The Comparison among the total polyphenol contents from crude extracts. Natal, female Swiss mice were divided into 11 groups (n¼6 per group). RN. 2014. – The aqueous extracts or acetone water extracts of Libidibia ferrea, Crude Extract Total polyphenol contenta Parapiptadenia rigida, and Psidium guajava were administered orally at doses of 50, 100 and 200 mg/kg, respectively. Indomethacin Aqueous Acetone–water (7:3, v/v) (10 mg/kg, p.o.) and carrageenan (0.25 mL, 1% in saline, i.p.; mg% mg% Sigma-Aldrich, São Paulo, Brazil) were administered 30 min later. After 4 h, the animals were sacrificed by thiopental injection Libidibia ferrea 6.41 (1.71) 30.47 (0.26) 6.88 (0.89) 32.50 (0.47) (20 mg/kg) for further investigation. The total leukocyte count Parapiptadenia rigida 4.54 (2.35) 22.44 (0.46) 7.65 (0.62) 35.82 (0.20) was determined in a Neubauer chamber. The results were expressed Psidium guajava 5.07 (0.64) 24.67 (0.14) 5.18 (1.25) 25.13 (0.28) as the total number of cells per peritoneal cavity (Mean and The data are represented as the mean (relative standard deviation) of three Standard Deviation). independent measurements. a Values are expressed as pyrogallol equivalents (PE) per g of plant extract. 2.3.4. Hot plate test (central analgesic activity) The method of Kuraishi et al. (1983) was used for this test. The hot plate test was carried out using a hot plate apparatus (Insight, São Paulo, Brazil), maintained at 5570.5 1C. Only mice that 3.1.3. HPLC analysis showed initial nociceptive responses (licking of the forepaws or The chromatographic fingerprints of the crude extracts are shown jumping) between 3 and 19 s were used for additional experi- in Fig. 1. Peaks gallic acid and catechin were present in all chromato- ments. The chosen mice were pre-treated with the aqueous grams, eluting at a retention time (Rt) of 9.3 min and 17.2 min at extracts and acetone–water extracts of Libidibia ferrea, Parapipta- 280 nm, respectively, thus indicating the presence of gallic acid and denia rigida, and Psidium guajava (50, 100, 200 mg/kg; p.o.) with catechin. vehicle (DMSO); and 30 min later, the measurements were taken. The content of gallic acid and catechin was determined from A morphine (10 mg/kg; i.p.)-treated animal group was included as extrapolation of the calibration curves (y¼1.232x0.025, R2¼ a positive control. The cut-off time was set at 30 s to minimize skin 0.9983, for gallic acid; and y¼0.5254x4.4547, R2¼0.9916, for damage. The reaction time, i.e. the amount of time it took for the catechin), which were made through the preparation of gallic acid animal to lick its forepaws or jump from the hot plate, was and catechin (Sigma Chemical Co., St. Louis, USA) solutions (Table 2). measured at 0, 30, 60, 90 and 120 min after the administration. The determination of contents was carried out in triplicate.

2.3.5. Acetic acid-induced abdominal writhing test (peripheral 3.2. In vitro microbiological tests analgesic activity) The method of Koster et al. (1959) was used for this test. The 3.2.1. Agar-well diffusion assay female Swiss mice were divided into five groups of six mice each and The zones of inhibition produced by all the plant extracts against were fasted overnight. The animals were treated with indomethacin bacteria are shown in Table 3. All the extracts showed activity against (10 mg/kg, p.o.), DMSO (10 mL/kg, p.o.) and the aqueous extracts or Gram-positive bacteria, but the Gram-negative bacteria were resis- acetone–water extracts of Libidibia ferrea, Parapiptadenia rigida or tant to most of the plant extracts. For the tests against the Gram- Psidium guajava (50, 100, 200 mg/kg; p.o.). The mice were treated positive bacteria, the highest inhibition zones were observed for the with acetic acid (0.6%, v/v in saline, 10 mL/kg, i.p.) 30 min after the aqueous extract of Psidium guajava (EAPg) and the acetone–water above treatment was carried out. The number of writhes was extract of Psidium guajava (EAWPg) against Staphylococcus aureus counted for 20 min. The results were expressed as the total whithes. ATCC 25923 (17.6770.58 mm and 2071.00 mm respectively). Regarding the Gram-negative bacteria, the most susceptible bacter- 2.4. Statistical analysis ium was Shigella flexneri and, again, EAPg (17.6770.58 mm) and EAWPg (18.3370.58 mm) were the most active. The data are presented as meansþstandard error of the mean (SEM), where appropriate. Analysis of variance (ANOVA) followed 3.2.2. MIC determination by Bonferroni's test was used to calculate the means (GraphPad In the MIC determination (Table 4), generally, the acetone–water PRISM 5.0 Software). A P-value of o0.05 was considered to extracts were more active than the aqueous extracts. The most indicate a significant difference. susceptible Gram-positive bacterium was Staphylococcus epidermidis, and the most susceptible Gram-negative bacterium was Shigella fl 3. Results exneri.Again,mostGram-negativestrainswereresistant,especially Escherichia coli, showing MIC values greater than 10 mg/mL. The 3.1. Plant analyses highest activity in the Gram-positive group was observed for EAWPg against Staphylococcus epidermidis (MIC¼39 μg/mL). For the Gram- fl 3.1.1. Phytochemical analysis negative group, EAWPg had the lowest MIC against Shigella exneri ¼ μ TLC for chemical identification of the constituents of the crude (MIC 625 g/mL). Regarding the MRSA strain, considerable activity ¼ μ extracts from Libidibia ferrea bark, Parapiptadenia rigida bark and was observed for EAPg (MIC 625 g/mL). Psidium guajava leaves revealed the presence of condensed tannins (catechins) and hydrolysable tannins (gallic acid). 3.2.3. Synergism for plant extracts and antimicrobials and for combinations of plant extracts 3.1.2. Estimation of TPC In the evaluation of synergism among extracts and antimicro- The TPC of the crude extracts were calculated. Comparison of bials, none of the combinations resulted in the production of the average crude extract yields showed that the yields from the inhibition zones for the antimicrobials oxacillin or penicillin. aqueous extracts were all less than the yields from the acetone– According to the adopted criteria for a synergistic effect (mean water extracts (Table 1). inhibition zone Z2mm),synergismwasobservedintwoofthe18 92 A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96

Fig. 1. Chromatographic fingerprints of crude extracts from Parapiptadenia rigida (1 and 2), Libidibia ferrea (3 and 4) and Psidium guajava (5 and 6), aqueous and acetone– water, obtained by HPLC and analyzed at 280 nm. Each chromatographic peak appears with its corresponding retention time. UV spectrum pattern identified as gallic acid (A) and catechin (B). Natal, RN. 2014. combinations evaluated. EAPg in the presence of the aqueous extract 3.3. In vivo tests of Parapiptadenia rigida (EAPr) resulted in an increase in the mean inhibition zone of 2 mm. The same effect occurred with the 3.3.1. Carrageenan peritonitis (anti-inflammatory activity) acetone–water extract of Libidibia ferrea (EAWLf) in the presence In order to evaluate a possible inhibitory effect of the aqueous of EAWPg. extract of Libidibia ferrea (EALf), EAPr, EAPg, and EAWLf, the A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96 93

Table 2 The absolute values and percentages of gallic acid and catechin quantified by HPLC in crude extracts. Natal, RN. 2014.

Crude extract Gallic acid Catechin

lg%a lg%a

AQ AC–WAQAC–WAQAC–WAQAC–W

Libidibia ferrea 0.013 (1.31) 0.010 (0.49) 0.05 (0.54) 0.04 (1.45) 0.027 (4.09) 0.011 (0.84) 1.07 (1.32) 0.44 (4.11) Parapiptadenia rigida 0.018 (0.20) 0.007 (0.02) 0.07 (0.07) 0.03 (1.45) 0.004 (1.35) 0.022 (2.11) 0.86 (1.45) 1.04 (2.41) Psidium guajava 0.055 (0.56) 0.065 (0.19) 0.22 (1.45) 0.26 (0.38) 0.006 (0.78) 0.009 (1.10) 0.88 (0.59) 0.92 (1.44)

The data are represented as the mean (relative standard deviation) of three independent measurements; AQ, aqueous crude extract; AC–W, acetone:water (7:3, v/v) crude extract. a Values are expressed as percentages (mg/g) of gallic acid and catechin in the crude extracts.

Table 3 Mean inhibition zones (mm) a of Gram-positive and Gram-negative bacteria against the aqueous and acetone–water extracts of Libidibia ferrea, Parapiptadenia rigida and Psidium guajava. Natal, RN. 2014.

Bacteria Plant species Control

Libidibia ferrea (bark) Parapiptadenia rigida (bark) Psidium guajava (leaves) Cloranfenicol (30 mg)

AQ AC–WAQAC–WAQAC–W

Gram-positive Staphylococcus aureus ATCC 25923 11.0070.00 15.0070.00 13.0070.00 14.3371.53 17.6770.58 20.0071.00 22.0070.70 Staphylococcus epidermidis INCQS 00016 16.0071,73 15.3370.58 14.0070.00 16.6770.58 15.3370.58 14.3370.58 24.0070.00 Enterococcus faecalis ATCC 29212 12.6770,58 11.6770.58 13.6770.58 11.0071.00 14.6770.58 14.3370.58 27.6770.58 MRSA 9.3370.58 12.3370.58 6.3370.58 13.0071.00 14.6770.58 15.0070.00 12.3370.58

Gram-negative Escherichia coli ATCC 25922 – 7.00 70.00 – 8.3370.58 ––24.0071.41 Salmonella enteritidis INCQS 00258 8.0071.00 8.3370.58 6.3370.58 – 8.3370.58 – 26.0070.00 Shigella flexneri 13.0071.00 12.3370.58 6.6771.15 13.3370.58 17.6770.58 18.3370.58 34.6770.58 Klebsiella pneumoniae – 10.0070.00 – 7.00 71.73 – 11.0071.00 20.6771.15

AQ, aqueous crude extract; AC–W, acetone–water (7:3, v/v) crude extract; (–), no inhibition zone. a Values are presented as mean7standard deviation of three replicates.

Table 4 Minimum inhibitory concentration (MIC; mg/mL)a of the Libidibia ferrea, Parapiptadenia rigida and Psidium guajava extracts against Gram-positive and Gram-negative bacteria. Natal, RN. 2014.

Bacteria Plant extracts Control

Libidibia ferrea (bark) Parapiptadenia rigida (bark) Psidium guajava (leaves) Cephalothin Gentamicin Vancomycin

AQ AC–WAQAC–WAQAC–W

Gram-positive Staphylococcus aureus ATCC 25923 500070 250070 10,00070 125070 62570 125070 0.62570ND ND Staphylococcus epidermidis INCQS 6257015070 156271325 9377442 312 703970 0.2570NDND 00016 Enterococcus faecalis ATCC 29212 62570 125070 250070 125070 625701947164 8 70NDND MRSA 500070500070 410,00070 250070 62570 125070ND ND o2.570

Gram-negative Escherichia coli ATCC 25922 410,00070 410,00070 410,00070 410,00070 4500070 45000701070NDND Salmonella enteritidis INCQS 00258 500070 250070500070500070500070500070ND 0.12570ND Shigella flexneri 500070 375071760 500070500070 250070 62570 ND 0.570ND Klebsiella pneumoniae 500070500070 375071760 500070500070500070 ND 0.570ND

AQ, aqueous crude extract; AC–W, acetone–water (7:3, v/v) crude extract; ND, not determined. a Values are presented as mean7standard deviation of two replicates.

acetone–water extract of Parapiptadenia rigida (EAWPr) and exudates, and the positive group (indomethacin) showed an EAWPg, at 50 mg/kg, 100 mg/kg and 200 mg/kg, respectively, on inhibitory effect on cell recruitment into the peritoneal cavity; cell recruitment into the peritoneal cavity, the carrageenan- thus, the carrageenan-induced peritonitis test used did not show induced peritonitis test was used. The negative control group augmentation of inflammatory cell migration (po0.01). All of the showed an increase in the numbers of leukocytes from peritoneal extracts significantly reduced the number of leukocyte migration 94 A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96

(npo0.05; nnnpo0.001; Fig. 2). The mechanism of action of 3.3.3. Acetic acid writhing reflex (analgesic activity) carrageenan on peritonitis involves synergism involving prosta- The effect of all extracts on the writhing response in mice is shown noids, leukotriene B4 and other chemotactic agents such as C5a in Fig. 3. Indomethacin significantly decreased the writhing response and IL-8, which promote the recruitment of leukocytes, mainly (po0.001). Similarly, EAPr (50 mg/kg) decreased the writhing neutrophils (Foster et al., 1986). response (po0.01). Other extracts that presented analgesic activity were EAWPr (100 mg/kg and 200 mg/kg) and EAWPg (50 mg/kg), which reduced the writhing response (po0.05). 3.3.2. Hot plate test (analgesic activity) Using the hot plate test, the effect of all extracts on analgesic activity was determined. None of the extracts presented central 4. Discussion analgesic activity (po0.05). In contrast, morphine showed central analgesic activity (po0.05) (Table 5). In our study, we found that the method of extraction, with water or an acetone–water mixture, maintained the presence of polyphenols,

25000 60

20000 *** 15000 40

10000 * * * * ** 20 * * 5000 ** ***

0

Number of leukocyte migration A B 3 4 5 6 1 1 2 3 4 5 6 0 C1 C2 C C C C D D2 D3 D4 D5 D6 E E E E E E Number of writhing response A B 3 5 C1 C2 C3 C4 C5 C6 D1 D2 D3 D4 D5 D6 E1 E2 E E4 E E6 Fig. 2. Cell migration induced by aqueous extracts Libidibia ferrea (EALf) at 50, 100 and 200 mg/kg (D1, D2, and D3, respectively), aqueous extracts of Parapiptadenia Fig. 3. Number of writhing responses by aqueous extracts Libidibia ferrea (EALf) at rigida (EAPr) at 50, 100 and 200 mg/kg (E1, E2, and E3, respectively), aqueous 50, 100 and 200 mg/kg (D1, D2, and D3, respectively), aqueous extracts of extracts of Psidium guajava (EAPg) at 50, 100 and 200 mg/kg (C1, C2, and C3, Parapiptadenia rigida (EAPr) at 50, 100 and 200 mg/kg (E1, E2, and E3, respectively), respectively), acetone–water extracts of Libidibia ferrea (EAWLf) at 50, 100 and aqueous extracts of Psidium guajava (EAPg) at 50, 100 and 200 mg/kg (C1, C2, and 200 mg/kg (D4, D5, and D6, respectively), acetone–water extracts of Parapiptadenia C3, respectively), acetone–water extracts of Libidibia ferrea (EAWLf) at 50, 100 and rigida (EAWPr) at 50, 100 and 200 mg/kg (E4, E5, and E6, respectively) and 200 mg/kg (D4, D5, and D6, respectively), acetone–water extracts of Parapiptadenia acetone–water extracts of Psidium guajava (EAWPg) at 50, 100 and 200 mg/kg rigida (EAWPr) at 50, 100 and 200 mg/kg (E4, E5, and E6, respectively) and (C4, C5, and C6, respectively). Natal, RN. 2014. (ANOVA Test: npo0.05); A, negative acetone–water extracts of Psidium guajava (EAWPg) at 50, 100 and 200 mg/kg control (DMSO); B, positive control (indomethacin). The bars represent the mean (C4, C5, and C6, respectively). Natal, RN. 2014. (ANOVA Test: npo0.05); A, negative number of leukocytesþstandard deviation, groups compared with A: negative control (DMSO); B, positive control (indomethacin). The bars represent the mean control (ANOVA: npo0.05; nnpo0.01; C4, C5, C6, D1, D2, D3, D4, D5, D6, E1, E2, E3, number of writhing responsesþstandard deviation, groups compared with E4, E5, E6, nnnpo0.001). A: negative control (ANOVA: npo0.05; nnpo0.01; C4-E6, nnnpo0.001).

Table 5 Number and perceptual pain latency time. Natal, RN 2014.

Pain latency initial value at time¼0 min Pain latency at the indicated time after administration

0 s 30 min 60 min 90 min 120 min

Control (DMSO) 13.4þ5.0 6.2þ6.8 4.8þ3.3 7.8þ5.1 6.3þ2.1 Morphine (10 mg/kg) 8.6þ2.3 27.2þ3.0* 23.2þ7.8* 25.3þ6.4* 27þ2.6* EAPg (50 mg/kg) 9.2þ4.1 9.2þ4.1 10þ2.9 15.2þ7.7 17 þ10.5 EAPg 100 mg/kg 9.8þ4.1 9.8þ4.5 11.2þ3.6 7.6þ4.4 9.2þ3.0 EAPg 200 mg/kg 10.2þ4.7 11þ4.7 7.6þ5.9 8þ6.2 5.6þ2.5 EAWPg 50 mg/kg 10.3þ4.5 15.2þ8.6 8.8þ6.1 6.6þ4.4 10þ7.5 EAWPg 100 mg/kg 14.6þ2.9 7þ2.9 5þ3.3 5þ31þ1.7 EAWPg 200 mg/kg 13.4þ3.3 15.2þ3 8.8þ3.1 5þ38þ3.5 EALf 50 mg/kg 14 þ4.7 12.3þ912þ9.2 15.2þ9.2 8þ3.5 EALf 100 mg/kg 16.4þ2.9 18þ2.9 20 þ8.7 8þ1.8 13 þ8.4 EALf 200 mg/kg 12þ3.7 18þ2.9 20þ8.7 8þ1.8 13 þ8.1 EAWLf 50 mg/kg 14.4þ1.8 12.2þ2.2 14.2þ97.2þ4.9 7.8þ2.0 EAWLf 100 mg/kg 16.2þ5.4 17.3þ9 20.8þ7.4 17.3 þ4.9 18þ5.2 EAWLf 200 mg/kg 15.4þ2.9 13.7þ8.1 20.3þ7 18.7þ6.4 20þ2.0 EAWPr 50 mg/kg 10 þ3.3 4.7 þ0.6 4.5þ3.1 6þ2.9 8.5þ4.0 EAPr 100 mg/kg 10 þ6.3 10.4þ711.2þ8.1 7.8þ6.8 10.4þ6.9 EAPr 200 mg/kg 8.6þ3.5 8þ3.4 7.4þ4.2 4.2þ3.3 6þ5 EAWPr 50 mg/kg 15.4þ3.6 20.8þ5.7 12.2þ1.9 7.8 þ3.6 8.8þ6.1 EAWPr 100 mg/kg 14.8þ4.8 15þ7.7 7.2 þ1.9 7.8 þ3.6 7þ4.7 EAWPr 200 mg/kg 12.8þ0.8 10.2þ4.4 12.8þ9.4 10.8þ3.0 13.4þ8.1

Symbols for pain latency: EALf, aqueous extract of Libidibia ferrea; EAPr, aqueous extract of Parapiptadenia rigida; EAPg, aqueous extract of Psidium guajava; EAWLf, acetone– water extract of Libidibia ferrea; EAWPr, acetone–water extract of Parapiptadenia rigida; EAWPg, acetone–water extract of Psidium guajava; DMSO, negative control; morphine (10 mg/kg), positive control. n Compared to the negative control group (ANOVA Test: npo0.05). A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96 95 including total polyphenols, gallic acid and catechin, confirming et al., 1992; Nakanishi et al., 2010), therefore, this biomolecules previous reports in which these components have been identified in may be related to the anti-inflammatory activity observed in the the plant species used in this study (Gasparin Verza et al., 2007; present study. Regarding Psidium guajava, there was no central Schmidt et al., 2011; Vasconcelos et al., 2011; Chang et al., 2013). analgesic activity, but EAPg at a dose of 50 mg/kg showed The literature regarding Libidibia ferrea and Parapiptadenia significant reduction in writhings, i.e., peripheral analgesia, in rigida is scarce, and this study may contribute to corroborate the mice. Among the mediators involved in the pain process, prosta- ethnopharmacological use of these plants as antimicrobial agents. glandin endoperoxide H synthase (PGHS) is a rate-limiting enzyme In the present study, the aqueous and water–acetone extracts of for the production of PGs and thromboxane from arachidonic acid. Parapiptadenia rigida were effective against Staphylococcus aureus The enzyme catalyzes both bisdioxygenation of arachidonic acid and Staphylococcus epidermidis, which is in accordance with other (cyclooxygenase activity) producing PGG2 and conversion of PGG results reported in the literature, although the authors used to PGH (PG hydroperoxidase activity) (Kawakami et al., 2009). methanol as a solvent (de Souza et al., 2004). Both EAPr and EAWPr showed significant anti-inflammatory Concerning Libidibia ferrea, in our study, the aqueous and activity. In turn, peripheral analgesic activity was found for EAPr acetone–water extracts were active against all Gram-positive (50 mg/kg) and EAWPr (100 and 200 mg/kg). The content of total bacteria and against some Gram-negative bacteria, particularly polyphenols and catechin was the highest for EAWPr. Literature Shigella flexneri. It has been described that, according to the agar data are scarce; however, the study by Schmidt et al. (2010) diffusion assay, the methanolic extract of the bark has antibacterial identified catechin in the ethanolic extract of Parapiptadenia rigida activity against Staphylococcus aureus, which supports our find- bark, which afforded anti-inflammatory effects on NF-kappaB DNA ings. However, this extract also has been reported to have activity binding and p38alpha MAPK activity (Schmidt et al., 2010). against three Escherichia coli strains, contradicting our results In addition, both EALf and EAWLf showed anti-inflammatory (Pereira et al., 2012). activity, significantly reducing leukocyte migration. Unlike the Despite the fact that there are quite few studies in the literature study by Carvalho et al. (1996) showing analgesic activity by the regarding the antibacterial activity of Libidibia ferrea and Para- acetic acid model, we found a reduction in the percentage of piptadenia rigida, there is a large volume of studies about Psidium contortions; however, the finding was not statistically significant. guajava. Here, Psidium guajava extracts showed antibacterial Furthermore, extracts and polysaccharide fractions of Libidibia activity against all the Gram-positive strains and against some ferrea pods exhibit potent anti-inflammatory activity via negative Gram-negative strains, especially Shigella flexneri. Similar findings modulation of histamine, serotonin, bradykinin, PGs (2) and NO for Staphylococcus aureus,MRSAandShigella flexneri were obtained by released in carrageenan-induced edema, showing involvement of other studies, which used dilution and agar-diffusion methods mast cells (Pereira Lde et al., 2012). (Sanches et al., 2005; Chomnawang et al., 2009; Birdi et al., 2010). The acetic acid-induced writhing response in rats affords the Opposing results have been described for Escherichia coli,whichis rapid evaluation of peripheral analgesic activity. Acetic acid causes susceptible to Psidium guajava leaf extracts according to diffusion and inflammatory pain by inducing capillary permeability and liberat- dilution assays (Holetz et al., 2002; Gonçalves et al., 2008; Masadeh et ing endogenous substances that excite pain in nerve endings, thus al., 2013). This discrepancy may be due to the type of extract employed increasing PG1 and PG2 peripherally (Kumar et al., 2001). The and the method used to evaluate the antibacterial activity. In these mechanism of analgesic activity of EAPr (50 mg/kg), EAWPr studies, distinct extraction methods were utilized and none of them (100 mg/kg and 200 mg/kg) and EAWPg (50 mg/kg) could prob- was similar to the one used in the present work. ably be due to blockade of the effect or the release of endogenous All the extracts had considerable amounts of phenolic com- substances that excite pain nerve endings similar to that of pounds, which may be involved in the antibacterial activity indomethacin and non-steroidal anti-inflammatory drugs. Thus, observed in this study. Some researchers have shown that the the reduction in writhing indicates that the extracts might exert antibacterial activity is proportional to the amount of phenolic anti-nociceptive activity by inhibition of PG synthesis or activity. compounds inside the plant (Baydar et al., 2004; Tomás-menor et al., 2013). Here, gallic acid and catechin were the major phenolic compounds observed in the extracts. The bactericidal effect 5. Conclusion demonstrated by these phenolic substances may involve many mechanisms such as alteration of the physicochemical properties In conclusion, the choice of extraction solvent is important to of the plasma membrane, pore formation, DNA gyrase and nucleic preserve the chemical components, such as gallic acid and catechins, acid synthesis inhibition and toxicity through the generation of which may be related to the antimicrobial, anti-inflammatory and hydrogen peroxide (Mori et al., 1987; Arakawa et al., 2004; analgesic activities found in these extracts. Gradisar et al., 2007). It is described that gallic acid inhibits the growth of many bacteria, such as methicillin-sensitive Staphylo- coccus aureus, MRSA, Escherichia coli, Pseudomonas aeruginosa and Salmonella thyphi (Chanwitheesuk et al., 2007; Borges et al., 2013). Acknowledgments Catechins also are able to inhibit the growth of many MRSA strains (Stapleton et al., 2004). Moreover, gallic acid and catechin have This work was supported by grants from CNPQ (483870/2011-0; been shown to be effective against Helicobacter pylori (Díaz-gómez 480128/2012-0; 302113/2012-6) and FACEPE (IBPG-0423-4.03/11, et al., 2013). Therefore, the antibacterial activity of the extracts in APQ-1296-4.03-12). this study against Gram-positive and some Gram-negative bacteria may be partially due to these compounds. Regarding the anti-inflammatory activity, EAPg and EAWPg showed a reduction in leukocyte migration. The contents of gallic References acid and catechin were higher in water and acetone–water extract preparations. In the study by Denny, et al. (2013), Psidium guajava Arakawa, H., Maeda, M., Okubo, S., Shimamura, T., 2004. Role of hydrogen peroxide exhibited anti-inflammatory activity and phenolic compounds, in bactericidal action of catechin. Biological & Pharmaceutical Bulletin 27, 277–281. including gallic acid and catechin. It was demonstrated that gallic Baydar, N.G., Ozkan, G., Sagdiç, O., 2004. Total phenolic contents and antibacterial acid and catechins possess anti-inflammatory properties (Kroes activities of grape (Vitis vinifera L.) extracts. Food Control 15, 335–339. 96 A.A. de Araújo et al. / Journal of Ethnopharmacology 156 (2014) 88–96

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