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Quantification of Polyphenols and Evaluation of Antimicrobial Journal of Ethnopharmacology 156 (2014) 88–96 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep Research Paper Quantification of polyphenols and evaluation of antimicrobial, analgesic and anti-inflammatory activities of aqueous and acetone–water extracts of Libidibia ferrea, Parapiptadenia 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 plant 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 tannins (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 plants 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 Fabaceae 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
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