Emir. J. Food Agric. 2011. 23 (6): 505-510 http://ejfa.info/

Antibacterial activity of Minquartia guianensis extracts and phytochemical evaluation

Lorena M. C. Cursino 1, Ivanildes Santos 2, Luis A. M. Mariúba 2, Manoel F. Jeffreys 1, Nerilson M. Lima 1, Jaciara L. Oliveira 1, ∗ Patricia P. Orlandi 2 and Cecilia V. Nunez 1

1Coordination of Research on Natural Products - CPPN, National Institute of Amazonian Research - INPA, Av André Araújo, 2936, 69060-001, Manaus, Brazil; 2Instituto Leônidas e Maria Deane – ILMD, Fundação Oswaldo Cruz – FIOCRUZ, Manaus, Brazil

Abstract: The species Minquartia guianensis (Olacaceae) is found in the Amazon region and also in Nicarágua, Panamá and Costa Rica. Indigenous people from Ecuador use the bark infusion for intestinal infections treatment caused by parasites, against muscular pain and cutaneous irritations. For this reason, the aim of this work was to evaluate the antibacterial activity of M. guianensis extracts over Gram-negative ( Shigella flexneri M90T, Salmonella choleraesuis 6958 , Escherichia coli E2348/69) and Gram-positive bacteria (Methicillin-Resistant Staphylococcus aureus 33591 , Methicillin-Sensible Staphylococcus aureus 25923, Bacillus cereus 9634 , Bacillus liquefaciens clinical isolated). These bacteria are diarrhea related, which causes several child death in tropical regions. The active extract is under fractionation (leaf DCM) and until now, four triterpene were isolated lupeol, taraxerol, lupenona and squalene, but it was not possible yet to assay the substances, because of their small amount.

Key words: Antibacterial Activity, Minquartia guianensis , Olacaceae, Gram-positive bacteria, Gram-negative bacteria

ط ا دات ا    ت Minquartia guianensis و  ت ا ت ا  

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ا :  ااع ت Minquartia guianensis ا   Olacaceae $# "! ا زون آ ا أ&* $# %ر(ا و  وآ)ر&% . &/.م )% ن ا;آادور ا 89 7ع 6ء ه3ا ات  2ج ا 0 ت ا & ا @8 ا?ت ، آ &/.م ا&*  2ج = م ا *2ت وا6//ت ا>& . و30ا ا/C، $ن ا0ف 8 هA3 ارا) ه F7  ط ا*دات ا% & /.D ت G@ Minquartia guianensis ا % & ا/ H  ام Shigella) (flexneri M90T, Salmonella choleraesuis 6958, Escherichia coli E2348/69 وا% & ا H  ام Methicillin-Resistant Staphylococcus aureus 33591, Methicillin-Sensible Staphylococcus) .(aureus 25923, Bacillus cereus 9634, Bacillus liquefaciens وA30 ا% & @ M2 L ة ;)0ل ، وا3ي &/C $# و$ة ا & 8 ا "ل $# ا"! اار& . &> ي ا N $# اPM اN O6 ااد ا  8 D./ ه3ا ات ، وGR ا ن Q@ Fل ار  8 آت ا & triterpene) 8) ه lupenona ، taraxerol ، lupeolو .squalene وF &%8 7ور GR اTن N6 هA3 ااد S ا F

∗Corresponding Author, Email : [email protected] Received 11 April 2011; Revised 09 May 2011; Accepted 10 May 2011

505 Lorena M. C. Cursino et al.

Introduction Several diseases are affecting people et al., 1994) and other triterpenes as squalene, around the world, among them intestinal lupe-3-one, taraxerone and lupeol (Cursino et infections are common. Studies with children al., 2009). in the hospital in João Pessoa, PB, Brazil, This article describes antibacterial activity showed a significant number of children are and phytochemical evaluation of M. guianensis under problems with intestinal infection extracts. (Tôrres et al., 2005). This problem is Materials and Methods widespread all over tropical regions and some Plant material of them are so acute that causes children death. Minquartia guianensis Aubl. (Olacaceae) The main causes of diarrhea are related to was collected in Reserva Florestal Ducke, bacterial infections (Viswanathan et al., 2008). Manaus, AM, in April/2005. A voucher Plant species are producers of several specimen (179.806) was deposited at INPA’s chemical metabolites and in the available Botanical Research Coordenation Herbarium. literature, there are several examples of plants The leaves and branches were dried, and their metabolites that showed antibacterial grounded and extracted by using activity. For example, ethanolic extracts dichloromethane (DCM), methanol (MeOH) Ocotea duckei showed activity against and water (H O), each extraction performed 3 Escherichia coli 2 (Antunes et al., 2006), times by using ultra-sound for 20 min. methanolic extract from Caesalpinia bonducella and triterpenes α-amiryn, β-amyrin, Assays lupeol, and lupeol acetate showed activity Bacterial strains against Staphylococcus aureus, Escherichia The extracts were tested against coli, Shigella flexneri (Saeed et al., 2001). The Gram-negative ( Shigella flexneri M90T, acetyl aleuritolic acid isolated from Salmonella choleraesuis 6958 , Escherichia coli Spirostachys africana was active against E2348/69) and Gram-positive bacteria Escherichia coli, Staphylococcus aureus , (Methicillin-Resistant Staphylococcus aureus Vibrio cholera, Salmonella typhi , Shigella 33591 , Methicillin-Sensible Staphylococcus dysentery, S. flexnerii and S. boydii (Mathabe aureus 25923, Bacillus cereus 9634 , Bacillus et al., 2008). liquefaciens clinical isolated). The bacteria Among plant kingdom, Olacaceae is a were kept on the defined bacterium medium family with 24 genera and 150 species, and in which contains proteose peptone, sodium Brazil there are 12 genera and 60 species (APG chloride, meat extract, bacteriological grade II, 2008). Several compounds were isolated agar and distilled water, at room temperature. from this family, such as: flavonoids, alkaloids, Agar-Well diffusion method terpenes and saponins (Forgacs, 1981; The assay was conducted by agar well Polonsky, 1984; Haron, 1997; Wiart , 2001). diffusion method (Perez et al., 1990). The The plant species chosen for this work is bacterial strains grown on nutrient agar at 37°C Minquartia guianensis Aubl. which is found in for 24 h were suspended in a saline solution the Amazon region and also in Nicaragua, (0.85% NaCl) and adjusted to a turbidity of 0.5 Panama and Costa Rica (MOBOT, 2010). Mac Farland standards (10 8 CFU/mL). The Their popular names are: acariquara, suspension was used to inoculate 90 mm acariquara-roxa, acari, acapu, acaximba and diameter Petri. Wells (6 mm diameter) were arariuba (Camargo, 2005). There are few punched in the agar and filled with 100 µL of 20 previous chemical work realized with this mg/mL extracts. The dissolution of the organic species. Rasmussen et al. (2000) described the extracts (DCM and MeOH) was aided by 1% minquartinoic acid which showed activity (v/v) DMSO and that of the aqueous extracts against malaria and leishmaniasis. Other with water, which did not affect the growth of compounds isolated from this species were microorganisms, in accordance with our control triterpenes, xantones, acetylenic acid (El-Seedi experiments. Plates were incubated in air at

506 Emir. J. Food Agric. 2011. 23 (6): 505-510 http://ejfa.info/

37°C for 24 h. The experiments were conducted mm) to define it then we can consider that the thrice. DMSO was taken as control for the extracts: H 2O leaves, MeOH and H 2O branch organic extracts. Sterile distilled water was extracts active against Shigella flexneri , DCM taken as control for aqueous extracts. Imipenem and MeOH leaf extracts and MeOH branch was used as standard antibiotic (32 µg). extract against Bacillus cereus and DCM and In order to visualize the activity, 8 mL of MeOH leaf extracts and MeOH branch extract triphenyltetrazolium chloride 0,1% with against Bacillus liquefaciens , and MeOH leaf bacteriological agar 1% were add after 18 h of extract against Methicillin-Resistant incubation and incubated again for 30°C. Staphylococcus aureus. Antibacterial activities were evaluated by By other hand if only extracts that showed measuring inhibition zone diameters after 4 h. at least half of inhibition antibiotic halo are considered so the active one will be: DCM leaf Resazurin MIC assay extract against Salmonella choleraesuis, H O The method used is the proposed by Mann 2 leaves, MeOH and H O branch extracts active and Markham (1998), with some 2 against Shigella flexneri , DCM and MeOH leaf modifications. Serial three-fold dilutions extracts and MeOH branch extract against starting at 20 mg/mL of each extract were Bacillus cereus , DCM leaf extract against prepared by vortexing in sloppy agar at room Methicillin-Sensible Staphylococcus aureus , temperature. The resazurin assay medium, LB, which now is the most active extract. was inoculated with the test organism to yield a So, in the second analyses, the most active final cell density ca. 1 log cycle lower than the extract is the DCM leaf extract against cell density required to reduce resazurin 5 Methicillin-Sensible Staphylococcus aureus , (usually 2.5 x 10 cell/mL). The inoculum density was confirmed by plate count. which sowed the same inhibition as that the A sterile 96-well microtitre tray was set up antibitic used. And the extracts which showed Bacillus liquefaciens with each of the tested bacteria as follows: 100 activity against , in the µL inoculum + 100 µL extract dilution; 100 µL first analysis, now classified as medium active. O leaf extract and H O inoculum + 100 µL extract diluent (positive The MeOH and H 2 2 branch extracts showed a not defined halo, control); sterile resazurin assay medium plus called intermediary halo, which can be infered 100 µL extract diluent (negative controls). The as bacteriostatic (Table 1). well contents were thoroughly mixed. Two The results obtained from M. guianensis trays were prepared for each organism and leaf extracts (Table 1) showed a mild tendency incubated at 37°C for 3 h. After incubation, 30 of the active extracts to be more active against µL resazurin solution (6 µg/mL) was added to Gram-positive bacteria. This can be explained all except negative control, to which 30 µL through difference of bacterial wall. As distilled water was added. After a second Gram-positive organisms have a thick layer of incubation for 4 h at 37°C, the wells were peptidoglycan in the cell wall (murein), these assessed visually for colour change, with the extracts can have some murein inhibitor highest dilution remaining blue, indicating the synthesis in their composition. As there is a MIC. huge mixture of substances in the extract Results and Discussion composition, it is not possible yet to explain Agar-Well diffusion method the action mechanism. Some authors define an active extract when As the leaves and branches extracts of it shows an inhibition halo > 13 mm (Vieira, Minquartia guianensis were active against 2010). But it is more interesting to compare the Staphylococcus aureus (Table 1), which is also inhibition halo against the antibiotic halo used related to cutaneous irritations it corroborates the as standard. folk uses of their barks in Ecuador (Marles et al., So it is need to define what activity is, 1989). because if we consider the first thought (> 13

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Table 1. Antibacterial inhibition halos found to M. guianensis extracts. Inhibiton halo (mm) Bacteria Leaves Extracts Branches Extracts Imipenem DCM MeOH H2O DCM MeOH H2O Gram-negative bacteria Salmonella choleraesuis 20 10 4 - 3 4 3 6958 Shigella flexneri M90T 30 9 - 20 IH - 20 20 IH Escherichia coli E2348/69 10 3 - - - - - Gram-positive bacteria Bacillus cereus 9634 30 23 16 7 8 16 7 Bacillus liquefaciens 50 21 20 6 11 21 - clinical isolated Methicillin-Sensible Staphylococcus aureus 10 10 5 1.5 3 5 - ATCC: 25923 Methicillin-Resistant Staphylococcus aureus 40 - 19 IH - - - - ATCC:33591 -: No active IH: Intermediary halo

Resazurin MIC assay The MIC leaves MeOH extract was 10 The minimum inhibitory concentration mg/mL against Methicillin-Sensible (MIC) obtained to the leaves DCM extract was Staphylococcus aureus, 20 mg/mL against 10 mg/mL against Methicillin-Sensible Methicillin-Resistant S. aureus , and 5 mg/mL Staphylococcus aureus , 5 mg/mL against for both Bacillus cereus and B. liquefaciens . Bacillus liquefaciens , and 2.5 mg/mL against The MIC branches MeOH extract was 2.5 B. cereus but this extract was inactive against mg/mL against Sensible Staphylococcus Methicillin-Resistant Staphylococcus aureus aureus, 20 mg/mL against Methicillin- (Table 2). Resistant S. aureus , and 5 mg/mL for both Bacillus cereus and B. liquefaciens .

Table 2. MIC found to M. guianensis extracts. MIC (mg/mL)* Bacteria Leaves Extracts Branches Extracts

DCM MeOH H2O DCM MeOH H2O Gram-positive bacteria Bacillus cereus 9634 2.5 5 NA NA 5 NA Bacillus liquefaciens 5 5 NA NA 5 NA clinical isolated Methicillin-Sensible Staphylococcus aureus 10 10 NA NA 2.5 NA ATCC: 25923 Methicillin-Resistant Staphylococcus aureus - 20 NA NA 20 NA ATCC:33591 -: No active NA: Not assayed

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Phytochemical evaluation Phenols, tannins and saponins were found In order to determine which chemical in the H 2O leaf extract. classes are present in the leaves extracts, a Both DCM extracts showed positive standard phytochemical screening was made, reaction to triterpenes and only DCM leaf following classical assays (Matos, 1997; Bessa extract showed coumarins. Alkaloids, et al., 2007). anthocyanins, anthocyanidins, chalcones and There were found phenols, tannins, aurons were not found in any extract analyzed flavonoids, saponins and triterpenes in MeOH (Table 3). leaf extract and MeOH and H 2O branch extract.

Table 3. Phytochemical prospection of M. guianensis extracts. Leaves Extracts Branches Phytochemical DCM MeOH H2O DCM MeOH H2O detected Phenols and tanins - +++ ++ - +++ +++ Alkaloids - - - - Saponins - +++ +++ - ++ +++ Coumarins + - - - Anthocyanins and ------anthocyanidins Chalcones and ------aurons Flavonoids + +++ - - +++ Triterpenes +++ + - + -: Negative; +: Low; ++ : Medium; +++ : Strong

As the most active extract was the DCM References leaf extract against several strains and showed reaction when analyzed to triterpenes, it was Antunes, R. M. P., E. O. Lima, M. S. V. chosen to perform a phytochemical Pereira et al. 2006. Atividade fractionation. By now, four triterpenes were antimicrobiana “ in vitro ” e determinação da isolated: lupeol, taraxerol, lupenone and concentração inibitória mínina (CIM) de squalene (Cursino et al., 2009). As there are fitoconstituintes e produtos sintéticos sobre some reports about antibacterial activity of the bactérias e fungos leveduriformes. Rev. triterpenes previously isolated, we believe that Bras. Farmacog. 16(4):517-524. part of the DCM leaves extract activity is due Bessa, T., M. G. H. Terrones and D. Q. Santos. to them. Further assays are needed to prove it. 2007. Avaliação fitotóxica e identificação As the amount of these triterpenes is not de metabólitos secundários da raiz de enough to perform the MIC, a new Cenchrus echinatus . Horizonte Cientifico fractionation of the leaves DCM extract is 1:1-17. going on to get more mass to perform these assays. Camargo, J. L. C. and I. D. K. Ferraz. 2005. “Acariquara-roxa, Minquartia guianensis Acknowledgment Aubl.”, Informativo Técnico Rede de The authors thank CT-Agro/CNPq/MCT Sementes da Amazônia. 10. and PPBio/INPA/CNPq/MCT for the financial support. Cursino, L. M. C., A. S. S. Mesquita, D. W. O. Mesquita, C. C. Fernandes, O. L. P. Pereira Jr, I. L. Amaral and C. V. Nunez. 2009. Triterpenos das folhas de Minquartia

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