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Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas ISSN: 0717-7917 [email protected] Universidad de Santiago de Chile Chile

Rojas, Janne; Velasco, Judith; Morales, Antonio; Díaz, Tulia; Meccia, Gina Evaluation of antibacterial activity on different solvent extracts of Euphorbia caracasana Boiss and Euphorbia cotinifolia L. (Euphorbiaceae) collected in Venezuela Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, vol. 7, núm. 4, julio, 2008, pp. 198-201 Universidad de Santiago de Chile Santiago, Chile

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ArtículoOriginal | Original Article

Evaluation of antibacterial activity on different solvent extracts of Euphorbia caracasana Boiss and Euphorbia cotinifolia L. (Euphorbiaceae) collected in Venezuela

[Evaluación de la actividad antibacteriana en extractos de diferentes solventes de Euphorbia caracasana Boiss y Euphorbia cotinifolia L. (Euphorbiaceae) recolectadas en Venezuela] Janne ROJAS1*, Judith VELASCO2, Antonio MORALES1, Tulia DÍAZ2, Gina MECCIA 1

1. Organic Biomolecular Research Group, Research Institute, Faculty of Pharmacy and Biomedical Sciences. University of Los Andes. Mérida, Venezuela. 2. Microbiology and Parasitology Department, Faculty of Pharmacy and Biomedical Sciences. University of Los Andes. Mérida, Venezuela. *Contacto: [email protected]

Recibido | Received 13/02/2008; Aceptado | Accepted 03/04/2008; Online 12/07/2008

Abstract In the present investigation the evaluation of the antibacterial activity of Euphorbia caracasana Boiss and Euphorbia cotinifolia L. has been evaluated against Gram positive and Gram negative bacteria using the disc diffusion agar method. E. caracasana and E. cotinifolia leaves were extracted with isopropyl alcohol. These extracts, after concentration, were fractionated by column chromatography and selected fractions, based on their TLC profiles, tested for antibacterial activity. Fractions eluted with dichloromethane/methanol 1:1 from the column chromatography of E. caracasana extract showed antibacterial activity against S. aureus ATCC 25923 at concentrations of 336 μg/mL and 33,6 μg/mL (dilution 1:10 of the same extract). Fractions eluted with n-hexane-dichloromethane 1:1 and dichloromethane from the same column, lacked of antibacterial activity. None of the fractions eluted from the column separation of E. cotinifolia extract showed antibacterial activity in any of bacteria tested. Keywords: Antibacterial activity, Euphorbia caracasana, Euphorbia cotinifolia, Euphorbiaceae, Staphylococcus aureus.

Resumen En la presente investigación se evaluó la actividad antibacteriana de Euphorbia caracasana Boiss y Euphorbia cotinifolia L. contra bacterias Gram positivas y Gram negativas usando el método de difusión en agar con discos de papel. Las hojas de E. caracasana y E. cotinifolia fueron extraídas con alcohol isopropílico. Los extractos, luego de concentrados, fueron separados por cromatografía en columna y las fracciones eluídas de ésta, fueron seleccionadas basandose en el perfil cromatográfico TLC, y analizadas para determinar su actividad antibacteriana. Solo las fracciones eluídas con diclorometano 1:1 de la cromatografía en columna del extracto de E. caracasana, mostraron actividad antibacteriana contra S. aureus ATCC 25923 a las concentraciones de 336 μg/mL y 33,6 μg/mL (dilución 1:10 del mismo extracto). Las fracciones eluídas con n-hexano-diclorometano 1:1 y diclorometano de la misma columna, no mostraron actividad antibacteriana. Ninguna de las fracciones eluídas de la separación cromatográfica de E. cotinifolia mostraron actividad antibacteriana en las bacterias ensayadas. Palabras clave: Actividad Antibacteriana, Euphorbia caracasana, Euphorbia cotinifolia, Euphorbiaceae, Staphylococcus aureus.

Antibacterial activity of E. caracasana Boiss and E. cotinifolia . Rojas et al.

Gram positive and Gram negative bacteria using the INTRODUCTION disc diffusion agar method. To the best of our Euphorbia genus belongs to the family knowledge there are no reports regarding the Euphorbiaceae. This family comprises about 300 antibacterial activity of these two species. genus and 5000 species distributed mainly in MATERIALS AND METHODS America and tropical Africa. (Webster, 1994) The plants of the family Euphorbiaceae contain skin Plant material irritating and tumour-promoting diterpenoids, which have tigliane, ingenane, and daphnane skeletons The leaves of E. caracasana were collected in (Evans & Taylor, 1983). Some species are used in March 2005 in sector La Conquista, Pueblo Llano, at folk medicine to treat skin diseases, gonorrhoea, 1800 m above sea level and leaves of E. cotinifolia migraines, intestinal parasites, and warts (Singla & were collected in April 2005 in Sector La Pedregosa, Pathak, 1990), In addition, several macrocyclic Mérida State at 1520 m. Voucher specimens (JR 19 diterpenoids with antibacterial, anticancer, PGE2- and JR 20, respectively) were deposited in the Dr inhibitory, anti-multidrug-resistant, prolyl Luis E Ruiz T. Herbarium, Faculty of Pharmacy and endopeptidase inhibitory, antifeedant, anti-HIV, and Biomedical Sciences, University of Los Andes, analgesic activity have recently been isolated from Venezuela. different Euphorbia species. (Hohmann et al, 2002; Hohmann et al, 2003). They include jatrophane, Extraction and column chromatographic ingol, myrsinane diterpenoids, leucocyanidol, separation quercitol, camphol, quercetrin, dihydroellagitannins The plant material (2.8 kg, E. caracasana and 2.0 and dimeric hydrolysable tannins-euphorbins. (Haba kg, E. cotinifolia), dried and powdered, was extracted et al, 2007; Jasbi, 2006). by soaking in isopropyl alcohol (8 L each) for 3 days The antibacterial activity of several Euphorbia at room temperature. After filtration, the extracts species has been evaluated in different occasions. were concentrated to dryness (190 g E. caracasana The aqueous extract of Euphorbia hirta, used in and 171 g E. cotinifolia, respectively) and further traditional medicine for the treatment of dysentery, fractionated on a column (30 cm x 6 cm) containing colic, ulcers, asthma and chronic bronchial infections silica gel (230-400 mesh). Elution was initially with showed activity against Escherichia coli, Proteus n-hexane (4 L), followed by mixtures of n-hexane- vulgaris, Pseudomonas aeruginosa and dichloromethane (CH2Cl2) 1:1 (4 L), CH2Cl2 (4 L), Staphylococcus aureus (Hore et al, 2006; Sudhakar et CH2Cl2-methanol (MeOH) 1:1 (3 L), and MeOH (3 al, 2006). The methanolic and acetone leaf extracts of L) of increasing polarity. Fractions (90), each of 200 Euphorbia fusiformis showed growth inhibition of mL, were collected and examined by TLC. Selected Staphylococcus aureus, Escherichia coli, fractions, based on their TLC profiles, were used for Pseudomonas aeruginosa, Klebsiella pneumoniae, the antibacterial assay. Proteus vulgaris, Salmonella typhii A and Salmonella typhii B. (Natarajan et al, 2005). Ethanolic extract of Thin layer chromatography (TLC) Euphorbia australis showed activity against Bacillus TLC analysis was carried out on all the fractions cereus, Escherichia coli, Klebsiella pneumoniae, eluted from the column. Oven dried (100oC for 30 Pseudomonas aeruginosa and Salmonella min) silica gel 60 F254 (Merck, UK) layers, 250 μm typhimurium (Palambo & Semple, 2001). Similarly thick were utilized and different mixtures of n- the ethanol, acetone and water extracts of Euphorbia hexane, CH2Cl2, and MeOH were used, depending on fruticosa (Alasbahi et al, 1999) and methanol extracts the polarity of the eluted fractions to be analyzed. of Euphorbia macroclada (Darwish et al, 2002) The plates were sprayed with sulphuric acid diluted showed inhibitory effects against Staphylococcus to 10 % v/v with water and heating in an oven at aureus. These results may support the popular use of 80oC for 10 min. these plants in traditional medicine for the treatment of fever, wound infections, and intestinal disorders. Bacterial strains In the present investigation the evaluation of the The microorganisms used were Staphylococcus antibacterial activity of Euphorbia caracasana and aureus (ATCC 25923), Enterococcus faecalis Euphorbia cotinifolia has been evaluated against (ATCC 29212), Escherichia coli (ATCC 25992),

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Klebsiella pneumoniae (ATCC 23357) and column, lacked of antibacterial activity. None of the Pseudomonas aeruginosa (ATCC 27853). fractions eluted from the column separation of E. cotinifolia extract showed antibacterial activity in any Antibacterial method of the bacteria tested. Antibacterial activity was determined using the Antibacterial activity has been reported for disc diffusion agar method described by Velasco et different Euphorbia species (Annapurna et al, 2004; al, (2005). The strains were maintained in agar Gonçalves et al, 2006; Sudhakar et al, 2006). The conservation at room temperature. Each bacterial ethanol extract of E. fusiformis showed antibacterial inoculum was incubated in 2.5 mL Müeller-Hinton activity against S. aureus (Natarajan et al, 2005). broth at 37 ºC for 18 hours. The bacterial inoculum Similar studies carried out with methanol extract of was diluted in sterile 0.85 % saline to obtain turbidity E. hirta and E. tirucalli revealed antibacterial activity visually comparable to a McFarland Nº 0.5 standard against Staphylococcus epidermidis (Parekh et al, (106-8 CFU/mL). Every inoculum was spread over 2005). plates containing Müeller-Hinton agar. Paper filter In recent years, multi-resistant bacterial strains discs (6 mm) saturated with 20 μL of every selected have increased dramatically, and thus the treatment fraction, eluted from the column chromatography of several infections has become very difficult, with the following solvent mixtures (n- reducing the therapeutic options. (Velazco et al, hexane/CH2Cl2 1:1, CH2Cl2 and CH2Cl2/MeOH 1:1) 2002; Velásquez et al, 2004; Hsueh et al, 2004; as well as dilutions 1:10, using the same solvents, Alavarez et al, 2006). However, the antibacterial were placed over the plates. These were preincubated activity of E. caracasana semipurified extract against at 4 ºC for 18 h and finally incubated at 37 ºC for 24 S. aureus, offers an option to the pharmaceutical h. The inhibitory zone around the disc was measured industry of new natural medicine sources with and expressed in mm. A positive control was also activity against these bacterial strains that represent assayed to check the sensitivity of the tested an important public health problem. organisms using the following antibiotics: Ampicillin-sulbactam® (10 μg/10 μg) for S. aureus Acknowledgments (inhibition zone 50 mm), Vancomycin® (30 μg) for The authors would like to acknowledge Consejo E. faecalis (inhibition zone 30 mm), Gentamicin® de Desarrollo Cientifico, Humanistico y Tecnológico (10 μg) for E. coli (inhibition zone 42 mm), (CDCHT) Mérida, Venezuela for the financial Cefotaxime® (30 μg) for K. pneumoniae (inhibition support (FA-304-03-08-A) of this investigation. zone 40 mm) and Cefoperazone® (75 μg) for P. aeruginosa (inhibition zone 36 mm). A negative REFERENCES control was also included in the test using a filter Alasbahi R, Safiyeva S, Craker L. 1999. Antimicrobial paper disc saturated with hexane, CH2Cl2 and MeOH activity of some Yemeni medicinal plants. J Herbs to check the possible activity of these solvents Spices Med Plants. 6:75-83. against the bacteria assayed. The experiments were Annapurna J, Chowdary I, Lalitha G, Ramakrishna S, repeated at least twice. Iyengar D. 2004. Antimicrobial Activity of Euphorbia nivulia leaf extract. Pharm Biol. 42: 91-93. Alvarez C, Cortes J, Arango A, Correa C, Leal A, Grebo. Results and discussion 2006. Resistencia antimicrobiana en unidades de E. caracasana and E. cotinifolia leaves were cuidado intensivo de Bogotá, Colombia, 2001-2003. extracted with isopropyl alcohol. These extracts, after Salud Pública. 8: 86-101. concentration, were fractionated by column Darwish R, Aburjai T, Al-Khalil S, Mahafzah A. 2002. chromatography and selected fractions, based on their Screening of antibiotic resistant inhibitors form local plant material against two strains of Staphylococcus TLC profiles, tested for antibacterial activity. aureus. J Ethnopharmacol. 79: 359-364. Fractions eluted with CH2Cl2/MeOH 1:1 from the Evans F, Taylor S. 1983. Pro-inflammatory, tumor column chromatography of E. caracasana extract promoting and antitumor diterpene of the plant showed antibacterial activity against S. aureus ATCC families Euphorbiaceae and Thymelaeaceae. In: Herz 25923 at concentrations of 336 μg/mL (inhibition W, Grisebach H, Kirby G. (Eds.), Progress in the zone 10 mm) and 33,6 μg/mL (dilution 1:10 of the Chemistry of Organic Natural Products, 44. Springer- same extract, inhibition zone 7 mm). Fractions eluted Verlag, New York, pp. 1-99. with n-hexane-CH2Cl2 1:1 and CH2Cl2 from the same

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