Antibacterial activity of extracts of six macroalgae from the northeastern brazilian coast

Avaliação de atividade antibacteriana de extratos de seis macroalgas do nordeste brasileiro

Antibacterial activity of extracts of six macroalgae from the northeastern brazilian coast

Avaliação de atividade antibacteriana de extratos de seis macroalgas do nordeste brasileiro

José Vitor M. Lima-Filho^I; Ana F.F.U. Carvalho^II; Sissi M. Freitas^II; Vânia M.M. Melo^II

^IDepartamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte

IIDepartamento de Biologia, Universidade Federal do Ceará, Fortaleza, CE, Brasil

^{Correspondence}

SHORT COMMUNICATION

ABSTRACT

Hexane, chloroform and ethanol extracts of six marine macroalgae (Rhodophyta and Chlorophyta) from North Ceará coast (Northeast Brazil) were evaluated for antibacterial activity by the single disk method. Best results were shown by the hexane extracts of Amansia multifida against enteric Gram-negative strains such as Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, S. cholerae-suis, Serratia marcescens, Vibrio cholerae and the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus.

Key words: antibacterial activity, algae, lipid-soluble extracts

RESUMO

Extratos lipídicos de seis macroalgas (Rhodophyta e Chlorophyta) coletadas na costa do Estado do Ceará foram avaliadas para atividade antibacteriana pelo método de difusão em disco. Os extratos de Amansia multifida (obtidos a partir dos solventes orgânicos hexana, clorofórmio e etanol) demonstraram os melhores resultados contra as espécies entéricas Gram negativas de Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, S. cholerae-suis, Serratia marcescens, Vibrio cholerae e bactérias Gram positivas Bacillus subtilis e Staphylococcus aureus.

Palavras-chave: atividade antibacteriana, algas, extratos lipídicos

More than 150 000 macroalga or seaweed species are found in the oceans of the globe but only a few of them were identified (9). Secondary or primary metabolites from these organisms may be potential bioactive compounds of interest for the pharmacological industry (1). Special attention has been reported for antiviral, antibacterial and/or antifungal activities related to marine algae against several pathogens (3,5,6,7,8,13,14,17,20). As an efficient strategy of investigation, organic solvents have been used to extract the possible lipid-soluble active principles from macroalgae (11). Since Brazil has an extensive coast where algae from virtually all groups are present, the goal of the present work was to test six macroalgae from Northeastern Brazil for antibacterial activity.

Samples of algae were collected during low tide at Pacheco Beach, Ceará State, from May to June 1995. Ecological damage during harvesting was prevented by not removing the algae stems. All samples were brought to laboratory in plastic bags containing sea water to prevent evaporation and then washed with distilled water to separate potential contaminants. Algae were identified following Harvey (9), and belonged to two families: Chlorophyceae (Ulva fasciata DELILE, Caulerpa cupressoides WEST IN VAHL, Caulerpa prolifera FORSSKAL) and Rhodophyceae (Gracilaria domingensis SONDER, Gracilaria sp., Amansia multifida LAMOUROUX).

Samples were dried in oven at 37ºC and ground in an electric coffee mill. Resulting powder was submitted to lipid-soluble extraction with hexane, chloroform and ethanol 1:15 (p:v) using a Soxhlet extractor at 55-60ºC. All samples were refluxed until saturation (24 h) and the respective extracts were dried in an oven at 50ºC (chloroform and ethanol extracts) or rotavapor (hexane extracts). Subsequently, the residual extracts were suspended in the respective solvents to final concentration of 1mg/20 µl.

Antibacterial activity was tested against the pathogenic Gram-negative strains of Citrobacter freundii, Escherichia coli (ATCC – 13863), Enterobacter aerogenes, Klebsiella pneumoniae (ATCC – 10031), Morganella morganii, Pseudomonas aeruginosa (ATCC – 25619), Salmonella typhi, S. enterica subsp. Typhimurium, S. enterica subsp. Enteritidis, S. cholerae-suis, Serratia marcescens and Vibrio cholerae or Gram-positive strains of Bacillus subtilis (ATCC – 6633), Staphylococcus epidermidis and S. aureus (ATCC – 6538). Single disk method as described by Bauer et al was used (4). The bacterial strains used in this work (others than ATCC strains) were isolated from human beings and belong to the laboratory collection of the Department of Biology from Federal University of Ceará. All bacteria were grown (10⁸ cells/ml) in nutrient broth incubated at 37ºC for 24 h and plated, using a sterile swab, onto Petri dishes containing Müeller-Hinton agar. At the same time, sterile discs of 5 mm diameter were embedded with 20 µl of the algal lipid-soluble extracts – experimental group – or with the solvent – control group – and added to the cultured dishes. Toxicity of the macroalgae extracts against microorganisms was determined after 24 h at 37ºC by measuring the diameter of the halo around the discs, using a pachymeter. All experiments were performed at least in duplicate. Representative halos were those measuring a diameter superior to 10 mm.

Results are described in Table 1. The largest halos were achieved by the hexane extract of the Rhodophiceae Amansia multifida against E. aerogenes, K. pneumoniae, P. aeruginosa, S. typhi, S. cholerae-suis, Ser. marcescens, Vibrio cholerae, B. subtilis and Staphylococcus aureus. In addition, Gracilaria sp. produced a discrete inhibition halo against B. subtilis. Presence of representative halos in Chlorophyceae hexane extracts was shown only by C. cupressoides against M. morganii, B. subtilis and S. epidermidis. Other algae hexane, chloroform and ethanol extracts achieved halos not superior to 10 mm or were not identified (data not shown). Likewise, solvents used alone (controls) produced no halo (data not shown).

Lipid-soluble extracts from marine macroalgae have been investigated as a source of substances with pharmacological properties. Moreover, several different organic solvents have been used to screening algae for antibacterial activity. Early, Olessen et al. (12) related antibacterial activity in the chloroform and acetone extracts of Falkenbergia billebrandii against S. aureus. Kamat et al. (10) reported antiviral activities in ethanol extracts from 17 macroalga species of Indian Coast from 31 species tested. Moreover, antiherpes and antiinfluenza activities have been recently reported (18,19). Sastry et al. (16) showed antibacterial activity against Gram-positive and Gram-negative pathogenic strains after successive extraction with benzene, chloroform and methanol. Likewise, Mahasneh et al. (11) have shown antibiotic activity in organic extracts of six species of marine algae against multi-antibiotic resistant bacteria.

Antimicrobial activity depends on both algal species and efficiency on extraction of their active(s) principle(s). For example, water extracts from U. fasciata, C. prolifera and G. domingensis were all effective against Proteus vulgaris but the same did not happen with A. multifida (21). Likewise, lipid-soluble extracts of A. multifida (chloroform-methanol 2:1, 30 min reflux) tested against P. aeruginosa, E. coli, B. subtilis and S. aureus have shown no activity (2). On the other hand, from the 6 algae used in the present study, only the hexane extract of the Rhodophyceae A. multifida was effective against all bacteria tested (Table 1).

The chemical nature of active principles in lipid-soluble extracts of marine algae is not so far totally identified. Rossel et al. (15) associated antibiotic activity from 10 Xantophyta to the presence of unsaturated fatty acids, organic acids and phenol compounds. Our preliminary results suggest that antibacterial activity observed in A. multifida hexane extract against Gram-positive as well Gram-negative bacteria could be due to more than one active principle. This hypothesis will be further investigated.

Finally we conclude that macroalgae from the Brazilian coast are potential sources of bioactive compounds and should be investigated for natural antibiotics.

Submitted: April 18, 2002; Returned to authors for corrections: August 19, 2002; Approved: December 05, 2002.

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Publication Dates

• Publication in this collection
  18 June 2003
• Date of issue
  Dec 2002

History

• Accepted
  05 Dec 2002
• Received
  18 Apr 2002
• Reviewed
  19 Aug 2002