Antimicrobial activity of aqueous and methanolic extracts from Arthrospira maxima

N. B. Medina-Jaritz1, D. R. Perez-Solis1, S. L. Ruiloba de Leon F.2 and R. Olvera-Ramírez*1 1 Department of Botany, National School of Biological Sciences, National Politecnic Institute, Prol. Carpio y Plan de

Ayala s/n, 11340, Mexico City, D.F., Mexico 2 Department of Microbiology, National School of Biological Sciences, National Politecnic Institute, Prol. Carpio y Plan

de Ayala s/n, 11340, Mexico City, D.F., Mexico

Arthrospira maxima is a filamentous, undifferentiated, non-toxigenic cyanobacteria, that has been used as food since ancient times. There have been numerous studies on its antioxidant and anti-inflammatory activities, although antimicrobial action is mentioned, it has not been ascertained. To evaluate the antimicrobial activity of Arthrospira maxima different concentrations of aqueous and methanolic extracts were tested by the agar diffusion technique against Proteus vulgaris, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Candida albicans. The aqueous extracts showed antibacterial activity against all organisms tested, except to Bacillus subtilis, while methanol extract showed antimicrobial activity against all microorganisms, even to Staphylococcus aureus.

Keywords antimicrobial assays; antibacterial; antifungal; cyanobacteria; Spirulina; methanolic extracts.

1. Introduction

Cyanobacteria or blue–green algae are photoautotrophic microorganisms largely distributed in nature. Some of them have been used as human food for many years because of their high protein content (35–65%) and nutritional value. Arthrospira (Spirulina) is the best known genus and it was consumed by the Aztecs in Mexico Valley and by the Chaad lake population in Africa. At present, some countries are culturing it on a large scale[1]. Spirulina platensis is a planktonic photosynthetic filamentous cyanobacterium that forms massive populations in tropical and subtropical bodies of water which have high levels of carbonate and bicarbonate, and alkaline pH values of up to 11. This cyanobacterium is recognizable by the main morphological feature of the genus, i.e. the arrangement of multicellular cylindrical trichomes in an open left-hand helix along the entire length of the filaments[1]. For centuries, native peoples have harvested Spirulina from Chad Lake in Africa and Texcoco Lake in Mexico for use as a source of food[1], a fact which means that Spirulina deserves special attention both as a source of single cell protein (SPC)[2] and because of its nutraceutical properties. The chemical composition of Spirulina indicates that it has high nutritional value due to its content of a wide range of essential nutrients, such as provitamins, minerals, proteins and polyunsaturated fatty acids such as gamma-linolenic acid[3]. More recently, Spirulina has been studied because of its therapeutic properties[4] and the presence of antioxidant compounds[3, 5] such as phenolics. The occurrence of phenolic compounds in plants is well documented and these compounds are known to possess antioxidant activity in biological systems but the antioxidant characteristics of algae and cyanobacteria are less well documented, although decreased cholesterol levels have been reported in hypercholesterolemic patients fed Spirulina6 and the antioxidant activity of phycobiliproteins extracted from S. platensis has also been demonstrated [5]. It has been claimed that consumption of Spirulina is beneficial to health due to its chemical composition including compounds like essential amino acids, vitamins, natural pigments, and essential fatty acids, particularly γ-linolenic acid, a precursor of the body’s prostaglandins[7, 8, 9, 10]. It has also been reported that some cyanobacteria produce substances that can either promote or inhibit microbial growth[11, 12, 13]. Secondary metabolites from cyanobacteria are associated with toxic, hormonal, antineoplastic and antimicrobial effects [14, 15]. The antimicrobial substances involved may target various kinds of micro-organisms, prokaryotes as well as eukaryotes. The properties of secondary metabolites in nature are not completely understood [16, 17]. Secondary metabolites influence other organisms in the vicinity and are thought to be of phylogenetic importance. Recently, there has been an increasing interest in cyanobacteria as a potential source for new drugs [18, 19]. Antimicrobial effects from cyanobacterial aqueous and organic solvent extracts are visualized in bioassays using selected micro-organisms as test organisms [20, 21, 22]. Methods commonly applied are based on the agar diffusion principle using pour-plate or spread plate (seeded plates) techniques. Antimicrobial effects are shown as visible zones of growth inhibition (inhibition halos). Bacterial bioassays comprise different target bacteria: Micrococcus luteus, Bacillus subtilis, B. cereus and Escherichia coli are commonly used to detect antibiotic residues in food[23, 24]. A large number of microalgal and cyanobacterial extracts and/or extracellular products have been found to have antibacterial or antifungal activity. Pedersen and Dasilva[25] have pointed out that the cyanobacterium Calothrix brevissima produce bromophenols, which possesses antibacterial activity. Mundt et al.[26] have proved that the cyanobacterium Oscillatoria redekei produce fatty acids which show antibacterial activity. Matern et al.[27] isolated two depsipeptide metabolites, scyptolin A and B from the axenically grown terrestrial cyanobacterium Scytonema hofmanni

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PCC 7110 and designated as hofmannolin. Bloor and England[28] reported that extracellular metabolites produced by Nostoc muscorum inhibited the growth of Bacillus circulans. Austin et al.[29] reported that the supernatants and extracts derived from a commercial heterotrophically grown spray-dried preparation of Tetraselmis suecica (Chlorophyceae) were observed to inhibit Aeromonas hydrophila, A. salmonicida, Lactobacillus sp., Serratia liguefaciens, Staphylococcus epidermidis, Vibrio anguillillarum, V. salmonicida and Yersinia ruckeri type I. Kim et al.[30] extracted Hizikia fusiforme by solvents (hexane ethyl ether and ethanol), these extracts were found to be active against E. coli and Bacillus subtilis. Ohta et al. [31] have proved that the methanol extracts from Chlorococcum strain HS-101 and Dunaliella primolecta strongly inhibited the growth of a strain of methicillin resistant Staphylococcus aureus, which cause serious problems in Japanese hospitals. Ostensvik et al.[32] have examined five strains of cyanobacteria for antibacterial activity and they found that the methanol extracts made from Tychonema bourrellyi, Aphanizomenon flos-aquae and Cylindrospermopsis raciborskii showed the most pronounced inhibitory effects against Bacillus cereus and B. subtilis. Blue green algae (cyanobacteria) have been recognized in the last decades as a source of novel cytotoxic and antifungal metabolites. Some of these metabolites have potential for development of new pharmaceutical compounds. De Cano et al.[12] showed the antifungal activity of phenolic compounds from the terrestrial cyanobacterium N. muscorum against Candida albicans.

2. Methods

2.1 Propagation of Arthrospira maxima

This strain was selected from the strain collection of the Plant Physiology Laboratory, Department of Botany, located in the National School of Biological Sciences, National Polytechnic Institute, and was cultivated in 1 L Erlenmeyer flasks bubbled with air in Zarrouk medium[33], under photoperiodic illumination (12/12 photosynthetically active radiation provided by “daylight” fluorescent lamps, at 25 to 28°C.

2.2 Aqueous extracts of Arthrospira maxima

The aqueous extract was obtained from 1.5 g or 2.0 g biomass of A. maxima of 30 days of growth (harvested by filtration on Whatman No. 1), and resuspended in 10 mL of phosphate buffer pH 7, under aseptic conditions. The cell rupture was reached by freeze-thaw cycles until a change of color was observed. The suspension was centrifuged at 3500 rpm for 45 minutes, the supernatant fluid was withdrawn with a pipette to a sterile container.

2.3 Methanol extracts of Arthrospira maxima

Biomass from a 40 mL culture of 15 days of growth was harvested by filtration (Whatman No. 1), and then was weighed, adding, for each gram, 60 mL of methanol and 2mL of acetic acid, and rested for 24 h. After this time the methanol and acetic acid was evaporated to dryness by heating in a water bath. The resulting concentrate was resuspended in 3 mL of methanol.

2.4 Antimicrobial activity tests.

Different concentrations of aqueous and methanol extracts were tested by the agar diffusion technique against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus vulgaris and Candida albicans. They were cultivated in Müller Hinton solid media which was supplemented with yeast extract. Sterilized Petri dishes containing about 20 mL medium had been inoculated with 0.1 mL of microbial suspension adjusted to tube 1 MacFarland’s nephelometer with saline solution. Sterilized paper discs (0.6 cm) impregnated with 0.1 mL of the extracted material and air dried, were placed over the agar surface; two discs of each concentration of the extracts were placed on the plates with the tested microorganisms, also a filter paper disk impregnated with Zarrouk media and another disc impregnated with water or methanol (depending on the test extract) were placed. Two plates were tested per target microorganism. The plates were left 2 h at 4 ◦C then incubated at 37 ◦C for 24 h and examined for zones of inhibition around the disc (fig. 1).

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3. Results and Discussion

The antimicrobial activity was evaluated as the diameters of the inhibition halos formed as a result of disc assay method. Data present in Table 1 show that the aqueous extract of A. maxima was antagonistic to all test microorganisms examined except B. subtilis. The highest antagonism of the aqueous extract was recorded against S. aureus. However, inside the inhibition halos there were some resistant colonies, indicating that these colonies represent a variant (mutant) strain. The methanol extracts showed better antimicrobial activity against all strains used, including Bacillus subtilis that was between the most susceptible strains to the methanol extract.

Table 1 Mean average and standard deviation of inhibition halos from aqueous and methanol extracts from Arthrospira maxima.

Extract Aqueous Methanolic Target microorganism

Inhibition halos (ᴓ mm) with 0.15 g/mL

Inhibition halos (ᴓ cm) with 0.2 g/mL

Inhibition halos (ᴓ cm) with 0.33 g/mL

Inhibition halos (ᴓ cm) with 0.5 g/mL

Inhibition halos (ᴓ cm) with 0.66 g/mL

ma sd ma sd ma sd ma sd ma sd E. coli 11.5* 0.6 11.8* 0.5 16.8 1.0 40.0 1.0 36.5 4.1 P. vulgaris 14.0* 2.4 13.0* 2.0 13.8 3.6 44.8 3.9 43.0 1.8 C. albicans 11.8* 0.5 11.8* 0.5 9.4 1.1 17.3 0.3 22.8 4.7 S. aureus >50.0 0.5 >50.0 0.5 0.0 0.0 43.0 1.8 41.3 1.3 B. subtilis 0.0 0.0 0.0 0.0 13.5 1.7 41.3 5.1 41.5 3.3 Control 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

*Some colonies growth in the inhibition areas

Data also show that the three concentrations of methanolic extract of A. maxima were antagonistic to all target microorganisms but S. aureus to 0.33 g/mL. The highest antagonism of the methanolic extract was recorded against B. subtilis, P. vulgaris and S. aureus (with 0.5 g/mL and 0.66 g/mL). The larger inhibition halos were with methanolic extract at 0.5 g / mL, in some cases the inhibitory activity decreased while concentration was increased; the inhibition of growth of P. vulgaris and E. coli was larger with methanolic extracts compared with aqueous extracts. Meanwhile, the inhibition halos against C. albicans were similar with both extracts (aqueous and methanolic). The plates seeded with S. aureus showed little growth across the board in both tests, indicating susceptibility to water extracts and methanolic extract at 0.33 g/mL. Clearly, there was a similar inhibitory effect between the last two concentrations and even in most cases the average concentration of 0.5 g / mL resulted in greater inhibition zones, when it was expected that higher concentrations would produce greater inhibition due to the cause-effect relationship between the level of exposure to a substance and the magnitude of the response to this, the toxicity being directly proportional to the dose in most cases. This result can not be fully elucidated, since no one knows the structure of the metabolite responsible for the antimicrobial activity, although it may be due to linoleic acid and other fatty acids whose antimicrobial activity has been tested[34] or synergistic effect between these fatty acids[35]. However, as Arthrospira as well has harmful or toxic compounds to microorganisms, has compounds that can serve as substrates (vitamins, proteins, carbohydrates, trace elements, fibers, lipids, organic acids, etc.) that stimulate the growth of microorganisms, it is possible that at 0.5g/mL, the metabolites

Fig. 1 The appearance of the target microorganisms-seeded Petri plate assay for antimicrobial activity of A. maxima extract showing the inhibition halos caused.

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responsible for the antimicrobial activity were qualitatively and quantitatively in the right proportion to deliver antimicrobial activity while at 0.66 the concentration of other metabolites stimulate the growth. The results above may be the basis for further studies that seek to purify and characterize the metabolite responsible for the activity shown, to propose alternatives therapies (less harmful) for the treatment of opportunistic infections and even for infections caused by pathogenic microorganisms.

Acknowledgements The support by SIP project 20101161 and COFAA (IPN) is gratefully acknowledged.

References

  [1]  Vonshak A, ed. Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology. London, UK; Taylor & Francis; 1997. 233.

[2] Anupama PR. Value-added food: single cell protein. Biotechnology Advances; 2000. 18: 459–479. [3] Miranda MS, Cintra RG, Barros SBM, Filho JM. Antioxidant activity of the microalga Spirulina maxima. Brazilian Journal of

Medical and Biological Research; 1998. 31: 1075–1079. [4] Belay A, Ota Y, Miyakawa K, Shimamatsu H. Current knowledge on potential health benefits of Spirulina. Journal of Applied

Phycology; 1993. 5: 235–241. [5] Estrada JE, Bescós P, Villar Del Fresno AM. Antioxidant activity of different fractions of Spirulina platensis protean extract. Il

Farmaco; 2001. 56: 497–500. [6] Ramamoorthy A, Premakumari S. Effect of supplementation of Spirulina on hypercholesterolemic patients. Journal of Food

Science and Technology; 1996. 33(2): 124–128. [7] Fox RD. Spirulina, production and potential. Edisud; 1996. Aix-en Provence, France. [8] Richmond, A. Spirulina. In: Borowitzka MA, Borowitzka LJ. eds., Micro-Algal Biotechnology. Cambridge University Press;

1987. 85-121. [9] Doumenge F, Durand-Chastel E, Toulemont A. Spirulina algue de vie. Bull. Inst. Oceanogr. Monaco; 1993. 2: 222. [10] Henrikson R. Microalga Spirulina. Barcelona. Urano; 1994. 220 pp. [11] de Caire GZ, de Cano MS, de Mulé MCZ, de Halperin DR, Galvagno MA. Action of cell-free extracts and extracellular

products of Nostoc muscorum on growth of Sclerotinia sclerotiorum. Phyton; 1987. 47: 43–46. [12] de Cano MMS, De Mule MCZ, De Caire CZ, De Halperin DR. Inhibition of Candida albicans and Staphylococcus aureus by

phenolic compounds from the terrestrial cyanobacterium Nostoc muscorum. J. Appl. Phycol.; 1990. 2: 79-82. [13] Kulik MM. The potential for using cyanobacteria (blue-green algae) and algae in the biological control of plant pathogenic

bacteria and fungi. European Journal of Plant Pathology; 1995. 101: 585-599. [14] Carmichael WW. Cyanobacteria secondary metabolites-the cyanotoxins. J. Appl. Bacterol.; 1992. 72: 445-459. [15] Patterson GML, Larsen LK, Moore RE. Bioactive Natural Products from Blue-green Algae. J. Appl. Phycol.; 1994. 6: 151-

157. [16] Metting B, Pyne JW. Biologically active compounds from microalgae. Enzyme and Microb. Technol.; 1986. 8: 386–394. [17] Inderjit L, Dakshini KMM. Algal allelopathy. The Botanical Review; 1994. 60: 182–196. [18] Glombitza KW, Koch M. Secondary metabolites of pharmaceutical potential. In: Cresswell RC, Rees TAV, Shah N, eds.

Algal and Cyanobacterial Biotechnology. Harlow, UK: Longman Scientific & Technical.;1989. 161–238. [19] Schwartz RE, Hirsch CF, Sesin DF. Pharmaceuticals from cultured algae. Journal of Industrial Microbiology; 1990. 5: 113–

124. [20] Kellam SJ, Cannel RJP, Owsianka, AM, Walker JM. Results of a large-scale screening programme to detect antifungal activity

from marine and freshwater microalgae in laboratory culture. British Phycological Journal; 1988. 23: 45–47. [21] Frankmo¨lle WP, Larsen LK, Caplan FR. Antifungal cyclic peptides from the terrestrial blue-green alga Anabaena laxa. The

Journal of Antibiotics; 1992. 45: 1451–1457. [22] Falch BS, König GM, Wright AD. Biological activities of cyanobacteria: evaluation of extracts and pure compounds. Planta

Medica;1995. 61: 321–328. [23] Crosby NT. Determination of Veterinary Residues in Food. New York. Ellis Horwood. 1991. 108-116. [24] McGill AS, Hardy R. Review of the methods used in the determination of antibiotics and their metabolites in farmed fish. In:

Michel C, Alderman DJ, eds. Chemotherapy in Aquaculture: from Theory to Reality. Paris, France: Office International Des Epizooties. 1992, 343–380.

[25] Pedersen M, Dasilva EJ. Simple brominated phenols in the bluegreen alga Calothrix brevissima. West. Planta; 1973. 115: 83-96.

[26] Mundt S, Kreitlow S, Jansen R. Oscillatoria redekei HUB 051. Journal of Applied Phycology. 2003; 15(2-3): 263-267. [27] Matern U, Oberer L, Erhard M, Herdman M, Weckesser J. Hofrnannolin a cyanopeptolin from Scytonema hofmanni PCC

7110. Phytochemistry; 2003. 64: 1061–1067. [28] Bloor S, England RR. Elucidation and optimization of the medium constituents controlling antibiotic production by the

cyanobacterium Nostoc muscorum. Enzyme Microb. Technol.; 1991. 13: 76-81. [29] Austin, B., E. Baudet & M. stobie, 1992. Inhibition of bacteria fish pathogens by Tetraselmis suecica. Journal of Fish Dieases.

15:53-61. [30] Kim SH, Lim SB, Ko YH, Oh, CK, Park CS. Extraction yields of Hizikia fusiforme by solvents and then antimicrobial effects.

Bull. Kor. Fish. Soc.; 1994. 27: 462-468. [31] Ohta S, Shiomi Y, Kawashima A, Aozasa O, Nakao IT, Nagate N, Kitamura K, Miyata H. Antibiotic effect of linolenic acid

from Chlorococcum strain HS-IO1 and Dunaliella primolecta on methicillin-resistant Staphylococcus aureus. Journal of Applied Phycology; 1995. 7: 121-127.

1270 ©FORMATEX 2011

Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)______________________________________________________________________________

[32] Ostensvik , Skulberg OM, Underdal B, Hormazabal V. Antibacterial properties of extracts from selected planktonic freshwater cyanobacteria. A comparative study of bacterial bioassays. The Society for Applied Microbiology; 1998. 84: 1117-1124.

[33] Zarrouk, C. Contribution a l’estudie d’una cyanophyceé. Influence de divers facteurs physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima. Ph. D. Thesis. Université de Paris. 1966.

[34] Benkendorff K, Davis AR, Rogers CN, Bremner JB. Free fatty acids and sterols in the benthic spawn of aquatic molluscs, and their associated antimicrobial properties. Journal of experimental marine biology and ecology; 2005. 316(1): 29-44.

[35] Mendiola JA, Jaime L, Santoyo S, Reglero G, Cifuentes A, Ibanez E, Senorans FJ. Screening of functional compounds in supercritical fluid extracts from Spirulina platensis. Food Chem.; 2007. 102: 1357-1367.

 

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