versión On-line ISSN 1851-5657
Phyton (B. Aires) v.73 Vicente López ene./dic. 2004
Active fractions from four species of marine algae
Oranday MA, MJ Verde, SJ Martínez-Lozano, NH Waksman
Department of Biochemistry. School of Biological Sciences, Department of Toxicology, School of Medicine, and Facultad de Ciencias Biológicas. Universidad Autónoma de Nuevo León 66450. Monterrey (México)
Received 19.IV.2003: accepted 28.V.2003
We wish to thank CONACYT
Abstract. A bioassay-directed is utilized to detect substances with biological activity from Gracilaria tikvahiae, Ulva lactuca, Ulva fasciata and Sargassum fluitans. In a preliminary assessment, polar and non polar extracts of four species of marine protoctist form were screened for antibacterial and antifungal properties against seven microorganisms by the diffusion method, non polar extracts of Sargassumfluitans,and polar extracts of Gracilaria tikvahiae inhibited the growth of more than four microorganisms. Extracts were separated using chromatography column and fractions were tested againstStapylococcusaureus andCandida albicans. The eighty fraction of petroleum ether of S. fluitans exhibited high activity against C.albicans, MIC 0.16 µg/mL.
In recent years several marine bacterial and protoctist forms have been confirmed as important source of new compounds potentially useful for the development of chemotherapeutic agents.Previous investigations of the production of antibiotic substances by aquatic organisms point to these forms as a rich and varied source of antibacterial and antifungal agents.
Antimicrobial substances produced by benthic tropical marine forms were studied by Allen & Dawson (1) and were screened by them against E. coli and S. aureus. Mesmar & Abussaud (2) reported that most effective antibacterial agents were species from the marine protoctist phylum Pr 15. Chlorophyta (green), followed by Pr 12. Phaeophyta (brown) and Pr 13. Rhodophyta (red). König & Wrigth (3) provide an overview of the literature related to the three forms that contain naturally active products. Species of the genera studied have been widely reported for their antimicrobial activities. Acrylic acid, the first compound with antibiotic activity of any of these marine forms, was obtained from the genus Gracilaria (4). Antiviral activity of the alcohol extract of Ulva fasciata was reported by Sharma & Bhakuni (1992). Antibiotic substances of Sargassum natans were isolated by Martínez & Casillas (7), from Sargassum, which they subsequently characterized (6). Glombitza & Sukopp (4) report polihidroxphenyl ether with antimicrobial activity from the brown alga Sargassum spinuligerum.
The nonpolar extracts obtained by successive extractions using benzene, chloroform and methanol exhibited high antimicrobial activity according to Sastry & Rad (9).
This study reports the screening for antimicrobial activity of the chlorophyte (Ulva fasciata, Ulva lactuca), rhodophyte (Gracilaria tikvahiae) and phaeophyte (Sargassum fluitans), all of which are marine forms.
MATERIALS & METHODS
Plant material. The marine material used in this study was collected from the coast of the Gulf of Mexico at N20º 28' 28", W.97º 51' 05" during August 1994. Samples of these forms were authenticated by Dra. Leticia Villarreal Rivera of our Department of Botany and have been deposited in the herbarium of the Faculty of Biology at Universidad Autónoma de Nuevo León.
Chemical extraction and fractionation.The extracts were obtained by macerating 30 g of the dried plant in cold petroleum ether for 48 h, with the resultant extract being filtered and then concentrated to dryness in a rotary evaporator under reduced pressure. Chloroform, acetone, ethanol, methanol or water extracts were obtained similarly. The extracts were diluted in different solvents for approximately 2.5 mg/ml, then sterilized by passage through 0.45 µm filter.
The active extract was analyzed chromatographically on silica gel 60 (Baker) eluted with benzene acetone gradient into several fractions giving 42 fractions tested against S. aureus and C. albicans.
Microorganisms. The strains used in this study (E.coli, S.aureus, S, enteriditis, S. epidermidis, P. aeruginosa, S, faecalis and C. albicans), were provided by Manuel Rodriguez Q. (Department of Microbiology Fac. Medicina, Universidad Autónoma de Nuevo León), the bacterial strains were grown and maintained on nutrient agar slants. Yeast was cultured on Sabouraud dextrose 45 agar slants. The inoculated agar was incubated a 37ºC.
Antimicrobial activity test. Test was performed by the diffusion method (Rios et al) using 1 ml. of inoculum contains 10 microorganisms per plate. Extract-impregnated discs (10 µg per disc) were placed on the agar Muller Hinton and incubated at 37ºC overnight. Sterile solvents were used as negative control and various dilutions of ampiciline and penprocilin were used as positive control. Experiments were carried out in quadruplicate. Antimicrobial activities of crude extracts were indicated by clear zones of growth inhibition The MIC value was taken as the lowest concentration of compound which inhibited the growth of the test microorganisms after 24 h of incubation at 37ºC
RESULTS & DISCUSSION
The comparative preliminary analysis of the macerated and soxhlet extracts (Table 1) showed that macerated extracts are the more active. A total the 42 crude extracts corresponding to species were tested (Table 2); antimicrobial activity was found in ether extracts of Sargassum fluitansagainst E. coli, S. aureus, Candida albicans and S. epidermidis; these pharmacological activities have been reported in lipophilic extract of phaeophytas by Tringali et al. acetone extracts of Gracilaria foliifera exhibited activity against microorganisms mentioned, and P. aeruginosa, antimicrobial activity of extract genus Gracilaria was already reported (Hoppe et al, Nagal et al). The results of genus Ulva showed moderate activity against C. albicans and S. aureus and was not more considered in the present study.
Table 1. Comparison of extraction soxhlet Vs dipping
Table 2 . Results of the antimicrobial screening of algae
Active extracts the S. fluitans and G. tikvahiae were separated using chromatography column, and fractions were tested against these microorganisms (Table 3), eigth fractions of petroleum ether of S. fluitans exhibited high activity against C. albicans, MIC 0.16 µg/mL
Table 3. Results of the antimicrobial screening of fractions separated using chromatography column
The present study confirms the traditional use the cold extract in marine plants. The four algae showed antimicrobial activity on several of the selected pathogenic microorganisms. The wider spectrum was presented for Sargassum fluitans and Gracilaria follifera showed grand activity, since it inhibited more 4 microorganisms. Most antimicrobial activity was found in ether extracts of Sargassum fluitans against E. coli, S. aureus, Candida albicans and S. epidermidis, these pharmacological activities have been reported in lipophilic extracts of phaeophytas by Tringali et al; acetone extracts of Gracilaria follifera exhibited activity against microorganisms mentioned, and P. aeruginosa, antimicrobial activity of extract genus Gracilaria was already reported (Hoppe et al, Nagal et al). The eight fraction of petroleum ether of S. fluitans exhibited high activity against C. albicans MIC 0.16 µg/mLThe fact that the algae tested demonstrated antimicrobial activity, emphasizes the important role of traditional medicine in the search for antibiotic compounds from natural sources.
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