Abstract
The antimicrobial activities of methanol, methanol–chloroform, diethyl ether, ethyl acetate, and butanol extracts of Gracilaria changii B.M. Xia & I.A. Abbott (Gracilariaciae) were studied. These extracts were tested against 22 bacteria, six yeasts strains, and 11 fungal isolates by the disk diffusion and broth dilution methods. The results indicated that all of the solvent systems used in the study had equal effectiveness against the tested microorganisms. All of the extract preparations of G. changii showed significant activities against seven bacterial and four yeast isolates tested. The minimum inhibitory concentration (MIC) values of Pseudomonas aeruginosa and Bacillus subtilis were 6.25 and 3.125 to mg/mL, respectively, while the MIC for Candida albicans was 3.125 mg/mL. No fungal isolates showed any susceptibility against the different crude preparations of G. changii. The activities were compared to known commercialized antibiotics such as chloramphenicol and miconazole nitrate.
Introduction
Antibiotics are defined as substances produced by microorganisms that, in high dilution, are antagonistic to the growth or life of other microorganisms (CitationO’Grady et al., 1997). As a consequence of an increasing demand for biodiversity in the screening programs seeking therapeutic drugs from natural products, there is greater interest in marine organisms, especially algae or seaweed. The ability of seaweed to produce secondary metabolites has been extensively documented (CitationCaballero & Melian, 1990; CitationFaulkner, 1993).
Malaysia is naturally endowed with a very rich diversity of algae. Among the algae with therapeutic properties in Malaysia, Gracilaria changii B.M. Xia & I.A. Abbott (Gracilariaciae) has yet to gain importance and popularity. G. changii is found predominantly in the mangrove areas of Malaysia. It grows abundantly in hot, humid equatorial countries such as Malaysia and Thailand. Although at one time it was considered a foodstuff because the alga is edible, the economic importance of the alga is now beginning to attract the attention of researchers. The Gracilaria spp. are used widely in traditional medicine in Malaysia. The Malay people administer the agar derived from Gracilaria internally to treat coughs and consumption (CitationBurkill, 1935). In addition, Gracilaria spp. boiled in vinegar is used as a poultice to treat swollen knees and unhealthy sores (CitationBurkill, 1935).
There are numerous reports of compounds with a broad range of biological activity in red, green, and brown algae (CitationMunro et al., 1987; Scheuer, 1990Citation), but not many on the local marine algae in Malaysia. Therefore, the present study evaluated the in vitro antimicrobial activity of different crude extract preparations of a local alga, G. changii.
Materials and methods
Microorganisms
Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Acinetobacter calcoaceticus, Staphylococcus saprophyticus, Serratia marcescens, Klebsiella pneumoniae, Acinetobacter anitratus, Salmonella paratyphyi B, Bacillus licheniformis, Micrococcus sp., Staphylococcus epidermidis, Citrobacter preundii, Erwinia enterocolitica, Salmonella typhi, Burkholderia pseudomallei, Bacillus subtilis, Erwinia sp., Bacillus cereus, Enterobacter aerogenes, Candida albicans strains 1, 2 and 3, Rhodotorula rubra, Cryptococcus neoformans, Saccharomyces cerevisiae, Trichoderma viride, Rhizopus sp., Mucor sp., Penicillium sp., Fusarium sp., Trichophyton rubrum, Microsporum canis, Trichophyton mentagrophytes, Fusarium oxysporium, Aspergillus niger, and Aspergillus flavus were the test organisms used and were obtained from the Fermentation and Enzyme Technology Laboratory, University of Science Malaysia. The bacterial isolates were maintained on nutrient agar slants at 37°C while fungi including the yeasts were maintained on Sabouraud dextrose–agar slants at room temperature. The stock culture was maintained on Nutrien agar slants (for bacteria) and Sabouraud dextrose–agar slants (for fungi) at 4°C.
Algal sample
A fresh sample of G. changii was collected from Pantai Morib, Selangor, Malaysia, in January 2003 and authenticated by Professor Phang Siew Moi (Institute of Biological Sciences, Faculty of Science, University of Malaya).
Extraction procedure
At the field the fresh algae were rinsed with seawater to remove debris and epiphytes before transport to the laboratory. In the laboratory, the algae were further washed with fresh water and brushed with a soft brush before being sun-dried. The-sun dried algae were cut into small pieces. Approximately 100 g of dried algae was added to 200 mL each of methanol and methanol–chloroform mixture (1:1-v/v) and soaked for 4 days. Removal of the alga from the solvents was done by filtration through cheesecloth and the filtrate was concentrated using a rotary evaporator. After evaporation, 2.708 g of polar fraction (2.708%) was obtained from the methanol extract and 2.712 g (2.712%) from the methanol–chloroform (1:1) extract. The polar fraction (methanol extract) was further partitioned in aqueous diethyl ether (1:1-v/v) and yielded 1.511 g (1.511%) of diethyl ether fraction. Subsequently the aqueous layer formed was further partitioned in ethyl acetate (100 mL) to yield about 0.987 g (0.987%) of ethyl acetate fraction. Consequently, the aqueous layer formed was further partitioned in butanol (100 mL) and yielded 0.415 g (0.415%) of butanol fraction (CitationDuan et al., 2006). The aqueous layer formed was stored in a refrigerator at 4°C. The entire fractions were evaporated to dryness in a rotary evaporator. The dried fractions were then re-dissolved in 10% DMSO (v/v) to yield solutions containing 100 mg extract/mL. All solvents used were HPLC grade.
Antimicrobial activity
The antimicrobial activity of the extracts was determined following the method described by CitationNCCLS (2002) with slight modifications to the medium used (Muller–Hinton agar replaced with Sabouraud dextrose agar or nutrient agar).
Disk diffusion technique
The test microbe was removed aseptically with an inoculating loop and transferred to a test tube containing 5 mL of sterile distilled water. Sufficient inoculums were added until the turbidity equaled 0.5 McFarland (108 cfu/mL) standards (bioMerieux, Marcy d’Etoile, France). The test tube suspension (1 mL) was added to 15–20 mL of nutrient agar or Sabouraud dextrose agar before setting aside the seeded agar plate (9 cm in diameter) to solidify for 15 min. Three disks of Whatman’s No. 1 filter paper, 6 mm in diameter, were used to screen the antimicrobial activity. Each sterile disk was impregnated with 20 μL of extract (corresponding to 100 mg crude extract/mL), chloramphenicol or miconazole nitrate (30 μg/mL, as positive control for bacteria and fungi, respectively), or 10% DMSO (v/v) (as negative control), before it was placed on the surface of the seeded plates. The plates were incubated at 37°C overnight and examined for zones of growth inhibition.
Determination of minimum inhibitory concentrations
The determination of minimum inhibitory concentration (MIC) was applied on extracts that proved their high effectiveness against microorganisms by the disk diffusion method. The highest dilution of a extract that still retains an inhibitory effect against the growth of a microorganism is known as the MIC (CitationMisra & Dixit, 1978). The complete protocol of the MIC test is found in the M7-T2 publication of the National Committee for Clinical Laboratory Standards (NCCLS, 2000). Briefly, different extract preparations were subjected to a serial dilution using sterile nutrient broth or Sabouraud dextrose broth medium as a diluents to give final crude extract concentrations between 1.275 and 200.000 mg/mL. The tubes were inoculated with the bacterial or yeast suspension (20 μL/mL broth), homogenized, and incubated at 37°C for 24 h. The lowest dilution of the extract that retained its inhibitory effect resulting in no growth (absence of turbidity) of a microorganism was recorded as the MIC value of the extract. The microorganism growth was indicated by the turbidity. Each test was performed in triplicate and repeated twice. A control experiment was run in parallel to study the impact of the solvent itself (without algal component) on growth of the test organisms. The solvent (10% DMSO) was diluted in a similar pattern with sterile nutrient broth or Sabouraud dextrose broth, as indicated above, and inoculation by microorganisms followed by incubation was done similarly.
Results
The crude extract preparations (methanol, methanol–chloroform, diethyl ether, ethyl acetate, and butanol) of G. changii exhibited zones of inhibition by the disk diffusion method against seven out of 22 bacterial and four out of six yeast isolates tested. The Gram-positive bacterium, B. subtilis, and the Gram-negative bacterium, P. aeruginosa, were found susceptible to the extracts and exhibited zones of growth inhibition at the concentration of 100 mg/mL ().
Table 1. Antimicrobial activitiesa of various extractsb of Gracilaria changii.
Among the Gram-negative bacteria tested, K. peneumoniae, S. typhi, and P. aeruginosa exhibited zones of inhibition between 10 and 14 mm in diameter. However, some of the Gram-negative and Gram-positive bacteria used were resistant to the crude extracts. The yeasts, C. albicans and R. rubra, were found with zones of inhibition of 10–14 mm). On the other hand, no antifungal activity was exhibited by all of the crude preparations of G. changii. As seen clearly from and , all of the solvents had equal efficiency in the present study.
shows the MIC values for each of the microorganisms which were inhibited by the algal extracts. A total of eight microorganisms showed MIC values of 3.125 mg/mL and four microorganisms showed MIC values of 6.25 mg/mL for all of the crude preparations tested.
Table 2. Minimum inhibitory concentration (mg/mL) of various extractsa of Gracilaria changii.
Discussion
The antimicrobial activities of methanol, methanol–chloroform, diethyl ether, ethyl acetate, and butanol extracts of G. changii were studied. In our study, G. changii had a wide variety of antimicrobial activity against pathogenic microorganisms.
The main objective of the study was to evaluate and compare the ability of different G. changii crude extract preparations against various test microorganisms. As for the effectiveness of the extraction method, some studies have shown that methanol extraction yielded higher antimicrobial activity thann-hexane and ethyl acetate (CitationFebles et al., 1995), whereas others found chloroform to be better than methanol and benzene (CitationSastry & Rao, 1994). It is clear that the use of organic solvents always provides a higher efficiency in extracting antimicrobial active compounds. Our studies showed that the active compounds were present in all of the organic solvents used in the present study. The different crude preparations showed different spectrum of activities, especially by the disk diffusion method, where the microorganisms tested produced zones of inhibition about 10–14 mm in diameter.
The agar dilution method showed MIC values of 3.125 to 6.25 mg/mL for each of the microorganisms which were inhibited. Furthermore, the microorganisms that were inhibited showed relatively lower MIC values ranging from 3.125 to 6.25 mg/mL. The broader spectrum of activities could be due to synergistic effects of the various components in the G. changii extract. The observation of a large zone of inhibition produced against P. aeruginosa, but with MIC value of 6.25 mg/mL only, can be explained on the basis that the diffusion method can be used only to detect antimicrobial activity and not to quantify the activity. C. albicans, which causes vulvovaginitis, oral thrush, diarrhea, and systemic infections in immunocompromised hosts, could be treated with the methanol extract, as the MIC for this fungus was found to be only 3.125 mg/mL. The G. changii extracts also could be used to treat R. rubra, S. typhi, and S. aureus infections.
Further purification of the active compounds and in vivo evaluation of their antimicrobial activity, along with toxicity studies of the extracts from G. changii, are therefore suggested as further studies.
Acknowledgement
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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