Evaluation of in vitro antimicrobial property of seaweed ( Halimeda tuna ) from Tuticorin coast , Tamil Nadu , Southeast coast of India

The seaweed (Halimeda tuna) was examined for antibacterial and antifungal activity in vitro using the well diffusion method, minimum inhibitory concentration, minimum bactericidal concentration and minimum fungicidal concentration. The activity was against 10 bacterial strains (Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi, Klebsiella oxytoca, Escherichia coli, Proteus mirabillis, Lactobacillus vulgaris, Pseudomonas sp., Klebsiella pneumonia and Vibrio cholerae) and nine fungal strains (Aspergillus niger, Aspergillus flavus, Alternaria alternaria, Candida albicans, Epidermophyton floccossum, Trichophyton mentagrophytes, Trichophyton rubrum, Pencillium sp. and Rhizopus sp.). The methanolic extracts in the present study exhibited a broad spectrum of antimicrobial activity compared to the ethanolic and chloroform extracts. Results of the present study confirm the potential use of seaweed extracts as a source of antimicrobial compound.


INTRODUCTION
Seaweeds are considered as a source of bioactive compounds as they are able to produce a great variety of secondary metabolites characterised by a broad spectrum of biological activities.Compounds with antioxidant, antiviral, antifungal and antimicrobial activities have been detected in brown, red and green algae (Yuan et al., 2005;Bansemir et al., 2006;Chew et al., 2008).Seaweeds are rich and varied source of bioactive natural products and have been studied as potential biotical and pharmaceutical agents.They are used in traditional remedies in many parts of the world.Extracted substances from seaweeds have antibacterial actions and other properties include antifungal activities and growth inhibition of plants (Abdussalam, 1990;Scheuer, 1990;Rizvi and Shameel, 2003;Su et al., 1973;Burkholder and Sharma, 1969;Chapman, 1980;Arasaki and Arasaki, 1983;Abbott, 1988).
Numerous substances were identified as antimicrobial agents from algae: chlorellin derivatives, acrylic acid, halogenated aliphatic compounds, terpenes, sulphur containing heterocyclic compounds, phenolic inhibitors, etc (Espeche et al., 1984).Antibacterial activity was, however, found to vary with season (Moreau et al., 1984).Therefore, the aim of the present investigation was to evaluate the antimicrobial activity of the seaweed Halimeda tuna against different human pathogenic bacterial and fungal strains.

Collection, extraction and preparation of sample
Fresh plants of H. tuna (Bryopsidales, Chlorophyta) (J.Ellis and Solander) J.V.Lamouroux 1816 were collected from the intertidal region (Lat.8° 40′ to 8° 55′ N and Long.78° 0′ to 78° 15′ E) on the Tuticorin, Tamil Nadu, Southeast coast of India (Figure 1).The plants were cleaned of epiphytes and extraneous matter, and the necrotic parts were also removed.Afterward, the plants were washed with seawater and then in fresh water and then transported to the laboratory in sterile polythene bags at 0°C temperature.
Samples were rinsed with sterile distilled water and were then shade-dried, cut into small pieces and powdered in a mixer grinder.The seaweed powdered samples were extracted (10 g/100 ml) in chloroform, ethanol and methanol thrice by soaking overnight at room temperature.The extracts from three consecutive soaking were pooled and evaporated under reduced pressure.The residues (crude extracts) obtained were finally dried under rotary vacuum evaporator (Rotavapor® R-210/R-215, Lark, Chennai), and the crude extracts were tested for their antibacterial activity against the pathogens used in the present investigation.

Aqueous extract
The powdered sample was mixed with distilled water and filtered.The filtrate was evaporated in vacuo at 40°C using a rotary evaporator.

Microorganisms used
The 10 bacterial strains used were: Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi, Klebsiella oxytoca, Escherichia coli, Proteus mirabillis, Lactobacillus vulgaris, Pseudomonas spp., Klebsiella pneumoniae and Vibrio cholerae.The stock culture was maintained on nutrient agar medium at 4°C.Nine fungal pathogens namely Aspergillus niger, Aspergillus flavus, Alternaria alternaria, Candida albicans, Epidermophyton floccossum, Trichophyton mentagrophytes, Trichophyton rubrum, Pencillium sp. and Rhizopus sp. were also used.The stock culture was maintained on Sabouraud dextrose agar (SDA) medium at 4°C.These bacterial and fungal strains were isolated and obtained from the Department of Microbiology, Rajah Muthiah Medical College and Hospital, Annamalai University, Annamalai Nagar, Tamil Nadu, India.
In vitro antibacterial activity was determined by using Mueller Hinton agar (MHA) and Mueller Hinton broth (MHB), while the in vitro antifungal activity was determined by using SDA, all of which were obtained from Himedia Ltd., Mumbai.

Preparation of inocula
Twenty-four hour old culture of selected bacteria was mixed with physiological saline and the turbidity was corrected by adding sterile physiological saline until a Mac Farland turbidity standard of 0.5 (106 colony forming units (CFU/ml).The isolates were sub cultured on Sabouraud dextrose agar and incubated at 35°C for seven to 14 days.The growth was scraped aseptically, crushed and macerated thoroughly in sterile distilled water and the fungal suspension was standardized spectrophotometrically to an absorbance of 0.600 at 450 nm.

Antibacterial and antifungal assays
The antibacterial and antifungal susceptibility test was followed by well diffusion method (Reinheimer et al., 1990).Petri plates were prepared by pouring 15 ml of Mueller Hinton agar for bacteria and Sabouraud dextrose agar for fungi and allowed to solidify.After swabbing the bacterial pathogens on the Muller Hinton agar plates, as well as the fungal pathogens on Sabouraud dextrose agar, 0.1 ml of seaweed extract was poured into the wells and the plates were incubated at 37°C for 24 h and 28°C for 72 to 96 h (fungi).The extract inhibiting the growth of pathogen was assessed based on the inhibition zone around the well and the results were recorded.

Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration of the plant extract was tested by the two-fold serial dilution method.The test extract was dissolved in 5% dimethyl sulfoxide (DMSO) to obtain 1000 µg/ml stock solutions.Then 0.5 ml of stock solution was incorporated into 0.5 ml of Mueller Hinton agar for bacteria and Sabouraud dextrose broth for mycelial fungi to get a concentration of 500 µg/ml, and serially diluted by double technique to achieve 250, 125, 62.5 and 31.25 µg/ml, respectively.Next, 50 µL of standardized suspension of the test organism was transferred on to each tube.The control tube contained only organisms and not the plant extract.The culture tubes were incubated at 37°C for 24 h (bacteria) and 28°C for 72 to 96 h (mycelia fungi).The lowest concentrations, which did not show any growth of tested organism after macroscopic evaluation was determined as MIC.

Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC)
All the tubes used in the MIC study that did not show any growth of the bacteria and fungi after the incubation period were first diluted (1:4) in fresh Mueller-Hinton broth for bacteria and Sabouraud dextrose broth for mycelial fungi and then subcultured onto the surface of the freshly prepared Mueller-Hinton agar (for bacteria) and Sabouraud dextrose agar (for fungi) plates and incubated in biological oxygen demand (BOD) incubators at 37°C for 24 h (bacteria) and 28°C for 72 to 96 h (mycelia fungi).The MBC and MFC were recorded as the lowest concentration of the extract that did not permit any visible bacteria and fungal colony growth on the appropriate agar plate after the period of incubation.

RESULTS
In the present investigation, extracts of a marine algal species were tested against the bacterial and fungal pathogens by well diffusion method.A total of 19 microorganisms which consisted of 10 bacteria and nine fungi were tested.When the chloroform, ethanol, methanol and aqueous extracts were assayed against the test organisms, the zones of inhibition obtained was between 2 to 20 mm.Methanolic extracts showed exhibited broad spectrum of antimicrobial activity compared to other ethanolic and choloroform extract.The results of preliminary screening tests are summarized in Tables 1 and 2, which revealed that H. tuna species possessed antibacterial and antifungal activity.MIC values of 31.25 to 500 µg/ml were obtained for the chloroform and ethanol extract, 15.62 to 250 µg/ml were obtained for the methanol extract in the tests with the bacterial agents, while the range of 15.62 to 250 µg/ml were obtained for the methanol extract with the fungi agents.Moreover, 15.62 to 500 µg/ml and 31.25 to 500 µg/ml obtained for the ethanol extract and chloroform extract was recorded against the fungal isolates.On the other hand, the MIC values obtained in antibacterial assays using aqueous extract were 125 to 500 µg/ml, while the values recorded in the antifungal assays were 62.5 to 250 µg/ml.
The MBC of the chloroform extracts showed *KET, Ketoconazole antifungal control.
that with the exception of the antibacterial assays against S. aureus, S. typhimurium, S. paratyphi, K. oxytoca, E. coli, all the extracts exhibited a MBC at a concentration of 500 µg/ml, while the aqueous extracts had a MBC value ranging from 250 to 500 µg/ml (Table 3).On the other hand, MFC of the methanol extracts showed that with the exception of the antifungal assays against A. mentagrophyte, all the extracts exhibited a MFC at a concentration of 500 µg/ml, while aqueous extracts had a MFC value ranged between 250 to 500 µg/ml (Table 4).

DISCUSSION
Some volatile compounds released from species and herb extracts show wide antimicrobial activities against fungi (Sindhu et al., 2009) and bacteria (Bandyopadhyay et al., 2007).Seaweeds are considered as source of bioactive compounds and produce a great variety of secondary metabolites characterized by a broad spectrum of biological activities.Compounds with cytostatic, antiviral, antihelminthic, antifungal and antibacterial activities have been detected in green, brown and red algae (Ganesan et al., 2008).In the present study, results show that all solvent extracts and aqueous extracts of H. tuna gave favorable results against all the tested microorganisms with MIC values between 15.62 and 500 µg/ml.The present study reveals that the seaweed extracts of H. tuna was very effective against S. aureus, S. typhimurium, S. paratyphi, K. oxytoca, E. coli, A. niger, A. flavus, A. alternaria, C. albicans, E. floccossum, than the other strains tested.The algal extracts such as Enteromorpha ramulosa (Smith) Carmichael and Dictyopteris membranacea (Stackhouse) Batters were active against Gram-positive and Gramnegative bacteria (González et al., 2001).
In the present study, fungal strains were more susceptible to the extracts than the bacterial strains.Among all the extracts used for the present study, the methanol extract showed a higher antibacterial and antifungal activity than that of other extracts and aqueous extract.This may be due to the solvent to extract the different constituents having antimicrobial activity.Kandhasamy and Arunachalam (2008)   were studied against both Gram negative and Grampositive pathogenic bacteria.Methanolic extracts of all the seaweed extracts exhibited broad spectrum of antibacterial activity.The crude aqueous extracts of the whole plant of Parthemia iphionoids showed limited antibacterial activity against E. coli, S. aureus, S. typhimurium and Bacillus cereus (Afifi et al., 1991).Methanol was the most effective solvent for extracting antibacterial compounds from the selected seaweeds (Vlachos et al., 1996).Previous reports on the most effective solvent for the extraction of antimicrobials have been varied; Gonzalez et al. ( 2001) selected methanol as solvent for extraction of antimicrobial compounds from red, green and brown seaweeds, Shanmughapriya et al. (2008) found methanol: toluene (3:1) as the best solvent for extracting antimicrobials from fresh algae, while Plaza et al. (2009) found significant differences in the antimicrobial activity depending on the solvent used.The higher zone of inhibition was recorded at 1000 µg/ml concentration than 500 µg/ml concentration of all the extracts.When the disc dosage level increases, the inhibitory effect also increased.Similar observations were made by Chattopadhyay et al. (2001Chattopadhyay et al. ( , 2002) ) while studying the antimicrobial activities of Alstonia macrophylla and Mallotus peltatus leaves.The overall antimicrobial activity assessed from the above results indicates the presence of active constituents in the extractions of seaweed which showed better antimicrobial activity against pathogens used.Differences between the results of the present investigation and results of other studies may be due to the production of bioactive compounds related to the seasons, method, organic solvents used for extraction of bioactive compounds and differences in assay methods.Finally, it can be concluded from the study that the extracts of H. tuna species used in the present investigation shows better antibacterial activity against pathogens used.They are potential sources of bioactive compounds and should be investigated for natural antibiotics.Hence, further research should be made to identify and purify these antibacterial substances.

Figure 1 .
Figure 1.Map showing the sampling area.