Investigation on antibacterial, antifungal and cytotoxic properties of chosen mangroves

Indian Journal of Geo-Marine Sciences Vol. 44(11), November 2015, pp. 1769-1777 Investigation on antibacterial, antifungal and cytotoxic properties of chosen mangroves R. Ramasubburayan 1,2, S. Prakash 3, P. Iyapparaj 4, S. Sumathi 2, B. Jude Thaddaeus 1, A. Palavesam 1,2 & G. Immanuel 1 * 1 CMST, Manonmaniam Sundarnar University, Rajakkamangalam 629 502, Tamilnadu, India. 2 Department of Animal Science, Manonmaniam Sundarnar University, Tirunelveli 627 012, Tamilnadu, India 3 Life Science Research Institute, SRM University, Kattankulathur, Kancheepuram 603 203, Tamilnadu, India. 4 CAS in Marine Biology, Annamalai University, Parangipettai 608 502, Tamilnadu, India *[E-Mail: gimmas@gmail.com] Received 18 July 2013; revised 19 August 2013 In the present study antibacterial, antifungal and cytotoxic properties of organic solvent extracts of three mangroves viz. Avicennia marina, Acanthus ilicifolius and Excoecaria agallocha were evaluated. Results showed significant variation in bioactivity with respect to mangroves and solvents used for extraction. Antibacterial and antifungal activity of crude extracts revealed that, methanol extracts had wide spectrum antagonistic activity than chloroform and acetone extracts. Antagonistic activity exhibited by methanol extracts were in the following order: E. agallocha > A. marina > A. ilicifolius. Artemia cytotoxicity assay results inferred that, methanol extracts of E. agallocha, A. marina and A. ilicifolius recorded higher toxicity at lower concentration. Phytochemical analysis of crude extracts showed marked variation in its presence. Findings of the present study indicated that, the synergistic effect of active principles within the crude extracts were responsible for promising biological activity. Thus mangroves proved to be a good source of antimicrobial and cytotoxic agent. [Key words: Mangrove, Antibacterial, Antifungal, Cytotoxicity] Introduction Bacterial resistance to antibiotics has become a worldwide problem in recent years. Indiscriminate use of antibacterial drugs has led to development of many multi-drug resistant (MDR) pathogenic bacterial strains. Development of such newer disease causing pathogens and evolution of existing microorganisms has resulted in severe consequences including increased cost of medicines and mortality of patients 1,2. For instance, it was reported that the problem of enteric fever caused by Salmonella typhi and S. paratyphi- A in India and its resistance to antibiotics such as chloramphenicol, ampicillin and cotrimoxazole has reached a level of crisis 3. Similarly Staphlococcus aureus, a virulent pathogen responsible for wide range of infections such as pneumonia, osteomyelitis and endocarditis has also developed resistant to most antibiotics 4. Besides, fungal infections have also reached a level of crisis in immunocompromised individuals in recent years. Of the different fungal species affecting humans, Candida and Aspergillus species are the most prevalent pathogens which are now reported to be resistant to numerous antifungal agents. Thus antibiotic-resistant pathogen has become an important and growing threat to public health. Rapid emergence of MDR human pathogenic organisms has now necessitated the search for new antimicrobial agents that will be effective against pathogenic microorganisms without any fear of development of resistance 5. Investigation on utilization of marine/terrestrial plant extracts as a source of antimicrobials is being tried now extensively to control the development of MDR bacterial strains and also to replace the usage of commercial antibiotics. One of the added advantage of using plant extracts as source of antimicrobial agent is that secondary metabolites (phytochemicals) of plants are comparable with antibiotics in several cases in vitro. Mangrove plants have been recognized as one of the most promising source of natural products and proved to be significant to humans. Indeed many mangrove plants have been used in folklore medicine worldwide. They are the rich source of phytochemicals and possess various biological activities which lead to discovery of herbal drugs and semi synthetic drugs 6. According to a review, about 349 metabolites have been isolated from mangrove species, out

1770 INDIAN J MAR SCI VOL 44, NO.11 NOVEMBER 2015 of which 200 metabolites are reported exclusively from true mangrove plants 7. Extracts from mangroves and mangrove associated species have proven to possess antimicrobial activities against human, animal and plant pathogens 6. For instance, leaf, fruit and bark extracts of several mangroves such as Rhizophora mangle, Ceriops tagal, Pemphis acidula and Sonneratia alba were reported to exhibit broad spectrum antimicrobial activity 8,9,10. Moreover, studies on mangrove plant parts and their major chemical classes have also shown that they possess some important biological activities such as antiplasmodial and antiviral activities 11,12. Present study is an attempt to evaluate the antibacterial, antifungal and cytotoxic efficiency of mangroves such as Avicennia marina, Acanthus ilicifolius and Excoecaria agallocha. Materials and Methods For the present study, leaves of mangroves such as A. marina, A. ilicifolius and E. agallocha were collected from Vellar estuary (Lat: 11 29' N; Long. 79 46') of Port Novo, Cuddalore District, Tamilnadu, India. Collected leaves were washed thoroughly in water and dried under shade. Then the dried leaves were pulverized and subjected to percolation by soaking individually in solvents of varying polarity viz. methanol, acetone and chloroform for one week. Extracts were then filtered with Whatman No.1 filter paper (Himedia, India), evaporated and concentrated under rotary vaccum evaporator. The crude extracts obtained were then used for further study. For the present study, human pathogens such as Salmonella typhi (MTCC 733), Enterococcus faecalis (MTCC 439), Bacillus subtilis (MTCC 1134), Streptococcus pneumoniae (MTCC 1936), Klebsiella pneumoniae (MTCC 7407), Pseudomonas aeruginosa (MTCC 2642), Escherichia coli (MTCC 1671), Streptococcus aureus (MTCC 96), S. mutans (MTCC 890) and Proteus vulgaris (MTCC 744) and the fungal pathogens such as Aspergillus fumigatus (MTCC 4333), A. niger (MTCC 961), Rhizomucor miehei (MTCC 546a), Candida glabrata (MTCC 3984), C. albicans (MTCC 183) and C. tropicalis (MTCC 184) were collected from Microbial type culture collection, Chandigarh, India. Antibacterial activity of the crude extracts of selected mangroves was tested through agar well diffusion method 13. For this, Muller hinton agar plates (MHA, Himedia, India) were prepared separately and overnight culture of test bacterial strains were seeded individually over the surface of MHA plates using sterile cotton swabs. Then wells of 6 mm diameter were made over the agar plates using sterile cork borer. Extracts were then prepared at a concentration of 500µg per well by dissolving in dimethyl sulfoxide (DMSO). Wells were then filled with 100µl of extract. Rifamycin (Himedia, India) was used as positive control (25µg), while DMSO was used as negative control. Plates were then incubated for 24h at 37ºC. Antibacterial activity of the extracts was determined by measuring the diameter of the inhibition zone around the well. The assay was carried out in triplicate. In the present study antifungal efficacy of the mangrove plant extracts was determined through agar well diffusion method 14. For this, Sabouraud dextrose agar plates (SDA) were prepared. Then with sterile cotton swabs 72h culture of test fungal strains were seeded individually over the surface of SDA plates. Well of 6mm diameter was made in the centre of the agar plate with a sterile cork borer. Test extracts were then prepared at a concentration of 500µg per well by dissolving in DMSO. Wells were then filled with 100µl of extract. Flucanazole (Himedia, India) was used as positive control (100µg) and DMSO was used as negative control. Plates were then incubated for 48-96h at 32ºC. Antifungal activity of the extracts was determined by measuring the diameter of the inhibition zone around the well. The assay was carried out in triplicate. The phytochemical contents of chosen mangrove plant extracts were identified by following the methods of Sofowara 15 and Kepam 16. Artemia cytotoxicity assay is one of the simple and inexpensive screening techniques mainly performed to assess the toxic nature of plant/microbial extracts. In the present study, the method described by Ortega - Morales et al. 17 was followed to find out the cytotoxicity of crude extracts of chosen mangroves. Before the start up of experiment, the cysts of brine shrimp (Artemia salina) were hatched in cone-shaped vessel containing filtered seawater under mild aeration for 24h. After 24h of hatching, active nauplii were collected from the hatching

RAMASUBBURAYAN et al: INVESTIGATION ON ANTIBACTERIAL, ANTIFUNGAL AND CYTOTOXIC PROPERTIES 1771 chamber and used for further assay. In order to perform the cytotoxicity assay, ten nauplii were collected through a thin capillary glass tube and placed in small test tubes containing 10ml of brine solution. Then various concentrations of crude extracts of selected mangroves were prepared by diluting in DMSO and maintained at room temperature for 24h under the light. Controls (DMSO) were maintained separately. After 24h of exposure, the number of live and dead larvae in each test concentrations was counted. Then LC 50 values were determined through probit analysis. The assay was carried out in triplicate. The results obtained in the present study were subjected to Two way ANOVA through SPSS package 10.0. LC 50 values were determined by EPA probit analysis software. Results Table 1 shows the results on antibacterial activity of different solvent extracts of selected mangroves. In the present study of the three different solvent extracts of A. marina tested, it was found that methanol extract had shown markedly higher antagonistic activity (100%) by growth inhibition against all the ten pathogenic bacterial strains. It recorded maximum zone of inhibition of 14mm against E. coli and minimum of 7mm against the bacterial strains such as P. vulgaris, E. faecalis and S. aureus, respectively. Acetone extract of A. marina recorded 80% inhibitory activity and recorded maximum inhibitory zone against S. typhi and E. coli (12mm) and minimum against S. pneumoniae (7mm). Nevertheless, chloroform extract of A. marina displayed only 60% antagonistic activity by growth inhibiting six out of ten pathogenic bacterial strains such as P. aeruginosa, S. pneumoniae, K. pneumoniae, E. coli, E. faecalis and S. aureus. The two way ANOVA for the data on antibacterial activity as a function of variation due to different bacterial strains (B) was statistically non significant (F B = 1.84; P > 0.05); whereas variation due to different solvents (S) of mangrove A. marina was statistically significant (F S = 2.57; P < 0.05). Table 1 - Antibacterial activity of different solvent extracts of chosen mangroves Zone of Inhibition (mm) Bacterial Organic Mangrove plants Pathogens solvents DMSO Rifamycin A. marina A. ilicifolius E. agallocha S. typhi M 8.00 ± 0.29 8.00 ± 0.15 10.00 ± 0.32 0.00 ± 0.00 8.00 ± 0.00 C 0.00 ± 0.00 10.00 ± 0.34 9.00 ± 0.33 A 12.00 ± 0.40 9.00 ± 0.29 12.00 ± 0.51 P. vulgaris M 7.00 ± 0.16 10.00 ± 0.30 16.00 ± 0.56 0.00 ± 0.00 12.00 ± 0.10 C 0.00 ± 0.00 9.00 ± 0.26 10.00 ± 0.40 A 8.00 ± 0.20 8.00 ± 0.21 11.00 ± 0.47 P. aeruginosa M 11.00 ± 0.40 13.00 ± 0.45 15.00 ± 0.50 0.00 ± 0.00 9.00 ± 0.10 C 8.00 ± 0.24 8.00 ± 0.20 0.00 ± 0.00 A 0.00 ± 0.00 0.00 ± 0.00 10.00 ± 0.39 S. pneumoniae M 8.00 ± 0.27 0.00 ± 0.00 14.00 ± 0.46 0.00 ± 0.00 10.00 ± 0.10 C 8.00 ± 0.21 0.00 ± 0.00 0.00 ± 0.00 A 7.00 ± 0.16 0.00 ± 0.00 9.00 ± 0.35 K. pneumoniae M 12.00 ± 0.41 0.00 ± 0.00 13.00 ± 0.41 0.00 ± 0.00 10.00 ± 0.10 C 9.00 ± 0.35 0.00 ± 0.00 10.00 ± 0.38 A 11.00 ± 0.40 0.00 ± 0.00 8.00 ± 0.26 B. subtilis M 9.00 ± 0.33 10.00 ± 0.33 12.00 ± 0.38 0.00 ± 0.00 7.00 ± 0.00 C 0.00 ± 0.00 9.00 ± 0.38 12.00 ± 0.47 A 10.00 ± 0.33 8.00 ± 0.24 8.00 ± 0.20 E. coli M 14.00 ± 0.51 12.00 ± 0.37 14.00 ± 0.45 0.00 ± 0.00 14.00 ± 0.11 C 12.00 ± 0.44 11.00 ± 0.45 8.00 ± 0.27 A 12.00 ± 0.46 10.00 ± 0.33 10.00 ± 0.38 S. mutans M 8.00 ± 0.27 8.00 ± 0.20 12.00 ± 0.37 0.00 ± 0.00 10.00 ± 0.08 C 0.00 ± 0.00 0.00 ± 0.00 9.00 ± 0.30 A 0.00 ± 0.00 0.00 ± 0.00 8.00 ± 0.23 E. faecalis M 7.00 ± 0.20 9.00 ± 0.22 11.00 ± 0.33 0.00 ± 0.00 13.00 ± 0.11 C 10.00 ± 0.41 10.00 ± 0.00 0.00 ± 0.00 A 8.00 ± 0.27 10.00 ± 0.32 7.00 ± 0.16 S. aureus M 7.00 ± 0.16 12.00 ± 0.35 10.00 ± 0.28 0.00 ± 0.00 9.00 ± 0.07 C 8.00 ± 0.20 0.00 ± 0.00 8.00 ± 0.23 A 8.00 ± 0.16 0.00 ± 0.00 9.00 ± 0.31 M Methanol; C Chloroform; A Acetone; DMSO Negative control; Rifamycin Positive control Each value is the Mean ± SD of three replicates

1772 INDIAN J MAR SCI VOL 44, NO.11 NOVEMBER 2015 Antibacterial activity of crude extracts of A. ilicifolius also depicted that, methanol extract had showed higher antagonistic activity (80%). It exhibited more pronounced growth inhibitory activity of 13mm against P. aeruginosa and 12mm against E. coli and S. aureus. Chloroform extract of A. ilicifolius exhibited moderate antagonistic activity (60%) by forming zone of inhibition ranged from 8 to 11mm; whereas acetone extract tested for antibacterial activity recorded marginally reduced growth inhibitory activity (50%) against the pathogenic bacterial strains tested. Two way ANOVA test conducted for the data on antibacterial activity as a function of variation due to different bacterial strains (B) and different solvents (S) of mangrove A.ilicifolius were statistically significant (F B = 5.67 and F S = 3.95; P < 0.05). Antibacterial activity of crude extracts of E. agallocha inferred that, methanol and acetone extracts exhibited higher degree of bioactivity (100%). However, among both the solvent extracts tested, methanol extract showed broad spectrum antagonistic activity with markedly elevated inhibitory zone ranged between 10 and 16mm; whereas acetone extract displayed comparatively lesser growth inhibitory activity with the zone of inhibition ranged from 7 to 12mm. Chloroform extract of E. agallocha recorded 70% bioactivity and expressed maximum inhibitory zone against B. subtilis (12mm) and minimum against E. coli and S. aureus (8mm each). Two way ANOVA test conducted for the data on antibacterial activity as a function of variation due to different bacterial strains (B) was statistically non significant (F B = 1.03; P > 0.05); whereas variation due to different solvents (S) was statistically significant (F S = 9.92; P < 0.01). Antibiotic Rifamycin (positive control) displayed superior antagonistic activity against the bacterial pathogens with prominent inhibition zone ranged between 7 and 14mm. DMSO was used as negative control which always showed no growth inhibitory activity against the pathogenic bacterial strains tested. The results on antifungal activity of crude extracts of chosen mangroves are shown in Table 2. Among various solvent extracts of A. marina tested, methanol extract showed 100% growth inhibition of fungal strains. It recorded maximum zone of inhibition of 11mm against C. albicans and minimum of 8mm against C. glabrata. On the other hand, acetone and chloroform extract of A. marina tested against fungal pathogens recorded considerably lesser antagonistic activity of 83.3 and 66.6% with the zone of inhibition ranged from 7 to 10 and 7 to 9mm, respectively. The two way ANOVA for the data on antifungal activity as a function of variation due to different fungal strains (F) was statistically non significant (F F = 1.03; P > 0.05); whereas the variation due to different solvents (S) of mangrove A. marina was statistically significant (F S = 2.63; P < 0.05). Table 2 - Antifungal activity of different solvent extracts of chosen mangroves Zone of Inhibition (mm) Fungal Organic Mangrove plants Pathogens Solvents DMSO Flucanozole A. marina A. ilicifolius E. agallocha C. albicans M 11.00 ± 0.53 9.00 ± 0.36 12.00 ± 0.54 0.00 ± 0.00 13.00 ± 0.11 C 9.00 ± 0.38 9.00 ± 0.35 9.00 ± 0.34 A 10.00 ± 0.41 10.00 ± 0.40 10.00 ± 0.42 C. glabrata M 8.00 ± 0.27 10.00 ± 0.41 10.00 ± 0.42 0.00 ± 0.00 9.00 ± 0.09 C 7.00 ± 0.24 7.00 ± 0.24 10.00 ± 0.39 A 8.00 ± 0.30 8.00 ± 0.25 9.00 ± 0.37 C. tropicalis M 9.00 ± 0.36 9.00 ± 0.32 11.00 ± 0.48 0.00 ± 0.00 12.00 ± 0.11 C 0.00 ± 0.00 0.00 ± 0.00 9.00 ± 0.29 A 7.00 ± 0.25 0.00 ± 0.00 7.00 ± 0.20 A. niger M 10.00 ± 0.49 9.00 ± 0.28 9.00 ± 0.37 0.00 ± 0.00 7.00 ± 0.10 C 8.00 ± 0.29 7.00 ± 0.20 9.00 ± 0.32 A 8.00 ± 0.34 7.00 ± 0.30 7.00 ± 0.27 A. fumigatus M 10.00 ± 0.46 8.00 ± 0.23 7.00 ± 0.23 0.00 ± 0.00 7.00 ± 0.08 C 0.00 ± 0.00 8.00 ± 0.32 8.00 ± 0.22 A 8.00 ± 0.28 9.00 ± 0.34 8.00 ± 0.32 R. miehei M 9.00 ± 0.40 0.00 ± 0.00 8.00 ± 0.30 0.00 ± 0.00 8.00 ± 0.00 C 8.00 ± 0.32 0.00 ± 0.00 0.00 ± 0.00 A 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 M- Methanol; C-Chloroform; A-Acetone; DMSO-Negative control; Flucanazole-Positive control Each value is the Mean ± SD of three replicates

RAMASUBBURAYAN et al: INVESTIGATION ON ANTIBACTERIAL, ANTIFUNGAL AND CYTOTOXIC PROPERTIES 1773 Antifungal activity of crude extracts of A. ilicifolius emphasized that, methanol extract showed 83.3% antagonistic activity by growth inhibition against fungal pathogens such as C. albicans, C. glabrata, C. tropicalis, A. niger and A. fumigatus with prominent inhibitory zones ranged between 8 and 10mm. But it failed to display antagonistic activity against R. miehei. Quite interestingly in the present study it was noticed that, both chloroform and acetone extract of A. ilicifolius exhibited equal fungal growth inhibitory activity (66.6%). Both the extracts displayed growth inhibition against all the tested pathogenic fungal strains, except C. tropicalis and R. miehei. The two way ANOVA for the data on antifungal activity as a function of variation due to different fungal strains (F) was statistically significant (F F = 9.38; P < 0.05); whereas variation due to different solvents (S) of mangrove A. ilicifolius was statistically non significant (F S = 2.03; P > 0.05). Antifungal activity of crude extracts of E. agallocha inferred that, methanol extract had shown higher degree of antagonistic activity (100%). It expressed maximum (12mm) inhibitory zone against C. albicans and minimum (7mm) against A. fumigatus. Acetone and chloroform extracts of E. agallocha tested against fungal strains attributed equal growth inhibitory activity (83.3%) and showed zone of inhibition ranged between 7 and 10mm. Both the extracts showed negative impact on the growth inhibitory activity against R. miehei. The two way ANOVA for the data on antifungal activity as a function of variation due to different fungal strains (F) and solvents (S) of mangrove E. agallocha were statistically significant (F F = 5.84 and F S = 3.02; P > 0.05). In the present study, antibiotic flucanazole (positive control) showed strong fungal growth inhibitory activity with the zone of inhibition ranged from 7 to 13mm. Phytochemical analysis of crude extracts of chosen mangroves showed presence of variety of phytochemicals viz. alkaloids, glycosides, saponins, phenols, flavanoids, terpenoids, steroids, tannins, carboxylic acids, quinones, coumarins and resins. However, attribution of phytochemicals in the mangrove extracts showed significant variation with respect to plant and solvent system used for extraction (Table 3). Table 3 - Phytochemical constituents of crude extracts of chosen mangroves Phytochemicals Mangroves Organic solvents Alkaloids Glycosides Saponins Phenols Flavonoids Terpenoids Steroids Tannins Carboxylic acids Quinones Coumarins Resins M - - + + - - + + - + + + A. marina C + - + - + + + - - - - - A + - - + - + - + - - + - M - + + + - - - + + - - - A. ilicifolius C - - + - - + + - - - - - A + - + - - - - + - - - + M + - + + + + - + + - + + E. agallocha C + - - + - - - + + + - - A + + - + - - + + - - - - M-Methanol; C-Chloroform; A-Acetone; + Present; - Absent Table 4 shows the results on Artemia cytotoxicity of crude extracts of chosen mangroves. Results inferred that, the cyotoxicity of different solvent extracts of A. marina and E. agallocha in terms of lethality (LC 50 ) were in the following order: Methanol > Acetone > Chloroform (326 ± 11, 375 ± 14, 487 ± 17 and 225 ± 9, 336.5 ± 14 and 411 ± 16µg ml -1 ). However, crude extracts of A. ilicifolius showed a little variation in lethality (LC 50 ) of brine shrimp A. salina and exhibited the results in the following array: Methanol > Chloroform > Acetone (426.2 ± 12, 572.6 ± 19 and 695 ± 22µg ml -1 ). DMSO was used as control which recorded cytotoxicity (LC 50 ) of >1000µg ml -1.

1774 INDIAN J MAR SCI VOL 44, NO.11 NOVEMBER 2015 Table 4 - Artemia cytotoxicity assay of crude extracts of chosen mangroves Mangrove Organic Solvents - LC 50 (µg ml -1 ) plants Methanol Chloroform Acetone A. marina 326 ± 11 487 ± 17 375 ± 14 A. ilicifolius 426.2 ± 12 572.6 ± 19 695 ± 22 E. agallocha 225 ± 9 411 ± 16 336.5 ± 14 Control (DMSO) > 1000 Each value is the Mean ± SD of three replicates Discussion Results obtained from the present study evidently substantiated that, almost all the mangrove plant extracts had significantly inhibited the growth of bacterial and fungal pathogens with varying level of inhibitory zones. In the present study, among various solvent extracts of A. marina tested, it was found that methanol extract had shown broad spectrum antagonistic activity with the higher level of zone of inhibition ranged from 7 to 14mm. This result was in agreement with Kumar et al. 18 who studied the in vitro antibacterial activity of A. marina and depicted that, methanol extract had pronounced growth inhibitory activity ranged between 12 and 18mm against bacterial pathogens such as S. aureus, S. epidermidis, B. subtilis, E. coli, P. aeruginosa and K. pneumoniae. Similarly, Dhayanithi et al. 19 tested various solvent extracts of A. marina against six different multi drug resistant S. aureus (MRSA) and inferred that methanol extract had higher degree of growth inhibitory activity with the zone of inhibition ranged from 10 to 17mm. Nevertheless, acetone and chloroform extract of A. marina in the present study exhibited comparatively less growth inhibitory activity (7 to 12mm) against the tested bacterial strains. Supporting the results of the present study, Subashree et al. 20 and Arivuselvan et al. 9, investigated the antibacterial activity of different solvent extracts of A. marina and evidenced that, acetone and chloroform exhibited marked reduction in antagonistic activity. In general, ability of an extract to exhibit antagonistic activity depends on the chemical diversity within the extract. Similarly in the present study, differences in the antibacterial activity attributed by different solvent extracts of A. marina may be due to variation in the composition of secondary metabolites. Antibacterial activity of crude extracts of A. ilicifolius inferred that, methanol extract had shown much influence on inhibition of bacterial pathogens with the zone of inhibition ranged between 8 and 13mm. This observation indicated that, the active principle responsible for antibacterial activity may reside within the methanolic extract of A. ilicifolius. In consistent to the results of the present study, Khajure and Rathod 21 depicted that, methanol extract of A. ilicifolius had effectively inhibited the growth of bacterial pathogens such as B. subtilis, S. aureus, P. aeruginosa and P. vulgaris with the zone of inhibition ranged between 12 and 17mm. As like that of A. marina, in the present study, chloroform and acetone extract of A. ilicifolius showed remarkable reduction in inhibitory activity (60 and 50%) against the tested pathogenic bacterial strains. The decrease in antibacterial activity noticed in chloroform and acetone extract of A. ilicifolius may be due to bacterial resistance towards the extracts or inadequate concentration of the extract to display inhibitory activity. Results on antibacterial activity of crude extract of E. agallocha revealed both methanol and acetone extract had expressed 100% growth inhibitory activity. However, methanol extract exhibited pronounced inhibitory activity. It recorded zone of inhibition ranged between 10 and 16mm. This agrees with the result of Patra et al. 22, who emphasized that, methanol extract of E. agallocha had better antagonistic activity against pathogenic bacterial strains with the zone of inhibition ranged between 3 and 20mm. On the other hand, acetone and chloroform extracts displayed moderate antagonistic activity against the tested bacterial pathogens. Findings of our results was supported by Vadlapudi et al. 23, who investigated antibacterial activity of methanol, chloroform and hexane extracts of E. agallocha against few pathogenic bacterial strains and inferred that, chloroform and hexane extracts had shown poor antimicrobial activity than methanol extract. As like that of antibacterial activity, results of antifungal activity have also emphasized that methanol extract of mangroves had better bioactivity. For instance, antifungal activity of A. marina revealed that, methanol extract had shown a wide spectrum antagonistic activity ranged between 8 and 11mm. But, acetone and chloroform extract of A. marina exhibited comparably lesser antifungal activity. In consonance with the results of the present study Kumar et al. 18 examined the antifungal activity of different organic solvent extracts of

RAMASUBBURAYAN et al: INVESTIGATION ON ANTIBACTERIAL, ANTIFUNGAL AND CYTOTOXIC PROPERTIES 1775 A. marina and substantiated that methanol extract has significantly inhibited the growth of all the tested fungal pathogens viz. A. niger, R. oyrzae, C. albicans and S. cerevisiae with markedly increased inhibitory zones ranged between 13 and 18mm. Similarly, Vadlapudi and Chandrasekhar Naidu 24 investigated the antifungal activity of methanol and chloroform extract of A. alba and depicted that methanol extract had higher degree of bioactivity than chloroform extract. Result on antifungal activity of crude extracts of A. ilicifolius also inferred that methanol extract had attributed good fungal growth inhibitory activity (8 to 10mm). Whilst, chloroform and acetone extracts of A. ilicifolius evidenced consistent reduction of antifungal activity (66.6%). Both the extracts recorded maximum zone of inhibition (9 and 10mm) against C. albicans and minimum (7mm) against C. glabrata and A. niger. In consistent to the present result, Ganesh and Vennila 25 investigated the effect of various solvent extract of A. ilicifolius on clinically important fungal strains such as A. niger and C. albicans and inferred that, methanol extract had recorded more pronounced bioactivity against C. albicans (10mm) than A. niger (7mm). Antifungal activity of E. agallocha inferred that, methanol extract had exhibited 100% antifungal activity with maximum of 12mm against C. albicans. A similar observation was made by Kumar et al. 18, who stated a promising antifungal activity of methanol extract of E. agallocha with maximum growth inhibitory activity against C. albicans (18mm). On the other hand, chloroform and acetone extracts of E. agallocha exhibited comparatively lesser level of antagonistic activity. The varying degree of bioactivity clearly suggested that different class of compounds with different polarity is responsible for the observed activity 26. The significant antibacterial and antifungal activity rendered by methanol extracts which is equal or lesser than the standard antibiotic rifamycin and flucanzole (positive control), tends to show that the active compounds of the plants are better extracted with methanol 27. Thus the finding of the present study clearly pointed out that methanol is found to be the best solvent to extract antimicrobial compounds from the leaves of mangroves. Mangroves are biochemically unique, producing a wide array of novel natural products. Presence of numerous phytochemicals within the extract takes advantage over a single molecule in treating disease by acting against pathogens. Results showed the presence of variety of phytochemicals within the extract, however, its presence showed considerable variation with respect to plant and solvent used for extraction. Among the extracts tested, results evidently substantiated that, methanol extracts of all the mangrove plants had numerous phytochemicals with noteworthy antimicrobial activity. Trease and Evans 28 pointed out that the highest antibacterial activities in mangrove halophytes were due to the presence of high content of phenols which include tannins, coumarins, and their glycosides and anthroquinones in mangrove halophytes. Similarly in the present study, plant extracts that exhibit pronounced antibacterial and antifungal activity were believed to be due to the presence of abundant phytochemicals. Artemia cytotoxicity assay using brine shrimp A. salina nauplii is mainly carried out to assess the toxic nature of plant extracts. Moreover, toxicity to brine shrimp coincides with cytotoxicity to mammalian cells in many cases 29. Among the different solvent extracts tested, methanol extract of mangrove exhibited higher cytotoxicity at lower concentration than chloroform and acetone extracts. For instance, methanol extract of A. marina, A. ilicifolius and E. agallocha recorded highest activity with the LC 50 value of 326 ± 11, 487 ± 17 and 375 ± 14 µg ml -1, respectively. In general it is revealed that, the degree of lethality is directly proportional to the concentration of the extract 30. Krishnaraju et al. 30 screened aqueous extracts of 118 Indian medicinal plants for their cytotoxicity using brine shrimp A. salina and showed variation in lethality (LC 50 ) ranged from 13.5 to >5000 µg ml -1. On the other hand, Manilal et al. 31, tested cytotoxicity of mangrove plant extracts such as A. marina, B. cylindrica and A. ilicifolius against brine shrimp A. salina and recorded the lethal dose (LD 50 ) value of 318, 410 and 475 µg ml -1, respectively. Conclusion Results of the present study emphasized that, the antimicrobial activity exhibited by methanol extracts of E. agallocha and A. marina was quite comparable with antibiotic Rifamycin and Flucanzole, this in turn imply that, the inhibitory substance responsible for biological activity is better extracted with methanol than

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