International Journal of Scientific Research and Review ISSN NO: 2279-543X

ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF MARINE PUFFER LAGOCEPHALUS LUNARIS COLLECTED FROM THOOTHUKUDI COAST

Selvi. S1 and Dr. P.J. Joslin2

1 Assistant Professor of Zoology, St. Mary’s College (Autonomous), Thoothukudi. Affiliated to Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli – 627012, Tamil Nadu, India. Corresponding Author E – mail : [email protected].

2 Associate Professor of Zoology, St. Mary’s College (Autonomous), Thoothukudi. Affiliated to Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli – 627012, Tamil Nadu, India.

ABSTRACT

Antibacterial and antifungal activities of tissue extracts of puffer fish Lagocephalus lunaris against various bacterial and fungal pathogens have been evaluated. Liver, skin, muscle, intestine and ovary extracts of L.lunaris were prepared by using acetic acid and methanol. The antibacterial and antifungal activities were performed by Agar diffusion technique. All the extracts showed inhibitory activity for antibacterial test. The ovary extract showed maximum activity with zone of inhibition 13.0 mm radius at 10 mg/ µl in Salmonella typhi and the muscle extract showed minimum zone of inhibition with 6.5 mm radius at 10 mg/ µl in Pseudomonas aeruginosa. Among the extracts tested except the muscle extract, all the other extracts exhibited antifungal activity. The skin extract showed maximum zone of inhibition against A. niger with 9.50 mm radius at 200 µg concentration. These results revealed that the extracts of puffer fish L.lunaris possess potent bioactive compounds which can be further explored and utilized as drug for the welfare of mankind.

Key words: Lagocephalus lunaris, antibacterial activity, antifungal activity, pseudomonas aeruginosa, zone of inhibition.

INTRODUCTION:

The marine biota is the largest source of novel discovery of natural products such as pharmacological metabolites and medicines. There has been an extensive research showing that vast bioactive substances were identified and characterized from marine organisms, indeed several of them showed promising results to treat human and diseases

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[1 and 2]. Marine organisms are a rich source of structurally novel and biologically active metabolites. Primary and secondary metabolites produced and stored by these organisms may be potential bioactive compounds of interest in the pharmaceutical industries. The number of natural products isolated from the marine organisms increases rapidly [3 and 4]. Many classes of natural products from marine sources exhibiting antitumour, anti-leukaemia, antibacterial and antiviral activities have been reported worldwide [5]. hold the credit of possessing rich protein sources. These marine proteins are not only correlated to the intact proteins, but also to the possibility of generating bioactive peptides [6].

Puffer fishes are commonly distributed in the tropics, but are relatively uncommon in temperate regions and completely absent from cold water. There are 189 species of puffer fishes and 28 genera in the family [7]. Puffer fishes are the second most poisonous vertebrate in the word, the first being a “Golden Poison Frog” [8]. Puffer fish is known to carry tetrodotoxin (TTX) ( [9,10 and11] ) which is known a non-protein organic compound and one of the strongest marine paralytic toxins today. The toxin has only occasionally been detected in the muscles of these fishes. The toxin is produced by several bacteria species including Mycobacterium arabino galatanolyticum, Serratia mascescens, Vibrio alginolyticus and Bacillus sp. ([12 and13]). Anbuchezhian et al.,s[14]. analyzed that antimicrobial peptide from the epidermal mucus of estuarine cat fishes. Antibacterial activity in tissue extracts has been demonstrated in several fish species [15]. Kumaravel et al., [16] determined the in-vitro antimicrobial activity of tissue extracts of puffer fish A. immaculatus against clinical pathogens. The antibacterial activities of fatty acids have been noted in several pioneering studies ([17]; [18];[19][20]). In India, studies on the antimicrobial activity of puffer fish are very limited and it remains unexploited. The aim of the present study is to evaluate the antibacterial and antifungal activities of liver, skin, muscle, intestine and ovary of L.lunaris collected from Thoothukudi coast. MATERIALS AND METHODS: Collection of Specimen: Specimens of the puffer fish L. lunaris were collected from fish landing centre at fishing harbour, Thoothukudi. They were kept in ice-box and transported to the laboratory and maintained in a deep freezer at -20oC until use. Extraction of samples: Specimen of L. lunaris was thawed and dissected out into tissues like liver, skin, muscle, intestine and ovary. Ten grams of each tissue was homogenised with 50ml of 0.1%

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acetic acid and were kept in water bath around 45oC for 10 minutes, cooled and centrifuged off. Then it was stored in the deep freezer at -20oC for further use. The preparation of methanol extract was followed by Chellaram et al., [21]. 10g of dry powdered tissue was soaked in methanol and maintained for 72 hours in an orbital shaker. The extract was filtered through Whatman No.1 filter paper, centrifuged at 15,000 rpm for 30 minutes and the solvent was concentrated by rotary evaporator with reduced pressure to give a dark brown gummy mass. The resultant residue was stored at 4oC for further analysis.

Tested strains:

Bacterial strains include Bacillus cereus(BC), Vibrio cholerae (01 (VC01)),Vibrio cholerae(0139)(VC0139), Escherichia coli(EC), Pseudomonas aeruginosa(PA), Aeromonas hydrophila(AH), Salmonella typhi(ST), Shigella flexneri(SF), Pseudomonas sp(P.sp) and Staphylococcus aureus (SA).

Aspergillus niger and Aspergillus flavus were used to test the antifungal activity. The filamentous fungi were grown on Sabouraud dextrose agar (SDA) slants at 28oC for 10 days. The spores were collected using sterile double distilled water and stored in refrigerator.

Antibacterial activity:

Antibacterial activity of crude acetic acid extracts of various tissues of L. lunaris was determined by following the method of Acar, [22]. 1.0 ml of 12 hr old nutrient broth was transferred into the sterilized petridishes. Petri plates were prepared by pouring approximately 20 ml of Muller Hinton Agar Medium and allowed to solidify. Sterilized paper discs prepared from Whatmann No 1 were used for loading acetic acid extract. The paper discs were loaded with different concentrations viz 10mg/10µl, 1mg/10µl and 0.1mg/10µl. The plates were incubated for 24hr at 37oC and solvent control was performed in each case. Areas of inhibited microbial growth were observed as clear zone around the paper disc after 24 hours. Penicillin, Streptomycin and Malachite green were used as positive controls.

Antifungal Activity:

Antifungal activity of the crude methanol acid extract of liver, skin, muscle, intestine and ovary of marine puffer fish L. lunaris was determined by the standard method [23] using Muller Hinton agar medium. Petri plates were prepared by pouring approximately 20ml of Muller Hinton agar medium and allowed to solidify. After solidification, culture of

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each fungal strain was swabbed with sterile cotton on the surface of medium. Wells of 5mm diameter were punched using sterilized cork bores. Samples of different concentrations viz 25µg, 50µg, 75µg were added separately in each well. Ketoconazole (1000 µg/ well) was used as a positive reference to determine the sensitivity of fungal species tested. The plates were incubated at 28oC for 3-5 days. After incubation, zone of inhibition were observed and noted.

RESULTS:

Antibacterial Activity: The crude tissue extracts of liver, skin, muscle, intestine and ovary of L. lunaris were screened against ten pathogenic bacteria for testing their antibacterial activity. The inhibition zones of the extracts were compared with standard antibiotics Penicillin, Streptomycin and Malachite green (Plate 6). All the extracts tested against bacterial strains showed antibacterial activity. The maximum zone was observed against Salmonella typhi in the ovary extract of L. lunaris and the minimum zone was observed against Pseudomonas aeruginosa in the muscle extract as shown in Table 1and plates 1 – 5.

Antifungal Activity: For the antifungal activity Aspergillus niger, (9.5mm radius) showed efficient antifungal activity for methanol extract of liver at 200 µg .The same extract showed the lowest inhibition with 1.0 mm radius against A. niger at 50 µg .Except the muscle extract, all the other extracts showed antifungal activity at high concentration (200 µg). The results are shown in Table 2 and plates 7 – 11. DISCUSSION: Most of the marine creatures have been found to exhibit antimicrobial activity against human bacterial pathogens as well as fungi and viruses and most of the antibacterial agents have derived from marine sources [24]. Fish mucus is believed to play an important role in the prevention of colonization by parasites, bacteria and fungi and thus acts as a chemical defense barrier. Many host defense peptides were found in various marine [25 and 26]. Antimicrobial peptides in marine organisms are existing candidates for the development of new antibacterial compounds due to their broad activity spectrum and difficulty for bacteria to develop resistance to them [27].

Bacteriostatic compounds from marine puffer fish L.lunaris collected from Thoothukudi coast inhibited the growth of ten bacteria viz B. cereus, V. cholerae (01), V. cholerae (0139), E. coli, P.aeruginosa, A. hydrophila, S.typhi, S.flexneri, Pseudomonas sp. and

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S. aureus. Varying degrees of antibacterial activity was found in crude acetic acid extracts of liver, skin, muscle, intestine and ovary. Acetic acid extract of ovary showed strong antibacterial activity against bacterial strains. The diameter of inhibition of bacterial growth is regarded as an estimate of strength of extracts. In the present study the tested bacterial strains exhibited different patterns of inhibition zones. The maximum zone was observed against the Salmonella typhi in the ovary extract of L.lunaris and minimum zone was observed against P. aeruginosa in the muscle extract. The findings of Mohana Priya et al., ([28]; [16]; [5];[29]) lended support to our results. Mohana Priya et al., [28] reported that maximum zones against E. coli, in the skin extracts of A. hispidus and the minimum zone was observed against Proteus vulgaris in the liver extract. Kumaravel et al.,[16] reported that liver and skin extracts of A. immaculatus showed antibacterial activity against S. aureus and V. cholerae. Khora et al.,[5] reported that the crude acetic acid extract of skin of puffer fish A. stellatus exhibited maximum inhibitory activity against E. coli and S. aureus and minimum activity against K. pneumoniae. Selvi and Joslin [29] reported that the acetic acid extract of ovary of A. immaculatus showed maximum inhibitory activity against Shigella flexneri and minimum activity against Pseudomonas sp. in the muscle extract.

In the present study the crude methanol extract of liver, skin, muscle, intestine and ovary were subjected to antifungal activity on two fungal strains such as A.niger and A. flavus. The antifungal activity of the tissue extracts of L. lunaris is quite encouraging, as the maximum zone of inhibition produced by skin extract against the fungal pathogen A. niger and the minimum activity was recorded by liver against A. niger. These results were supported by Mohana Priya et al., [28] and Khora et al.,[5]. Mohana Priya et al., [28] reported that the maximum zone was observed against the A. niger in the skin extract of A. hispidus and the minimum zone was observed against Trichophyton viridae in the liver extract. Khora et al., [5] reported that antifungal activity of acetic acid extract of skin of A. stellatus has shown activity against fungal organisms. Maximum activity was observed against A. niger and A. flavus and minimum activity against T. virdae.

Table – 1 Activity of acetic acid extracts of liver, skin, muscle, intestine and ovary of Lagocephalus lunaris against bacterial strains

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Bacterial 10 mg/ 10l. 1 mg/ 10l. 0.1 mg/ 10l. Strains

Control Liver Skin Muscle Intestin e Ovary Liver Skin Muscle Intestin e Ovary Liver Skin Muscle Intestin e Ovary BC - +++ ++ +++ +++ +++ +++ + ++ ++ ++ ++ + ++ ++ +

VC(01) - +++ ++ ++ +++ +++ ++ + ++ ++ ++ ++ + + + ++

VC(0139) - +++ ++ +++ ++ ++ ++ + ++ ++ ++ + + + + +

EC - +++ + ++ ++ +++ +++ + ++ +++ ++ ++ + + ++ +

PA - +++ + ++ +++ ++ ++ + ++ ++ ++ ++ + ++ ++ +

AH - +++ + ++ +++ ++ +++ + ++ ++ ++ ++ + + + +

ST - +++ + +++ ++ +++ +++ + ++ ++ +++ ++ + ++ + ++

SF - +++ + +++ +++ ++ ++ + ++ ++ ++ + + ++ + ++

P.sp - +++ + ++ +++ ++ +++ + ++ ++ ++ ++ + + ++ +

SA - +++ + +++ ++ +++ +++ + ++ + ++ ++ + ++ +s ++

Zone of inhibition mm (radius), + 5.5 – 8.5, ++ 8 .5 – 11.5, +++ 11.5 – 14.5

Table - 2 Activity of methanol extract of liver, skin, muscle, intestine and ovary of Lagocephalus lunaris on Aspergillus niger and Aspergillus flavus

+ - 1 - 4 mm ++ - 4 - 7 mm +++ - 7 - 10 mm ++++ - 10 - 13 mm

Concentration in µg

Positive Control 50 100 150 200

Methanol A. A. A. A. A. A. A. A. A. A. niger flavus niger flavus niger flavus niger flavus niger flavus Liver + + + + + ++ +++ ++ +++ +++ Skin ------++ - +++ +++ Muscle ------++++ ++++ Intestine +++ ++++ ++ - ++ - ++ - ++ -

Ovary - - - - ++ - +++ - ++++ +++

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Plate -1 Plate -2

Plate - 3 Plate – 4

Plate - 5 Muller Hinton Agar plates showing antibacterial activity of acetic acid extract of various tissues of Lagocephalus lunaris against bacterial strains

Plate 1 – Liver Plate 2 - Skin Plate 3 – Muscle Plate 4 – Intestine Plate 5 - Ovary

ZI – Zone of inhibition a – 10mg/10µl b –1mg/10µl c – 0.1mg/10µl d – control

Plate - 6 Muller Hinton Agar plates showing antibacterial activity of commercial antibiotics penicillin, streptomycin and malachite green on bacterial strains ZI - Zone of inhibition a - penicillin b - streptomycin c - malachite green

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Plate - 7 Plate - 8

Plate – 9 Plate - 10

Plate - 11

Muller Hinton Agar plates showing anti fungal activity of methanol extract of various tissues of Lagocephalus lunaris against Aspergillus niger and Aspergilus flavus

Plate 7 – Liver Plate 8 - Skin Plate 9 – Muscle Plate 10 – Intestine Plate 11 - Ovary

ZI - Zone of Ihibition a - 50µg/10µl b - 100µg/10µl c - 150µg/10µl d - 200µg/10µl e - control

CONCLUSION: The tissue extracts of L..lunaris showed varying degrees of antibacterial and antifungal activity on the microorganisms tested. It is interesting to note that crude extract of various tissues showed prominent activity against various pathogenic microbes. In conclusion, it can be stated that the puffer fish L.lunaris extracts have strong antibacterial and antifungal activities. Hence this information may help to develop potential bioactive compounds from L.lunaris in pharmaceutical industry for the development of drugs. REFERENCES:

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