INTRODUCTION
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| Terrestrial environment have relatively exhausted its resource for the pharmaceutical purpose. Recent decades, Marine ecosystem has been drawn attention of many scientists due to its biological and chemical diversity [1]. Marine environment acts as a huge reserve to exploit natural products for the treatment of human and fish disease [2]. In last century, there has been a huge progress in the field of marine natural products and its chemistry [3, 4], So far approximately 7000 marine natural products have been reported from the marine organisms mainly invertebrates [5]. Among them, Fishes are the largest class of invertebrates. Due to the aquatic environment, fishes have some unique characteristics [3, 4]. Fishes are evolving with innate immune response to protect themselves against the infection [6]. |
| Puffer fish belong to the family Tetraodontidae, order Tetraodontiformes commonly found in tropical seas is accumulating tetrodotoxin (TTX) in the body by the bioaccumulation via food chain. Tetrodotoxin, a sodium channel blocking compound mainly causes respiratory paralysis and intoxications [7, 8]. Traditionally, Japanese and Chinese are used to eat as delicacy and some part of the puffer fish as health tonic. Japanese researchers before World War 2 have used the crude puffer fish extract for treating migraines and menstrual cramps [9]. It has been shown a reducing symptom of withdrawal of heroin without any side effects. During last decades, TTX has been used as a tool for analyzing and characterizing the voltage gated sodium channel [10]. Recently, China and Canada have performed clinical trials for using TTX as an analgesic for reducing the pain in the cancer patients [11]. |
| In India, studies on the puffer fish are very limited and it remains unexploitated. The aim of the present study is to evaluate the bioactive compound present in the puffer fish A. hispidus which is very common from the southeast coast of India. |
MATERIALS AND METHODS
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| Specimen collection |
| Puffer fish specimens of A. hispidus (Muller, 1841) Family- Tetraodontidae, Order- Tetraodontiformes was collected from the fish landing station at Mandapam region near Ramasewarm at the latitude of 9 ·17 ?? N – 70.9 E ?? longitudes from the southeast coast of India in the month of September 2012. Then they were washed with sea water and transported to the laboratory in dry ice and was stored in the deep freezer at -20o C [12]. |
| Extraction of samples |
| Specimen A. hispidus was thawed and dissected into tissues like Muscle, Liver, and Skin. Ten grams of each tissue was homogenized with 50ml of 0.1% acetic acid and were boiled in a hot water bath around 45o C for 10mins and cooled and centrifuged off. Then it was condensed using rotary evaporator at 60o C. Then it was stored at the deep freezer for further use at -20o C [13]. |
| Tested strains: |
| Gram positive strains Bacillus cereus, Staphylococcus aureus and Gram negative bacteria Escherichia coli, Klebsiella pneumoniae Pseudomonas aeruginosa, Proteus vulgaris, Vibrio chlorae were used. Fungal strains Candida albicans, Aspergillus niger, Aspergillus fumigatus, Trichophyton rubrum and Trichophython viridae were used. |
| Antibacterial activity test |
| Antibacterial test of tissue extracts was performed in vitro using the agar disc diffusion method according to Kirby with little modifications [14]. Sterile disks of 5mm in diameter were impregnated with 20μl of tissue extracts kept on the Muller Hinton agar plates previously swabbed with bacterial culture and incubated at 37o C for 24hrs. The diameter of the inhibition zones of the plates around the discs were measured and expressed in mm. All tests were performed in triplicate. Ampicillin was used as the positive control. |
| Antifungal activity test |
| Antifungal activity of tissue extracts was screened using the agar disc diffusion method according to Mederios et al., 2011 with little modifications [15]. Sterile discs of 5mm in diameter were impregnated with 20μl of tissue extracts kept on the Muller Hinton Agar plates previously swabbed with fungal culture and incubated at 37o C for 72 hrs the diameter of the inhibition zones of fungal growth around the discs were measured and expressed in mm. Flucanozole was used as the positive control. |
| Hemolytic activity |
| Hemolytic assay was performed by the microtitre plate method of Prasad with little modifications [16]. Heparin used as an anticoagulant. A chicken blood was collected from the local slaughter house at Vellore and was centrifuged at 5000 rpm for 5mts, the supernatant was discarded and the pellet was suspended in normal saline (pH 7.4). The process was repeated thrice to obtain the erythrocyte suspension. The hemolytic test was performed in 96 well microtitre plates. Two fold dilution of the crude extract were made with 100 μl of saline. Then 100 μl erythrocyte suspensions were added to the wells. 100 μl of distilled water maintained as positive control. Then, the plate was gently shaken and it was allowed to stand for 3hrs at the room temperature and the results were observed. The uniform red color suspension in the well considered as positive hemolysis and a button formation in the bottom of the wells was considered as lack of hemolysis. |
| Cytotoxicity test |
| Hela cells were purchased from the NCCS (National Centre for Cell Sciences) Pune, India. The cells were sub cultured in a fresh flask containing Dulbecco Minimum Essential Medium (DMEM) with 10% fetal Bovine serum. The cells were rinsed in serum free medium and trypsin (0.1%) was added to remove the cell from substrate. The cells were counted and seeded on to a 96well microtitre plate at the rate of 200μg/ml and incubated overnight at 370 C in CO2 incubator. 10mg/ml stock of the test samples was prepared in DMEM. The stock samples were serially diluted in DMEM 10% FBS. 100μl of the diluted cells were added to cells in triplicate wells containing 100μl of the medium. The well with 100μl of these DMEM was served as a negative control and 5μg/ml Doxorubicin was used as a positive control. |
| Wells without any cells were used as a blank. The plates were incubated at 370 C in CO2 incubators for 48hrs. |
| After 48hrs of incubation the plates were taken for the MTT cell viability test. 20μl of 5mg/ml MTT in PBS was added to each well and incubated at 370 C for 4hrs. Then medium was aspirated and replaced with 200μl of Dimethyl sulfoxide to each well. Then the plates were incubated for 10mts and then read at microplate reader at 570nm [17]. |
RESULTS
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| Antibacterial assay |
| The crude tissue extracts (Liver, Muscle, skin) of A. hispidus were screened against seven human pathogenic bacteria for testing their antibacterial activities. The inhibition zones of the extracts were compared with std Ampicilin for bacterial culture. The maximum zone was observed against the E. coli in the skin extract of A.hispidus and the minimum zone was observed against P. vulgaris in the liver extract as shown in Table 1 & Figure 1. |
| Antifungal activity: |
| The crude tissue extracts like liver, muscle, and skin of A. hispidus were screened against five human pathogenic fungi for testing their antifungal activities. The inhibition zones of the extracts were compared with standard Flucanozole for fungal culture. The maximum zone was observed against the A. niger in the skin extract of A. hispidus and the minimum zone was observed against T. virdae in the liver extract as shown in table 2 & Figure 2. |
| Hemolytic activity: |
| The crude tissues extracts of A. hispidus showed pronounced hemolytic activity on chicken blood. The highest hemolytic activity was shown by crude skin extract, and the liver and muscle extract showed moderate hemolytic activity. |
| Cytotoxicity test: |
| The viability of HeLa cells were observed upon adding the crude tissue extracts. The maximum activity against the HeLa cell line in the skin tissue extract of A. hispidus. When compared to other tissue extracts the IC50 values of the skin extract against the HeLa cell line was found to be 1.78mg/ml. |
DISCUSSION:
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| Recent decades, Marine biotoxins has received much attention by various investigators all over the globe for their pharmaceutical potential. Fishes are living in challenging aquatic environment which contains a wide range of adverse effective compounds. The mucus secretions in the epidermis acts as a biological barrier for the fish protect from the environment [18]. Previous reports revealed that the components present in the epidermal secretion of the mucus acts as a barrier against pathogens and therefore may have a potential source of antimicrobial compounds [19]. Many researchers has been reported an endogenous peptides with antimicrobial activity from fish mainly from the skin and its secretions[20]. The antimicrobial property of mucus against the various pathogens has been demonstrated previously in rock fish [21], rainbow trout [22], and various species of cat fishes[23]. In this study, a pronounced antimicrobial activity has shown against various bacterial and fungal strains. The tissue extracts of A. hispidus shown activity against the bacterial and fungal strains. The antibacterial activity was observed maximum against E. coli by skin extract and minimum was found against P. vulgaris by liver extract. The antifungal activity maximum was observed against A. niger and the minimumv against T. viridae. It goes along with the report of Kumaravel et al., 2011 puffer fish. Arothron immaculatus from Parangipettai coast showed antibacterial activity against pathogens and that extracts not shown any activity against fungal pathogens [24]. In this study, the extracts have been shown activity against fungal strains. These variations occurred may be due to the physical parameters of the environment and the chemical composition in the fish. Chicken blood showed high hemolytic activity on the skin tissue extracts when compared to liver, muscle extract as same as reported by Bragadeeswaran et al., 2010 has studied the hemolytic potential of tetrodotoxin producing bacteria in A. hispidus [25]. The pronounced cytotoxicity of skin extracts showed inhibitory activity against Hela cells. Antitumor activity of the masked puffer fish Arothron diadematus was studied by the Fouda et al., 2005 [26]. The results clearly indicate the toxins present in the fish are having bioactive compounds that may be used for therapeutic needs. |
CONCLUSION
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| In the present study the various parts of puffer fish has been examined for their bioactivity and in particularly the crude skin extract showed a broad spectrum of activities. These results revealed that A. hispidus skin has some valuable bioactive compound. However, it would have to be further subjected to analyze the extract compound present in it. |
Tables at a glance
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| Table 1 |
Table 2 |
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Figures at a glance
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| Figure 1 |
Figure 2 |
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