Antimicrobial activity of Tetrodotoxin extracted from liver, skin and muscles of puffer fish, Lagocephalus sceleratus inhabiting Mediterranean Sea, Egypt

Introduction:

       The bio resources present in the marine ecosystem have potent biomolecules which includes many natural organic compounds. These compounds are reported to have biological activities like anti- tumor, anti- viral and analgesic (Rajamanikandan et al., 2011). Marine fish are also of high nutritive value. Proteins from fish have shown biological activities like anti- bacterial, anti- inflammatory and antioxidant (Khora, 2013).

           Fish live in intimate contact with an environment containing pathogenic organisms. The slow adaptive immune response of fish makes innate immunity, which is fast acting and temperature independent predominant system of fish host defence (Ellis, 2012). The defence includes many elements such as antimicrobial peptides, lipids and polypeptides (Ravichandran et al., 2010).

          Tetrodotoxin (TTX) is a strong marine toxin; also there is no known antidote for TTX, which is a powerful sodium channel inhibitor (Hwang and Noguchi, 2007). Although TTX was first isolated from puffer fish, it has been found in other marine organisms and a few terrestrial species (e.g., newt and frog) (Miyazawa and Noguchi, 2001). TTX is thermo-stable and also cannot be removed by washing (Noguchi and Ebesu, 2001).

            TTX is a new phenomenon for Mediterranean countries as L. sceleratus (Gmelin, 1789) invaded the Mediterranean Sea. L. sceleratus has had negative ecological, economical, and health effects in the Mediterranean basin (Aydin, 2011; Streftaris and Zenetos, 2006). It was reported that this species was high in protein and had a favourable fatty acid profile (Aydin et al., 2013).

            Nevertheless, puffer fish may be a threat to public health if they contain TTX. According to current European Union (EC, 2004) and Turkish regulations, the landing, sale and consumption of puffer fish is prohibited. Although this fish is poisonous, it is consumed in some countries, including Mediterranean countries, such as Turkey, Egypt and Lebanon (Aydin, 2011; Chamandi et al., 2009 and El-Sayed et al., 2003).

          The current study was undertaken to evaluate the toxicity of three organs: liver, skin and muscles. In addition, the extracted toxins were partially purified and identified using TLC, UV spectrophotometer, and LC–MS–MS; also studies the antimicrobial activities against different bacteria and fungi strains.

Material and methods:

1. Sampling

A total of 20 specimens (12 males and 8 females) puffer fish, L. sceleratus (Gmelin, 1789), ranging between 14.34 – 45.78 cm in total length and 50.90 – 3820g in weight, the Puffer Fish were captured by commercial bottom trawlers using purse seine nets at 24 and 50 meter depths in the Egyptian coast of eastern Mediterranean Sea from El-Hammam to Rosetta (31° 00’& 31° 50’ N) and (27° 00’& 31° 40’ E) in two seasons winter (cold season) in January 2016 and summer (warm season) in July 2016. Directly after collection, the fresh samples were weighed and immediately frozen at -20˚C. Then they were transported to the Laboratory of Marine Biology in Zoology Department, Faculty of Science, Al-Azhar University, Cairo, where they were taxonomically identified according to Randall (1983).

2. Isolation and partial purification of toxin

        Organs pooled from all specimens were collected (2430 g liver, 12,321 g muscles, 2356 g skin), A 10 g portion of the muscle, liver and skin were extracted with 25 ml 1% acetic acid (Merck) and homogenized separately. Each homogenate heated for 10 minutes in a boiling water bath (Memmert, Schwabach, Germany), with occasionally stirring. The mixture was cooled to the room temperature (23–24˚C) and then filtered with Whatman 110 mm filter paper (Sigma-Aldrich, Lyon, France) then residue on the filter paper was washed with portions of 0.1% acetic acid followed by centrifugation at 5000 rpm for 15 minutes. The above step was carried out thrice to extract the toxin. Supernatant obtained was concentrated using rotary evaporator, the final solution, of which 1 ml was equivalent to 0.2 g of tissue and stored at  -20º C for further use (Khora, 1991).

3. Tested organisms

         Bacterial species such as Staphylococcus aureus, Bacillus subtilis, Streptococcus agalactiae and Enterococcus faecalis (gram positive bacteria) and Vibiro cholerae, Aeromonas veronii, Escherichia coli, Proteus vulgaris, Salmonella typhi and Shigella dysenteriae (gram negative bacteria) were maintained in Luria Bertani broth. Fungal species such as Candida albicans, Aspergillus fumigatus and Trichophyton rubrum were maintained in Potato dextrose broth.

4. Antimicrobial activity:

         Antimicrobial activity was determined using the agar well diffusion assay method as described by (Holder and Boyce, 1994). The tested organisms were sub cultured on nutrient agar medium for bacteria and Saboroud dextrose agar for fungi. Ampicillin for gram positive bacteria and Gentamycin for gram negative bacteria were used as a positive control for bacterial strains; fluconazole was used as a positive control for fungi. The plates were done in triplicate. Bacterial cultures were incubated at 37°C for 24 h while the other fungal cultures were incubated at (25-30°C) for 3-7 days Antimicrobial activity was determined by measurement zone of inhibition. Then, the percentage of inhibition of cell growth is calculated as follows:

Percentage of inhibition = 100 – (Sample / Control) x 100

5. Tetrodotoxin (TTX) standard

         The TTX standard was purchased from Sigma-Aldrich, USA. Standard solutions of TTX were prepared with methanol (Merck) and stored at -20 ˚C until used. All chemicals were used for analytical grade, solvents for HPLC and TLC from El-Nasr Co., Egypt. Also, all analytical experiments for TTX were conducted in Analytical Chemistry Laboratory of Analytical Chemistry Department, Faculty of pharmacy, Al-Azhar University, Cairo except TTX levels (µg/g) using LC–MS–MS were conducted in Analytical Central Lab. which belong to Faculty of pharmacy, Cairo University.

6. Thin-layer chromatography (TLC) of toxins

         TLC was performed on silica gel 60-F254 precoated plates (Merck) with the butanol : acetic acid : water (2:1:1 v/v) solvent system. After development, the plates were sprayed with 10% KOH, then heated at 110˚C for 10 min, according to (Onoue et al., 1984). The experiment was conducted by Dr. Ahmed Serag, in Department of Analytical Chemistry, Faculty of pharmacy, Al-Azhar University, Cairo.

7. Sample extraction for the LC–MS–MS analysis

       Analysis was carried out by the method of (Silva et al., 2012). A 1 g tissue sample of muscle, liver and skin was extracted with 3 ml of 1% acetic acid. Then the solution was homogenized using the homogenizer at 2400 rpm for 10 min (IKA T25 Digital Ultra Turrax, Staufen, Germany), followed by an ultrasonic bath (Bandelin Sonorex RK 100, Berlin, Germany) for 10 min at 100 Hz. Two additional homogenization (2400 rpm, 10 min each) were done and the extract was centrifuged at 4500 rpm for 20 min at 4˚C (Hettich Zentrifugen, Universal 32R, Tuttlingen, Germany). Supernatants were combined and adjusted to a final volume of 7 ml. After that, 1 ml of the extract was cleaned by running through a 500 mg/3ml C18 solid-phase extraction (SPE) cartridge (Supelco, Bellefonte, PA, USA). The sample was eluted with 10 ml of 100% methanol and diluted with the same solvent to a final volume of 12 ml. Finally, each sample was concentrated by drying (Buchi, Rotavapor R-3, Flawil, Switzerland) and re-suspended in 1 ml of methanol, and the sample (100 µl) was filtered through a 0.45 µm membrane filter (Clarinet, Agela Technologies, Wilmington, USA) before LC–MS/ MS analysis.

8. UV Spectrophotometer

       Shimadzu UV-Visible 1650 Spectrophotometer, (Tokyo, Japan), equipped with 10 mm matched quartz cells.

 9. HPLC (High Performance Chromatograph)

           HPLC, constaMetric® 4100 LDC analytical pump (Milton Roy, USA), equipped with Spectra system UV3000 diode-array UV-Visible detector and spectra system AS3000 auto sampler. The chromatographic analysis was carried out using (ChromQuest 4.2.34, version 3.1.6) data analysis program.

9.1. Chromatographic conditions:

          At ambient temperature, isocratic separation was carried out on supelco C18 column ( 25 cm X 4.6mm , 5μm particle size) using mobile phase consists of acetonitrile: methanol: water (50:30:20, by volume). The mobile phase was degassed by a degasser before pumped at flow rate of 1ml/min. The injected volume of the standard solution was 20 μl and UV detection at 220 nm.

10. Statistical analyses

         Statistical analysis: Data were computerized and analyzed using SAS Version 9 statistical package (SAS, 2002), the analysis was revised and graphics were drawn by Excel for Microsoft office 2010. The obtained data were assessed by calculation of the mean (M) and standard deviation (SD) was calculated using multiple linear regressions (Finney, 1971).

Results:

1. Antimicrobial activity

         The antibacterial activity of the crude extracts of puffer fish L. sceleratus were tested against the bacterial strains. The results of the inhibition activity of the crude TTX extract isolated from liver, skin and muscles of L. sceleratus as shown in the Table 1 as compared with the standard ampicillin in gram positive bacteria. The maximum activity of the crude TTX of liver followed with skin and muscles extract were shown against the Bacillus subtilis as 17.51, 16.27 and 15.28 respectively and 13.83, 12.75 and 11.30 respectively in Staphylococcus aureus. On the other hand the minimum activity against Streptococcus agalactiae as 10.84, 11.44 and 12.68 respectively with the crude TTX of skin followed with muscles and liver extract that shown in the Figure 1.

        The current results of the inhibition activity of the crude TTX extract isolated from liver, skin and muscles of L. sceleratus as shown in the Table 1 as compared with the standard Gentamycin in gram negative bacteria. The maximum activity of the crude TTX of liver followed with skin and muscles extract were shown against the Escherichia coli as 22.24, 20.16 and 19.16 respectively and the crude TTX of liver followed with muscles and skin extracts as 20.49, 19.67 and 18.02 respectively in Aeromonas veronii. On the other hand the minimum activity against Vibiro cholerae as 8.08 in muscle, 8.45 in skin and 9.22 in liver extracts that shown in the Figure 2.

         The antifungal activity of the crude extracts of puffer fish L. sceleratus were tested against the fungal strains. The results of the inhibition activity of the crude extract of L. sceleratus as shown in the Table 1 as compared with the standard fluconazole. The maximum activity of the crude TTX of liver followed with skin and muscles extract was shown against the Aspergillus fumigatus as 19.21, 17.11 and 16.27 respectively andthe activity of the crude TTX of skin followed with liver and muscles extract was shown against Candida albicans as 13.17, 12.50 and 10.66 respectively. On the other hand the minimum activity against Trichophyton rubrum as 5.33 in skin and 6.21 in muscles extracts and no activity with the crude TTX of liver  extract that shown in the Figure 3.

2. LC–MS–MS (Liquid Chromatograph / mass spectrometry) analysis

       The maximum amount of TTX in females livers tissues followed with males livers tissues using LC–MS–MS were 30.33 and 25.37 µg/g respectively (Table 2).TTX in muscle tissues was found above 2 µg/g only in a female fish, only in the winter (2.26 µg/g), TTX levels in the skins exceeded 3 µg/g for females in winter, although for males were < 2 µg/g in winter and summer. TTX levels in the liver (of those fish analyzed) exceeded 2 µg/g for females in the summer and become highly toxic as (21.04µg/g) than males were (4.04µg/g) in the same season as showed in Figures 4 and 5.

3. HPLC (High Performance Liquid Chromatograph) analysis

        HPLC techniques allow the separation and sensitive detection of individual TTX and its analogs irrespective of their number and group. Therefore, HPLC methods have opened up a new dimension in TTX analysis. Additionally, accurate HPLC determination of the various TTX components in the samples is a necessity. Using these methods, the toxic principles produced peaks identical to those of authentic TTX and its derivatives. The HPLC method utilizes a computer controlled by a high pressure pump with a syringe loading sample injector or an auto-sampler system, a stainless steel column, a reaction pump for delivering reagents, and a monitor and chromato-recorder for calculation of the peak area. The treated toxins are identified by comparing their retention times with those of authentic TTX (Figures 6 and 7).

4. TLC (Thin Layer Chromatograph) analysis

       TLC is a useful technique in those laboratories where HPLC and other costly analytical systems are not available. The developed toxins were visualized as yellow spots under UV light at 365 nm.

5. UV spectrum (Ultra Violet spectrophotometer) analysis

         In UV spectroscopy, TTX is generally determined by irradiating a crude toxin with UV light. A small amount of TTX is dissolved in 2 ml of 2 M NaOH and heated in a boiling water bath for 45 min. After cooling to room temperature, the UV spectrum of the solution is examined for characteristic absorptions, associated with C9-base, 2-amino-6-hydroxymethyl-8- hydroxyquinazoline, possibly formed from TTX and/or related substances, if present. In the analysis, the UVspectrum of the alkali decomposed compounds of TTX appears as a shoulder at near 276 nm, indicating the formation of C9-base specific to TTX or related substances (Figure 8 and 9). A similar result was observed by Saito et al., 1987used this method in experiments analyzing TTX and its derivatives in toxic puffer fish.

Discussion:

         The antibacterial activity of fish has been known for many years and has been demonstrated in the mucous of several fishes. It was reported that epithelial tissues produce antimicrobial molecules which serve as the first line of a host defence against microbial invasion in vertebrates (Ganz, 1999). In this study, antimicrobial activity of skin extract of L. sceleratus has shown activity against various bacterial and fungal organisms. The antibacterial activity was found maximum against Enterococcus faecalis and Bacillus subtilis in gram positive bacteria, Escherichia coli and Aeromonas veronii in gram negative bacteriaand minimum against Streptococcus agalactiae in gram positive bacteria and Vibiro cholerae in gram negative bacteria, antifungal activity was observed maximum against Aspergillus fumigatus and Candida albicans and minimum activity against Trichophyton rubrum by the skin extract of puffer fish L. sceleratus. Similar results were also reported from the puffer fish A. hispidus from the Mandapam coast (Mohana and Khora, 2013) from A. immaculatus from the Parangipettai coast (Kumaravel et al, 2011) which reports antibacterial activity against various pathogens. The results clearly indicate the bio toxin present in the fish is having potential that may be used for pharmaceutical needs. Though this toxin is a health threat in its original form due to its binding ability, yet it shows potentials as a drug in lower doses.

            Noguchi and Arakawa (2008) reported that gonads of L. sceleratus were extremely toxic (>1000 MU/g), liver, skin and muscles were less toxic (10–100 MU/g), intestinal was moderately toxic (100–1000 MU/g), and this fish muscle was generally toxic. Many researchers reported that highest concentrations of TTX level were in gonads, following in liver, intestines, skin and muscle tissue in puffer fish (Chua & Chew, 2009; El-Sayed et al., 2003). Azman et al. (2014) reported that TTX levels of liver, muscle and skin of L. sceleratus males were 24.7µg/g, 30.0µg/g and 0.51µg/g respectively. The finding of liver and skin were nearly similar with this work but TTX levels in muscle tissue were more than the result of Azman in cold season.

       El-Sayed et al. (2003) investigated TTX level in 45 silver stripe blaasop puffer fish samples. Highest values were found in liver, muscle tissue and skin, as (converted from MU/g to µg/g) 246 MU/g (54.12 µg/g), 127 MU/g (27.94 µg/g) and 119 MU/g (26.18 µg/g) respectively. They found higher levels of toxins, compared to these results. It was considered that this case was related to the sensitivity of GC mass spectroscopy that used in research also, the different in species and founa between the previous study in Red Sea and the current study in Mediterranean Sea.

       Dao et al. (2012) investigated the TTX level of L. sceleratus by HPLC and found TTX content in liver, skin and muscle tissue (MU/g) as 20.8 (4.58 µg/g), 27.8 (6.11 µg/g) and 58.7 (12.91 µg/g), respectively, but they especially found high levels of toxins in skin and muscle tissue compared to our study. This can be due to the environmental conditions, geographical differences and feeding regime. First TTX content in puffer fish investigated with chromatographic method was carried out by Rodríguez et al. (2012) in the European waters. They investigated TTX levels of L. sceleratus caught from the Aegean Sea by LC–MS–MS. TTX content ranged from 3.37 to 32.15, 0.53 to 3.40 and 0.66 to 3.07 (µg/g) in liver, skin and muscle, respectively. The results of this study are nearly similar with our study in terms of TTX levels in liver, skin, and muscle tissue, with the consideration that the sex and seasonal factors not taken in Rodríguez study to differentiation in TTX levels between tested organs.

         Many researchers reported that the toxicity of puffer fish was dependent on reproduction season, since the reproductive period for most species of puffer fish was from late spring to summer. Thus, toxin levels in the fall and winter season were the lowest (El-Sayed et al., 2003; Hwang & Noguchi, 2007). However, Yu and Yu (2002) reported that Takifugu niphobles and Takifugu alboplumbeus species did not show toxic effects in the reproduction period. It was similarly indicated by Katikou et al. (2009) and Rodríguez et al. (2012), TTX level of L. sceleratus species, caught from the Mediterranean Sea, was higher in the autumn and winter season. It is thought that this situation may be specific in the Mediterranean and Aegean Sea. The effects of seasonal variations toxin levels should be investigated with more scientific research.

        The Minimum Lethal Dosage (MLD) of TTX for the average weight female (50 kg) was estimated to be 2 mg (Katikou et al., 2009). This is equivalent to 10 MU TTX per g of edible part (Miyazawa & Noguchi, 2001). Because males and females cannot generally be distinguished by fishers or consumers easily, fish meat should not be eaten in the winter without prior testing, although more data collection is strongly recommended before making any such change. Further studies should also focus on the gain into fisheries economy or a drug for the pharmacy sector.