The specimens were collected from Coral reefs at Mandapam near Kurusadai island (78 ^o 11' to 79 ^o 15' E longitude and from 8 ^o 49' to 9 ^o 15' N latitude) in the Gulf of Mannar, Tamilnadu, India. The animals were brought to the laboratory as live and immediately washed, kept in the sterile clean saline water and stored at -20 ^o C until further use.

The crude extract was collected according to the standard methods [₃]. Briefly 100 g of the freeze–dried animal pieces was homogenized in an electrically driven tissue grinder/ blender for 5 min, the homogenate was dissolved in 100 ml normal saline (0.9% NaCl) and this was centrifuged at 10,000g for 10 min. Finally, the supernatant was collected into sterile test tubes. The desired test concentrations of the extract were always prepared from this stock by serial dilution.

Amount of protein in the samples were estimated according to the method of [₄] with bovine serum albumin was used as a standard.

Molecular weights of the partially purified active fractions of crude extracts were analyzed by SDS-PAGE [₅]. Running gels contained 10.0% acrylamide, and electrophoresis was conducted at 50 V for 3 h in Tris–glycine buffer, maintained the pH 8.3. After run, gel was stained with 0.3% Coomassie blue(R-250) and their molecular weights were determined in gel documentation system (Lark, India) by comparison with protein Standards (GeNei ^TM ) Myosin, Rabbit Muscle 205 kDa; Phosphorylase b 97 kDa; Bovine Serum Albumin 66 kDa; Ovalbumin 43 kDa; Carbonic anhydrase 29 kDa.

The acute toxicity study was carried out for the isolated toxin extract using adult sea shore crabs Ocypode macrocera (10–20 g total body weight). This assay was carried out by the injection of 0.1 ml of the crude extract into the third walking leg of the shore crabs. To determine the LD50, ten crabs were injected with dilutions of the extract (0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml) and observed for 2 hrs. Triplicates of each concentration were used. The LD50 was obtained by the Lehman method [₆]. The crude extract, which displayed activity at the sodium-channel, can be already detected by this test. Positive reactions were observed as tetanic contractions of the extremities of the shore crab. The LD50 of these extracts were calculated graphically using standard method [₇].

The cytotoxicity assay was evaluated using the standard method of [₈] with Artemia salina nauplii (24 h post hatching). Fifty organisms were used for each concentration of crude extract, which were 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/ml subjected to 24 h exposures. Triplicates of each concentration were used for assessed the toxicity [₉].

The hemolytic activity of the crude toxin was assessed by the micro hemolytic method [₁₀]. Chicken, Cow, Goat and Human blood (A1+ve) was collected from slaughter house and Government hospital, Parangipettai, Tamilnadu, with EDTA solution (2.7g/ 100 ml) as anticoagulant and brought to the laboratory. The blood was centrifuged at 5,000 rpm for 5 minutes; the supernatant are discarded, the pellet was suspended in normal saline (pH 7.4). The mixture was further centrifuged at 5,000 rpm for 5 min, the supernatant was discarded and pellet resuspended in normal saline (pH 7.4.). This procedure is repeated thrice. From these, 1% erythrocyte suspension was prepared by adding 99 ml normal saline to 1ml of packed RBC.

The micro hemolytic test was performed in 96 well ‘U' bottom microtitre plates. Different rows were selected for these different blood samples. Serial dilution of the crude toxin were made in 100 µl then also discarded. 100µl of 1% RBC was added into all the wells with appropriate controls were included in the test. 1% RBC suspension, 100 µl of distilled water was added, which served as a positive control and 100 µl of normal saline, which served as, negative control. The plate was gently shaken and allowed to stand for three hour at room temperature and the results were recorded. Uniform red color suspension in the wells considered as positive hemolysis and a butten formation in the bottom of the wells was considered as lack of hemolysis. Reciprocal of the highest dilution of the crude extract showing the hemolytic pattern was taken as one Hemolytic Unit (HU).

A total of 30 g of crude extracts were obtained from the samples. The protein concentration of crude was 130 µg/mg and 70 µg/mg respectively.

The protein patterns and molecular weight of the crude extracts of S. mertensii and S. haddoni were analyzed. The molecular weight was estimated as 216 kDa (SmNt1), 193 kDa (SmNt2), 95 kDa (SmNt3), 60 kDa (SmNt4) & 46 kDa (SmNt5) and 103 kDa (ShNt1), 82 kDa (ShNt2) & 63 kDa (ShNt3) (Fig.1) respectively.

Figure 1

Figure 1: SDS-PAGE analysis of crude venom from S and . Purified Protein Standards (GeNei ) fractions. A 10% polyacrylamide mini- slab gel was prepared, and 10 µl of each sample was injected onto each well. Lanes: 1. Protein Standards (GeNei ); 2. S; 3.

The results showed that crude extract of both species (S. mertensii and S. haddoni) produced an evident biological activity on LD 50 test used crabs (Ocypode macrocera). After injection of crude extract (1 mg/ml) into their third walking leg, strong concentration of the walking appendages was observed followed by intense spasmodic movement. The legs turned tremble with involuntary lateral movements, shivering and stiffness appendages, change in carapace color, complete loss of control and paralysis. Mortality was observed in 30 sec., at 0.8 mg/ml, the crabs died within 5 min. After injections with 0.6mg/ml crabs died within 15 min. and 0.4 mg/ml crabs died within 25 min respectively. Over dose range of 0.4-1 .0 mg/ml all crabs showed spasmodic movements. The LD 50 for the two crude extract was obtained with 0.47 mg/kg and 0.71 mg/kg of toxin protein was used.

The bioassay results (Artemia salina nauplii) were represented in fig.2 and 3. Both sea anemone extracts displayed toxicity, with LC50 <1mg/ml and they showed significant cytotoxicity (LC 50 = 0.65mg/ml) and (LC 50 = 0.90mg/ml).

Figure 2

Figure 2: Shown the bioassay of crude toxin of brine shrimp lethality

Figure 3

Figure 3: Shown the bioassay of crude toxin of brine shrimp lethality

The hemolytic assay of sea anemones was represented in fig.4 and 5. The S. mertensii shown the maximum hemolytic activity in goat erythrocyte (10 ^-8 ), the minimum hemolytic activity in chicken erythrocyte (10 ^{- 5} ). The medium hemolytic activity was observed in cow and human erythrocytes (10 ^-6 & 10 ^-7 ). The S haddoni shown the maximum hemolytic activity in goat erythrocyte (10 ^-7 ) and minimum hemolytic activity was observed in chicken erythrocyte (10 ^-4 ). The medium hemolytic activity was found in cow and human erythrocytes (10 ^-5 &10 ^-6 ).

Figure 4

Figure 4: Shown the hemolytic activity of .

Figure 5

Figure 5: Shown the hemolytic activity of