Biological properties of a venom extract from the sea anemone, Bunodosoma cavernata

PII: S0041-0101(98)00003-8

Toxicon Vol. 36, No. 12, pp. 2013±2020, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0041-0101/98 $19.00 + 0.00

SHORT COMMUNICATION BIOLOGICAL PROPERTIES OF A VENOM EXTRACT FROM THE SEA ANEMONE, BUNODOSOMA CAVERNATA A. E. ENO,1* R. S. KONYA2 and J. O. IBU3 Department of Physiology, College of Medical Sciences, University of Calabar, PMB 1115 Calabar, Cross River State, Nigeria, 2Department of Zoology, Faculty of Science, University of Port Harcourt, Rivers State, Nigeria; and 3Department of Human Physiology, Faculty of Medicine, University of Jos, Jos, Plateau State, Nigeria 1

(Received 12 November 1996; accepted 9 December 1997)

A. E. Eno, R. S. Konya and J. O. Ibu. Biological properties of a venom extract from the sea anemone, Bunodosoma cavernata. Toxicon 36, 2013±2020, 1998.ÐCrude extract was prepared from the sea anemone, Bunodosoma cavernata. The protein content of the extract was estimated to be 0.52 mg protein/ml. The extract was standardized based on the percentage inhibition of histamine-induced contraction of the guinea pig ileum, to determine the biological unit of activity (AU) of the extract. As extracts prepared on di€erent occasions lost potency on storage, the stability of the extract was also investigated. Extracts prepared from fresh animals were about 15% more potent than those prepared from freeze-dried animals. However, freeze-dried animal extracts maintained their potency for about 6 months under storage at ÿ208C. Lethality studies gave an LD50 of 40 mg protein/kg mice i.p. Also, the crude extract dose-dependently hemolyzed human erythrocytes at room temperature. This activity was favoured by higher temperatures, which peaked at about 608C, and by pH in the alkaline range. We conclude that the crude extract from B. cavernata, though highly toxic, may also contain some biologically active agents which include a haemolytic factor and antihistamine(s), as indicated by its histamine-blocking action. # 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION

Sea anemones contain a variety of interesting biological active compounds including some potent toxins (Mathias et al., 1960; Beress, 1982). For this reason many investigators have focused attention on the biological activities of the protein molecules of various species of sea anemones. To date, peptides and proteins ®gure prominently amongst the various classes isolated and characterized (Platou et al., 1986; Norton, * Author to whom correspondence should be addressed. 2013

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1991). The proteins were ®rst isolated as cardiac stimulants (Norton et al., 1976) and neurotoxins (Beress et al., 1975) and these two activities remain the primary focus of attention. The most thoroughly characterized proteins from sea anemones are the 5000 Da toxins that act by binding to the voltage-gated sodium channel (Simpson et al., 1990), although some toxins from the sea anemones Bunodosoma granulifera and Stichodactyla helianttus act on potassium channels (Karlsson et al., 1993). Unfortunately, information about the biological activities of Bunodosoma cavernata is hardly documented although the species is found in abundance in Nigerian coastal waters. It has been suggested that anemone toxins resemble one another both toxicologically and biochemically (Toom et al., 1975). Therefore, it is expected that some biologically potent activities reported in other species of sea anemones are present in extracts from B. cavernata. The main objective of this paper is to provide a starting point for our understanding of the nature and biological activity of the crude extract obtained from this anemone, B. cavernata.

MATERIALS AND METHODS Preparation of the crude extract The extract was prepared by the method of Walker (1977). 100 g of the freeze-dried animal specimens was homogenized in an electrically driven tissue grinder/blender for 5 min. The homogenate was then dissolved in 100 ml saline (0.9% NaCl) and then centrifuged (10 000g) for 10 min. The supernatant was collected into small tubes. The desired test concentrations of the extract were always prepared from this stock by serial dilutions with saline. Estimation of the protein content of the crude extract The determination of the protein content of the crude extract from B. cavernata was performed using egg albumin (BDH) as standard, according to the method described by Lowry et al. (1951). Electrolyte contents of the sea anemone extract and the sea water The crude sea anemone extract (50 ml) of concentration 0.52 mg protein/ml (estimated according to Lowry et al., 1951) was prepared with deionized water instead of saline. Also, 50 ml of ®ltered brackish water was obtained from the animal location (near the estuary). Both solutions (extract and brackish water) were taken to the National Fertilizer Company of Nigeria (NAFCON) laboratories for the analysis of the ionic composition of the solutions. Determination of the biological unit of activity of the crude extract from Bunodosoma cavernata Stability of the extract. Investigations on the stability of the extract were carried out based on results of preliminary experiments on the guinea pig ileum. In those experiments, the extract caused a dose-dependent inhibition of histamine-induced contractions of the ileum. As a result of that study, the stability of the extract was determined. A segment of guinea pig ileum (3 cm long) was suspended vertically in an organ bath (25 ml) containing atropinized (2.9  10ÿ6 M) Tyrode solution of the following composition (mM concentration): NaCl, 140.1; CaCl2, 0.9; KCl, 2.7; NaHCO3, 12.0; MgCl2, 0.5; NaH2PO4, 0.3 and glucose, 5.5. The solution was bubbled with air and maintained at 37218C. Both ends of the tissue were tied with cotton threads and one end was tied to a rigid support inside the organ bath while the other end was attached to an isometric force transducer (FT 0.03). A resting tension of 1 g was maintained throughout the experiments. Two doses of histamine were selected based on the histamine dose±response curve. Histamine, 1.5 and 3.0 mg/ml were found to produce 25 and 75% of the maximum responses of the ileum, respectively. They were given alternately at 5 min intervals, ®rst, without the extract in the bathing solution, then in the presence of the extract. Di€erent preparations of extracts from the same batch of animals were used for the assay. These were extracts prepared from the fresh animals, newly (1±7 days) freeze-dried animals and 2±8 months old (freeze-dried) animals. The extract concentrations were the same (10.4 mg protein/ml) in all the assays. Each extract was assayed ten times, to determine the potency of the extract in the inhibition of histamine-induced contractions of the ileum. Standardization of the extract. The standardization procedure was as described by Aldeen et al. (1981). The crude extract inhibited histamine-induced contraction of guinea pig ileum. To standardize the extract, an arbi-

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trary unit of biological activity was de®ned. A sample of the extract was added to 1 litre of Tyrode solution and a guinea pig ileum preparation was exposed to the solution for 40 min. If the extract solution produced 90% inhibition of the response of the ileum to histamine (1.5 mg/ml), then the sample was said to contain 100 units of activity (100 AU). No reference is made in this de®nition to the volume of the sample. A solution of extract was prepared which produced about 90% inhibition of histamine-induced contraction of the ileum. To samples of this nature, were added known volumes of Tyrode solution to give a series of dilutions, each of which was then assayed for histamine blocking activity on a separate piece of ileum taken from one guinea pig. Eight such complete experiments were done on di€erent samples of the crude extract and the results from 64 separate ileal preparations were determined. Haemolytic activity of the crude extract. Human blood was used in the in vitro experiments. The blood was obtained from the University of Port Harcourt Medical Centre. They were collected in heparinized vacutainers and stored for 2 days at 48C before use. The procedure for the determination of the percentage haemolysis was a described by Jiang et al. (1989). 5 ml whole blood was centrifuged (6.000 rpm for 5 min) to obtain packed blood cells. This was washed three times in 20 nM HEPES (N-2-hydroxyethyl-piperazine-N1-2-ethanesulphonic acid) bu€er (HEPES), 130 mM NaCl, pH adjusted to 7.4, at room temperature. Following the ®nal centrifugation step, 0.05 ml aliquots of packed blood cells (RBC) were added to 7.5 ml of the incubation medium (HEPES bu€er with indicated pH and temperature). The preparations were incubated for 2 h with or without the crude extract, centrifuged and the haemoglobin released estimated by reading the absorbance of the supernatant at 540 nm. The 100% haemolysis point was determined by incubating 0.05 ml of the erythrocytes in 7.5 ml distilled water. Blanks containing no extract were subtracted from all samples. The percentages maximum haemolysis produced was calculated using the formula: Test Haemolysis ÿ Control Haemolysis  100 100%Haemolysis ÿ Control Haemolysis The standardization procedure described above for the extract using the inhibition of histamine-induced contractions of the ileum was also employed in the haemolytic assay (i.e. 90% haemolysis = 100 AU). Based on the ED50 value, a low dose (28 AU) was selected and its haemolytic action tested, ®rst at various temperatures (20±808C), pH 7.4 and then at room temperature and a pH of the incubating medium ranging from 5±9. E€ect of the extract on mice Toxicity test. Male white Wistar mice (15±20 g) were randomly assigned to 10 cages of 40 animals per cage. Each group was injected intraperitoneal with one of the following: 20, 25, 30, 35, 40, 45, 50, 55, 60 and 65 mg protein/kg of the crude sea anemone extract. The maximum volume injected was 0.2 ml per dose. The groups were returned to their home cages after injection and given free access to food and water. The mortality in each group (cage) was assessed 24 h after the administration of the extract. Percentage mortalities were converted to probits and plotted against the log10 of the dose of the extract. The results were subjected to statistical analysis of the regression line.

STATISTICAL ANALYSIS

Results were expressed as mean values 2S.E.M. based on 5±10 experiments. Signi®cance was determined by the Student's t-test. A probability level of 5% or better was considered signi®cant. RESULTS AND DISCUSSION

The protein content of the crude extract was estimated to be 0.52 mg/protein/ml and the estimated electrolyte contents of both the extract and the brackish water are shown in Table 1. Some elements like zinc, phosphorus, potassium and iron occurred in much higher concentrations in the animal extract than in the immediate environment (brackish water). This suggests that these elements are acquired by the animal from other sources, possibly food materials or by bioaccumulation. Results of the acute toxicity studies showed a dose±mortality relationship which was apparently sigmoidal (not shown). A plot of the probit values (% mortality) vs log-dose of extract gave a straight line from which the LD50 was extrapolated. This value was

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A. E. ENO et al. Table 1. Electrolyte contents of the sea anemone extract and the brackish water at the animal location. Values were determined in the laboratories of the National Fertilizer Company of Nigeria `NAFOCON' Port Hacourt, Nigeria Estimated values pH Conductivity Chloride Zinc Phosphorus Potassium Sodium Calcium Iron Silica Orthophosphate Total phosphate Total solids Total dissolved solids Total suspended solids Bicarbonates Sulphates Temperature Salinity Turbidity

crude extract

brachish water

units

6.0 230 7807.5 4.06 547.4 618.2 1476.9 16 84.42 ÿ ÿ ÿ ÿ ÿ

7.88 38100 14990 0.067 ÿ 300 3568.2 974.0 0.783 1.52 1.36 2.40 35074 33568

ÿ m-mhos/cm mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l

ÿ

1506

mg/l

ÿ ÿ 24 ÿ ÿ

173.0 3762.08 24 21 26

mg/l mg/l 8C NTU

Fig. 1. Graph showing the inhibition (%) of histamine (1.5 mg/ml)-induced contractions of the guinea pig ileum by the crude extract (40 AU) against time (min) of exposure of the tissue preparation to the extract. Inhibitory activity was gradual and maximum inhibition was attained after 35±40 min. Data are the mean values2S.E.M. n = 6.

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about 40 mg protein/kg mice i.p. Other jelly®sh classi®ed as highly toxic are Physalis, LD50 0.75 mg/kg, mice (Garriott and Lane, 1969) and Stomolophus meleagris 0.85 mg/ kg, mice (Toom and Chan, 1972). The stability of the extract following prolonged storage was tested in the isolated guinea pig ileal preparation. The extract (1±4 mg protein/ml) produced spontaneous contraction of the ileum; for this reason, atropinized Tyrode solution was used as the bathing solution. Also, the extract dose-dependently inhibited histamine-induced contraction of the ileum. The inhibitory response developed very slowly and reached a maximum in about 35±40 min (Fig. 1). Extracts prepared from newly (1±7 days) freezedried animals, under storage at ÿ208C, and those prepared after storage for up to 6 months appeared to be equipotent in inhibiting histamine-induced contractions of the ileum (Fig. 2). The newly freeze-dried animal extract (10.4 mg protein/ml) produced 65% (1.5 mg/ml histamine) and 72% (3 mg/ml histamine) inhibition of histamine-induced contraction of the ileum, while the 6 months old, freeze dried animal extract (10.4 mg protein/ml) produced 63% (1.5 mg/ml histamine) and 69% (3 mg/ml histamine) inhibition of the ileum. There was no signi®cant di€erence between the results obtained from both extracts. However, animal extracts (10.4 mg protein/ml) stored beyond 6 months showed signi®cant reduction in their biological activity (potency) in the ileum (P < 0.05). However, extracts (10.4 mg protein/ml) prepared from fresh sea anemones produced 78 2 2% (1.5 mg/ml histamine) and 87 2 2% (3 mg/ml histamine) inhibition of histamine-induced contractions of the ileum (Fig. 2). These results are consistent with

Fig. 2. Histogram showing the inhibition (%) of histamine (3 mg/ml, hatched column: 1.5 mg/ml, open column)-induced contractions of the guinea pig ileum by the crude extract (10.4 mg protein/ ml). To determine the potency of extracts following storage at ÿ208C extracts prepared from 2± 8 months old freeze-dried animal specimens of same concentrations were used. Responses obtained from freeze-dried animal extracts before storage, were used as controls. Data are the mean values2S.E.M. n = 10.

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Fig. 3. The relation between AU, inhibition of histamine-induced contractions and haemolysis. Extract was diluted to give solutions with di€erent numbers of AU, these solutions were assayed for histamine inhibition (.) (n = 64) and for haemolysis (w) (n = 35). Note that AU refers to the number of units and not to their ®nal concentrations in the assay test solutions which were di€erent for the two assays (see Section 2).

the ®ndings of Aldeen et al. (1981), which suggested that sea anemone extracts prepared on di€erent occasions varied in their histamine-blocking activity, no doubt re¯ecting di€erences in the eciency of extraction and the use of several di€erent batches of the animals and, possibly, variations in sensitivity of ilea to histamine. For these reasons, the standardization of the crude extract was inevitable. That is, the concentration of the extract preparation was de®ned by the activity (% inhibition of histamine-induced contraction of the ileum and/or % haemolysis of red blood cells) rather than the active principles in the extract. From the results (Fig. 3), the dilution±response relationships for both histamine and haemolysis assays were apparently sigmoidal and straight line regressions were ®tted to the middle regions, i.e. points between 30 and 75 AU by calculation. The regressions were for the % inhibition of histamine-induced contractions of the ileum (a = 35.4; b = 176: r = 0.93; P < 0.05) and for the percentage haemolysis of red blood cells (a = 0.128; b = 1.13: r = 0.91; P < 0.05). From the AU de®nition, 90% inhibition of the ileum or haemolysis of erythrocytes was equivalent to 100 AU. The use of 90% inhibition was dictated by practical considerations and by the observation that the responses to the extract of di€erent ileal preparations were less variable when a high rather than a low concentration of the extract was used. However, it appears more satisfactory to base the activity unit scale on a point in the linear region of the sigmoid concentration response curve rather than at the upper end of the curve as suggested by Aldeen et al. (1981). Although the haemolysis assay of the extract gave a graded response, it also showed both temperature [Table 2(A)] and pH [Table 2(B)] dependence. The haemolysis was favoured by low temperatures, peaking at 608C optimum and in a slightly alkaline

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Table 2. E€ect of the crude extract (28 AU) on in vitro haemolysis (%) of human red blood cells at varying temperatures (20±808C) of the incubating medium, pH 7.4, (A) and at room temperature pH range 5.0±9.0 (B). Data are mean value2S.E.M. n = 10 A temperature of incubation medium (8C)

% haemolysis

20 30 40 50 60 70 80

11.620.8 32.121.8 35.920.7 46.621.6 52.421.2 42.421.8 24.721.7 B

pH of incubation medium

% haemolysis

5.0 5.5 6.0 6.5 7.0 7.5 8.0 9.0

10.321.5 12.721.4 17.322.9 23.523.8 32.422.8 35.127.4 40.422.0 51.123.0

medium. This is in agreement with earlier reports on the haemolytic activity of other species of anemones (Endean and Noble, 1971; Toom et al., 1975; Walker, 1977). In conclusion, it appears that B. cavernata extracts are highly toxic and very unstable on storage. Therefore, it is probably more satisfactory to de®ne the concentration of the extract preparation by its biological activity. Anti-histamine(s) and haemolytic factors are probably among the active agents present in the extract. However, it is premature to speculate on the activity of the crude extract. Further progress must await re®nement and isolation of the active agents. Acknowledgements ÐWe thank Professor Jones Akpan (Professor of Pharmacology) of the College of Medical Sciences, University of Calabar, Nigeria, for his skillful assistance and very stimulating discussions. Also, we gratefully acknowledge the technical assistance of Mr Famous Ojorikre and Mr Isikaku both of the Zoology department, University of Port Harcourt, Nigeria.

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Karlsson, E., Harvey, A. L., Aneiros, A. and Castaneda, O. (1993) Potassium channel toxins from marine animals. Toxicon 31, 497±540. Mathias, A. P., Ross, D. M. and Schachater, M. (1960) The distribution of 5-hydroxytryptamine, homarine and other substances in sea anemones. J. Physiol. 151, 296±311. Lowry, O. H., Rosebroughm, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 263±270. Norton, T. R., Shibata, S., Kashiweagi, M. and Bentley, J. (1976) Isolation and characterization of the cardiotonic polypeptide anthopleurin A. from the sea anemone, Anthopleura xanthogrammica. J. Pharm. Sci. 65, 1365±1374. Norton, R. S. (1991) Structure and structure±function relationship of sea anemone proteins that interact with the sodium channel. Toxicon 29, 1051±1084. Platou, E. S., Refsum, H. and Hotvedt, R. (1986) Class III antiarrhythmic action linked with positive inotropy: Antiatthythmic, electrophysiological and haemodynamic e€ects of the sea anemone polypeptide ATX II in the dog heart. J. Cardiovasc. Pharmacol. 8, 459±465. Simpson, R. J., Reid, G. E., Moritz, R. L., Morton, C. and Notronn, R. S. (1990) Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone, Actinia tenebrosa. Eur. J. Biochem. 190, 319±328. Toom, P. M. and Chan, D. S. (1972) Preliminary Studies of nematocysts from the jelly®shÐStomolophus meleagris. Toxicon 10, 605±612. Toom, P. M., Larsen, J. B., Chan, D. S., Peeper, D. A. and Price, W. (1975) Cardiac e€ects of Stomolophus meleagris toxin. Toxicon 13, 159±164. Walker, M. J. A. (1977) Pharmacological and biochemical properties of a toxin-containing material from the jelly®sh, Cyanea capillata. Toxicon 15, 3±14.