biological activity of sea anemone proteins: i....

Indian Journal of Experimental Biology

Vol. 47, December 2010, pp 1225-1232

Biological activity of sea anemone proteins: I. Toxicity and histopathology

Vinoth S Ravindran1*, L Kannan

2 & K Venkateshvaran

3†

1, 2Centre of Advanced Study in Marine Biology, Annamalai University, Portonovo 608 502, India 3Aquatic Biotoxinology Laboratory, Central Institute of Fisheries Education, Versova, Mumbai 400 054, India

Received 1 May 2008: revised 12 July 2010

The crude as well as partially purified protein fractions from anemone species viz. Heteractis magnifica, Stichodactyla

haddoni and Paracodylactis sinensis, collected from the Gulf of Mannar, south east coast of India were found to be toxic at

different levels to mice. The mice showed behavioral changes such as loss of balance, opaque eyes, tonic convulsions,

paralysis, micturiction, flexing of muscles, prodding (insensitive to stimulii), foaming from mouth and exophthalmia. The

toxic proteins upon envenomation produced several chronic and lethal histopathological changes like formation of pycnotic

nuclii and glial nodules in the brain; heamolysis, thrombosis and myocardial haemorrhage in the heart; granulomatous

lesions, and damage to the hepatic cells in the liver and haemorrhage throughout the kidney parenchyma and shrinkage of

glomerular tufts in the kidney. The toxins proved to be neurotoxic, cardiotoxic, nephrotoxic and hepatotoxic by their action

on internal organ systems. The toxins were also thermostable till 60oC and had considerable shelf life.

Keywords: Mammalian toxicity, Sea anemone protein, Thermostable

Sea anemones are ocean dwelling sedentary organism

belonging to Phylum Cnidaria, Class Anthozoa.

They produce toxic polypeptides and proteins.

More than 40 toxic peptides have been isolated

from different sea anemones1,2

. The understanding

of envenomation requires recognition of the gross

manifestations seen in organs removed from the body

and an appreciation of the underlying microscopic

changes3 in correlation with clinical signs and

symptoms4,5

. Hence histopathological investigations

are inevitable to reveal the effect of the toxin on

specific organs.

Studies regarding the histopathalogy of the

animals, envenomed by sea anemone toxins are very

meager. Limited reports are there on the

histopathalogical effects caused by the sea anemone

toxins. Fatal damage to the liver cells of a young man,

who was stung by an anemone, has been reported6.

Incidentally, it was the only known case of death

caused by sea anemone envenomation. The toxin of

the sea anemone Anthopleura middori caused severe

damage to the brain, heart, kidney and liver cells of

the male albino mice7. The report also exhibits the

toxin to have caused haemorrhage in the brain,

haemolysis in the heart and kidney and severe damage

to the hepatocytes in the liver. Inspite of all these

explorations from these organisms, the sea anemones

from the Indian waters have been poorly studied for

their toxicity. The present study has been therefore,

undertaken to obtain information on the toxicity of

three sea anemone species viz. Heteractis magnifica

(Quoy and Gaimard, 1833), Stichodactyla haddoni

(Saville-Kent, 1893) and Paracodylactis sinensis

Carlgren 1934, collected from the Gulf of Mannar,

southeast coast of India.

Materials and Methods

Extraction of crude—The anemones were collected

from the field and 500 g fresh weight of body tissue

was macerated and extracted with 500 ml of

methanol, which was evaporated to dryness in a

Rotary Flash Evaporator at 37°C and the extracts8

were stored at -20°C until further analysis.

Partial purification of the crude extract—Partial

purification of the crude extract (5mg/ml) was

carried out using DEAE Cellulose Anion Exchange

Chromatography9. Ten fractions were collected in a

step-wise gradient with 0.1-1M NaCl in phosphate

buffer saline (PBS). The collected fractions were

stored at -20°C for further use.

——————

*Correspondent author (present address):

Suganthi Devadason Marine Research Institute,

44 Beach Rd. Tuticorin 628 001, India

Telephone: (0) 9655638373; 0461-2336487/88

Fax: 0461-2325692

E-mail: [email protected]

INDIAN J EXP BIOL, DECEMBER 2010

1226

Protein estimation—Protein estimation10

was done,

using bovine serum albumin (BSA) as the standard.

The absorbance was read spectrophotometrically at

280 nm.

Mice bioassay for lethality—Kausauli strain male

albino mice of 20±2 g body weight procured from

M/s. Haffkine Biopharma, Mumbai, were maintained

in a healthy condition in the animal house, following

the codal formalities of Central Institute of Fisheries

Education, Mumbai, in accordance with the norms of

Animal Welfare Ethics.

The crude toxin dissolved @ 5 mg/ml in PBS

was injected ip to the test mice in doses containing

1.25, 2.50, 3.75 and 5.0 mg of toxin. Also, 1.0 ml of

each fraction (protein content mentioned in Table 2)

was injected ip to different test mice. Triplicate sets

were maintained for each dose. The injected mice

were kept under observation in mice rearing cages.

The time of injection and the time of death were

recorded using a seconds-timer stop-watch, besides

recording the behavioral changes before death.

Mice that died upon envenomation were autopsied

to observe gross anatomical changes such as

hemorrhage, blood clots, septicemia, dark or pale

discoloration of internal organs, etc., if any.

Stability of toxin—Bioassays were conducted to

ascertain the stability of the crude anemone proteins

from H. magnifica, S. haddoni and P. sinensis, as

affected by (i) heating at different temperatures

(50, 60, 80 and 100°C) (ii) at different levels of pH

(3.0-8.0) and (iii) storing at –20°C for more than one

year. In the first case, samples were heated to the

above said temperature on a water bath for 5 min9.

Each of these samples was immediately tested for the

toxicity by ip injection with the lethal dose to male

albino mice as described above. In the second case,

pH of the samples was adjusted using 0.1N HCl

or 0.1N NaOH to pH levels 3.0, 4.0, 5.0, 6.0, 7.0 and

8.0. From the adjusted toxin solutions, lethal dose

was immediately injected ip to mice to ascertain

the toxicity.

Histopathology—Brain, heart, liver and kidney

were dissected out from mice that died upon

envenomation while ascertaining the toxicity of the

anemone extracts. The dissected organs were fixed in

10% formalin for a minimum period of 24 h and

processed for histopathological observations5.

Prepared sections were examined and photographed

under a microscope (Labomed, CX II).

Results Crude extract—The amount of crude extract

obtained from 500 g fresh weights in each case

was 9.73 g in H. magnifica, 7.84 g in S. haddoni and

5.37 g in P. sinensis.

Protein content—Protein content in the crude

extract was highest in H. magnifica followed by

S. haddoni and P. sinensis. Amount of protein in the

purified fractions also followed a similar trend as

the crude (Table 1).

Mice bioassay for lethality—Crude extract of the

anemones containing 1.25, 2.50, 3.75 and 5.0 mg of

protein when injected ip to mice showed symptoms of

toxicity but the levels at which they were lethal varied

from species to species. The dose at which the crude

and fractionated proteins were toxic have been

tabulated (Table 2).

Behavioral changes in mice—The changes in

behaviour of the envenomed were: lying on belly with

widespread forelimbs, running around the cage in an

exited manner, escape reaction, prolonged palpitation,

closed eyes, grooming, shivering of fore limbs, loss of

balance, opaque eyes, squeaking, tonic convulsions,

gasping for breath, arching of body backwards,

paralysis, micturiction, flexing of muscles, prodding

(insensitive to stimulii), diarrohea, lethargy, dragging

of hind limbs, rolling of tail, foaming from mouth and

exophthalmia. However, interestingly, when certain

fractions such as fraction 5 from H. magnifica,

fraction 6 from S. haddoni and fraction 5 from

P. sinensis were injected, the mice became very brisk

and active after envenomation and all these fractions

were not lethal.

Stability test—Lethal activity of the crude proteins

was not affected on storage for 14 months at -20°C.

The crude extracts were found to be thermolabile. They

were stable up to 60°C after which they lost their

Table 1—Protein content (µg/ml) of crude and partially purified

fractions of sea anemones

[Values are mean±SE of triplicate sets]

Fraction no. H. magnifica S. haddoni P. sinensis

Crude 981.1±0.32 820.4±0.37 605.3±0.31 1 70.5±0.11 30.3±0.24 110.6±0.33

2 160.7±0.25 30.6±0.19 10.3±0.14

3 70.3±0.18 140.8±0.27 50.4±0.18

4 80.6±0.17 10.2±0.13 10.3±0.11

5 20.3±0.13 20.5±0.11 20.2±0.16

6 30.7±0.21 60.4±0.23 10.2±0.13

7 10.9±0.17 10.1±0.09 10.6±0.06

8 10.7±0.08 10.2±0.10 10.7±0.08

9 20.4±0.15 10.3±0.06 10.4±0.11

10 10.2±0.10 10.4±0.11 10.2±0.11

RAVINDRAN et al.: BIOLOGICAL ACTIVITY OF SEA ANEMONES : TOXICITY & HISTOPATHOLOGY

1227

activity. The crude toxin of all the three anemone

species lost their activity in higher acidic pH.

Mice injected with these toxins showed some

toxic symptoms initially, but recovered within a

few minutes.

Histopathological observations—Histopathological

observations indicated that, irrespective of the species

of anemone and the toxin obtained from it, effects

were more or less similar on the organs viz. brain,

heart, liver and kidney of the targeted test animals.

Therefore, the following description of results, as also

the ensuing discussion, are on the basis of the effects

of anemone toxins on the organ systems and not specific to the species studied.

Effect of the sea anemone toxins on different

organs of mice—The concentration, at which the

crude protein was lethal, in case of each anemone

toxin, had the following effects in the different organs

of mice, which were observed at autopsy. Even, the

fractioned proteins of the three target anemone

species that showed lethality produced marked histopathological changes, in different organs.

Brain: In the brain, the capillaries were enlarged,

particularly in the cerebrum (Fig. 1a). On the other

hand, mild congestion of capillaries and pycnotic

nuclii were found in the cerebellum (Fig. 1b). In

fractionated proteins, brain tissues showed glial nodule formation in the cerebrum (Fig. 1c).

Heart: In the heart, endocardium was found to

contain large amount of haemolysed blood (Fig. 2a).

Myocardial haemorrhage in focal areas without

cellular reactions was observed (Fig. 2b). Thrombosis

could be observed in the myocardium (Fig. 2c).

The heart tissues showed myofibril separation

(Fig. 2d) upon treatment with fractionated proteins.

Liver: Liver cells showed degenerative changes and the hepatic cells lost their structure and showed marked congestion (Fig. 3a, b). Blood vessels contained partially haemolysed blood (Fig. 3b). Pycnotic nuclei could be observed (Fig. 3c).

Perivascular region showed cellular reactions, with granulomatous lesions. In the case of treatment with fractionated proteins, pycnotic as well as karyorhectic nuclei formation could be observed (Fig. 3c). The sinusoids were distended and disrupted with accumulated erythrocytes. Coagulative necrosis

and degenerative changes were found in the hepatic cells in the focal areas of the liver. Centrilobular necrosis could also be seen (Fig. 3d).

Kidney: Kidney showed wider areas of haemorrhage throughout the parenchyma (Fig. 4a). Further degeneration of kidney parenchyma,

congestion of blood vessels in the cortical region and large areas of haemolysis were also noticed (Figs. 4b and 4c). In the case of fractionated proteins, kidney tissues showed shrinkage of glomerular tuft.

Discussion

During the present study, toxic proteins have been isolated from the body of the sea anemones viz. H. magnifica, S. haddoni and P. sinensis using methanol as the extraction medium

8, as it is considered

as a universal solvent which could extract even the basic proteins without exempting any one of them.

In the present study, protein level in the crude extract was found to be maximum in H. magnifica, followed by S. haddoni and P. sinensis (Table 1). On purification, the protein content considerably

decreased in all the three anemone species. Similar reduction in the protein content on purification through different columns has been reported

11,12 in

the toxin of Aiptasia pallida.

Table 2—Toxicity of crude extracts of the anemones injected ip at different doses to male albino mice

Amount of protein (µg/kg) Species Quantity injected

(ml) In the injected sample (µg/ml) Toxic dose (µg/kg)

Death time (sec)

(mean±SE)

0.25 245.27 - - -

0.50 490.55 - - -

0.75 735.83 - - -

H. magnifica

1.0 980.1 49,005 68.3±2.60

0.25 205.1 - - -

0.50 410.2 - - -

0.75 615.3 30,765 135±2.64

S. haddoni

1.0 820.4 - - -

0.25 151.32 - - -

0.50 302.7 15,135 86.33±1.45

0.75 453.98 - - -

P. sinensis

1.0 605.3 - - -


1228

The results of the present study revealed that

the crude protein extracted from all the three sea

anemone species were lethal to mice (Table 2).

Upon partial purification also, various protein

fractions were found to be lethal (Table 3). In all

the three anemone species, the purified fractions

exhibited lethality at minimum protein levels

(10.1±0.09 to 160.7±0.25 µg/ml). As in the present

investigation, instances of toxicity of various sea

anemones have been well established. A number of

sea anemones exhibited toxicity on insects, crabs,

fishes and mice. Different toxins have been reported

to exhibit toxicity at various doses13

as in the present

study. The potency of the presently extracted toxins is

comparable with other toxins extracted from various

other tropical and temperate sea anemones14-19

.

The behavioural changes and/or the symptoms

of envenomation that were exhibited by the test

mice in the present study may be due to the action of

the anemone toxins on the organ systems. Symptoms

such as loss of balance, arching of body backwards,

tonic convulsions, paralysis, rolling of tail, dragging

of hind limbs, foaming from mouth reveals the

action of the toxins on the CNS and brain. Similar

observations have also been reported in mice

envenomed with the toxins of R. macrodactylus17

and

Bundosoma caissarum20

. However detailed individual

studies are warranted to establish the mechanism

of action and the related symptoms on envenomation.

In the present study, the toxicity lowered upon

repeated freezing and thawing. This is in conformity

with earlier reports17

in tenebrosin-C, extracted from

Actinia tenebrosa. The toxins isolated from the sea

anemones H. magnifica, S. haddoni and P. sinensis

were stable upto 60°C and for 14 months. Similar

instances of considerable thermostability, as in the

present study, have been reported in various other

anemone toxins21,22

. Long shelf life under low

temperatures as in the present study is a characteristic

feature of many anemone toxins14

. Instance of

susceptibility to acidic pH as in the present

investigation has also been reported in the toxin of

Aiptasia pallida23

. The considerable stability of the

presently extracted anemone toxins, especially the

non-toxic fractions in a wide range of temperatures

(-20° to 60°C); at alkaline pH; and storage for

14 months at a constant temperature of -20°C is quite

desirable for drug development.

Observed histopathological changes in the test

mice suggest that all the main organs viz. brain, heart,

liver and kidney were affected by the anemone toxins

(crude toxin and fractionated proteins). Analysis of

various histopathological sections showed that the

action on the organs of mice did not vary with

the toxic proteins extracted from different species.

Fig. 1—Effect of crude and fractionated proteins of sea anemone

species on brain of male albino mice (a) enlarged capillaries,

(b) pycnotic nuclei (c) Glial nodule in cerebellum [× 400]


1229

Hence, few histopathological observations have been

explained to show the effects that anemone toxins can

cause to the mammalian organs. It is also noteworthy

to mention that such histopathological studies with the

sea anemone toxins are meager.

Capillaries of the brain in the cerebrum and the

cerebellum have been affected by the anemone toxins.

This can be attributed to the fact that the sea

anemones have been reported to possess neurotoxins of polypeptide nature

23.

Fig. 2—Effect of crude and fractionated proteins of sea anemone species on the heart of male albino mice (a) haemolysed blood in

endocardium, (b) haemorrhage in focal areas, (c) large areas of thrombosis in myocardium, (d) myofibrilar separation [× 400]

Fig. 3—Effect of crude and fractionated proteins of sea anemones on the liver of male albino mice (a) degenerative changes in the hepatic

cells (b) haemolysed blood in central veins (c) pycnotic and karyorhectic nuclei (d) centrilobular necrosis [× 400]


1230

Histopathological diagnoses of the heart imply

acute cardiotoxicity of these anemone toxins.

Cardiotoxicity is a common characteristic of

coelentrate toxins24,25

. Instances of cardiotoxicity by

anemone toxins have been reported earlier from

sea anemones such as Bundosoma granulifera26

,

Tealia felina27

Actinia equina,28,29

and A. tenebrosa 30

.

Severe damage to the kidney cells observed in the

present study suggests the anemone toxins affect

kidneys. Depressing effect of toxins after digestion,

causing hypotension resulting in renal ischemia and

thus making the kidney more susceptible to the toxic

action has been reported31

. The damage to the heart

tissue could have also affected the kidney, as, any

haemoglobin released upon haemolysis in the heart

should finally go into the tubular filtrate and if the

amount of haemoglobin filtered greatly exceeded the

amount of haemoglobin that can be reabsorbed by

the proximal tubules, and then the unabsorbed

haemoglobin would precipitate, can block the tubules

and eventually result in renal damage32

. The present

study also revealed haemolysis in the heart.

Damage caused to the hepatocytes in the present

study could be attributed to the storage of toxin in

the liver for detoxification. Liver degeneration and

Fig. 4—Effect of crude and fractionated proteins of sea anemone

species on the kidney of male albino mice (a) shrinkage of

glomerular tuft and hemorrhage through out kidney parenchyma

[× 100] (b) degenerative changes in the kidney parenchyma and

accumulation of nuclei [× 400] (c) congestion in the cortical

region and haemolysis [× 100]

Table 3—Toxicity of 1 ml of various purified protein fractions of

sea anemones on male albino mice

Protein content Species Fraction

no. Toxic dose

(µg/kg)

Death time (sec)

(mean±SE)

1 3525 --

2 8035 66±2.51

3 3515 --

4 4030 --

5 1015 --

6 1535 --

7 545 341.2±4.52

8 535 --

9 1020 47.9±2.30

H. magnifica

10 510 66.5±2.71

1 1515 74.7±4.65

2 1530 --

3 7040 1454.9±3.17

4 510 71.4±2.51

5 1025 51.3±2.16

6 3020 --

7 505 --

8 510 61.2±1.90

9 515 --

S. haddoni

10 520 64.8±3.12

1 5530 --

2 515 203.2±4.18

3 2520 52.9±2.19

4 515 57.1±1.85

5 1010 --

6 510 --

7 530 --

8 535 --

9 520 1269±6.40

P. sinensis

10 510 --


1231

massive hepatocellular destruction has been reported

in a young man who had encountered an anemone and

had no signs of liver disease previously6. Also, there

are reports of elevated liver function rates after

envenomation in animals and humans33, 34

which could

eventually damage the liver tissues.

From the present study, it is evident that the crude

as well as the fractionated proteins obtained from

the sea anemones viz. H. magnifica, S. haddoni and

P. sinensis are neurotoxic, cardiotoxic, nephrotoxic

and hepatotoxic to mice. Future studies could

reveal these toxic proteins as active agents altering

biological functions and over there, the toxicity and

effects of these toxins on the organ systems have

to be taken into account.

Acknowledgement This paper is a tribute to the co-author (Late)

Dr.K.Venkateshvaran, Principal Scientist, CIFE,

Mumbai. The authors thank the authorities of Centre

of Advanced Study in Marine Biology, Annamalai

University, Paragipettai and Central Institute of

Fisheries Education, Mumbai, for facilities.

References 1 Haefner B, Drugs from the deep sea: Marine natural products

as drug candidates, DDT, 12 (2003) 536.

2 Auguoto B, Koldo M, Ariana B, Gutierrez A, Ana R V, Juan

M & Gonzalez M. Purification, cloning and characterization

of fragaceotoxin C, a novel actinoporin from the sea

anemone Actina fragacea, Toxicon 54 (2009) 869.

3 Lewis M G & Rowden G, Histopathology a step by step

approach (Little Brown Company, Waltham, Massachusetts,

U.S.A) 1984,15.

4 Curran C R, Colour atlas of histopathalogy 3rd edition

(Harvey Miller Publishers, Oxford University Press, New

York) 1985, 292.

5 Lefkowitch H J, Histopathlogy of disease (Churchill Living

Stone Publisher, New York) 1987, 233.

6 Garcia P J, Schein R M H, & Burnett J W, Fulminant

hepatic failure from a sea anemone sting, Annals Inter Med,

8 (1994) 665.

7 Sivappa M U, Bioactive potential of the sea anemone

Anthopleura middori, M.F.Sc, Dissertation, Central Institute

of Fisheries Education, Mumbai, 2005.

8 Sunahara S, Muramoto K, Tenma K & Kamiya H, Amino

acid sequence of two sea anemone toxins from Anthopleura

fuscoviridis Toxicon, 25 (1987) 211.

9 Shiomi K, Takamiya M, Yamanaka H & Kikuchi T,

Purification of a lethal factor in the skin secretion from the

oriental catfish Plotosus lineatus, Nippon suisan Gakkaishi,

53 (1987) 1275.

10 Lowry O H, Rosebrough N J, Farr A L & Randall R J,

Protein measurement with folin phenol reagent, J Biol Chem,

193 (1951) 265.

11 Lin X Y, Ishida M, Nagashima Y & Shiomi K, A

Polypeptide Toxin in the sea Anemone Actinia equine

homologous with other sea anemone sodium channel toxins:

Isolation and amino acid sequence, Toxicon, 34 (1996) 57.

12 Grotendorst G R & Hessinger D A, Purification and partial

characterization of the phospholipase A2 and co-lytic from

sea anemone (Aiptasia pallida) nematocyst venom, Toxicon,

37 (1999) 1779.

13 Beress L, Phylum Coelenterata (cnidaria) Poisonous and

venomous marine animals of the world 2nd edn, edited by

Halstead B W (Darwin Press, Princeton, NJ) 1988,152.

14 Bruhn T,schaller C, Sanchmide C, Ravens U,Heubach J F,

Kckhardt K, Schmidtmayer J, Schmidt H, Aneiros A,

Wachter E & Bruhn T, Schaller C, Schulze C, Sanchez-

Rodriguez J, Dannmeier C, Ravens U, Beress L, Isolation,

characterization of five neurotoxic and cardiotoxic

polypeptides from the sea anemone Anthopleura

elegantissima, Toxicon, 39 (2001) 693.

15 Sencic L & Macek P, New method for isolation of

venom from the sea anemone Actinia cari. Purification and

characterization of cytolytic toxins, Comp Biochem Physiol,

97B (1990) 687.

16 Honma T, Iso T, Ishida M, Nagashima Y & Shiomi K,

Occurrence of type III sodium channel peptide toxins in two

species of sea anemones, Toxicon, 41 (2003) 637.

17 Mahnir V M, Kozlovskaya E P & Kalinovsky A I, Sea

anemone Radianthus macrodactylus - A new source of

Palytoxin, Toxicon, (30) (1992)1449.

18 Macek P & Lebez D, Isolation and characterization of three

lethal and hemolytic toxins from the sea anemone Actinia

equina L, Toxicon, 26 (1988) 441.

19 Wang Y, Chua K L & Khoo H E, A new cytolysin from

the sea anemone, Heteractis magnifica: isolation, cDNA

cloning functional expression, Biochim Biophys Acta, 1478

(2000) 9.

20 Gondran M, Eckeli A L, Migues P V, Gabilan N H &

Rodrigues A L S, The crude extract from the sea anemone,

Bunodosoma caissarum elicits convulsions in mice: Possible

involvement of the glutamatergic system, Toxicon, 40

(2002)1667.

21 Monastyrnaya M M, Zykova T A, Apalikova O V, Shwets T

V & Kozloskaya E P, Biologically active polypeptides

from the tropical sea anemone Radianthus macrodactylus,

Toxicon, 40 (2002) 1197.

22 Castaneda O, Sotolongo V, Amor A M, Stoklin R, Anderson

A J, Harvey A L, Engstrom A, Wernstedt C & Karlsson E,

Characterization of a potassium channel toxin from the

Caribbean sea anemone Stichodactyla helianthus, Toxicon,

33 (1995)603.

23 Erxleben C & Rathmayer W, Effects of the sea anemone

Anemonia sulcata toxin II on skeletal muscle and on neuro

muscular transmission, Toxicon, 22 (1984)387.

24 Turner R J & Freeman S E, Effects of Chironex fleckeri toxin

on the isolated perfused guinea-pig heart, Toxicon, 7

(1969)277.

25 Lee C Y, Lee S Y, Chen Y M, Leu H M & Perlan I,

Coronary vasospasm as the primary cause of death due to

equinatoxin a toxic protein from Actinia equina, Toxicon,

23 (1985) 586.

26 Salinas E M, Cebada J, Valdes A, Garateix A, Aneiros A & Alvarez J L, Effects of a toxin from the mucus of the caribbean sea anemone (Bunodosoma graunlifera) on the ionic currents of single ventricular mammalian cardiomyocytes, Toxicon, 35 (1997)1699.


1232

27 Konya R S & Elliott R C, The coronary vasoconstrictor

action of extract IV from the dahlia sea anemone Tealia

felina L, Toxicon, 34 (1996) 277.

28 Sket D, Draslar K, Ferlan I & D Lebez, Equinatoxin, a lethal

protein from Actinia equina, Pathophysiological action,

Toxicon, 12 (1974) 63.

29 Bunc M, Drevensek G, Budihna M & Suput D, Effects of

equinatoxin II from Actinia equina (L.) on isolated rat heart:

The role of direct cardiotoxic effects in equinatoxin II

lethality, Toxicon, 37 (1999)109.

30 Galettis P & Norton R S, Biochemical and pharmacological

studies of the mechanism of action of tenebrosin-C, A

cardiac stimulatory and haemolytic protein from the sea

anemone, Actinia tenebrosa, Toxicon, 28 (1990) 695.

31 Chen C F, Yen T S, Chen W Y, Chapman B J & Munday K A, The renal, cardiovascular and hemolytic actions in the rat of a toxic extract from the bile of the grass carp (Ctenopharyngodon idellus), Toxicon, 22 (1984)433.

32 Chen C F, Yen T S, Cheng J T, Hsieh B S & Hsu H C, Acute renal failure due to ingestion of raw bile of grass carp Ctenopharyngodon idella, J Formosan Med Assn, 75 (1976)149.

33 Muhrich K H, Sengottuvelu S, Manson P N, Myers R A M, Burnett J W & Marzella L, Pathophysiology of sea nettle (Chyrsaora quinquecirrha), envenomation in a rat model and the effects of hyperbaric oxygen and verapamil treatment, Toxicon, 29 (1991) 857.

34 Burnett J W, Human injuries following jellyfish stings, Maryland Med J, 41(1992)509.

biological activity of sea anemone proteins: i....

Documents