Occurrence of non-protein low molecular weight cardiotoxin in Indian King Cobra (Ophiophagus hannah) Cantor 1836, venom.

Pathophysiology due to snakebite is a combined effect of various actions of the complex venom constituents. Importance of protein toxins in snake envenomation is well known. The present investigation reports the existence of nonprotein/nonpetide low molecular weight toxin in Indian King Cobra venom, which plays an important role in envenomation consequences in experimental animal models. A group of non-peptidic toxins (OH-NPT1) was isolated from Indian King Cobra Ophiophagus hannah by thin layer chromatography and silica gel column chromatography. UV, IR, NMR and (ESI) TOF-MS studies characterized the OH-NPT1 as a mixture of aliphatic acids having molecular weights 256, 326 and 340Da. The minimum lethal dose of OH-NPT1 was found to be 2.5 microg/20g (iv) and 4microg/20g (ip) in male albino mice. The cardiotoxic property of OH-NPT1 was established through studies on isolated guinea pig heart and auricle preparations, ECG studies in albino rat and estimation of LDH1/LDH and CPK-MB/CPK ratio in Swiss albino mice. Commercial antiserum failed to neutralize the lethality and cardiotoxicity of the toxin. However, calcium and magnesium effectively neutralized the lethal action.

Lethal, oedema, haemorrhagic activity of spotted butterfish (Scatophagus argus, Linn) sting extract and its neutralization by antiserum and pharmacological antagonists.

An attempt has been made in this communication to develop antiserum in rabbit against Scatophagus. argus sting extract. Antiserum did not neutralized the sting extract induced proinflammatory and haemorrhagic activity but successfully neutralized lethality upto 2LD50. Cyproheptadine, indomethacin and BW 755C pretreatment significantly reduced sting extract induced proinflammatory activity. The haemorrhagic activity of sting extract was significantly inhibited by temperature, UV-exposure, EDTA, cyproheptadine, indomethacin and BW 755C pretreatment. The results conclude that the local effects of S.argus venom is likely to be mediated through release of mediators and may be encountered by pharmacological antagonists better than the antiserum.

Pharmacological studies on the venomous spotted butterfish (Scatophagus argus Linn) sting extract on experimental animals.

A sting of the fish S. argus, a venomous edible spotted butterfish, produces tremendous local pain, severe swelling, rise of body temperature, throbbing sensation etc. To establish the pharmacological activities of S. argus sting extract, the present investigation, was carried out on experimental animals. The LD50 of extract was found to be 9.3 mg/kg (iv) in male albino mice. The extract showed loss of sensation, urination and salivation in mice. It potentiated pentobarbitone induced sleeping time in male albino mice and produced hypothermia. Extract produced a fall of cat and guinea pig blood pressure, which was completely abolished by mepyramine. It produced a transient reduction of respiratory rate in rat, but decreased respiratory amplitude in cat, which was abolished after vagotomy. On isolated toad heart, the extract increased both the amplitude and rate of contraction. On isolated guinea pig heart, the sting extract decreased both the rate and amplitude of contraction leading to cardiac arrest, but it had no effect on isolated guinea pig auricle. The extract produced a reversible blockade of electrically induced twitch response of isolated chick biventer cervices preparation, but it had no effect on the isolated rat phrenic nerve diaphragm preparation. It produced a slow contractile response on isolated guinea pig ileum, rat uterus and rat fundal strip preparations but produced slow relaxation on isolated rat duodenum preparation. The contractile response on isolated guinea pig ileum and rat fundal strip was antagonised by SC19220. It did not produce any significant cutaneous haemorrhage in mice and did not produce any haemolysis on saline washed erythrocytes. The sting extract significantly increased capillary permeability of guinea pig dorsal flank and produced oedema in mice hind paw.

Isolation of a haemorrhagic protein toxin (SA-HT) from the Indian venomous butterfish (Scatophagus argus, Linn) sting extract.

A haemorrhagic protein toxin (SA-HT) was isolated and purified from the spine extract of the Indian venomous butterfish, S. argus Linn, by two step ion exchange chromatography. The toxin was homogeneous in native and SDS-PAGE gel. SDS-molecular weight of the toxin was found to be 18.1 +/- 0.09 kDa. SA-HT produced severe haemorrhage on stomach wall but devoid of cutaneous haemorrhage. UV, EDTA, trypsin, protease, cyproheptadine, indomethacin, acetylsalicylic acid and BW755C treatment significantly antagonized the haemorrhagic activity of SA-HT. The toxin produced dose and time dependent oedema on mice hind paw, which was significantly encountered by cyproheptadine, indomethacin and BW755C. SA-HT increased capillary permeability on guinea pig dorsal flank. On isolated guineapig ileum, rat fundus and uterus, SA-HT produced slow contraction which was completely antagonised by prostaglandin blocker SC19220. On isolated rat duodenum, SA-HT produced slow relaxation. SA-HT significantly increased plasma plasmin, serum MDA level and decreased serum SOD level indicating the possible involvement of cyclooxygenase and lipooxygenase pathway.

Isolation and partial structural evaluation of a cardiotoxic factor from Indian common murrel (Channa striatus L.) skin extract.

It was earlier reported from this laboratory that, Channa striatus, L a common edible fish, whose skin extract (CSSE) was pharmacologicaclly potent and contains several bioactive compounds. In the present communication a cardiotoxic factor was isolated and purified by thin layer chromatography followed by silica gel and neutral alumina column chromatography. Spectroscopic studies (UV, IR, 1H and 13C NMR, FAB-MS) indicated that the lethal cardiotoxic factor (CSS-CTF II) was an aromatic alkaloid compound with -NH, > C = C < and -OH functional groups. The molecular weight was found to be 413 dalton. LD50 of CSS-CTF II was found to be 42.5 mg/kg (i.v) in Swiss albino male mice. Pharmacological studies showed that CSS-CTF II possesses hypotensive and cardiotoxic activities and produced death through apnoea in experimental animals but had no effect on nerve muscle preparations. The haematological and biochemical data also indicated the toxic nature of CSS-CTF II, through significant fall in haemoglobin, total RBC, WBC, platelet count and increased cardiac marker enzyme CPK and CPK-MB value in experimental animals. The present investigation thus established the toxic nature of CSS-CTF II isolated from edible fish C. striatus skin extract. Further work is needed to identify CSS-CTF II's mechanism of action and its antagonism for therapeutic purpose.

A lethal neurotoxic protein from Indian king cobra (Ophiophagus hannah) venom.

A lethal neurotoxin protein (Toxin CM36) was isolated and purified from the Indian King Cobra (Ophiophagus hannah) venom by CM-Sephadex ion exchange chromatography and HPLC. The purified toxin had a SDS-molecular weight of 15 +/- 0.5 kD. The UV absorption spectra of Toxin CM36 showed a peak at 280 nm and an Emax at 343.8 nm, when excited at 280 nm fluorescence. Toxin CM36 had an LD50 of 3.5 microg/20 g (i.v.) in male albino mice. It exhibited neurotoxicity and produced irreversible blockade of isolated chick biventer cervicis and rat phrenic nerve diaphragm. The neurotoxicity was found to be Ca2+ dependent. Toxin CM36 had no significant effect on isolated guineapig heart and auricle. It also had no effect on blood pressure of cat and rat but produced respiratory apnoea in rat and guineapig. Toxin CM36 lacked phospholipase activity.

Snake venom as therapeutic agents: from toxin to drug development.

Snake bite injuries and death are socio-medical problems of considerable magnitude. In India a large number of people suffer and die every year due to snake venom poisoning. Snake venom, though greatly feared, is a natural biological resource, containing several components that could be of potential therapeutic value. Use of snake venom in different pathophysiological conditions has been mentioned in Ayurveda, homeopathy and folk medicine. It is well known that snake venom is complex mixture of enzymes, peptides and proteins of low molecular mass with specific chemical and biological activities. Snake venom contains several neurotoxic, cardiotoxic, cytotoxic, nerve growth factor, lectins, disintrigrins, haemorrhagins and many other different enzymes. These proteins not only inflict death to animals and humans, but can also be used for the treatment of thrombosis, arthritis, cancer and many other diseases. An overview of various snake venom components that have prospects in health and diseases are discussed in this review.

Pharmacological and haematological study of shol fish (Channa striatus) skin extract on experimental animal.

Snake head fish Channa striatus (locally called 'shol') skin extract (SFSE) was examined for certain pharmacological and haematological effects on experimental animals. LD50 of SFSE was found to be 6 mg/20gm (iv) in male albino mice. SFSE potentiated pentobarbitone induced sleeping time in male albino mice and produced hypothermia. Low dose of SFSE decreased respiratory rate in rat and guineapig and high dose produced apnoea leading to death. On isolated toad and guineapig heart, SFSE significantly decreased rate and amplitude of contraction leading to temporary blockade, which returned after repeated wash. On isolated nerve muscle preparations, SFSE produced irreversible blockade of twitch response. SFSE induced quick contraction on isolated guineapig ileum, which was antagonised by atropine and cyproheptadine. SFSE did not possess haemolytic and haemorrhagic activity but produced anaemia in male albino mice. A neurotoxic compound (fluoroscent and ninhydrin positive) was isolated from SFSE by thin layer chromatography. This compound (CS-NT) was lethal in male albino mice, produced death by apnoea in rat and produced irreversible blockade of isolated nerve-muscle preparation. This study confirms that the skin of Channa striatus possesses toxic, and lethal components, which needs further detailed study.

Isolation, purification and partial chemical characterization of a lethal factor from common indian toad (Bufo melanostictus, Schneider) skin extract.

Indian toad (Bufo melanostictus, Schneider) skin extract (TSE) is pharmacologically potent and probably contains several bioactive compounds [Das et. al., Indian J Pharmacol, 28 (1996) 72]. A lethal factor was isolated and purified by neutral alumina column chromatography followed by HPLC. Spectroscopic (UV, IR, FAB-MASS) study indicated that the lethal factor (TSE-LF) was a 254 Da long chain compound with carbonyl, hydroxyl and ester as functional groups. LD50 of TSE-LF was found to be 3.5 mg/kg (iv). Biological study showed that TSE-LF possesses hypotensive, cardiotoxic, neurotoxic activity and produced death by apnoea in experimental animal. Cyproheptadine antagonised TSE-LF induced contraction of isolated smooth muscle indicating involvement of histamine/serotonin receptors. TSE-LF induced neurotoxic action on chick biventer cervices was mediated through Ca2+ ion.

Haematinic effect of Hygrophila spinosa T. Anderson on experimental rodents.

Ethanolic extract of the aerial parts of H. spinosa a semiwoody herb was examined on male albino rats for certain haematological changes. The extract (100 & 200 mg/kg, po) significantly increased the haemoglobin, haematocrit, RBC and total WBC, as compared with vehicle treated control rat haemogram. In anemic male albino rats, the extract significantly increased haemoglobin, haematocrit and RBC count. Serum iron and serum total iron binding capacity were significantly decreased in H. spinosa extract treated anemic rats as compared with those in the vehicle treated anemic control rats. These findings demonstrated the haematinic effect of H. spinosa extract on experimental animals.

Bidder's organ extract induced anaphylaxis in experimental animals.

Bidder's organ (BO, a vestigeal organ), present in toad Bufo melanostictus (Schenider), is a characteristic feature of all male bufo. Its possible anaphylactic properties are investigated on experimental animals. BO extract produced both in vivo and in vitro anaphylactic reaction in guineapig. Dyspnoea and bronchoconstriction was a major cause of anaphylactic death. Blood histamine level was significantly increased in the anaphylactic animals. BO extract significantly released histamine from chopped lung preparation, an action antagonised by disodium chromoglycate. BO extract degranulated peritoneal mast cell in vitro. Passive cutaneous anaphylactic reactions were enhanced by BO extract and were significantly inhibited by disodium chromoglycate. Anaphylotoxin (identity not known) present in bidder's organ is probably involved in toad defence.

A lethal cardiotoxic protein isolated from Bidder's organ of common Indian toad, Bufo melanostictus Schneider.

A lethal cardiotoxic (BO-CT; Bidder's organ cardiotoxin) protein was purified from the Bidder's organ of the common Indian toad B. melanostictus by gel filtration on Sephadex G-200. The homogeneity of cardiotoxin was tested by gel electrophoresis. The molecular weight of lethal BO-CT was 62 KDa and was devoid of glycoprotein. LD50 of the BO-CT was 50 micrograms/20 g (i.v.) in male albino mice. On isolated heart and auricle BO-CT initially increased the rate and amplitude of contraction and finally produced irreversible blockade of contraction. BO-CT induced auricular blockade, was not influenced by verapamil, propranolol and atropine. On isolated chick biventer cervicis preparation BO-CT produced irreversible blockade of electrically induced twitch response followed by contracture. This action was not antagonized by 4-aminopyridine and neostigmine. BO-CT induced contracture on chick biventer cervicis was increased by Ca2+, decreased by Na+ and abolished by K+. Cardiotoxic and neuromuscular activity of BO-CT was heat stable and abolished by proteolytic enzyme.

Isolation, purification and partial characterization of a haemolytic protein from Bidder's organ of toad Bufo melanostictus (Schneider).

A haemolytic protein toxin (BO-HT) from Bidder's organ of toad, B. melanostictus, purified by DEAE-cellulose column chromatography was electrophoretically homogeneous and was glycoprotein in nature (PAS-positive). The molecular weight was estimated to be 14.4 kDa by SDS-polyacrylamide gel electrophoresis. The sensitivity of the haemolysin of different RBC ghost cell preparation was in the order: buffalo > goat > ox > guinea pig > mice > human > chick > rabbit > rat. The haemolytic activity was increased with the decrease in RBC concentration and was produced over a wide range of temperature. Maximum haemolytic effect was produced at 2 hr of incubation. The toxin showed maximum activity at 3 and minimum at 10 pH. Divalent cations (Ca2+, Zn2+, Cu2+, Mg2+) showed inhibitory effect on BO-HT induced haemolysis, whereas sucrose, EDTA, cholesterol, 2-mercaptoethanol and oxygen did not alter the haemolytic activity. Haemolytic activity was reduced by proteolytic enzymes (trypsin, protease) and was totally antagonized by the toad serum.

Detection and distribution of scorpion (Heterometrus bengalensis) venom in rabbit tissues by enzyme-linked immunosorbent assay (ELISA).

ELISA was carried out to detect distribution of scorpion venom in experimental animal tissues. The venom content of different tissues was in the order, liver greater than kidney greater than spleen greater than lung greater than heart greater than diaphragm greater than brain. Tissue distribution of venom antigen in the envenomental subject by ELISA will provide a better approach for serotherapy.

Histamine, 5-HT & hyaluronidase in the venom of the scorpion Lychas laevifrons (Pock).

Factors involved in the pathophysiological changes such as severe pain, burning sensation, redness, swelling and edema in case of the scorpion L. laevifrons were investigated. The presence of pain-producing autacoids histamine 2.1 +/- 0.18 micrograms/mg and 5-HT 0.23 +/- 0.1 micrograms/ml was confirmed by thin layer chromatography and bioassay. Histamine releasing substance was detected in vitro in the chopped guineapig lung. Venom also contained hyaluronidase 5 x 10(-4) N-acetyl-D-glucosamine released/h/mg, which facilitates spread of the toxic principles in the tissues. It is concluded that histamine, 5-HT, histamine-releasing factor and hyaluronidase are partly involved in the pathophysiological changes induced by the venom. It is suggested that mepyramine and cyproheptadine may prove useful in the management of scorpion envenomation.

Isolation and purification of a lethal scorpion toxin with neuromuscular blocking activity.

Toxin-L a lethal neuromuscular blocking agent was isolated from the venom of the scorpion, Lychas laevifrons (Pocock), by the CM-cellulose ion-exchange chromatography. It was a homogenous, thermolabile and low molecular weight protein. The toxin produced irreversible blockade of indirect stimulation induced twitch responses on innervated rat phrenic nerve-diaphragm and chick biventer cervicis preparation. The toxin did not produce any contractile response on toad rectus abdominis muscle preparation. On chronically denervated rat diaphragm, the toxin failed to alter the responses induced by direct stimulation, exogenous acetylcholine, potassium chloride and caffeine. Acetylcholine and carbachol induced contractions on isolated chick biventer cervicis remained unaltered by the toxin. Neostigmine failed to alter toxin induced neuromuscular blockade on innervated rat diaphragm. The toxin released a significant amount of acetylcholine from innervated rat diaphragm. It may be concluded that the toxin acts presynaptically through the release of acetylcholine, thereby producing neuromuscular blockade.

Isolation, purification and immunological evaluation of toxin Hb from scorpion Heterometrus bengalensis (C.L. Koch) venom.

Toxin-Hb, a lethal toxic antigenic protein, isolated from the venom of H. bengalensis by CM-cellulose ion-exchange chromatography was a heat labile basic protein with a molecular weight of 10 kDa. It produced irreversible blockade on the isolated rat phrenic nerve diaphragm and chick biventer cervicis. LD50 of toxin Hb was 0.48 mg/kg (iv) in mice. Antiserum was raised in mice by hyperimmunization against toxin Hb. Antitoxin Hb antiserum was immunologically potent as revealed by immunogel-diffusion and immunoelectrophoresis. Five fold protection against the lethal action of toxin Hb was achieved by the antiserum. It also effectively antagonised toxin Hb induced neuromuscular blockade on isolated rat phrenic nerve diaphragm and chick biventer cervicis preparations.

Pharmacology of scorpion (Lychas laevifrons Pock) venom with special reference to its neuromuscular activity.

L. laevifrons venom caused irreversible blockade of electrically induced twitch responses on phrenic nerve diaphragm and chick biventer cervicis preparation. The venom lowered cat blood pressure, caused a brief cardiac arrest and increased cutaneous capillary permeability. It contracted several smooth muscle preparations. The quick contraction produced on guinea pig ileum was partly antagonized by mepyramine and completely by methysergide. The residual slow contraction was antagonized by SC 19220, a prostaglandin blocker. Haemolysis was not produced by the venom on human RBC. LD50 of crude venom in mice was 13.8 mg/kg (iv).