Effects of ethanolic extract of Iris germanica on lipid profile of rats fed on a high-fat diet.

Ethanolic extract of Iris germanica rhizomes was investigated for hypolipidemic activity. I. germanica belong to the family Irdaceae and has been used to treat liver and spleen ailments in traditional system of medicine. Two groups of Wistar rats were fed with high-fat diet and ethanolic extract of I. germanica were administered orally in one group of rats, while other received saline for 10 weeks. Complete lipid profiles of experimental animals were determined by assessing serum levels of total lipids, triglycerides, cholesterol, high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol. Results indicate that ethanolic extract of I. germanica significantly lowered the lipid components especially, the cholesterol and triglycerides.

Primary structure and electrophysiological characterization of two almost identical isoforms of toxin from Isometrus vittatus (family: Buthidae) scorpion venom.

Two almost identical proteins with 70 amino acid residues each, closely packed by four disufide bridges, and molecular masses of 7899.5 and 7884.7 were isolated and sequenced from the venom of the scorpion Isometrus vittatus from Pakistan. They differ by an acidic amino acid residue (glutamic or aspartic) at the same position 55 of the peptide chain, however, they exhibit the same length, the same charge and are undistinguishable when separated by C(18) reverse phase HPLC. The mixture of the two proteins called IsomTx1 depolarizes the cockroach isolated axon; artificial repolarization is followed by sustained repetitive activity, artificial hyperpolarization facilitates bursting activity observed as an answer to rapid depolarization to -60 mV. The depolarization is antagonized by TTX. In voltage-clamp experiments IsomTx1 increases axonal sodium permeability which has a particular importance between resting and threshold potentials and moderately slows down the fast inactivation. These characteristics closely resemble those of other anti-insect scorpion toxins classified as contractive toxins from Androctonus and Buthotus venoms.

Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A2 (PLA2-H1).

A toxic phospholipase A2 (PLA2-H1), isolated from the venom of the sea snake Hydrophis cyanocinctus, was tested for its ability to induce myonecrosis and histopathological changes in albino rats and mice. Induction of myonecrosis was demonstrated by their ability to release creatine kinase (CK) from damaged muscle fibers and direct histopathological examination of the injected muscles (i.m.). PLA2-H1 exhibits intense myonecrosis characterized by the changes including, necrosis and edematous appearance with cellular infiltrate, vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells. No myoglobinuria was noted in any group of animals. The purified PLA2-H1 was also administered intraperitoneally into the experimental animals and tissue samples were taken at several time intervals. Light microscopic examination of the kidney sections revealed severe damage, evident by focal tubular necrosis, complete disquamation of epithelial lining and epithelial degeneration of tubules in all test animals. Light micrographs of liver sections after 24 h of injection shows fatty infiltration in parenchyma and squashed hepatocytes, while after 48 h, fatty vacuolation of parenchyma in a generalized pattern was observed. Furthermore, sections of the lungs of the same group of animals (48 h) show dilated bronchia and marked infiltration of inflammatory cells within alveoli. Our results suggest that the purified PLA2-H1 induced moderate myotoxicity in muscles and mild histopathological changes in other vital organs without myoglobinuria.

Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom.

The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid sequence (ADDKNPLEEAFREADYEVFLEIAKNGL) and the chemical composition of the purified LNV-LAO shows close structural homology with other L-amino acid oxidases isolated from different snake venoms. The secondary structural contents analysis of LAO, established by means of circular dichroism, revealed ca. 49% alpha-helix, 19% beta-sheet, 10% beta-turn, and 22% random coil structure. The purified LNV-LAO not only retained its specific enzymatic activity (73.46 U/mg), determined against L-leucine as a substrate, but also exhibited potent haemolytic (1-10 microg/ml), edema- (MED 4.8 microg/ml) and human platelet aggregation-inducing (ED50 33 microg/ml) properties. Unlike other haemorrhagic snake venom L-amino acid oxidases, the LNV-LAO does not produce haemorrhage. In addition to these local effects, the purified LNV-LAO showed apoptosis-inducing activity in the MM6 cell culture assay. After 18 h treatment with 25-100 microg/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by means of fluorescent microscopy and agarose gel electrophoresis.

Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization and N-terminal sequence of two phospholipases A2.

Two phospholipases A2 (PLA2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA2s show close sequence homologies to those of PLA2s from other Elapid snakes (60-84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60-65 degrees C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA2 (H1) shows higher toxicity to albino rats (LD50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.

Enzymatic activities of some snake venoms from families Elapidae and Viperidae.

Alkaline phosphomonoesterase, phosphodiesterase, L-amino acid oxidase, hyaluronidase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A2 and proteinase activities were determined in eight snake venoms, including three from sea snake, of families Elapidae and Viperidae from Pakistan. The species includes three sea snakes Hydrophis cyanocinctus, Enhydrina schsitosa, Microcephalophis gracilis gracilis and two land snakes Naja naja naja, Bungarus caeruleus of family Elapidae while three land snakes Vipera russelli russelli, Echis carinatus and Eristocophis macmahoni of family Viperidae. The venoms of family Elapidae are characterized by low levels to traces of proteinase, L-amino acid oxidase and arginine ester hydrolase activities with the exception of Naja naja naja and a moderate to high levels of phospholipase A2 activities. The venoms of family Viperidae, on the other hand, are characterized by the presence of moderate to high levels of 5'-nucleotidase, proteinase, phosphodiesterase and phosphomonoesterase activities.

Effects of drugs against cytotoxic compoundsfrom marine animals iv-hepatotoxic activity ofsea-snake hydrophis cyanocinctus venom.

Hepatotoxic activity and pathogenesis of sea snake Hydrophis cyanocinctus venom have been studied in guinea pigs and albino rats by histopathological examination and determining total Ca(2+). Concentration in liver tissue. Efficacy of two Ca(2+). antagonist drugs, verapamil and diltiazem was also studied against the cytotoxic activity of this venom. Administration of both lethal and sublethal doses of Hydrophis cyancinctus crude venom induced increased cellularity within the portal area in liver accompanied by increased tissue Ca(2+) concentration. This elicit a close correlation between tissue necrosis and increased Ca(2+) content in tissues. Both verapamil and diltiazem administration after an injection of H. cyanocinctus venom showed significant decrease in Ca(2+) concentration and tissue necrosis. Increase in survival time was also noted in almost all animals receiving Ca(2+) - antagonist drugs. It is thus suggested that cytotoxic compounds present in this marine venom cause tissue and cellular necrosis by increasing Ca(2+) influx within the cells which in turn stimulates Ca(2+) -dependent processes resulting in both organ dysfunction and sometimes fatality in few cases. But both these effects could be inhibited by using antagonizing drugs which could reduce the pathological conditions induced by such compounds by blocking the cation influx and its dependent processes.

Isolation and biological properties of scorpionvenom proteins: iv-comparative study on four proteinases isolated from the venom of isometrus vittatus.

1) Four proteinases were isolated from the venom of a local scorpion species Isonietrus vittatus collected from Sindh region, Pakistan and named PRO,(IA) PRO,(IB) PRO(IVA) and PRO(IVB). 2) Successive chromatography on Sephadex G-50, CM-Cellulose and Sephadex G-100 yield 25.5 mg, 20.5 mg, 24.0 mg and 20.0 mg of PRO,(IA) PRO(IB), PRO,(IVA) and PRO(IVB) respectively. 3) The purified toxins were homogenous by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) giving single peak and band with an apparent molecular weight of 80.0 kda, 75.5 kda, 19.0 kda and 17.0 kda by gel filtration and 81.0 kda, 74.7 kda, 18.25 kda and 17.2 kda by SDS-PAGE. 4) PRO,(IA) PRO,(IVA) and PRO(IVB) have LD(50s) of 1.27 mg/kg, 1.91 mg/kg, 1.68 and 1.70 mg/kg respectively. The former two also caused marked alteration of serum enzyme and chemical constituent levels at sublethal dose whereas PRO(IVA) and PRO(IVB) were found to be less effective. 5) Enzymatic and biological activities of all four were inhibited by heating at 62 degrees C. Both activities were also inhibited by 1.8 mM EDTA and 1.2mM PMSF. Activities were enhanced by Ca(2+) but retarded and inhibited by Cu(4+) and Mg(2+), respectively.

Changes in serum components induced by venoms of marine animals.

Serum enzyme and chemical component levels were investigated in a rat model after experimental envenomation by venoms of coelentrate, Physalia species, four sea-snakes, Hydrophis spiralis, H. Cyanocinctus, H. Lapemoides and Lapemis curtus and a gastropod molluse Conus coronatus. The LD(50)s through i.v. route were found to be 4.2 mg/kg for Physalia, 0.4 mg/kg for H.spiralis 0.60 mg/kg for H.cyanocinctus, 0.60 mg/kg for H. lapemoides, 0.70 mg/kg for L.curtus and 2.9 mg/kg for C.coronatus. Marked elevation of serum enzyme levels was induced by sea-snake and Physalia venom while C.coronatus venom showed no significant change in serum levels. The results also indicate gross morphological changes in liver, spleen, gall bladder, lungs and heart by Physalia and sea snake venoms.

Toxicology of Physalia's (Portuguese man-o' war) venom.

The venomous pelagic coelentrate Physalia species (Blue bottle) or the Portuguese man-o'war has recently been studied in detail due to their hazardous effects on community. It was observed that the venom of the animal is lethal to man and can also produce cutaneous stings of varying severity. Physalia species also possess significant cardiotoxicity to man, rats, mice and are lethal to lower animals. Thu effect has been attributed to the abnormality in ionic transport across membranes. The cutaneous pain and musculotoxic action in human produced by Physalia venom may be induced by one or several high molecular weight polypeptides. Blockage of specified neurons and neuromuscular junction besides alteration in end plate potential (EPP) and end plate current (EPC) was also attributed to high molecular weight toxic fractions. Survey of enzymatic contents of Physalia venom reveals the presence of a wide variety of enzymes whose presence and actions were similar to that of complex enzyme mixture of snake venom. Besides these clinical manifestations neurotoxicity cytotoxicity and other physiopharmacological effects were also demonstrated.