The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes

Malayan cobra (Naja naja sputatrix) venom was found to exhibit an in vitro anticoagulant activity that was much stronger than most common cobra (genus Naja) venoms. The most potent anticoagulants of the venom are two lethal phospholipase A2 enzymes with pI's of 6.15 and 6.20, respectively. The anticoagulant activity of the venom is due to the synergistic effect of the venom phospholipase A2 enzymes and polypeptide anticoagulants. Bromophenacylation of the two phospholipase A2 enzymes reduced their enzymatic activity with a concomitant drop in both the lethal and anticoagulant activities.     

四味镇痛中药对内腓肽降解酶作用的实验研究

目的 :研究四味镇痛中药对内腓肽降解酶的作用。方法 :从大鼠的肾脏制备含有内腓肽降解酶的物质 ,建立内腓肽降解酶活性检测模型 ;并用此模型观察四味镇痛中药提取物对内腓肽降解酶特别是对中性内肽酶活性 (NEP2 4 .11)的抑制作用。结果 :(1)钩藤和羌活的水提物显示很强的NEP2 4 11酶抑制作用 ;(2 )无论是有机提取物还是水提物 ,延胡索和川芎对NEP2 4 11的抑制作用都弱于钩藤和羌活 ;(3)钩藤和羌活不仅是氨肽酶和中性内肽酶的双重抑制剂 ,而且是氨肽酶、中性内肽酶和血管紧张素转化酶的三重抑制剂。结论 :四味镇痛中药具有不同的镇痛机制 ,其中 ,钩藤和羌活通过抑制氨肽酶和中性内肽酶而发挥镇痛作用     

Inhibition of Naja nigricolis (Reinhardt) venom protease activity by Luffa egyptiaca (Mill) and Nicotiana rustica (Linn) extracts

Luffa egyptiaca and Nicotiana rustica are used in traditional medicine to treat snakebites and were evaluated for inhibitory activities on Naja nigricolis venom protease. The aqueous and ethanolic extracts of L. egyptiaca significantly reduced the maximum velocity (Vmax) and the computed index of physiological efficiency (Kcat) of the enzyme in a dose dependent fashion. The protease activity was non-competitively inhibited by the aqueous extract of N. rustica with the Vmax significantly decreased and the K(M) remained unchanged. However, the N. rustica ethanol extract completely inhibited the protease activity. Ethyl acetate fractions partitioned from ethanol extracts of both plants were also found to completely inhibit the N. nigricolis venom protease activity at 0.1 and 0.05%. The use of these plants could be important in the treatment of snakebites.     

Characterization and immunological comparison of isoenzymes of phospholipases A2 from snake venoms of different genera and families.

Phospholipases A2 (PLA2) were isolated and purified from the Taiwan cobra (Naja naja atra) and several snake species of the same or different genera by semipreparative cation-exchange and reversed-phase HPLC. They were shown to possess different enzymatic activity toward the synthetic substrate L-alpha-lecithin by the fatty-acid titration method. The immunological cross-reactivity of these structurally similar isotoxins was investigated using immunodiffusion, precipitin reaction and enzyme-linked immunosorbent assay (ELISA). PLA2 from the venoms of the same species such as the Taiwan cobra (Naja naja atra) and the Indian cobra (Naja naja naja) showed a high degree of antigenic resemblance whereas no immunoreactivity was observed among those PLA2 from different genera. Quantitative immunoreactivity assays by ELISA revealed the partial cross-reactivity between the antibody against PLA2 of Taiwan cobra and those isoenzymes from snakes of remotely-related species. The immunological relatedness between PLA2 of the representative snake species of different genera and families is shown to be correlated with the extent of sequence homology among these enzymes.     

Comparison of ELISA with activity and ligand-binding methods for the determination of thymidylate synthase concentration.

The determination of enzyme levels in cellular extracts by active site titrations or by catalytic activity measurements is relevant in both science and medicine. However, these techniques assume that enzymes exhibit the same response in crude sample matrices as they do in the purified state. We report here an example of how an enzyme-linked immunosorbent assay (ELISA) was used to determine the true enzyme concentration which was compared to the effective enzyme concentration obtained by ligand binding and catalytic assay methods in a crude bacterial cell extract. Rabbit antibodies specific for Lactobacillus casei thymidylate synthase (TS) were used to develop a highly specific and sensitive heterogeneous noncompetitive ELISA assay with a typical detection limit of 1.4 fmol of TS (100 pg) and a dynamic working range of 3 orders of magnitude. The antibodies showed identical responses for TS, its inhibitory binary complex with 5-fluoro-2'-deoxyuridylate, and its inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate in the immunoassay. L. casei cell-free extracts were subjected to extraction with CM-Sephadex and the various fractions were analyzed by ELISA, active-site titrations, and catalytic assays which demonstrated that assays which assumed full catalytic or ligand-binding competence underestimated the true enzyme level.     

Rapid TLC/GC-MS identification of acetylcholinesterase inhibitors in alkaloid extracts

Alkaloid extracts from 12 plant species of the families Amaryllidaceae, Fumariacae and Papaveraceae were studied with respect to their acetylcholinesterase inhibitory activity and alkaloid patterns. Fifty-three alkaloids were identified by GC-MS, including known acetylcholinesterase (AChE) inhibitors such as galanthamine, epigalanthamine, sanguinine and epinorgalanthamine in extracts of Amaryllidaceae plants and protopine in extracts of Fumariaceae and Papaveraceae plants. The galanthamine-containing extracts of the amaryllidaceous plants were found to be the most active while the extract of Corydalis bulbosa was the most active among the extracts of the tested plants from the Fumariaceae and Papaveraceae plants. TLC bioautographic assay, preparative TLC and GC-MS analysis were combined to identify the active compounds in the studied extracts. Galanthamine was isolated from the known AChE inhibitors in the extracts of Amaryllidaceae plants. Corydaline, bulbocapnine and stylopine were found to be active in the extracts of plant species of the families Fumariaceae and Papaveraceae. Available standards of deshydrocorydaline--a precursor of corydaline, corydaline and stylopine--were tested for AChE inhibitory activity. Deshydrocorydaline and corydaline showed potent inhibitory activity comparable with that of the positive control galanthamine.     

Identification of serotonin-sensitive aryl acylamidase activity with cobra venom acetylcholinesterase

Acetylcholinesterase purified from cobra (Naja naja) venom exhibits a serotonin-sensitive aryl acylamidase activity. Both acetylcholinesterase and aryl acylamidase activities co-eluted in column chromatographic procedures (Sephadex G-75 and Zinc-Sepharose), co-migrated on polyacrylamide gel electrophoresis, co-immunoprecipitated by anti-snake venom antibody and showed the same heat denaturation profile at 40 degrees C. Further, several potent acetylcholinesterase inhibitors at different concentrations inhibited the cholinesterase and aryl acylamidase activities to the same extent. It is concluded that in cobra venom, acetylcholinesterase is associated with a serotonin-sensitive aryl acylamidase activity similar to earlier observations made with acetylcholinesterase from different sources.     

In vitro antibacterial activity of Psidium guajava Linn. leaf extract on clinical isolates of multidrug resistant Staphylococcus aureus

In the present study, antibacterial activity of aqueous and organic extracts of Psidium guajava leaves was evaluated against multidrug resistant (MDR) clinical isolates of Staphylococcus aureus strains collected from hospitals in northern (Malabar region) Kerala. The strains which exhibited resistance against all the antibiotics tested was selected for antibacterial assays. Minimum inhibitory concentration (MIC) for methanolic and aqueous extracts was found to be 625 ug/ml and 7.5 mg/ml, respectively. Minimum bactericidal concentration (MBC) recorded for methanolic and aqueous extracts was 1.25 and 12.5 mg/ml, respectively. Methanolic extract at minimum bactericidal concentration inhibited the growth of MDR strain by 80%. Time-kill assay revealed that methanolic extract (4 mg/ml) killed MDR bacteria within 10 hr. Total polypeptide profiling of bacterial cultures by SDS-PAGE indicated a high degree of protein degradative activity of the extract. Finally, a human RBC based haemolytic assay showed absence of haemolysis even at concentrations higher than that of MBC, advocating thereby its safety in therapeutic use.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

黄顶菊茎叶浸提液对白菜和水稻幼苗化感作用的初步研究

研究了不同浓度的黄顶菊茎叶浸提液对白菜和水稻幼苗的化感作用.结果表明:黄顶菊茎叶浸提液显著地抑制了白菜和水稻幼苗的根长和苗高,降低了白菜和水稻的根系活力,提高了丙二醛含量,并且增大了受体细胞膜透性,使细胞内含物外渗,导致电导率值增高;同时,黄顶菊茎叶浸提液对白菜和水稻的过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)的活性均有显著的抑制作用,导致受体对外界不良环境的抵抗力降低.由此可见黄顶菊通过影响植物体内的一系列生理过程,降低植物对环境的抗胁迫能力,使受体植物的生长发育受到抑制,从而表现出强烈的化感作用.综合各项指标可以看出,白菜较水稻对黄顶菊化感作用较敏感.     

Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.     

A novel alpha-glucosidase inhibitor from pine bark

Inhibitors of carbohydrate-hydrolysing enzymes play an important role for the treatment of diabetes. To our knowledge, a number of inhibitors such as, 1-deoxynojirimycin, acarbose and voglibose have been identified as a result of screening of secondary metabolites up to now. In this note, we report the inhibitory effect on carbohydrate hydrolysis of ethanol extracts from more than 1400 species of plants with the aim of identifying a potential antihyperglycemic drug. Pinus densiflora bark extracts showed the highest inhibition activity against several carbohydrate-hydrolysing enzymes.     

两种莪术类药用植物姜黄素提取物对小鼠抗氧化活性研究

本研究通过对两种莪术类药用植物姜黄素提取物,结合3种姜黄素的含量分析其对小鼠的抗氧化药效。通过高效液相色谱法测定总姜黄素提取物中3种姜黄素的含量。选取48只雌性小鼠,随机分成4组分别灌胃提取液(含1组喂基础饲料)。正常饲喂14 d后,小鼠采血测定SOD (超氧化物歧化酶)、CAT (过氧化氢酶)、GSH-Px (谷胱甘肽过氧化物酶)、XOD (黄嘌呤氧化酶)及NO (一氧化氮)、MDA (丙二醛)含量。结果表明,经姜黄素提取液灌胃的小鼠,SOD、CAT、XOD活性升高,NO含量下降;GSH-PX活性普遍升高,MDA含量普遍下降;2种莪术抗氧化活性具有差异性,浓度或含量与抗氧化活性不具有正相关关系;产地不同,也是影响体内抗氧化活性的因素之一。     

A comparison of eel electroplax and snake venom acetylcholinesterase

The effects of some cholinergic ligands, harmala alkaloids and local anesthetics on the activity of eel electroplax and Naja naja siamensis venom acetylcholinesterase have been studied. In most cases, eel electroplax was found to be more susceptible towards inhibition than the venom acetylcholinesterase. No major difference was observed with respect to the type of inhibition in both enzymes. The activation of the two enzyme preparations by inorganic cations (Ca2+, Mg2+ and Na+) showed a similar pattern. In both preparations, the onset of activation was detectable at much lower concentration with the divalent metal ions than with the monovalent Na+. Antagonism between Ca2+ and decamethonium, tubocurarine and tetracaine in both enzymes approached competitive kinetics. The onset of substrate inhibition is delayed by Ca2+ (30 mM) in both enzymes. It is suggested that the Ca2+ binding site overlaps with the substrate inhibitory site. It is concluded that cobra venom acetylcholinesterase has similar allosteric binding sites to those of eel electroplax.     

Hemolytic,cytotoxic and complement inactivating properties of extracts of different species of Aspergillus

Some of the biological properties of saline extracts of the mycelia of several species of the Aspergillus genus, namely, A. fumigatus, A. flavus, A. niger, A. nidulans, A. parasiticus and A. glaucus, were studied. Only the extract prepared from A. fumigatus was found to be hemolytic for sheep red blood cells. In contrast, all the extracts with the only exception of that of A. glaucus, had cytotoxic effects on Vero cells. Both, the hemolytic and cytotoxic constituents of the extracts were removable by adsorption with activated carbon. Heating of the extracts at 100°C for 30 minutes also resulted in detoxification. In vivo studies, performed only with detoxified extracts of A. fumigatus, showed these were capable of depleting complement levels in guinea pigs. Complement inactivation was also found to occur in vitro and was caused by all the extracts tested. Also triggered by the extracts was the conversion of serum C3 but not of purified C3, indicating that other serum factors are essential in the process. Despite the similarity in this respect with cobra venom factor, differences in activity after heating-negative in cobra venom factor-indicate that the complement inactivating substance/s present in the Aspergillus extracts differ from those of the snake product.     

Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja)

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits.     

High aryl acylamidase activity associated with cobra venom acetylcholinesterase: Biological significance

Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is aryl acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.     

New biological activity against phospholipase A2 by Turmerin, a protein from Curcuma longa L

Turmerin is a protein from Turmeric (Curcuma longa L.) with a relative molecular mass of 14 kDa. The protein inhibits the enzymatic activity and neutralizes the pharmacological properties, such as cytotoxicity, oedema and myotoxicity of multitoxic phospholipase A2 (NV-PLA2) of cobra (Naja naja) venom at a 1:2.5 molar ratio of NV-PLA2:Turmerin. A Lineweaver-Burk plot indicates that Turmerin follows a linear mixed type of inhibition.     

内蒙古产白刺、小果白刺和霸王α-葡萄糖苷酶抑制活性

首次利用体外α-葡萄糖苷酶抑制模型对内蒙古产3种蒺藜科植物的9个提取物进行活性评价,并与阳性对照Acarbose比较,发现3种植物均有抑制α-葡萄糖苷酶活性。其中白刺石油醚提取物对α-葡萄糖苷酶的抑制活性(IC50=81.80 mg/L)最高,其余依次为小果白刺乙酸乙酯提取物(IC50=610.29 mg/L),霸王石油醚(IC50=627.22 mg/L)和乙酸乙酯提取物(IC50=838.40 mg/L),它们的抑制活性远大于阳性对照Acarbose(IC50=1103.01 mg/L)。结果发现,不同植物不同溶剂提取物的α-葡萄糖苷酶抑制活性不同。同一植物不同溶剂提取物相比较,甲醇提取物的α-葡萄糖苷酶抑制活性不及乙酸乙酯和石油醚提取物。     

Screening of Echinops ellenbeckii and Echinops longisetus for biological activities and chemical constituents

Members of the genus Echinops in the family of Asteraceae are widely used in Ethiopian herbal medicine for the treatment of various diseases and illness such as migraine, diarrhea, heart pain, different forms of infections, intestinal worm infestation and hemorrhoid. Hydroalcoholic extracts of the root, flower head, leaf and stem of Echinops ellenbeckii O. Hoffm. and Echinops longisetus A. Rich were investigated for their chemical constituents and biological activities. The presence of alkaloids, saponins, phytosterols, polyphenols and carotenoids in the different parts of the plants was observed whilst anthraquinones were not detected. The leaf extracts of both plants and stem extract of E. longisetus showed strong inhibitory activity against cultures of Staphylococcus aureus. None of the extracts were found to be active against Gram-positive organisms. The flower extract of E. ellenbeckii showed strong inhibitory activity against Candida albicans. Root and flower extracts of the plants investigated showed lethal activity against earthworms. Moreover, the extracts of the roots of both plants showed molluscicidal activity against schistosome-transmitting snail hosts. The biological activities observed were dose dependent.