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Objectives: Antivenom serums have been used extensively for over a century and are the only effective treatment option for snake bites and other dangerous animal envenomations. In therapeutic serum centers, a wide range of antivenoms is made from animal serum, mainly equine and sheep, that are immunized with single or multiple venoms. This work aimed to use caprylic acid (CA) to purify therapeutic snake antivenom.Methods : : Plasma was obtained from equine immunized with a mixture of venoms. Immunized plasma was obtained by precipitation of different concentrations (2-5%) of CA. This methodology was compared to that based on ammonium sulfate (AS) precipitation. Sediment plasma proteins were purified by ion-exchange chromatography. Protein assay, SDSPAGE, and agar gel diffusion were performed.Results : : The total protein precipitation with AS was higher than precipitation with CA, but the best results were obtained when CA was added to the plasma until a final CA concentration of 5% was reached. Chromatography and electrophoresis indicated a stronger band for the 5% CA, and the gel diffusion assay showed antigen-antibody interaction in the purified serum.Conclusion : : The use of CA compared to the routine method for purifying hyperimmune serums is a practical and cost-effective method for preparing and producing therapeutic serums. It constitutes a potentially valuable technology for alleviating the critical shortage of antivenom in Iran.

Snakebite affects around 3 or 4 million humans annually leading to more than 100,000 deaths. Coagulopathy is one of the significant causes of both morbidity and mortality in these patients. Accordingly, it is of utmost importance to diagnose and treat coagulation disorder due to bites; in addition, it is accompanied by various clinical aspects, such as pre-coagulation, fibrinogen coagulation time, fibrinolytic, platelet activation, anticoagulant, thrombotic, and bleeding. The main cause of coagulopathy caused by snakebite is the presence of compounds found in snake venom. These compounds are mostly proteins with enzymatic activity and high stability; moreover, they rapidly react with factors in the blood circulatory system and disrupt their correct functioning. Regarding the snake venom compounds, especially their proteins, it should be mentioned that different snakeschr('39') venoms have different proteins, which can have a role in coagulation or anticoagulation depending on its amount. The coagulant proteins are subclassified as clotting factor activators and thrombin-like enzymes. The anticoagulant proteins can prevent blood clotting leading to coagulopathy and include phospholipases A2, fibrinolytic, protein C activator, and L-amino acid oxidase (enzymatic anticoagulants) or C-type lectin-like proteins, three-finger toxins (TFTs), and proteinase inhibitors (nonenzymatic anticoagulants). All of these factors cause coagulopathy due to snake bites, which is a clinically important phenomenon and should be carefully examined; otherwise, it would be difficult to make the diagnosis and treatment process. If untreated, coagulopathy can develop quickly and lead to the patientchr('39')s death.

The pathogenesis of Mycobacterium tuberculosis (Mtb) is related to its low molecular weight proteins mainly ESAT6 and CFP10 that are highly specific and potentially useful for the diagnosis of tuberculosis. This research focused on isolation, purification, and characterization of low molecular weight proteins from Mtb. Cultures of Mtb were inactivated by heating at 68 °C for 90 min and 100 °C for 3 hrs, respectively. Inactivated cultures were filtered and the proteins in the supernatant fluid precipitated with two rounds of ammonium sulfate, at 4 °C. The collected precipitates were dialyzed and subjected to gel chromatography (G-50) and the obtained fractions were analyzed for protein concentrations and molecular weight. ESAT6 and CFP10 protein complex in the purified fraction was confirmed by Western blotting. Guinea pig sensitization assay was used for estimating the potency of the purified fraction compared to the standard PPD. The maximum amount of low molecular weight proteins were precipitated by 20% ammonium sulfate. SDS-PAGE analysis revealed protein bands of approximately 10-15 kDa. The purity of the proteins was ≥95%, as confirmed by SDS-PAGE. The presence of the ESAT-6/CFP10 complex was confirmed by Western blot analysis. The purified fractions showed no cross-reaction with BCG or M. avium strain. ESAT-6/CFP-10 purified by the ammonium sulfate method appeared to be suitable for the development of a diagnostic kit for the detection of Mtb.

Objectives: Nowadays cancer treatment is an important challenge in the medical world that needs better therapies. Many active secretions produced by insects such as honey bees used to discover new anticancer drugs. Bee venom (BV) has a potent anti inflamma- tory, anti cancer and tumor effects. The aim of present study is evaluation of anticancer effects induced by Apis mellifera venom (AmV) on cell Lines.Methods: AmV was selected for study on cancer cell lines. Total protein, molecular weight and LD50 of crude venom were determined. Then, cells were grown in Dulbecco’s Modified Eagle medium supplemented with 10% fetal bovine serum and 1% antibiotics. The A549, HeLa and MDA-MB-231 cell Lines were exposed by different concentration of AmV. The morphology of cells was determined and cell viability was studed by MTT assay. Evaluation of cell death was determined by and DNA fragmentation.Results: The results from MTT assay showed that 3.125 µg/mL of A549, 12.5 for HeLa and 6.25 µg/mL of MDA-MB-231 killed 50% of cells (p < 0.05). Morphological analysis and the results from hoescht staining and DNA fragmentation indicated that cell death induced by AmV was significantly apoptosis.Conclusion: The data showed that using lower dosage of AmV during treatment period cause inhibition of proliferation in time and dose dependant manner. Findings indicated that some ingredients of AmV have anticancer effects and with further investigation it can be used in production of anticancer drugs.

Introduction Bee venom (BV) contains compounds that have biological and biochemical activitis. Therefore, purification of BV and investigation of its biochemical properties can be useful in understanding its therapeutic aspects. In the present study, the biochemical effects of Apis mellifera venom on the blood and liver of laboratory mice were investigated. Materials and Methods Protein concentration of Apis mellifera crude venom was determined. Purification of venom was performed with Sephadex G-50 chromatography. SDS-PAGE was used to determine the protein profile of BV and its fractions. The hemolytic activity of venom was investigated. Biochemical tests were performed in one control group (with a normal diet and without injection of venom) and three experimental groups (with injections of different venom doses). Results Crude venom protein concentration was about 4.1 mg/ml. The chromatogram showed three large peaks and melittin account for about 50% of the BV. By electrophoresis, it was found that the venom has compounds with a molecular weight of 3 to 90 kD, including melittin, phospholipase, hyaluronidase, acid phosphomonoesterase. The hemolytic test showed HD50 of melittin at a concentration of 0.5 μg/ml. Biochemical tests on the control and the three experimental groups showed that when venom dose increased in the three experimental groups; the group with the highest venom dose (0.1 mg/ml) showed the highest decrease in serum parameters compared to the control group. Conclusion BV, especially melittin, has a significant hemolytic activity and biologically very active, so it can be used to treat some diseases.

Background: In recent years, the number of people with cancer has increased. For this reason, different drugs have been suggested for the treatment of cancer, but none of them has resulted in complete remission. Many bio-toxins are biologically active compounds with anticancer activity. In the meantime, bee venom (BV) has a potent anti cancer and tumor effects. The aim of present study is evaluation of BV anticancer effects on breast cancer cell line (MDA-MB-231). Methods: Crude bee venom was obtained from Apis mellifera and the amount of its protein was determined. MDA-MB-231 cancer cell line was cultured and then the cells were exposed to 1.56, 3.125, 3.25, 6.25, 12.5, 25 and 50 µg/ml of bee venom for 24 and 48 h, respectively. The morphology and apoptosis were evaluated and cell viability was determinedby MTT assay. Results: MTT assay showed that BV at concentration 6.25 µg/ml for MDA-MB-231 killed 50% of cells (p

Background: Some venoms and their isolated compounds have been shown to have antibacterial properties. Snake, scorpion and bee venoms are a complex mixture of proteins such as phospholipase and melittin, which have an effect on bacterial growth inhibition. This study aimed to investigate of antibacterial effect of three different venoms against selected bacterial strains. Materials & Methods: Crude venoms obtained from snake (Echis carinatus), scorpion (Mesosobuthus epues) and bee (Apis mellifera) were selected. The crude venoms from these species was purified by using gel filtration chromatography and the molecular weights of the compounds in these venoms estimated by using SDS-PAGE. The approximate lethal dose values of venoms were determined. Antibacterial activity of venoms against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli were evaluated. Venoms and its isolated fractions and standard antibiotic were tested by using the disc diffusion method. Results: E. carinatus crude venom and fraction 2 were effective against S. aureus and E. coli. M. eupeus crude venom and fraction 1 and 4 were effective against B. subtilis. A. mellifera crude venom demonstrated antibacterial activity against E. coli, S. aureus and Fraction 3 of this venom has an inhibition effect for E. coli and S. aureus. Conclusion: Snake, scorpion and bee venoms inhibit the growth and survival of bacterial strains and that these venoms can be used as a complementary antimicrobial agent against pathogenic bacteria.

Objectives: Venomous fishes have different pharmacological effects and are useful. Among the venomous fish, stonefishes; especially Pseudosynanceia melanostigma has various pharmacological effects on the nervous, muscular and cardiovascular system of humans. In this study, toxicological characteristics, some blood effects, pharmacological and enzymatic properties of Pseudosynanceia melanostigma venom was investigated. Methods: Crude venom purified by using gel filtration chromatography and the molecular weights of the venom and its fractions were estimated. The approximate LD values of this venom were determinedand the effects of LD50 dose on the blood of rabbits were studied. Hemolytic and Hemorrhagic activity of the venom sample was determined. In this case coagulation tests were performed. Results: The LD50 of the Pseudosynanceia melanostigma crude venom was also determined to be 194.54 μg/mouse. The effect of two doses of LD50 showed a non-significant differences decrease in RBCs and MCV. In other cases, the results showed significant differences in WBC, Plt, Hb, MCH, MCHC and HCT; also it's showed a significant decrease. WBC count showed a significant increase with two doses of LD50 groups. The prothrombin time and partial prothrombin time were increased after venom treatment. As well as bleeding and clotting time were increased. According to the results, a minimum dose for Haemorrhagic effect 40 μg was obtained. Conclusion: Venom of Pseudosynanceia melanostigma has inhibitory effect on platelet aggregation that can be used to design and develop of anticoagulant drugs.

Thiouracilis a historically relevant anti-thyroid preparation. Because of its structure you can find it in various chemical reactions differently. In this study, the reaction of Thiouracil with Chloroacetic acid and the formation of their additive products has been investigated. This reaction is aconcerted process, and it has not been determined yet by exhaustive mechanisms. From the potential energy profile, two possible mechanisms as well as two NH bonds dissociations are examined. Density Functional Theory (DFT) was used to compare these mechanisms. Calculation results for comparing these two pass ways were indicated byB3LYP/6-311g (d,p) levels of theory. The activation energies to 2-(6-oxo-1,6-dihydropyrimidin-2-ylthio) acetic acid and 2-(4-oxo-1,4-dihydropyrimidin-2-ylthio) acetic acid formation were obtained 55.78 and 72.9 kcal.mol-1, respectively. These calculations were also carried out for ethyl and methyl Thiouracil derivatives. The calculation results indicate that removal of hydrogen from nitrogen to sulfur group at the ortho position is more favorable

Background & Objective: Currently selection and use of nanoparticle adjuvant is very important. Due to the lack of effective anti-snakebite serums to treat it and high cost of purchasing external anti-snakebite serums, preparation of suitable internal nanoparticles to produce hyper-immunization plasma has become very important. In order to minimize the disadvantages associated with traditionally used adjuvants in ovines and to produce potent antivenum, a comparison was made between various adjuvants for their immune-potentiation capacity and safety. Material & methods: The present study was conducted in 15 sheep, divided into three groups and hyper-immunized using crude venom of cobra snake (Naja naja oxiana) along with three adjuvants, Montanide, aluminum phosphate and poly methyl methacrylate through subcutaneous route at intervals of a week. Periodic standard safety assessments were done. Results: The neutralization activity (LD50) of pooled sera samples by 9th week, obtained with aluminum phosphate, Montanide and poly methyl methacrylate groups were 1.5, 2.2 and 2 LD50/ml respectively. The sheep of poly methyl methacrylate group showed minimum local reactions at injection site, while sheep from other two groups exhibited moderate reactions. However, these were transient and reabsorbed or healed subsequently. Conclusion: Poly methyl methacrylate nanoparticle adjuvant could be a possible alternative to the emulsion adjuvants for primary phase of immunization in antivenomous preparation considering its acceptable immunopotentiation capacity and safety in donor animals.

Objectives: Mellitine, a major component of bee venom (BV, Apis mellifera), is more active against gram positive than gram negative bacteria. Moreover, BV has been reported to have multiple effects, including antibacterial, antivirus, and anti-inflammation effects, in various types of cells. In addition, wasp venom has bee

Objectives: Bee venom (BV) is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera) on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Methods: Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50), and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blood samples were obtained from 10 rabbits, and the prothrombin time (PT) and the partial thromboplastin time (PTT) tests were conducted. The approximate lethal dose (LD) values of BV were determined. Results: Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations (SDs) = ± 0.71; however, clotting was observed in the control group 13.8 s, SDs = ± 0.52. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa), respectively. The LD50 of the crude BV was found to be 177.8 μg/mouse. Conclusion: BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase A2 (PLA2) and melittin, and that can increase the blood clotting times in vitro.

Objective(s): Echis carinatus is one of the venomous snakes in Iran. The venom of Iranian Echis carinatus is a rich source of protein with various factors affecting the plasma protein and blood coagulation factor. Some of these proteins exhibit types of enzymatic activities. However, other items are proteins with no enzymatic activity. Materials and Methods: In order to study the mechanism and effect of the venom on human plasma proteins, the present study has evaluated the effect of crude venom and all fractions. A procoagulant factor (prothrombin activator) was isolated from the venom of the Iranian snake Echis carinatus with a combination of gel filtration (Sephadex G-75), ion-exchange chromatography (DEAE- Sepharose) and reverse phase HPLC. Furthermore, proteolytic activity of the crude venom and all fractions on blood coagulation factors such as prothrombin time (PT) was studied. Results: In the present study, the PT test was reduced from 13.4 s to 8.6 s when human plasma was treated with crude venom (concentraion of venom was 1 mg/ml). The purified procoagulant factor revealed a single protein band in SDS polyacrylamide electrophoresis under reducing conditions and its molecular weight was estimated at about 65 kDa. A single-band protein showed fragment patterns similar to those generated by the group A prothrombin activators, which convert prothrombin into meizothrombin independent of the prothrombinase complex. Conclusion: This study showed that the fraction which separated from Iranian snake Echis carinatus venom can be a prothrombin activators. It can be concluded that this fraction is a procoagulant factor.

Background/objective: Mycobacterium bovis, responsible for bovine tuberculosis is a member of Mycobacterium tuberculosis complex. This bacterium is responsible for infection in wide range of hosts. Bovine tuberculosis is well known as Zoonotic tuberculosis in human and is transmitted via consumption of unpasteurized milk, contaminated meat products and also by ingestion of Mycobacteria from the environment. The objectives of the present study was to compare the genomic pattern of M. bovis obtained from human subjects in Zanjan province, with those of atypic cattle in Iran by Restriction fragment length polymerization and DNA hybridization methods. Methods: DNA was isolated from 2 M. bovis strains isolated from suspected patients by van Sooligen method. Finger printing methods using RFLP and DNA hybridization with probes DR and PGRS was performed. The obtained patterns were compared with the genomic pattern of 161 M. bovis strains isolated from infected cattle lymph nodes, present in the Tuberculin reference Laboratory at Razi vaccine and Serum Research Institute, Karaj. Results: Comparisons of the genetic pattern of the 2 M. bovis strains from Zanjan province with 16 distinct patterns obtained with PGRS probe and 20 patterns with DR probes from 161 M. bovis isolates indicated no correlation of the patterns of Zanjan isolates with those present at Razi Tuberculin Reference Laboratory. Conclusion: With respect to the age of patients, absence of epidemic and lack of cluster in the mentioned province and other provinces, our results indicate recurrence of the infection due to M. bovis isolates which were present in Zanjan province in previous years. These isolates had no association with the bovine tuberculosis isolates present in Razi Tuberculin Reference laboratory.

Objective/background: Tuberculosis (TB) is one of the most common infectious diseases in Iran and around the world. Diagnosis of this disease in many cases is difficult and often requires the use of paraclinical methods. Current diagnostic methods are either too slow or lack enough sensitivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. They can be helpful for mycobacteria diagnosis. Antigen 60 (A60) is a thermostable antigen found in the cytosol of Mycobacterium bovis and Mycobacterium tuberculosis. This antigen is used in ELISA systems design for diagnosis of tuberculosis. The aim of this study is purification of A60 from bacterial cytoplasm and to evaluate the efficiency of this antigen and compare it with the production human tuberculin and standard human tuberculin. Methods: Using gel filtration chromatography with a sepharose 4B column, A60 was purified from other bacterial components. A60 was recognized by agar gel immunodiffusion with anti-BCG and anti-A60 antiserum, where it formed an immunoprecipitation line with anti-BCG antiserum and anti-A60 antiserum. Molecular weight components of the A60 were obtained using electrophoresis. Results: Seven fractions were obtained by chromatography. In analyzing with dot blotting, both the cytoplasm and cell wall of BCG had A60. This test showed that the first fraction creates maximum color intensity and as a result, the highest amount of A60 was in fraction one. In agar gel immunodiffusion, the cytoplasm sample and all fractions obtained from chromatography showed a positive reaction with anti-A60 antiserum, and fraction one had the most among sediment of the other fractions. Molecular weight components of the A60 were identified to be approximately 35 kDa, 38 kDa, 40 kDa, and 65 kDa. Conclusion: Results of reactions of the injected A60 and standard human tuberculin shows the effectiveness of this antigen in comparison with standard human tuberculin. Detection of antibody in the serum of patients is a rapid and repeatable method. A60 with 89% sensitivity and 94% specificity could be an appropriate matter for the diagnosis of tuberculosis. Because this method can be performed without radioactive materials or advanced and expensive equipment, it will provide results quickly.