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2.
Article in Chinese | WPRIM | ID: wpr-970599

ABSTRACT

This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.


Subject(s)
Animals , Zebrafish , Bufanolides/analysis , Bufonidae , Chromatography, High Pressure Liquid , Quality Control , Cell Line, Tumor
3.
Article in Chinese | WPRIM | ID: wpr-1008624

ABSTRACT

Bufonis Venenum, an animal medicinal material, is widely used for treating cardiovascular diseases and pain induced by rheumatics or malignant tumors. In view of the high activity and high toxicity, it is of great significance to pay attention to the quality control of Bufonis Venenum to ensure the safety and effectiveness of its preparations. China's drug standards involve 102 preparations(474 batch numbers) containing Bufonis Venenum approved for sale, including 14 preparations in the Chinese Pharmacopoeia(2020 edition) and 68 preparations in the standards issued by the Ministry of Health Drug Standard of the People's Republic of China. Bufonis Venenum is mostly used in pill and powder preparations in the form of raw powder, with the main functions of clearing heat, removing toxin, relieving swelling and pain, replenishing qi, activating blood, opening orifice, and awakening brain. Except the high level of quality control for Bufonis Venenum in the preparations in the Chinese Pharmacopoeia(2020 edition), the quality control standards of Bufonis Venenum in other preparations are low or even absent. Therefore, it is urgent to conduct research on the improvement of quality standards for the preparations containing Bufonis Venenum. This study retrieved the reports focusing on the quality evaluation and quality control of the preparations containing Bufonis Venenum from CNKI, PubMed, and Web of Science. Qualitative and quantitative analysis methods for 64 preparations containing Bufonis Venenum have been reported, mainly including thin-layer chromatography, HPLC fingerprint, and multi-component content determination. The index components mainly involved bufadienolides, such as gamabufalin, arenobufagin, bufotalin, bufalin, cinobufagin, and resibufogenin. According to the literature information, this paper suggests that attention should be paid to the correlations between the analysis methods and detection indexes of medicinal materials, decoction pieces and preparations, the monitoring of indole alkaloids, and the content uniformity inspection for further improving the quality standards for the preparations containing Bufonis Venenum.


Subject(s)
Animals , Humans , Bufonidae , Powders , Bufanolides/pharmacology , Quality Control , Chromatography, High Pressure Liquid , Pain/drug therapy
4.
Braz. j. biol ; 82: 1-6, 2022. map, ilus, tab
Article in English | LILACS, VETINDEX | ID: biblio-1468461

ABSTRACT

Euphlyctis cyanophlyctis (the skittering frog) is one of the most widespread species in Pakistan. Present study was aimed to know the presence of Euphlyctis cyanophlyctis in urban and rural areas of Lower Dir, the North-western Pakistan. A total of 33 frogs were collected, including 15 from rural and 18 from urban areas. The frogs were caught by hands covered with gloves instead of using nets. The collection was managed from August to October 2016 and from April to May 2018. Morphometric analysis, coloration as well as photographs of the frogs have been provided in detail. Skittering frogs were seen frequent in swampy areas near the water bodies. These frogs were mostly seen after sunset.


Euphlyctis cyanophlyctis (a rã que desliza) é uma das espécies mais comuns no Paquistão. O presente estudo teve como objetivo conhecer a presença de Euphlyctis cyanophlyctis em áreas urbanas e rurais de Lower Dir, noroeste do Paquistão. Um total de 33 sapos foram coletados, incluindo 15 de áreas rurais e 18 de áreas urbanas. As rãs foram apanhadas com as mãos cobertas com luvas em vez de redes. A coleta foi gerenciada de agosto a outubro de 2016 e de abril a maio de 2018. Análises morfométricas, coloração e também fotografias das rãs foram fornecidas em detalhes. Rãs saltitantes foram vistas freqüentemente em áreas pantanosas próximas aos corpos d’água. Essas rãs eram vistas principalmente após o pôr do sol.


Subject(s)
Animals , Bufonidae/anatomy & histology , Bufonidae/growth & development
6.
Article in Chinese | WPRIM | ID: wpr-888004

ABSTRACT

Bufonis Venenum,the dried secretion of Bufo bufo gargarizans or B. melanostictus,is toxic and hard with the efficacy of removing toxicity for detumescence and relieving pain. The processing of Bufonis Venenum dates back to the Song dynasty. In addition to the wine-processing,milk-processing and talcum powder-processing,there were some other kinds of processing methods in ancient times,such as baking,calcining,water-soaking and vinegar-processing. Modern studies have shown that the Bufonis Venenum has the main chemical components of bufadienolides,indole alkaloids sterols,and other compounds. It has the pharmacological effects of antitumor,cardiac,antibacterial,and analgesic activities,local anesthesia,and so on. This paper reviews the processing evolution,chemical components and pharmacological effects of Bufonis Venenum,providing references for its special processing and modern research as well as the theoretical basis for the research on its processing mechanism and quality control.


Subject(s)
Animals , Bufanolides/pharmacology , Bufonidae , Quality Control
7.
Article in Chinese | WPRIM | ID: wpr-888184

ABSTRACT

Indolealkylamines(IAAs) are the main hydrophilic substances in toad skin, mainly including free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine. In this study, the LPS-activated neutrophils were used to investigate the structure-activity relationship and anti-inflammatory mechanism of the above-mentioned five monomers from the toad skin in vitro. The neutrophils were divided into the control group, model group(1 μg·mL~(-1) LPS), positive drug group(100 μg·mL~(-1) indometacin), as well as the low-(50 μg·mL~(-1)), medium-(100 μg·mL~(-1)) and high-dose(200 μg·mL~(-1)) free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine groups. The levels of IL-6, TNF-α and IL-1β in the neutrophil supernatant of each group was measured by enzyme-linked immunosorbent assay(ELISA) after LPS stimulation, followed by the detection of apoptosis in each group after Annexin V/PI staining. The protein expression levels of caspase-3, Bax, Bcl-2, beclin1, LC3-I, and LC3-Ⅱ were assayed by Western blot. The results showed that IAAs reduced the excessive secretion of inflammatory cytokines caused by LPS compared with the model group. Besides, the activity of each free IAAs(N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine and dehydrobufotenine), especially bufotenine, was stronger than that of the binding bufothionine. As revealed by Annexin V/PI staining, LPS delayed the early apoptosis of neutrophils compared with the control group, while bufotenine promoted the apoptosis of neutrophils in a dose-dependent manner, which might be related to the elevated expression of apoptosis-related protein Bax/Bcl-2. In addition, LPS activated the autophagy pathways in neutrophils. This study confirmed the efficacy of IAAs in reducing the secretion of inflammatory cytokines in neutrophils induced by LPS for the first time. For instance, bufotenine exerts the anti-inflammatory effect possibly by inducing the apoptosis of neutrophils.


Subject(s)
Animals , Anti-Inflammatory Agents/pharmacology , Apoptosis , Bufonidae , Lipopolysaccharides/toxicity , Neutrophils , Skin
8.
J. venom. anim. toxins incl. trop. dis ; 27: e20200073, 2021. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1154769

ABSTRACT

he resistance against antimalarial drugs represents a global challenge in the fight and control of malaria. The Brazilian biodiversity can be an important tool for research and development of new medicinal products. In this context, toxinology is a multidisciplinary approach on the development of new drugs, including the isolation, purification, and evaluation of the pharmacological activities of natural toxins. The present study aimed to evaluate the cytotoxicity, as well as the antimalarial activity in silico and in vitro of four compounds isolated from Rhinella marina venom as potential oral drug prototypes. Methods: Four compounds were challenged against 35 target proteins from P. falciparum and screened to evaluate their physicochemical properties using docking assay in Brazilian Malaria Molecular Targets (BraMMT) software and in silico assay in OCTOPUS® software. The in vitro antimalarial activity of the compounds against the 3D7 Plasmodium falciparum clones were assessed using the SYBR Green I based assay (IC50). For the cytotoxic tests, the LD50 was determined in human pulmonary fibroblast cell line using the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Results: All compounds presented a ligand-receptor interaction with ten Plasmodium falciparum-related protein targets, as well as antimalarial activity against chloroquine resistant strain (IC50 = 3.44 µM to 19.11 µM). Three of them (dehydrobufotenine, marinobufagin, and bufalin) showed adequate conditions for oral drug prototypes, with satisfactory prediction of absorption, permeability, and absence of toxicity. In the cell viability assay, only dehydrobufotenin was selective for the parasite. Conclusions: Dehydrobufotenin revealed to be a potential oral drug prototype presenting adequate antimalarial activity and absence of cytotoxicity, therefore should be subjected to further studies.(AU)


Subject(s)
Bufanolides/administration & dosage , Bufonidae , Biodiversity , Malaria/immunology , Antimalarials , In Vitro Techniques , Computer Simulation
9.
Acta amaz ; 49(2): 145-151, abr. - jun. 2019. tab
Article in English | LILACS, VETINDEX | ID: biblio-1119166

ABSTRACT

The skin secretion from toads of the Bufonidae family has great potential in the search for new active compounds to be used as drug candidates in treating some diseases, among them cancer. In this context, this study aimed to evaluate the cytotoxic and antimutagenic activity of the parotoid gland secretion extracts of Rhinella marina and Rhaebo guttatus, as well as biochemically analyze transaminases and serum creatinine for liver and renal damage, respectively. Cytotoxicity was performed by the colorimetric method based on MTT (3- [4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) with different concentrations of the extracts in Walker or splenic tumor cell cultures from rats and mice. The micronucleus test was performed with male Swiss mice treated orally with the extracts for 15 days, and then intraperitoneally with N-ethyl-N-nitrosurea (50 mg kg-1). Micronucleated polychromatic erythrocytes (MNPCE) were evaluated in bone marrow. The extracts showed cytotoxic activity in the evaluated cells. There was a significant reduction in the frequency of MNPCE (R. marina = 56% and R. guttatus = 75%, p < 0.001), indicating antimutagenic potential of the extracts. The groups treated only with extract showed an increase in MNPCE frequency, evidencing mutagenic potential. Biochemical analyzes showed no significant difference between treatments. Thus, under our experimental conditions, the extracts of R. marina and R. guttatus skin secretions presented chemopreventive potential for cancer. (AU)


A secreção cutânea de anuros da família Bufonidae tem grande potencial na busca de novos compostos ativos para utilização como fármacos candidatos no tratamento de algumas doenças, entre elas o câncer. Neste contexto, este estudo teve como objetivo avaliar a atividade citotóxica e antimutagênica dos extratos da secreção da glândula parótida de Rhinella marina e Rhaebo guttatus, bem como a análise bioquímica de transaminases e creatinina séricas, para avaliar dano hepático e renal, respectivamente. A avaliação de citotoxicidade foi realizada pelo método colorimétrico baseado no MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide), com diferentes concentrações dos extratos em culturas de células do Tumor de Walker ou células esplênicas de rato e camundongo. O teste do micronúcleo foi realizado com camundongos Swiss machos que receberam tratamento oral com os extratos durante 15 dias, seguido de tratamento intraperitoneal com N-etil-N-nitrosuréia (50 mg kg-1). A frequência de eritrócitos policromáticos micronucleados (PCEMN) foi determinada em medula óssea. Os extratos apresentaram ação citotóxica nas células avaliadas. Houve uma redução significativa na frequência de PCEMN (R. marina = 56% e R. guttatus = 75%, p < 0,001), observando-se um potencial antimutagênico dos extratos. Os grupos tratados somente com os extratos apresentaram um aumento na frequência de PCEMNs, evidenciando um potencial mutagênico. As análises bioquímicas não apresentaram diferença significativa entre os tratamentos. Assim, nas condições experimentais testadas, as secreções cutâneas de R. marina e R. guttatus apresentaram potencial quimiopreventivo para câncer.(AU)


Subject(s)
Animals , Mice , Bufonidae/physiology , Antimutagenic Agents/analysis , Cytotoxins/analysis , Parotid Gland/chemistry , Chemoprevention/veterinary , Bioprospecting
10.
J. venom. anim. toxins incl. trop. dis ; 25: e20190029, 2019. tab
Article in English | LILACS, VETINDEX | ID: biblio-1020025

ABSTRACT

Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins. Methods: Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis. Results: Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomic-identified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes. Conclusions: Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.(AU)


Subject(s)
Animals , Steroids , Bufonidae/parasitology , Proteomics , Alkaloids
11.
Rev. bras. parasitol. vet ; 27(3): 390-395, July-Sept. 2018. graf
Article in English | LILACS | ID: biblio-1042480

ABSTRACT

Abstract Although a group of soft ticks (Argasidae) associated with amphibians was recently discovered in Brazilian rainforests, parasitism by these ticks on cold-blooded animals remains less common than on mammal and bird species. In this study, we identified ticks that were collected from toads that had been caught in December 2016 and January 2017, at Itinguçú waterfall (22°54'05" S; 43°53'30" W) in the municipality of Itaguaí, state of Rio de Janeiro. Tick specimens were identified using a morphological and molecular approach. In total, twelve larvae of Ornithodoros ticks were collected from three individuals of Rhinella ornata and were identified as Ornithodoros faccinii. Our results include a longer 16S rRNA mitochondrial sequence for O. faccinii that supports its phylogenetic relatedness to Ornithodoros saraivai, and we report this tick species parasitizing Rhinella toads for the first time in Brazil.


Resumo Embora um grupo de carrapatos moles (Argasidae) associado a anfíbios tenha sido recentemente descoberto nas florestas brasileiras, o parasitismo por esses carrapatos em animais de sangue frio permanece menos comum do que nas espécies de mamíferos e aves. Neste estudo, identificamos carrapatos que foram coletados de sapos capturados em dezembro de 2016 e janeiro de 2017, na cachoeira de Itinguçú (22°54'05" S; 43°53'30" W) no município de Itaguaí, estado do Rio de Janeiro. Os espécimes de carrapatos foram identificados usando uma abordagem morfológica e molecular. No total, doze larvas de carrapatos Ornithodoros foram coletadas de três indivíduos de Rhinella ornata e foram identificadas como Ornithodoros faccinii. Nossos resultados incluem uma maior seqüência mitocondrial 16S rRNA para O. faccinii que suporta sua relação filogenética com Ornithodoros saraivai e relatamos esta espécie de carrapato parasitando sapos Rhinella pela primeira vez no Brasil.


Subject(s)
Animals , Tick Infestations/parasitology , Bufonidae/parasitology , Ornithodoros/genetics , Phylogeny , Brazil , RNA, Ribosomal, 16S , Ornithodoros/anatomy & histology , Ornithodoros/classification
12.
Pesqui. vet. bras ; 35(6): 579-582, June 2015. graf
Article in English | LILACS | ID: lil-766187

ABSTRACT

We sampled ticks from specimens of the rococo toad Rhinella schneideriby flannel dragging on two Islands located in the São Francisco River near the Três Marias hydroelectric dam, southeastern Brazil. A total of 120 toads was examined, of which 63 (52.5%) were parasitized only by Amblyomma rotundatumtotaling 96 larvae, 163 nymphs and 134 females. The burden of parasitism ranged from one to 43 ticks, with a mean intensity of infestation of 6.2±5.5 ticks per host. The tick A. rotundatumexhibited highly aggregated distribution. Peak abundance of larvae and nymphs occurred in the dry season (May to September), whereas peak abundance of females occurred in the wet season (October to April). We collected most ticks near the head and hind limbs of R. schneideri. The finding of two engorged A. rotundatumnymphs in the same resting places of two toads and the absence of this species in the dragged areas suggest a nidicolous behavior at the studied site.


Sapos da espécie Rhinella schneideri foram capturados e examinados para coleta das fases parasitárias, assim como arrasto de flanela para coleta das fases de vida livres de carrapatos em duas ilhas localizadas no rio São Francisco , próximas à represa Três Marias, região sudeste do Brasil. No total, 120 indivíduos foram examinados, dos quais 63 (52,5%) estavam parasitados por Amblyomma rotundatum totalizando 96 larvas, 163 ninfas e 134 fêmeas. A abundância do parasitismo variou de 1 a 43 carrapatos, com uma intensidade média de infestação de 6,2±5,5 carrapatos/hospedeiro. A infestação por A. rotundatumapresentou uma distribuição altamente agregada. O pico de abundância de larvas e ninfas ocorreu na estação seca (maio a setembro), enquanto o pico de abundância de fêmeas ocorreu na estação chuvosa (outubro a abril). A maioria dos carrapatos foi coletada na região da cabeça e membros posteriores. A presença de duas ninfas ingurgitadas de A. rotundatum nos mesmos lugares de descanso de dois sapos e a ausência desta espécie na coleta por arrasto de flanela sugere um comportamento nidicola no local estudado.


Subject(s)
Animals , Bufonidae/parasitology , Host-Parasite Interactions , Tick Infestations/veterinary , Parasite Load/veterinary , Parasitic Diseases/parasitology , Hemorrhage/veterinary , Weight Loss
13.
Article in Chinese | WPRIM | ID: wpr-320806

ABSTRACT

Toad venom is the Bufo bufo gargarizans or B. melanostictus after the ears of the gland secretion, used in the treatment of various cancers in recent years. Research shows that the main anti-tumor components in bufadienolide. Bufadienolide have free type structure and conjunct type structure. To identify and clarify the difference between bufogenin and bufotoxin contained in Bufonis Venenum, which was from B. bufo gargarizans, an UPLC-TQ-MS method has been established. UPLC-TQ-MS method was used to identify and quantify the major bufadienolides in Bufonis Venenum. UPLC-TQ-MS assay with positive ion mode was performed on a Waters ACQUITY UPLC BEH C, (2.1 mm x 100 mm, 1.7 µm) with the mobile phase consisting of 0. 1% aqueous formic and acidacetonitrile in gradient elution at a flow rate of 0.4 mL · min⁻¹ and the column temperature was set at 35 °C. By comparing their retention time and high resolution mass data of Bufonis Venenum extracts, 37 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were free-type bufadienolides (bufogenin), 11 of them were conjugated bufadienolides. There were significant differences in the main composition between fresh and processed Bufonis Venenum. The study found that the chemical composition of toad venom through great changes after processing, conjunct type content is much less, free type content as well change.


Subject(s)
Animals , Amphibian Venoms , Chemistry , Metabolism , Bufonidae , Classification , Metabolism , Chromatography, High Pressure Liquid , Methods , Molecular Structure , Tandem Mass Spectrometry , Methods
14.
J. venom. anim. toxins incl. trop. dis ; 21: 25, 31/03/2015. graf
Article in English | LILACS, VETINDEX | ID: biblio-954750

ABSTRACT

Background The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity ofRhinella schneideri poison (RsP) components on the complement system.Methods The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.Results The fractionation protocol was able to isolate the component S5 from theRsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b- 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.Conclusions This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.(AU)


Subject(s)
Animals , Poisons , Biological Products , Bufonidae , Chemotaxis
15.
J. venom. anim. toxins incl. trop. dis ; 21: 1-12, 31/03/2015. graf, tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1484627

ABSTRACT

Background The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity ofRhinella schneideri poison (RsP) components on the complement system.Methods The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.Results The fractionation protocol was able to isolate the component S5 from theRsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b- 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.Conclusions This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.


Subject(s)
Animals, Poisonous , Bufonidae , Amphibian Venoms
16.
Braz. j. biol ; 74(2): 438-443, 5/2014. tab, graf
Article in English | LILACS | ID: lil-719226

ABSTRACT

Life tables and survival curves of tadpoles from Rhinella icterica species were studied in the laboratory, under abiotic conditions controlled by a purification filter, a timer and a chiller. The survival curve for larval stage confirms a great mortality trend in the initial stages, which decreases when reaching the mature morphological condition (r = –0.94). Stages 37, 38, 39, 40 and 41 showed gradual values for their age structures, while stages 42, 43 and 44 presented high variations. Based on the results under laboratory conditions, it can be concluded that the maturity of R. icterica tadpoles development between 37 and 44 stages has a negative correlation and their predicted life expectancy is a logarithmic growth curve (y=–761.96Ln(x)+5298.5).


Tabelas de vida e curvas de sobrevivência em estágios larvais de girinos da espécie Rhinella icterica foram estudadas em laboratório, sob condições abióticas controladas com uso de filtro de depuração, timer e chiller. A curva de sobrevivência por estágio larval confirma a tendência de maior mortalidade para os estágios iniciais e menor mortalidade para o avanço da condição morfológica madura (r = –0,94). Os estágios 37, 38, 39, 40 e 41 apresentaram valores gradativos para sua estrutura etária, enquanto nos estágios 42, 43 e 44 ocorreram variações acentuadas. Com base nos resultados obtidos, nas condições de laboratório, pode-se concluir que a maturidade de desenvolvimento de girinos de R. icterica entre os estágios 37 e 44, apresenta correlação negativa e a predição de esperança de vida a uma curva logarítmica de crescimento (y=–761,96Ln(x)+5298,5).


Subject(s)
Animals , Bufonidae/physiology , Longevity/physiology , Bufonidae/classification , Bufonidae/growth & development , Laboratories , Models, Biological
17.
J. venom. anim. toxins incl. trop. dis ; 20: 1-14, 04/02/2014. ilus, graf
Article in English | LILACS, VETINDEX | ID: biblio-1484603

ABSTRACT

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action...


Subject(s)
Animals , Alkaloids/pharmacology , Bufotenin/pharmacology , Rabies/drug therapy , Amphibian Venoms/adverse effects , Amphibian Venoms/pharmacology , Bufonidae , Mass Spectrometry/methods
18.
Acta Pharmaceutica Sinica ; (12): 1446-1450, 2014.
Article in Chinese | WPRIM | ID: wpr-299113

ABSTRACT

To identify the active components in Bufo melanostictus Schneider and clarify the difference between fresh and dried Venenum Bufonis, a UPLC-Orbitrap MS method has been established. The separation was performed with gradient elution of acetonitrile and water (with 0.1% formic acid) as mobile phase. By comparing their retention time and high resolution mass data of Venenum Bufonis extracts, 39 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were bufadienolides. There were significant differences in the main composition between fresh and dried Venenum Bufonis. There are fewer bufadienolides in fresh toad venom.


Subject(s)
Animals , Amphibian Venoms , Chemistry , Bufanolides , Chemistry , Bufonidae , Chromatography, High Pressure Liquid , Tandem Mass Spectrometry
19.
Int. j. morphol ; 31(2): 681-686, jun. 2013. ilus
Article in English | LILACS | ID: lil-687124

ABSTRACT

Morphometry structure of Sicilian green toad from the Nature Reserve "Monte Pellegrino" (north-western Sicily) was studied. A total of 666 individuals (354 males and 312 females) were captured in 2003 during reproduction period. Meaningful differences have statistically emerged among the two sexes and the body weight is the best parameter (up to 92 percent correct classification), followed by the length of the body (up to 90 percent correct classification). The simultaneous use of all the examined characters only increases of 1percent the probability for correct discrimination of the sex. In comparison to the dimension of other green toad populations studied by other authors, the body size of this sicilian population results elevated, similar to that of Corsica and of Sardinia. It is possible that the high dimension of the Sicilian population is the result of low competition with other amphibians species and/or to the absence of a winter latency.


Se realizó un estudio morfométrico de la estructura del sapo verde siciliano de la Reserva Natural "Monte Pellegrino" (norte-oeste de Sicilia). Un total de 666 individuos (354 machos y 312 hembras) fueron capturados desde el año 2003 durante la época de reproducción. Se encontraron diferencias estadísticamente significativas entre los dos sexos, donde el peso corporal fue el mejor parámetro (hasta 92 por ciento de clasificación correcta), seguido por la longitud corporal (hasta 90 por ciento de clasificación correcta). El uso simultáneo de todos los caracteres examinados solo aumentó un 1 por ciento la probabilidad de una correcta discriminación del sexo. En comparación con la dimensión de otras poblaciones de sapos verdes estudiados por otros autores, el tamaño del cuerpo de esta población siciliana resulta elevado, similar a las poblaciones de Córcega y Cerdeña. Es posible que la gran dimensión de la población siciliana pueda deberse al bajo nivel de competencia con anfibios otras especies y/o a la ausencia de una latencia de invierno.


Subject(s)
Humans , Male , Female , Biometry , Bufonidae/anatomy & histology , Italy
20.
Acta Pharmaceutica Sinica ; (12): 250-254, 2013.
Article in Chinese | WPRIM | ID: wpr-235676

ABSTRACT

To study the bioactive polypeptides included in Bufo skin and its secretions the plasmid skin cDNA library of adult Japanese toad Bufo japonicus formosus was prepared. The pSD64TR has been used as the vector and the cloning sites are Xho I and EcoR I. To screen cDNAs encoding bioactive components, the plasmid cDNA library was transformed into E. coli DH5 competent cells, and positive colonies were screened by colony PCR (polymerase chain reaction). The suspension of a single colony in LB medium was used as the template, SP6 (the upstream primer of the plasmid cDNA library) and a primer with Xho I site and polyT were used as the primers. As the result, 465 positive colonies out of 1 344 were obtained and their plasmid were collected and sequenced. By homologous analysis, it was found that one of the cDNAs encoding a peptide with high homolog with transgelin-2, which was registered in GenBank (accession number: JX197456), and it was indicated as a partial cDNA sequence with a deletion at the 5' end. The transcript is 997 bp consisting of 31 bp 5', 618 bp 3' untranslated region (UTR) and an open reading frame (ORF) of 348 bp encoding a polypeptide of 115 amino acids. In the putative protein product, there is a calponin homology domain, two cysteine residues for a disulfide bond and three a-helix domains, and five potential phosphorylation sites. The homologous analysis indicates 90% similarity with Xenopus (Silurana) tropicalis and 89% with Xenopus laevis, and 71%-85% with other species.


Subject(s)
Animals , Amino Acid Sequence , Base Sequence , Bufonidae , Genetics , Metabolism , Cloning, Molecular , Gene Library , Microfilament Proteins , Chemistry , Genetics , Metabolism , Muscle Proteins , Chemistry , Genetics , Metabolism , Open Reading Frames , Phosphorylation , Phylogeny , Plasmids , Genetics , Sequence Homology, Amino Acid , Skin , Metabolism , Xenopus , Genetics
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