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1.
Zhongguo Zhong Yao Za Zhi ; 49(4): 932-941, 2024 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-38621900

RESUMO

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.


Assuntos
Bufanolídeos , Animais , Bufo bufo , Distribuição Tecidual , Bufonidae , Espectrometria de Massas por Ionização por Electrospray
2.
Toxicon ; 227: 107092, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36967019

RESUMO

The rise and spread of resistant Plasmodium falciparum strains are responsible for an increase in therapeutic failures in many of the regions endemic with malaria. The need for new therapeutic candidates is now more urgent than ever. Animal venoms have long been considered as interesting resources to exploit in terms of potential therapeutic candidates. Among these, the cutaneous secretions of toads constitute a rich and diverse source of bioactive molecules. We focused on two different species: Bufo bufo and Incilius alvarius. The dried secretions underwent a solvent-based extraction and were submitted to a systematic bio-guided fractionation approach using preparative thin-layer chromatography. Initial crude extracts were tested in vitro for their antiplasmodial activity. Based on these results, only crude extracts displaying IC50 < 100 µg/mL were considered for further fractionation. All extracts and fractions, including those that did not display antiplasmodial properties, were characterized by chromatographic (LC-UV/MS) and spectrometric techniques (HRMS). Antiplasmodial activity was evaluated in vitro using a chloroquine-sensitive strain (3D7) and a resistant one (W2). Toxicity was assessed on normal human cells for the samples displaying IC50 < 100 µg/mL. Crude extracts from Bufo bufo secretions exhibited no appreciable antiplasmodial activities. However, the methanol and dichloromethane extracts from Incilius alvarius secretions gave IC50 of (34 ± 4) µg/mL and (50 ± 1) µg/mL respectively when tested on W2 strain. No significant effect was observed on 3D7. This poison would warrant further investigation in terms of its antiplasmodial potential. Following preliminary characterization, it was revealed that the fractions of interest contained mainly bufotoxins, bufagins and alkaloids.


Assuntos
Antimaláricos , Malária , Venenos , Animais , Humanos , Antimaláricos/toxicidade , Antimaláricos/análise , Bufo bufo , Extratos Vegetais/uso terapêutico , Malária/tratamento farmacológico , Plasmodium falciparum , Bufonidae
3.
PLoS One ; 18(3): e0283136, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36989260

RESUMO

The common toad (Bufo bufo) has been the subject of many folk tales and superstitions in Western Europe, and as a result, it is characterised by numerous common names (zoonyms). However, the zoonyms of the toad and its associated traditions have remained unexplored in the Balkans, one of Europe's linguistic hotspots. In the present study, it was attempted to fill this knowledge gap by focusing on Greece, where more than 7.700 individuals were interviewed both in the field and through online platforms, in order to document toad zoonyms from all varieties and dialects of Greek, as well as local non-Greek languages such as Arvanitika, South Slavic dialects, and Vlach. It was found that the academically unattested zoonyms of the toad provide an unmatched and previously unexplored linguistic and ethnographic tool, as they reflect the linguistic, demographic, and historical processes that shaped modern Greece. This is particularly pertinent in the 21st century, when a majority of the country's dialects and languages are in danger of imminent extinction-and some have already gone silent. Overall, the present study shows the significance of recording zoonyms of indigenous and threatened languages as excellent linguistic and ethnographic tools that safeguard our planet's ethnolinguistic diversity and enhance our understanding on how pre-industrial communities interacted with their local fauna. Furthermore, in contrast to all other European countries, which only possess one or only a few zoonyms for the toad, the Greek world boasts an unmatched 37 zoonyms, which attest to its role as a linguistic hotspot.


Assuntos
Idioma , Linguística , Animais , Humanos , Grécia , Bufonidae , Bufo bufo , Demografia , Península Balcânica
4.
Zhongguo Zhong Yao Za Zhi ; 47(11): 2924-2931, 2022 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-35718513

RESUMO

On the premise of strictly controlling the harvesting conditions of Bufonis Venenum, we studied the relationship between the quality and resource distribution of Bufonis Venenum in China, aiming to provide the data for comprehensively understanding the geographical distribution and characteristics of Bufonis Venenum in China. In this study, 105 samples of Bufonis Venenum were collected from 42 counties and cities in 19 provinces in China, and the material basis and index components were determined by mass spectrometry and high performance liquid chromatography. The obtained data formed the quality database of Bufonis Venenum from different producing areas in China. The analysis of the material basis showed that Bufonis Venenum was mainly produced in two characteristic regions(north area and south area) divided by Qinling Mountains, northern edge of Huaiyang hills and the connecting area of Huang-Huai Plain, Huangshan Mountains, and Tianmu Mountains. Eight differential components were identified in the Bufonis Venenum samples from the south area and the north area. All the Bufonis Venenum samples from the north area showed the content of index components above the requirements of Chinese Pharmacopoeia(2020 edition), while those from the south area had the content of index components lower than the standards of Chinese Pharmacopoeia(2020 edition). The quality evaluation showed uneven distribution of Bufonis Venenum quality, which was high in the north and low in the south. The results provided a research basis for the breeding base selection of Bufo bufo gargarizans.


Assuntos
Bufanolídeos , Bufo bufo , Animais , Bufanolídeos/análise , Bufonidae , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas
5.
Int J Biol Macromol ; 175: 67-78, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33548318

RESUMO

Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, "cholesterol-bile acid-bufadienolidies pathway" was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3ß-hydroxysteroid dehydrogenase (3ßHSD) and steroid 5ß-reductase (SRD5ß) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5ß cDNA of B. bufo gargarizans (Bbg-SRD5ß) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5ß. Bbg-SRD5ß had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5ß in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5ß was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5ß can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5ß-Y132G was 1.8 times higher than that of wild type Bbg-SRD5ß. Although Bbg-SRD5ß was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5ß is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5ß.


Assuntos
Venenos de Anfíbios/química , Bufo bufo/metabolismo , Oxirredutases/isolamento & purificação , Sequência de Aminoácidos , Venenos de Anfíbios/metabolismo , Animais , Bufanolídeos/química , Bufanolídeos/metabolismo , Bufonidae/metabolismo , Clonagem Molecular/métodos , DNA Complementar/metabolismo , Fases de Leitura Aberta , Oxirredutases/genética , Oxirredutases/metabolismo , Esteroides/metabolismo
6.
Zhongguo Zhong Yao Za Zhi ; 44(9): 1850-1856, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31342712

RESUMO

As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.


Assuntos
Venenos de Anfíbios/química , Bufanolídeos/análise , Bufo bufo , Animais , Cromatografia Líquida de Alta Pressão , Medicina Tradicional Chinesa , Controle de Qualidade
7.
Artigo em Chinês | WPRIM | ID: wpr-773157

RESUMO

As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.


Assuntos
Animais , Venenos de Anfíbios , Química , Bufanolídeos , Bufo bufo , Cromatografia Líquida de Alta Pressão , Medicina Tradicional Chinesa , Controle de Qualidade
8.
Fitoterapia ; 131: 215-220, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30385402

RESUMO

Two new 19-norbufadienolides (1 and 2) and one new 14,15-epoxy bufadienolide (3) alongside 16 known bufadienolides (4-19) were isolated from Bufonis Venenum that originated from the skin and parotid venom glands of an Asiatic toad (Bufo bufo gargarizans Cantor). The structures of these bufadienolides were elucidated based on the interpretation of their HRESIMS and NMR data. Compound 1, which had a unique peroxide, was established through extensive single-crystal X-ray diffraction. The two 19-norbufadienolides exhibited more potent cardiotonic activity in the isolated toad heart model and lower cytotoxicity against U87, U251, and LN-18 cell lines than other bufadienolides, such as bufalin and bufotalin. The results suggested that 19-norbufadienolides might be more suitable for developing cardiotonic agents with low cytotoxicity.


Assuntos
Venenos de Anfíbios/química , Bufanolídeos/química , Bufo bufo , Cardiotônicos/farmacologia , Animais , Bufanolídeos/isolamento & purificação , Bufanolídeos/farmacologia , Cardiotônicos/isolamento & purificação , Linhagem Celular Tumoral , Coração/efeitos dos fármacos , Humanos , Técnicas In Vitro , Estrutura Molecular
9.
J Ethnopharmacol ; 187: 74-82, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27063985

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom and toad skin have been widely used for treating various cancers in China. Bufadienolides are regarded as the main anticancer components of toad venom, but the difference on composition and anticancer activities of bufadienolides between toad venom and toad skin remains unclear. METHODS: Fractions enriched with free and conjugated bufadienolides were prepared from toad venom and toad skin. Bufadienolides in each fraction were comprehensively profiled by using a versatile UHPLC-TOF-MS method. Relative contents of major bufadienolides were determined by using three bufogenins and one bufotoxin as marker compounds with validated UHPLC-TOF-MS method. Furthermore, cytotoxicity of the fractions was examined by MTT assay. RESULTS: Two fractions, i.e., bufogenin and bufotoxin fractions (TV-F and TV-C) were isolated from toad venom, and one bufotoxin fraction (TS-C) was isolated from toad skin. Totally 56 bufadienolides in these three fractions were identified, and 29 were quantified or semi-quantified. Bufotoxins were identified in both toad venom and toad skin, whereas bufogenins exist only in toad venom. Bufalin-3-conjugated bufotoxins are major components in toad venom, whereas cinobufotalin and cinobufagin-3-conjugated bufotoxins are main bufotoxins in toad skin. MTT assay revealed potent cytotoxicity of all the fractions in an order of TV-F>TV-C>TS-C. CONCLUSIONS: Our study represents the most comprehensive investigation on the chemical profiles of toad venom and toad skin from both qualitative and quantitative aspects. Eight bufotoxins were identified in toad skin responsible for the cytotoxicity for the first time. Our research provides valuable chemical evidence for the appropriate processing method, quality control and rational exploration of toad skin and toad venom for the development of anticancer medicines.


Assuntos
Venenos de Anfíbios/química , Antineoplásicos/isolamento & purificação , Bufanolídeos/isolamento & purificação , Pele/química , Animais , Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Bufo bufo , Células CACO-2 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Células Hep G2 , Humanos , Células MCF-7 , Espectrometria de Massas
10.
Nat Prod Commun ; 10(8): 1353-4, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26434114

RESUMO

A new C23 steroid, (3ß,5ß,14ß)-methyl (3-hydroxy-14,15-epoxy-20-oxo-21-norcholan-24-oate) (1), together with four known ones (2-5), were isolated from the venom of Bufo bufo gargarizans. Their structures were elucidated on the basis of extensive spectroscopic analysis. The cytotoxicity of these compounds was also evaluated against human hepatocarcinoma HepG2 cells. Compound 3 showed significant cytotoxicity with an IC50 value of 16.8 +/- 0.7 µM.


Assuntos
Venenos de Anfíbios/química , Antineoplásicos/química , Esteroides/química , Venenos de Anfíbios/farmacologia , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Bufo bufo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Estrutura Molecular , Esteroides/isolamento & purificação , Esteroides/farmacologia
11.
Gene ; 571(2): 172-7, 2015 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-26091834

RESUMO

Toad Bufo bufo gargarizans Cantor is still used in China as traditional Chinese medicine. However, present investigations on its skin secretions were mainly focused on the bufadienolides, the proteins/peptides contained in the secretions are largely unknown. A cDNA encoding a novel cathelicidin termed BG-CATH was identified by analysis of the toad skin transcriptome. The BG-CATH precursor was predicted to have 2 possible cleavage sites following dibasic cleavage signals at its C-terminal, which will generate two mature peptides, BG-CATH37 and BG-CATH(5-37). Phylogenetic analysis suggests that amphibian cathelicidins might evolve from common ancestors. The two predicted mature cathelicidins from B. bufo gargarizans were synthesized and both of them showed weak antimicrobial activities against human pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (MIC ≥ 200 µg/mL). However, BG-CATH37 and BG-CATH(5-37) had strong antimicrobial activities against aquatic bacteria of Vibrio splendidus, Streptococcus iniae and Aeromorus hydrophila, which were common microorganisms in the habitat of B. bufo gargarizans (MIC 3.125-40 µg/mL). BG-CATH37 and BG-CATH(5-37) showed no hemolytic activity even at high concentrations (400 µg/mL). CD spectra analysis suggested that structure rigidity of BG-CATH37 and BG-CATH(5-37) might play an important role to regulate their biological activities. Selective antimicrobial activity against habitat microorganisms might reflect the adaptation of amphibians to their living environments.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Bufo bufo/genética , Bufo bufo/microbiologia , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Sequência de Bases , Bufo bufo/metabolismo , Clonagem Molecular , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Catelicidinas
12.
Fitoterapia ; 105: 7-15, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26022446

RESUMO

Twelve new bufadienolides (1-12), along with fourteen known analogues (13-26) were isolated from the skins of Bufo bufo gargarizans Cantor. Their chemical structures were elucidated on the basis of NMR, HRESIMS and X-ray diffraction analysis. Compound 1 was an unusual bufadienolide with 3,19-epoxy moiety and A/B trans ring junction. Compounds 2-4 were rare bufadienolides possessing 10-H or 10-carboxyl units. All the isolated compounds were tested for their cytotoxic effects on HepG2, A549 and HeLa cells. Six new compounds (2, 3, 5, 6, 10 and 12) displayed significant anti-proliferative activities with IC50 values ranging from 0.049 to 1.856 µM. Arenobufagin (24) exhibited the most potent cytotoxic activity with IC50 value 0.011 µM. In addition, the present data provided more insight into the structure-activity relationships of bufadienolides.


Assuntos
Bufanolídeos/química , Bufo bufo , Pele/química , Animais , Bufanolídeos/isolamento & purificação , Células HeLa/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Humanos , Estrutura Molecular
13.
Zhongguo Zhong Yao Za Zhi ; 39(5): 841-5, 2014 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-25204176

RESUMO

Twelve compounds were isolated from the venom of Bufo bufo gargarizans. On the basis of their physical and chemical properties and spectral data, their structures were identified as resibufagenin (1), bufotalin (2), desacetylcinobufagin (3), 19-oxodesacetylcinobufotalin (4), cinobufotalin (5), 1beta-hydroxylbufalin (6), 12alpha-hydroxybufalin (7), bufotalinin (8), Hellebrigenin (9), telocinobufagin (10), hellebrigenol (11) and cinobufagin-3-hemisuberate methyl ester (12), respectively. Compounds 7 and 12 are new natural products.


Assuntos
Bufanolídeos/química , Peçonhas/química , Animais , Bufo bufo , Medicina Tradicional Chinesa , Estrutura Molecular
14.
J Pharm Biomed Anal ; 97: 54-64, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24814996

RESUMO

Traditional Chinese medicines (TCMs) have played a significant role in the process of discovering natural bioactive compounds, especially in anticancer therapeutics. However, the components of TCMs are complex mixtures with wide variation in polarity and content, which leads to inefficiency in the process of active compound discovery from TCMs. In this paper, the popular strategy of utilizing "pre-fractionated natural product libraries" has been improved by a new class separation approach to accelerate the process. As an example, the skin of Bufo bufo gargarizans Cantor, a well-known TCM, mainly contains two distinct bufadienolide classes: amino acid-conjugated bufadienolides (AACBs) and free form bufadienolides (AAUBs). We utilized hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE) to resolve the two types of bufadienolides, which co-eluted on C18 columns. By this strategy, twelve bufadienolides of the two types were purified via prep-HPLC from one active fraction, and eight of them were identified by (1)H NMR and (13)C NMR. These results indicated that the class separation method not only overcame the limited orthogonality in a 2D-RPLC×RPLC system but also accelerated the process of active compound discovery.


Assuntos
Bufanolídeos/isolamento & purificação , Extração em Fase Sólida/métodos , Animais , Bufanolídeos/química , Bufo bufo , Cromatografia de Fase Reversa , Espectroscopia de Ressonância Magnética , Medicina Tradicional Chinesa , Pele/química
15.
Artigo em Chinês | WPRIM | ID: wpr-330350

RESUMO

Twelve compounds were isolated from the venom of Bufo bufo gargarizans. On the basis of their physical and chemical properties and spectral data, their structures were identified as resibufagenin (1), bufotalin (2), desacetylcinobufagin (3), 19-oxodesacetylcinobufotalin (4), cinobufotalin (5), 1beta-hydroxylbufalin (6), 12alpha-hydroxybufalin (7), bufotalinin (8), Hellebrigenin (9), telocinobufagin (10), hellebrigenol (11) and cinobufagin-3-hemisuberate methyl ester (12), respectively. Compounds 7 and 12 are new natural products.


Assuntos
Animais , Bufanolídeos , Química , Bufo bufo , Medicina Tradicional Chinesa , Estrutura Molecular , Peçonhas , Química
16.
Zh Evol Biokhim Fiziol ; 49(2): 128-36, 2013.
Artigo em Russo | MEDLINE | ID: mdl-23789398

RESUMO

By the method of synchronous multielectrode cartography (24-unipolar leads), distribution of durations and time of end of repolarization were studied on ventricular epicardium of pikes (Esox lucius) and frogs (Rana esculenta) and in ventricular intramural layers of toads (Bufo bufo) at the ectopic heart excitation. The time of arrival of the excitation wave and of the end of repolarization in each lead was determined from the minimum of time derivative of potential at the period of the QRS complex and by minimum of the T wave, respectively. It has been established that at the ventricle electrostimulation, alongside with deceleration and a change of sequence of the myocardium activation, there occurs redistribution of local repolarization durations: in areas of late activation the repolarization being longer than in zones of early activation (p < 0.05). At stimulation, the apicobasal gradient of repolarization is predominantly changed due to electrophysiological processes in the apical areas. In all studied species. at ectopical excitation of the heart ventricle the sequence of its repolarization repeats the depolarization sequence due to delay of activation (in fish) and redistribution of repolarization durations (in amphibians).


Assuntos
Potenciais de Ação/fisiologia , Sistema de Condução Cardíaco/fisiologia , Ventrículos do Coração , Função Ventricular/fisiologia , Animais , Bufo bufo , Estimulação Elétrica , Eletrocardiografia , Eletrodos Implantados , Esocidae , Rana esculenta , Especificidade da Espécie , Fatores de Tempo
17.
Int J Oncol ; 41(4): 1431-42, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22858649

RESUMO

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho­mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.


Assuntos
Autofagia/efeitos dos fármacos , Bufanolídeos/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Animais , Autofagia/genética , Bufanolídeos/química , Bufo bufo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-akt/farmacologia , Serina-Treonina Quinases TOR/farmacologia , Peçonhas/química
18.
Biol Trace Elem Res ; 150(1-3): 178-94, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22700180

RESUMO

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85 µg/L) for up to 40 days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1 µg/g in kidneys and gills (0.64-0.95 and 0.52-0.76; n = 4), whereas the levels stayed below 0.5 in liver (0.14-0.29; n = 4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn-thioneins in liver and Zn,Cu-thioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd-thionein and in pellet extract of kidneys as Zn,Cu,Cd-thioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56-5.0 µg/g >0.03-0.72 µg/g; n = 11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r > 0.80; α = 0.05, n = 5), and between Cd and Se in kidney (r = 0.76; n = 5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd-thioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.


Assuntos
Bufo bufo/metabolismo , Cádmio/toxicidade , Exposição Ambiental , Metalotioneína/metabolismo , Necturus maculosus/metabolismo , Oligoelementos/metabolismo , Poluentes Químicos da Água/toxicidade , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Bufo bufo/crescimento & desenvolvimento , Cádmio/análise , Cádmio/farmacocinética , Cavernas , Cobre/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Exposição Ambiental/efeitos adversos , Monitoramento Ambiental/métodos , Feminino , Água Doce/química , Masculino , Metalotioneína/química , Necturus maculosus/crescimento & desenvolvimento , Especificidade de Órgãos , Estações do Ano , Selênio/metabolismo , Eslovênia , Distribuição Tecidual , Oligoelementos/análise , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/farmacocinética , Zinco/metabolismo
19.
Zhongguo Zhong Yao Za Zhi ; 36(16): 2207-10, 2011 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-22097331

RESUMO

Eight compounds were isolated from Bufonis periostracum by repeated column chromatography on silica gel, ODS and Sephadex LH-20 and their structures were characterized as palmitatic acid cholesteryl ester (1), cholesterol (2), 5alpha, 8alpha-epidioxycholesta-6-en-3beta-ol (3), cholest-5-en-3beta, 7beta-diol (4), cholest-7-en-3beta, 5alpha, 6beta-triol (5), 3-octaddecyloxy-1, 2-propanediol (6), isisamide (7) and bufothionine (8) on the base of spectral analysis. Compounds 1-8 were isolated from Bufonis periostracum for the first time and compounds 3, 5, 6, 7 were obtained from Bufo bufo gargarizans and Bufo genus for the first time. The bioassays showed all tested samples displayed no antitumor activity against the cell lines such as A549, BeL 7402, HGC-27 and HL-60, except the control compound bufalin.


Assuntos
Antineoplásicos/farmacologia , Bufonidae/metabolismo , Animais , Bufo bufo/metabolismo , Humanos
20.
Int Immunopharmacol ; 11(3): 342-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21185919

RESUMO

The skin of the toad Bufo bufo gargarizans Cantor is known to be rich in bufadienolides, peptides and alkaloids. It has been found to be a source of some extracts and biologically active compounds with antitumor activity. Cinobufacini (Huachansu), a Chinese medicine prepared from the dried toad skin, has been widely used in clinical therapy for various cancers in China. Bufadienolides, such as bufalin, cinobufagin, resibufogenin, and telocinobufagin, are the major active compounds derived from the toad skin. They are the maker biologically active compounds of cinobufagin while the antitumor activity of cinobufagin may be due to this kind of components. Experimental research has suggested that cinobufacini and its active compounds (e.g. bufalin and cinobufagin) exhibit significant antitumor activity, including inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance, and regulation of the immune response. Clinical data have indicated that cinobufacini may have effective anticancer activity with low toxicity and few side effects. Data to date suggest it may also enhance quality of life for patients with cancer. Thus, this review briefly summarizes recent studies on the anticancer activity of cinobufacini and some of its active compounds from the skin of the toad Bufo bufo gargarizans Cantor. This might provide additional evidence for further study of the extracts and active compounds from the toad skin in cancer treatment.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Bufo bufo/metabolismo , Pele/química , Animais , Linhagem Celular Tumoral , Humanos , Medicina Tradicional Chinesa , Estrutura Molecular
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