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.
Animals ; Antineoplastic Agents ; pharmacology ; Bufo bufo ; metabolism ; Bufonidae ; metabolism ; Humans

Cinobufacino injection is purified from water extraction of the skin of Bufo bufo gargarizans, which has been widely used for various cancers in clinic with significant anti-tumor effects. Bufadienolides were regarded as the main active constituents of cinobufacino injection in previous reports. In present study, 6 bufadienolides were isolated and purified from Cinobufacino injection. Their structures were identified as 3-epi-ψ-bufarenogin (1), ψ-bufarenogin (2), 3-epi-arenobufagin (3), arenobufagin (4), 3-epi-gamabufotalin (5), and 3-oxo-arenobufagin (6), separately. Among them, 1 and 3 were new compounds, 5 and 6 were new natural products. Compounds 1, 2 and compounds 3, 4 were two pairs configuration isomers at C-3, separately.
Animals ; Bufanolides ; chemistry ; isolation & purification ; Bufo bufo ; Injections ; Skin ; chemistry

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.
Animals ; Bufanolides ; chemistry ; Bufo bufo ; Medicine, Chinese Traditional ; Molecular Structure ; Venoms ; chemistry

The classical cable function has been used to represent the response of peripheral nerves stimulated by external parallel electric field. Experiments show that peripheral nerves can be activated by perpendicular electric field induced by magnetic pulses, indicating that the activation mechanism needs to be thoroughly investigated. Several excitation properties of peripheral nerves in transverse-field were explored in the present paper. With a human's median nerve in vivo, stimulation threshold, excitation position and the relation between excitation threshold and fiber's radius were studied. The relation between stimulation threshold and stimulation duration was researched with sciatic nerves from toad in vitro. The experimental results verify that the modified cable function is much efficient than the classical cable function. The research will improve the nerve magnetic stimulating technique and be beneficial to further application.
Animals ; Bufo bufo ; Electric Stimulation ; Electromagnetic Fields ; Electrophysiology ; Humans ; Magnetics ; Median Nerve ; physiology ; Peripheral Nerves ; physiology ; Sciatic Nerve ; physiology

Action Potentials ; Animals ; Bufo bufo ; Caffeine ; pharmacology ; Female ; Heart ; drug effects ; physiology ; In Vitro Techniques ; Male ; Sciatic Nerve ; physiology

The dielectric spectroscopy of the skeletal muscle cells is approached by extracellular perfusion, using curve fitting and numerical calculation with nonlinear Cole-Cole equation, and the dielectric parameter of skeletal muscle cells is compared after perfusion, The results show: (1) the permittivy at low frequency (epsilon1) and the conductivity at hight frequency (kappa(h)) reduces in keeping with extracellular perfusate; (2) the effect of extracellular perfusion on the characteristic frequency (f(c1),f(c2)) is small; (3) the extracellular perfusion mostly influences the peaks of deltaepsilon" and tgdelta for the skeletal muscle cells.
Animals ; Bufo bufo ; Electric Conductivity ; Electricity ; Electrophysiology ; Extracellular Fluid ; physiology ; Muscle, Skeletal ; cytology ; physiology ; Spectrum Analysis ; methods

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.
Amphibian Venoms ; chemistry ; Animals ; Bufanolides ; analysis ; Bufo bufo ; Chromatography, High Pressure Liquid ; Medicine, Chinese Traditional ; Quality Control

Cinobufacino injection is a significant anti-tumor medicine for the treatment of various tumors in clinic, which was made from water extraction of the skin of Bufo bufo gargarizans. In present paper, HPLC-DAD-FT-ICR-MS method was used to identify the major bufadienolides in cinobufacino for the first time. Solid-phase extraction with dichloromethane and silica was used to enrich the total bufadienolides in cinobufacino. Based on the UV and high resolution MS/MS data, 33 bufadienolides were analyzed and characterized. Among them, eight compounds were identified by comparing with standard references unambiguously. This study elucidated the major bufadienolides in cinobufacino, which provided material foundation of cinobufacino and will be benefit for the further pharmacological research.
Amphibian Venoms ; chemistry ; Animals ; Bufanolides ; analysis ; chemistry ; Bufo bufo ; Chromatography, High Pressure Liquid ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

OBJECTIVETo establish a HPLC method for bufothionine in the skin of Bufo bufo gargarizans and Huachansu injection.

METHODThe samples were separated using a Lichrosob C18 column with CH3CN-H2O (10:90) as mobile phase. Flow rate was at 1.0 mL x min (-1) and the detection wavelength was at 225 nm.

RESULTThe calibration curve of bufothionine was linear over the range of 0.0772-0.4632 microg and the average recovery was 99. 2%. The contents of bufothionine were fluctuated from 36.4-641.8 microg x g(-1) in the skin of Bufo bufo gargarizans and 22.47-33.16 microg x mL(-1) in Huachansu injection, respectively.

CONCLUSIONThe contents of bufothionine were greatly different between cultured and wild species. The method was suitable for the quality control of the skin of Bufo bufo gargarizans and its preparation.

Animals ; Bufo bufo ; China ; Chromatography, High Pressure Liquid ; instrumentation ; methods ; Injections ; Materia Medica ; administration & dosage ; chemistry ; isolation & purification ; Phenothiazines ; analysis ; Quality Control ; Reproducibility of Results ; Skin ; chemistry

The classical cable function has been used to represent the response of peripheral nerves stimulated by external parallel electric field. It can not describe the excitation of peripheral nerves stimulated by perpendicular electric field. In this paper, responses of the nodes of Ranvier to transverse-field are deeply investigated by mathematic simulation and in-vitro experiments. The paper demonstrates that, under perpendicular electric field stimulation, the responses evoke a two-stage process including an initial polarization and the actual change of the transmembrane potential. It is the net inward current along a radial direction of the node of Ranvier that causes the peripheral nerve excitation. Based on the two-stage process, a novel model is introduced to describe peripheral nerves stimulated by transverse-field, and the classical cable function is modified. The new model and the improved cable function are verified by several in-vitro experiments. They can be used to represent peripheral nerves responses by arbitrary electric field stimulation.
Animals ; Bufo bufo ; Electric Conductivity ; Electric Stimulation ; Electromagnetic Fields ; Electrophysiology ; Membrane Potentials ; Models, Biological ; Nerve Fibers ; physiology ; Peripheral Nerves ; physiology ; Ranvier's Nodes ; physiology ; Sciatic Nerve ; physiology