1. The metabolic fate of cinobufagin, a major component of the cardiotonic Senso, has been studied after i.v. administration to rats. Two metabolites were detected in rat serum and shown by n.m.r. and mass spectroscopy to be desacetylcinobufagin (I) and 3-epidesacetylcinobufagin (II). 2. Inhibitory activities of I and II on guinea-pig heart Na+/K+-ATPase were less than that of cinobufagin. 3. Serum or plasma levels of cinobufagin, I and II in rats, cats and dogs were determined by h.p.l.c. after i.v. and/or oral administration of cinobufagin. After i.v. administration, cinobufagin, I and II appeared in rat serum, but in dog and cat plasma only unchanged cinobufagin was present. After oral administration, only II was detected in rat serum, but neither cinobufagin nor either of its metabolites (I and II) were found in cat and dog serum.

Cinobufagin, one of the active principles extracted from toad venom, was studied on the isolated vas deferens of the guinea pig. The preparation was suspended in a bath containing 10 ml Krebs solution. Isometric tension of vas deferens was recorded on polygraph with a force displacement transducer. Cinobufagin caused a long lasting contraction of the vas deferens of the guinea pig at concentrations of 15-50 mumol/L. The contraction was inhibited following reserpinization and cold storage treatment. The cinobufagin induced contraction was partly blocked by pretreatment with phentolamine and verapamil. These results suggest that the cinobufagin induced contraction of vas deferens might be related to an action which promotes release of NA from the adrenergic nerve terminals.

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTS), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTS, we extended the original set of spin-labelled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance (CW-EPR) spectra of spin-labelled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker-length, where the longest linker placed the spin label at the entrance to the binding site. Temperature-related changes in spectral line-shapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D₂O-electron spin echo envelope modulation (D₂O-ESEEM) in pulse-EPR measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.

As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.

Keywords: Bufonis Venenum; bufalin(BF); cinobufagin(CB); quantitative analysis of multi-components by single-marker(QAMS); resibufogenin(RB).

Cinobufagin (CBG) has been shown to have antinociceptive properties. Nevertheless, the antinociceptive effect and mechanism of CBG are still unclear. The present study was designed to investigate the antinociceptive effect of CBG in the thermal and chemical pain models and further to explore the molecular target and potential signal pathway. As shown in the hot-plate test, formalin test, and acetic acid writhing test in mice, administration of CBG produced significant antinociceptive activity in a dose-dependent manner, and the antinociceptive effect was blocked by intraperitoneal pretreatment of methyllycaconitine citrate (an α7 nicotinic receptor antagonist) and intrathecal delivery of an α7 nicotinic receptor antagonist siRNA (α7-siRNA). Immunofluorescence demonstrated that the α7 nicotinic receptor and IκB/NF-κB were coexpressed in primary cultured lumbar DRG neurons. In the chemical pain models and primary cultured DRG neurons, Western blot analysis showed that the formation of p-IκB and p-NF-κB was regulated by CBG, and the effect of CBG was inhibited by α7-siRNA, and ELISA analysis indicated that CBG also regulated the expression of inflammatory cytokines through the α7 nicotinic receptor in DRG. These results suggest that CBG may activate an α7 nicotinic receptor, thereby triggering the inhibition of the DRG NF-κB signaling pathway, resulting in an antinociceptive effect in mice.

Aim of the study was to observe and analyze the clinical effect of intravenous infusion of zoledronic acid combined with oral medication of cinobufagin in treating metastatic bone tumors. The 120 patients who have been treated in the hospital for metastatic bone tumor from June 2014 to June 2017 were selected as research objects. They were randomly divided into research group and control group, each containing 60. The research group was treated with intravenous infusion of zoledronic acid combined with oral medication of cinobufagin. In the control group, only zoledronic acid intravenous infusion was administered. The overall treatment effect of the two groups was observed. The pain of two groups was evaluated using numerical rating scale (NRS). The results showed that compared with the control group, the research group achieved better clinical effect and had a higher quality of life, and the intergroup difference was of statistical significance, P<0.05. There was no difference in rate of adverse reactions between the two groups, P>0.05, without statistical significance. The combined therapy of intravenous infusion of zoledronic acid and oral medication of cinobufagin can obtain better therapeutic effect in treating metastatic bone tumors.

The title compound, C24H33NO4·H2O, the reaction product of de-acetyl-cinobufagin with ammonium acetate, consists of three cyclo-hexane rings (A, B and C), one five-membered ring (D), one six-membered lactone ring (E) and an epoxide ring (F). The stereochemistry of the ring junctures are A/B cis, B/C trans, C/D cis and D/F cis. Cyclo-hexane rings A, B and C have normal chair conformations. The five-membered ring D adopts an envelope conformation (with the C atom bearing the lactone ring as the flap) and the lactone ring E is planar. In the crystal, hy-droxy and water O-H⋯O and amine N-H⋯O hydrogen bonds involving carbonyl, hy-droxy and water O-atom acceptors link the mol-ecules into a three-dimensional network.

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.

A reversed-phase high performance liquid chromatographic method for the quantitative determination of three bufogenins in toad venom was established. An Alltech Alltima C18 column (250 mm x 4.6 mm, 5 microm) was used. The mobile phase was acetonitrile-0.1 mol/L acetate buffer (pH 3.2) (50:50, v/v), and the flow rate was 0.8 mL/min. The detection wavelength was set at 299 nm. The method has good linearity in the ranges of 0. 25 -0.875 microg for cinobufagin (r = 0.999 0), 0.25 -0.875 microg for resibufogenin (r = 0.999 1), and 0.15 - 0.525 microg for bufalin (r = 0. 999 0). The average recoveries were 100.3%, 100.0% and 98.0% for cinobufagin, resibufogenin, and bufalin, respectively. The results indicate that the method is simple, accurate, reproducible, and can be used for the quality control of bufogenins in toad venom.

OBJECTIVE

To explore the regulation of transdermal absorption of Secretio Bufonis (SB) and the effect of low frequency complex impulse current (LFCIC) on it.

METHODS

By modifying three-chamber flow diffusion pool to develop a prototype LFCIC device for transdermal delivery, using high performance liquid chromatograph (HPLC) to determine the quantitative transdermal absorption of the amount of ingredients of SB, including bufalin, cinobufagin and resibufogenin, etc. and the transdermal absorption velocity was calculated.

RESULTS

The chief ingredients of SB could be absorbed through skin, but the volume was low. Additional application of LFCIC could enhance the cumulative infiltration volume and velocity of transdermal diffusion. Difference appeared 2 hrs after and significant difference appeared 4 hrs after the application, and 13.8 Hz showed the optimal effect of transdermal delivery.

CONCLUSIONS

Chief ingredients of SB could be absorbed through transdermal medication, and LFCIC can evidently enhance the amount and velocity of transdermal absorption of SB.

Hydroxylation is an important route to synthesize more hydrophilic compounds of pharmaceutical significance. Microbial hydroxylation offers advantages over chemical means for its high specificity. In this study, a fungal strain Alternaria alternata AS 3.4578 was found to be able to catalyze the specific 12beta-hydroxylation of a variety of cytotoxic bufadienolides. Cinobufagin and resibufogenin could be completely metabolized by A. alternata to generate their 12beta-hydroxylated products in high yields (>90%) within 8 h of incubation. A. alternata could also convert 3-epi-desacetylcinobufagin into 3-epi-12beta-hydroxyl desacetylcinobufagin as the major product (70% yield). C-3 dehydrogenated products were detected in these reactions in fair yields, while their accumulation was relatively slow. The 12beta-hydroxylation of bufadienolides could be significantly inhibited by the substitution of 1beta-, 5-, or 16alpha-hydroxyl groups, and the 14beta,15beta-epoxy ring appeared to be a necessary structural requirement for the specificity. For the biotransformation of bufalin, a 14beta-OH bufadienolide, this reaction was not specific, and accompanied by 7beta-hydroxylation as a parallel and competing metabolic route. The biotransformation products were identified by comparison with authentic samples or tentatively characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectrometry analyses.

A new bufadienolide, 12-oxo-desacetylcinobufagin, was obtained from the biotransformation of cinobufagin by Alternaria alternata in fairly low yield (0.34%). Its structure was established on the basis of (1)H-NMR, (13)C-NMR, 2D-NMR, and MS spectral data.

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.

The aim of this paper is to report the development of a method for the determination of the binding rates of cinobufagin (CBG) and resibufogenin (RBG) with different plasma proteins. Equilibrium dialysis method was used to imitate the binding process between cinobufagin, resibufogenin and plasma proteins. HPLC was employed to determine the concentration of cinobufagin and resibufogenin inside and outside of the dialysis membrane, and then on the basis of which protein binding rates were calculated. The calibration curves of both cinobufagin and resibufogenin with human, rats' and Beagle dogs' plasma were linear in the range of 0.50-40.00 microg x mL(-1), while the buffer solution was 0.25-4.00 microg x mL(-1). The extract recovery of cinobufagin was in the range of (68.73 +/- 2.16)%--(79.27 +/- 1.62)%, while that of resibufogenin was (71.59 +/- 4.31)%--(83.47 +/- 2.63)%. The average plasma protein binding rates with cinobufagin were 85.63%, 80.21% and 70.10% in human, rats' and Beagle dogs' plasma, whereas those binding rates with resibufogenin were 84.51%, 75.11% and 70.60%, respectively. The results suggested that the protein binding rates of cinobufagin and resibufogenin were of middle strength, which in rats', Beagle dogs' and human plasma were decreased in the following order: human plasma>> rats' plasma>> Beagle' dogs plasma.

The dog heart-lung preparations were prepared. The "equilibrium point", which could be defined as the point at which the cardiac output (CO)-curve and the venous return (VR)-curve crossed, when the CO and VR were plotted against the right atrial pressure, was recorded directly by utilizing an X-Y recorder. The CO-curve was obtained, as a locus of the equilibrium point, by raising and lowering the level of blood in the venous reservoir (competence test). The meaning of the procedure was shown to increase or decrease the mean systemic pressure, and to cause the corresponding parallel shift in the VR-curve. The VR-curve was obtained by changing myocardial contractility. When heart failure was induced by pentobarbital or by chloroform, the equilibrium point shifted downwards to the right, depicting the VR-curve. During development of the failure, the slopes of CO-curves decreased gradually. Effects of cinobufagin and norepinephrine were also analyzed. Utilization of the X-Y recorder enabled us to settle the uniform experimental conditions more easily, and to follow the effects of drugs continuously on a diagram equating the CO- and VR-curves (Gyton's scheme).

Mucor spinosus has been employed for the biotransformation of cinobufagin (1) to afford three metabolites. On the basis of their physico-chemical data, the structures of the transformation products have been characterized as 1beta-hydroxy-cinobufagin (2), 12beta-hydroxy-cinobufagin (3) and 1beta,12beta-dihydroxy-cinobufagin (4), of which metabolites 2 and 4 are new compounds. In vitro cytotoxic activities of the biotransformation products and the substrate-cinobufagin have been assayed against four tumor cell lines of Bel 7420, BGC 823, HeLa and HL 60; they all showed cytotoxic activities.

OBJECTIVE

The objective of this study was to design and prepare a novel solid dispersion using spray congealing to achieve fast and synchronous dissolution of bufalin, cinobufagin, and resibufogenin, three therapeutically complementary drugs.

METHODS

The solid dispersion was characterized with dissolution, X-ray diffractometry, and fourier transform infrared spectroscopy after preparation and storage for four weeks at different temperatures and relative humidity.

RESULTS

It was found that all drugs were molecularly dispersed within matrix and had a significant enhancement (∼4-fold higher) of dissolution rate. Furthermore, synchronized release of different drugs from a single carrier was achieved due to the highly molecular dispersibility and the excellent solubilization properties of F127. In addition, the solid dispersion was physically stable for at least four weeks at controlled conditions. But for samples under stress conditions, the results showed that drug-rich phase was formed and storage temperature was the dominant factor in determining stability of the solid dispersion (SD).

CONCLUSIONS

These findings highlight the fitness of spray congealing to co-deliver multiple drugs, which open new perspectives for the development of more advanced combination of multiple therapeutic agents, presumably improving the bioavailability and therapeutic efficacy.

To observe the effect of cinobufagin on transient outward potassium current (I_A) in rat dorsal root ganglion cells of cancer-induced bone pain (CIBP) and explore the possible analgesic mechanism of cinobufagin.

Whole cell patch clamp technique was used to examine the effect of cionbufagin on I_A in acutely isolated dorsal root ganglion (DRG) cells from normal SD rats and rats with bone cancer pain.

The DRG cells from rats with CIBP showed obviously decreased I_A current density, an activation curve shift to the right, and an inactivation curve shift to the left. Cinobufagin treatment significantly increased the I_A current density and reversed the changes in the activation and inactivation curves in the DRG cells.

I_A current is decreased in DRG neurons from rats with CIBP. Cinobufagin can regulate the activation and inactivation of I_A current in the DRG cells, which may be related to its analgesic mechanism.