Design, synthesis and antitumor activity of 3-arenobufagin esters / 药学实践杂志

Objective To search for novel potent 3-ester derivatives of arenobufagin and test their antitumor activities in vitro. Methods Target compounds were synthesized by esterification of arenobufagin with acids. CellTiter method was used to assay the in vitro antitumor activities. Results 3-Ester derivatives exhibited excellent antitumor activities against all the cancer cells. Conclusion Among the 3-ester derivatives, compound 2a had the best activities with the IC50 of 4.0−91.7 nmol/L and appeared to be a valuable candidate for further study.

Establishment of HPLC fingerprint of Bufonis Venenum pulp and determination of eight components / 中草药

Objective: To establish the HPLC fingerprint of Bufonis Venenum pulp and the determination methods of eight components in Bufonis Venenum pulp, and compare the differences of components of Bufonis Venenum pulp in different origins for quality evaluation. Methods: The mobile phase was acetonitrile and water with a gradient elution program. The detection wavelength was set at 296 nm. The flow rate was 1.2 mL/min. The column temperature was set at 30 ℃, and 10 μL of the test solution was injected. HPLC fingerprint of Bufonis Venenum pulp was established and eight components were determined. The results were analyzed by cluster analysis and principal component analysis. Results: There were 12 common peaks in the HPLC fingerprints of 18 batches of samples, and the similarity of each sample was different. The linear relationship of eight components was good (r2 > 0.999 5), RSD of precision and repeatability was less than 0.5%, and the stability was also good within 30 h (RSD < 0.7%). The average recoveries of gamabufotalin, arenobufagin, telocinobufagin, bufotaline, cinobufotalin, bufalin, cinobufagin and resibufogenin were 103.7%, 103.0%, 102.9%, 103.0%, 103.9%, 100.3%, 103.4%, and 103.2% respectively, and RSD was all less than 1.2%. The results of the content determination, cluster analysis and principal component analysis of eight components showed that Bufonis Venenum pulp in different habitats were different from each other. Conclusion: The method is simple and reliable, which can provide some basis and reference for quality control of Bufonis Venenum pulp.

Study on chemical constituents and pharmacodynamic changes of Bufonis Venenum with different drying methods / 中草药

Objective To study the changes of chemical constituents and pharmacodynamics with different drying methods (sun- drying, 50 ℃ vacuum-drying, 50 ℃ and 80 ℃ heat-drying, freeze drying method) in Bufonis Venenum. Methods HPLC method and TLC method was established for studying the changes of chemical constituents from B. Venenum before and after being dried, and determine the inhibitory effect of dried samples on five different tumor cell lines proliferation by MTT assay. Results The B. Venenum processed by five different methods were different in character, while no significant differences in the types and content of chemical constituents; The total content of resibufogenin and cinobufagin was more than 6%, which was consistent with the 2015 edition of Chinese Pharmacopeia; 50 ℃ and 80 ℃ heat-drying of B. Venenum showed more effective inhibitiory effect than other dry methods. Conclusion The appearance of B. Venenum met the criterion of pharmacoperia by sun-drying and 50 ℃ heat-drying method. Although the color of B. Venenum under the vacuum-drying and freeze drying method did not meet the requirements of Chinese Pharmacopeia, the main effective components have a few changes.

Arenobufagin is a novel isoform-specific probe for sensing human sulfotransferase 2A1

Human cytosolic sulfotransferase 2A1 (SULT2A1) is an important phase II metabolic enzyme. The detection of SULT2A1 is helpful for the functional characterization of SULT2A1 and diagnosis of its related diseases. However, due to the overlapping substrate specificity among members of the sulfotransferase family, it is difficult to develop a probe substrate for selective detection of SULT2A1. In the present study, through characterization of the sulfation of series of bufadienolides, arenobufagin (AB) was proved as a potential probe substrate for SULT2A1 with high sensitivity and specificity. Subsequently, the sulfation of AB was characterized by experimental and molecular docking studies. The sulfate-conjugated metabolite was identified as AB-3-sulfate. The sulfation of AB displayed a high selectivity for SULT2A1 which was confirmed by reaction phenotyping assays. The sulfation of AB by human liver cytosols and recombinant SULT2A1 both obeyed Michaelis-Menten kinetics, with similar kinetic parameters. Molecular docking was performed to understand the interaction between AB and SULT2A1, in which the lack of interaction with Met-137 and Tyr-238 of SULT2A1 made it possible to eliminate substrate inhibition of AB sulfation. Finally, the probe was successfully used to determine the activity of SULT2A1 and its isoenzymes in tissue preparations of human and laboratory animals.

Simultaneous determination of nine bufadienolides in Liushen Pill by HPLC-MS / 中草药

Objective: To establish an HPLC-MS method for determining nine bufadienolides (gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin, and resibufogenin) in Bufonis Venenum and Liushen Pill. Methods: HPLC analysis was performed on ODS-2 column (250 mm×4.6 mm, 5 μm) with the mobile phase made up of acetonitrie and 0.15% phosphoric in water. Flow rate was set at 1 mL/min and column temperature was 40℃. The detection wavelength was 296 nm. Nine bufadienolides were determined by HPLC-ESI-TOF-MS with the mobile phase made up of acetonitrie and 0.2% formic acid in water. Eletrospray ionization source was applied and operated in the positive ion mode. Results: Nine bufadienolides were separated at baseline with good linearity (r≥0.9996). The recovery rates were 92%-105% (RSD<5%). Conclusion: The method established is rapaid, simple, and accurate, which is helpful to control the quality of Liushen Pill.