Systematic comparison of two kinds of Bufonis Venenum derived from different Bufo gargarizans subspecies based on metabolomics and antitumor activity / 中国中药杂志

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.

Research progress in quality control of Bufonis Venenum in preparations / 中国中药杂志

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.

Processing evolution,chemical components and pharmacological effects of Bufonis Venenum / 中国中药杂志

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.

Design of dual targeting immunomicelles loaded with bufalin and study of their anti-tumor effect on liver cancer / 中西医结合学报

OBJECTIVE@#Bufalin is an effective drug for the treatment of liver cancer. But its high toxicity, poor water-solubility, fast metabolism and short elimination half-life limit its use in tumor treatment. How to make the drug accumulate in the tumor and reduce side effects while maintaining its efficacy are urgent problems to be solved. The goal of this study is to solve these problems.@*METHODS@#A copolymer with tunable poly-N-isopropylacrylamide and polylactic acid was designed and synthesized. The corresponding dual targeting immunomicelles (DTIs) loaded with bufalin (DTIs-BF) were synthesized by copolymer self-assembly in an aqueous solution. The size and structure of DTIs-BF were determined by ZetaSizer Nano-ZS and transmission electron microscopy. Then, its temperature sensitivity, serum stability, critical micelle concentration (CMC), entrapment efficiency (EE), drug release and non-cytotoxicity of blank block copolymer micelles (BCMs) were evaluated. Next, the effects of DTIs-BF on cellular uptake, cytotoxicity, and tumor cell inhibition were evaluated. Finally, the accumulation of DTIs-fluorescein isothiocyanate (FITC) and the in vivo anti-tumor effect were observed using an interactive video information system.@*RESULTS@#DTIs-BF had a small size, spherical shape, good temperature sensitivity, high serum stability, low CMC, high EE, and slow drug release. The blank BCMs had very low cytotoxicity. Compared with free bufalin, the in vitro cellular internalization and cytotoxicity of DTIs-BF against SMMC-7721 cells were significantly enhanced, and the effects were obviously better at 40 °C than 37 °C. In addition, the therapeutic effect on SMMC-7721 cells was further enhanced by the programmed cell death specifically caused by bufalin. When DTIs-FITC were injected intravenously in BALB/c nude mice bearing liver cancer, the accumulation of FITC was significantly increased in tumors.@*CONCLUSION@#DTIs-BF is a potentially effective nano-formulation and has broad prospects in the clinical treatment of liver cancer.

A target lipidomics approach to investigate the acute inflammatory irritation induced by indolealkylamines from Chansu water fraction in rats / 中国天然药物

Chansu has demonstrated adverse reactions in clinical settings, which is associated with its toxicity and limits its clinical applications. But there are methodological limitations for drug safety evaluation. In the current study, ultra-high performance liquid chromatography, lipidomic profiling, and molecular docking were used to systemically assess Chansu-induced acute inflammatory irritation and further identify the underlying drug targets. Compared with the EtOAc extract, Chansu water fraction containing indolealkylamines caused acute inflammatory irritation in rats, including acute pain (spontaneous raising foot reaction), and inflammation (paw edema). At the molecular level, lipids analysis revealed significantly higher levels of pro-inflammatory mediators of the COX and LOX pathways. However, anti-inflammatory mediators from the CYP 450, ALA, and DHA pathways markedly decreased after exposure to Chansu water fraction. Moreover, four indolealkylamines from Chansu showed a high theoretical affinity to a known irritation target, 5-HT

Dehydrobufotenin extracted from the Amazonian toad Rhinella marina (Anura: Bufonidae) as a prototype molecule for the development of antiplasmodial drugs

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)

Anti-tumor effect and its related mechanisms of cinobufotalin combined with cisplatin on H22 liver cancer mice / 中国中药杂志

In order to observe the anti-tumor effect of cinobufotalin on H22 liver cancer mice and to explore its regulatory mechanism, 50 Kunming mice were subcutaneously inoculated with H22 intraperitoneal passage cells under the armpit to establish H22 hepatocellular carcinoma model. They were then randomly divided into model group, cinobufotalin low dose group, cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, which received 0.01% ethanol solution, 1 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cisplatin, 5 mg·kg~(-1)cisplatin + 5 mg·kg~(-1) cinobufotalin respectively for 10 days. The general condition of mice during the intervention was observed, and the inhibition rate, tumor mass, thymus index, histopathological changes of the tumors, apoptotic rate of the tumors, the expressions of phosphatidylinositol 3-kinase(PI3 K), protein kinase B(Akt), apoptosis related gene(Fas), Fas ligand(FasL) mRNA and protein phosphorylated Akt(pAkt) protein in the tumors of each group were compared. The results showed that during the modeling period, the mice showed a decline in food intake, dark fur, poor mental status, and gradually worsened over time. The mental status of mice in each intervention group was improved gradually, especially in the cisplatin+cinobufotalin group. As compared with the model group, the tumor mass of each intervention group was lower(P<0.05). As compared with the cinobufotalin low dose group, the tumor mass was lower and inhibition rate was higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, the tumor mass was lower and the inhibition rate was higher in cisplatin+cinobufotalin group(P<0.05). As compared with the model group, the thymus index was higher in cinobufotalin high dose group and cisplatin + cinobufotalin group, while was lower in cisplatin group(P<0.05). As compared with the cinobufotalin low dose group, the thymus index was higher in the cinobufotalin high dose group and lower in the cisplatin group(P<0.05). As compared with the cinobufotalin high dose group, the thymus index was lower in cisplatin group(P<0.05). As compared with cisplatin group, the thymus index was higher in cisplatin+cinobufotalin group(P<0.05). Pathological staining showed that a large number of heterogeneous cells and mitotic phenomena were observed in the model group. Cell fragments and neutrophils were observed in the tumor tissues of the intervention groups, showing diffuse necrosis, and the diffuse necrosis was more obvious in the cisplatin+cinobufotalin group. As compared with the model group, the apoptotic rate of the tumors and the relative expressions of Fas mRNA and protein were higher in the intervention groups, while the relative expressions of PI3 K, FasL mRNA and protein and the relative expression of pAkt protein were lower in the intervention groups(P<0.05). As compared with the cinobufotalin low dose group, the apoptotic rate of the tumors and relative expression of Fas and protein were higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, apoptotic rate of the tumors and the relative expression of Fas mRNA and protein were higher in the cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower in the cisplatin+cinobufotalin group(P<0.05). In summary, cinobufotalin has significant anti-tumor effect on H22 liver cancer mice, and can enhance the immune function of mice and synergistically enhance the effect of chemotherapy. Its mechanism may be associated with regulating PI3 K/Akt/Fas/FasL signaling pathway related genes and protein expression.

Effect of cinobufagin on transient outward potassium current in dorsal root ganglion cells of rats with cancer-induced bone pain / 南方医科大学学报

OBJECTIVE@#To observe the effect of cinobufagin on transient outward potassium current () in rat dorsal root ganglion cells of cancer-induced bone pain (CIBP) and explore the possible analgesic mechanism of cinobufagin.@*METHODS@#Whole cell patch clamp technique was used to examine the effect of cionbufagin on in acutely isolated dorsal root ganglion (DRG) cells from normal SD rats and rats with bone cancer pain.@*RESULTS@#The DRG cells from rats with CIBP showed obviously decreased current density, an activation curve shift to the right, and an inactivation curve shift to the left. Cinobufagin treatment significantly increased the current density and reversed the changes in the activation and inactivation curves in the DRG cells.@*CONCLUSIONS@# current is decreased in DRG neurons from rats with CIBP. Cinobufagin can regulate the activation and inactivation of current in the DRG cells, which may be related to its analgesic mechanism.

Correlative study of bufogenin constituents in venom of Bufo bufo gargarizans / 中国中药杂志

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.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.</p><p><b>METHODS</b>The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.</p><p><b>RESULTS</b>The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.</p>

Mechanism of colon cancer cell apoptosis induced by telocinobufagin: role of oxidative stress and apoptosis pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis.</p><p><b>METHODS</b>MTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting.</p><p><b>RESULTS</b>Telocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells.</p><p><b>CONCLUSION</b>Telocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.</p>

Bufalin reverses hepatocyte growth factor-induced resistance to afatinib in H1975 lung cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of bufalin in reversing hepatocyte growth factor (HGF)-induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism.</p><p><b>METHODS</b>The afatinib-resistant H1975 lung cancer cells (H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad-HGF-GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway-related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot.</p><p><b>RESULTS</b>The results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67 ± 8.76)%, significantly lower than the growth rate of (63.45 ± 12.65)% in the H1975 cells treated with HGF alone (P < 0.05). The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber (48.98 ± 11.43), significantly lower than the 118.92 ± 37.29 of afatinib-treated or the 88.84 ± 19.53 of bufalin-treated cells (P < 0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p-EGFR, p-cMet, p-AKT, p-ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down-regulated markedly, and the expression of E-cadherin was up-regulated markedly.</p><p><b>CONCLUSIONS</b>Combination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial-mesenchymal transition.</p>

Chemical constituents of bufadienolides in cinobufacino injection / 药学学报

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.

Comparison of the chemical composition between fresh and dried venenum bufonis by UPLC-Orbitrap MS / 药学学报

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.

Proteomics research of bufalin-induced apoptosis in osteosarcoma cell lines / 中国中药杂志

<p><b>OBJECTIVE</b>To study the apoptosis inducing effects of bufalin on various human osteosarcoma cells and the concerning molecular mechanisms.</p><p><b>METHOD</b>MTT assay was used to detect the growth inhibition rates of osteosarcoma cells U-20S, U-20S/MTX300, SaOS-2, IOR/OS9 treated with bufalin in different concentrations and times. The apoptosis of cells was observed flow cytometry 48 h following bufalin treatment. The proteomic techniques were used to separate and compare the treated and control groups 48 h after bufalin-incubation. Then, the proteomic results were validated by western blot.</p><p><b>RESULT</b>Bufalin inhibited the growth of human osteosarcoma cells U20S, U20S/MTX300 (methotrexate resistant cells), SAOS2, IOR/OS9 in a dose- and time-dependent manner. The 72 h IC50 were (37.43 +/- 4.1), (32.24 +/- 5.3) nmol x L(-1) in U20S,U20S/MTX300 cells,respectivly. Flow cytometry showed that the apoptosis cells were increased following bufalin treatment. The protein expression profile showed 24 differentiated expression proteins. Among these proteins, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27) decreased significantly and the result was then validated by western blot. Ectopic expression of Hsp27 could reduce the bufalin-induced apoptosis remarkably in U20S and U20S/MTX300 cells.</p><p><b>CONCLUSION</b>Bufalin could inhibit the cell growth and induce apoptosis on human osteosarcoma cells. The effect of bufalin may be related to the joint intervention with multiple protein targets. Among them, downregulation of Hsp27 plays a critical role in the bufalin-induced apoptosis in human osteosarcoma cells.</p>

Reversal effect of cinobufacini on multidrug resistance of Raji/ADR cells and its mechanisms / 中国实验血液学杂志

The aim of this study was to explore the reversing effect of cinobufacini on multidrug resistance of Raji/ADR cells and its mechanisms. The growth inhibitory rate, half inhibitory concentration (IC50), reversing multiples to adriamycin- resistance were detected by MTT, and the curve of growth inhibitory rate was drawn; the MDR-1 and MRP-1 gene transcription was determined by RT-PCR; the expressions of P-gp and MRP-1 proteins were assayed by Western blot and flow cytometry. The results showed that the inhibitory rates of cinobufacini on Raji and Raji/ADR cells at 72 h were 75.6% and 69.3% respectively, the IC50 were 3.9 mmol/L and 4.6 mmol/L without significant difference (P > 0.05). The reversing multiples to adriamycin-resistance were 255.7 multiples, the transcription of mdr-1 and mrp-1 genes and the expression of P-gp and MRP-1 proteins significantly decreased (P < 0.05) in Raji/ADR cells after the treatment with cinobufotalin. It is concluded that cinobufotalin can reverse the adriamycin-resistance in Raji/ADR cells and the expression of P-gp and MRP-1 proteins were down-regulated through the transcriptional pathway. The cinobufotalin is an effective reversal agent for the multidrug resistance of tumors.

Bufadienolides from venom of Bufo bufo gargarizans / 中国中药杂志

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.

Research progress of in vitro and in vivo anti-tumor effects and formulation of bufalin / 中国中药杂志

Bufalin is an active compound of the traditional Chinese medicine Chansu, which exhibits significant anti-tumor activities in many solid tumors and leukemia cell lines. Bufalin could introduce apoptosis, reverse drug-resistance, and prevent migration and invasion of tumor cells. This paper reviewed the latest research progress of the in vitro and in vivo anti-tumor effect and mechanism of bufalin on a series of cancers, such as hepatocellular carcinoma, lung cancer, colon cancer, gastric cancer, leukemia, bladder cancer, and its formulation study is also summarized for the reference of its further study and application.

Identification of bufadienolides profiling in cinobufacino by HPLC-DAD-FT-ICR-MS method / 药学学报

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.

A new bufadienolide with cytotoxic activity from the Chinese traditional drug Ch'an Su / 中国天然药物

AIM@#To study the bufadienolides in the Chinese traditional drug "Ch'an Su" and their cytotoxic activity.@*METHOD@#Various chromatographic techniques were used to isolate the constituents, and their structures were elucidated through physical and spectroscopic data.@*RESULTS@#Twenty compounds were isolated, and eighteen were evaluated in vitro for their cytotoxic activity against A-549 and K-562 cells.@*CONCLUSION@#Compound 1 (bufalin 3β-acrylic ester) was a new bufadienolide and exhibited the most potent activity against the two tumor cell lines with IC50 values of 7.16 and 6.83 nmol · L(-1). The relationships between structure and activity are discussed.