The drug-resistance of malaria parasites is the main problem in the disease control. The huge Brazilian biodiversity promotes the search for new compounds, where the animal kingdom is proving to be a promising source of bioactive compounds. The main objective of this study was to evaluate the antiplasmodial and cytotoxic activity of the compounds obtained from the toad venoms of Brazilian Amazon. Toad venoms were collected from the secretion of Rhinella marina and Rhaebo guttatus in Mato Grosso State, Brazil. The powder was extracted at room temperature, yielding 2 extracts (RG and RM) and a substance ('1') identified as a bufadienolide, named telocinobufagin. Growth inhibition, intraerythrocytic development, and parasite morphology were evaluated in culture by microscopic observations of Giemsa-stained thin blood films. Cytotoxicity was determined against HepG2 and BGM cells by MTT and neutral red assays. The 2 extracts and the pure substance ('1') tested were active against chloroquine-resistant Plasmodium falciparum strain, demonstrating lower IC₅₀ values. In cytotoxic tests, the 2 extracts and substance '1' showed pronounced lethal effects on chloroquine-resistant P. faciparum strain and low cytotoxic effect, highlighting toad parotoid gland secretions as a promising source of novel lead antiplasmodial compounds.
Amphibian Venoms* ; Animals ; Biodiversity ; Brazil* ; Bufo marinus ; Malaria ; Neutral Red ; Parasites ; Plasmodium falciparum

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.
Amphibian Venoms ; chemistry ; Animals ; Bufanolides ; chemistry ; Bufonidae ; Chromatography, High Pressure Liquid ; Tandem Mass Spectrometry

To evaluate the effectiveness and safety of Huachansu in the treatment of cancer-related pain,four Chinese databases( CNKI,VIP,Wan Fang,Sino Med) and three English databases( Cochrane Library,Medline,PubMed) were systematically and comprehensively retrieved since the establishment of each database to October 2018. Randomized controlled trials( RCTs) for the treatment of cancer-related pain with Huachansu were screened out according to pre-established inclusion criteria and exclusion criteria. Rev Man5. 3 software was used for Meta-analysis. A total of 241 articles were retrieved,and finally 10 studies were included. The total sample size was 1 293,including 648 in the experimental group and 645 in the control group. The overall quality of the included studies was generally low. The results of Meta-analysis showed that Huachansu combined with Western medicine acesodynes was superior to the single use of Western medicine acesodynes in the treatment of short-term pain relief,improvement of quality of life and reduction of constipation,nausea and vomiting,dizziness,drowsiness,anorexia and other adverse reactions. And it also has the advantage of a shorter onset time and longer duration time of analgesia,but cannot reduce the incidence of dysuria. Based on the findings,Huachansu had a certain effect in the treatment of cancer-related pain,and a significant positive effect on the improvement of quality of life and the reduction of adverse reactions. No serious adverse reactions occurred. However,due to the small number of studies included,the low quality of the included studies,published biases and other restrictions,the evidence in this study has a low quality,and the conclusion shall be adopted with caution. The effectiveness and safety of Huachansu in the treatment of cancer-related pain remained to be further confirmed in the future with a well-designed,rigorous,and standardized report,with a large sample size,multiple centers,and sufficient follow-up time for randomized controlled trials.
Amphibian Venoms ; therapeutic use ; Cancer Pain ; drug therapy ; Humans ; Quality of Life ; Randomized Controlled Trials as Topic

Toad toxin has digitalis-like cardioactive effects that include bradycardia, varying degrees of atrioventricular block, ventricular tachycardia, ventricular fibrillation and sudden cardiac death. We report here on a 54-year-old man who had varying degrees of atrioventricular block and nonsustained ventricular tachycardia two hours after he ate a bowl of toad soup.
Amphibian Venoms* ; Atrioventricular Block ; Bradycardia ; Death, Sudden, Cardiac ; Digitalis* ; Humans ; Hyperkalemia* ; Middle Aged ; Poisoning* ; Tachycardia, Ventricular ; Ventricular Fibrillation

OBJECTIVETo kinds of establish a HPLC method for determining contents of indole alkaloids and bufadienolides contained in toad medicines, and analyze two kinds of components contained in toad venom, toad skin and toad periostracum.

METHODAs for alkaloids, Nucleosil C18 column was adopted with acetonitrile and water containing 0.5% potassium dihydrogen phosphate (6: 94, adjust pH to 3.2 with phosphate acid) as the mobile phase. The flow rate was 0.8 mL x min(-1), the detection wavelength was 275 nm, and the column temperature was 30 degrees C. As for bufadienolides, Alltima C18 column was adopted with acetonitrile and water containing 0.3% acetic acid (B) as the mobile phase. The gradient process was as follows: a linear gradient from 28% to 54% acetonitrile in the first 15 min, then kept at 54% for additional 20 min. The flow rate was 0.6 mL x min(-1), the detection wavelength was 296 nm, and the column temperature was 30 degrees C.

RESULTThe linear ranges were 0.079 6-0.796 microg for serotonin, 0.097 2-1.945 microg for N-methylserotonin, 0.074 4-0.744 microg for N,N-dimethylserotonin, 0.103-2.05 microg for N,N,N-trimethylserotonin, and 0.067 2-0.672 microg for bufothionine, respectively. The average recoveries of serotonin and N-methylserotonin were 98.6% and 91.3%, respectively. The linear ranges of gamabufotalin, bufotalin, bufalin, cinobufagin and resibufogenin were 0.004 83-0.614, 0.007 9-1.006, 0.007 95-1.016, 0.009 7-1.24 and 0.009 6-1.22 microg, respectively, and their average recoveries were 101.6%, 102.5%, 101.0%, 99.1% and 98.9%, respectively.

CONCLUSIONToad venom has the highest contents of indole alkaloids and bufadienolides, followed by toad skin, and toad periostracum showed the lowest contents and even no detection result.

Amphibian Venoms ; chemistry ; Animals ; Bufanolides ; chemistry ; isolation & purification ; Bufonidae ; Chromatography, High Pressure Liquid ; Indole Alkaloids ; chemistry ; isolation & purification ; Skin ; chemistry

Toad venom is the Bufo bufo gargarizans or B. melanostictus after the ears of the gland secretion, used in the treatment of various cancers in recent years. Research shows that the main anti-tumor components in bufadienolide. Bufadienolide have free type structure and conjunct type structure. To identify and clarify the difference between bufogenin and bufotoxin contained in Bufonis Venenum, which was from B. bufo gargarizans, an UPLC-TQ-MS method has been established. UPLC-TQ-MS method was used to identify and quantify the major bufadienolides in Bufonis Venenum. UPLC-TQ-MS assay with positive ion mode was performed on a Waters ACQUITY UPLC BEH C, (2.1 mm x 100 mm, 1.7 µm) with the mobile phase consisting of 0. 1% aqueous formic and acidacetonitrile in gradient elution at a flow rate of 0.4 mL · min⁻¹ and the column temperature was set at 35 °C. By comparing their retention time and high resolution mass data of Bufonis Venenum extracts, 37 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were free-type bufadienolides (bufogenin), 11 of them were conjugated bufadienolides. There were significant differences in the main composition between fresh and processed Bufonis Venenum. The study found that the chemical composition of toad venom through great changes after processing, conjunct type content is much less, free type content as well change.
Amphibian Venoms ; chemistry ; metabolism ; Animals ; Bufonidae ; classification ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Molecular Structure ; Tandem Mass Spectrometry ; 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

OBJECTIVETo study the preparation of Venenum Bufonis beta-cyclodextrin inclusion complexes.

METHODAn optimal condition was established by the uniform design. Under the optimal conditions the Venenum Bufonis beta-cyclodextrin inclusion complexes were prepared with 5 different methods.

RESULTThe ball grinding method was superior to other four methods. The bufadienolide inclusion rate of Venenum Bufonis beta-cyclodextrin prepared with ball grinding method was 85.42%.

CONCLUSIONBall grinding method is the best method for the preparation of Venenum Bufonis beta-cyclodextrin inclusion complexes.

Amphibian Venoms ; administration & dosage ; chemistry ; Animals ; Bufanolides ; Bufo bufo ; Cholenes ; analysis ; Cyclodextrins ; Drug Carriers ; Drug Stability ; Materia Medica ; administration & dosage ; chemistry ; Solubility ; Technology, Pharmaceutical ; methods ; beta-Cyclodextrins

OBJECTIVETo investigate the mechanisms underlying the effect of Chansu Injection (CHS) in reversing multi-drug resistance (MDR) of HL60/ADM cells.

METHODSMTT assay was used to investigate the effect of CHS on adramycin (ADM) sensitivity of HL-60/ADM cells. Flow cytometry was used to observe the effect of CHS on the cell cycle of HL60/ADM cell. The expressions of NF-κB, MRP, GST-π, and iNOS were detected by immunocytochemistry.

RESULTSTreatment with CHS lowered the IC(50) of ADM in HL60/ADM cells from 34.1971 µmol/L to 17.4393 µmol/L, and caused an increase in G(0)/G1 and G(2)/M phase cells with decreased S phase cells. CHS decreased the expressions of MRP mRNA and GST-π and MRP proteins but increased the expressions of iNOS and NF-κB proteins in the cells.

CONCLUSIONCHS can partly reverse MDR in HL60/ADM cells possibly by down-regulating MRP and GST-π, up-regulating NF-κB and iNOS, and promoting cell apoptosis, thereby increase ADM sensitivity of HL-60/ADM cells.

Amphibian Venoms ; pharmacology ; Bufanolides ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; HL-60 Cells ; Humans

OBJECTIVETo study the bioaffinity between 8 bufadienolides(Bu) and tumor cells and analyze the correlation between the bioaffinity and the anti-tumor activities of Bu.

METHODMix and cultivate the chloroform extract of Chansu and MGC-803. Measure the content of 8 Bu in supernatant and cells using HPLC and calculate their affinity rate.

RESULTThe coefficient correlation between the decrease of Bu in cell supernatant after affinity and its MGC-803 restrictive activities, and between the cotent percentage of the free Bu in free cells with its MGC-803 restrictive activities, and between the difference between the decrease and the percentage and its MGC-803 restrictive activities is r = 0.82 (P < 0.05), r = -0.04 and r = 0.83 (P < 0.05) respectively.

CONCLUSIONEight Bu have different levels of affinity with MGC-803 which correlate with their anti-tumor activities.

Amphibian Venoms ; chemistry ; Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; Anura ; Bufanolides ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Chromatography, High Pressure Liquid ; methods ; Neoplasms ; drug therapy