Study on quality control method of toad venom based on characteristic chromatogram and QAMS / 中国中药杂志

Toad venom (Chansu) is prepared from the dried secretion of parotid gland and skin gland from Bufo bufo gargarizans or B. melanostictus. Up to now, much attention shall be paid to the poor quality of commercial toad venom because of the adulteration. So, it is urgent to establish a scientific and perfect quality control method to improve the quality of toad venom and guarantee its safety and effectiveness in clinical application. The different batches of toad venom samples were assayed by high performance liquid chromatography (HPLC) and the quantitative analysis of multi-components by single marker (QAMS) was used to detect the contents of five bufagenins. As a result, the reference characteristic chromatogram was established, displaying serotonin, gamabufotalin, arenobufagin, hellebrigenin, telocinobufagin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin as characteristic peaks. Taking cinobufagin as an internal reference substance, QAMS was verified for the determination of five bufagenins (gamabufotalin, bufotalin, bufalin, cinobufagin, resibufogenin) in toad venom samples. The durability and applicability of the relative correction factor (RCF) were also studied systematically. RCFs of cinobufagin to gamabufotalin, bufotalin, bufalin and resibufogenin were determined as 1.05, 0.895, 1.09 and 0.913, respectively. The characteristic chromatogram and QAMS established in this study could effectively control the quality of toad venom and provide scientific evidence for the improvement of the quality standard of the toad venom to be described in Chinese Pharmacopoeia (2020 edition).

Synergy with the active ingredients of toad venom and sorafenib suppresses hepatocellular carcinoma HepG2 cells proliferation through down-regulating Akt/NF-κB signaling pathways / 中国药理学通报

Aim To investigate the effect of the active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib on the growth of hepatocellular carcinoma HepG2 cells,and to explore the possible mechanism.Methods The rates of inhibition after treated with drugs 12,24,48 h were detected by MTT assay.The changes of cell morphology were detected by Hoechst 33342 fluorescent staining.The changes of cell cycle were detected by flow cytometry.The expressions of proteins such as Akt,p-Akt (Ser473),IκB,NF-κB,p-NF-κB p65,Bcl-2,Bax,cyclin A,PCNA were detected by Western blot.Results Bufalin,cinobufagin and sorafenib could inhibit the proliferation of HepG2 cells,presenting a dose-and time-dependent manner.Meanwhile,it could significantly increase the inhibitory rate of cells compared with those of single treatment,and they performed a synergistic activity in sorafenib combined with cinobufagin or bufalin by Jin Formula after 24 h treatment (P ＜ 0.01).The results of fluorescence staining showed the observation of the morphological features of nuclear condensation.Sorafenib induced the cell cycle G0/G1 phase arrest (P ＜0.01),and bufalin,cinobufagin and the combination treatment generated the cell cycle S phase arrest (P ＜0.01).The results of Western blot showed that the expressions of Akt,NF-κB were not obviously changed between control and all other treatment.The expression levels of p-Akt (Ser473),p-NF-κB p65,Bcl-2,PC-NA and cyclin A in combination treatment significantly decreased,and the expression levels of IκB and Bax significantly increased compared to those in single treatment (P ＜ 0.01).Conclusion The active ingredients of toad venom (bufalin and cinobufagin) combined with sorafenib performs a synergetic effect on the anti-cancer of HepG2 cells by down-regulating Akt/ NF-κB signaling pathway.

Identification of Proteins in Toad Venom by NanoLC-LTQ-Orbitrap Velos Pro / 中国药学杂志

OBJECTIVE: To proteins in toad venom. To investigate the by proteomic approach. METHODS: The total proteins from the ear-side gland of toad were digested by trypsin, and the peptides were further analyzed by the NanoLC-linear trap quadropole (LTQ)-Orbitrap Velos Pro.. The raw data acquired by mass spectrometer were imported into MaxQuant software for the identification of peptides and proteins. The proteins were categorized based on gene ontology annotation in biological process, cellular component and molecular function. RESULTS: A total of 407 protein groups and 880 peptides were identified. There were 76 pathways associated with the identified proteins in toad venom, including the 5-HT receptor mediated signaling pathway, beta adrenergic receptor signaling pathway, blood coagulation, cadherin signaling pathway, cholesterol biosynthesis, etc.. CONCLUSION: This study lays the foundation for further exploration of the proteins in toad venom and their functions.

Advance on quality control of toad venom and its key influence factors / 中国中药杂志

Toad venom (Chansu) is prepared from the dried secretion of Bufo bufo gargarizans or B. melanostictus. It is not only one of the famous and expensive traditional Chinese medicines(TCMs) from animal origin, but also one of 28 kinds of toxic TCMs to be required for special management issued by the State Council of the People's Republic of China. Chansu contains the rich bufadienolides and indole alkaloids, and displays various bioactivity including cardiotonic, anti-tumor, analgesia, and local anesthesia. Based on the published references in the recent years, the advance on the identification of adulterants and quality evaluation as well as the influence factors on the quality of toad venom was summarized to improve the quality standards and promote the level of quality control of toad venom and its preparations.

Antiplasmodial and Cytotoxic Activities of Toad Venoms from Southern Amazon, Brazil

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.

Stimulating effects of toad venom (CHANSU) in the rat hind paw / 中国药学杂志

OBJECTIVE: The stimulating effects of toad venom (Chansu) on rats were investigated in this study. METHODS: After treatment with toad venom (sc, 10 and 50 mg · mL-1), foot swelling and foot-ground reaction force were observed to reflect the inflammation and pain, respectively. The levels of inflammatory mediators (PGE2, PGF2α, PGE1, and 8, 9-DiHETrE) were further determined using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: No raising foot was observed in the normal rats group, but this foot-ground reaction force was significant in the toad venom 10 and 50 mg · mL-1 groups. The number of raising foot after toad venom treatments were (14 ± 5) and (99 ± 8) times in 10 min (P 2 and PGF2α, and reduced the relative content of anti-inflammatory PGE1 and 8, 9-DiHETrE in the rat hind-paw. CONCLUSION: Toad venom can dose-dependently produce stimulating effects in the rat paw.

Research advances of antitumor activity of toad venom / 中国生化药物杂志

Toad venom as a Chinese traditional natural medicine with a kind of effective chemical components has been widely used for many disease.Recently more and more research was focused on its remarkable antitumor efforts, new studies have found that the mechanism was closely associated with endoplasmic reticulum stress,tumor-related inflammation, autophagy, mitochondria-induced apoptosis pathway, heat chock protein and immuneomodulatory.In this article we reviewed the antitumor research advances of toad venom and its active compounds from several aspects mentioned above.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.

Clinical and electrocardiographic evaluation during experimental toad poisoning in dogs

Accidents involving toad poisoning are frequent and dogs are the most common victims; they become poisoned by biting or ingesting a toad. When released in the organism, the venom is absorbed by both the oral mucosa and the digestive tract, initiating its toxic action. The aim of this work was to evaluate the clinical and electrocardiographic aspects of dogs subjected to experimental toad poisoning, as well as their response to treatment with propranolol. Twenty dogs were divided into two groups, a control group (n = 5) and a poisoned group (n = 15). After general anesthesia, the control group received a placebo, while the poisoned group received a venom aliquot through an orogastric tube. Results were tested through multivariate analysis (p < 0.05). The animals in the poisoned group had gastrointestinal symptoms including emesis, intense salivation, hyperemic or congested oral mucosa and pasty diarrhea. Non-responsive mydriasis, nystagmus, depression, stupor, tachypnea, opisthotonus and ataxia were also manifested by 100 percent of the poisoned animals. Affected dogs had an increase in blood pressure, statistically significant throughout study. Five poisoned animals developed ventricular tachycardia and were treated with propranolol (0.5 mg/kg IV). All propranolol-treated animals returned to normal sinus rhythm, which evidences the efficacy of this drug to treat ventricular arrhythmias caused by toad venom.

Dosagem de marcadores cardíacos CK-MB e TnIc e eletrólitos no envenenamento experimental por veneno de sapo em cães / Determination of cardiac markers and CK-MB TnIc and electrolyte in experimental poisoning by poison frog in dogs

Dentre os sinais sistêmicos causados pelo envenenamento por veneno de sapo (bufotoxina) em cães, os efeitos cardiotóxicos são um dos mais importantes. O objetivo deste estudo foi avaliar as potenciais alterações no músculo cardíaco de cães envenenados experimentalmente por veneno de sapo e observar as alterações eletrolíticas que podem ocorrer nesse tipo de envenenamento. Utilizaram-se 20 cães divididos em grupo controle (n=5) e grupo envenenado (n=15). O veneno de sapo foi extraído por meio de compressão manual das glândulas paratóides. Após anestesia geral, os cães do grupo controle receberam placebo (solução fisiológica) e os do grupo envenenado uma alíquota do veneno por sonda orogástrica. As colheitas de sangue para dosagem dos marcadores cardíacos foram realizadas seis e 24 horas após o envenenamento. As colheitas de sangue para dosagem dos eletrólitos foram realizadas antes e duas, quatro, seis e 12 horas após o envenenamento. A análise estatística empregada foi o teste não-paramétrico de Mann-Withney (P<0,05). Os cães envenenados por veneno de sapo apresentaram elevação dos níveis dos marcadores cardíacos CK-MB e TnIc, confirmando a cardiotoxicidade do veneno. Hipocalemia e hipocalcemia foram também observadas nos cães envenenados.

Among the systemic signs of toad venom (bufotoxin) poisoning in dogs, the cardiotoxic effects are one of the most important. Thus, the objective of this experiment was to evaluate potential changes in the cardiac muscle in dogs poisoned experimentally by toad venom and to observe the eletrolyte alterations which may occur in this condition. Twenty dogs divided into control group (n=5) and poisoned group (n=15) were utilized. The toad venom was extracted by manual compression of the paratoidic glands. After general anesthesia, dogs in the control group received placebo and dogs in the poisoned group received the venom by orogastric catheter. Samples for dosage were collected 6 hours and 24 hours after poisoning and 0, 2, 4, 6 and 12 hours after poisoning for electrolytes dosage. The Man-Withney test was used for statistical analysis (P<0.05). The poisoned dogs showed (saline) elevated levels of cardiac markers CK-MB and TnIc, confirming the cardiotoxic effect of the bufotoxin. Hypokalemia and hypocalcemia were also observed.

Toad poisoning in three dogs: case reports

Toad poisoning is frequent in dogs, but has been infrequently addressed in published case reports and review articles. Dogs can be poisoned when they bite a toad or otherwise ingest the venom. The venom effects manifest soon after the accident, since the toxin is rapidly absorbed by the mucous membrane of the digestive system. Hospital records of three dogs, diagnosed with toad poisoning, were retrospectively reviewed from January 2005 to July 2007. Poisoned dogs may present only local irritation or systemic signs in the gastrointestinal, cardiac and neurological systems. All three cases presented herein had clinical signs of gastrointestinal alterations including vomiting, sialorrhea and diarrhea. Two dogs developed abnormal cardiac rhythm and two exhibited neurological signs. A poisoned animal requires emergency care and symptomatic therapy with intense monitoring of its clinical parameters. Although there have been reports on the low mortality of dogs poisoned by toads, one animal died even after appropriate therapy. The severity of clinical signs and the risk of death must be considered by the veterinarian.