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Acute promyelocytic leukaemia (APL) is a malignant hematological tumour characterized by the presence of promyelocytic leukaemia-retinoic acid receptor A (PML-RARA) fusion protein. Cinobufagin (CBG) is one of the main effective components of toad venom with antitumor properties. However, only a few reports regarding the CBG treatment of APL are available.We explored the effect and mechanism of action of CBG on NB4 and NB4-R1 cells.We evaluated the viability of NB4 and NB4-R1 cells treated with 0, 20, 40, and 60 nM CBG for 12, 24, and 48 h. After treatment with CBG for 24 h, Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl-2), β-catenin, cyclin D1, and c-myc expression was detected using western blotting and real-time polymerase chain reaction. Caspase-3 and PML-RARA expression levels were detected using western blotting.CBG inhibited the viability of NB4 and NB4-R1 cells. The ICCBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the β-catenin signalling pathway. This study proposes a novel treatment strategy for patients with APL, particularly those with ATRA-resistant APL.

Toad venom, a dried product of secretion from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, has had the therapeutic effects of hepatocellular carcinoma confirmed. Bufalin and cinobufagin were considered as the two most representative antitumor active components in toad venom. However, the underlying mechanisms of this antitumor effect have not been fully implemented, especially the changes in endogenous small molecules after treatment. Therefore, this study was designed to explore the intrinsic mechanism on hepatocellular carcinoma after the cotreatment of bufalin and cinobufagin based on untargeted tumor metabolomics. Ultraperformance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was performed to identify the absorbed components of toad venom in rat plasma. In vitro experiments were determined to evaluate the therapeutic effects of bufalin and cinobufagin and screen the optimal ratio between them. An in vivo HepG2 tumor-bearing nude mice model was established, and a series of pharmacodynamic indicators were determined, including the body weight of mice, tumor volume, tumor weight, and histopathological examination of tumor. Further, the entire metabolic alterations in tumor after treating with bufalin and cinobufagin were also profiled by UHPLC-MS/MS. Twenty-seven active components from toad venom were absorbed in rat plasma. We found that the cotreatment of bufalin and cinobufagin exerted significant antitumor effects both in vitro and in vivo, which were reflected in inhibiting proliferation and inducing apoptosis of HepG2 cells and thereby causing cell necrosis. After cotherapy of bufalin and cinobufagin for twenty days, compared with the normal group, fifty-six endogenous metabolites were obviously changed on HepG2 tumor-bearing nude mice. Meanwhile, the abundance of α-linolenic acid and phenethylamine after the bufalin and cinobufagin intervention was significantly upregulated, which involved phenylalanine metabolism and α-linolenic acid metabolism. Furthermore, we noticed that amino acid metabolites were also altered in HepG2 tumor after drug intervention, such as norvaline and Leu-Ala. Taken together, the cotreatment of bufalin and cinobufagin has significant antitumor effects on HepG2 tumor-bearing nude mice. Our work demonstrated that the in-depth mechanism of antitumor activity was mainly through the regulation of phenylalanine metabolism and α-Linolenic acid metabolism.

Owing to inefficiency of chemotherapy towards cancer treatment, formulation and application of herbal drug compounds will open new avenues with this regard. In this study, the anticancer effects of itexin, cinobufacini, and Physalis alkekengi (P. alkekengi) were assessed. Herein, synergistic effects of vitexin, cinobufacini, and P. alkekengi hydroalcoholic extract were assessed against estrogen-receptor (EGFR2)-positive breast cancer mouse model. Sixty ER + breast cancer BALB/c mice (six groups each including ten members) were included. The anticancer effects of P. alkekengi hydroalcoholic extract, vitexin, and cinobufacini were administered against EGFR2 cancerous cells for 14 days. The tumor size, cytotoxic effects, and expression of Beclin-1, LC3-II, and ATG5 autophagy-related genes were investigated using RT-qPCR technique. The data was analyzed using chi-square, ANOVA, and multinomial logistic regression tests. The 50% lethal dose (LD50) of P. alkekengi and vitexin against the breast cancer cells included 12 mg/kg, respectively, while cinobufacini LD50 was 24 mg/kg but had no toxicity against CRL7242 breast normal cells. Furthermore, 24 mg/kg of the P. alkekengi, vitexin, and cinobufacini significantly increased the ATG5, Beclin-1, and LC3-II gene expression. Considering anticancer effects of P. alkekengi, vitexin, and cinobufacini against breast cancer through induction of the autophagy pathway, the compound formulations can be applied as anticancer therapies.

Malaria remains to date one of the most devastating parasitic diseases worldwide. The fight against this disease is rendered more difficult by the emergence and spread of drug-resistant strains. The need for new therapeutic candidates is now greater than ever. In this study, we investigated the antiplasmodial potential of toad venoms. The wide array of bioactive compounds present in Bufonidae venoms has allowed researchers to consider many potential therapeutic applications, especially for cancers and infectious diseases. We focused on small molecules, namely bufadienolides, found in the venom of Rhinella marina (L.). The developed bio-guided fractionation process includes a four solvent-system extraction followed by fractionation using flash chromatography. Sub-fractions were obtained through preparative TLC. All samples were characterized using chromatographic and spectrometric techniques and then underwent testing on in vitro Plasmodium falciparum cultures. Two strains were considered: 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant). This strategy highlighted a promising activity for one compound named resibufogenin. With IC

Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, “cholesterol-bile acid-bufadienolidies pathway” was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3β-hydroxysteroid dehydrogenase (3βHSD) and steroid 5β-reductase (SRD5β) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5β cDNA of B. bufo gargarizans (Bbg-SRD5β) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5β. Bbg-SRD5β had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5β in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5β was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5β can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone owithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5β-Y132G was 1.8 times higher than that of wild type Bbg-SRD5β. Although Bbg-SRD5β was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5β is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5β.

Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L−1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g−1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.

Aqueous extract of toad skin (named as Cinobufacini or Huachansu) provides plentiful sources of bioactive peptides that remain undetected and unidentified. High-resolution mass spectrometry-based peptidomics platforms have developed into a major approach to the discovery of natural peptides, with data-dependent acquisition modes providing a wealth of peptide profiling information. In this study, we used a gel- and HLB (a solid phase extraction cartridge)-based two-dimensional separation and purification system and nano-liquid chromatography-tandem mass spectrometry-based peptidomic studies with homology matching for the identification of peptides from Cinobufacini. We evaluated 232 multi-charged peptides and found several specific peptides, some of which were validated by target parallel reaction monitoring mode. These peptides are the first to be identified in Cinobufacini and are completely different from ones identified in toad venom. So, this mapping provides key peptide information for the quality control of Bufo bufo gargarizans skin and its preparation.

Cinobufagin (Huachansu), an aqueous extract from the dried skin of the toad Bufo bufo gargarizans Cantor (frog skin), is a biologically active ingredient of a traditional Chinese medicine cinobufacini that can treat multiple bone pathological conditions such as bone pain, bone tumors, and osteosarcoma.The study aimed to explore the roles and molecular mechanisms of cinobufagin underlying osteosarcoma development and doxorubicin (ADR) resistance.Cell viability, migration, and invasion were examined by CCK-8, wound healing, and Transwell invasion assays, respectively. RNA sequencing analysis was performed in MNNG/HOS cells treated with or without cinobufagin. The relationships of cinobufagin, forkhead box O1 (FOXO1), and Fc fragment of IgG binding protein (FCGBP) were examined by luciferase reporter, immunofluorescence (IF), RT-qPCR, and chromatin immunoprecipitation (ChIP) assays together with weighted gene co-expression network analysis (WGCNA) analysis. Epithelial-mesenchymal transition (EMT) marker levels were examined through the Western blot assay. The function and molecular basis of cinobufagin in osteosarcoma were further investigated by mouse xenograft experiments.Cinobufagin reduced cell viability, weakened ADR resistance, and inhibited cell migration/invasion/EMT in osteosarcoma cells. Cinobufagin enhanced FOXO1-mediated transcription of downstream genes including FCGBP. FCGBP knockdown partly abrogated the effect of cinobufagin on osteosarcoma cell development. Cinobufagin inhibited the growth of mouse osteosarcoma xenografts in vivo. Cinobufagin reduced the expression of Ki-67 and MMP9 and facilitated caspase-3 expression in osteosarcoma xenografts.Cinobufagin suppressed tumor progression and reduced ADR resistance by potentiating FOXO1-mediated transcription of FCGBP in osteosarcoma.

Amphibians produce defensive chemicals which provide protection against both predators and infections. Within species, populations can differ considerably in the composition and amount of these chemical defenses. Studying intraspecific variation in toxins and linking it to environmental variables may help us to identify the selective drivers of toxin evolution, such as predation pressure and infection risk. Recently, there has been a renewed interest in the unique toxins produced by salamanders from the genus Salamandra: the samandarines. Despite this attention, intraspecific variation has largely been ignored within Salamandra‐species. The aim of this study was to investigate whether geographic variation in profiles of samandarines exists, by sampling 4 populations of Salamandra atra over its range in the Dinaric Alps. In addition, we preliminary explored whether potential variation could be explained by predation (counting the number of snake species) and infection risk (cultivation and genomic analyses of collected soil samples). Salamanders from the 4 populations differed in toxin composition and in the size of their poison glands, although not in overall toxin quantity. Nor predation nor infection risk could explain this variation, as populations barely differed in these variables. Sampling over a much broader geographic range, using better estimators for predation and infection risk, will contribute to an improved understanding of how environment may shape variation in chemical defenses. Nevertheless, as the 4 populations of S. atra did differ in their toxin profiles, we propose that this species provides an interesting opportunity for further ecological and evolutionary studies on amphibian toxins.

Toad venom (Chansu) is used in the treatment of infectious and inflammatory diseases in China and East/Southeast Asian countries. However, the anti-inflammatory components of toad venom have not yet been systematically evaluated and clearly defined. To investigate the anti-inflammatory effects of toad venom and identify new anti-inflammatory ingredients, we used zebrafish, an alternative drug screening model, to evaluate the anti-inflammatory effects of 14 bufadienolides previously isolated from toad venom. Most of the bufadienolides were found to exert significant anti-inflammatory effects on lipopolysaccharide-, CuSO4-, or tail transection-induced zebrafish inflammatory models. Moreover, gammabufotalin ( 6) inhibits lipopolysaccharide-induced inflammation by suppressing the myeloid differentiation primary response 88/nuclear factor-kappa B and STAT3 signal pathways. This study confirms the potential of zebrafish in drug screening, clarifies the anti-inflammatory effects of bufadienolides from toad venom, and indicates that gammabufotalin may be developed as a novel therapeutic agent for inflammatory diseases in the future.

The aim of this review is to provide a critical and comparative account of the total synthetic approaches toward histrionicotoxins, alkaloids isolated from skin extracts of Colombian poison arrow frog Dendrobates histrionicus. We have summarized the maneuvers in each paper by graphically detailing the synthesis and associated reaction niceties of only the key intermediates by different researchers. Fascinating structural feature of histrionicotoxins is "8-hydroxy-l-azaspiro[5.5]undecane core" with two side chains at C-2 and C-7 which differ in their length and nature of unsaturation. All synthetic approaches to histrionicotoxins aim at the construction of key intermediate "azaspiro[5.5]undecane ring" and installation of side chains at C-2 and C-7.

Metabolic research is a key method to understand the fate of traditional Chinese medicine (TCM) prescription in vivo and help to explain its pharmacological effects. Huachansu (HCS) tablets are prepared from the dried skin of Bufo gargarizans (Cantor, 1842), which have the effects of detumescence and analgesia, and used in the treatment of middle and advanced tumors. However, an in-depth understanding of the chemical components of HCS tablet and its metabolism in vivo is lacking. In this study, an integrated analytical strategy based on UPLC-ESI-QTOF/MS

Anti-tumor candidate drugs from natural products have gained increasing attention. Cinobufagin is a natural product isolated from the traditional chinese medicine Chansu. Herein, we find that cinobufagin inhibits the proliferation and colony-forming ability of human hepatoma HepG2 and SK-HEP-1 cells. Furthermore, cinobufagin induces G2-phase cell cycle arrest and DNA damage in cancer cells. Thymidylate synthase (TYMS), the major target of chemotherapeutic drugs 5-FU or other fluoropyrimidines, which catalyzes the conversion of dUMP to dTMP and provides the sole de novo source of thymidylate for DNA synthesis. We demonstrate that cinobufagin suppresses TYMS expression via proteasome-dependent degradation in human hepatoma cells, moreover, depletion of TYMS restrains the proliferation and colony formation of tumor cells, and the results of western blotting and immunofluorescence assay indicate DNA damage is induced in tumor cells transfected with TYMS-targeting siRNA (siTYMS), additionally, knockdown of TYMS enhances the inhibitory effect of cinobufagin on the proliferative potential of HepG2 and SK-HEP-1 cells. It is worth noting that cinobufagin in combination with 5-FU exhibits antagonism or synergism combined effects on the proliferation of human hepatoma cells, indicating that Chansu-related preparations such as cinobufacini injection and Huachansu capsules applied to clinical practice should be used with caution in combination with 5-FU for the treatment of liver cancer. Collectively, cinobufagin exerts good anti-hepatoma activity through inhibition of growth and induction of DNA damage by promoting the degradation of TYMS. Our results provide evidence that cinobufagin might be a potential agent for the treatment of cancers such as hepatocellular carcinoma. It can also promote the scientific development of Chansu, and has great significance for enriching the application of TCM in the development of new anti-tumor drugs.

Toad venom contains a large number of bufadienolides, which have a variety of pharmacological activities, including antitumor, cardiovascular, anti-inflammatory, analgesic and immunomodulatory effects. The strong antitumor effect of bufadienolides has attracted considerable attention in recent years, but the clinical application of bufadienolides is limited due to their low solubility and poor bioavailability. In order to overcome these shortcomings, many strategies have been explored, such as structural modification, solid dispersion, cyclodextrin inclusion, microemulsion and nanodrug delivery systems, etc. In this review, we have tried to summarize the pharmacological activities and structure–activity relationship of bufadienolides. Furthermore, the strategies for solubility and bioavailability enhancement of bufadienolides also are discussed. This review can provide a basis for further study on bufadienolides.

Toad venom is a traditional Chinese medicine that has a long history in treating infectious and inflammatory diseases, such as carbuncle, pharyngitis. As one of the major active components in toad venom, resibufogenin (RBG) possesses a variety of pharmacological activities, including lowering blood pressure, reducing proteinuria and preventing oxidative stress. But only its antitumor activity attracts widespread attention in these years. This study aimed to explore the nonnegligible anti-inflammatory activity of RBG in vivo and in vitro. In endotoxemia mice, a single intraperitoneal administration of RBG significantly lowered serum TNF-α, IL-6 and MCP-1 levels. In LPS-stimulated macrophages, RBG decreased LPS-induced pro-inflammatory mediators' productions (e.g., iNOS, IL-6, TNF-α and MCP-1) through suppressing their transcriptions. Mechanism study showed that RBG hindered IκBα phosphorylation and prevented nuclear translocation of p65, thus inactivating nuclear factor-κB (NF-κB) signaling. Concurrently, RBG also dampened activator protein-1 (AP-1) signaling through inhibiting the phosphorylation levels of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Besides LPS (TLR4 ligand) model, RBG also inhibited Pam3CSK4 (TLR2 ligand)- or poly I:C (TLR3 ligand)-induced inflammatory reactions, suggesting that its target(s) site is(are) not on the cytomembrane. These findings not only support the pharmacological basis for the traditional use of toad venom in inflammatory diseases, but also provide a promising anti-inflammatory candidate.

Background: Huachansu injection (HCS) is a widely used traditional Chinese medicine for advanced non-small cell lung cancer (NSCLC) to alleviate the adverse drug reactions (ADRs) and enhance the clinical efficacy of chemotherapy. Objective: To evaluate the efficacy and safety of HCS as an adjunctive treatment to platinum-based chemotherapy (PBC) for advanced NSCLC. Methods: A systematic review and meta-analysis were conducted according to PRISMA guidelines. A total of nine databases were searched to select randomized controlled trials (RCTs) of HCS plus PBC to treat NSCLC from inception to October 10, 2020. RCTs on HCS plus PBC vs PBC alone for advanced NSCLC were included. Dichotomous data were pooled as risk ratio (RR) with 95% confidence intervals. RCTs compared to HCS plus PBC vs PBC alone were included. Primary outcomes were objective response rate (ORR) and disease control rate (DCR), and secondary outcomes were survival rate, quality of life (QOL), and adverse drug reactions (ADRs). GRADE software was used to access the quality of evidence. Results: A total of 32 RCTs, including 2753 patients, were included. Compared to PBC alone, HCS plus PBC improved the ORR, DCR, 1- and 2-year survival rates, and QOL and alleviated neutropenia, thrombocytopenia, nausea, vomiting, anemia, liver injury, renal injury, and alopecia. Conclusions: Compared to PBC alone, HCS plus PBC improved the clinical efficacy and alleviated the ADRs in advanced NSCLC patients. Considering the limitations of the included RCTs, high-quality trials with longer follow-ups are needed to further confirm the results.

Bufalin is an endogenous cardiotonic steroid, first discovered in toad venom but also found in the plasma of healthy humans, with anti-tumour activities in different cancer types. The current review is focused on its mechanisms of action and highlights its very large spectrum of effects both in vitro and in vivo. All leads to the conclusion that bufalin mediates its effects by affecting all the hallmarks of cancer and seems restricted to cancer cells avoiding side effects. Bufalin decreases cancer cell proliferation by acting on the cell cycle and inducing different mechanisms of cell death including apoptosis, necroptosis, autophagy and senescence. Bufalin also moderates metastasis formation by blocking migration and invasion as well as angiogenesis and by inducing a phenotype switch towards differentiation and decreasing cancer cell stemness. Regarding its various mechanisms of action in cancer cells, bufalin blocks overactivated signalling pathways and modifies cell metabolism. Moreover, bufalin gained lately a huge interest in the field of drug resistance by both reversing various drug resistance mechanisms and affecting the immune microenvironment. Together, these data support bufalin as a quite promising new anti-cancer drug candidate.

Bufotenines, a natural component from toad venom, showed great potential for development as a novel anti-inflammation and analgesia agent, but the potential toxicity limited its clinic use. In this paper, bufotenines-loaded liposome was prepared and optimized. Then, the therapeutic effects and drug safety of bufotenines-liposome were investigated against inflammation and pain on animal models, with a focus on gastrointestinal toxicity. Bufotenines and its liposome significantly increased paw withdrawal mechanical threshold (PWMT) in Von Frey test and hot paw withdrawal latency (HPWL) in hot-plate test. Moreover, intestinal absorption in vitro and pathological analysis in vivo showed that total bufotenines-loaded liposome significantly reduced the gastrointestinal irritation through reducing exposure of total bufotenines on intestinal tissue. High-sensitivity lipidomics analysis revealed the effect of total bufotenines-loaded liposome were be related to the down-regulation of inflammatory mediators from cyclooxygenase (COX) and lipoxygenase (LOX), the up-regulation of cytochrome P450 (CYP450), and other pathways, thus regulating lipid metabolism pathway and ultimately reducing gastrointestinal irritation. This study shows that liposome-loaded bufotenines has anti-inflammatory, analgesic effects and achieves toxicity reduction. These results provide systematic evidences for efficacy and safety of toad venom active ingredients.

Cinobufagin, a bufadienolide of toad venom of Bufo bufo gargarizans, is used as a cardiotonic, central nervous system (CNS) respiratory agent, as well as an analgesic and anesthetic. However, several research showed that bufadienolide has a few side effects on the CNS, such as breathlessness or coma. Although cinobufagin was shown to display pharmacological effects in various models, the toxic effect of cinobufagin is elusive in brain cell models. The aim of this study was to explore whether cinobufagin affected viability, Ca2+ homeostasis, and reactive oxygen species (ROS) production in Gibco® Human Astrocyte (GHA) and HCN-2 neuronal cell line. In GHA cells but not in HCN-2 cells, cinobufagin (20-60 μM) induced [Ca2+ ]i rises. In terms of cell viability, chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid reduced cinobufagin-induced cytotoxicity in GHA cells. In GHA cells, cinobufagin-induced Ca2+ entry was inhibited by 2-aminoethoxydiphenyl borate or SKF96365. In a Ca2+ -free medium, treatment with thapsigargin or U73122 abolished cinobufagin-evoked [Ca2+ ]i rises. Furthermore, treatment with N-acetylcysteine reversed ROS production and cytotoxicity in cinobufagin-treated GHA cells. Together, in GHA cells but not in HCN-2 cells cinobufagin caused cytotoxicity that was linked to preceding [Ca2+ ]i rises by Ca2+ influx via store-operated Ca2+ entry and phospholipase C-dependent Ca2+ release from the endoplasmic reticulum. Moreover, cinobufagin induced ROS-associated cytotoxicity.

Toads in the family Bufonidae contain bufadienolides in their venom, which are characterized by their chemical diversity and high pharmacological potential. American trypanosomiasis is a neglected disease that affects an estimated 8 million people in tropical and subtropical countries. In this research, we investigated the chemical composition and antitrypanosomal activity of toad venom from Rhinella alata collected in Panama. Structural determination using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy led to the identification of 10 bufadienolides. Compounds identified include the following: 16β-hydroxy-desacetyl-bufotalin-3-adipoyl-arginine ester (1), bufotalin (2), 16β-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester (3), bufotalin-3-pimeloyl-arginine ester (4), 16β-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester (5), bufotalin-3-suberoyl-arginine ester (6), cinobufagin-3-adipoyl-arginine ester (7), cinobufagin-3-pimeloyl-arginine ester (8), cinobufagin-3-suberoyl-arginine ester (9), and cinobufagin (10). Among these, three new natural products, 1, 3, and 5, are described, and compounds 1–10 are reported for the first time in R. alata. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid attached to C-3, and a hydroxyl group at C-14 in the structure of bufadienolides that is important for their biological activity. Bufadienolides showed cytotoxic activity against epithelial kidney Vero cells, however, bufagins (2 and 10) displayed low mammalian cytotoxicity. Compounds 2 and 10 showed activity against the cancer cell lines MCF-7, NCI-H460, and SF-268.

Dendrobatid frogs sequester alkaloid defenses from dietary arthropods. Here, we provide experimental evidence that mother strawberry poison frogs (Oophaga pumilio) provision alkaloids to tadpoles. Captive-raised females were fed the synthetic alkaloid decahydroquinoline (DHQ), which we subsequently quantified in their skin, eggs, and developing tadpoles. DHQ quantity was positively associated with tadpole mass/development, suggesting high sequestration rates by tadpoles. These data confirm that tadpoles obtain nutrition and alkaloids by feeding exclusively on maternally provisioned eggs.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a major dose- and therapy-limiting side effect that is particularly difficult to treat. Huachansu, an aqueous extract from toad skin, is a widely used anti-cancer natural product in China. Clinical findings have established the safety and effectiveness of Huachansu in combination with chemotherapy to promote the therapeutic efficacy while alleviate the side effects, especially cancer-related pain symptoms. Unfortunately, experimental data on the effects and mechanisms of Huachansu in combination with chemotherapy is not available. In this study, the effects of Huachansu were tested in vivo on a rat model of oxaliplatin-induced CIPNP. The results show, a single injection of Huachansu 2.5 g/kg produced a short-term analgesic effect on pre-established oxaliplatin-induced CIPNP after 60 min, as indicated by decreased mechanical and thermal hypersensitivity in comparison to oxaliplatin-treated rats. Repeated doses of Huachansu, given during CIPNP induction, prevented the development of oxaliplatin-induced CIPNP. This prophylactic effect of Huachansu was associated with suppressed oxaliplatin-induced TRPV1 up-regulation in the dorsal root ganglia and spinal astrocyte activation. These findings reveal Huachansu therapeutic potential in treating and preventing CIPNP.

Oral venom systems evolved multiple times in numerous vertebrates, enabling the exploitation of unique predatory niches. Yet how and when they evolved remains poorly understood. Up to now, most research on venom evolution has focused strictly on toxins. However, using toxins present in modern-day animals to trace the origin of the venom system is difficult, since they tend to evolve rapidly, show complex patterns of expression, and were incorporated into the venom arsenal relatively recently. Here we focus on gene regulatory networks associated with the production of toxins in snakes, rather than the toxins themselves. We found that overall venom gland gene expression was surprisingly well conserved when compared to salivary glands of other amniotes. We characterized the “metavenom network,” a network of ∼3,000 nonsecreted housekeeping genes that are strongly coexpressed with toxins and are primarily involved in protein folding and modification. Conserved across amniotes, this network was coopted for venom evolution by exaptation of existing members and the recruitment of new toxin genes. For instance, starting from this common molecular foundation, Heloderma lizards, shrews, and solenodon evolved venoms in parallel by overexpression of kallikreins, which were common in ancestral saliva and induce vasodilation when injected, causing circulatory shock. Derived venoms, such as those of snakes, incorporated novel toxins, though they still rely on hypotension for prey immobilization. These similarities suggest repeated cooption of shared molecular machinery for the evolution of oral venom in mammals and reptiles, blurring the line between truly venomous animals and their ancestors.

Toad venom, a traditional natural medicine, has been used for hundreds of years in China for treating different diseases. Many studies have been performed to elucidate the cardiotonic and analgesic activities of toad venom. Until the last decade, an increasing number of studies have documented that toad venom is a source of lead compound(s) for the development of potential cancer treatment drugs. Research has shown that toad venom contains 96 types of bufadienolide monomers and 23 types of indole alkaloids, such as bufalin, cinobufagin, arenobufagin, and resibufogenin, which exhibit a wide range of anticancer activities in vitro and, in particular, in vivo for a range of cancers. The main antitumor mechanisms are likely to be apoptosis or/and autophagy induction, cell cycle arrest, cell metastasis suppression, reversal of drug resistance, or growth inhibition of cancer cells. This review summarizes the chemical constituents of toad venom, analyzing their anticancer activities and molecular mechanisms for cancer treatments. We also outline the importance of further studies regarding the material basis and anticancer mechanisms of toad venom.

Huachansu (HCS) injection has been used for the clinical treatment of hepatocellular carcinoma (HCC) patients in China for more than thirty years. However, the underlying mechanisms of HCS injection in inhibiting HCC remains unclear. In this study, we aimed to evaluate the effects of HCS injection on HCC both in vitro and in vivo and to systematically explore the underlying mechanisms of HCS's actions against HCC using an ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based cellular metabolomics and lipidomics approach. MTT and DAPI staining were performed to evaluate the effects of HCS injection on cell proliferation and apoptosis, respectively, in human HepG2 HCC cells with and without HCS treatment. An H22 hepatoma-bearing mouse model was employed to investigate the anti-HCC action of HCS injection in vivo. A UPLC-HRMS-based cellular metabolomics and lipidomics method was employed to characterize cellular metabolite fingerprinting, to identify potential biomarkers associated with the effect of HCS injection on the inhibition of HCC and to explore the anti-HCC mechanisms of HCS injection from the perspective of cellular metabolism. As a result, HCS injection inhibited the proliferation of HepG2 cells and induced their apoptosis. Body weight loss, sarcoma weights and volumes of H22 hepatoma-bearing mice in the HCS treatment group were significantly attenuated compared to those in the model group. Twenty-five biomarkers responsible for the intervention effect of HCS were screened and annotated using UPLC-HRMS-based cellular metabolomics and lipidomics. Among these biomarkers, two cellular metabolites, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and glutathione, were confirmed unambiguously using the reference substances. Metabolic pathway analysis revealed that the anti-HCC effects of HCS injection primarily involved the metabolism of glutathione, cysteine and methionine, sphingolipid, fatty acid biosynthesis, amino sugar and nucleotide sugars. As evidenced by western blotting experiments, the changes in UDP-GlcNAc content in response to HCS injection were linked to the inhibition of UDP-GlcNAc pyrophosphorylase 1 (UAP1) expression in HepG2 cells. In conclusion, HCS injection has a curative anti-HCC effect, and a UPLC-HRMS-based cellular metabolomics and lipidomics approach combined with further confirmatory experiments is a promising means of discovering its underlying mechanisms.

to evaluate the effectiveness and safety of Huachansu (HCS) injection plus chemotherapy in the treatment of gastric cancer.A thorough and systematic retrieval of randomized controlled trials (RCTs) concerning HCS injection for treating gastric cancer was conducted in several electronic databases from inception to May 10, 2018. The quality of the RCTs was assessed by the Cochrane risk of bias tool. And the data about objective remission rate, performance status, adverse drug reactions (ADRs) and other outcomes were extracted and analyzed by Review Manager 5.3 and Stata 13.0 software.A total of 14 RCTs with 976 participants were involved in the current Meta-analysis. The results suggested that HCS injection combined with chemotherapy was associated with better effects than receiving conventional chemotherapy alone in respect of improving the objective response rate [RR = 1.18, 95% CI (1.03, 1.37), Z = 2.32, P = 0.02], and performance status [RR = 1.84,95% CI (1.43, 2.36), Z = 4.74, P0.000 01]. In addition, HCS injection combined with chemotherapy could relieve pain for patients with gastric cancer.This Meta-analysis revealed that HCS injection plus chemotherapy might more effective than chemotherapy in treating gastric cancer. Nevertheless, more large-scale and rigorously designed RCTs should be performed to validate this finding.

Rhinella schneideri is a common toad found in South America, whose paratoid toxic secretion has never been explored as an insecticide. In order to evaluate its insecticidal potential, Nauphoeta cinerea cockroaches were used as an experimental model in biochemical, physiological and behavioral procedures. Lethality assays with Rhinella schneideri paratoid secretion (RSPS) determined the LD50 value after 24 h (58.07&micro, g/g) and 48 h exposure (44.07 &micro, g/g) (R2 = 0.882 and 0.954, respectively). Acetylcholinesterase activity (AChE) after RSPS at its highest dose promoted an enzyme inhibition of 40%, a similar effect observed with neostigmine administration (p &lt, 0.001, n= 5). Insect locomotion recordings revealed that RSPS decreased the distance traveled by up to 37% with a concomitant 85% increase in immobile episodes (p &lt, 0.001, n = 36). RSPS added to in vivo cockroach semi-isolated heart preparation promoted an irreversible and dose dependent decrease in heart rate, showing a complete failure after 30 min recording (p &lt, 0.001, n &ge, 6). In addition, RSPS into nerve-muscle preparations induced a dose-dependent neuromuscular blockade, reaching a total blockage at 70 min at the highest dose applied (p &lt, 6). The effect of RSPS on spontaneous sensorial action potentials was characterized by an increase in the number of spikes 61% (p &lt, 0.01). Meanwhile, there was 42% decrease in the mean area of those potentials (p &lt, 0.05, n &ge, 6). The results obtained here highlight the potential insecticidal relevance of RSPS and its potential biotechnological application.

Chemical compounds from skin secretions from toads of Bufonidae family have been long-studied. In the search for new molecules with pharmacological action, the 3β-OH groups of bufadienolides are commonly derivatised using acetyl groups. This work described the isolation and/or structural elucidation of isolated and derivatised compounds from the venom of the Brazilian anuran Rhinella marina, and their evaluation in in vitro assays. In the methanolic extract of the R. marina venom, compound cholesterol (1) was isolated from the CRV-52 fraction by classic column chromatography, dehydrobufotenine (2) by Sephadex LH-20 from the CRV-28 fraction, and a mix of suberoyl arginine (3) and compound 2 was obtained from the CRV-6-33 fraction. The compounds marinobufagin (4), telocionbufagin (5) and bufalin (6) were isolated by classic column chromatography, followed by separation via HPLC in the CRV-70 fraction, and the compound marinobufotoxin (9) was isolated by classic column chromatography in the CRV-6 fraction, here being isolated for the first time in R. marina specimens. Compounds 4 and 5 were submitted for acetylation with acetic anhydride, in the presence of pyridine and 4-dimethyilaminopiridine (DMAP), in order to obtain the compounds 3-acetyl-marinobufagin (7) and 3-acetyl-telocinobufogin (8). The isolated and derivatised compounds were identified by 1H and 13C NMR, and their molecular mass confirmed by mass spectrometry. All compounds (except 1 and 3) were tested in cytotoxic assays by the MTT method and presented cytotoxic potential against human cancer cell lines, as well as against non-tumoral human embryonic kidney HEK-293 cells. With the exception of compound 2, all molecules presented IC50 values

Since Rhinella sp. toads produce bioactive substances, some species have been used in traditional medicine and magical practices by ancient cultures in Peru. During several decades, the Rhinella horribilis toad was confused with the invasive toad Rhinella marina, a species documented with extensive toxinological studies. In contrast, the chemical composition and biological effects of the parotoid gland secretions (PGS) remain still unknown for R. horribilis. In this work, we determine for the first time 55 compounds from the PGS of R. horribilis, which were identified using HPLC-MS/MS. The crude extract inhibited the proliferation of A549 cancer cells with IC50 values of 0.031 &plusmn, 0.007 and 0.015 &plusmn, 0.001 &micro, g/mL at 24 and 48 h of exposure, respectively. Moreover, it inhibited the clonogenic capacity, increased ROS levels, and prevented the etoposide-induced apoptosis, suggesting that the effect of R. horribilis poison secretion was by cell cycle blocking before of G2/M-phase checkpoint. Fraction B was the most active and strongly inhibited cancer cell migration. Our results indicate that the PGS of R. horribilis are composed of alkaloids, bufadienolides, and argininyl diacids derivatives, inhibiting the proliferation and migration of A549 cells.

Two new indole alkaloids, Bufotenidine B (2) and Bufocarboline A (6), along with seven known indole alkaloids (1, 3–5, and 7–9) and three organic acids (10–12), were isolated from the water extract of toad venom. The structures of the new alkaloids were elucidated by extensive spectroscopic methods. The absolute configurations of 4, 6, and 8 were determined for the first time by electronic circular dichroism (ECD) calculations. The cytotoxic activity of all compounds was tested against human malignant melanoma cells A375 by the MTT method, and no antitumor activity was observed.

Bufonis Venenum(toad venom) is prepared from the dried secretion of Bufo bufo gargarizans or B. melanostictus. Toad venom powder is one of the processed slices of crude material toad venom. In the present study, the global quality control method and standard of toad venom and its processed slice, toad venom powder were established, including TLC identification, characteristic chromatogram and QAMS by HPLC. The relative correction factor(RCF) was re-calculated and validated. The average RCFs of cinobufagin to gamabufotalin, bufotalin, bufalin and resibufogenin were considered for the determination of five bufadienolides in the samples. The total amount in the different batches of the dried samples varied from 4.06% to 17.0%. Referring to the revised methods for crude materials, the quality standard of toad venom powder was drafted including appearance description, TLC examination, characteristic chromatogram, water content and the total amount of five bufadienolides. The present investigation provided scientific evidences for the quality standard improvement of toad venom to be described in the next edition of Chinese pharmacopoeia(2020 edition).