Bufo Gargarizans Research Articles

A new bufadienolide from Bufo gargarizans Cantor

Bufo gargarizans Cantor (B. gargarizans) is the most widely distributed and abundant species of toad in China. Bufadienolides and indole alkaloids have cardiotonic and anti-tumor activities and are important pharmacological components of B. bufo gargarizans. In this experiment, a novel compound (1) and two known compounds (2 and 3) were isolated and identified from the dry skin of B. bufo gargarizans, both of which are bufadienolides. Various column chromatography methods were used to separate and purify the extract from the dried skin of B. bufo gargarizans. Accurate molecular weights were measured by HR-ESI-MS, and the chemical structure of the compounds was determined by NMR spectrometers. The structures were named as (2β,5β,16α)-2,5,16-trihydroxide bufa-14,20,22 dienolide (1), gamabufotalin (2) and desacetylbufotalin (3). In vitro cytotoxic activity assay indicated that compound 1 showed a moderate cytotoxicity against A549 cells with IC50 value of 12.65 μM.

3α-Hydroxybufadienolides in Bufo gallbladders: structural insights and biotransformation

Bufadienolides, naturally occurring steroids primarily found in toads, have garnered attention for their pharmacological properties and ecological significance. In this study, we isolated and identified 21 bufadienolides from the gallbladders of Bufo gargarizans, comprising four new compounds and 17 known ones. Notably, the predominance of 15 bufadienolides with a 3α-OH configuration in toad bile differs significantly from the 3β-OH bufadienolides found in venom secreted by toad glands. Moreover, our investigation into the biotransformation of 3β-OH and 3α-OH bufadienolides in the liver and kidney tissues of toads revealed an irreversible conversion from 3β-OH to 3α-OH bufadienolides, suggesting a crucial role in toad self-detoxification. These findings provide valuable insights into the structural diversity of bufadienolides and advance our understanding of their medical and ecological significance.Graphical

Molecular phylogeny of the family Rhabdiasidae (Nematoda: Rhabditida), with morphology, genetic characterization and mitochondrial genomes of Rhabdias kafunata and R. bufonis

BackgroundThe family Rhabdiasidae (Nematoda: Rhabditida) is a globally distributed group of nematode parasites, with over 110 species parasitic mainly in amphibians and reptiles. However, the systematic position of the family Rhabdiasidae in the order Rhabditida remains unsolved, and the evolutionary relationships among its genera are still unclear. Moreover, the present knowledge of the mitochondrial genomes of rhabdiasids remains limited.MethodsTwo rhabdiasid species: Rhabdias kafunata Sata, Takeuchi & Nakano, 2020 and R. bufonis (Schrank, 1788) collected from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in China, were identified based on morphology (light and scanning electron microscopy) and molecular characterization (sequencing of the nuclear 28S and ITS regions and mitochondrial cox1 and 12S genes). The complete mitochondrial genomes of R. kafunata and R. bufonis were also sequenced and annotated for the first time. Moreover, phylogenetic analyses based on the amino acid sequences of 12 protein-coding genes (PCGs) of the mitochondrial genomes were performed to clarify the systematic position of the family Rhabdiasidae in the order Rhabditida using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic analyses based on the 28S + ITS sequences, were also inferred to assess the evolutionary relationships among the genera within Rhabdiasidae.ResultsThe detailed morphology of the cephalic structures, vulva and eggs in R. kafunata and R. bufonis was revealed using scanning electron microscopy (SEM) for the first time. The characterization of 28S and ITS regions of R. kafunata was reported for the first time. The mitogenomes of R. kafunata and R. bufonis are 15,437 bp and 15,128 bp long, respectively, and both contain 36 genes, including 12 PCGs (missing atp8). Comparative mitogenomics revealed that the gene arrangement of R. kafunata and R. bufonis is different from all of the currently available mitogenomes of nematodes. Phylogenetic analyses based on the ITS + 28S data showed Neoentomelas and Kurilonema as sister lineages, and supported the monophyly of Entomelas, Pneumonema, Serpentirhabdias and Rhabdias. Mitochondrial phylogenomic results supported Rhabdiasidae as a member of the superfamily Rhabditoidea in the suborder Rhabditina, and its occurrance as sister to the family Rhabditidae.ConclusionsThe complete mitochondrial genome of R. kafunata and R. bufonis were reported for the first time, and two new gene arrangements of mitogenomes in Nematoda were revealed. Mitogenomic phylogenetic results indicated that the family Rhabdiasidae is a member of Rhabditoidea in Rhabditina, and is closely related to Rhabditidae. Molecular phylogenies based on the ITS + 28S sequence data supported the validity of Kurilonema, and showed that Kurilonema is sister to Neoentomelas. The present phylogenetic results also indicated that the ancestors of rhabdiasids seem to have initially infected reptiles, then spreading to amphibians.Graphical

Unraveling the antimicrobial potential of Lactiplantibacillus plantarum strains TE0907 and TE1809 sourced from Bufo gargarizans: advancing the frontier of probiotic-based therapeutics.

In an era increasingly defined by the challenge of antibiotic resistance, this study offers groundbreaking insights into the antibacterial properties of two distinct Lactiplantibacillus plantarum strains, TE0907 and TE1809, hailing from the unique ecosystem of Bufo gargarizans. It uniquely focuses on elucidating the intricate components and mechanisms that empower these strains with their notable antibacterial capabilities. The research employs a multi-omics approach, including agar diffusion tests to assess antibacterial efficacy and adhesion assays with HT-29 cells to understand the preliminary mechanisms. Additionally, gas chromatography-mass spectrometry (GC-MS) is employed to analyze the production of organic acids, notably acetic acid, and whole-genome sequencing is utilized to identify genes linked to the biosynthesis of antibiotics and bacteriocin-coding domains. The comparative analysis highlighted the exceptional antibacterial efficacy of strains TE0907 and TE1809, with mean inhibitory zones measured at 14.97 and 15.98 mm, respectively. A pivotal discovery was the significant synthesis of acetic acid in both strains, demonstrated by a robust correlation coefficient (cor ≥ 0.943), linking its abundance to their antimicrobial efficiency. Genomic exploration uncovered a diverse range of elements involved in the biosynthesis of antibiotics similar to tetracycline and vancomycin and potential regions encoding bacteriocins, including Enterolysin and Plantaricin. This research illuminates the remarkable antibacterial efficacy and mechanisms intrinsic to L. plantarum strains TE0907 and TE1809, sourced from B. gargarizans. The findings underscore the strains' extensive biochemical and enzymatic armamentarium, offering valuable insights into their role in antagonizing enteric pathogens. These results lay down a comprehensive analytical foundation for the potential clinical deployment of these strains in safeguarding animal gut health, thereby enriching our understanding of the role of probiotic bacteria in the realm of antimicrobial interventions.

Modulating pancreatic cancer microenvironment: The efficacy of Huachansu in mouse models via TGF-β/Smad pathway

Ethnopharmacological relevanceHuachansu (HCS) is a traditional Chinese medicine obtained from the dried skin glands of Bufo gargarizans and clinical uses of HCS have been approved in China to treat malignant tumors. The traditional Chinese medicine theory states that HCS relieves patients with cancer by promoting blood circulation to remove blood stasis. Clinical observation found that local injection of HCS given to pancreatic cancer patients can significantly inhibit tumor progression and assist in enhancing the efficacy of chemotherapy. However, the material basis and underlying mechanism have not yet been elucidated. Aim of the studyTo investigate the therapeutic potential of HCS for the treatment of pancreatic cancer in in situ transplanted tumor nude mouse model. Furthermore, this study sought to elucidate the molecular mechanisms underlying its efficacy and assess the impact of HCS on the microenvironment of pancreatic cancer. To identify the antitumor effect of HCS in in situ transplanted tumor nude mouse model and determine the Chemopreventive mechanism of HCS on tumor microenvironment (TME). MethodsUsing the orthotopic transplantation nude mouse model with fluorescently labeled pancreatic cancer cell lines SW1990 and pancreatic stellate cells (PSCs), we examined the effect of HCS on the pancreatic ductal adenocarcinoma (PDAC) microenvironment based on the transforming growth factor β (TGF-β)/Smad pathway. The expression of TGF-β, smad2, smad3, smad4, collagen type-1 genes and proteins in nude mouse model were detected by qRT-PCR and Western blot. ResultsHCS significantly reduced tumor growth rate, increased the survival rate, and ameliorated the histopathological changes in the pancreas. It was found that HCS concentration-dependently reduced the expression of TGF-β1 and collagen type-1 genes and proteins, decreased the expression of Smad2 and Smad3 genes, and downregulated the phosphorylation level of Smad2/3. Additionally, the gene and protein expression of Smad4 were promoted by HCS. Further, the promoting effect gradually enhanced with the rise of HCS concentration. ConclusionsThe results demonstrated HCS could regulate the activity of the TGF-β/Smad pathway in PDAC, improved the microenvironment of PDAC and delayed tumor progression. This study not only indicated that the protective mechanism of HCS on PDAC might be attributed partly to the inhibition of cytokine production and the TGF-β/Smad pathway, but also provided evidence for HCS as a potential medicine for PDAC treatment.

Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase

Ample evidence indicates that bufalin (BFN), a cardiotonic steroid in Bufo toad toxin, possesses a potent anticancer activity mainly by stimulating apoptosis in cancer cells. Human red blood cells (RBCs) undergo eryptosis which contributes to a plethora of pathological conditions. No reports, however, have examined the potential toxicity of BFN to RBCs. This study aims to characterize the biochemical mechanisms governing the influence of BFN on the physiology and lifespan of RBCs.Isolated RBCs from healthy volunteers were exposed to anticancer concentrations of commercially available BFN from the skin of Bufo gargarizans (10–200 μM) for 24 h at 37 °C. Photometric assays were used to estimate hemolysis and hemolytic markers, and flow cytometry was used to detect eryptotic markers. Phosphatidylserine externalization was captured by fluorescein isothiocyante-labeled annexin V, cellular dimensions by light scatter patterns, and intracellular Ca2+ and reactive oxygen species (ROS) by fluorogenic dyes Fluo4/AM and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. BFN caused Ca2+-independent hemolysis and release of LDH, AST, CK, and K+, and increased annexin V-bound cells, cytosolic Ca2+, cell shrinkage, and ROS levels. BFN also disrupted Na+ and Mg2+ trafficking, and was sensitive to PEG 8000, sucrose, SB203580, and NSC 23766. In whole blood, BFN depleted hemoglobin stores, increased fragmented RBCs, and was selectively toxic to reticulocytes, lymphocytes, and platelets. In conclusion, BFN elicits premature RBC death, subject to regulation by p38 MAPK and Rac1 GTPase, and is detrimental to other peripheral blood cells. Altogether, these novel findings prompt cautious consideration of the toxin in anticancer therapy.

Synthesis and tissue distribution of bufadienolides in Bufo bufo gargarizans

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.

Kinship influences the metamorphic traits of Asiatic toad (Bufo gargarizans) larvae, in Central China

Kin recognition has been selected by natural selection because it can enhance the chances of cooperation among close relatives, but it could also be an accidental consequence of other life-history processes. In this study, we carried out experiments by using Asiatic toad tadpoles (Bufo gargarizans) to evaluate the hypothesis that: (1) individuals can use waterborne chemical cues or combine other possible cues to communicate kinship identity when exposed to kin during early ontogeny; (2) unfamiliar siblings (or familiar non-siblings) should have similar growth with unfamiliar non-siblings because the former is not able to identify their relatives (or may not consider familiar non-siblings as kin); (3) the familiar siblings may obtain a more equal resource share in populations of siblings, thus size variation is weak. The first and second predictions were partially better supported: kinship had a positive effect on tadpole growth, obtained larger mass at metamorphosis, and increased survival, but development time was longer than that of unfamiliar non-siblings. Further, the growth of unfamiliar siblings and familiar non-siblings was different from that of familiar siblings, suggesting that Asiatic toad tadpoles lose kin-recognition ability when isolated across the embryonic stage or may not be considered as siblings when exposed to water from non-sibling groups during early embryonic development. Thus, besides chemical cues, Asiatic toad tadpoles need to combine other possible modalities to communicate kinship identity. The third prediction was supported weakly. Therefore, our results suggest that exposure to kin during early embryonic development is essential for altering kin-recognition ability. More importantly, familiar sibling groups can be expected to develop slower and spend more time in the water, allowing more time for feeding and growth, which is consistent with kin selection theory, suggesting that natural selection should drive the evolution of mechanisms that minimize the competitive intensity between sibling groups.

3D Morphological Scanning and Environmental Correlates of Bufo gargarizans in the Yellow River Basin

Morphology plays a crucial role in understanding the intricacies of biological forms. Traditional morphometric methods, focusing on one- or two-dimensional geometric levels, often fall short of accurately capturing the three-dimensional (3D) structure of organisms. The advent of 3D scanning techniques has revolutionized the study of organismal morphology, enabling comprehensive and accurate measurements. This study employs a 3D structured light scanning system to analyze the morphological variations in the Chinese toad (Bufo gargarizans Cantor, 1842) along the Yellow River Basin. The 3D digital model obtained from the scan was used to calculate various morphological parameters including body surface area, volume, fractal dimensions, and limb size. The research explores geographic variability patterns and identifies environmental drivers affecting the 3D phenotypic variation of B. gargarizans. Results reveal a bimodal pattern of variation in the toad population, with higher elevations exhibiting smaller body sizes, greater appendage proportions, and more complex body structures. Linear regression analyses highlight the influence of elevation and annual mean temperature on the morphological variation of B. gargarizans, with elevation playing a significant role. This study underscores the significance of 3D morphometric analysis in unraveling the intricacies of organismal morphology and understanding the adaptive strategies of species in diverse environments.

Six bufadienolides derivatives are the main active substance against human colorectal cancer HCT116 cells of Huachansu injection

BackgroundHuachansu injection (HCS) is an aqueous extract preparation of Traditional Chinese Medicine (TCM) toad skin (Bufo melanosticus or B. bufo gargarizans). As a national second-class and self-developed TCM preparation, it is widely used to treat human colorectal cancer (CRC), yet its substantial toxic side effects, such as vascular irritation reaction, drug fever, and allergic reactions, pose a considerable challenge during clinical use. In-depth research on the material basis of HCS' traditional efficacy is essential for developing more effective and less toxic medicine. Identifying the main components responsible for the efficacy of HCS is crucial in determining its material basis and explaining any adverse reactions. PurposeThe purpose of this study is to clarify the main active components of HCS as a growth inhibitory agent against CRC HCT116 cells and to further understand the material basis of its anti-CRC properties. MethodsSulforhodamine B (SRB) assay was used to screen the compound with strong inhibitory activity against HCT116 cells from 44 compounds isolated from aqueous extracts of toad skin (B. melanosticus). The screened compounds as standard and MeOH extracts from HCS were determined by HPLC coupled with triple quadrupole mass spectrometry. The partial MeOH extract was subjected to HPLC system using solvents in a gradient of increasing polarity, yielding 12 fractions in total (HCS1 to HCS12). Besides, the screened active compounds were knocked out from HCS to obtain a fraction as a negative control, and the knocked-out components were collected as one fraction using semi-preparative HPLC. Those fractions were submitted to HPLC-MS/MS or HPLC-DAD system to determine the presence or absence of the screened compounds. In addition, cytotoxicity of those prepared fractions against CRC HCT116 cells was evaluated to determine the effect of the screened compounds in the anti-CRC of HCS. ResultsSix compounds, namely arenobufagin, telocinobufagin, gamabufalin, hellebrigenin, 19-hydroxylbufagin and argentinogenin, were screened for their inhibitory activity (with an IC50 value of less than 400 nM), and were present in HCS at a total-mass-percentage of 1.35×10−3 % in HCS MeOH extract. In addition, those 6 compounds were mainly distributed in HCS8, which also showed the strongest inhibitory activity against HCT116 cells. And the results of multicomponent knockout assay indicated the negative control lost its anti-CRC activity significantly, while the activity can be restored after those 6 compounds were added to the negative control. ConclusionsThose six bufadienolides derivatives are responsible for the anti-CRC HCT116 cells activity of HCS, and are the main components of HCS on anti-CRC. Through this research, we can more clearly understand the main anti-CRC active components in HCS, and the work also lays the foundation for the development of more effective and less toxic medicine from HCS.

Cloning, characterization and specificity of a new aromatic-L-amino-acid decarboxylases from Bufo bufo gargarizans

5-Hydroxytryptamine (5-HT) and its derivative bufotenine, which possess important physiological functions, are the primary active components in the secretions of toad parotid and skin gland. However, the biosynthetic pathway of these substances remains unclear in toads. To characterize toad's Aromatic-L-amino-acid decarboxylase (AADC), the key enzyme in the predicted 5-HT derivatives biosynthetic pathway, the full-length cDNA of AADC from Bufo bufo gargarizans (BbgAADC) was cloned from the parotoid gland of B. bufo gargarizans. The recombinant BbgAADC exhibited optimal expression in E. coli BL21 (DE3) containing pCold-BbgAADC after induction for 16 h at 15 °C with 0.3 mM IPTG, resulting in substantial yields of soluble proteins. The enzymological properties of BbgAADC were assessed, and it was determined that the optimal reaction temperature was 37 °C, the optimal pH was 8.6, and the optimum molar ratio of pyridoxal-5′-phosphate (PLP) to BbgAADC was found to be 3.6:1. Additionally, high substrate specificity was observed, as BbgAADC could catalyze the production of 5-HT from 5-hydroxytryptophan (5-HTP) but not dopamine or tryptamine from levodopa or tryptophan, respectively. The Km of the recombinant protein BbgAADC was 0.2918 mM and the maximum reaction rate (Vmax) was 1.182 μM·min−1 when 5-HTP was used as substrate. The Kcat was 0.0545 min−1, and Kcat/Km was 0.1868 mM−1·min−1. To elucidate the mechanism of BbgAADC, molecular docking was performed with PLP and 5-HTP, or the external aldimine formed by 5-HTP and PLP. The results indicated that the active sites for BbgAADC to bind with PLP were K303, H192, N300, A148, F309, T246, A273, and T147. W71, Y79, F80, P81, T82, H192, T246, N300, H302, F309, and R477 served as catalytically active sites for the binding of BbgAADC to 5-HTP. Furthermore, R447, W71, S149, N300, A148, and T147 of BbgAADC were involved in the decarboxylation reaction of the aldimine formed by PLP and 5-HTP.

Chemical Composition, Pharmacological Effects and Clinical Applications of Cinobufacini.

Chinese medicine cinobufacini is an extract from the dried skin of Bufo bufo gargarizans Cantor, with active ingredients of bufadienolides and indole alkaloids. With further research and clinical applications, it is found that cinobufacini alone or in combination with other therapeutic methods can play an anti-tumor role by controlling proliferation of tumor cells, promoting apoptosis, inhibiting formation of tumor neovascularization, reversing multidrug resistance, and regulating immune response; it also has the functions of relieving cancer pain and regulating immune function. In this paper, the chemical composition, pharmacological effects, clinical applications, and adverse reactions of cinobufacini are summarized. However, the extraction of monomer components of cinobufacini, the relationship between different mechanisms, and the causes of adverse reactions need to be further studied. Also, high-quality clinical studies should be conducted.

Evolutionary analysis of major histocompatibility complex variants in chytrid-resistant and susceptible amphibians

An amphibian emerging infectious disease (EID), chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), originated in Asia but primarily led to declines and extinctions in amphibian populations outside of Asia. Host major histocompatibility complex (MHC) molecules exhibit high polymorphism, and the evolution of MHC can be influenced by recombination and pathogens. Previous studies have indicated that host MHC class II is associated with Bd resistance. In this study, I conducted recombination and selection tests on functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) and an Australasian Bd-susceptible species (Litoria caerulea). Recombination at the same site was identified in both species, supporting the hypothesis that recombination contributes to MHC IIß1 diversity in amphibians. Positive selection was observed in MHC IIß1 alleles in both species. In L. caerulea, at least four amino acid sites were identified under significant positive selection in the MHC IIß1, whereas these sites were either negatively selected or conserved in B. gargarizans. This suggests these sites might be selected for Bd resistance. Hydrophobicity was detected in certain amino acid sites relating to Bd resistance, suggesting this physicochemical property may be a factor selected to counteract Bd infection. These findings of this study provide an evolutionary basis for understanding how amphibian MHC IIß1 may undergo selection in response to chytrid infection.

Molecular identification of animal species of Bufonis Venenum from secretions

The animal species is one of the key factors affecting the quality of Bufonis Venenum. The quality of Bufonis Venenum derived from Bufo bufo gargarizans is significantly higher than that from B. melanostictus. Since Bufonis Venenum is from secretions, the conventional identification methods are difficult to identify the animal species due to the lack of the appearance and morphology of the animals. The rapid development of molecular identification technology has provided new methods for the identification of Bufonis Venenum. However, because of the low content and serve degradation of residual DNA in secretions, the research on the molecular identification of Chinese medicinal materials from secretions remains to be carried out. To understand the animal species of Bufonis Venenum, this study collected 83 samples of Bufonis Venenum, including 7 commercially available samples, 5 reference medicinal materials, and 71 animal samples from which Bufonis Venenum was prepared according to the method in the 2020 edition of the Chinese Pharmacopoeia. Different DNA extraction methods were used and compared, and the mitochondrial 16S rRNA gene fragments were amplified, on the basis of which the phylogenetic trees were built. Finally, molecular identification of the animal species of the samples was performed. The results showed that the DNA extracted from Bufonis Venenum by the reagent kit had good quality, and 16S rRNA sequences were successfully amplified from 80 out of the 83 samples. In addition, 71 16S rRNA sequences of the animal species of Bufonis Venenum were downloaded from GenBank. The phylogenetic trees constructed based on the neighbor-joining(NJ) method and the Bayesian inference(BI) method showed that the samples derived from B. bufo gargarizans and B. melanostictus were clustered into separate monophyletic clades, with the support of 100%(NJ) and 1.00(BI), respectively. The animal species of both commercially available samples and reference medicinal materials were B. bufo gargarizans. In conclusion, DNA can be extracted from Bufonis Venenum derived from secretions, and the 16S rRNA gene sequences can be amplified, which can be used for molecular identification of the animal species of Bufonis Venenum. The findings provide a reference for the quality control of Bufonis Venenum and the identification of animal species of medicinal materials derived from secretions.

Combinational Antitumor Strategies Based on the Active Ingredients of Toad Skin and Toad Venom.

A multidrug combination strategy is an important mean to improve the treatment of cancer and is the mainstream scheme of clinical cancer treatment. The active ingredients of traditional Chinese medicine, represented by toad skin and toad venom, have the advantages of high efficiency, low toxicity, wide action and multiple targets and have become ideal targets in combined treatment strategies for tumors in recent years. Toad skin and toad venom are traditional Chinese animal medicines derived from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider that have shown excellent therapeutic effects on the treatment of various cancers and cancer pain as adjuvant antitumor drugs in clinical practice. The involved mechanisms include inducing apoptosis, arresting the cell cycle, inhibiting cell proliferation, migration and invasion, inhibiting tumor angiogenesis, reversing the multidrug resistance of tumor cells, and regulating multiple signaling pathways and targets. Moreover, a multidrug combination strategy based on a nanodelivery system can realize the precise loading of the active ingredients of toad skin or toad venom and other antitumor drugs and carry drugs to overcome physiological and pathological barriers, complete efficient enrichment in tumor tissues, and achieve targeted delivery to tumor cells and the controlled release of drugs, thus enhancing antitumor efficacy and reducing toxicity and side effects. This article reviewed the clinical efficacy and safety of the combination of toad skin and toad venom with chemotherapeutic drugs, targeted drugs, analgesics and other drugs; evaluated the effects and mechanisms of the combination of toad skin and toad venom with chemotherapy, targeted therapy, radiotherapy or hyperthermia, traditional Chinese medicine, signaling pathway inhibitors and other therapies in cell and animal models; and summarized the codelivery strategies for the active ingredients of toad skin and toad venom with chemotherapeutic drugs, small-molecule targeted drugs, monoclonal antibodies, active ingredients of traditional Chinese medicine, and photodynamic and photothermal therapeutic drugs to provide a basis for the rational drug use of toad skin and toad venom in the clinic and the development of novel drug delivery systems.

Advances on Delivery System of Active Ingredients of Dried Toad Skin and Toad Venom.

Dried toad skin (TS) and toad venom (TV) are the dried skin of the Bufo bufo gargarizans Cantor and the Bufo melanostictus Schneider, which remove the internal organs and the white secretions of the skin and retroauricular glands. Since 2005, cinobufacini preparations have been approved by the State Food and Drug Administration for use as adjuvant therapies in the treatment of various advanced cancers. Meanwhile, bufalenolides has been identified as the main component of TS/TV, exhibiting antitumor activity, inducing apoptosis of cancer cells and inhibiting cancer cell proliferation or metastasis through a variety of signaling pathways. However, clinical agents frequently face limitations such as inherent toxicity at high concentrations and insufficient tumor targeting. Additionally, the development and utilization of these active ingredients are hindered by poor water solubility, low bioavailability, and rapid clearance from the bloodstream. To address these challenges, the design of a targeted drug delivery system (TDDS) aims to enhance drug bioavailability, improve targeting within the body, increase drug efficacy, and reduce adverse reactions. This article reviews the TDDS for TS/TV, and their active components, including passive, active, and stimuli-responsive TDDS, to provide a reference for advancing their clinical development and use.

Identification of anti-tumor constituents from toad skin and toad venom by UPLC-QTOF/MS in-depth chemical profiling combined with bioactivity-based molecular networking

Toad skin (TS) and toad venom (TV) are two different traditional Chinese medicine (TCM) prepared from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. Both of them were used in the treatment of ulcers, and now they are regarded as animal drugs with anti-tumor activity. However, the comparison analysis between their compositions and anti-tumor active ingredients has not been elucidated clearly. In this study, technology of ultra-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and bioactivity-based molecular networking were applied to investigate the anti-tumor active ingredients of TS and TV. A method of collision energies-relied mass spectrometry combined with molecular networking was initially developed for identifying their chemical components. A combination of Cytoscape and molecular networking was then employed to analyze the prototype constitutes of TS absorbed in rat serum. In addition, cytotoxicity test was applied to evaluate their anti-proliferative activities against human gastric cancer (GC) cells. As a result, a total of 162 compounds were tentatively characterized, and four structural types existed in both TS and TV, including bufogenins, bufotoxins, indolealkylamines (IAAs), and acyl-arginine. Cyclic dipeptides could only be detected in TS, and the total number of bufogenins and bufotoxins in TV was higher than that of TS. Both of them significantly inhibited the viability of SGC7901 cells, and 70% methanol eluate of TS was the most potential active fraction. 44 prototype compounds in TS were identified, from which three types of potential tumor cytotoxic components, including bufogenins, IAAs, and cyclic dipeptides, were predicted. In human GC cells SGC7901, a mixture of nine bufogenins, including bufotalin, cinobufotalin, gamabufotalin, telocinobufagin, cinobufagin, bufarenogin, arenobufagin, hellebrigenin, and desacetylcinobufotalin, was conducted as bioactive components, which exerted a significant inhibitory effect on the cell viability of SGC7901 cells with a half maximal inhibitory concentration (IC50) value of 0.23 (0.19–0.27, 95%CI) μg/mL. Comparison of their chemical composition and anti-tumor activity reveals that TS might be able to partially replace TV, whilst bufogenins are the potential active ingredients.

Why Bufo gargarizans tadpoles grow bigger in Pb-contaminated environments? The gut microbiota matter

The impacts of lead/Pb2+ on ecosystems have received widespread attention. Growth suppression is a major toxic effect of Pb compounds on aquatic animals, however, some studies have also reported their growth-promoting effects. These complex outcomes may be explained by anions that accompany Pb2+ or by the multiple toxic mechanisms/pathways of Pb2+. To examine these hypotheses, we tested how Bufo gargarizans tadpoles responded to Pb(NO3)2 (100 and 200 μg/L Pb2+) using transcriptomics and microbiomics, with NaNO3 and blank groups as controls. Tadpoles exposed to Pb(NO3)2 showed delayed development while increased somatic growth in a dose-dependent manner, which can be attributed to the effects of NO3- and Pb2+, respectively. Tadpole transcriptomics revealed that exposure to NO3- downregulated the MAPK pathway at transcriptional level, explaining the development-suppressing effect of NO3-; while Pb2+ upregulated the transcription of detoxification pathways (e.g., xenobiotics metabolism by cytochrome P450 and glutathione metabolism), indicating cellular stress and thus contradicting the growth advantage of Pb2+-exposed tadpoles. Pb2+ exposure changed the tadpole gut microbiota drastically, characterized by increased polysaccharides and carbohydrate utilization while decreased fatty acid and amino acid consumption according to microbial functional analysis. Similar gut microbial variations were observed in field-collected tadpoles from different Pb2+ environments. This metabolic shift in gut microbiota likely improved the overall food utilization efficiency and increased the allocation of fatty acids and amino acids to the host, explaining the growth advantage of Pb2+-exposed tadpoles. In summary, our results suggest multiple toxic pathways of Pb2+, and the gut microbiota may affect the pollution outcomes on animals.

Gut microbiota perturbations during larval stages in Bufo gargarizans tadpoles after Cu exposure with or without the presence of Pb

Cu and Pb are ubiquitous environmental contaminants, but there is limited information on their potential impacts on gut microbiota profile in anuran amphibians at different developmental stages during metamorphosis. In this study, Bufo gargarizans tadpoles were chronically exposed to Cu alone or Cu combined with Pb from Gs26 throughout metamorphosis. Morphology of tadpoles, histological characteristic and bacterial community of intestines were evaluated at three developmental stages: Gs33, Gs36, and Gs42. Results showed that Cu and Cu + Pb exposure caused various degrees of morphological and histological changes in guts at tested three stages. In addition, bacterial richness and diversity in tadpoles especially at Gs33 and Gs42 were disturbed by Cu and Cu + Pb. Beta diversity demonstrated that the bacterial community structures were influenced by both heavy metals exposure and developmental stages. Alterations in taxonomic composition were characterized by increased abundance of Proteobacteria and Firmicutes, reduction of Fusobacteriota, as well as decreased Cetobacterium and increased C39 at all three stages. Overall, response of gut bacterial diversity and composition to Cu stress depends on the developmental stage, while the altered patterns of bacterial community at Cu stress could be modified further by the presence of Pb. Moreover, predicted metabolic disorders were associated with shifts in bacterial community, but needs integrated information from metagenomic and metatranscriptomic analyses. These results contribute to the growing body of research about potential ecotoxicological effects of heavy metals on amphibian gut microbiota during metamorphosis.

Grouping rule in tadpole: is quantity more or size assortment more important?

The ability to perceive group size and discriminate the ontogeny of conspecifics would play a crucial role in the grouping behavior of animals. However, the relative importance of numerical quantity and size-assortative preferences in shaping grouping rules remains poorly understood. In this study, I examined the responses of Miyako toad (Bufo gargarizans miyakonis) tadpoles to number quantity and size discrimination by choice tests at different ontogenetic stages (small, medium, and large). The results revealed that small-sized tadpoles in early developmental stages significantly preferred larger numbers (4) compared to smaller ones (1). However, this preference was not observed in later developmental stages (medium and large). And interestingly, when there was no quantity bias, size discrimination was not observed in tadpoles, irrespective of their ontogeny. These findings suggest that Miyako toad tadpoles discern quantity, i.e., the number of conspecifics, but exhibit ontogeny-dependent utilization of this ability. Understanding the interplay between numerical quantity and size-assortative preferences in grouping behavior will provide esteemed insights into the adaptive value of number sense in vertebrates and shed light on evolutionary processes.