Evaluation of analgesic and anti-inflammatory actions of indolealkylamines from toad venom in mice using lipidomics and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom is one of widely used traditional Chinese medicines due to its analgesic and anti-inflammatory activities. However, hydrophilic alkaloids from toad venom, which may have certain pharmacological activities, have not been systematic studied. AIM OF THE STUDY: The aim of the study was to identify the indolealkylamines (IAAs) from toad venom and investigate the analgesic and anti-inflammatory actions. MATERIALS AND METHODS: The alkaloids were extracted and identified by high-resolution mass spectrometry. The analgesic abilities were determined using hot-plate test, formalin test and von Frey test. High-sensitivity lipidomics was used to investigate the regulatory function of IAAs on inflammatory eicosanoids. Besides, network pharmacology and molecular docking were used to demonstrate the candidate targets of IAAs. RESULTS: 22 constituents have been characterized by high performance liquid chromatography (HPLC)-Triple TOF 5600, including six specific IAAs (serotonin, N-methyl serotonin, bufotenine, bufotenidine, bufothionine and dehydrobufotenine). Pharmacological studies showed that the IAAs from toad venom exerted significant analgesic activities at doses of 5, 15 and 45 mg/kg in vivo. Moreover, lipids analysis revealed IAAs might down-regulate inflammatory mediators from COX, LOX, DHA and LA pathways in formalin models, thus showing anti-inflammatory effect. The potent pharmacological function might because of the binding of IAAs and protein targets, such as sigma-1 receptor. CONCLUSION: The studies provided a systemic evidence for the analgesic and anti-inflammatory activities of IAAs from toad venom. It suggested that IAAs might be a potential candidate to reduce inflammatory pain disorders.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

The Paraguayan Rhinella toad venom: Implications in the traditional medicine and proliferation of breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Toads belonging to genus Rhinella are used in Paraguayan traditional medicine to treat cancer and skin infections. AIM OF THE STUDY: The objective of the study was to determine the composition of venoms obtained from three different Paraguayan Rhinella species, to establish the constituents of a preparation sold in the capital city of Paraguay to treat cancer as containing the toad as ingredient, to establish the effect of the most active Rhinella schneideri venom on the cell cycle using human breast cancer cells and to assess the antiprotozoal activity of the venoms. METHODS: The venom obtained from the toads parotid glands was analyzed by HPLC-MS-MS. The preparation sold in the capital city of Paraguay to treat cancer that is advertised as made using the toad was analyzed by HPLC-MS-MS. The effect of the R. schneideri venom and the preparation was investigated on human breast cancer cells. The antiprotozoal activity was evaluated on Leishmania braziliensis, L. infantum and murine macrophages. RESULTS: From the venoms of R. ornata, R. schneideri and R. scitula, some 40 compounds were identified by spectroscopic and spectrometric means. Several minor constituents are reported for the first time. The preparation sold as made from the toad did not contained bufadienolides or compounds that can be associated with the toad but plant compounds, mainly phenolics and flavonoids. The venom showed activity on human breast cancer cells and modified the cell cycle proliferation. The antiprotozoal effect was higher for the R. schneideri venom and can be related to the composition and relative ratio of constituents compared with R. ornata and R. scitula. CONCLUSIONS: The preparation sold in the capital city of Paraguay as containing the toad venom, used popularly to treat cancer did not contain the toad venom constituents. Consistent with this, this preparation was inactive on proliferation of human breast cancer cells. In contrast, the toad venoms of Rhinella species altered the cell cycle progression, affecting the proliferation of malignant cells. The findings suggest that care should be taken with the providers of the preparation and that the crude drug present a strong activity towards human breast cancer cell lines. The antiprotozoal effect of the R. schneideri venom was moderate while the venom of R. ornata was devoid of activity and that of R. scitula was active at very high concentration.

Toxins and pharmacologically active compounds from species of the family Bufonidae (Amphibia, Anura).

ETHNOPHARMACOLOGICAL RELEVANCE: Among amphibians, 15 of the 47 species reported to be used in traditional medicines belong to the family Bufonidae, which demonstrates their potential in pharmacological and natural products research. For example, Asian and American tribes use the skin and the parotoid gland secretions of some common toads in the treatment of hemorrhages, bites and stings from venomous animals, skin and stomach disorders, as well as several types of cancers. OVERARCHING OBJECTIVE: In addition to reviewing the occurrence of chemical constituents present in the family Bufonidae, the cytotoxic and biomedical potential of the active compounds produced by different taxa are presented. METHODOLOGY: Available information on bioactive compounds isolated from species of the family Bufonidae was obtained from ACS Publications, Google, Google Scholar, Pubmed, Sciendirect and Springer. Papers written in Chinese, English, German and Spanish were considered. RESULTS: Recent reports show more than 30% of amphibians are in decline and some of bufonid species are considered to be extinct. For centuries, bufonids have been used as traditional folk remedies to treat allergies, inflammation, cancer, infections and other ailments, highlighting their importance as a prolific source for novel drugs and therapies. Toxins and bioactive chemical constituents from skin and parotid gland secretions of bufonid species can be grouped in five families, the guanidine alkaloids isolated and characterized from Atelopus, the lipophilic alkaloids isolated from Melanophryniscus, the indole alkaloids and bufadienolides known to be synthesized by species of bufonids, and peptides and proteins isolated from the skin and gastrointestinal extracts of some common toads. Overall, the bioactive secretions of this family of anurans may have antimicrobial, protease inhibitor and anticancer properties, as well as being active at the neuromuscular level. CONCLUSION: In this article, the traditional uses, toxicity and pharmacological potential of chemical compounds from bufonids have been summarized. In spite of being reported to be used to treat several diseases, neither extracts nor metabolites from bufonids have been tested in such illness like acne, osteoporosis, arthritis and other illnesses. However, the cytotoxicity of these metabolites needs to be evaluated on adequate animal models due to the limited conditions of in vitro assays. Novel qualitative and quantitative tools based on MS spectrometry and Nuclear Magnetic Resonance spectroscopy is now available to study the complex secretions of bufonids.

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.

On-line comprehensive two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography for preparative isolation of toad venom.

An on-line comprehensive preparative two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography (2D NPLC×RPLC) system was constructed with a newly developed vacuum evaporation assisted adsorption (VEAA) interface, allowing fast removal of NPLC solvent in the vacuum condition and successfully solving the solvent incompatibility problem between NPLC and RPLC. The system achieved on-line solvent exchange within the two dimensions and its performance was illustrated by gram-scale isolation of crude extract from the venom of Bufo bufo gargarizans. Within separation time of ∼20h, 19 compounds were obtained with high purity in a single run. With the VEAA interface, the 2D system exhibited apparent advantages in separation efficiency and automation compared with conventional methods, indicating its promising application in the routine separation process for complicated natural products.

Two novel antimicrobial peptides from skin venoms of spadefoot toad Megophrys minor.

Amphibian skin contains rich bioactive peptides. Especially, a large amount of antimicrobial peptides have been identified from amphibian skin secretions. Antimicrobial peptides display potent cytolytic activities against a range of pathogenic bacteria and fungi and play important defense roles. No antimicrobial peptides have been reported from toads belonging to the family of Pelobatidae. In this work, two novel antimicrobial peptides (Megin 1 and Megin 2) were purified and characterized from the skin venoms of spadefoot toad Megophrys minor (Pelobatidae, Anura, Amphibia). Megin 1 had an amino acid sequence of FLKGCWTKWYSLKPKCPF-NH2, which was composed of 18 amino acid residues and contained an intra-molecular disulfide bridge and an amidated C-terminus. Megin 2 had an amino acid sequence of FFVLKFLLKWAGKVGLEHLACKFKNWC, which was composed of 27 amino acid residues and contained an intra-molecular disulfide bridge. Both Megin 1 and Megin 2 showed potential antimicrobial abilities against bacteria and fungi. The MICs of Megin 1 against Escherichia coli, Bacillus dysenteriae, Staphylococcus aureus, Bacillus subtilis, and Candida albicans were 25, 3, 6.25, 3, and 50 µg·mL(-1), respectively. The corresponding MICs for Megin 2 were 6.25, 1.5, 12.5, 1.5, and 12.5 µg·mL(-1), respectively. They also exerted strong hemolytic activity against human and rabbit red cells. The results suggested that megin peptides in the toad skin of M. minor displayed toxic effects on both eukaryotes and prokaryotes. This was the first report of antimicrobial peptides from amphibians belonging to the family of Pelobatidae.

Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

Traditional Chinese medicine: a treasured natural resource of anticancer drug research and development.

To discover and develop novel natural compounds, active ingredients, single herbs and combination formulas or prescriptions in traditional Chinese medicine (TCM) with therapeutic selectivity that can preferentially kill cancer cells and inhibit the amplification of cancer without significant toxicity is an important area in cancer therapy. A lot of valuable TCMs were applied as alternative or complementary medicines in the United States and Europe. But these TCMs, as one of the main natural resources, were widely used to research and develop new drugs in Asia. In TCMs, some specific herbs, animals, minerals and combination formulas were recorded and exploited due to their active ingredients and specific natural compounds with antitumor activities. The article focused on the antitumor properties of natural compounds and combination formulas or prescriptions in TCMs, described its influence on tumor progression, angiogenesis, metastasis, and revealed its mechanisms of antitumor and inhibitory action. Among the nature compounds, triptolide, berberine, matrine, oxymatrine, kurarinone and deoxypodophyllotoxin (DPT) with specific molecular structures have been separated, purified, and evaluated their antitumor properties in vitro and in vivo. Cancer is a multifactorial and multistep disease, so the treatment effect of combination formulas and prescriptions in TCMs involving multi-targets and multi-signal pathways on tumor may be superior than that of agents targeting a single molecular target alone. Shi Quan Da Bu Tang and Yanshu injection, as well known combination formulas and prescriptions in TCMs, have shown an excellent therapeutic effect on cancer.

Two-dimensional countercurrent chromatography × high performance liquid chromatography for preparative isolation of toad venom.

In this work, a new on-line two-dimensional chromatography coupling of flow programming counter-current chromatography and high-performance liquid chromatography (2D CCC×HPLC) was developed for preparative separation of complicated natural products. The CCC column was used as the first dimensional isolation and a preparative ODS column operated in reversed-phase (RP) mode as the second dimension. The CCC was operated at a controlled flow rate to ensure that each fraction eluted within one hour, corresponding to the isolation time of the 2nd dimensional preparative HPLC. The eluent from the 1st dimensional CCC was diluted using a makeup pump and trapped onto holding column, before been eluted and transferred to the 2nd dimensional HPLC. The performance of the holding column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure, for the solution to the issue of online trapping of low pressure eluent from a CCC column. Satisfactory trapping efficiency and tolerable CCC pressure can be achieved using a commercially available 15mm×30mm i.d. ODS pre-column. The present integrated system was successfully applied in a one-step preparative separation of 12 compounds, from the crude methanol extract of venom of Bufo bufo gargarizans. Compounds 1-12 were isolated in overall yield of 1.0%, 0.8%, 2.0%, 1.3%, 2.0%, 1.5%, 1.9%, 3.6%, 6.1%, 4.8%, 3.5% and 4.1%, with HPLC purity of 99.9%, 99.7%, 90.6%, 99.9%, 77.0%, 99.9%, 90.4%, 99.9%, 52.0%, 99.9%, 99.3%, and 85.0%, respectively. All the results demonstrate that the flow programming CCC×HPLC method is an efficient and convenient way for the separation of compounds from toad venom and it can also be applied to isolate other complex multi-component natural products.

Histrionicotoxin alkaloids finally detected in an ant.

Workers of the ant Carebarella bicolor collected in Panama were found to have two major poison-frog alkaloids, cis- and trans-fused decahydroquinolines (DHQs) of the 269AB type, four minor 269AB isomers, two minor 269B isomers, and three isomers of DHQ 271D. For the first time in an ant, however, the DHQs were accompanied by six histrionicotoxins (HTXs), viz., 283A, 285A, 285B, 285C, 287A, and 287D. This co-occurrence of the HTX and DHQ alkaloids is the usual pattern seen in dendrobatid frogs. This finding contrasts with our earlier study, where workers of a Brazilian ant, Solenopsis (Diplorhoptrum) sp., were found to have a very similar DHQ complex but failed to show HTXs. Several new DHQ alkaloids of MW 271 (named in the frog as 271G) are reported from the above ants that have both m/z 202 and 204 as major fragment ions, unlike the spectrum seen for the poison-frog alkaloid 271D, which has only an m/z 204 base peak. Found also for the first time in skin extracts from the comparison frog Oophaga granulifera of Costa Rica is a trace DHQ of MW 273. It is coded as 273F in the frog; a different isomer is found in the ant.

[In vitro anti-proliferation effect of peptides from cinobufacini injection].

Cinobufacini is an aqueous extract of Bufo bufo gargarizans Cantor dried skin, which has been widely used for cancer therapy in China. So far, its active components are still not very clear. In previous reports, bufadienolides with low-concentration were usually studied because of their anticancer effects. However, the high polarity constituents in cinobufacini are less investigated. The present study found that more than 50% contents of cinobufacini were water-soluble peptides. Then, in vitro anticancer experiments were carried out, including human stomach cancer cell lines BGC823 and MCG803, human colon cancer cell lines DLD-1 and HT-29, and human pancreatic cancer cell line MIAPACA-2. The IC50 for these cell lines model were ranged from 25-123 microgmL(-1). The results indicated that these peptides showed similar activity with cinobufacini injection. As a conclusion, this study provides a new and further understanding of anticancer components in cinobufacini injection.

Immunomodulatory effects of cinobufagin isolated from Chan Su on activation and cytokines secretion of immunocyte in vitro.

The objective of this study was to evaluate the immunomodulatory effects of cinobufagin (CBG) isolated from Chan Su (Venenum Bufonis) in vitro. In this paper, our results show that CBG significantly stimulated cell proliferation of splenocytes and peritoneal macrophages (PMΦ) and markedly enhanced the phagocytic activation of PMΦ. CBG also significantly increased CD4(+)CD8(+) double-positive T-cell populations and the percentage of S-phase cells of splenic lymphocytes. The levels of several Th1 cytokines, including interferon-γ and tumor necrosis factor-α, are significantly increased after CBG treatment, whereas the levels of the Th2 cytokine interleukin-4 and interleukin-10 are significantly decreased. As a result, the ratio of Th1/Th2 also increased. Taken together, these results indicated that CBG had potential immune system regulatory effects and suggested that this compound could be developed as a novel immunotherapeutic agent to treat immune-mediated diseases such as cancer.

Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin.

A protein (BMP1) was purified from common Indian toad (Bufo melanostictus, Schneider) skin through DEAE cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the BMP1 was found to be 79 kDa. BMP1 (0.5 and 1 mg/kg/day, i.p.) significantly decreased the number of viable Ehrlich ascites carcinoma (EAC) cells, thereby increased the lifespan of EAC bearing mice (p<0.001). MTT values reduced significantly with the treatment of BMP1 (0.5 and 1.0 mg/kg/day, i.p. for 3 days) on EAC cells indicated its antiproliferative activity. This was also supported by flow-cytometric data on the cell cycle arrest at G1 in EAC cells. BMP1 (1 mg/kg) reduced the solid tumor weight and volume of about three times further support the antiproliferative nature. Fluorescence and confocal microscopic study on EAC cells after BMP1 (0.5 mg/kg/day, i.p. for 3 days) treatment indicated certain features of apoptosis, like nuclear fragmentation, membrane blebbing, and vacuolization of cells. DNA fragmentation was clearly observed in alkaline comet assay. Apoptosis induced by BMP1 was further confirmed through flow-cytometric analysis of annexin-V binding study, sub-G1 arrest in the cell cycle and found to be mediated through caspase 3 dependent pathway. LD50 of BMP1 was found to be 12.2 mg/kg, i.p. in male Swiss albino mice. BMP1 treatment at 0.5 mg/kg and 1.0 mg/kg for 10 days did not alter any hematological and biochemical parameters in mice, but after 30 days of treatment produce significant rise in total leucocyte count, neutrophil percentage, serum urea, creatinine, GOT, LDH and decrease in lymphocyte percentage as compared to respective control. In conclusion, BMP1, a protein molecule isolated from Indian toad (B. melanostictus, Schneider) skin, showed antiproliferative and apoptogenic activity on EAC cancer cell with limited toxicity.

Phantasmidine: an epibatidine congener from the ecuadorian poison frog Epipedobates anthonyi.

The skin of the Ecuadorian poison frog Epipedobates anthonyi contains the potent nicotinic agonists epibatidine (1) and N-methylepibatidine (3). In addition, a condensed tetracyclic epibatidine congener has been identified with activity at nicotinic acetylcholine receptors, but different selectivity than epibatidine. This rigid tetracycle has been named phantasmidine (4). Phantasmidine has a molecular formula of C(11)H(11)N(2)OCl, shares a chloropyridine moiety with 1, and also contains furan, pyrrolidine, and cyclobutane rings. A combination of GC-MS and GC-FTIR analysis with on-column derivatization, 1D NMR spectroscopy with selective irradiation, and spectral simulation, along with 2D NMR, were used to elucidate the structure from a total sample of approximately 20 microg of HPLC-purified 4 and its corresponding acetamide (5). After synthesis, this novel rigid agonist may serve as a selective probe for beta4-containing nicotinic receptors and potentially lead to useful pharmaceuticals.

N-methyldecahydroquinolines: an unexpected class of alkaloids from Amazonian poison frogs (Dendrobatidae).

The dominant alkaloids previously identified in skin extracts of Amazonian dendrobatid frogs of the genus Ameerega are histrionicotoxins and 2,5-disubstituted decahydroquinolines. Analysis of alkaloids in skin extracts of Ameerega picta from Bolivia revealed that the alkaloid 257A, previously reported as a 2,5-disubstituted decahydroquinoline, is an N-methyl-2,5-disubstituted decahydroquinoline. We characterized alkaloids of another 12 of the more than 25 species recently assigned to the genus Ameerega, and five additional N-methyldecahydroquinolines were identified. In some cases, the relative configuration of the N-methyldecahydroquinolines was determined by comparison with the N-methylated products prepared from the corresponding 2,5-disubstituted decahydroquinolines of known relative configuration. A dietary source for N-methyldecahydroquinolines is unknown; however, myrmicine ants are the likely source for the 2,5-disubstituted decahydroquinolines. The alkaloids in skin extracts of three species of another genus of Amazonian poison frog, Adelphobates, were also characterized, but N-methyldecahydroquinolines were not detected.

Epiquinamide: a poison that wasn't from a frog that was.

In 2003, we reported the isolation, structure elucidation, and pharmacology of epiquinamide (1), a novel alkaloid isolated from an Ecuadoran poison frog, Epipedobates tricolor. Since then, several groups, including ours, have undertaken synthetic efforts to produce this compound, which appeared initially to be a novel, beta2-selective nicotinic acetylcholine receptor agonist. Based on prior chiral GC analysis of synthetic and natural samples, the absolute structure of this alkaloid was established as (1S,9aS)-1-acetamidoquinolizidine. We have synthesized the (1R*,9aS*)-isomer (epi-epiquinamide) using an iminium ion nitroaldol reaction as the key step. We have also synthesized ent-1 semisynthetically from (-)-lupinine. Synthetic epiquinamide is inactive at nicotinic receptors, in accord with recently published reports. We have determined that the activity initially reported is due to cross-contamination from co-occurring epibatidine in the isolated material.

Alkaloids from amphibian skin: a tabulation of over eight-hundred compounds.

A diverse array of biologically active, lipid-soluble alkaloids have been discovered in amphibian skin. Such alkaloids include the following: the steroidal samandarines from salamanders, the batrachotoxins, histrionicotoxins, gephyrotoxins, and epibatidine from neotropical poison frogs (Dendrobatidae), the pumiliotoxins, allopumiliotoxins, homopumiliotoxins, and decahydroquinolines from certain genera of anurans from four families (Dendrobatidae, Mantellidae, Bufonidae, and Myobatrachidae), a variety of izidines (pyrrolizidines, indolizidines, quinolizidines, lehmizidines), pyrrolidines, piperidines, various tricyclics (related in structures to the coccinellines), and spiropyrrolizidines from the first three of these four families, the pseudophrynamines from one genus of Australian frogs, and a variety of unclassified alkaloids as yet of undetermined structure. With the exception of the samandarines and the pseudophrynamines, all alkaloids appear to be derived from dietary sources. Although only a few of the over 800 amphibian skin alkaloids have been detected in arthropods, putative arthropod sources for the batrachotoxins and coccinelline-like tricyclics (beetles), the pumiliotoxins (ants, mites), the decahydroquinolines, izidines, pyrrolidines, and piperidines (ants), and the spiropyrrolizidines (millipedes) have been discovered. Ants are likely sources for histrionicotoxins, lehmizidines, and tricyclic gephyrotoxins. Epibatidines represent an important alkaloid class without a putative dietary source. The structures for many of these alkaloids have been rigorously established, while the structures of others represent tentative proposals, based only on mass spectral and FTIR spectral data, along with analogies to structures of well-defined alkaloids.

Studies on the poisonous skin secretion of individual red bellied toads, Melanophryniscus montevidensis (Anura, Bufonidae), from Uruguay.

Toads belonging to the genus Melanophryniscus contain toxic alkaloids in their skin. From six locations in south-eastern Uruguay 81 specimens of Melanophryniscusmontevidensis were collected. In whole animal methanolic extracts of individual specimens, alkaloids of the pumiliotoxin (PTX) group and indolizidines were identified by gas chromatography/mass spectrometry; the predominant component PTX 251D was assayed quantitatively. The PTX-content of the various toad populations was found to be highly variable among individual specimens as well as among the populations. Very high levels of PTX 251D were detected in toads of the western part of the collection area, whereas very low levels of this alkaloid were assayed in toads near the Brazilian border. Remarkably high concentrations of the non-alkaloid hydroquinone were found to be present in all toads. The analysis of extracts from 125 arthropod samples (Arachnida and Insecta, including termites, ants and beetles), which may represent a potential food source, revealed no alkaloids of the PTX group.

Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi.

Amphibian skin secretions contain many bioactive compounds. In the present work, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by successive ion-exchange and gel-filtration chromatography. Baserpin is a single chain glycoprotein, with an apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin-trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpin superfamily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6 x 10(6) M(-1) s(-1)), bovine chymotrypsin (8.9 x 10(6) M(-1) s(-1)) and porcine elastase (6.8 x 10(6) M(-1) s(-1)), whereas it shows no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors.