Use of whole-body cryosectioning and desorption electrospray ionization mass spectrometry imaging to visualize alkaloid distribution in poison frogs.

Ambient mass spectrometry is useful for analyzing compounds that would be affected by other chemical procedures. Poison frogs are known to sequester alkaloids from their diet, but the sequestration pathway is unknown. Here, we describe methods for whole-body cryosectioning of frogs and use desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to map the orally administered alkaloid histrionicotoxin 235A in a whole-body section of the poison frog Dendrobates tinctorius. Our results show that whole-body cryosectioning coupled with histochemical staining and DESI-MSI is an effective technique to visualize alkaloid distribution and help elucidate the mechanisms involved in alkaloid sequestration in poison frogs.

Quantitative analysis of fourteen bufadienolides in Venenum Bufonis crude drug and its Chinese patent medicines by ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Venenum Bufonis, a product of the secretions of Bufo gargarizans Cantor or B. melanostictus Schneider, possessed an array of pharmacological activities, such as cardiotonic, anti-tumor, antinociceptive, anti-inflammatory, anesthetic and antimicrobial activities. However, there were few efficient methods for quality evaluation of Venenum Bufonis medicinal materials and its related Chinese patent medicines. AIM OF THE STUDY: To establish an effective method for quality assessment of crude drugs and Chinese proprietary medicines of Venenum Bufonis, and explore the relationship of primary compounds - target - pathway - disease through a series of network databases. MATERIALS AND METHODS: An ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-QqQ-MS/MS) method was developed and validated to simultaneously determine 14 bufadienolides for quantitative analysis of 71 batches of crude drugs and 20 kinds of Chinese patent medicines of Venenum Bufonis. Multiple reaction monitoring with good specificity and accuracy was applied to monitor the 14 bufadienolides in positive mode. RESULTS: The methodology was validated with good specificity, precision, stability, repeatability and recovery. The low limits of quantification were in the range of 0.1-2.7 ng/mL. The relative standard deviation values for intra- and inter-day precisions ranged from 0.98% to 6.3% and from 2.39% to 6.76%, respectively. The recovery was varied from 87.78% to 110.57% for crude drugs and 88.32%-100.96% for Chinese proprietary medicine (Shexiang Baoxin Pill). The contents of 14 analytes in 71 batches of crude drugs and 20 sorts of Chinese proprietary medicines were procured, the results showed that the contents of crude drugs collected from the market exhibited great variations. Furthermore, 13 batches of crude drugs were identified as counterfeit with no bufadienolides detected. In addition, the total contents of bufadienolides in the same drug showed great difference among products from various manufacturers or brands. Subsequently, 9 bufadienolides with the higher contents were applied to screen the anti-tumor effect by network pharmacology, and 8 pathways which had prior correlation with bufadienolides were disclosed. CONCLUSION: This method could be used for quality assessment of crude drugs and Chinese patent medicines of Venenum Bufonis, and the data could be served as the fundamental basis for drug research and development of Venenum Bufonis.

A strategy for the metabolomics-based screening of active constituents and quality consistency control for natural medicinal substance toad venom.

Natural medicinal substances (NMS) have great inconsistency due to chemical variability, seriously limiting their development as therapeutics. Here, we chose toad venom with anti-tumor activities as a model and developed a pipeline of metabolomics-based screening and quality consistency control (MSQCC) as one potential solution to this long-standing problem. Our study firstly exemplified the power of the co-correlation screen of metabolomic and biological profiles of 180 fractions prepared from natural heterogeneous venom samples, to identify a series of bufadinolides as quality control markers for cancer cell inhibition. The next quantitative monitoring of these markers revealed great batch-to-batch variation of toad venoms. Finally, we developed a marker-based blending program (Markers-NMBT) to normalize heterogeneity of NMS. It created the blends for the conversion of the unqualified venoms with high variation in the contents of bufadienolides, into qualified products consistent with the reference. Thus, this work provides a strategy for rapid, large-scale discovery, quantification and application of quality control markers to ensure batch-to-batch consistency, and can be a crucial technology in the development of modern NMS preparations.

[Study on quality control method of toad venom based on characteristic chromatogram and QAMS].

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

Metabolomics method based on ultra high performance liquid chromatography with time-of-flight mass spectrometry to analyze toxins in fresh and dried toad venom.

Drying is a critical step to prolong the storage time in natural medicine processing but it changes the chemical characteristics of the product. In this study, research was performed to characterize the metabolomic changes in toad venom induced by vacuum-drying at 60°C and air-drying at room temperature by ultra high performance liquid chromatography coupled with pattern recognition approaches. In total 52 metabolites, down-regulated or up-regulated, were identified as potential chemical markers. Compared with fresh toad venom, vacuum-drying at 60°C succeeded in raising the conjugated-type bufadienolide content significantly, while the content of free-type bufadienolides were slightly reduced. On the other hand, toad venom air-dried at room temperature presented a relatively low amount of bufadienolides compared with fresh venom. For example, the content of several known anti-tumor components (gamabufotalin, bufotalin, cinobufagin, etc.) were significantly reduced. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide bioassay further showed that venom air-dried at room temperature had weaker anti-tumor activity on human hepatocellular carcinoma SMMC-7721 proliferation in vitro than samples vacuum-dried at 60°C. These results showed that the great metabolomic changes of toad venom occurred during the drying process, suggesting that a proper drying procedure is important for sustaining the chemical quality of natural medicines.

Evidence of maternal provisioning of alkaloid-based chemical defenses in the strawberry poison frog Oophaga pumilio.

Many organisms use chemical defenses to reduce predation risk. Aposematic dendrobatid frogs sequester alkaloid-based chemical defenses from a diet of arthropods, but research on these defenses has been limited to adults. Herein, we investigate chemical defense across development in a dendrobatid frog, Oophaga pumilio. This species displays complex parental care: at hatching, mothers transport tadpoles to phytotelmata, and then return to supply them with an obligate diet of nutritive eggs for about six weeks. We collected eggs, tadpoles, juveniles, and adults of O. pumilio, and detected alkaloids in all life stages. The quantity and number of alkaloids increased with frog and tadpole size. We did not detect alkaloids in the earliest stage of tadpoles, but alkaloids were detected as trace quantities in nutritive eggs and as small quantities in ovarian eggs. Tadpoles hand-reared with eggs of an alkaloid-free heterospecific frog did not contain alkaloids. Alkaloids that are sequestered from terrestrial arthropods were detected in both adults and phytotelm-dwelling tadpoles that feed solely on nutritive eggs, suggesting that this frog may be the first animal known to actively provision post-hatch offspring with chemical defenses. Finally, we provide experimental evidence that maternally derived alkaloids deter predation of tadpoles by a predatory arthropod.

The glycogen synthase kinase-3ß/nuclear factor-kappa B pathway is involved in cinobufagin-induced apoptosis in cultured osteosarcoma cells.

Cinobufagin, a major component of cinobufacini (huachansu), is an important cardenolidal steroid. Several studies have suggested that cinobufagin has potent anti-cancer effects. The present study examines the apoptosis-inducing activity and the underlying mechanism of action of cinobufagin in osteosarcoma (OS) cells. Our results showed that cinobufagin potently inhibited the proliferation of U2OS, MG63 and SaOS-2 cells. Significant increases in G2/M cell-cycle arrest and apoptosis in OS cells were also observed. The expression levels of several apoptotic proteins were assessed after cinobufagin treatment in U2OS cells. Among them, xIAP, cIAP-1, survivin and Bcl-2 levels decreased remarkably, while the levels of Bax and cleaved-PARP increased. Furthermore, we validated the inhibition of GSK-3ß/NF-κB signaling following cinobufagin treatment. Western blots showed a decrease in nuclear p65 protein expression after exposure to different concentrations of cinobufagin, while the phosphorylation of GSK-3ß was simultaneously increased. Transduction with constitutively active forms of GSK-3ß could protect against the downregulation of p65 and upregulation of cleaved-PARP that are induced by cinobufagin treatment. However, combined treatment with cinobufagin and SB216367 resulted in a significant reduction in p65 and an increase in cleaved-PARP in U2OS cells. Altogether, these results show that cinobufagin is a promising agent for the treatment of OS. These studies are the first to reveal the involvement of the GSK-3ß/NF-κB pathway in cinobufagin-induced apoptosis.

[Quod medicina aliis, aliis est acre venenum**--venoms as a source of anticancer agents]. / Quod medicina aliis, aliis est acre venenum*--jady jako zródlo substancji o dzialaniu przeciwnowotworowym.

Natural product derived from plants and animals were used in folk medicine for centuries. The venoms produced by animals for hunting of self-defence are rich in bioactive compounds with broad spectrum of biological activity. The papers presents the most promising compounds isolated from venoms of snakes, scorpions and toads. For these compounds both: mechanism of anticancer activity as well as possibilities of clinical use are presented.

PTM-driven differential peptide display: survey of peptides containing inter/intra-molecular disulfide bridges in frog venoms.

Amphibian defensive skin secretions are complex species-specific mixtures of biologically active molecules, including many uncharacterized peptides. Many of these peptides are post-translationally modified and amongst the modifications discovered so far on amphibian defense peptides, disulfide bonds are quite frequently encountered. The presence of this PTM often complicates the MS-based sequencing. Here we demonstrate a method to target peptides containing inter/intra-molecular S-S bonds applying a PTM-driven differential display. Upon reduction of the disulfide bond both molecular mass and retention time of a peptide are altered. Assembling the LC-MS data by plotting the m/z data against retention time generates a peptide display and overlaying peptide displays of untreated and DTT-reduced material yields a differential display. From such an overlay, peptides originally carrying a disulfide bond are recognized due to the shift in both retention time and m/z values, whereas non cystine containing peptides remain unaltered in the differential display. The success of this approach is demonstrated by the visualization of the cystines-containing peptides in the skin secretion of Odorrana schmackeri, Phyllomedusa burmeisteri, Phyllomedusa rohdei, Kassina senegalensis, and Bombina variegata. The venoms from these different species yield complicated differential displays, showing interesting peptides, allowing one to target them for more detailed structural characterization.

Discovery of skin alkaloids in a miniaturized eleutherodactylid frog from Cuba.

Four phylogenetically independent lineages of frogs are currently known to sequester lipid-soluble skin alkaloids for which a dietary source has been demonstrated. We report here a remarkable fifth such instance, in Eleutherodactylus iberia and Eleutherodactylus orientalis, two species of miniaturized frogs of the family Eleutherodactylidae from Cuba. Six pumiliotoxins and two indolizidines were found in E. iberia, one of the smallest frogs in the world and characterized by a contrasting colour pattern for which we hypothesize an aposematic function. Analyses of stomach content indicated a numerical prevalence of mites with an important proportion of oribatids-a group of arthropods known to contain one of the pumiliotoxins detected in E. iberia. This suggests that miniaturization and specialization to small prey may have favoured the acquisition of dietary skin alkaloids in these amphibians.

Roughing it: a mantellid poison frog shows greater alkaloid diversity in some disturbed habitats.

Four five-skin alkaloid extracts of the Madagascan poison frog Mantella baroni from three disturbed collection sites were compared with four five-skin extracts from three undisturbed sites. The number of alkaloids (diversity) was significantly different in M. baroni between undisturbed and disturbed collection sites, with more alkaloids generally being found in frogs from disturbed sites. Two undisturbed sites did not differ from two disturbed sites, but the third disturbed site (coded 6) had more than twice the alkaloid diversity found in frogs from the third undisturbed site (coded 5a/5b). There was no difference in the quantity of alkaloids in M. baroni between undisturbed and disturbed collection sites. The hypothesis that an undisturbed habitat confers a benefit to poison frogs dwelling therein, in allowing for the sequestration of greater alkaloid diversity and amounts, is challenged by our results. In the course of our study, we found that collections of frogs separated by an interval of three months at an undisturbed site differed by only 4% in alkaloid composition over this period, whereas frogs collected at a disturbed site and collected approximately three months later already had a 26% difference in alkaloid composition between the two collections. This constancy of skin alkaloid composition likely reflects a constancy of dietary prey items consumed by frogs at undisturbed sites.

Sex-related differences in alkaloid chemical defenses of the dendrobatid frog Oophaga pumilio from Cayo Nancy, Bocas del Toro, Panama.

Poison frogs contain an alkaloid-based chemical defense that is sequestered directly from a diet of alkaloid-containing arthropods. Geographic and temporal variation in alkaloid defense is common in poison frogs and is generally attributed to differences in the availability of alkaloid-containing arthropods. Variable chemical defense in poison frogs may have important consequences for predator-prey interactions, requiring a full understanding of the factors involved in explaining such variation. In the present study, we examine alkaloid variation in the dendrobatid poison frog Oophaga pumilio between males and females on Cayo Nancy (Isla Solarte), located in the Bocas del Toro archipelago of Panama. On average, females contained a significantly larger number and quantity of alkaloids when compared to males. Alkaloid composition varied significantly between males and females, illustrating that chemical defense in this population of O. pumilio is sex-dependent. The variation in alkaloids between sexes is attributed to differences in feeding and behavior between males and females. The majority of alkaloids present in the skin of O. pumilio appear to be of oribatid mite origin, supporting the importance of these dietary arthropods in the chemical defense of poison frogs.

Anfíbios e répteis do Parque Nacional da Serra da Bodoquena, Mato Grosso do Sul, Brasil / Amphibians and reptiles of the Serra da Bodoquena National Park, Mato Grosso do Sul, central Brazil

O Estado de Mato Grosso do Sul situa-se na porção mediana da grande diagonal de formações abertas da América do Sul, uma região que abriga uma grande diversidade de anfíbios e répteis, sendo algumas endêmicas. Apesar de sua importância biogeográfica, essa diagonal tem sido pouco considerada em estudos com abrangência regional. Dentre os componentes fitofisionômicos que compõem essa região e cuja representatividade se faz presente no Estado, as matas estacionais deciduais são elementos marcantes na região da Serra da Bodoquena. Devido a peculiaridade desse ambiente, muitas áreas ainda necessitam de uma maior avaliação quanto à sua biodiversidade. Informações sobre a herpetofauna do Parque Nacional da Serra da Bodoquena são escassas e pontuais e sugerem uma semelhança com a fauna de cerrado. Este trabalho apresenta registros da herpetofauna do Parque Nacional da Serra da Bodoquena e regiões de entorno, abrangendo áreas em bom estado de conservação e locais com atividade agropecuária. As amostragens foram realizadas em dois períodos, contemplando as estações seca e chuvosa na região. O inventário consistiu em busca ativa limitada por tempo, além de registros oportunísticos. Informações sobre a riqueza de espécies da herpetofauna da Caatinga, Cerrado e Chaco foram compiladas da literatura e comparadas com aquela registrada no presente trabalho. Foram registradas 63 espécies, sendo 38 anfíbios e 25 répteis. Muitas das espécies estavam associadas a ambientes particulares, como matas de galeria e afloramentos rochosos. A maior similaridade da herpetofauna do Parque Nacional da Serra da Bodoquena deu-se com aquela encontrada no Cerrado, provavelmente porque a área está inserida nesse bioma. Considerando-se a diversidade de hábitats da área, provavelmente o número de espécies deverá aumentar com a realização de novos inventários na região. Agregar novas informações sobre a herpetofauna da Serra da Bodoquena é fundamental para o plano de manejo...

The State of Mato Grosso do Sul, central Brazil, is located in the center of the broad South American open-dry diagonal areas. This region harbors a great diversity of amphibians and reptiles, with some endemic species. Despite such biogeographical scenario, biodiversity of this region is poorly known at local scales. Dry forests are important physiognomies in Mato Grosso do Sul and represent an important landscape element at Serra da Bodoquena, resulting in local characteristics that require biodiversity surveys. There are few and punctual information on the herpetofauna from the Serra da Bodoquena National Park and they suggest a similarity with the Cerrado herpetofauna. The present study represents the first record of the herpetofauna from the Serra da Bodoquena National Park and adjacent areas. Samples were conducted in pristine and disturbed areas in two distinct climatic periods, covering both rainy and dry seasons, by means of active search limited by time as well as opportunistic records. Data on the herpetofauna of the Caatinga, Cerrado, and Chaco biomes were compiled and compared with the species sampled in the present study. We registered 63 species, including 38 amphibians and 25 reptiles. Several species were associated to particular habitats, such as gallery forests or rocky outcrops. The herpetofauna from the Serra da Bodoquena National Park showed a greater similarity with that of the Cerrado, probably because the area is inserted in this biome. Considering the habitat diversity found at Serra da Bodoquena, the number of species must increase with other surveys in the area. Natural history and ecological studies on amphibian and reptiles of the Serra da Bodoquena must be encouraged in order to contribute for the National Park management.

Structural and functional analogs of the novel mammalian neuropeptide, neuromedin S (NmS), in the dermal venoms of Eurasian bombinid toads.

We report the isolation and structural characterization of two neuromedin S (NmS) analogs, (NmS-17 and NmS-33), from the dermal venoms of Eurasian bombinid toads. NmS is a novel neuromedin U (NmU)-related peptide with potent anorexigenic and circadian rhythm-modulating properties recently discovered in mammals. Cloning of NmS precursor-encoding cDNAs from skin venom-derived libraries revealed the presence of a high degree of transcript splice variation comparable to that found previously for NmU in both amphibian skin and mammalian brain. Synthetic replicates of both amphibian NmS peptides evoked robust and dose-dependent transient increases in intracellular calcium ion concentrations in CHO cells that had been stably transfected with either FM-3/GPR66 or FM-4/TGR-1 human NmU receptors. The potency and efficacy of these amphibian skin peptides at such receptors were comparable to those observed with human NmS and rat NmS. These data show that NmS and NmU genes had already diverged at the level of the Amphibia and that differential splicing of their transcribed mRNAs has been highly conserved throughout tetrapod vertebrate evolution indicative of fundamental biological function. NmS is additionally a novel neuropeptide homolog that can be added to the biologically active peptide arsenal of amphibian venom/defensive skin secretions.

Rapid separation and determination of resibufogenin and cinobufagin in toad venom and Liushen tablet by beta-cyclodextrin modified micellar electrokinetic chromatography.

A rapid cyclodextrin modified micellar electrokinetic chromatography (CD-MEKC) method was proposed for the determination of resibufogenin and cinobufagin in the Chinese herbal extracts from toad venom and its medicinal preparation (Liushen tablet). The two components have the close structural similarity and similar hydrophobicity, which result in poor resolution in normal MEKC. The addition of neutral beta-CD to the MEKC system was found to improve the separation of the studied compounds. The effects of several CD-MEKC parameters on the resolutions were evaluated systematically. Based on the investigation, a background electrolyte solution consisting of 10 mM borate buffer adjusted to pH 8.5, 40 mM sodium dodecyl sulfate (SDS), 12 mM beta-CD and 10% (v/v) of methanol was found to be optimal conditions for the fast separation. The contents of resibufogenin and cinobufagin were successfully determined within 5 min, with satisfactory repeatability and recovery.

Efficacy of digoxin specific Fab fragments (Digibind) in the treatment of toad venom poisoning.

Chan Su, a traditional Chinese medication, and Love Stone, a topical aphrodisiac, are both made from dried venom of the toad bufo bufo gargarizans and contain bufalin, cinobufotalin, cinobufagin, and other cardioactive steroids of the bufadienolide class. Deaths have occurred following ingestion of these products and the clinical course resembles digoxin toxicity. The purpose of this study was to determine the efficacy of digoxin specific Fab fragments in treating Chan Su poisoning. An ethanolic extract was prepared from Chan Su. Digoxin specific Fab fragments were reconstituted in normal saline to a concentration of 80 mg/ml. An approximate LD90 dose was determined in preliminary experiments. Mice were then randomly divided into a treatment group of 15 mice and a control group of 30 mice. The treatment group was pretreated with 20 ml/kg of digoxin specific Fab fragment solution by intraperitoneal injection at t = 0, followed by 10 ml/kg of digoxin specific Fab fragments intraperitoneal at t = 30 min. The control group was pretreated with equal volumes of intraperitoneal normal saline at the same times. Immediately following the 30 min injection, both groups were given the estimated LD90 dose of Chan Su extract by subcutaneous injection. An endpoint of survival at 6 h was chosen after preliminary results showed that all deaths occurred in the first 4 h. All 30 of the control mice had seizures followed by death compared to 11 seizures and 7deaths in the 15 treatment mice. These results were statistically significant by Fisher's exact test (p = 0.00003 for mortality and p = 0.009 for seizures). Digoxin specific Fab fragments are effective in the treatment of Chan Su poisoning in mice and may be effective for poisoning by other cardioactive steroids of the bufadienolide class.

Absence of tetrodotoxins in a captive-raised riparian frog, Atelopus varius.

Bufonid frogs of the genus Atelopus contain two classes of skin toxins, namely the steroidal bufadienolides and the water-soluble tetrodotoxins. Frogs of the Panamanian species Atelopus varius have now been raised in captivity and levels in skin extracts of bufadienolides and of tetrodotoxin-like compounds assessed, using inhibition of [3H]ouabain binding and inhibition of [3H]saxitoxin binding, respectively. Levels of ouabain equivalents, corresponding to bufadienolides, were comparable to those found in wild-caught frogs from the same population in Panama, while tetrodotoxin-like activity was undetectable. The results strongly implicate environmental factors, perhaps symbiotic microorganisms, in the genesis of tetrodotoxins in the skin of frogs of the genus Atelopus, while indicating that the frog itself produces the skin bufadienolides.

Treatment of toad venom poisoning with digoxin-specific Fab fragments.

Toxicity from toad venom poisoning is similar to digoxin toxicity and carries a high mortality rate. We report on six previously healthy men who developed vomiting and bradycardia after ingesting a purported topical aphrodisiac. Each patient had positive apparent digoxin levels and the first four patients died of cardiac dysrhythmias. The last two patients recovered following treatment with digoxin Fab fragments. We analyzed samples of the purported aphrodisiac and found that it was identical to Chan Su, a Chinese medication made from toad venom. To our knowledge, this is the first reported use of digoxin Fab fragments to treat toad venom poisoning.