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Malaria remains a major health issue worldwide that affects many people, particularly in developing nations. Since, the malarial parasite has developed resistance against nearly every antimalarial drug now in use, it is imperative to search for novel antimalarial medications. Toxins produced by skin glands of toads have been shown to possess antiparasitic properties against a variety of protozoan parasites because of the bufadienolides they contain. Even though several studies have been conducted to show that toad skin secretions have antimalarial properties, very little information is known about the precise mechanism by which they work against Plasmodium infection. Thus, the goal of this study was to evaluate the antiplasmodial activity of crude skin extracts from Common Asian Toads, Duttaphrynus melanostictus, of different sizes and illustrate how they work against Plasmodium falciparum 3D7 cells. The findings demonstrated a negative correlation between the toad size and percent yield of the extracts. HPTLC and UPLC-MS/MS analysis of the extracts exhibited varied composition of bufadienolides depending on the size of the animal. The extract obtained from small toads containing resibufagin and marinobufagin lactate demonstrated highest antiplasmodial activity and showed lowest cytotoxicity on peripheral blood mononuclear cells. It was discovered that the extract was effective against the trophozoite stage of the parasite. The extract was reported to inhibit Na+/H+ ATPase of Plasmodium by binding to sodium-enzyme complex at ATP binding site. The study offers baseline data that can be used to assess the antimalarial potential of individual components in the skin extract derived from small toads. [ABSTRACT FROM AUTHOR]

Bryophyllum pinnatum (Lam.) Oken, a multipurpose medicinal herb, has drawn much interest for its therapeutic qualities from both traditional and modern medicine systems. Many active secondary metabolites, such as bufadienolides, triterpenes, phenols, alkaloids, glycosides, lipids, flavonoids, and organic acids, are responsible for the plant's curative properties. B. pinnatum exhibits a noteworthy significance in oncological research by exhibiting its ability to modify numerous pathways, which may suggest a potential anticancer impact. The herb is recommended for treating lithiasis, a common cause of renal failure, due to its effectiveness in dissolving stones and avoiding crystal formation. The plant has a major impact on diabetes, especially type II diabetes. Moreover, the versatility of B. pinnatum extends to its examination in connection to COVID-19. However, caution is warranted, as B. pinnatum has been reported to possess toxicity attributed to the presence of bufadienolides in its metabolic profile. A comprehensive investigation is essential to thoroughly understand and confirm the synthesis of potentially hazardous compounds. This is crucial for minimizing their presence and ensuring the safe consumption of B. pinnatum among diverse populations of organisms. This review highlights the various medical uses of B. pinnatum, including its ability to effectively treat kidney and liver diseases, as well as its anti-leishmanial, neuropharmacological, antibacterial, immunosuppressive, anti-tumour, and cytotoxic effects. While extensively employed in both traditional and scientific domains, the plant's complete medicinal potential, molecular mechanisms, safety profile, and pharmacodynamics remain ambiguous, rendering it an ideal candidate for pioneering research endeavours. [ABSTRACT FROM AUTHOR]

In this study, a combination approach involving macroporous resin (MR) column chromatography and gradient countercurrent chromatography (CCC) was employed to enrich and purify bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch. Initially, a D101 MR‐packed column chromatography was utilized for fractionation and enrichment of the bufadienolides, which were effectively eluted from the column using a 60% ethanol solution. CCC was subsequently introduced to separate the enriched product using the ethyl acetate/n‐butanol/water (EBuWat, 4:1:5, v/v) and EBuWat (5:0:5, v/v) solvent systems in a gradient elution mode. As results, five bufadienolides, including 6.1 mg of hellebrigenin‐3‐O‐β‐D‐glucoside (1), 2.2 mg of tigencaoside A (2), 8.3 mg of deglucohellebrin (3), 3.5 mg of 14 β‐hydroxy‐3β‐[β‐D‐glucopyranosyl‐(1→6)‐(β‐D‐glucopyranosyl)oxy]‐5α‐bufa‐20,22‐dienolide (4), and 3.0 mg of 14β‐hydroxy‐3β‐[(β‐D‐glucopyranosyl)oxy]‐5α‐bufa‐20,22‐dienolide (5), were effectively separated from 300 mg of the enriched product. The respective high‐performance liquid chromatography purities were as follows: 95.2%, 75.8%, 85.7%, 82.3%, and 92.8%. This study provides valuable insights for the efficient enrichment and separation of bufadienolides from Helleborus thibetanus Franch. [ABSTRACT FROM AUTHOR]

The traditional Chinese medicine toad venom (Venenum bufonis) has been extensively used to treat various diseases, including cancers, in China and other Southeast Asian countries. The major constituents of toad venom, e.g., bufadienolides and alkaloids, exhibit broad-spectrum pharmacological effects in cancers. Herein, two new bufadienolides (1 and 2), along with eleven known compounds (3–13) were successfully isolated from Bufo melanostictus Schneider. Their structures were elucidated by extensive spectroscopic data and X-ray diffraction analysis. Furthermore, four lactam derivatives were synthesized through the transformation of bufadienolides lactones. The inhibitory effects of these compounds against human prostate cancer cell lines PC-3 and DU145 were evaluated. The outcomes indicated a notable trend, with a substantial subset displaying nanomolar range IC50 values against PC-3 and DU145 cells, underscoring their pronounced cytotoxicity. Moreover, a noteworthy distinction surfaces, wherein lactones consistently outperformed their lactam counterparts, further validating their heightened potency for the treatment of prostate cancer. This study contributes significant preclinical evidence substantiating the therapeutic viability of bufadienolides and toad venom as intervention strategies for prostate cancer. [ABSTRACT FROM AUTHOR]

Abstract 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 Abstract

Toad Venom (TV) is the dried product of toxic secretions from Bufo bufo gargarizans Cantor (BgC) or B. melanostictus Schneider (BmS). Given the increasing medical demand and the severe depletion of wild toads, a number of counterfeit TVs appeared on the market, posing challenges to its quality control. In order to develop an efficient, feasible, and comprehensive approach to evaluate TV quality, a thorough analysis and comparison of chemical compounds among legal species BgC and BmS, as well as the main confusion species B. andrewsi Schmidt (BaS) and B. raddei Strauch (BrS), were conducted by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), high performance liquid chromatography (HPLC), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Nano LC-MS/MS analyses. We identified 126 compounds, including free or conjugated bufadienolides, indole alkaloids and amino acids, among the four Bufo species. The content of main bufadienolides, such as gamabufotalin, bufotalin, bufalin, cinobufagin, and resibufogenin, and the total protein contents varied widely among 28 batches of TV due to their origin species. The sum of the five bufadienolides within the BgC, BmS, BaS, and BrS samples were 8.15–15.93%, 2.45–4.14%, 11.15–13.50%, and 13.21–14.68%, respectively. The total protein content of BgC (6.9–24.4%) and BaS (19.1–20.6%) samples were higher than that of BmS (4.8–20.4%) and BrS (10.1–13.7%) samples. Additionally, a total of 1357 proteins were identified. There were differences between the protein compositions among the samples of the four Bufo species. The results indicated that BgC TV is of the highest quality; BaS and BrS TV could serve as alternative resources, whereas BmS TV performed poorly overall. This research provides evidence for developing approaches to evaluate TV quality and selecting the proper Bufo species as the origin source of TV listed in the Chinese pharmacopoeia. [ABSTRACT FROM AUTHOR]

Three new bufadienolides with a Δ14,15 double bond, named bufogarlides A-C (1-3), together with three known analogs (4-6), were isolated from the skins of Bufo bufo gargarizans. Their structures were identified by analyses of spectroscopic data (1 D and 2 D NMR, HR-ESIMS), and comparison with the literature data. All the isolates were evaluated for their cytotoxic activities against ovarian carcinoma cell lines A2780 and SKOV3. Among them, compound 5 showed the highest potential for the growth inhibition of cancerous cells A2780 and SKOV3 with the IC50 values of 21.09 and 67.08 nM, respectively. [ABSTRACT FROM AUTHOR]

Amphibians are widely known as a prolific source of bioactive metabolites. In this work, we isolated and characterized compounds with antiparasitic activity from the oocytes of the toad Rhinella alata collected in Panama. Bio-guided isolation and structural elucidation were carried out using chromatographic and spectroscopic techniques, respectively. The organic extract was subjected to solid phase extraction followed by HPLC purification of the fraction with in vitro activity against Trypanosoma cruzi trypomastigotes. Seven steroids (1–7) of the bufadienolide family were isolated, and their structures were determined using NMR and MS analyses; of these 19-formyl-dyscinobufotalin, (3) is reported as a new natural product. Compounds 1 and 3–7 resulted in a good anti-trypanosomal activity profile. Among these, 16β-hydroxyl-hellebrigenin (1) and bufalin (7) showed significant selectivity values of >5 and 2.69, respectively, while the positive control benznidazole showed a selectivity of 18.81. Furthermore, molecular docking analysis showed compounds 1, 3 and 7 interact through H-bonds with the amino acid residues GLN-19, ASP-158, HIS-159 and TRP-177 from cruzipain at the catalytic site. Given the lack of therapeutic options to treat American trypanosomiasis, this work can serve as the basis for further studies that aim for the development of bufadienolides or their derivatives as drugs against Chagas disease. [ABSTRACT FROM AUTHOR]

Toads of the genus Atelopus are chemically defended by a unique combination of endogenously synthesized cardiotoxins (bufadienolides) and neurotoxins which may be sequestered (guanidinium alkaloids). Investigation into Atelopus small-molecule chemical defenses has been primarily concerned with identifying and characterizing various forms of these toxins while largely overlooking their ecological roles and evolutionary implications. In addition to describing the extent of knowledge about Atelopus toxin structures, pharmacology, and biological sources, we review the detection, identification, and quantification methods used in studies of Atelopus toxins to date and conclude that many known toxin profiles are unlikely to be comprehensive because of methodological and sampling limitations. Patterns in existing data suggest that both environmental (toxin availability) and genetic (capacity to synthesize or sequester toxins) factors influence toxin profiles. From an ecological and evolutionary perspective, we summarize the possible selective pressures acting on Atelopus toxicity and toxin profiles, including predation, intraspecies communication, disease, and reproductive status. Ultimately, we intend to provide a basis for future ecological, evolutionary, and biochemical research on Atelopus.

Bufadienolides, naturally found in toad venoms having steroid‐like structures, reveal antiproliferative effects at low doses. However, their application as anticancer drugs is strongly prevented by their Na+/K+‐ATPase binding activities. Although several kinds of research were dedicated to moderating their Na+/K+‐ATPase binding activity, still deeper fundamental knowledge is required to bring these findings into medical practice. In this work, we reviewed data related to anticancer activity of bufadienolides such as bufalin, arenobufagin, bufotalin, gamabufotalin, cinobufotalin, and cinobufagin and their derivatives. Bufotoxins, derivatives of bufadienolides containing polar molecules mainly belonging to argininyl residues, are reviewed as well. The established structures of bufotoxins have been compiled into a one‐page figure to review their structures. We also highlighted advances in the structure‐modification of the structure of compounds in this class. Drug delivery approaches to target these compounds to tumor cells were discussed in one section. The issues related to extraction, identification, and quantification are separated into another section. [ABSTRACT FROM AUTHOR]

Abstract Rhabdophis tigrinus (R. tigrinus) is a common colubrid snake that possesses a series of paired sac-like nuchal glands behind the head. When pressure is applied to the nuchal area, the thin skin over the nuchal glands can rupture and release secretions. In Japan, 19 cases of ophthalmia caused by the nuchal gland secretion of R. tigrinus have been reported. However, only one case has been documented in an English report. A 72-year old woman was sprayed by the nuchal gland fluid of R. tigrinus in her right eye. She presented with symptoms of eye pain and blurred vision. A slit-lamp examination revealed diffuse superficial keratitis, corneal stromal edema with Descemet membrane folds, and conjunctival injection. The best-corrected visual acuity (BCVA) of her right eye was 0.6. She was prescribed 0.5% moxifloxacin and 0.1% fluorometholone eye drops four times a day, and the symptoms resolved without sequelae within 5 days. The BCVA in the right eye improved to 1.0. In previous reports, ophthalmic examinations revealed conjunctivitis, keratitis, and corneal edema with Descemet membrane folds. Topical antibiotics and corticosteroid were prescribed in most cases, and eyes healed within 5-7 days without any sequelae. While corneal edema may resolve spontaneously in a few days when inhibition of the toxin has ceased, the use of topical steroids is recommended, as it can increase the activity of Na/K pumps that remained functional, thereby accelerating recovery. In fact, our patient used a topical steroid and recovered without sequelae.

Urginea maritima L. (squill) species is widely spread at the Mediterranean region as two main varieties, i.e., white squill (WS) and red squill (RS), that are recognized for several health potentials. The major secondary metabolite classes of the squill are cardiac glycosides, mainly, bufadienolides, flavonoids, and anthocyanins. Herein, a multiplex MS and NMR metabolomics approach targeting secondary and aroma compounds in WS and RS was employed for varieties classification. Solid-phase micro extraction-gas chromatography/mass spectroscopy (SPME-GC/MS), ultra-high-performance liquid chromatography/mass spectrometry (UPLC/MS), as well as nuclear magnetic resonance (NMR) provided fingerprinting and structural confirmation of the major metabolites for both types of the squill. For comparison of the different platforms' classification potential, multivariate data analysis was employed. While Bufadienolides, viz. "hydroxy-scilliglaucosidin-O-rhamnoside, desacetylscillirosidin-O-rhamnoside and bufotalidin-O-hexoside" as well as oxylipids, were enriched in WS, flavonoids, i.e., dihydro-kaempferol-O-hexoside and its aglycon, taxifolin derivative, were predominant in RS. A cytotoxicity screening against three cancer cell lines, including breast adenocarcinoma (MCF-7), lung (A-549), and ovarian (SKOV-3) cell lines was conducted. Results revealed that WS was more effective on A-549 and SKOV-3 cell lines (WS IC50 0.11 and 0.4 µg/mL, respectively) owing to its abundance of bufadienolides, while RS recorded IC50 (MCF7 cell line) 0.17 µg/mL since is is rich inflavonoids. [ABSTRACT FROM AUTHOR]

Bufadienolides, cyclopentanophenantrene derivatives that possess similar structures to cardiac glycosides, have been established to reveal cytotoxicity. Their higher concentrations as anticancer means are expected to affect the normal function of the heart, liver, and kidney. In this work, we established the mass ratio of bufadienolides isolated from the venom of Bufo viridis. Arenobufagin, gamabufotalin, telocinobufagin, and marinobufagin were found as the major compounds in the sum. Their effects on biological parameters, used as indicators of the normal functioning of organs, were determined in rabbits. The blood samples, taken on the 10th and 30th days following the drug administration, showed these paramaters reaching the control levels for 30 days in all studied doses: 0.15, 0.45, and 0.6 mg/kg. The 0.15 and 0.6 mg/kg doses were established not to cause significant changes in the activities of alanine aminotransferase and aspartate aminotransferase, and the quantities of cholesterol and fasting blood sugar in blood samples. Significant changes were found in quantities of glucose (a reduction) and urea (an increase) on the 10th day of the administration. But their levels reached the control level for the next 20 days. We observed significant differences in thromboelastographic parameters in mice on the 10th day after the drug administration in a 0.15 mg/kg dose. In mice, LD50 levels of intravenous, subcutaneous, and oral administrations made 14.5, 110, and 215 mg/kg, respectively. These results enable the suggestion of optimum concentration that does not cause severe damage to the functions of organs. [ABSTRACT FROM AUTHOR]

The traditional Chinese medicine toad venom (Venenum bufonis) has been extensively used to treat various diseases, including cancers, in China and other Southeast Asian countries. The major constituents of toad venom, e.g., bufadienolides and alkaloids, exhibit broad-spectrum pharmacological effects in cancers. Herein, two new bufadienolides (1 and 2), along with eleven known compounds (3–13) were successfully isolated from Bufo melanostictus Schneider. Their structures were elucidated by extensive spectroscopic data and X-ray diffraction analysis. Furthermore, four lactam derivatives were synthesized through the transformation of bufadienolides lactones. The inhibitory effects of these compounds against human prostate cancer cell lines PC-3 and DU145 were evaluated. The outcomes indicated a notable trend, with a substantial subset displaying nanomolar range IC50 values against PC-3 and DU145 cells, underscoring their pronounced cytotoxicity. Moreover, a noteworthy distinction surfaces, wherein lactones consistently outperformed their lactam counterparts, further validating their heightened potency for the treatment of prostate cancer. This study contributes significant preclinical evidence substantiating the therapeutic viability of bufadienolides and toad venom as intervention strategies for prostate cancer.

Toad Venom (TV) is the dried product of toxic secretions from Bufo bufo gargarizans Cantor (BgC) or B. melanostictus Schneider (BmS). Given the increasing medical demand and the severe depletion of wild toads, a number of counterfeit TVs appeared on the market, posing challenges to its quality control. In order to develop an efficient, feasible, and comprehensive approach to evaluate TV quality, a thorough analysis and comparison of chemical compounds among legal species BgC and BmS, as well as the main confusion species B. andrewsi Schmidt (BaS) and B. raddei Strauch (BrS), were conducted by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), high performance liquid chromatography (HPLC), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Nano LC-MS/MS analyses. We identified 126 compounds, including free or conjugated bufadienolides, indole alkaloids and amino acids, among the four Bufo species. The content of main bufadienolides, such as gamabufotalin, bufotalin, bufalin, cinobufagin, and resibufogenin, and the total protein contents varied widely among 28 batches of TV due to their origin species. The sum of the five bufadienolides within the BgC, BmS, BaS, and BrS samples were 8.15–15.93%, 2.45–4.14%, 11.15–13.50%, and 13.21–14.68%, respectively. The total protein content of BgC (6.9–24.4%) and BaS (19.1–20.6%) samples were higher than that of BmS (4.8–20.4%) and BrS (10.1–13.7%) samples. Additionally, a total of 1357 proteins were identified. There were differences between the protein compositions among the samples of the four Bufo species. The results indicated that BgC TV is of the highest quality; BaS and BrS TV could serve as alternative resources, whereas BmS TV performed poorly overall. This research provides evidence for developing approaches to evaluate TV quality and selecting the proper Bufo species as the origin source of TV listed in the Chinese pharmacopoeia.

Trypanosomiasis is one of the neglected tropical diseases which curtails the productivity of the victim and causes mortality in extreme cases. Non-availability of vaccines, low therapeutic index of trypanocidal drugs and development of resistance lead to the need for research focused on developing alternative treatment options against this disease. The aim of the current work is to use computer-assisted drug discovery techniques to examine the anti-trypanosomal action of bufadienolides, which are commonly detected in the skin secretions of toads. A library of 16 compounds from the bufadienolides family was tested for toxicity and docked against the Trypanosoma brucei hydrolase. The oral bioavailability and good suitability of the compounds for transdermal delivery was also indicated by the ADMET study. The findings based on molecular docking and dynamics simulation along with toxicity assessment showed that three compounds, marinobufagin, resibufogenin and resibufaginol, have the lowest toxicity and maximum binding energy against Trypanosoma brucei hydrolase. According to the results obtained by molecular docking interactions between drugs and target proteins are observed and contain a number of hydrogen bonds near the residues in the active pocket of the enzyme. The results of this study contribute to the understanding of the SAR of bufadienolides and provide a foundation for further research and drug development efforts. The computational analysis, including the density functional theory study, the knowledge of inhibitory mechanisms and aids in the design and optimization of novel anti-trypanosomal drugs.

The genus Kalanchoe Adans. (Crassulaceae) is native to Madagascar and comprises 145 species, being naturalized in the tropics and cultivated worldwide. In addition to having ornamental value, several Kalanchoe species are commonly used in popular medicine for the treatment of inflammatory conditions, wounds, gastric ulcers, and other diseases. The great importance of the genus is reflected on its acknowledgment by traditional and alternative health systems and organizations, as well as on the growing number of papers reporting pharmacological properties of extracts and isolated compounds from Kalanchoe. Among these properties, we highlight anti-inflammatory, antitumor, wound healing, antiulcer, and muscle relaxing properties. These activities are attributed mostly to flavonoids and bufadienolides, the main secondary metabolites reported in Kalanchoe extracts. While bufadienolides are generally related to cytotoxic and muscle relaxing activities, flavonoids are commonly reported as anti-inflammatory and wound healing agents. This review provides up to date information and perspectives on bioactive compounds from the Kalanchoe genus that are potentially useful for the development of new drugs. It includes not only a discussion on the advantages of the Kalanchoe species as source of bioactive compounds, but also the gaps, opportunities, and challenges to translate the acquired knowledge into innovation for drug development. [ABSTRACT FROM AUTHOR]

Bufadienolides are steroids that inhibit Na+/K+-ATPase; recent evidence shows that bufalin inhibits the activity of porcine aminopeptidase N (pAPN). We evaluated the selectivity of some bufadienolides on metallo-aminopeptidases. Among the enzymes of the M1 and M17 families, pAPN and porcine aminopeptidase A (pAPA) were the only targets of some bufadienolides. ѱ-bufarenogin, telocinobufagin, marinobufagin, bufalin, cinobufagin, and bufogenin inhibited the activity of pAPN in a dose-dependent manner in the range of 10−7–10−6 M. The inhibition mechanism was classical reversible noncompetitive for telocinobufagin, bufalin and cinobufagin. Bufogenin had the lowest K i value and a non-competitive behavior. pAPA activity was inhibited by ѱ-bufarenogin, cinobufagin, and bufogenin, with a classical competitive type of inhibition. The models of enzyme-inhibitor complexes agreed with the non-competitive type of inhibition of pAPN by telocinobufagin, bufalin, cinobufagin, and bufogenin. Since APN is a target in cancer therapy, we tested the effect of bufadienolides on the MeWo APN+ human melanoma cell line; they induced cell death, but we obtained scant evidence that inhibition of APN contributed to their effect. Thus, APN is a selective target of some bufadienolides, and we suggest that inhibition of APN activity by bufadienolides is not a major contributor to their antiproliferative properties in MeWo cells. • Telocinobufagin, marinobufagin, bufalin, cinobufagin and bufogenin inhibited pAPN through a noncompetitive mechanism. • Docking studies suggest that these molecules interacted with the S1 and S1´ sites of pAPN without chelation of zinc ion. • ѱ-bufarenogin, cinobufagin, and bufogenin, also inhibited pAPA with a classical competitive type of inhibition. • Bufadienolides and bestatin reduced human melanoma MeWo cell line viability/metabolism and increased cell death. • Some bufadienolides modified the percentages of MeWo cells in each cell cycle phase and/or cells with DNA fragmentation. [ABSTRACT FROM AUTHOR]

Introduction: Lipid metabolism participates in various biological processes such as proliferation, apoptosis, migration, invasion, and maintenance of membrane homeostasis of prostate tumor cells. Bufadienolides, the active ingredients of Chansu, show a robust anti-proliferative effect against prostate cancer cells in vitro, but whether bufadienolides could regulate the lipid metabolism in prostate cancer has not been evaluated. Objectives: Our study explored the regulatory effects of bufadienolides on lipid metabolism in human prostate carcinoma cells (PC-3). Methods: Untargeted lipidomics and transcriptomics were combined to study the effect of different bufadienolides interventions on lipid and gene changes of PC-3 cells. The key genes related to lipid metabolism and prostate cancer development were verified by qPCR and western blotting. Results: Lipidomic analysis showed that the active bufadienolides significantly downregulated the content of long-chain lipids of PC-3 cells. Based on transcriptomic and qPCR analyses, many genes related to lipid metabolism were significantly regulated by active bufadienolides, such as ELOVL6, CYP2E1, GAL3ST1, CERS1, PLA2G10, PLD1, SPTLC3, and GPX2. Bioinformatics analysis of the Cancer Genome Atlas database and literature retrieval showed that elongation of very long-chain fatty acids protein 6 (ELOVL6) and phospholipase D1 (PLD1) might be important regulatory genes. Western blot analysis revealed that active bufadienolides could downregulate PLD1 protein levels which might promote anti-prostate cancer effect. Conclusions: All these findings support that bufadienolides might induce lipid metabolic remodeling by regulating long-chain lipids synthesis and phospholipid hydrolysis to achieve an anti-prostate cancer effect, and PLD1 would probably be the key protein. [ABSTRACT FROM AUTHOR]

• Verified the irrationality of traditional value orientation of bufonis venenum. • The key "toxicity and activity" bufadienolide Q-markers were screened and compared. • The correlation between toxicity and activity of bufonis venenum were identified. • Deacetylcinobufagin with low toxicity and strong activity was found and verified. Bufonis Venenum (BV) is a traditional animal-based Chinese medicine with therapeutic effects against cancer. However, its clinical use is significantly restricted due to associated cardiovascular risks. BV's value in China's market is typically assessed based on "content priority," focusing on indicator components. However, these components of BV possess both antitumor activity and toxicity, and the correlation between the antitumor activity and toxicity of BV has not yet been elucidated. This study employs an integrated multi-omics approach to identify bufadienolide Q-markers and explore the correlation between BV's antitumor activity and toxicity. The aim is to establish a more comprehensive method for BV's quality. Normal zebrafish and HepG2 xenograft zebrafish were chosen as activity and toxicity evaluation models. Ultra-high performance liquid chromatography (UHPLC) coupled with a linear ion trap orbitrap (LTQ-Orbitrap) mass spectrometry was used to quantify eight batches of BV and key "toxic and effective" components were screened out. Transcriptomic and metabolomic analyses were performed to elucidate the regulatory mechanisms underlying the antitumor activity and cardiovascular toxicity of the key components in BV. Eight key "toxic and effective" compounds were identified: resibufogenin, cinobufagin, arenobufagin, bufotalin, bufalin, gamabufotalin, desacetylcinobufagin, and telocinobufagin. The findings showed that bufalin and cinobufagin interfered with calcium homeostasis through CaV and CaSR, induced cardiotoxicity, and upregulated CASP9 to activate myocardial cell apoptosis. However, desacetylcinobufagin exhibited greater potential in terms of anti-tumor effects. Combining the results of untargeted and targeted metabolomics revealed that desacetylcinobufagin could have a callback effect on differential lipids and correct abnormal energy and amino acid metabolism caused by cancer, similar to cinobufagin and bufalin. Microscale thermophoresis (MST) ligand binding measurements also showed that the binding of desacetylcinobufagin to GPX4 has a more potent ability to induce ferroptosis in tumor cells compared to cinobufagin. An innovative evaluation method based on the zebrafish was developed to investigate the relationship between the toxicity and efficacy of BV. This study identified toxicity and activity Q-markers and explored the mechanism between the two effects of BV. The research data could offer valuable insights into the efficacy of BV. Additionally, desacetylcinobufagin, an active ingredient with low toxicity, was found to enhance the quality of BV. [Display omitted] [ABSTRACT FROM AUTHOR]

Amphibians are widely known as a prolific source of bioactive metabolites. In this work, we isolated and characterized compounds with antiparasitic activity from the oocytes of the toad Rhinella alata collected in Panama. Bio-guided isolation and structural elucidation were carried out using chromatographic and spectroscopic techniques, respectively. The organic extract was subjected to solid phase extraction followed by HPLC purification of the fraction with in vitro activity against Trypanosoma cruzi trypomastigotes. Seven steroids (1–7) of the bufadienolide family were isolated, and their structures were determined using NMR and MS analyses; of these 19-formyl-dyscinobufotalin, (3) is reported as a new natural product. Compounds 1 and 3–7 resulted in a good anti-trypanosomal activity profile. Among these, 16β-hydroxyl-hellebrigenin (1) and bufalin (7) showed significant selectivity values of >5 and 2.69, respectively, while the positive control benznidazole showed a selectivity of 18.81. Furthermore, molecular docking analysis showed compounds 1, 3 and 7 interact through H-bonds with the amino acid residues GLN-19, ASP-158, HIS-159 and TRP-177 from cruzipain at the catalytic site. Given the lack of therapeutic options to treat American trypanosomiasis, this work can serve as the basis for further studies that aim for the development of bufadienolides or their derivatives as drugs against Chagas disease.

Several Asian natricine snakes of the genus Rhabdophis feed on toads and sequester steroidal cardiac toxins known as bufadienolides (BDs) from them. A recent study revealed that species of the Rhabdophis nuchalis Group ingest lampyrine fireflies to sequester BDs. Although several species of fireflies are distributed in the habitat of the R. nuchalis Group, only lampyrine fireflies, which have BDs, are included in the diet of these snakes. Thus, we hypothesized that the R. nuchalis Group chemically distinguishes fireflies that have BDs from those that do not have BDs. We also predicted that the R. nuchalis Group detects BDs as the chemical cue of toxin source. To test these predictions, we conducted 3 behavioral experiments using Rhabdophis chiwen , which belongs to the R. nuchalis Group. In the first experiment, R. chiwen showed a moderate tongue flicking response to cinobufagin, a compound of BDs. On the other hand, the snake showed a higher response to the chemical stimuli of lampyrine fireflies (BD fireflies) than those of lucioline fireflies (non-BD fireflies). In the second experiment, in which we provided live BD and non-BD fireflies, the snake voluntarily consumed only the former. In the third, a Y-maze experiment, the snake tended to select the chemical trail of BD fireflies more frequently than that of non-BD fireflies. These results demonstrated that R. chiwen discriminates BD fireflies from non-BD fireflies, but the prediction that BDs are involved in this discrimination was not fully supported. To identify the proximate mechanisms of the recognition of novel toxic prey in the R. nuchalis Group, further investigation is necessary. [ABSTRACT FROM AUTHOR]

Rhabdophis tigrinus (R. tigrinus) is a common colubrid snake that possesses a series of paired sac-like nuchal glands behind the head. When pressure is applied to the nuchal area, the thin skin over the nuchal glands can rupture and release secretions. In Japan, 19 cases of ophthalmia caused by the nuchal gland secretion of R. tigrinus have been reported. However, only one case has been documented in an English report. A 72-year old woman was sprayed by the nuchal gland fluid of R. tigrinus in her right eye. She presented with symptoms of eye pain and blurred vision. A slit-lamp examination revealed diffuse superficial keratitis, corneal stromal edema with Descemet membrane folds, and conjunctival injection. The best-corrected visual acuity (BCVA) of her right eye was 0.6. She was prescribed 0.5% moxifloxacin and 0.1% fluorometholone eye drops four times a day, and the symptoms resolved without sequelae within 5 days. The BCVA in the right eye improved to 1.0. In previous reports, ophthalmic examinations revealed conjunctivitis, keratitis, and corneal edema with Descemet membrane folds. Topical antibiotics and corticosteroid were prescribed in most cases, and eyes healed within 5-7 days without any sequelae. While corneal edema may resolve spontaneously in a few days when inhibition of the toxin has ceased, the use of topical steroids is recommended, as it can increase the activity of Na/K pumps that remained functional, thereby accelerating recovery. In fact, our patient used a topical steroid and recovered without sequelae. [ABSTRACT FROM AUTHOR]

Cardiotonic steroids (CTS) were first documented by ancient Egyptians more than 3000 years ago. Cardiotonic steroids are a group of steroid hormones that circulate in the blood of amphibians and toads and can also be extracted from natural products such as plants, herbs, and marines. It is well known that cardiotonic steroids reveal effects against congestive heart failure and atrial fibrillation; therefore, the term "cardiotonic" has been coined. Cardiotonic steroids are divided into two distinct groups: cardenolides (plant-derived) and bufadienolides (mainly of animal origin). Cardenolides have an unsaturated five-membered lactone ring attached to the steroid nucleus at position 17; bufadienolides have a doubly unsaturated six-membered lactone ring. Cancer is a leading cause of mortality in humans all over the world. In 2040, the global cancer load is expected to be 28.4 million cases, which would be a 47% increase from 2020. Moreover, viruses and inflammations also have a very nebative impact on human health and lead to mortality. In the current review, we focus on the chemistry, antiviral and anti-cancer activities of cardiotonic steroids from the naturally derived (toads) venom to combat these chronic devastating health problems. The databases of different research engines (Google Scholar, PubMed, Science Direct, and Sci-Finder) were screened using different combinations of the following terms: "cardiotonic steroids", "anti-inflammatory", "antiviral", "anticancer", "toad venom", "bufadienolides", and "poison chemical composition". Various cardiotonic steroids were isolated from diverse toad species and exhibited superior anti-inflammatory, anticancer, and antiviral activities in in vivo and in vitro models such as marinobufagenin, gammabufotalin, resibufogenin, and bufalin. These steroids are especially difficult to identify. However, several compounds and their bioactivities were identified by using different molecular and biotechnological techniques. Biotechnology is a new tool to fully or partially generate upscaled quantities of natural products, which are otherwise only available at trace amounts in organisms. [ABSTRACT FROM AUTHOR]

Twelve bufadienolides and six 19-norbufadienolides were isolated from the aerial parts of Helleborus foetidus. They consist of aglycons and glucosides and include nine previously undescribed compounds and a compound reported for the first time as a genuine natural product. Their structures were established by extensive spectroscopic analysis and the structure and absolute configuration of two previously unreported 3,4-epoxy derivatives were confirmed by single crystal X-ray diffraction analysis. The compounds were tested for their cytotoxicity on MCF-7 human breast cancer cells. They show differential cytotoxic activity with IC 50 values in the range of 2.4 nM - >10 μM. The potency of the activity strongly correlates with the presence of a C-19 aldehyde group. The data complement the scientific basis underpinning the use of H. foetidus in anthroposophic medicine for the integrative treatment of cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Toads belonging to the Bufonidae family have a pair of paratoid glands that store highly toxic a biological secretion with varied chemical composition, that act as a chemical defense against microbial infections and predators. The paratoid gland secretion (PGS) of bufonids is rich in bioactive steroids, alkaloids, proteins, bufadienolides and bufotoxins. In the present investigation we performed a systematic analysis of the chemical profile of PGS obtained from the Bufonidae toad Rhinella jimi ("Cururu" toad) collected at three different regions of Piauí state, Northeastern Brazil. Our aim was to investigate the PGS variation related to the season of animals collection, geographic distribution and gender of the animals. The methanolic extracts of PGS were analyzed by UPLC-QToF-MS/MS. Principal component analysis (PCA) were applied to the data set obtained by the UPLC-QToF-MS/MS analyses. Among 23 compounds identified, dehydrobufotenine, suberoyl arginine, 3-(N- suberoyl-argininyl) telocinobufagin, 3-(N- suberoyl-argininyl) marinobufagin, telocinobufagin, marinobufagin and 3-(N -suberoyl-argininyl) bufalin were detected in all PGS. Minimal variations in the composition of paratoid secretions of R. jimi were observed related to distinct geographical and seasonal parameters. R. jimi female animals presented the most diverse chemical composition in its PGS. With this comparative study, unprecedented for the species, it was possible to observe that the secretions of the paratoid glands produced by R. jimi from different regions of the state of Piauí, at different times of the year, presented consistent chemical composition, with discrete particularities in the number and nature chemistry of its constituents. [Display omitted] • This is the first comparative study of Rhinella jimi PGS observing gender, seasonality and geographic occurrence. • The amount of compounds evidenced in the females of R jimi was higher than that found in the males. • The climatic conditions of the seasons may have influenced the chemical composition of the PGS. • The PGS collected in the central region of the state of Piauí showed the highest amount of compounds identified. • Seven compounds were identified in all secretion of paratoid glands samples. [ABSTRACT FROM AUTHOR]

Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, an infectious disease of amphibians, which has contributed to population declines in hundreds of species worldwide. Common toads (Bufo bufo) exhibit low resistance and relatively high tolerance to Bd infection, which may partly be attributable to bufadienolide toxins secreted in their granular skin glands. Bufadienolides are known to provide an effective defense against several pathogens, parasites, and predators. The toxin production of bufonids is a plastic trait, inducible by several environmental factors. Here, we experimentally infected juvenile common toads with Bd and investigated if the toadlets could clear the infection over time, whether the infection induced bufadienolide production, and whether the infection caused decreased body mass. We found that prevalence remained 100% throughout the entire experimental period, but infection intensity did not increase and it was significantly lower on day 30 than on day 20. At the same time, compared to controls, infected toadlets produced lesser amounts of bufadienolides and their body mass was also lower. These results suggest that although young toadlets may not be able to clear Bd infection on their own, they may be able to keep infection intensities under control. Nonetheless, even if toadlets do not succumb to the disease, the costs of chronic infection may still compromise their fitness. [ABSTRACT FROM AUTHOR]

Phytochemical investigation of the whole plants of Helleborus niger L. (Ranunculaceae) resulted in the isolation of five undescribed compounds, including one bufadienolide (1), two bufadienolide rhamnosides (2 and 3), and two ecdysteroids (12 and 13), along with eight known compounds (4 – 11). The chemical structures of 1 – 3 , 12 , and 13 were determined by spectroscopic studies, including 2D NMR, and chromatographic and spectroscopic analyses of the hydrolyzed products. Compounds 1 – 13 were evaluated for their cytotoxic activity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, SBC-3 human small-cell lung cancer cells, and TIG-3 human normal diploid lung cells. Compounds 1 – 12 showed cytotoxic activity against HL-60, A549, and SBC-3 cells, with IC 50 values ranging from 0.0016 to 6.1 μM. Bufadienolide rhamnoside 2 exhibited potent cell proliferation inhibitory activity against SBC-3 cells after 24–48 h of treatment and apoptosis-inducing activity in SBC-3 cells via an intrinsic pathway after 72 h of treatment. The JFCR39 panel screening of 2 suggests that the molecular target of 2 is Na+,K+-ATPase. [Display omitted] • Five undescribed steroid derivatives were isolated from Helleborus niger. • Their structures were determined by spectroscopic analysis and hydrolysis studies. • Isolated compounds showed cytotoxic activity against HL-60, A549, and SBC-3 cells. • An undescribed bufadienolide rhamnoside induced apoptosis in SBC-3 cells. [ABSTRACT FROM AUTHOR]

Urginea maritima L. (squill) species is widely spread at the Mediterranean region as two main varieties, i.e., white squill (WS) and red squill (RS), that are recognized for several health potentials. The major secondary metabolite classes of the squill are cardiac glycosides, mainly, bufadienolides, flavonoids, and anthocyanins. Herein, a multiplex MS and NMR metabolomics approach targeting secondary and aroma compounds in WS and RS was employed for varieties classification. Solid-phase micro extraction-gas chromatography/mass spectroscopy (SPME-GC/MS), ultra-high-performance liquid chromatography/mass spectrometry (UPLC/MS), as well as nuclear magnetic resonance (NMR) provided fingerprinting and structural confirmation of the major metabolites for both types of the squill. For comparison of the different platforms’ classification potential, multivariate data analysis was employed. While Bufadienolides, viz. “hydroxy-scilliglaucosidin-O-rhamnoside, desacetylscillirosidin-O-rhamnoside and bufotalidin-O-hexoside” as well as oxylipids, were enriched in WS, flavonoids, i.e., dihydro-kaempferol-O-hexoside and its aglycon, taxifolin derivative, were predominant in RS. A cytotoxicity screening against three cancer cell lines, including breast adenocarcinoma (MCF-7), lung (A-549), and ovarian (SKOV-3) cell lines was conducted. Results revealed that WS was more effective on A-549 and SKOV-3 cell lines (WS IC50 0.11 and 0.4 µg/mL, respectively) owing to its abundance of bufadienolides, while RS recorded IC50 (MCF7 cell line) 0.17 µg/mL since is is rich inflavonoids.

Secondary metabolism (the formation of substances of specialized metabolism) is one of the distinctive specificities of the plant organism. Data on complete sequences of plant genomes indicate that from 15 to 25% of all genes are involved in this process (the biosynthesis of enzymes, transporters and transfactors serving it), while many aspects of secondary metabolism, including its physiological functions, are still unclear. The use of plant cell and organ cultures is a promising approach to solving fundamental and applied problems in specialized metabolism. The mechanisms of formation of secondary metabolites in plant systems in vitro with different levels of cell differentiation can differ significantly from those in an intact plant, which, when comparing these biological systems, can be effectively used to study the principles of regulation, organization and functioning of secondary metabolism. To date, more than 100 000 compounds of specialized metabolism (alkaloids, isoprenoids, phenolic compounds and a number of "minor" groups of secondary metabolites) are known, which, as a rule, are biologically active substances. This fact determines their practical significance and wide application in medicine: today, about a third of all medicinal substances are of plant origin. An acute shortage of medicinal plant materials makes the use of plant cell and organ cultures a very promising source of biologically active substances. Cardiac glycosides are one of the most important groups of secondary metabolites, which have been used in medicine for the treatment of cardiac diseases for more than two centuries. Interest in them is now growing significantly due to the discovery of new properties, including antiviral and antitumor activity. The review analyzes the main results of works devoted to the study of the formation of cardiac glycosides in plant cell and organ cultures producing these compounds. [ABSTRACT FROM AUTHOR]

Kalanchoe species are succulents with anti-inflammatory, antioxidant, and analgesic properties, as well as cytotoxic activity. One of the most popular species cultivated in Europe is Kalanchoe daigremontiana Raym.-Hamet and H. Perrier. In our study, we analyzed the phytochemical composition of K. daigremontiana water extract using UHPLC-QTOF-MS and estimated the cytotoxic activity of the extract on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The expression levels of 92 genes associated with cell death were estimated via real-time PCR. The antioxidant activity was assessed via flow cytometry on human keratinocyte HaCaT cell line. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (ferric-reducing antioxidant power) assays were also applied. We identified twenty bufadienolide compounds in the water extract and quantified eleven. Bersaldegenin-1,3,5-orthoacetate and bryophyllin A were present in the highest amounts (757.4 ± 18.7 and 573.5 ± 27.2 ng/mg dry weight, respectively). The extract showed significant antiproliferative and cytotoxic activity, induced depolarization of the mitochondrial membrane, and significantly arrested cell cycle in the S and G2/M phases of SKOV-3 cells. Caspases-3, 7, 8, and 9 were not activated during the treatment, which indicated non-apoptotic cell death triggered by the extract. Additionally, the extract increased the level of oxidative stress in the cancer cell line. In keratinocytes treated with menadione, the extract moderately reduced the level of oxidative stress. This antioxidant activity was confirmed by the DPPH and FRAP assays, where the obtained IC50 values were 1750 ± 140 and 1271.82 ± 53.25 μg/mL, respectively. The real-time PCR analysis revealed that the extract may induce cell death via TNF receptor (tumor necrosis factor receptor) superfamily members 6 and 10. [ABSTRACT FROM AUTHOR]

Context Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. Objective The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate – a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. Materials and methods The cytotoxic activity of the compound (at 0.1–20.0 μg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. Results The compound had strong effect on the cells (IC50 = 0.55 μg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. Conclusions Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.

Toads of the genus Atelopus are chemically defended by a unique combination of endogenously synthesized cardiotoxins (bufadienolides) and neurotoxins which may be sequestered (guanidinium alkaloids). Investigation into Atelopus small-molecule chemical defenses has been primarily concerned with identifying and characterizing various forms of these toxins while largely overlooking their ecological roles and evolutionary implications. In addition to describing the extent of knowledge about Atelopus toxin structures, pharmacology, and biological sources, we review the detection, identification, and quantification methods used in studies of Atelopus toxins to date and conclude that many known toxin profiles are unlikely to be comprehensive because of methodological and sampling limitations. Patterns in existing data suggest that both environmental (toxin availability) and genetic (capacity to synthesize or sequester toxins) factors influence toxin profiles. From an ecological and evolutionary perspective, we summarize the possible selective pressures acting on Atelopus toxicity and toxin profiles, including predation, intraspecies communication, disease, and reproductive status. Ultimately, we intend to provide a basis for future ecological, evolutionary, and biochemical research on Atelopus.

The genus Kalanchoe Adans. (Crassulaceae) is native to Madagascar and comprises 145 species, being naturalized in the tropics and cultivated worldwide. In addition to having ornamental value, several Kalanchoe species are commonly used in popular medicine for the treatment of inflammatory conditions, wounds, gastric ulcers, and other diseases. The great importance of the genus is reflected on its acknowledgment by traditional and alternative health systems and organizations, as well as on the growing number of papers reporting pharmacological properties of extracts and isolated compounds from Kalanchoe. Among these properties, we highlight anti-inflammatory, antitumor, wound healing, antiulcer, and muscle relaxing properties. These activities are attributed mostly to flavonoids and bufadienolides, the main secondary metabolites reported in Kalanchoe extracts. While bufadienolides are generally related to cytotoxic and muscle relaxing activities, flavonoids are commonly reported as anti-inflammatory and wound healing agents. This review provides up to date information and perspectives on bioactive compounds from the Kalanchoe genus that are potentially useful for the development of new drugs. It includes not only a discussion on the advantages of the Kalanchoe species as source of bioactive compounds, but also the gaps, opportunities, and challenges to translate the acquired knowledge into innovation for drug development.

Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate – a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. The cytotoxic activity of the compound (at 0.1–20.0 μg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. The compound had strong effect on the cells (IC50 = 0.55 μg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides. [ABSTRACT FROM AUTHOR]

Cardiotonic steroids (CTS) were first documented by ancient Egyptians more than 3000 years ago. Cardiotonic steroids are a group of steroid hormones that circulate in the blood of amphibians and toads and can also be extracted from natural products such as plants, herbs, and marines. It is well known that cardiotonic steroids reveal effects against congestive heart failure and atrial fibrillation; therefore, the term "cardiotonic" has been coined. Cardiotonic steroids are divided into two distinct groups: cardenolides (plant-derived) and bufadienolides (mainly of animal origin). Cardenolides have an unsaturated five-membered lactone ring attached to the steroid nucleus at position 17; bufadienolides have a doubly unsaturated six-membered lactone ring. Cancer is a leading cause of mortality in humans all over the world. In 2040, the global cancer load is expected to be 28.4 million cases, which would be a 47% increase from 2020. Moreover, viruses and inflammations also have a very nebative impact on human health and lead to mortality. In the current review, we focus on the chemistry, antiviral and anti-cancer activities of cardiotonic steroids from the naturally derived (toads) venom to combat these chronic devastating health problems. The databases of different research engines (Google Scholar, PubMed, Science Direct, and Sci-Finder) were screened using different combinations of the following terms: “cardiotonic steroids”, “anti-inflammatory”, “antiviral”, “anticancer”, “toad venom”, “bufadienolides”, and “poison chemical composition”. Various cardiotonic steroids were isolated from diverse toad species and exhibited superior anti-inflammatory, anticancer, and antiviral activities in in vivo and in vitro models such as marinobufagenin, gammabufotalin, resibufogenin, and bufalin. These steroids are especially difficult to identify. However, several compounds and their bioactivities were identified by using different molecular and biotechnological techniques. Biotechnology is a new tool to fully or partially generate upscaled quantities of natural products, which are otherwise only available at trace amounts in organisms.

Drimia Jacq., a bulbous geophyte belonging to family Hyacinthaceae Batsch ex Borkh. is well known for its medicinal values. Leaves and bulbs of eight genotypes of Drimia were sequentially extracted with three solvents (DCM, MeOH and H 2 O) and analysed for their phytochemicals (TPC, TFC and TTC) and in vitro antioxidant activities (DPPH, ABTS radical scavenging activities and phosphomolybdenum assay). Bulbs of D. indica (triploid form) extracted with MeOH showed the highest TPC (22.19 ± 1.00 mg GAE/g DW). Higher TFC was recorded in MeOH extract of bulbs of D. raogibikei (58.65 ± 1.95 mg QE/g DW) whereas significantly maximum TTC (857.17 ± 38.62 mg GAE/g DW) was found in MeOH extract of D. razii. DPPH radical scavenging activity was highest in DCM bulb extract of D. coromandeliana (12.22%). DCM extract of D. polyantha showed greatest (64.26%) ABTS radical scavenging activity. Phosphomolybdenum reduction potential was maximum in MeOH leaf extract of D. indica (triploid form) (52.86 ± 0.20 mg AAE/g DW). RP-HPLC estimation of cardiac glycoside (Scillaren A) from the bulbs of eight genotypes of Drimia revealed the highest (5.54 ± 0.03 mg/g DW) quantity in DCM extracts of D. coromandeliana. Presence of a bufadienolide (Scillaren A) was confirmed by FTIR and HR-LCMS. Additionally, HR-LCMS analysis revealed the presence of three cardenolides (convallatoxin, digoxigenin monodigitoxoside and peruvoside) and 160 major metabolites in the bulbs of Drimia species. Present study is the first comparative account of phytochemicals, antioxidants, cardiac glycosides and LC-MS profiling of major metabolites from eight Indian Drimia genotypes. [Display omitted] • Leaf and bulb extracts (DCM, MeOH and H 2 O) of eight Drimia genotypes were investigated for their phytochemicals (TPC, TFC and TTC) and antioxidant activities. • Drimia indica (diploid) leaves showed considerable antioxidant potential with higher level of TPC and TFC. • First comparative account of phytochemicals, antioxidants, cardiac glycosides of Indian Drimia. • DCM extract was found suitable for extraction of bufadienolides. • Drimia coromandeliana and D. razii are elite sources for bufadienolide, i.e. Scillaren A. • First comparative chemical profiling of bulbs of Indian Drimia species using HR-LCMS. [ABSTRACT FROM AUTHOR]

Background: Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide amphibian biodiversity loss. Many amphibians exhibit skin-based chemical defences, which may play an important role against invading pathogens, but whether the synthesis of these chemical compounds is enhanced or suppressed in the presence of pathogens is largely unknown. Here we investigated direct and indirect effects of larval exposure to the globally distributed and highly virulent Bd-GPL strain on skin secreted chemical defences and life history traits during early ontogeny of agile frogs (Rana dalmatina) and common toads (Bufo bufo). Results: Exposure to Bd during the larval stage did not result in enhanced synthesis of the antimicrobial peptide Brevinin-1 Da in R. dalmatina tadpoles or in increased production of bufadienolides in B. bufo tadpoles. However, exposure to Bd during the larval stage had a carry-over effect reaching beyond metamorphosis: both R. dalmatina and B. bufo froglets contained smaller quantities of defensive chemicals than their Bd-naïve conspecifics in the control treatment. Prevalence of Bd and infection intensities were very low in both larvae and metamorphs of R. dalmatina, while in B. bufo we observed high Bd prevalence and infection intensities, especially in metamorphs. At the same time, we did not find a significant effect of Bd-exposure on body mass or development rate in larvae or metamorphs in either species. Conclusions: The lack of detrimental effect of Bd-exposure on life history traits, even parallel with high infection intensities in the case of B. bufo individuals, is surprising and suggests high tolerance of local populations of these two species against Bd. However, the lowered quantity of defensive chemicals may compromise antimicrobial and antipredatory defences of froglets, which may ultimately contribute to population declines also in the absence of conspicuous mass-mortality events. [ABSTRACT FROM AUTHOR]

Abstract Many organisms use inducible defenses as protection against predators. In animals, inducible defenses may manifest as changes in behavior, morphology, physiology, or life history, and prey species can adjust their defensive responses based on the dangerousness of predators. Analogously, prey may also change the composition and quantity of defensive chemicals when they coexist with different predators, but such predator‐induced plasticity in chemical defenses remains elusive in vertebrates. In this study, we investigated whether tadpoles of the common toad (Bufo bufo) adjust their chemical defenses to predation risk in general and specifically to the presence of different predator species; furthermore, we assessed the adaptive value of the induced defense. We reared tadpoles in the presence or absence of one of four caged predator species in a mesocosm experiment, analyzed the composition and quantity of their bufadienolide toxins, and exposed them to free‐ranging predators. We found that toad tadpoles did not respond to predation risk by upregulating their bufadienolide synthesis. Fishes and newts consumed only a small percentage of toad tadpoles, suggesting that bufadienolides provided protection against vertebrate predators, irrespective of the rearing environment. Backswimmers consumed toad tadpoles regardless of treatment. Dragonfly larvae were the most voracious predators and consumed more predator‐naïve toad tadpoles than tadpoles raised in the presence of dragonfly cues. These results suggest that tadpoles in our experiment had high enough toxin levels for an effective defense against vertebrate predators even in the absence of predator cues. The lack of predator‐induced phenotypic plasticity in bufadienolide synthesis may be due to local adaptation for constantly high chemical defense against fishes in the study population and/or due to the high density of conspecifics.

Chemical compounds from skin secretions from toads of Bufonidae family have been long-studied. In the search for new molecules with pharmacological action, the 3 β -OH groups of bufadienolides are commonly derivatised using acetyl groups. This work described the isolation and/or structural elucidation of isolated and derivatised compounds from the venom of the Brazilian anuran Rhinella marina , and their evaluation in in vitro assays. In the methanolic extract of the R. marina venom, compound cholesterol (1) was isolated from the CRV-52 fraction by classic column chromatography, dehydrobufotenine (2) by Sephadex LH-20 from the CRV-28 fraction, and a mix of suberoyl arginine (3) and compound 2 was obtained from the CRV-6-33 fraction. The compounds marinobufagin (4), telocionbufagin (5) and bufalin (6) were isolated by classic column chromatography, followed by separation via HPLC in the CRV-70 fraction, and the compound marinobufotoxin (9) was isolated by classic column chromatography in the CRV-6 fraction, here being isolated for the first time in R. marina specimens. Compounds 4 and 5 were submitted for acetylation with acetic anhydride, in the presence of pyridine and 4-dimethyilaminopiridine (DMAP), in order to obtain the compounds 3-acetyl-marinobufagin (7) and 3-acetyl-telocinobufogin (8). The isolated and derivatised compounds were identified by 1H and 13C NMR, and their molecular mass confirmed by mass spectrometry. All compounds (except 1 and 3) were tested in cytotoxic assays by the MTT method and presented cytotoxic potential against human cancer cell lines, as well as against non-tumoral human embryonic kidney HEK-293 cells. With the exception of compound 2 , all molecules presented IC 50 values < 4 μM, and none caused hemolysis of human erythrocytes, demonstrating a promising cytotoxic potential of natural and chemically-modified bufadienolides. This study presents a detailed contribution of bioactive chemicals from Brazilian Amazon Rhinella species, and indicates promising areas for further studies and pharmaceutical investments. [Display omitted] • Seven compounds were identified in the extract from Rhinella marina venom. • Compounds 4 and 5 were acetylated resulting in the compounds 7 and 8. • Marinobufotoxin (9) was isolated for the first time in Rhinella marina. • The tested compounds demonstrated potent activity against tumour cell lines. [ABSTRACT FROM AUTHOR]

A new bufadienolide (1), two new bufadienolide glycosides (2 and 3), a new ecdysteroid (4), and four known compounds (5–8), were isolated from the whole plants of Helleborus niger L. (Ranunculaceae). The structures of the new compounds (1–4) were determined by spectroscopic analysis, including 2D NMR spectral data, and hydrolytic studies. Compounds 1–6 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC50 values ranging from 0.0055 to 1.9 µM. HL-60 cells treated with either 3 or 4 showed apoptosis characteristics, such as nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3/7. [ABSTRACT FROM AUTHOR]

Several Asian natricine snakes possess special organs called nuchal glands. Previous studies showed the nuchal glands of Rhabdophis tigrinus contain cardiac steroidal toxins known as bufadienolides (BDs) that are sequestered from toads consumed as prey. Recently, a congener has been found to ingest fireflies and sequester defensive BDs from them. As amphibians are probably the plesiomorphic diet of Rhabdophis species, it is presumed that a dietary transition of toxin source from toads to fireflies has occurred within the radiation of Rhabdophis. Because snakes heavily rely on chemical cues to recognize their prey, we predicted that species of Rhabdophis are capable of detecting BDs. To test the prediction, we conducted chemical preference tests using adults and hatchlings of R. tigrinus. We presented seven stimuli (water, cologne, earthworm, cinobufagin [a compound of BDs], toad, frog, and lampyrine firefly) to them. Both adult and hatchling R. tigrinus showed a higher response to toads and frogs than the controls (water and cologne), but, contrary to our prediction, they did not respond to cinobufagin. Adult R. tigrinus did however show a higher response to lampyrine fireflies than the controls. Our data imply that chemical resemblance between toads and fireflies elicited the reaction in adult R. tigrinus, but the chemical substances remain unknown. To identify the proximate mechanisms of the unique shift from toad-eating to firefly-eating in the snakes, further investigation is necessary. [ABSTRACT FROM AUTHOR]

Kalanchoe species are succulents with anti-inflammatory, antioxidant, and analgesic properties, as well as cytotoxic activity. One of the most popular species cultivated in Europe is Kalanchoe daigremontiana Raym.-Hamet and H. Perrier. In our study, we analyzed the phytochemical composition of K. daigremontiana water extract using UHPLC-QTOF-MS and estimated the cytotoxic activity of the extract on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The expression levels of 92 genes associated with cell death were estimated via real-time PCR. The antioxidant activity was assessed via flow cytometry on human keratinocyte HaCaT cell line. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (ferric-reducing antioxidant power) assays were also applied. We identified twenty bufadienolide compounds in the water extract and quantified eleven. Bersaldegenin-1,3,5-orthoacetate and bryophyllin A were present in the highest amounts (757.4 ± 18.7 and 573.5 ± 27.2 ng/mg dry weight, respectively). The extract showed significant antiproliferative and cytotoxic activity, induced depolarization of the mitochondrial membrane, and significantly arrested cell cycle in the S and G2/M phases of SKOV-3 cells. Caspases-3, 7, 8, and 9 were not activated during the treatment, which indicated non-apoptotic cell death triggered by the extract. Additionally, the extract increased the level of oxidative stress in the cancer cell line. In keratinocytes treated with menadione, the extract moderately reduced the level of oxidative stress. This antioxidant activity was confirmed by the DPPH and FRAP assays, where the obtained IC50 values were 1750 ± 140 and 1271.82 ± 53.25 μg/mL, respectively. The real-time PCR analysis revealed that the extract may induce cell death via TNF receptor (tumor necrosis factor receptor) superfamily members 6 and 10.

The secreted poisonin bufonids (Anura: Bufonidae) include proteins, biogenic amines, toxic bufadienolides and alkaloids. The chemical composition of the methanolic extract of parotoid gland secretions by the Amazonian toad Rhinella margaritifera was evaluated in a UFLC-DAD-micrOTOF system. Of the twenty three compounds found in the methanolic extract, eighteen were identified by the mass/charge ratio as: five arginine diacids, six bufagenins (telocinobufagin, marinobufagin, bufotalin, cinobufotalin, bufalin and cinobufagin), six bufotoxins, and an alkaloid (dehydrobufotenin). Image 1 • Rhinella margaritifera has a wide distribution throughout the Amazon basin. • The R. margaritifera poison presented arginines, bufagenins, bufotoxins and alkaloid. • Bufotalin was the major compound found in the R. margaritifera poison. [ABSTRACT FROM AUTHOR]

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nuchodorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins. [ABSTRACT FROM AUTHOR]