RESUMEN
Bufadienolides (BDs) are a class of naturally occurring toxins present in amphibian toads. Serving as the chemical weapons, they exist not only in the adult toads but also in toad eggs. Guided by mass spectrometry (MS)-based component analysis and feature-based molecular networking (FBMN), 30 bufadienolide-fatty acid conjugates (BDFs) were isolated from the fertilized eggs of toad Bufo gargrizans, including 25 previously undescribed compounds (1-25). Their chemical structures were elucidated by extensive spectroscopic analysis, chemical methods, and GC-MS. The toxicities of all BDFs and their corresponding free BDs were assessed using the zebrafish model. The structure-toxicity relationship analysis showed that the modification of BDs by hydroxy fatty acids can cause a significant increase of the toxicity. Furthermore, all the isolated compounds were evaluated for their antiproliferative activities in pancreatic cancer cell lines ASPC-1 and PANC10.05. The structure-activity relationship (SAR) analysis revealed that BDFs with hellebrigenin as the bufogenin moiety (6 and 7) exhibited the most potent antiproliferative effect. Further investigation into their functional mechanism demonstrated that 6 and 7 induced apoptosis in pancreatic cancer cells PANC10.05 and significantly suppressed the expression of the apoptosis-related gene c-MYC. In addition, 6 and 7 effectively inhibited the expression of the PI3K/Akt/mTOR pathway in PANC10.05. Moreover, we assessed the efficacy of 6 and 7 on cancer cells from various tissues and observed their broad-spectrum antiproliferative activity.
Asunto(s)
Bufanólidos , Bufonidae , Proliferación Celular , Ácidos Grasos , Pez Cebra , Animales , Bufanólidos/química , Bufanólidos/farmacología , Bufanólidos/toxicidad , Bufanólidos/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Humanos , Línea Celular Tumoral , Ácidos Grasos/química , Ácidos Grasos/farmacología , Ácidos Grasos/toxicidad , Relación Estructura-Actividad , Cigoto/efectos de los fármacos , Cigoto/química , Estructura MolecularRESUMEN
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-ß-D-glucoside (1), 2.2 mg of tigencaoside A (2), 8.3 mg of deglucohellebrin (3), 3.5 mg of 14 ß-hydroxy-3ß-[ß-D-glucopyranosyl-(1â6)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (4), and 3.0 mg of 14ß-hydroxy-3ß-[(ß-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.
Asunto(s)
Bufanólidos , Distribución en Contracorriente , Helleborus , Distribución en Contracorriente/métodos , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Helleborus/química , Porosidad , Resinas Sintéticas/química , Cromatografía Líquida de Alta Presión , Raíces de Plantas/químicaRESUMEN
Cinobufagin, a bufadienolide of toad venom of Bufo bufo gargarizans, is used as a cardiotonic, central nervous system (CNS) respiratory agent, as well as an analgesic and anesthetic. However, several research showed that bufadienolide has a few side effects on the CNS, such as breathlessness or coma. Although cinobufagin was shown to display pharmacological effects in various models, the toxic effect of cinobufagin is elusive in brain cell models. The aim of this study was to explore whether cinobufagin affected viability, Ca2+ homeostasis, and reactive oxygen species (ROS) production in Gibco® Human Astrocyte (GHA) and HCN-2 neuronal cell line. In GHA cells but not in HCN-2 cells, cinobufagin (20-60 µM) induced [Ca2+ ]i rises. In terms of cell viability, chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid reduced cinobufagin-induced cytotoxicity in GHA cells. In GHA cells, cinobufagin-induced Ca2+ entry was inhibited by 2-aminoethoxydiphenyl borate or SKF96365. In a Ca2+ -free medium, treatment with thapsigargin or U73122 abolished cinobufagin-evoked [Ca2+ ]i rises. Furthermore, treatment with N-acetylcysteine reversed ROS production and cytotoxicity in cinobufagin-treated GHA cells. Together, in GHA cells but not in HCN-2 cells cinobufagin caused cytotoxicity that was linked to preceding [Ca2+ ]i rises by Ca2+ influx via store-operated Ca2+ entry and phospholipase C-dependent Ca2+ release from the endoplasmic reticulum. Moreover, cinobufagin induced ROS-associated cytotoxicity.
Asunto(s)
Venenos de Anfibios/química , Astrocitos/metabolismo , Encéfalo/metabolismo , Bufanólidos/farmacología , Señalización del Calcio/efectos de los fármacos , Homeostasis/efectos de los fármacos , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Astrocitos/efectos de los fármacos , Encéfalo/patología , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Bufo bufo , Calcio/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Neuronas/efectos de los fármacos , Tapsigargina/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Three new bufadienolides, namely, paulliniogenin A (1), paulliniogenin B (2), and 16ß-formyloxybersamagenin 1,3,5-orthoacetate (3), together with two known bufadienolides and six known phenolic substances, were isolated from the stem bark of Bersama abyssinica subsp. abyssinica and B. abyssinica subsp. paullinioides. The structures of the compounds were elucidated based on their NMR and HRMS data analyses. The relative configurations were defined by single-crystal X-ray crystallography and NOESY correlations. Cytotoxicity against the L929 and KB3.1 cancer cell lines of the isolated compounds was investigated using an MTT assay. Paulliniogenin A (1) and 16ß-hydroxybersamagenin-1,3,5-orthoacetate (4) showed cytotoxicity against the KB3.1 cell line with IC50 values of 1.4 ± 0.77 and 1.6 ± 0.81 µM, respectively. Moreover, paulliniogenin A (1) and paulliniogenin B (2) demonstrated weak activity against Staphylococcus aureus.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Bufanólidos/farmacología , Magnoliopsida/química , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Bufanólidos/aislamiento & purificación , Línea Celular Tumoral , Humanos , Kenia , Ratones , Estructura Molecular , Fenoles , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Corteza de la Planta/químicaRESUMEN
The bulbs of the South African Drimia altissima (Asparagaceae or Hyacinthaceae sensu APGII) have yielded a range of previously undescribed bufadienolides, drimianins A-G (1-7), the known bufadienolides bovogenin A (8), 3ß-O-ß-d-glucopyranosylbovogenin A (9), scillaren F (10), and altoside (11), the known homoisoflavonoid (3S)-3-(4'-methoxybenzyl)-5,6,7-trimethoxychroman-4-one (urgineanin C), the sesquiterpenoids 1ß,6α-dihydroxy-4(15)-eudesmene and 6α-hydroxy-4(15)-eudesmen-1-one, polybotrin, adenosine, and 9R-hydroxy-(10E,12Z)-octadecadienoic acid ethyl ester. The bufadienolides isolated were tested at 10 µM in the NCI-60 cancer cell screen, and nine of these were selected for further screening at five concentrations. Drimianins C (3) and E (5) showed activity at the nanomolar level against a number of human cancer cell lines in the NCI-60 screen.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Bufanólidos/farmacología , Drimia/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Bufanólidos/aislamiento & purificación , Línea Celular Tumoral , Humanos , Estructura Molecular , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Raíces de Plantas/química , SudáfricaRESUMEN
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.
Asunto(s)
Bufanólidos/farmacología , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Extractos Vegetales/farmacología , Neoplasias del Cuello Uterino/metabolismo , Animales , Bufanólidos/aislamiento & purificación , Bufanólidos/uso terapéutico , Bufonidae , Puntos de Control del Ciclo Celular/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Daño del ADN/fisiología , Femenino , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Chansu, dried secretions from Bufonidae, has long been used for cancer treatment as a traditional Chinese medicine. In searching for effective anti-hepatoma agents from Chansu, our preliminary drug screening found that a bufadienolide, namely 1ß-hydroxyl-arenobufagin (1ß-OH-ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1ß-OH-ABF have not been defined. AIM OF THE STUDY: To evaluate the anti-hepatoma activity of 1ß-OH-ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo, as well as explore the underlying mechanisms. MATERIALS AND METHODS: The anti-proliferative effects of 1ß-OH-ABF on liver cancer Hep3B, HepG2, HuH7, SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1ß-OH-ABF. The collapse of the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining assay. Western blotting was used to examine the expression levels of targeted proteins. The role of mTOR in 1ß-OH-ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1ß-OH-ABF in vivo. RESULTS: We found that 1ß-OH-ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 1ß-OH-ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway, which is accompanied by the loss of ΔΨm, upregulation and translocation of Bax, as well as cleavages of caspase-9, caspase-3 and PARP. Mechanistically, 1ß-OH-ABF markedly decreases the expression level of p-AKT/AKT and p-mTOR (Ser2248 and Ser2481)/mTOR in a time-dependent manner. Inhibition of mTOR by siRNA strengthens 1ß-OH-ABF-mediated apoptosis. Critically, 1ß-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. CONCLUSION: 1ß-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1ß-OH-ABF may serve as a potential agent for the treatment of liver cancer.
Asunto(s)
Antineoplásicos/farmacología , Bufanólidos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Carcinoma Hepatocelular/patología , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/patología , Mitocondrias/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Pez CebraRESUMEN
Bufalin is an anticancer drug extract from traditional Chinese medicine. Several articles about bufalin have been published. However, the literature on bufalin has not yet been systematically studied. This study aimed to identify the study status and knowledge structures of bufalin and to summarize the antitumor mechanism. Data were retrieved and downloaded from the PubMed database. The softwares of BICOMB, gCLUTO, Ucinet 6.0, and NetDraw2.084 were used to analyze these publications. The bufalin related genes were recognized and tagged by ABNER software. Then these BF-related genes were performed by Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, and protein-protein interaction (PPI) network analysis. A total of 474 papers met the search criteria from 2000 to 2019. By biclustering clustering analysis, the 50 high-frequency main MeSH terms/subheadings were classified into 5 clusters. The clusters of drug therapy and the mechanism of bufalin were hotspot topics. A total of 50 genes were identified as BF-related genes. PPI network analysis showed that inducing apoptosis was the main effect of bufalin, and apoptosis-related gene Caspase 3 was the most reported by people. Bufalin could inhibit the proliferation, invasion, and metastasis of cancer cells through multiple signaling pathways, such as PI3K/AKT, Hedgehog, MAPK/JNK, Wnt/[Formula: see text]-catenin, TGF-[Formula: see text]/Smad, Integrin signaling pathway, and NF-KB signaling pathway via KEGG analysis. Through the quantitative analysis of bufalin literature, we revealed the research status and hot spots in this field and provided some guidance for further research.
Asunto(s)
Antineoplásicos , Bufanólidos/farmacología , Biología Computacional , Minería de Datos , Medicamentos Herbarios Chinos/farmacología , Neoplasias/genética , Neoplasias/patología , Fitoterapia , Apoptosis/genética , Bufanólidos/aislamiento & purificación , Bufanólidos/uso terapéutico , Caspasa 3/metabolismo , Proliferación Celular/genética , Medicamentos Herbarios Chinos/aislamiento & purificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Terapia Molecular Dirigida , FN-kappa B/genética , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.
Asunto(s)
Bufanólidos/aislamiento & purificación , Bufonidae/fisiología , Glándula Parótida/metabolismo , Animales , Bufonidae/clasificación , Cromatografía Liquida , Evolución Molecular , Japón , Espectrometría de Masas , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Glándula Parótida/química , Especificidad de la EspecieRESUMEN
From the leaves of South African medicinal plant Melianthus comosus, four previously undescribed bufadienolides, 16ß-formyloxymelianthugenin (1), 2ß-acetoxymelianthusigenin (2), 2ß-hydroxy-3ß,5ß-di-O-acetylhellebrigenin (3), and 2ß-acetoxy-5ß-O-acetylhellebrigenin (4) were isolated together with two known bufadienolides. The structural elucidation of the compounds was based on 1D and 2D NMR spectroscopy, high-resolution mass spectrometry, and other spectroscopic methods. The relative configurations were determined by single-crystal X-ray crystallography analysis and NOESY correlations. The isolated compounds displayed strong cytotoxicity against MCF-7 breast cancer cells, sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Compound 1 showed the most potent activity, with IC50 values of 0.07⯵M towards CCRF-CEM, 0.06⯵M towards CEM/ADR5000 and 0.36⯵M towards MCF-7 followed by compound 4 with IC50 values of 0.13⯵M towards CCRF-CEM, 0.08⯵M towards CEM/ADR5000 and 0.53⯵M towards MCF-7.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Bufanólidos/farmacología , Hojas de la Planta/química , Plantas Medicinales/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Conformación Molecular , Sudáfrica , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
CONTEXT: Kalanchoe species (Crassulaceae) are widely used in traditional medicine as remedies in infectious diseases and cancer treatment. OBJECTIVE: Cytotoxic and antimicrobial activities of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier, K. pinnata (Lam.) Pers., and K. blossfeldiana Poelln. extracts were determined. The relationship between biological activities and the extracts bufadienolides content was also investigated. MATERIALS AND METHODS: Fresh leaves of Kalanchoe species were macerated with 95% ethanol or water. The quantitative analysis of bufadienolides in the extracts was carried out with mass spectrometry. Cytotoxicity tests were performed on human cancer cell lines - HeLa, SKOV-3, MCF-7, and A375 by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and Real-Time Cell Analysis system. The microbiological study was done using a few bacteria strains (ß-hemolytic Streptococcus, Corynebacterium diphtheriae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus hirae, Escherichia coli) and Candida albicans. RESULTS: The K. blossfeldiana ethanol extract and K. daigremontiana water extract exhibited the most potent cytotoxic activity (IC50 < 19 µg/mL for HeLa and SKOV-3 cells). The strongest antibacterial effects showed ethanol extract of K. blossfeldiana and K. pinnata (MIC values were 8.45, 8.45, 0.25 and <33.75 µg/mL for S. aureus, S. epidermidis, and E. hirae, respectively). The highest total amount of bufadienolides was in K. daigremontiana ethanol extract. In contrast, K. blossfeldiana ethanol extract did not show the presence of these compounds. CONCLUSIONS: Kalanchoe blossfeldiana ethanol extract is a potential candidate for cancer and bacterial infection treatment. Additionally, the biological effects of Kalanchoe extracts are not dependent on the presence and amount of bufadienolides in the plant extracts.
Asunto(s)
Antiinfecciosos/farmacología , Antineoplásicos Fitogénicos/farmacología , Bufanólidos/farmacología , Kalanchoe/química , Extractos Vegetales/farmacología , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Hojas de la PlantaRESUMEN
Melianthus major is a medicinal plant endemic to South Africa. Its leaf extract led to the isolation of five new bufadienolides, 2ß-acetoxy-3,5-di-O-acetylhellebrigenin (1), 2ß-acetoxy-3-O-acetylhellebrigenin (2), 2ß-acetoxy-14-deoxy-15ß,16ß-epoxymelianthugenin (4), 2ß-acetoxy-14-deoxy-15ß,16ß-epoxymelianthusigenin (5), and 2ß-hydroxymelianthusigenin (6), and four known analogues. The structures of the compounds were elucidated using NMR and HRESIMS data analyses. The relative configurations were defined by single-crystal X-ray crystallography and NOESY correlations. The isolated compounds exhibited strong cytotoxicity against MCF-7 breast cancer cells and sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Compound 1 showed the most potent activity, with IC50 values of 0.1 µM toward CCRF-CEM and CEM/ADR5000 and 0.3 µM toward MCF-7.
Asunto(s)
Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Bufanólidos/aislamiento & purificación , Bufanólidos/farmacología , Magnoliopsida/química , Hojas de la Planta/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Análisis Espectral/métodosRESUMEN
The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.
Asunto(s)
Bufanólidos/toxicidad , Bufonidae , Neurotoxinas/toxicidad , Glándula Parótida/química , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Bufanólidos/aislamiento & purificación , Bloqueadores de los Canales de Calcio/aislamiento & purificación , Bloqueadores de los Canales de Calcio/toxicidad , Supervivencia Celular/efectos de los fármacos , Pollos , Inhibidores de la Colinesterasa/aislamiento & purificación , Inhibidores de la Colinesterasa/toxicidad , Relación Dosis-Respuesta a Droga , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Neurotoxinas/aislamiento & purificación , Vías Secretoras , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal 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.
Asunto(s)
Evolución Biológica , Dieta , Conducta Alimentaria , Conducta Predatoria , Serpientes/fisiología , Toxinas Biológicas/química , Animales , Anuros , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Bufonidae , Glicósidos Cardíacos , Colubridae , Mecanismos de Defensa , Glicosilación , Insectos , Larva , Peso Molecular , Oligoquetos , Estereoisomerismo , Toxinas Biológicas/aislamiento & purificaciónRESUMEN
American trypanosomiasis is a parasitic neglected disease, responsible for the death of approximately 10,000 people every year. Amphibians are recognized for producing in their cutaneous glands substances with pharmacological potential against a variety of pathologies. Here we investigated the antiprotozoal activity against Trypanosoma cruzi of bufadienolides isolated from the parotoid glands secretions of the toad Rhinella centralis from Panama. NMR and mass spectrometry analysis led to the identification of the active compound 19-hydroxy-bufalin, for which its antitrypanosomal activity and occurrence in the genus Rhinella are reported for the first time. This compound showed low cytotoxicity and significant selectivity which confers to it a potential role for the treatment of Chagas disease.
Asunto(s)
Venenos de Anfibios/toxicidad , Bufanólidos/toxicidad , Bufonidae , Trypanosoma cruzi/efectos de los fármacos , Animales , Bufanólidos/aislamiento & purificaciónRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the leading cause of cancer incidence and mortality worldwide. Arenobufagin (Arg), a representative natural bufadienolide compound, is one of the major bioactive components isolated from toad venom ("Chan Su"named in Chinese to treat multifarious clinical neoplasms in China). However, the underlying molecular mechanisms that Arg inhibited the metastasis of lung cancer cells remain poorly understood. MATERIALS AND METHODS: The mobility capacities of lung cancer cells treated with Arg were evaluated using wound healing assay. The anti-migratory and anti-invasive effects of Arg on lung cancer cells were investigated by transwell invasion assay and matrigel invasion assay. iTRAQ-labeled LC-MS proteomics was used to analyze the potential proteins related to metastasis in lung cancer cells treated with Arg and differentially-expressed proteins related to EMT and NFκB signaling cascade were further confirmed by Western blotting assay. The changed subcellular localization of p65 in lung cancer A549 and H1299 cells treated with Arg was detected by immunofluorescence staining. Molecular docking and molecular dynamic (MD) simulation assay were performed to verify the binding between Arg and IKKα/IKKß. siRNA knockdown was used to check whether Arg inhibited EMT of lung cancer cells via targeting NFκB signaling cascade, which was further verified by in vivo study of lung cancer cell xenograft mice model and pulmonary metastasis mice model accompanying with immunohistochemical and hematoxylin-eosin (HE) staining. RESULTS: Arg suppressed the wound closure of lung cancer cells using wound healing assay. Moreover, Arg significantly inhibited the migration and invasion of lung cancer cells by transwell invasion assay and matrigel invasion assay. 24 unique differentially-expressed proteins related to metastasis in lung cancer cells treated with Arg were identified using iTRAQ-labeled LC-MS proteomics and 14 differentially-expressed proteins related to EMT were further confirmed by Western blotting assay. Arg significantly decreased the phosphorylation of IKKß, IκBα and p65 in the cytoplasm of lung cancer cells by Western blotting assay, and remarkably reduced the release of p65 from the cytoplasm to the nucleus. Arg could be bound in the ATP binding pocket of IKKα and IKKß by molecular docking assay, and MD simulation assay further demonstrated that Arg binding to the ATP-binding pocket of IKKß was very stable in 300 ns MD simulation, compared with the binding of Arg and IKKα. IKKß/NFκB signaling cascade was also involved in the inhibitory effect of Arg on EMT of lung cancer cells by siRNA knockdown assay. The study of lung cancer cell xenograft mice model and pulmonary metastasis mice model in vivo indicated that Arg inhibited EMT and suppressed migration and invasion of lung cancer cells via downregulating IKKß/NFκB signaling cascade. CONCLUSION: In the present study, we explored the molecular mechanism of Arg prohibiting the metastasis of lung cancer cells in vitro and in vivo, which displayed Arg could target IKKß to inactive NFκB signaling cascade and further change the expression of proteins related to EMT. These results highlight the potential of toad venom as a potential chemotherapeutic agent and warrant its development as the clinical therapy for lung cancer.
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Venenos de Anfibios/química , Bufanólidos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Células A549 , Animales , Bufanólidos/aislamiento & purificación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Quinasa I-kappa B/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , FN-kappa B/metabolismo , Invasividad Neoplásica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Three new bufadienolides 14ß, 16ß-dihydroxy-3ß-[ß-D-glucopyranosyl-(1â6)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (1), 14ß-hydroxy-3ß-[ß-D-glucopyranosyl-(1â4)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (2) and hellebrigenin-3-O-ß-D-glucosyl-(1â4)-ß-D-glucoside (3), together with eight known bufadienolides (4-11) were isolated from the roots and rhizomes of Helleborus thibetanus. Their structures were elucidated by extensive spectroscopic methods and acid hydrolysis. Compounds 1-7 were evaluated for their cytotoxic activity against HCT116, A549 and HepG2 tumor cell lines. Compound 1 exhibited moderate cytotoxicity against HepG2 cells with IC50 value of 15.1 ± 1.72 µM. Compounds 5 and 6 exhibited moderate cytotoxicity against HCT116 cells with IC50 values of 15.12 ± 0.58 µM and 13.17 ± 2.34 µM, respectively.
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Bufanólidos/aislamiento & purificación , Citotoxinas/aislamiento & purificación , Helleborus/química , Raíces de Plantas/química , Rizoma/química , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Bufanólidos/química , Bufanólidos/farmacología , Línea Celular Tumoral , Citotoxinas/química , Citotoxinas/farmacología , Glicósidos/química , Glicósidos/aislamiento & purificación , Humanos , Estructura MolecularRESUMEN
From ancient times, medicinal plants have been usually utilized to treat many disorders, but today, interest in these herbs is again aroused, because of their fewer side effects and low-cost. In traditional medicine, for many diseases, various medicinal herbs have been suggested so far. Drimia maritime, also named squill, is an important medicinal plant for the treatment of many diseases, especially respiratory diseases. In the current evidence-based study, we conducted a review of the general characteristics, ingredients, administration form, and side effects of squill in traditional medicine. For this purpose, traditional Persian medicine literatures and electronic databases were examined including PubMed, Scopus, and Google Scholar. Many compounds are isolated from D.maritima, including scillaren, scillirubroside, scillarenin, and bufadienolide glycosides. Oxymel is the most commonly used form of squill for various diseases, especially respiratory diseases. Besides, squill has been used in the treatment of cardiovascular, digestive, and dermatological disorders, it is also used against various cancer cells for its antioxidant and cytotoxic properties. Moreover, there is relatively reliable evidence of its benefits for bacterial and helminthic infections, rheumatism, edema, gout, abortion induction, healing of wounds and urine induction. It seems that supplementary studies are required to explore the bioactive agents and their effective mechanisms.
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Drimia/química , Medicina Basada en la Evidencia/métodos , Fitoterapia/métodos , Preparaciones de Plantas/uso terapéutico , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Bufanólidos/uso terapéutico , Glicósidos Cardíacos/química , Glicósidos Cardíacos/aislamiento & purificación , Glicósidos Cardíacos/uso terapéutico , Humanos , Preparaciones de Plantas/química , Preparaciones de Plantas/aislamiento & purificaciónRESUMEN
Bufadienolide compounds have been used for growth inhibition and apoptosis induction in tumor cells. Those families of cardiotonic steroids can bind the Na,K-ATPase, causing its inhibition. The use of bufadienolides is widely described in the literature as an anticancer function. The aim of this study was to evaluate the effects of bufadienolides and alkaloid isolated from venom samples from R. marina on tumor cells. We performed cytotoxicity assay in MDA-MB-231 and TOV-21G cells and evaluated the activity of Caspases (3 and 9), Na, K-ATPase, PMCA and SERCA. Four compounds were extrated from the venom of R. marina. The compound 1 showed higher cytotoxicity in MDA-MB-231cells. Compound 1 also showed activation of Caspase 3 and 9. This compound caused an inhibition of the activity and expression of Na, K-ATPase, and also showed activation of both caspase-9 and caspase-3 in MDA-MB-231 cells. We also observed that Compound 1 had a direct effect on some ATPases, such as Na, K-ATPase, PMCA and SERCA. Compound 1 was able to inhibit the activity of the purified Na, K-ATPase enzyme from the concentration of 5⯵M. It also caused inhibition of PMCA at all concentrations tested (1â¯nM-30⯵M). However, the compound 1 led to an increase of the activity of purified SERCA between the concentrations of 7.5-30⯵M. Thus, we present a Na, K-ATPase and PMCA inhibitor, which may lead to the activation of caspases 3 and 9, causing the cells to enter into apoptosis. Our study suggests that compound 1 may be an interesting molecule as an anticancer agent.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Bufanólidos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Bufo marinus , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadRESUMEN
Comprehensive analysis and identification of chemical components are very important to evaluate the efficacy and safety of traditional Chinese medicine (TCM). Meanwhile, the discovery of new natural compounds is of great significance for drug exploitation and development. Although two-dimensional liquid chromatography (2D LC) systems expand the peak capacity and improve selectivity and resolution, interpreting the post-processing data is tedious and time-consuming. In this study, an off-line two-dimensional liquid chromatography/ultra-high performance supercritical fluid chromatography tandem quadrupole time-of-flight mass spectrometry (2D LC/UHPSFC-Q-TOF/MS) system was established for systematic chromatographic separation and identification of bufadienolides. Subsequently, the Global Natural Product Social Molecular Networking (GNPS) was applied for dereplication of chemical components of adjacent fractions with high efficiency and accuracy. The key parameters which affected separation and detection with respect to chromatographic conditions and mass spectrometry conditions were optimized. The extract of Venenum Bufonis was fractionated into forty fractions by first-dimensional reversed phase high-performance liquid chromatography (HPLC), which were further analyzed by the second-dimensional UHPSFC-Q-TOF/MS in positive ion mode. The data of forty fractions was imported into GNPS and processed automatically within about five hours. Furthermore, the chemical components with similar featured fragments were classified into the same cluster, which was helpful for components identification. A total of 229 bufadienolides were characterized and two subclasses of compounds (bufogenins conjugated with carboxylic acid and N-heterocyclic bufogenins) were found in Venenum Bufonis for the first time. In addition, UHPSFC exhibited powerful separation ability of isomers in Venenum Bufonis. In this analysis, two new compounds were isolated and fully characterized by NMR verifying the feasibility of this combined analytical strategy. This integrated strategy can improve the efficiency in the detection of new compounds and offer greater observation of isomers from medicinal herbs and other natural sources.