RESUMEN
Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.
Asunto(s)
Bufanólidos , Sistema Enzimático del Citocromo P-450 , Bufanólidos/química , Bufanólidos/metabolismo , Bufanólidos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/metabolismo , Hidroxilación , Línea Celular Tumoral , Bufonidae/metabolismo , Proliferación Celular/efectos de los fármacosRESUMEN
Arenobufagin, one of the bufadienolides isolated from traditional Chinese medicine Chan'su, exhibits potent antitumor activity. However, serious toxicity and small therapeutic window limits its drug development. In the present study, to our knowledge, novel 3,11-bispeptide ester arenobufagin derivatives have been firstly designed and synthesized on the base of our previous discovery of active 3-monopeptide ester derivative. The inâ vitro antiproliferative activity evaluation revealed that the moiety at C3 and C11 hydroxy had an important influence on cytotoxic activity and selectivity. Compound ZM350 notably inhibited tumor growth by 58.8 % at a dose 10â mg/kg in an A549 nude mice xenograft model. Therefore, compound ZM350 also presented a concentration-dependent apoptosis induction and low inhibitory effect against both hERG potassium channel and Cav1.2 calcium channel. Our study suggests that novel 3,11-bispeptide ester derivatives will be a potential benefit to further antitumor agent development of arenobufagin.
Asunto(s)
Antineoplásicos , Bufanólidos , Animales , Ratones , Humanos , Línea Celular Tumoral , Cardiotoxicidad/tratamiento farmacológico , Ratones Desnudos , Antineoplásicos/farmacología , Bufanólidos/química , Apoptosis , Ensayos de Selección de Medicamentos Antitumorales , Proliferación CelularRESUMEN
From the leaves of Kenyan medicinal plant Bersama abyssinica Subspecies abyssinica, four previously undescribed compounds namely, three bufadienolides, 10ß-formylpaulliniogenin B, 10ß-formylpaulliniogenin A and 1ß-acetoxy-3ß,5ß-dihydroxy-15-methoxy-16,19-dioxobufa-14(15),20,22-trienolide, and a phenolic compound 2,6,4'-trihydroxybenzophenone-4-O-(6â´-cinnamoyl)-ß-D-glucoside were isolated together with four known compounds. The structural elucidation of the compounds was based on 1D and 2D NMR spectroscopy and HRMS data analyses. The relative configurations were defined by NOESY correlations. Cytotoxic activities on L929 and KB3.1 cell lines of the isolated compounds were investigated using MTT assay. The 1ß-acetoxy-3ß,5ß-dihydroxy-15-methoxy-16,19-dioxobufa-14(15),20,22-trienolide showed significant cytotoxic activity against KB3.1 cell lines with IC50 of 3.9 ± 0.99 µM.
Asunto(s)
Antineoplásicos , Bufanólidos , Magnoliopsida , Plantas Medicinales , Bufanólidos/análisis , Bufanólidos/química , Línea Celular Tumoral , Kenia , Magnoliopsida/química , Hojas de la Planta/químicaRESUMEN
OBJECTIVE: To explore the active compounds and targets of cinobufotalin (huachansu) compared with the osteosarcoma genes to obtain the potential therapeutic targets and pharmacological mechanisms of action of cinobufotalin on osteosarcoma through network pharmacology. METHODS: The composition of cinobufotalin was searched by literature retrieval, and the target was selected from the CTD and TCMSP databases. The osteosarcoma genes, found from the GeneCards, OMIM, and other databases, were compared with the cinobufotalin targets to obtain potential therapeutic targets. The protein-protein interaction (PPI) network of potential therapeutic targets, constructed through the STRING database, was inputted into Cytoscape software to calculate the hub genes, using the NetworkAnalyzer. The hub genes were inputted into the Kaplan-Meier Plotter online database for exploring the survival curve. Functional enrichment analysis was identified using the DAVID database. RESULTS: 28 main active compounds of cinobufotalin were explored, including bufalin, adenosine, oleic acid, and cinobufagin. 128 potential therapeutic targets on osteosarcoma are confirmed among 184 therapeutic targets form cinobufotalin. The hub genes included TP53, ACTB, AKT1, MYC, CASP3, JUN, TNF, VEGFA, HSP90AA1, and STAT3. Among the hub genes, TP53, ACTB, MYC, TNF, VEGFA, and STAT3 affect the patient survival prognosis of sarcoma. Through function enrichment analysis, it is found that the main mechanisms of cinobufotalin on osteosarcoma include promoting sarcoma apoptosis, regulating the cell cycle, and inhibiting proliferation and differentiation. CONCLUSION: The possible mechanisms of cinobufotalin against osteosarcoma are preliminarily predicted through network pharmacology, and further experiments are needed to prove these predictions.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Bufanólidos/farmacología , Osteosarcoma/tratamiento farmacológico , Antineoplásicos/química , Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Bufanólidos/química , Biología Computacional , Bases de Datos de Compuestos Químicos , Bases de Datos Farmacéuticas , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Medicina Tradicional China , Farmacología en Red , Osteosarcoma/genética , Mapas de Interacción de Proteínas/efectos de los fármacos , Mapas de Interacción de Proteínas/genéticaRESUMEN
Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, "cholesterol-bile acid-bufadienolidies pathway" was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3ß-hydroxysteroid dehydrogenase (3ßHSD) and steroid 5ß-reductase (SRD5ß) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5ß cDNA of B. bufo gargarizans (Bbg-SRD5ß) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5ß. Bbg-SRD5ß had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5ß in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5ß was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5ß can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5ß-Y132G was 1.8 times higher than that of wild type Bbg-SRD5ß. Although Bbg-SRD5ß was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5ß is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5ß.
Asunto(s)
Venenos de Anfibios/química , Bufo bufo/metabolismo , Oxidorreductasas/aislamiento & purificación , Secuencia de Aminoácidos , Venenos de Anfibios/metabolismo , Animales , Bufanólidos/química , Bufanólidos/metabolismo , Bufonidae/metabolismo , Clonación Molecular/métodos , ADN Complementario/metabolismo , Sistemas de Lectura Abierta , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Esteroides/metabolismoRESUMEN
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.
Asunto(s)
Bufanólidos/química , Ecdisteroides/química , Helleborus/química , Plantas/química , Humanos , Estructura MolecularRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini is extracted from the skins and parotid venom glands of the toad for treating symptoms like swelling and pain in ancient times. Nowadays, cinobifucini injection has also achieved satisfactory therapeutic effects on hepatocellular carcinoma (HCC) in China. AIM OF THE STUDY: Our previous work found that bufothionine, an alkaloid abundant in cinobufacini injection, induced mitochondria-mediated apoptosis. In this work, the underlying effects of bufothionine on autophagy in HCC and its possible dependent pathway were investigated. METHODS: CCK-8 and Hoechst staining assays were performed to verify effects of drugs on proliferation and apoptosis of SMMC7721 cell. H22-tumor-bearing mice model was established by inoculating ascites fluid. HE staining was used to observe pathological changes in liver and tumor tissues. ELISA and Western blot experiments were conducted to investigate IL-6/JAK2/STAT3 signaling pathway. The effects of drugs on expressions of autophagic relative proteins were investigated by Western blot in vitro and in vivo. RESULTS: In vitro, CCK-8 and Hoechst staining assays showed that bufothionine inhibited SMMC7721 cell proliferation and promoted apoptosis at 100 µM. In vivo, bufothionine relieved symptoms of H22-tumor-bearing mice and exerted anti-inflammation activity. ELISA and Western blot demonstrated that bufothionine significantly reduced serum IL-6 concentration, suppressed p-Stat3tyr705, p-Stat3ser727 and Jak2 expressions in tumor tissues and upregulated Atg5, Atg7 and LC3â ¡ expressions in SMMC7721 cell and H22 tumor. CONCLUSION: This is the first report showing that bufothionine might induce autophagy in HCC by inhibiting JAK2/STAT3 pathway, presenting a possible anti-cancer mechanism of bufothionine in cinobufacini injection.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Autofagia/efectos de los fármacos , Bufanólidos/farmacología , Alcaloides Indólicos/farmacología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias/patología , Compuestos de Quinolinio/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Bufanólidos/química , Bufanólidos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Alcaloides Indólicos/uso terapéutico , Interleucina-6/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Neoplasias/metabolismo , Compuestos de Quinolinio/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismoRESUMEN
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
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
BACKGROUND: Homoisoflavonoids have been shown to have potent anti-proliferative activities in endothelial cells over other cell types and have demonstrated a strong antiangiogenic potential in vitro and in vivo in animal models of ocular neovascularization. Three species of Rhodocodon (Scilloideaea subfamily of the Asparagaceae family), endemic to Madagascar, R. cryptopodus, R. rotundus and R. cyathiformis, were investigated. PURPOSE: To isolate and test homoisoflavonoids for their antiangiogenic activity against human retinal microvascular endothelial cells (HRECs), as well as specificity against other ocular cell lines. METHODS: Plant material was extracted at room temperature with EtOH. Compounds were isolated using flash column chromatography and were identified using NMR and CD spectroscopy and HRESIMS. Compounds were tested for antiproliferative effects on primary human microvascular retinal endothelial cells (HRECs), ARPE19 retinal pigment epithelial cells, 92-1 uveal melanoma cells, and Y79 retinoblastoma cells. HRECs exposed to compounds were also tested for migration and tube formation ability. RESULTS: Two homoisoflavonoids, 3S-5,7-dihydroxy-(3'-hydroxy-4'-methoxybenzyl)-4-chromanone (1) and 3S-5,7-dihydroxy-(4'-hydroxy-3'-methoxybenzyl)-4-chromanone (2), were isolated along with four bufadienolides. Compound 1 was found to be non-specifically antiproliferative, with GI50 values ranging from 0.21-0.85 µM across the four cell types, while compound 2 showed at least 100-fold specificity for HRECs over the other tested cell lines. Compound 1, with a 3S configuration, was 700 times more potent that the corresponding 3R enantiomer recently isolated from a Massonia species. CONCLUSION: Select homoisoflavonoids have promise as antiangiogenic agents that are not generally cytotoxic.
Asunto(s)
Asparagaceae/química , Bufanólidos/química , Isoflavonas/química , Isoflavonas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Humanos , Vasos Retinianos/citologíaRESUMEN
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.
Asunto(s)
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
Toad venom (Chansu), a traditional Chinese medicine (TCM), has been widely used for treating various cancer. However, it is considerably difficult to evaluate the quality of Chansu due to its complex chemical compositions. Hence, finding the characteristic ingredients and developing a scientific and comprehensive quality evaluation method are essential for guaranteeing the safety and efficacy of Chansu. In this paper, the chemical composition database of Chansu was successfully established and HPLC-ESI-Q-TOF-MS/MS was applied for chemical profiling of the ingredients in Chansu. In total, 157 compounds were identified, including 22 amino acids, 8 alkaloids, 54 bufogenins, 63 bufotoxins, and 10 other compounds. Furthermore, HPLC fingerprints and quantitative analysis of its multicomponent were successfully developed to evaluate the quality consistency of Chansu from different origins. The results suggested that the HPLC fingerprint of Chansu could be divided into an amino acid and alkaloid region, as well as a bufogenins and bufotoxins region. The fingerprint profile of Chansu from different geographical origins were different, indicating that its quality was affected by the geographical factors. In addition, seven characteristic peaks were selected as the quantitative markers to evaluate the quality of the Chansu. The Kruskal-Wallis test illustrated that the contents of seven bufogenins in Chansu were significantly (p < 0.01) different among different origins. The total contents of the seven compounds ranged from 100.40 to 169.22 mg/g in 20 batches of Chansu samples. This study demonstrated that integrating HPLC-ESI-Q-TOF-MS/MS, HPLC fingerprints, and multicomponent quantitative analysis coupled with chemometrics was a comprehensive and reliable strategy for evaluation of Chansu in both qualitative and quantitative aspects. In addition, our study represented the most comprehensive characterization on the chemical compositions of Chansu, which could provide important reference information for the discovery of potential bioactive compounds.
Asunto(s)
Venenos de Anfibios/química , Alcaloides/química , Bufanólidos/química , Cromatografía Líquida de Alta Presión , Bases de Datos de Compuestos Químicos , Medicina Tradicional China , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Bufanólidos/aislamiento & purificación , Animales , Productos Biológicos/análisis , Bufanólidos/química , Bufonidae , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Espectroscopía de Resonancia Magnética , Espectrometría de Masas en TándemRESUMEN
Chan Su is a traditional medicine prepared from toxic secretions from the auricular and skin glands of Chinese toads. Previous studies show that active components in Chan Su can inhibit the proliferation of tumor cells. To study the effect of Chan Su peptides on angiogenesis, fresh Chan Su was collected and its component peptides were isolated by an extraction and precipitation method. A high-performance liquid chromatography (HPLC) fingerprint of the Chan Su component peptides revealed that there were more than 18 peptide component peaks. We demonstrate that Chan Su peptides inhibit angiogenesis in vitro by inhibiting human umbilical vein endothelial cell (HUVEC) proliferation and tube formation in a dose-dependent manner. Western blots indicated that Chan Su peptides inhibited the protein expression of VEGF165 and Ras, leading us to conclude that Chan Su peptide components exert anti-angiogenic effects by suppressing the VEGF165-VEGFR2-Ras signalling pathway. Finally, we identified the partial amino acid sequences of seven Chan Su peptides using the shotgun proteomics method.
Asunto(s)
Venenos de Anfibios/química , Inhibidores de la Angiogénesis/aislamiento & purificación , Bufanólidos/química , Medicina Tradicional China , Animales , Anuros , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Células Endoteliales de la Vena Umbilical Humana , Humanos , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Proteínas ras/antagonistas & inhibidoresRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The animal medicine of Venenum Bufonis (VB), a product of the secretions of Bufo gargarizans Cantor or B. melanostictus Schneider, has long been used as a traditional Chinese medicine (TCM) for the treatment of sunstroke and faint, acute filthy disease - abdominal pain or vomiting and diarrhea, etc. AIM OF THE REVIEW: This review is aimed at providing the comprehensive and up-to-date information of VB as regards its ethnopharmacological uses, constituents and their metabolism, pharmacokinetics, pharmacology and toxicology, all of which could be used as fundamental data for future research as well as development of new drugs. MATERIALS AND METHODS: The information and data about the studies of VB were collected from scientific journals, material medica, historical documents, library, and electronic databases (PubMed, Google Scholar, Science Direct, Researchgate, Web of Science and CNKI). RESULTS: To date, about 142 bufadienolides and 16 indole alkaloids have been isolated from VB in total. The extract and isolated compounds showed a wide range of in vitro and in vivo pharmacologic effects, such as cardiotonic, anti-tumor, antinociceptive, anti-inflammatory, anesthetic and antimicrobial activities. Especially, bufadienolides have been extensively studied due to its powerful anti-tumor activities against various cancer cells. Furthermore, their metabolites and metabolic pathways were concluded in detail, and the main metabolic pathways of bufadienolides were hydroxylation, 3-isomerization, 3-keto, 16-hydrolyzation, 3-O-sulfate and 3-O-glucuronide. CONCLUSIONS: Although VB possesses significant anti-tumor effect against various cancer cell lines, the development of new drugs still remains to be a challenge due to its pharmacodynamic effects in vivo, druggability and toxicology. The main problem lies in its side effects in vivo, poor bioavailability, fast metabolism, cardiotoxicity and neurovirulence. Besides, studies on its metabolism and toxicology in vitro and in vivo, as well as clinical trials should be further conducted for the new drug development and the establishment of optimal dosage of consumption of its administration.
Asunto(s)
Bufanólidos , Medicina Tradicional China , Animales , Bufanólidos/química , Bufanólidos/farmacología , Bufanólidos/uso terapéutico , Humanos , Fitoquímicos/análisisRESUMEN
The aim of this study was to investigate the effects of bufalin on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Bufalin is a cardiotonic steroid and a key active ingredient of the Chinese medicine ChanSu. The extracts of Chansu are used for various cancer treatments in China. In the present study, bufalin induced cell morphological changes, decreased total cell viability and induced G2/M phase arrest of cell cycle in NPC-TW 076 cells. Results also indicated that bufalin induced chromatin condensation (cell apoptosis) and DNA damage by DAPI staining and comet assay, respectively. The induced apoptotic cell death was further confirmed by annexin-V/PI staining assay. In addition, bufalin also increased ROS and Ca 2+ production and decreased the levels of ΔΨm . Furthermore, the alterations of ROS, ER stress and apoptosis associated protein expressions were investigated by Western blotting. Results demonstrated that bufalin increased the expressions of ROS associated proteins, including SOD (Cu/Zn), SOD2 (Mn) and GST but decreased that of catalase. Bufalin increased ER stress associated proteins (GRP78, IRE-1 α , IRE-1 ß , caspase-4, ATF-6 α , Calpain 1, and GADD153). Bufalin increased the pro-apoptotic proteins Bax, and apoptotic associated proteins (cytochrome c, caspase-3, -8 and -9, AIF and Endo G) but reduced anti-apoptotic protein Bcl-2 in NPC-TW 076 cells. Furthermore, bufalin elevated the expressions of TRAIL-pathway associated proteins (TRAIL, DR4, DR5, and FADD). Based on these findings, we suggest bufalin induced apoptotic cell death via caspase-dependent, mitochondria-dependent and TRAIL pathways in human nasopharyngeal carcinoma NPC-TW 076 cells.
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Apoptosis/efectos de los fármacos , Apoptosis/genética , Bufanólidos/farmacología , Mitocondrias/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Extractos de Tejidos/farmacología , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Humanos , Carcinoma Nasofaríngeo/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/metabolismo , Extractos de Tejidos/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Two new 19-norbufadienolides (1 and 2) and one new 14,15-epoxy bufadienolide (3) alongside 16 known bufadienolides (4-19) were isolated from Bufonis Venenum that originated from the skin and parotid venom glands of an Asiatic toad (Bufo bufo gargarizans Cantor). The structures of these bufadienolides were elucidated based on the interpretation of their HRESIMS and NMR data. Compound 1, which had a unique peroxide, was established through extensive single-crystal X-ray diffraction. The two 19-norbufadienolides exhibited more potent cardiotonic activity in the isolated toad heart model and lower cytotoxicity against U87, U251, and LN-18 cell lines than other bufadienolides, such as bufalin and bufotalin. The results suggested that 19-norbufadienolides might be more suitable for developing cardiotonic agents with low cytotoxicity.
Asunto(s)
Venenos de Anfibios/química , Bufanólidos/química , Bufo bufo , Cardiotónicos/farmacología , Animales , Bufanólidos/aislamiento & purificación , Bufanólidos/farmacología , Cardiotónicos/aislamiento & purificación , Línea Celular Tumoral , Corazón/efectos de los fármacos , Humanos , Técnicas In Vitro , Estructura MolecularRESUMEN
The mass spectrometry (MS) has been widely used for profiling chemical components of traditional Chinese medicine (TCM). However, there are few studies reporting quality control of TCM based on mass spectrometry fingerprint (MSF) due to its complicated operation and high cost. The aim of this study was to extend the application of MSF for quality evaluation of TCM. In this study, an MSF based on single quadrupole mass spectrometry method was established, and was successfully used for the quality control of Venenum bufonis (VB), a famous TCM which was used clinically for cancer treatment in China. The results showed that the superiority of MSF for more chemical information exposure and the finding of more potential chemical markers (eight versus four) compared with the traditional photo-diode array (a kind of ultra violet detector, PDA). Besides, the performance of MSF was also validated by similarity and principle component analysis (PCA) of MS data acquired on two other mass spectrometry (low-resolution, triple quadrupole, QQQ, and high-resolution, quadruple time-of-flight, Q-TOF), showing high consistency with QQQ and Q-TOF, but robustness with few parameters' settings. Based on our study, MSF could be widely applied for the quality control of TCM.
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Bufanólidos/análisis , Medicamentos Herbarios Chinos/análisis , Espectrometría de Masas/métodos , Análisis de Componente Principal/métodos , Bufanólidos/química , Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/química , Extractos Vegetales/análisis , Extractos Vegetales/química , Control de CalidadRESUMEN
Bufalin is a major cardiotonic compound in the traditional Chinese medicine, Chansu, prepared from toad skin secretions. Cell culture studies have suggested an anticancer potential involving multiple cellular processes, including differentiation, apoptosis, senescence, and angiogenesis. In prostate cancer cell models, P53-dependent and independent caspase-mediated apoptosis and androgen receptor (AR) antagonism have been described for bufalin at micromolar concentrations. Because a human pharmacokinetic study indicated that single nanomolar bufalin was safely achievable in the peripheral circulation, we evaluated its cellular activity within range with the AR-positive and P53 wild-type human LNCaP prostate cancer cells in vitro Our data show that bufalin induced caspase-mediated apoptosis at 20 nmol/L or higher concentration with concomitant suppression of AR protein and its best-known target, PSA and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Bufalin exposure induced protein abundance of P53 (not mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased senescence-like phenotype (SA-galactosidase). Small RNAi knocking down of P53 attenuated bufalin-induced senescence, whereas knocking down of P21CIP1 exacerbated bufalin-induced caspase-mediated apoptosis. In vivo, daily intraperitoneal injection of bufalin (1.5 mg/kg body weight) for 9 weeks delayed LNCaP subcutaneous xenograft tumor growth in NSG SCID mice with a 67% decrease of final weight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apoptosis or suppression of AR and PSA. Our data suggest potential applications of bufalin in therapy of prostate cancer in patients or chemo-interception of prostate precancerous lesions, engaging a selective activation of P53 senescence. Mol Cancer Ther; 17(11); 2341-52. ©2018 AACR.