Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death, DNA damage and cell cycle arrest in human cervical cancer HeLa cells.

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.

1ß-OH-arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway.

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.

Bufalin: A Systematic Review of Research Hotspots and Antitumor Mechanisms by Text Mining and Bioinformatics.

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.

Cytotoxic Bufadienolides from the Leaves of Melianthus major.

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.

Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content.

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.

Cytotoxic bufadienolides from the leaves of a medicinal plant Melianthus comosus collected in South Africa.

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 µM towards CCRF-CEM, 0.06 µM towards CEM/ADR5000 and 0.36 µM towards MCF-7 followed by compound 4 with IC50 values of 0.13 µM towards CCRF-CEM, 0.08 µM towards CEM/ADR5000 and 0.53 µM towards MCF-7.

Arenobufagin, isolated from toad venom, inhibited epithelial-to-mesenchymal transition and suppressed migration and invasion of lung cancer cells via targeting IKKß/NFκB signal cascade.

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.

Therapeutic Aspects of Squill; An Evidence-Based Review.

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.

A high-efficiency strategy integrating offline two-dimensional separation and data post-processing with dereplication: Characterization of bufadienolides in Venenum Bufonis as a case study.

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.

Bufalin Induces Apoptotic Cell Death in Human Nasopharyngeal Carcinoma Cells through Mitochondrial ROS and TRAIL Pathways.

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.

A Bufadienolide-Enriched Fraction of Bryophyllum pinnatum Inhibits Human Myometrial Contractility In Vitro.

Bryophyllum pinnatum has been used since the 1970s to prevent premature labour, first in anthroposophic hospitals and, more recently, also in the main Swiss perinatal centres. However, it is not known which compounds in B. pinnatum leaves contribute to the tocolytic effect. Here we studied the effects of a flavonoid-enriched fraction, the corresponding flavonoid aglycon mixture, a bufadienolide-enriched fraction, and B. pinnatum leaf press juice on human myometrial contractility in vitro. The strength (area under the curve and amplitude) and frequency of contractions were recorded using strips of human myometrium mounted in an organ bath system. Cell viability assays were performed with the human myometrium hTERT-C3 and PHM1 - 41 cell lines. Repeated addition of the flavonoid-enriched fraction, flavonoid aglycon mixture, bufadienolide-enriched fraction, or B. pinnatum leaf press juice led to a progressive decrease of contraction strength, without jeopardising the vitality of myometrium strips. The bufadienolide-enriched fraction was the most active, since 1 µg/mL of the bufadienolide-enriched fraction lowered the area under the curve to 40.1 ± 11.8% of the initial value, whereas 150 µg/mL of the flavonoid-enriched fraction, 6.2 µg/mL of the flavonoid aglycon mixture, and 10 µg/mL of the B. pinnatum leaf press juice were required to achieve comparable inhibition. A progressive increase of contraction frequency was observed, except in the case of the flavonoid aglycon mixture, which did not affect frequency. None of the test substances decreased myometrial cell viability, even at concentrations of 500 µg/mL of the flavonoid-enriched fraction, 40 µg/mL of the flavonoid aglycon mixture, 3.8 µg/mL of the bufadienolide-enriched fraction, and 75 µg/mL of the B. pinnatum leaf press juice, i.e., higher than those used in the myometrium experiments. Given the concentrations of flavonoids in the flavonoid-enriched fraction and B. pinnatum leaf press juice, and of bufadienolides in the bufadienolide-enriched fraction and B. pinnatum leaf press juice, it appears that bufadienolides may be mainly responsible for the relaxant effect.

Two new 19-norbufadienolides with cardiotonic activity isolated from the venom of Bufo bufo gargarizans.

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.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) µM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC50: 10.88 µM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC50: 7.5 µM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer.

Arenobufagin Induces Apoptotic Cell Death in Human Non-Small-Cell Lung Cancer Cells via the Noxa-Related Pathway.

Arenobufagin, an active component isolated from the traditional Chinese medicine Chan Su, exhibits anticancer influences in several human malignancies. However, the effects and action mechanisms of arenobufagin on non-small-cell lung cancer (NSCLC) are still unknown. In this study, we reported that arenobufagin acted through activation of Noxa-related pathways and promoted apoptotic cell death in human NSCLC cells. Our results revealed that arenobufagin-induced apoptosis was caspase-dependent, as evidenced by the fact that caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) were cleaved, and pretreatment with a pan-caspase inhibitor Z-VAD-FMK inhibited the pro-apoptosis effect of arenobufagin. Mechanistically, we further found that arenobufagin rapidly upregulated the expression of the pro-apoptosis protein Noxa, and abrogated the anti-apoptosis protein Mcl-1, a major binding partner of Noxa in the cell. More importantly, the knockdown of Noxa greatly blocked arenobufagin-induced cell death, highlighting the contribution of this protein in the anti-NSCLC effects of arenobufagin. Interestingly, arenobufagin also increased the expression of p53, a direct transcriptional activator for the upregulation of the Noxa protein. Taken together, our results suggest that arenobufagin is a potential anti-NSCLC agent that triggers apoptotic cell death in NSCLC cells through interfering with the Noxa-related pathway.

Phytochemical Investigations of Three Rhodocodon (Hyacinthaceae Sensu APG II) Species.

The genus Rhodocodon (Hyacinthaceae sensu APG II) is endemic to Madagascar, and its phytochemistry has not been described previously. The phytochemistry of three species in this genus has been investigated, and eight compounds, including three bufadienolides (compounds 1, 4, and 5), a norlignan (2), and four homoisoflavonoids (compounds 3 and 6-8), have been isolated and identified. Compounds 1-3 and 6-8 have not been described previously. The COX-2 inhibitory activity of compound 6 and compound 7 acetate (compound 7A) was investigated on isolated colorectal cancer cells. Compounds 6 and 7A inhibited COX-2 by 10% and 8%, respectively, at a concentration of 12.5 µM compared to 12% for 1 mM aspirin (the positive control).

Antioxidant efficacy of Kalanchoe daigremontiana bufadienolide-rich fraction in blood plasma in vitro.

CONTEXT: The main source of bufadienolides is toad venom; however, plants such as members of Kalanchoe Adans. (Crassulaceae) genus may also synthesize these bioactive substances. OBJECTIVE: This is the first study on antioxidant effects and cytotoxicity of bufadienolide-rich fraction isolated from Kalanchoe daigremontiana Raym.-Hamet & H. Perrier. MATERIALS AND METHODS: The methanolic fraction was extracted from the plant roots and contained 0.48 mg bufadienolides/mg of dry mass (11α,19-dihydroksytelocinobufagin, bersaldegenin-1-acetate, bersaldegenin-1,3,5-orthoacetate, 19-(acetyloxy)-3ß,5ß,11α,14-tetrahydroxyl-12-oxo-bufa-20,22-dienolide and 19-(acetyloxy)-1ß,3ß,5ß,14-tetrahydroxyl-bufa-20,22-dienolide, mainly). The cytotoxicity of K. daigremontiana fraction was evaluated in an in vitro experimental model of blood platelets. The viability of blood platelets was determined on the basis of a release of lactate dehydrogenase. RESULTS: The fraction scavenged DPPH• radicals, with EC50 of 21.80 µg/mL. Studies on an experimental model of blood plasma under peroxynitrite-induced oxidative stress revealed that the plant preparation had moderate antioxidant properties. Levels of 3-nitrotyrosine and thiol groups indicated that the protective effect of K. daigremontiana was significant mainly for its concentration of 50 µg/mL. No effect was found in prevention of oxidation of low-molecular plasma thiols (glutathione, cysteine and cysteinylglycine). Simultaneously, measurements of lipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS) indicated that the examined fraction might be effective antioxidant at broader concentration range, that is 1-5 and 25-50 µg/mL for hydroperoxides and TBARS generation, respectively. No cytotoxicity was observed at the concentration range of 1-50 µg/mL. CONCLUSIONS: Based on the obtained results, we suggest that antioxidant activity may additionally contribute to beneficial properties of K. daigremontiana-derived extracts.

Chemical profiling and cytotoxicity assay of bufadienolides in toad venom and toad skin.

ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom and toad skin have been widely used for treating various cancers in China. Bufadienolides are regarded as the main anticancer components of toad venom, but the difference on composition and anticancer activities of bufadienolides between toad venom and toad skin remains unclear. METHODS: Fractions enriched with free and conjugated bufadienolides were prepared from toad venom and toad skin. Bufadienolides in each fraction were comprehensively profiled by using a versatile UHPLC-TOF-MS method. Relative contents of major bufadienolides were determined by using three bufogenins and one bufotoxin as marker compounds with validated UHPLC-TOF-MS method. Furthermore, cytotoxicity of the fractions was examined by MTT assay. RESULTS: Two fractions, i.e., bufogenin and bufotoxin fractions (TV-F and TV-C) were isolated from toad venom, and one bufotoxin fraction (TS-C) was isolated from toad skin. Totally 56 bufadienolides in these three fractions were identified, and 29 were quantified or semi-quantified. Bufotoxins were identified in both toad venom and toad skin, whereas bufogenins exist only in toad venom. Bufalin-3-conjugated bufotoxins are major components in toad venom, whereas cinobufotalin and cinobufagin-3-conjugated bufotoxins are main bufotoxins in toad skin. MTT assay revealed potent cytotoxicity of all the fractions in an order of TV-F>TV-C>TS-C. CONCLUSIONS: Our study represents the most comprehensive investigation on the chemical profiles of toad venom and toad skin from both qualitative and quantitative aspects. Eight bufotoxins were identified in toad skin responsible for the cytotoxicity for the first time. Our research provides valuable chemical evidence for the appropriate processing method, quality control and rational exploration of toad skin and toad venom for the development of anticancer medicines.

New Bufadienolides Isolated from the Roots of Kalanchoe daigremontiana (Crassulaceae).

An aqueous extract from the roots of Kalanchoe daigremontiana turned out to be a rich source of bufadienolides. The existing literature data relate mainly to the aerial parts of Kalanchoe but there is no information about the metabolic profile of the roots, which are also used in traditional medicine. Our investigation concerning the roots of K. daigremontiana led to the isolation and characterization of eight new bufadienolides, namely 1ß,3ß,5ß,14ß,19-pentahydroxybufa-20,22-dienolide (1), 19-(acetyloxy)-1ß,3ß,5ß,14ß-tetrahydroxybufa-20,22-dienolide (2), 3ß-O-α-l-rhamno-pyranosyl-5ß,11α,14ß,19-tetrahydroxybufa-20,22-dienolide (3), 19-(acetyloxy)-3ß,5ß,11α,14ß-tetrahydroxybufa-20,22-dienolide (4), 3ß,5ß,11α,14ß,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (5), 19-(acetyloxy)-3ß,5ß,11α,14ß-tetrahydroxy-12-oxo-bufa-20,22-dienolide (6), 19-(acetyloxy)-1ß,3ß,5ß,11α,14ß-pentahydroxy-12-oxo-bufa-20,22-dienolide (7) and 1ß-(acetyloxy)-3ß,5ß,11α,14ß,19-pentahydroxy-12-oxo-bufa-20,22-dienolide (8), together with seven known compounds: 11α,19-dihydroxytelocinobufagin (9), bersaldegenin-1-acetate (10), daigredorigenin-3-acetate (11), bersaldegenin-1,3,5-orthoacetate (12), bryotoxin B (13), bryophyllin B (14) and bersaldegenin (15). The structures were established applying extensive 1D- and 2D-NMR and MS spectroscopic analyses.

Identification of the hydroxylated derivatives of bufalin: phase I metabolites in rats.

Bufalin was a typical bioactive bufadienolide, existed in the traditional Chinese medicine Chan Su with the high content of 1-5%. The in vivo metabolites (1-5) of bufalin were prepared by various chromatographic techniques from the bile samples of SD rats, which were administrated with bufalin orally. Their structures were determined on the basis of the widely spectroscopic data, including HRESIMS, 1D-, and 2D NMR. And 1-3, 5 were new compounds. In the in vitro cytotoxicity assay, metabolites (1-5) showed weaker cytotoxic effects than bufalin against human cancer cell lines A549 and H1299, which indicated that the metabolism was a significant pathway for the detoxification of bufalin. Structures analyses indicated that metabolites 1-5 were hydroxylated derivatives of bufalin. This study suggested that Phase I metabolism catalyzed by CYP450 enzymes was one of the metabolic ways of bufalin, which may promote the excretion of bufalin.

Qualitative and Quantitative Analysis of the Major Constituents in Shexiang Tongxin Dropping Pill by HPLC-Q-TOF-MS/MS and UPLC-QqQ-MS/MS.

Shexiang Tongxin dropping pill (STP) is a traditional Chinese medicine formula that consists of total saponins of ginseng, synthetic Calculus bovis, bear gall, Venenum bufonis, borneol and Salvia miltiorrhiza. STP has been widely used in China and Southeast Asia for the treatment of cardiovascular diseases. In this study, a qualitative analytical method using high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was developed for identification of the major constituents in STP. Based on the retention time and MS spectra, 41 components were identified by comparison with reference compounds and literature data. Moreover, using ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring mode, we quantified 13 of the identified constituents (ginsenoside Rg1, ginsenoside Rk3, cinobufagin, arenobufagin, bufalin, resibufogenin, tanshinone IIA, taurine, tauroursodeoxycholic acid, taurocholic acid, cholic acid, deoxycholic acid, and chenodeoxycholic acid). These results suggest that this new approach is applicable for the routine analysis and quality control of STP products and provides fundamental data for further in vivo pharmacokinetical studies.