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Brigitte Kopp - One of the best experts on this subject based on the ideXlab platform.
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Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na^+/K^+-ATPase
Molecular Cancer, 2013Co-Authors: Laetitia Moreno Y Banuls, Adriana Katz, Walter Miklos, Alessio Cimmino, Elena Ainbinder, Martin Zehl, Ernst Urban, Antonio Evidente, Brigitte Kopp, Walter BergerAbstract:Background Surface-expressed Na^+/K^+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC_50 in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two Bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the Bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.
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Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase.
'Springer Science and Business Media LLC', 2013Co-Authors: Moreno L. Y Banuls, Adriana Katz, Walter Miklos, Alessio Cimmino, Elena Ainbinder, Martin Zehl, Ernst Urban, Antonio Evidente, D.m. Tal, Brigitte KoppAbstract:BACKGROUND: Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50 in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human alpha1beta1 NaK. METHODS: The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two Bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human alpha1beta1, alpha2beta1 and alpha3beta1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. RESULTS: Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for alpha1beta1 than for the alpha2beta1 and alpha3beta1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. CONCLUSIONS: Altogether, these data show that the binding affinity of the Bufadienolides and cardenolides under study is usually higher for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth
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Bufadienolides and their antitumor activity.
Natural product reports, 2011Co-Authors: Huimin Gao, Brigitte Kopp, Ruxandra Popescu, Zhimin WangAbstract:Covering: 1998 to July 2010 Since 1998, a great number of Bufadienolides have been reported from plants, animals and plant cell suspensions, as well as from fungi and bacteria cultures. This review summarizes both new Bufadienolides and those obtained from new sources, together with data regarding the antitumor activity of bufadienolide derivatives.
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comparison of toad venoms from different bufo species by hplc and lc dad ms ms
Journal of Ethnopharmacology, 2010Co-Authors: Huimin Gao, Martin Zehl, Zhimin Wang, Alexander Leitner, Brigitte KoppAbstract:Abstract Ethnopharmacological relevance Toad venom, called Chansu in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers for a long time in clinic. Aim of the study The aim of the study is to investigate the chemical differences among a variety of toad venoms from different geographic locations and related Bufo species. Materials and methods Ten batches of commercial toad venom collected from different regions in China, one batch of fresh toad venom obtained from Bufo bufo gargarizans , and six batches of related Bufo species were analyzed by HPLC and LC-DAD-MS/MS. Individual components were identified by comparison of retention times, UV spectra, and mass spectra with authentic compounds, standard addition, as well as summarized MS fragmentation rules. Based on the profile of identified constituents and the content of cinobufagin and resibufogenin, the chemical differences observed among different samples are discussed. Results Overall, 43 compounds were identified in the methanolic extracts of the different samples of toad venom. Besides of suberoyl arginine, several free Bufadienolides, bufadienolide sulfates, and suberoyl esters of Bufadienolides were found. The total amounts of cinobufagin and resibufogenin, which are the only two control markers according to the current Chinese Pharmacopoeia, varied widely from 0.7% to 10.9% in the commercial Chansu samples collected in the different locations in China. Low levels of resibufogenin, but no cinobufagin was observed in the samples from Bufo melanosticus and Bufo marinus , and even neither of both compounds was found in the sample from Bufo viridis . Conclusions The chemical profiles of the different commercial and collected toad venoms from related Bufo species differed significantly, not only in the absolute and relative contents, but also in the number and type of the constituents. The main reason for this variation are species-specific differences, but additional factors, such as the harvest and post-harvest processing, and adaption to environmental factors in different geographic locations, also seem to contribute.
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The T-cell suppressive effect of Bufadienolides: structural requirements for their immunoregulatory activity.
International immunopharmacology, 2001Co-Authors: Peter Terness, Brigitte Kopp, Dan Navolan, C Dufter, Gerhard OpelzAbstract:Abstract Many studies indicate that substances similar to cardenolides and Bufadienolides naturally occur in mammals. The majority of previous studies focused on their cardiovascular, renal, and central nervous action. We analyzed the immunoregulatory property of 52 Bufadienolides. Human T-cells were stimulated “in vitro” with mitogens or alloantigens in the presence of Bufadienolides. The most active compound totally inhibited T-cell activity at a concentration of 0.75 pmol/10 5 cells. This effect is 16 384× stronger than that of cortisol and 256× stronger than that of cyclosporin A or tacrolimus. Preactivated T cells were downregulated and, most importantly, suppressed viable T cells could not be restimulated. Lack of the 17β-lactone ring dramatically reduced the activity of Bufadienolides. Substitution at C3 also affected their function: components with a 3-OH group were up to 1000× stronger than those without. The replacement of 14β-OH with an epoxy-group slightly decreased the activity. Because there is evidence that the latter change abolishes the cardiac activity, this finding is relevant for therapeutic applications in which immunosuppression without the risk of cardiotoxicity is attempted. One of the substances analyzed in this study was Proscillaridin A. A similar bufadienolide occurs naturally in mammals. We speculate that Bufadienolides represent an important bioregulatory link between the cardiovascular, nervous and immune systems.
Frank C. Schroeder - One of the best experts on this subject based on the ideXlab platform.
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chemical defense of an asian snake reflects local availability of toxic prey and hatchling diet
Journal of Zoology, 2013Co-Authors: Deborah A. Hutchinson, Alan H. Savitzky, Gordon M. Burghardt, Jerrold Meinwald, Frank C. Schroeder, C Nguyen, Akira MoriAbstract:Species that sequester toxins from prey for their own defense against predators may exhibit population-level variation in their chemical arsenal that reflects the availability of chemically defended prey in their habitat. Rhabdophis tigrinus is an Asian snake that possesses defensive glands in the skin of its neck (‘nuchal glands’), which typically contain toxic bufadienolide steroids that the snakes sequester from consumed toads. In this study, we compared the chemistry of the nuchal gland fluid of R. tigrinus from toad-rich and toad-free islands in Japan and determined the effect of diet on the nuchal gland constituents. Our findings demonstrate that captive-hatched juveniles from toad-rich Ishima Island that had not been fed toads possess defensive Bufadienolides in their nuchal glands, presumably due to maternal provisioning of these sequestered compounds. Wild-caught juveniles from Ishima possess large quantities of Bufadienolides, which could result from a combination of maternal provisioning and sequestration of these defensive compounds from consumed toads. Interestingly, juvenile females from Ishima possess larger quantities of Bufadienolides than do juvenile males, whereas a small sample of field-collected snakes suggests that adult males contain larger quantities of Bufadienolides than do adult females. Captive-born hatchlings from Kinkasan Island lack Bufadienolides in their nuchal glands, reflecting the absence of toads on that island, but they can sequester Bufadienolides by feeding on toads (Bufo japonicus) in captivity. The presence of large quantities of Bufadienolides in the nuchal glands of R. tigrinus from Ishima may reduce the risk of predation by providing an effective chemical defense, whereas snakes on Kinkasan may experience increased predation due to the lack of defensive compounds in their nuchal glands.
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Chemical investigations of defensive steroid sequestration by the Asian snake Rhabdophis tigrinus
Chemoecology, 2012Co-Authors: Deborah A. Hutchinson, Alan H. Savitzky, Akira Mori, Gordon M. Burghardt, Jerrold Meinwald, Frank C. SchroederAbstract:Rhabdophis tigrinus is an Asian natricine snake that possesses unusual defensive glands on the dorsal surface of its neck. These nuchal glands typically contain cardiotonic steroidal toxins known as Bufadienolides, which are also abundant in the skin of toads. Feeding experiments demonstrated that toads consumed as prey are the ultimate sources of the Bufadienolides in nuchal glands of R. tigrinus . Indeed, snakes on a toad-free Japanese island (Kinkasan, Miyagi Prefecture) lack these compounds in their nuchal glands, confirming that these snakes are unable to synthesize defensive Bufadienolides. However, when snakes from Kinkasan are fed toads in the laboratory, they accumulate Bufadienolides in their nuchal glands, indicating that they have not lost the ability to sequester defensive compounds from prey. In contrast, R. tigrinus from a toad-rich island (Ishima, Tokushima Prefecture) possess large quantities of Bufadienolides, reflecting the abundance of toads from which these compounds can be sequestered. Feeding experiments involving gravid R. tigrinus demonstrated that Bufadienolides can be provisioned to offspring so that hatchlings are chemically defended before their first toad meal. Maternal provisioning of Bufadienolides can take place through two routes: by deposition in yolk and by diffusion in utero, even late in gestation. We applied Bufadienolides to the surface of eggs from Kinkasan and found that the embryos are able to take up these compounds into their nuchal glands, demonstrating the feasibility of uptake across the eggshell. Female R. tigrinus provision Bufadienolides to their offspring in direct proportion to their own level of chemical defense. By feeding toad-derived bufotoxins to R. tigrinus hatchlings, we determined that the sequestration of these compounds involves at least three types of modification: hydrolytic cleavage of suberylarginine side chains, hydroxylation, and epimerization.
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Maternal provisioning of sequestered defensive steroids by the Asian snake Rhabdophis tigrinus
Chemoecology, 2008Co-Authors: Deborah A. Hutchinson, Alan H. Savitzky, Akira Mori, Jerrold Meinwald, Frank C. SchroederAbstract:Rhabdophis tigrinus obtains defensive steroids (Bufadienolides) from its diet and sequesters those compounds in specialized structures on its neck known as nuchal glands. Hatchling snakes lacking these steroids must acquire them from toads consumed as prey. Here we show that females provision Bufadienolides to their offspring in amounts correlated to the quantity in their own nuchal glands; thus, chemically protected mothers produce defended offspring. Bufadienolides can be provisioned to embryos via deposition in yolk and by transfer across the egg membranes within the oviducts. Maternally provisioned Bufadienolides persist in the nuchal glands of juvenile snakes from the time of hatching in late summer until the following spring, when toads of ingestible size become abundant. Therefore, maternal provisioning may provide chemical protection from predators for young R. tigrinus in the absence of dietary sources of Bufadienolides.
Deborah A. Hutchinson - One of the best experts on this subject based on the ideXlab platform.
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chemical defense of an asian snake reflects local availability of toxic prey and hatchling diet
Journal of Zoology, 2013Co-Authors: Deborah A. Hutchinson, Alan H. Savitzky, Gordon M. Burghardt, Jerrold Meinwald, Frank C. Schroeder, C Nguyen, Akira MoriAbstract:Species that sequester toxins from prey for their own defense against predators may exhibit population-level variation in their chemical arsenal that reflects the availability of chemically defended prey in their habitat. Rhabdophis tigrinus is an Asian snake that possesses defensive glands in the skin of its neck (‘nuchal glands’), which typically contain toxic bufadienolide steroids that the snakes sequester from consumed toads. In this study, we compared the chemistry of the nuchal gland fluid of R. tigrinus from toad-rich and toad-free islands in Japan and determined the effect of diet on the nuchal gland constituents. Our findings demonstrate that captive-hatched juveniles from toad-rich Ishima Island that had not been fed toads possess defensive Bufadienolides in their nuchal glands, presumably due to maternal provisioning of these sequestered compounds. Wild-caught juveniles from Ishima possess large quantities of Bufadienolides, which could result from a combination of maternal provisioning and sequestration of these defensive compounds from consumed toads. Interestingly, juvenile females from Ishima possess larger quantities of Bufadienolides than do juvenile males, whereas a small sample of field-collected snakes suggests that adult males contain larger quantities of Bufadienolides than do adult females. Captive-born hatchlings from Kinkasan Island lack Bufadienolides in their nuchal glands, reflecting the absence of toads on that island, but they can sequester Bufadienolides by feeding on toads (Bufo japonicus) in captivity. The presence of large quantities of Bufadienolides in the nuchal glands of R. tigrinus from Ishima may reduce the risk of predation by providing an effective chemical defense, whereas snakes on Kinkasan may experience increased predation due to the lack of defensive compounds in their nuchal glands.
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Chemical investigations of defensive steroid sequestration by the Asian snake Rhabdophis tigrinus
Chemoecology, 2012Co-Authors: Deborah A. Hutchinson, Alan H. Savitzky, Akira Mori, Gordon M. Burghardt, Jerrold Meinwald, Frank C. SchroederAbstract:Rhabdophis tigrinus is an Asian natricine snake that possesses unusual defensive glands on the dorsal surface of its neck. These nuchal glands typically contain cardiotonic steroidal toxins known as Bufadienolides, which are also abundant in the skin of toads. Feeding experiments demonstrated that toads consumed as prey are the ultimate sources of the Bufadienolides in nuchal glands of R. tigrinus . Indeed, snakes on a toad-free Japanese island (Kinkasan, Miyagi Prefecture) lack these compounds in their nuchal glands, confirming that these snakes are unable to synthesize defensive Bufadienolides. However, when snakes from Kinkasan are fed toads in the laboratory, they accumulate Bufadienolides in their nuchal glands, indicating that they have not lost the ability to sequester defensive compounds from prey. In contrast, R. tigrinus from a toad-rich island (Ishima, Tokushima Prefecture) possess large quantities of Bufadienolides, reflecting the abundance of toads from which these compounds can be sequestered. Feeding experiments involving gravid R. tigrinus demonstrated that Bufadienolides can be provisioned to offspring so that hatchlings are chemically defended before their first toad meal. Maternal provisioning of Bufadienolides can take place through two routes: by deposition in yolk and by diffusion in utero, even late in gestation. We applied Bufadienolides to the surface of eggs from Kinkasan and found that the embryos are able to take up these compounds into their nuchal glands, demonstrating the feasibility of uptake across the eggshell. Female R. tigrinus provision Bufadienolides to their offspring in direct proportion to their own level of chemical defense. By feeding toad-derived bufotoxins to R. tigrinus hatchlings, we determined that the sequestration of these compounds involves at least three types of modification: hydrolytic cleavage of suberylarginine side chains, hydroxylation, and epimerization.
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Maternal provisioning of sequestered defensive steroids by the Asian snake Rhabdophis tigrinus
Chemoecology, 2008Co-Authors: Deborah A. Hutchinson, Alan H. Savitzky, Akira Mori, Jerrold Meinwald, Frank C. SchroederAbstract:Rhabdophis tigrinus obtains defensive steroids (Bufadienolides) from its diet and sequesters those compounds in specialized structures on its neck known as nuchal glands. Hatchling snakes lacking these steroids must acquire them from toads consumed as prey. Here we show that females provision Bufadienolides to their offspring in amounts correlated to the quantity in their own nuchal glands; thus, chemically protected mothers produce defended offspring. Bufadienolides can be provisioned to embryos via deposition in yolk and by transfer across the egg membranes within the oviducts. Maternally provisioned Bufadienolides persist in the nuchal glands of juvenile snakes from the time of hatching in late summer until the following spring, when toads of ingestible size become abundant. Therefore, maternal provisioning may provide chemical protection from predators for young R. tigrinus in the absence of dietary sources of Bufadienolides.
Xinmiao Liang - One of the best experts on this subject based on the ideXlab platform.
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efficient purification of active Bufadienolides by a class separation method based on hydrophilic solid phase extraction and reversed phase high performance liquid chromatography
Journal of Pharmaceutical and Biomedical Analysis, 2014Co-Authors: Yanfang Liu, Aijin Shen, Chaoran Wang, Jingyu Yan, Weijie Zhao, Xinmiao LiangAbstract:Abstract Traditional Chinese medicines (TCMs) have played a significant role in the process of discovering natural bioactive compounds, especially in anticancer therapeutics. However, the components of TCMs are complex mixtures with wide variation in polarity and content, which leads to inefficiency in the process of active compound discovery from TCMs. In this paper, the popular strategy of utilizing “pre-fractionated natural product libraries” has been improved by a new class separation approach to accelerate the process. As an example, the skin of Bufo bufo gargarizans Cantor, a well-known TCM, mainly contains two distinct bufadienolide classes: amino acid-conjugated Bufadienolides (AACBs) and free form Bufadienolides (AAUBs). We utilized hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE) to resolve the two types of Bufadienolides, which co-eluted on C18 columns. By this strategy, twelve Bufadienolides of the two types were purified via prep-HPLC from one active fraction, and eight of them were identified by 1 H NMR and 13 C NMR. These results indicated that the class separation method not only overcame the limited orthogonality in a 2D-RPLC × RPLC system but also accelerated the process of active compound discovery.
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Purification of Bufadienolides from the skin of Bufo bufo gargarizans Cantor with positively charged C18 column
Journal of pharmaceutical and biomedical analysis, 2014Co-Authors: Zhimou Guo, Xiuli Zhang, Yanfang Liu, Aijin Shen, Chaoran Wang, Weijie Zhao, Xinmiao LiangAbstract:As a kind of promising anticancer compounds, the preparation of Bufadienolides is a hot study spot. However, due to the complexity of biological sample, the purification of Bufadienolides from a crude sample (toad skin) is a tough work. In this paper, we reported a new way based on positively charged C18 material (XCharge C18) to quickly separate and purify Bufadienolides from toad skin. By this method, the different ionic feature of the amino acid conjugated Bufadienolides (AACBs) and the free form Bufadienolides (AAUBs) was firstly utilized to obtain distinct separation selectivity on the XCharge C18 column. Additionally, the peak tailing problem of AACBs on conventional C18 was resolved and better resolutions were achieved on the XCharge C18, thus, two kinds of Bufadienolides on one column were successfully purified respectively. Taking F13 as an example, the method was validated by liquid chromatography-mass spectrometry (LC-MS), and then 4 AACBs as well as 4 AAUBs were simultaneously purified by preparative XCharge C18. In addition, the application of this method in other fractions was also validated. The results suggested that the developed method is a practical and promising tool for efficient separation and purification of Bufadienolides from toad skin.
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systematic screening and characterization of novel Bufadienolides from toad skin using ultra performance liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry
Rapid Communications in Mass Spectrometry, 2010Co-Authors: Yanfang Liu, Xiuli Zhang, Yuansheng Xiao, Xingya Xue, Xinmiao LiangAbstract:during the discovery process of novel compounds, it is of significant importance to differentiate novel from known compounds in crude extracts before starting the time-consuming process of purification. Bufadienolides are the main active components of the skin of the toad bufo bufo gargarizans cantor (toad skin), an important traditional chinese medicine. the fragmentation behavior and mass spectra profiles of Bufadienolides standards were investigated using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (uplc/esi-q-tofms). several fragmentation rules were summarized and applied to characterize novel and known Bufadienolides in toad skin. characteristic substituent groups could be identified by both diagnostic ions and their relative abundance. bufadienolide stereoisomers could be differentiated from positional isomers by comparing fragment abundance profiles. this was used to characterize new stereoisomers; for known Bufadienolides. a total of 39 Bufadienolides were screened out using a systematic method developed in our laboratory. in addition to 19 known Bufadienolides, 20 putative novel compounds, including 8 stereoisomers, were characterized. uplc/q-tofms was demonstrated to be a powerful tool for the characterization of low-abundance Bufadienolides in complex samples. this study provides guidelines for the targeted isolation of novel Bufadienolides from natural products. copyright (c) 2010 john wiley & sons, ltd.
Jin-ao Duan - One of the best experts on this subject based on the ideXlab platform.
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Total synthesis, chemical modification and structure-activity relationship of Bufadienolides.
European journal of medicinal chemistry, 2020Co-Authors: Yue Zhong, Jin-ao Duan, Chao Zhao, Tian-yuan Fan, Min Chen, Zhi-hao ShiAbstract:Abstract Bufadienolides are a type of natural cardiac steroids and originally isolated from the Traditional Chinese Medicine Chan’Su, they have been used for the treatment of heart disease in traditional remedies as well as in modern medicinal therapy with potent anti-tumor activities. Due to their unique molecular structures with unsaturated six-membered lactones attached to the steroid core, Bufadienolides have received great attention in the synthetic organic community. This review presents total synthetic efforts to some representative Bufadienolides, chemical modification of Bufadienolides will also be given to discuss their structure-activity relationship in anti-tumor.
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high resolution mass profile of Bufadienolides and peptides combing with anti tumor cell screening and multivariate analysis for the quality evaluation of bufonis venenum
Molecules, 2019Co-Authors: Jing Zhou, Zhenhua Zhu, Hengbin Wang, Yanqing Yan, Niancui Luo, Jin-ao DuanAbstract:In order to evaluate the quality of Bufonis Venenum commercial herbs, a three-step qualitative and quantitative research study was performed. Firstly, we tried to identify small molecules and peptides in Bufonis Venenum using pre-fractionation chromatography and high-resolution mass spectrometry. The database search of the small molecules and peptides of Bufonis Venenum revealed that the dried venom consisted of free/conjugated-type Bufadienolides and peptides with a mass range of 0.4-2 kDa. Secondly, we used partial least squares (PLS) multivariate statistical analysis to screen Bufadienolides markers (VIP > 1.5) responsible for the anti-tumor cell activity of Bufonis Venenum, including 21 identified Bufadienolides and 7 unknown compounds. It is noticeable that these bufadienolide markers could not be recognized by traditional HPLC-UV based spectrum-effect relationship analysis (correlation coefficient ranging from -0.24 to 0.40). Finally, we proposed a weight coefficient-based corrected total contents of 9 Bufadienolides as a quality evaluation indicator, which had good correlation with inhibitory effects on tumor cells of commercial Bufonis Venenum. The correlation coefficient increased from 0.4 to 0.6. Thus, our pre-fractionation chromatography and mass spectrometry strategy had significant advancement over the traditional spectrum-effect relationship method for chemical marker identification. These results could be crucial and helpful in the development of a quality evaluation method that could reflect the pharmacological activity of Bufonis Venenum.
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RESEARCH ARTICLE Molecular Structure-Affinity Relationship of Bufadienolides and Human Serum Albumin In Vitro and Molecular Docking Analysis
2016Co-Authors: Jing Zhou, Junfeng Zhang, Jin-ao DuanAbstract:The development of Bufadienolides as anti-tumor agents is limited due to poor pharmacoki-netic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine Bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecu-lar docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respective-ly. The structural differences between the various Bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that de-creased the binding affinity for HSA, other polar groups tended to increase the affinity, espe-cially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH> 5-OH> 16-OH; in addi-tion, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the bind
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Effect of drying methods on the free and conjugated bufadienolide content in toad venom determined by ultra-performance liquid chromatography-triple quadrupole mass spectrometry coupled with a pattern recognition approach
Journal of Pharmaceutical and Biomedical Analysis, 2015Co-Authors: Jing Zhou, Honglan Wang, Hongmei Wen, Hongyue Ma, Jin-ao Duan, Dawei Qian, Rui Liu, Yan Gong, Qinan WuAbstract:Abstract Drying is a useful technique for extending the shelf-life of biological products and enabling long-term storage; however, improper drying can reduce the chemical quality of the products. In this study, we used ultra-performance liquid chromatography–triple quadrupole/mass spectrometry (LC–MS/MS) and multivariate statistical analysis to investigate the effects of four drying methods (V: vacuum-drying at 60 °C, F: freeze-drying, H: air-drying at 60 °C and R: air-drying at room temperature) on the levels of 36 Bufadienolides in toad venom. Vacuum-drying at 60 °C produced the highest quality dried toad venom in terms of total bufadienolide content, whereas traditional air-drying at room temperature (RT) to dehydrate the toad venom led to a dramatic loss in free and conjugated Bufadienolides, reaching up to 60% and 70%, respectively. Assaying for free Bufadienolides ranked the drying methods as V ≈ F > H > R, whereas assaying for conjugated Bufadienolides slightly changed the order to V > F ≈ H > R. Furthermore, we identified 21 Bufadienolides as biomarkers responsible for the decline in the quality of dried toad venom, whose loss varied from 1.5-fold to 100-fold. Of these biomarkers, group I Bufadienolides that contain 16-OAc (e.g., cinobufagin and its hydroxyl or arginine ester derivatives) were characteristic components and were reduced to trace levels (loss of more than 10-fold) following traditional air-drying at RT. This might be attributed to the fact that most enzyme-sensitive Bufadienolides were biotransformed or degraded at room temperature but were retained using other drying methods.
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Molecular structure-affinity relationship of Bufadienolides and human serum albumin in vitro and molecular docking analysis.
PloS one, 2015Co-Authors: Jing Zhou, Honglan Wang, Junfeng Zhang, Jin-ao DuanAbstract:The development of Bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine Bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various Bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the Bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of Bufadienolides with improved pharmacological profiles as novel anti-tumor drugs.
