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Robert A Newman - One of the best experts on this subject based on the ideXlab platform.
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water soluble egg membrane enhances the immunoactivating properties of an aloe vera based extract of nerium oleander leaves
Clinical Cosmetic and Investigational Dermatology, 2016Co-Authors: Kathleen F Benson, Robert A Newman, Gitte S JensenAbstract:OBJECTIVE To evaluate a blend of two natural ingredients on immune parameters relevant for their current topical use and potential support of microcirculation in skin tissue. MATERIALS AND METHODS A blend (BL) of Aloe vera-based Nerium oleander extract (NAE-8i, Oleandrin-free) and hydrolyzed water-soluble egg membrane (WSEM) was applied to human whole-blood cultures for 24 hours, with each separate ingredient serving as a control. Immune-cell subsets were analyzed for expression levels of the activation markers CD69 and CD25. Culture supernatants were analyzed for cytokines, chemokines, and immunoregulating peptides. RESULTS BL increased CD69 expression on lymphocytes, monocytes, and CD3-CD56+ natural killer cells, and CD25 expression on natural killer cells. The number of CD69+CD25+ lymphocytes increased in cultures treated with BL and the separate ingredients. BL triggered production of multiple cytokines and chemokines, where CC chemokines MIP1α and MIP3α, as well as cytokines involved in wound healing - Groα, Groβ, ENA78, and fractalkine - reached levels manyfold above treatment with either NAE-8i or WSEM alone. CONCLUSION Data on BL showed that WSEM strongly enhanced NAE-8i's effects on immunoactivation in vitro. This has potential relevance for support of immunity in skin tissue, including antibacterial and antiviral defense mechanisms, wrinkle reduction, and wound care.
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Oleandrin-mediated inhibition of human tumor cell proliferation:
2016Co-Authors: Norma Llansa, Robert A NewmanAbstract:Cardiac glycosides such as Oleandrin are known to inhibit the Na,K-ATPase pump, resulting in a consequent increase in calcium influx in heart muscle. Here, we investigated the effect of Oleandrin on the growth of human and mouse cancer cells in relation to Na,K-ATPase subunits. Olean-drin treatment resulted in selective inhibition of human cancer cell growth but not rodent cell proliferation, which corresponded to the relative level of Na,K-ATPase α3 sub-unit protein expression. Human pancreatic cancer cell lines were found to differentially express varying levels of α3 protein, but rodent cancer cells lacked discernable expression of this Na,K-ATPase isoform. A correlation was observed between the ratio of α3 to α1 isoforms and the level of Oleandrin uptake during inhibition of cell growth and initiation of cell death; the higher the α3 ex-pression relative to α1 expression, the more sensitive the cell was to treatment with Oleandrin. Inhibition of pro-liferation of Panc-1 cells by Oleandrin was significantly re-duced when the relative expression of α3 was decreased by knocking down the expression of α3 isoform with α3 siRNA or increasing expression of the α1 isoform through transient transfection of α1 cDNA to the cells. Our data suggest that the relative lack of α3 (relative to α1) in rodent and some human tumor cells may explain their un-responsiveness to cardiac glycosides. In conclusion, the relatively higher expression of α3 with the limited expres-sion of α1 may help predict which human tumors are likely to be responsive to treatment with potent lipid-soluble car-diac glycosides such as Oleandrin. [Mol Cancer Ther 2009;8(8):2319–28
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dual activities of the anti cancer drug candidate pbi 05204 provide neuroprotection in brain slice models for neurodegenerative diseases and stroke
Scientific Reports, 2016Co-Authors: Michael J Van Kanegan, Denise E Dunn, Linda S Kaltenbach, Bijal D Shah, Daniel D Mccoy, Peiying Yang, Jiangnan Peng, Li Shen, Robert H Cichewicz, Robert A NewmanAbstract:We previously reported neuroprotective activity of the botanical anti-cancer drug candidate PBI-05204, a supercritical CO2 extract of Nerium oleander, in brain slice and in vivo models of ischemic stroke. We showed that one component of this neuroprotective activity is mediated through its principal cardiac glycoside constituent, Oleandrin, via induction of the potent neurotrophic factor brain-derived neurotrophic factor (BDNF). However, we also noted that the concentration-relation for PBI-05204 in the brain slice oxygen-glucose deprivation (OGD) model is considerably broader than that for Oleandrin as a single agent. We thus surmised that PBI-05204 contains an additional neuroprotective component(s), distinct from Oleandrin. We report here that neuroprotective activity is also provided by the triterpenoid constituents of PBI-05204, notably oleanolic acid. We demonstrate that a sub-fraction of PBI-05204 (Fraction 0–4) containing oleanolic and other triterpenoids, but without cardiac glycosides, induces the expression of cellular antioxidant gene transcription programs regulated through antioxidant transcriptional response elements (AREs). Finally, we show that Fraction 0–4 provides broad neuroprotection in organotypic brain slice models for neurodegeneration driven by amyloid precursor protein (APP) and tau implicated in Alzheimer’s disease and frontotemporal dementias, respectively, in addition to ischemic injury modeled by OGD.
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cellular location and expression of na k atpase α subunits affect the anti proliferative activity of Oleandrin
Molecular Carcinogenesis, 2014Co-Authors: Peiying Yang, Carrie Cartwright, Ekem Efuet, Stanley R Hamilton, Ignacio I Wistuba, David G Menter, Crandell Addington, Imad Shureiqi, Robert A NewmanAbstract:The purpose of this study was to investigate whether intracellular distribution of Na+, K+-ATPase α3 subunit, a receptor for cardiac glycosides including Oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na+, K+-ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of Oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na+, K+-ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, Oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na+, K+-ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy. © 2012 Wiley Periodicals, Inc.
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nerium oleander derived cardiac glycoside Oleandrin is a novel inhibitor of hiv infectivity
Fitoterapia, 2013Co-Authors: Shailbala Singh, Robert A Newman, Peiying Yang, Sachin Shenoy, Pramod N Nehete, Bharti P Nehete, Danielle Fontenot, Guojun Yang, Jagannadha K SastryAbstract:We evaluated the effectiveness of Anvirzel™, an aqueous extract of Nerium oleander on HIV infection of human peripheral blood mononuclear cells. Oleandrin, the principle cardiac glycoside (CG) in Anvirzel™ has been shown to exhibit anti-cancer properties but its efficacy against HIV is unknown. Treatment with Anvirzel™ significantly reduced the infectivity of virus produced from infected cells without any change in the total amount of virus produced. This is in contrast to treatment with AZT, a potent inhibitor of HIV replication that has been shown to significantly reduce virus production. Relative to untreated cultures, virus in cultures treated with Oleandrin had significantly reduced expression of the envelope protein gp120, the sole determinant of virus infectivity, suggesting a novel mechanism underlying the impaired infectivity. These results support the potential utility of the Nerium oleander aqueous extract, containing the CG Oleandrin as a novel candidate anti-HIV therapeutic.
Peiying Yang - One of the best experts on this subject based on the ideXlab platform.
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dual activities of the anti cancer drug candidate pbi 05204 provide neuroprotection in brain slice models for neurodegenerative diseases and stroke
Scientific Reports, 2016Co-Authors: Michael J Van Kanegan, Denise E Dunn, Linda S Kaltenbach, Bijal D Shah, Daniel D Mccoy, Peiying Yang, Jiangnan Peng, Li Shen, Robert H Cichewicz, Robert A NewmanAbstract:We previously reported neuroprotective activity of the botanical anti-cancer drug candidate PBI-05204, a supercritical CO2 extract of Nerium oleander, in brain slice and in vivo models of ischemic stroke. We showed that one component of this neuroprotective activity is mediated through its principal cardiac glycoside constituent, Oleandrin, via induction of the potent neurotrophic factor brain-derived neurotrophic factor (BDNF). However, we also noted that the concentration-relation for PBI-05204 in the brain slice oxygen-glucose deprivation (OGD) model is considerably broader than that for Oleandrin as a single agent. We thus surmised that PBI-05204 contains an additional neuroprotective component(s), distinct from Oleandrin. We report here that neuroprotective activity is also provided by the triterpenoid constituents of PBI-05204, notably oleanolic acid. We demonstrate that a sub-fraction of PBI-05204 (Fraction 0–4) containing oleanolic and other triterpenoids, but without cardiac glycosides, induces the expression of cellular antioxidant gene transcription programs regulated through antioxidant transcriptional response elements (AREs). Finally, we show that Fraction 0–4 provides broad neuroprotection in organotypic brain slice models for neurodegeneration driven by amyloid precursor protein (APP) and tau implicated in Alzheimer’s disease and frontotemporal dementias, respectively, in addition to ischemic injury modeled by OGD.
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cellular location and expression of na k atpase α subunits affect the anti proliferative activity of Oleandrin
Molecular Carcinogenesis, 2014Co-Authors: Peiying Yang, Carrie Cartwright, Ekem Efuet, Stanley R Hamilton, Ignacio I Wistuba, David G Menter, Crandell Addington, Imad Shureiqi, Robert A NewmanAbstract:The purpose of this study was to investigate whether intracellular distribution of Na+, K+-ATPase α3 subunit, a receptor for cardiac glycosides including Oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na+, K+-ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of Oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na+, K+-ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, Oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na+, K+-ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy. © 2012 Wiley Periodicals, Inc.
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nerium oleander derived cardiac glycoside Oleandrin is a novel inhibitor of hiv infectivity
Fitoterapia, 2013Co-Authors: Shailbala Singh, Robert A Newman, Peiying Yang, Sachin Shenoy, Pramod N Nehete, Bharti P Nehete, Danielle Fontenot, Guojun Yang, Jagannadha K SastryAbstract:We evaluated the effectiveness of Anvirzel™, an aqueous extract of Nerium oleander on HIV infection of human peripheral blood mononuclear cells. Oleandrin, the principle cardiac glycoside (CG) in Anvirzel™ has been shown to exhibit anti-cancer properties but its efficacy against HIV is unknown. Treatment with Anvirzel™ significantly reduced the infectivity of virus produced from infected cells without any change in the total amount of virus produced. This is in contrast to treatment with AZT, a potent inhibitor of HIV replication that has been shown to significantly reduce virus production. Relative to untreated cultures, virus in cultures treated with Oleandrin had significantly reduced expression of the envelope protein gp120, the sole determinant of virus infectivity, suggesting a novel mechanism underlying the impaired infectivity. These results support the potential utility of the Nerium oleander aqueous extract, containing the CG Oleandrin as a novel candidate anti-HIV therapeutic.
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in vitro and in vivo neuroprotective activity of the cardiac glycoside Oleandrin from nerium oleander in brain slice based stroke models
Journal of Neurochemistry, 2011Co-Authors: Denise E Dunn, Peiying Yang, Mary Johansen, Robert A NewmanAbstract:The principal active constituent of the botanical drug candidate PBI-05204, a supercritical CO(2) extract of Nerium oleander, is the cardiac glycoside Oleandrin. PBI-05204 shows potent anticancer activity and is currently in phase I clinical trial as a treatment for patients with solid tumors. We have previously shown that neriifolin, which is structurally related to Oleandrin, provides robust neuroprotection in brain slice and whole animal models of ischemic injury. However, neriifolin itself is not a suitable drug development candidate and the FDA-approved cardiac glycoside digoxin does not cross the blood-brain barrier. We report here that both Oleandrin as well as the full PBI-05204 extract can also provide significant neuroprotection to neural tissues damaged by oxygen and glucose deprivation as occurs in ischemic stroke. Critically, we show that the neuroprotective activity of PBI-05204 is maintained for several hours of delay of administration after oxygen and glucose deprivation treatment. We provide evidence that the neuroprotective activity of PBI-05204 is mediated through Oleandrin and/or other cardiac glycoside constituents, but that additional, non-cardiac glycoside components of PBI-05204 may also contribute to the observed neuroprotective activity. Finally, we show directly that both Oleandrin and the protective activity of PBI-05204 are blood brain barrier penetrant in a novel model for in vivo neuroprotection. Together, these findings suggest clinical potential for PBI-05204 in the treatment of ischemic stroke and prevention of associated neuronal death.
Subagus Wahyuono - One of the best experts on this subject based on the ideXlab platform.
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5α-Oleandrin reduce Bcl-2 protein and increase Bax protein expression on Hela cervical cancer cell
Trisakti University Press, 2017Co-Authors: Mae Sri Hartati Wahyuningsih, Sofia Mubarika, Subagus Wahyuono, Ibnu G. Ganjar, Tatsuo TakeyaAbstract:BACKGROUND The leaves of Nerium indicum Mill. have been utilized traditionally to cure cancer. By using Bioassay guided extraction and isolation method, three compounds (NiO-1, NiO-2, NiO-3) were isolated from an active fraction of the CHCl3 extract of N. indicum leaves. NiO-2 identified as 5α- Oleandrin is the best cytotoxic compound on HeLa cervical cancer cell in vitro among the other two. However mechanism of action of the compound hasn’t been evaluated yet. The aims of this study were to determine the mechanism of action at molecular level. METHODS The action mechanism of 5α-Oleandrin on HeLa cervical cancer cells was analyzed by staining the cells with Hoechst 33342; the agarose gel electrophoresis was aimed to determine the DNA fragmentation and the western blotting was aimed to determine the Bcl-2 and Bax protein expression. RESULTS Incubation of HeLa cervical cancer cell with 5α-Oleandrin at the concentration 3,47x10-4 mM (24 hours) followed by staining with Hoechst 33342, a broken up light blue color of nucleus was observed (compared with intensive color of untreated control). By gel electrophoresis (at the same concentration of the tested compound), a smear band at about 200 bp was observed. In addition, cells treated with 5α-Oleandrin displayed a decreasing of the Bcl-2 protein expression and increasing of the Bax protein expression CONCLUSION 5α-Oleandrin induced HeLa cervical cancer cells DNA fragmentation observed by the presence of a smear band at about 200 bp indicative the apoptotic occurrence. 5α-Oleandrin induces apoptosis by reducing the Bcl-2 protein expression but the Bax protein expression increases
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detection of apoptosis mechanism on renal cancer cell treated by 16 17 dehydrodeacetyl 5α Oleandrin compound isolated from nerium indicum mill leaves
INDONESIAN JOURNAL OF PHARMACY, 2008Co-Authors: Mae S H Wahyuningnsih, Sofia Mubarika, Ibnu Gholib Gandjar, Subagus Wahyuono, Awm Boersma, K. NooterAbstract:The 16,17-dehydrodeacetyl-5α-Oleandrin was isolated from an active fraction of Nerium indicum Mill leaves (fam. Apocynaceae). This compound was cytotoxic against various cancer cells, and selective on A498 cells (Renal cancer). However, the apoptosis mechanism was still unknown yet. Therefore, the aim of this study was to know the apoptotic mechanism of 16,17-dehydrodeacetyl-5α-Oleandrin on A498 cells by FITC labeled annexin V and immunucytochemical assays. The detection of apoptotic mechanism on A498 cells was performed with FITC-conjugated annexin V using Flow Cytometry. The p53 protein expression were detected using immunocytochemical. Treatment with 16,17-hydrodeacetyl-5α-Oleandrin (3.88 x 10-4 mM) using FITC-annexin V increased the percentage of the dead cells in the 24 th and 48th hours incubation period. The 16,17-dehydrodeacetyl-5α-Oleandrin (1,94x10-4 and 3,88x10-4 mM) raised significanly p53 protein expression (p<0,05). The percentage of the p53 protein expression increased throughout the time of samples incubation. Key words: 16,17-dehydrodeacetyl-5α-Oleandrin,fluorescein isothiocyanate-annexin V, immunocytochemically, A498 cells.
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Deteksi mekanisme apoptosis sel kanker ginjal pada pemberian senyawa 16,17-dehidrode-asetil-5alpha-Oleandrin hasil isolasi daun Nerium indicum Mill
[Yogyakarta] : Universitas Gadjah Mada, 2008Co-Authors: Mae S.h. Wahyuningsih, Sofia Mubarika, Ibnu Gholib Gandjar, Subagus WahyuonoAbstract:16,17-dehidrodeasetil-Sa-Oleandrin nierupakan senyawa golongan kardenolida yang diisolasi dari fraksi aktif daun Nerium indicum Mill (fam. Apocynaceae). Senyawa ini bersifat sitotoksik terhadap berbagai sel kanker dan selektif terhadap sel A498 (kanker ginjal) in-vitro, namun mekanisme apoptosisnya belum diketahui. Penelitian ini bertujuan untuk mendeteksi mekanisme apoptosis sel kanker ginjal pad a pemberian senyawa 16,17dehidrodeasetil-Sa-Oleandrin menggunakan metode fluorescein isothiocyanate-annexin V dan immunositokimia. Deteksi mekanisme apoptosis terhadap sel A498, dilakukan dengan metode FITC terlabel Annexin V menggunakan Flow Cytometry (FCM). Sedangkan ekspresi protein pS3 sel A498 dilakukan dengan metode immunositokimia . Hasil penelitian menunjukkan bahwa pemberian senyawa 16,17dehidrodeasetil-Sa-Oleandrin dosis 3,88x10-4 mM dengan menggunakan metode FITC-annexin V dapat menaikkan persentase jumlah kematian sel antara inkubasi 24 dan 48 jam. Senyawa 16,17-dehidrodeasetil-Sa-Oleandrin dosis 1,94x10-4 mM dan 3,88x10-4 mM, dapat menaikkan ekspresi protein pS3 secara bermakna (
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Structure identification of potential compound as selective renal anticancer isolated from Nerium indicum Mill. Leaves
'Indonesian Journal of Pharmacy', 2008Co-Authors: Mae S.h. Wahyuningsih, Sofia Mubarika, Ibnu Gholib Gandjar, Mark T. Hamann, Subagus WahyuonoAbstract:Oleandrin is one of cardenolida compound isolated from an active fraction of Nerium indicum Mill leaves. (fam. Apocynaceae), which have cytotoxic effect on several human cancer cells, and also to normal cells in vitro. Another compound which was potential as renal anticancer has also been isolated from N. indicum. However, its chemical structure has not been discovered. The aim of this study was to identify the potential compound as selective to renal cancer present in the leaves of N. indicum. The potential compound was isolated from the active fraction using Preparative TLC and structure elucidation was done by using spectroscopic methods (UV, IR, MS and NMR). Base on this spectra and by comparison with Oleandrin data, it was indicated that the potential compound as Renal anticancer was as 16,17-dehydrodeacetyl-5a-Oleandrin
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Cytotoxicity of Oleandrin isolated from the leaves of Nerium indicum Mill. on several human cancer cell lines
INDONESIAN JOURNAL OF PHARMACY, 2005Co-Authors: Mae Sri Hartati Wahyuningsih, Sofia Mubarika, R.l.m. Bolhuis, K. Nooter, Ibnu Gholib Gandjar, Subagus WahyuonoAbstract:Finding anticancer drugs from natural resources still proceeds. Oleandrin isolated from Nerium indicum Mill. inhibited the growth of mieloma cell line in vitro better than that of vincristine sulphate. This study was aimed to determine the cytotoxic effect of Oleandrin on various human cancer cell lines. Cytotoxic test of Oleandrin on seven human cancer cell lines was done by SRB-method. The analysis was conducted by comparing the ID50 of Oleandrin with that of doxorubicin and cisplatin as positive controls. This result indicated that Oleandrin possessed the best cytotoxic effect on breast cancer (MCF7) with ID50 at 8.85 nM. Keywords : Oleandrin, cytotoxicity, human cancer cells, ID50
Gilbert Pepin - One of the best experts on this subject based on the ideXlab platform.
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an unusual case of death suffocation caused by leaves of common ivy hedera helix detection of hederacoside c α hederin and hederagenin by lc ei ms ms
Journal of Analytical Toxicology, 2003Co-Authors: Yvan Gaillard, Pierre Blaise, Alexandre Darre, Thierry Barbier, Gilbert PepinAbstract:We report one fatal case of asphyxia caused by leaves of common ivy. Macroscopic examination of the corpse during the autopsy disclosed an incredible quantity of leaves of Hedera helix in the mouth and throat of the decedent. In order to rule out the possibility of poisoning by the toxic saponins contained in the plant, we have developed an efficient LC-EI/MS-MS assay of hederacoside C, alppha-hederin, and hederagenin in biological fluids and plant material. Sample cleanup involved solid-phase extraction of the toxins on C18 cartridges followed by LC analysis under reversed-phase conditions in the gradient elution mode. Solute identification was performed using full scan MS-MS spectrum of the analytes. Oleandrine was used as internal standard. Under these conditions, saponins in powdered dried leaves of Hedera helix were measured at a concentration of 21.83 mg/g for hederacoside C, 0.41 mg/g for alpha-hederin and 0.02 mg/g for hederagenin. No toxin was detected in cardiac blood, femoral blood, or urine of the deceased, but hederacoside C was quantitated at 857 ng/mL in the gastric juice. These findings led us to conclude that the man committed suicide and that the death was caused by suffocation by leaves of common ivy.
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poisoning by plant material review of human cases and analytical determination of main toxins by high performance liquid chromatography tandem mass spectrometry
Journal of Chromatography B: Biomedical Sciences and Applications, 1999Co-Authors: Yvan Gaillard, Gilbert PepinAbstract:The authors have reviewed the main toxic plants responsible for human deaths throughout the world. Forty plants (genera or species) were listed in order to establish an inventory of the active molecules that could be identified, the already published analytical methods and the reported human fatal cases. In a second step, the authors have developed a general method for the detection of various toxins in whole blood by high-performance liquid chromatography coupled to mass spectrometry or tandem mass spectrometry. Sample preparation was realized by liquid-liquid extraction at pH 9.5 for Oleandrine, taxol and the alkaloids. These latter compounds were divided into two groups following their chemical properties and could be subsequently purified by acid/base clean up. Cyanogenic compounds and atractyloside were isolated by precipitation of the protein content with acetone and purified for atractyloside by washing with chloroform. Separation of the drugs occurred under reversed-phase conditions on a C18 analytical column 150x2 mm I.D. (5 microm particle size) using two different mobile phases. The first one, formiate buffer 2 mM acidified at pH 3.0, was used for the separation of atractyloside, Oleandrine, taxol, the cyanogenic molecules and some alkaloids. The second mobile phase, formiate buffer 10 mM made basic at pH 8.2 was used for the majority of other alkaloids. A gradient elution mode was chosen using acetonitrile or acetonitrile-methanol (50:50, v/v) as the eluting solvent. Detection under positive ionization mode was the mode of choice for all compounds except for atractyloside (negative ions) and for taxol (mixed mode available). Application to real forensic cases has been demonstrated.
Amitava Dasgupta - One of the best experts on this subject based on the ideXlab platform.
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activated charcoal is effective but equilibrium dialysis is ineffective in removing oleander leaf extract and Oleandrin from human serum monitoring the effect by measuring apparent digoxin concentration
Therapeutic Drug Monitoring, 2003Co-Authors: Amitava Dasgupta, Shinian Cao, Alice WellsAbstract:Accidental poisoning from oleander leaf or oleander tea can be life threatening. The authors studied the effectiveness of activated charcoal and equilibrium dialysis in removing oleander leaf extract and commercially available Oleandrin as well as oleandrigenin, the active components of oleander plant, from human serum. Oleander leaf extract was prepared in distilled water and drug-free serum was supplemented with the extract. Then serum was treated with activated charcoal at room temperature and an aliquot was removed at 0 minutes, 10 minutes, 20 minutes, and finally 30 minutes to study the presence of oleander extract by measuring the apparent digoxin concentration using the FPIA for digoxin. The authors observed effective removal of oleander extract by activated charcoal. When the authors supplemented other drug-free serum pools with pure Oleandrin or oleandrigenin and then subsequently treated them with activated charcoal, the authors observed complete removal of digoxin-like immunoreactivity at the end of 30 minutes' treatment. When drug-free serum pool supplemented with either oleander leaf extract, Oleandrin, or oleandrigenin was passed through a small column packed with activated charcoal, the authors observed almost no apparent digoxin concentration following the passage through the column indicating that activated charcoal is very effective in removing oleander from human serum in vitro. In contrast, when serum pools containing either oleander leaf extract or Oleandrin were subjected to equilibrium dialysis against phosphate buffer at pH 7.4, the authors observed no significant reduction in apparent digoxin concentration even after 24 hours. The authors conclude that activated charcoal is effective but equilibrium dialysis is ineffective in removing oleander leaf extract from human serum.
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neutralization of cardiac toxins Oleandrin oleandrigenin bufalin and cinobufotalin by digibind monitoring the effect by measuring free digitoxin concentrations
Life Sciences, 1998Co-Authors: Amitava Dasgupta, Lyska EmersonAbstract:Abstract Oleandrin plant poisoning is common in children and the plant extract is used in Chinese medicines. The toxicity is due to Oleandrin and the deglycosylated metabolite oleandrigenin. Bufalin and cinobufotalin (toad cardiac toxins) are also widely used in Chinese medicines like Chan SU, and Lu-Shen -WU. Severe toxicity from bufalin after consumption of toad soup has been reported. Taking advantage of structural similarities of these toxins with digitoxin, we demonstrated that these compounds can be rapidly detected in blood by the fluorescence polarization immunoassay for digitoxin. The cross reactivities of these compounds with digoxin assay were much lower. For example, when a drug free serum was supplemented with 10 μg/ml of Oleandrin, we observed 127.7 ng/ml of digitoxin equivalent but only 2.4 ng/ml of digoxin equivalent concentration. Digibind neutralized all cardiac toxins studied as evidenced by significant fall of free concentrations. When aliquots of serum pool containing 50.0 ug/ml of Oleandrin were supplemented with 0, 10.0, 25.0, 50.0, 100, and 200 μg/ml of digibind, the mean free concentrations were 30.6, 23.3, 16.0, 10.7, 7.8 and 5.5 μg/ml respectively. Similarly, with 50.0 μg/ml of oleandrigenin (total concentration: 36.2 ng/ml), the free concentration was 14.5 ng/ml digitoxin equivalent in the absence of digibind and 5.4 ng/ml in the presence of 200 μg/ml of digibind. In another specimen containing 500 ng/ml bufalin (total concentration: 156.9 ng/ml), the free concentration was 8.6 ng/ml in the absence of digibind and none detected in the presence of 100.0 μg/ml digibind. Because such neutralization may also occur in vivo, digibind may be useful in treating patients exposed to these toxins.
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unexpected suppression of total digoxin concentrations by cross reactants in the microparticle enzyme immunoassay elimination of interference by monitoring free digoxin concentration
American Journal of Clinical Pathology, 1998Co-Authors: Amitava Dasgupta, Jerry ScottAbstract:The microparticle enzyme immunoassay (MEIA for digoxin (Abbott Laboratories, Abbott Park, Ill) requires no sample pretreatment and is widely used in clinical toxicology laboratories for monitoring serum digoxin concentrations. One advantage of the new MEIA is the lower cross-reactivities with such cross-reactants as digitoxin, Oleandrin, and bufalin compared with the fluorescence polarization immunoassay (FPIA)for digoxin. Digitoxin, Oleandrin, and bufalin showed positive cross-reactivity with MEIA and FPIAs for digoxin in the absence of the primary analyte, digoxin. A surprising finding was that digoxin concentrations were falsely decreased by these cross-reactants when serum pools containing digoxin were supplemented with various concentrations of these cross-reactants and when digoxin concentrations were measured by the MEIA. In contrast, digoxin concentrations were falsely elevated when measured by the FPIA. For example, when a serum pool containing 2.15 nmol/L of digoxin was supplemented with 129.5 nmol/L of bufalin, the apparent digoxin concentrations were 1.45 nmol/L with the MEIA and 3.00 nmol/L with the FPIA. Taking the advantage of only 25% protein binding ofdigoxin and more than 95% protein binding of digitoxin and bufalin, we demonstrated that monitoring free digoxin instead of total digoxin eliminated negative interference ofdigoxin by these cross-reactants in the MEIA and positive interference in the FPIA. Although Oleandrin is also strongly bound to serum protein, high concentrations of Oleandrin still modestly affect the free digoxin assay for both MEIA and FPIAs.
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rapid detection of oleander poisoning using fluorescence polarization immunoassay for digitoxin effect of treatment with digoxin specific fab antibody fragment ovine
American Journal of Clinical Pathology, 1997Co-Authors: Amitava Dasgupta, Amy P HartAbstract:Poisoning from the oleander plant is common. Taking advantage of the high cross-reactivity of Oleandrin, the major cardiac glycoside found in the oleander plant, we demonstrated that the serum digitoxin assay can be successfully used for the rapid diagnosis of oleander poisoning. Digitoxin is rarely used for treatment of cardiac disorders in the United States and has a therapeutic range of 19.7 to 39.3 nmol/L. In a typical oleander poisoning, serum Oleandrin concentrations may reach 174 mmol/L or more. A serum specimen supplemented with 174 mmol/L of Oleandrin containing no digitoxin showed an apparent digitoxin concentration of 1,272.1 nmol/L, a very high value compared with the range of the serum digitoxin assay, which is 2.6 to 104.8 nmol/L. Moreover, the response of the serum digitoxin assay with serum specimens containing various concentrations of Oleandrin (and no digitoxin) is linear. Therefore, the Oleandrin concentration in serum can be calculated from the apparent digitoxin concentration to access the severity of poisoning. Recently, the usefulness of the digoxin-specific Fab antibody fragment in the treatment of oleander poisoning has been described; however, no laboratory test was performed to demonstrate the progress of therapy. We demonstrated that the digoxin-specific Fab antibody can bind Oleandrin in vitro, thus reducing the pharmacologically active free Oleandrin. Because Fab and Oleandrin bound to Fab are absent in the protein-free ultrafiltrates, monitoring the activity of free Oleandrin in the ultrafiltrates can be used for monitoring the effectiveness of therapy.
