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Combination of Pretreatments with Acetic Acid and Sodium Methoxide for Efficient Digoxin Preparation from Digitalis Glycosides in Digitalis lanata Leaves

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Pharmacology & Pharmacy 2016年第5期7卷 200-207页

作者： Yasuhiko Higashi Yukari Ikeda youichi fujii Department of Analytical Chemistry Faculty of Pharmaceutical Sciences Hokuriku University Kanazawa Japan

We previously developed an HPLC method for determination of lanatoside C, digoxin and α-acetyldigoxin in digitalis glycosides isolated from Digitalis lanata leaves. Here, we present an improved HPLC-UV method to determine those compounds and deslanoside. We used the improved method to examine the effects of various pretreatments on the amounts of the four compounds isolated from the leaves, with the aim of maximizing the yield of digoxin. Leaves were extracted with 50% methanol, followed by clean-up on a Sep-Pak C18 cartridge prior to HPLC analysis. The amounts of lanatoside C, digoxin and α-acetyldigoxin per 100 mg of the leaves without pretreatment were 115.6, 7.45 and 23.8 μg, respectively (deslanoside was not detected). Pretreatment with acetic acid, which activated deglucosylation mediated by digilanidase present in the leaves, increased the amounts of digoxin and α-acetyldigoxin, while lanatoside C and deslanoside were not detected. Pretreatment with sodium methoxide, which hydrolyzed lanatoside C to deslanoside, increased the yields of deslanoside and digoxin, while lanatoside C and α-acetyldigoxin were not detected. The combination of both pretreatments afforded only digoxin in a yield of 115.1 μg/100 mg leaves. Use of the combined pretreatments appears to be effective for maximizing the yield of digoxin from the leaves.

关键词： Digoxin Lanatoside C α-Acetyldigoxin Deslanoside HPLC Digitalis lanata Pretreatment

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Development of Highly Specific Enzyme Immunoassay for Monitoring Serum Digitoxin Level in Patients

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Journal of Analytical Sciences, Methods and Instrumentation 2016年第2期6卷 15-22页

作者： Yasuhiko Higashi Yukari Ikeda Mayumi Douno youichi fujii Department of Analytical Chemistry Faculty of Pharmaceutical Sciences Hokuriku University Kanazawa Japan

We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.

关键词： Digitoxin Digitoxin 3’-Hemisuccinate-β-D-Galactosidase Digitoxin 3’-Hemisuccinate-Bovine Serum Albumin Antiserum Enzyme Immunoassay Cross-Reactivity

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