INTRODUCTION: Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol-3-angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis. OBJECTIVE: To develop a rapid and reliable method for quantification of ingenol in various plant extracts. METHODOLOGY: Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) after methanolysis and solid-phase extraction (SPE) purification. The 18 O-labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation. RESULTS: The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates. CONCLUSION: The newly established UHPLC-MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.
This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.
- MeSH
- chitin metabolismus MeSH
- chitinasy chemie izolace a purifikace metabolismus MeSH
- chromatografie DEAE-celulózová MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- Euphorbia chemie enzymologie MeSH
- hydrolýza MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- bolest farmakoterapie MeSH
- desenzibilizace imunologická MeSH
- Euphorbia MeSH
- finanční podpora výzkumu jako téma MeSH
- financování organizované MeSH
- kafr terapeutické užití MeSH
- kalmodulin MeSH
- kapsaicin farmakokinetika terapeutické užití MeSH
- kationtové kanály TRPV antagonisté a inhibitory účinky léků MeSH
- lidé MeSH
- Piper nigrum MeSH
- Check Tag
- lidé MeSH
