Contribution of NMR spectrometry within a pharmaceutical development resulting from extract of plants
Résumé
For a pharmaceutical application, the Laboratories Pierre Fabre developed an original extract of Eucalyptus globulus sheets. The phase impossible to circumvent so that a new drug can succeed is obtaining an AMM (Autorisation de Mise sur le Marché) implying a qualitative knowledge of a maximum of chemical components from this extract. The most active compounds of the extract are known thanks to previous works. In order toperfect the chemical knowledge of the extract, a total analytical methodology was developed within the ISA. To start this work, an important interest was carried to the compounds of the family of the acylphloroglucinols and flavonoïds specific to the eucalyptus kind. Semi-preparative and preparative chromatography enabled the purification of these compounds obtained from the extract and/or from enriched fractions of low content products. 1 to 5 mg of products are collected and characterized with Nuclear Magnetic Resonance, High resolution mass spectroscopy, Ultra-violet and Infrared spectroscopies. Among the 9 identified compounds, 3 had never been identified before in the vegetable kingdom (diformylphloroglucinol, macrocarpals P and E') and 1 (known) never mentioned in the sheets of E.globulus (grandinol). The 5 others compounds were previously identified in the E. globulus, they correspond to acylphloroglucinols which account for approximately 44% of the extract. This work shows the interest of NMR spectroscopy which allowed, thanks to 2D NMR experiments (HMBC and ROESY) to establish chemical structures never identified even on small quantities. Moreover, where the mass spectrometry indicated the same empirical formula NMR spectrometry highlighted for the macrocarpal E (already known in plane structure) the existence of macrocarpal E' (epimer on C9'). Thanks to this study, the knowledge of the extract of Eucalyptus Globulus sheets at least doubled. At the end of this work, we estimate the knowledge of extract at about 57%.