Fungal production and solid state chemistry of eritadenine : an integrated approach to development of an active pharmaceutical ingredient

Abstract: The present thesis demonstrates an integrated approach to the development of a potential active pharmaceutical ingredient, eritadenine, a cholesterol reducing compound originating from the shiitake mushroom (Lentinus edodes). The main areas covered in the thesis are a method for quantification of eritadenine, production of eritadenine by submerged cultivation of fungal mycelia and investigation of the influence of process parameters on mycelial growth and production, and finally solid state characterizations of eritadenine. The usage of the fungus as a source of eritadenine requires an analytical tool for quantification of the compound. An HPLC method was hence developed for identification and quantification of eritadenine, using chemically synthesized eritadenine as a reference. The amount of eritadenine in fruit bodies of selected strains of shiitake was determined and with the method developed in this study, eritadenine concentrations up to ten times higher than previously reported were detected. Since both fruit bodies and mycelia of shiitake have been shown to contain eritadenine submerged cultivation of shiitake mycelia was investigated as an alternative source for this compound. The mycelia were cultivated in various submerged conditions, both in shake flasks and in bioreactors. It was found that both the mycelia and the culture media contained eritadenine, of which the major part was detected in the culture media. While the biomass concentrations were higher in shake flasks, the eritadenine concentrations were considerably higher in the bioreactors, which were assigned to morphological variations. In an attempt to improve the mycelial growth and eritadenine production, a growth promotive substance in the form of a water extract of DDGS, a by-product from drygrind ethanol facilities, was added to the culture media. It was demonstrated that an amendment of the cultivation media with this extract caused a considerable growth promotive effect on shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production. If eritadenine will be used as a pharmaceutical agent, understanding about the solid state chemistry of the compound is required. Raman spectroscopy is a valuable technique for investigation of structural properties; hence, a Raman reference spectrum with line assignments for the solid state of synthetic eritadenine was established. To further investigate the solid state chemistry of eritadenine, its synthetic analogue was slowly crystallized from water and different ethanol concentrations, at different temperatures. Solids formed from slow cooling of either water or aqueous ethanol showed crystallinity. No polymorphism was detected, irrespective of solvent system or temperature. However, dissimilar thermal behaviours were observed, deducing crystals derived from water as dihydrates and crystals derived from aqueous ethanol as 2.5 hydrates.