Studies on tropane alkaloid extraction by volatile organic solvents: dichloromethane vs. chloroform

In order to investigate the production of tropane alkaloids by hairy roots of Atropa baetica, transgenic for the gene h6h encoding the enzyme hyoscyamine 6beta-hydroxylase, solvent extraction with chloroform and with dichloromethane of the metabolites present in the liquid medium and in the root tissue was compared. The extraction of scopolamine from the liquid medium was equally effective with either solvent, giving maximum values of around 850 microg/flask. For the roots, three different extraction methods were employed: A, employing chloroform:methanol: (25%) ammonia (15:5:1) for initial extraction, followed by treatment with sulfuric acid and ammonia, and using chloroform for the final extraction and washes; B, as A but using dichloromethane for extraction and washes; and C, as B but substituting chloroform for dichloromethane in the extraction cocktail. Scopolamine was the most abundant metabolite (present in amounts of 3250-3525 microg/g dry weight) and presented similar extraction efficiencies with all of the extraction methods employed. The highest amounts of hyoscyamine and the intermediate 6beta-hydxoxyhyoscyamine were present on day 31 (800 and 975 microg/g dry weight, respectively) and no statistical differences between the three extraction methods employed were detected. This study confirms that, for the extraction of tropane alkaloids, dichloromethane can replace the commonly employed chloroform, the use of which incurs major health, security and regulation problems.