ABSTRACT
Atropine is an antimuscarinic alkaloid identified in Atropa belladonna. In pharmacopeias, percolation is standardized as an extraction method for A. belladonna leaves, along with liquid-liquid extraction as a cleanup procedure and titration as an analytical method for assaying the atropine in the leaves. In this study, a faster, solvent-saving, and more reliable method for quality control of A. belladonna samples was developed. Ultrasound-assisted extraction was proposed and optimized by fractional factorial design followed by Box-Behnken design. For modeling atropine content, the following optimal conditions were established: particle size, 180 µm; percentage methanol in water, 50%; volume of solvent, 15 ml; time of extraction, 60 min; and number of extractions, two. This led to a significant improvement in atropine extraction (P < 0.001). For cleanup, solid-phase extraction was used as an alternative to liquid-liquid extraction, giving similar results, with higher reproducibility. Finally, for the atropine assay, a UPLC method was validated as a substitute for the classic titration method. Taken together, the development of an ultrasound-assisted extraction-solid-phase extraction-UPLC approach allowed the determination of atropine content in A. belladonna leaves in a time- and solvent-saving manner, with high reliability.
Subject(s)
Atropa belladonna/chemistry , Atropine/analysis , Chromatography, High Pressure Liquid/methods , Plant Extracts/chemistry , Plant Leaves/chemistry , Solid Phase Extraction/methods , Muscarinic Antagonists/analysis , Solvents/chemistrySubject(s)
Muscarinic Antagonists/administration & dosage , Muscarinic Antagonists/analysis , Pulmonary Disease, Chronic Obstructive/drug therapy , Tropanes/administration & dosage , Administration, Inhalation , Drug Chronotherapy , Germany , Humans , Randomized Controlled Trials as Topic , Scopolamine Derivatives/administration & dosage , Scopolamine Derivatives/adverse effects , Tiotropium Bromide , Tropanes/adverse effectsABSTRACT
Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D. leichhardtii following transformation by Agrobacterium rhizogenes strain A4. Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil. The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees. PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations. A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine. However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants. These results demonstrate that the A. rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D. myoporoides x D. leichhardtii obtained by conventional breeding.