Identification of Benzodiazepines in Artemisia dracunculus and Solanum tuberosum Rationalizing Their Endogenous Formation in Plant Tissue Dominique Kavvadias,* Ahmed A. Abou-Mandour,† Franz-C. Czygan,† Helmut Beckmann,‡ Philipp Sand,‡ Peter Riederer,‡ and Peter Schreier* ,1 *Institute of Food Chemistry, †Institute of Pharmaceutical Biology and Clinical Neurochemistry, and ‡Clinic and Policlinic for Psychiatry and Psychotherapy, University of Wu ¨ rzburg, Wu ¨ rzburg D-97074, Germany Received February 4, 2000 Sterile cultivated plant cell tissues and cell regener- ates of several species were tested for their binding affinity to the central human benzodiazepine receptor. Binding activity was found in extracts of Artemisia dracunculus cell tissue (IC 50  7 g/ml) and, to a lesser extent, in plant regenerates of potato herb (Solanum tuberosum). Preparative HPLC led to the isolation of fractions with a significant displacing potency in the benzodiazepine receptor binding assay. Using on-line HPLC-electrospray-tandem mass spectrometry (HPLC- ESI-MS/MS) in the “selected reaction monitoring” (SRM) mode, delorazepam and temazepam were found in amounts of about 100 to 200 ng/g cell tissue of Arte- misia dracunculus, whereas sterile potato herb con- tained temazepam and diazepam ranging approxi- mately from 70 to 450 ng/g cell tissue. It is the first report on the endogenous formation of benzodiaz- epines by plant cells, as any interaction of microorgan- isms and environmental factors was excluded. © 2000 Academic Press Key Words: Artemisia dracunculus; benzodiazepine receptor; temazepam; diazepam; delorazepam; plant cell tissue; Solanum tuberosum. Benzodiazepines are among the most frequently pre- scribed drugs (1). Their sedative-hypnotic, anxiolytic, tranquilizing and anticonvulsant effects are mediated by binding to a specific subtype of the GABA A receptor, i.e., the 1-type GABA A receptor, which is mainly ex- pressed in cortical areas and in the thalamus of all vertebrates (2– 4). Its characterization in 1977 sug- gested the existence of endogenous ligands for the binding sites which could be responsible for the phys- iological regulation of sleep, muscle tensions and anx- iety. Several purines such as inosine and hypoxanthine (5), methylisoguanosine (6) and other substances such as nicotinamide (7), prostaglandin A (8) as well as peptides found in animal brain (9) and human cerebro- spinal fluid (10) have been identified as endogenous benzodiazepine receptor ligands. However, none of these compounds possess the same high affinity as the benzodiazepines. For years, benzodiazepines were regarded exclu- sively as synthetic products, until diazepam, loraz- epam and N-desmethyldiazepam were found in brain and other peripheral tissues of untreated human sub- jects and animals (11). This class of substances could therefore act as an endogenous regulator (ligand) bind- ing to the specific 1-subunit of GABA A receptor. To date, however, the origin of benzodiazepines found in human and animal serum, brain and other mamma- lian tissues is still unknown. Dietary uptake origin from plants and accumulation in organism has been suggested (12–14). This hypothesis was supported by the occurrence of several benzodiazepines in plants (12, 13). Substances belonging to the 5-phenyl-1,4- benzodiazepinone group have been identified in wheat grains (diazepam, N-desmethyldiazepam, delorazepam, deschlorodiazepam, lormetazepam, isodiazepam and lorazepam and delormetazepam) as well as in potato tuber (diazepam, N-desmethyldiazepam, delorazepam, lorazepam and delormetazepam). HRGC-MS analyses revealed concentrations ranging from 0.2 to 4.0 ng/g (12, 13). However, the endogenous synthesis of benzo- diazepines has remained in question as contamination with benzodiazepines or other influences of the envi- ronment as well as interaction with microorganisms could be involved. In order to exclude any interference with the puta- tive biosynthetic pathway or any source of contamina- tion, we tested several sterile cultivated plant cell tis- sues and regenerates which grew under controlled air, 1 To whom correspondence should be addressed. Fax: +49-931- 8885484. E-mail: schreier@pzlc.uni-wuerzburg.de. Biochemical and Biophysical Research Communications 269, 290 –295 (2000) doi:10.1006/bbrc.2000.2283, available online at http://www.idealibrary.com on 290 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.