Antioxidant diterpenoids from callus and regenerated plants of Rosmarinus officinalis

The phenolic diterpenoids, carnosic acid and carnosol, are powerful antioxidant and antimutagenic compounds in animal systems. Though the levels of these natural products are high in cultivated and wild rosemary, the yields are inconsistent and highly variable. This dissertation research was designed to determine if these phenolic diterpenoids are produced in undifferentiated cells and regenerated shoots of rosemary. The research was conducted in two phases. In the first phase, solid and liquid media formulations and tissue culture systems were devised for the production of callus and regenerated plants of rosemary. In the second phase, these cultured tissues were extracted and analyzed for their phenolic diterpenoid composition. The diterpenoid components and yields of extracts from rosemary tissues grown in vitro and in terra were compared.; The addition of high levels of BAP to MS basal media with IAA and kinetin and the maintenance of cultures at elevated temperature and high illumination resulted in the relatively rapid regeneration of rosemary plants with one long initial passage and no required changes in media formulation. Two different media formulations were found to produce healthy green or white callus. Elevated temperatures and light levels enhanced the growth of regenerated shoots, while lower light and temperature levels ensured the maintenance of callus.; Four phenolic diterpenoids, carnosic acid, carnosol, rosmanol, and 12-methoxy carnosic acid, were extracted from shoots and flowers of soil grown rosemary as well as from rosemary callus and regenerated shoots grown in tissue culture. The yields of these compounds were substantially lower in the extracts of the plant material grown in vitro than in the extracts of cultivated rosemary shoots and flowers. Extracts containing detectable levels of these compounds were obtained by devising improved isolation procedures, including a protocol that resulted in canosic acid being the major component of the extracts. The ability to obtain relatively high proportions of carnosic acid facilitated the crystallization of this powerful antioxidant compound.