A combined HPLC-UV/PAD and HPLC-ESI/MS method allowing the fast detection and identification/structural characterisation of lignans of different structural subclasses is described. Twenty-four lignans of different skeletal types were analysed and the combined information derived from their UV and ESI/MS spectra led to the identification of group characteristics that can be used to establish the structure of unknown lignans in plant samples. This method was successfully applied to the identification of lignans in crude extracts of Linum usitatissimum L. and L. bienne Mill.

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Rosmarinic acid biosynthesis can be stimulated in cell cultures of Coleus blumei by culturing the cells in medium with 4% sucrose. In enzyme extracts of these cells two new enzymes of rosmarinic acid biosynthesis were discovered. Hydroxyphenylpyruvate reductase reduces 4-hydroxyphenylpyruvate and 3,4-dihydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate, respectively, using NADH. Rosmarinic acid synthase transfers the caffeoyl moiety of caffeoyl-CoA to the non-phenolic OH-group of 3,4-dihydroxyphenyllactic acid, in course of which rosmarinic acid is formed.

From aseptically grown Artembia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg 1 1 BAP, 0.05 mg 1-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydrolysate; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg 1-1 gibberellic acid, 0.5 g 1 -~ casein hydrolysate, 10 mg 1-1 or 20 mg 1-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.

Lignans in higher plants represent an ideal class of natural products to be investigated for the origin of stereochemical diversity since chiral lignans occur in pure enantiomeric form as well as in enantiomeric mixtures. Seeds of Linum usitatissimum contain 8S, 8'S-(+)- and 8R, 8'R-(-)-secoisolariciresinol [SS-(+)- and RR-(-)-secoisolariciresinol, respectively] as diglucosides (SS- and RR-secoisolariciresinol diglucosides) whereas aerial parts of flowering L. usitatissimum accumulate only lignans derived from RR-(-)-secoisolariciresinol. Pinoresinol-lariciresinol reductase (PLR) catalyzes two early steps in lignan biosynthesis. Up to now, only a cDNA encoding a PLR ( PLR-LU1) which is enantiospecific for the conversion of 8S, 8'S-(-)-pinoresinol (SS-pinoresinol) via 8S, 8'S-(-)-lariciresinol (SS-lariciresinol) to SS-(+)-secoisolariciresinol was cloned. Here we present the cloning of a cDNA encoding a RR-pinoresinol-RR-lariciresinol reductase ( PLR-LU2) from the leaves of L. usitatissimum which converts only RR-pinoresinol to RR-secoisolariciresinol. In leaves and stems of L. usitatissimum accumulating the 8R, 8'R-enantiomers of lignans, only PLR-LU2 was transcriptionally active. Both PLR-LU1 and PLR-LU2 transcripts were observed in seeds and contribute to the synthesis of SS- and RR-secoisolariciresinol, respectively. Thus, the enantiomeric composition of lignans in the organs of L. usitatissimum appears to be determined by the relative action of two PLRs with opposite enantiospecificities rather than by a single enzyme of low enantiospecificity.