Untersuchungen zur Bildung von Hydroxyzimtsäureestern in Ocimum basilicum, Cichorium intybus und Actaea racemosa

An important group of plant compounds are phenolics, especially hydroxycinnamic acid esters like rosmarinic acid, chlorogenic acid, but also chicoric acid and cimicifugic acids. Most of these hydroxycinnamic acid esters are synthesised by BAHD-type hydroxycinnamoyltransferases. Chicoric acid, dicaffeoyltartaric acid, was first isolated from Cichorium intybus L. (Asteraceae). The formation of chicoric acid is a two-step reaction. Caffeiic acid is transferred from caffeoyl-CoA to tartaric acid to form caftaric acid, a further addition of caffeic acid leads to chicoric acid. This was observed in crude protein extracts from Equisetum arvense L. (Equisetaceae) and Arachis glabrata L. (Fabaceae). The main enzyme of this reaction was not isolated yet. The common basil (Ocimum basilicum L., Lamiaceae) contains, amongst others, rosmarinic and chicoric acids. In this thesis, crude protein extracts of basil cell cultures were used to investigate the formation of chicoric acid. For the enzyme assays m-, L-, as well as D-tartaric acid were used with caffeoyl-CoA as donor substrate. None of these assays showed a formation of chicoric acid. There was even no chicoric acid present in cell cultures we established although rosmarinic acid could be found. Elicitation of the cell cultures up to 96 h with 100 µM methyl jasmonate resulted in an increase of rosmarinic acid content from 2.6 % to 5.3 % of cell dry weight. However, the biosynthesis of chicoric acid was not induced. Supplementary feeding of tartaric acid to the cell cultures could not induce the biosynthesis of chicoric acid. Furthermore, investigations on cell cultures of Cichorium intybus revealed the absence of chicoric acid. Further research is necessary because the involved enzymes of chicoric acid biosynthesis are still unknown. Cimicifugic acids got the name from Cimicifuga racemosa (L.) NUTT., nowadays Actaea racemosa L.. This perennial plant from the family Ranunculaceae forms a distinct rhizome. Black cohosh is used against peri- and postmenopausal afflictions. Nevertheless, the biological effect and the biosynthesis of cimicifugic acids are not elucidated yet. An uncharacterised nucleotide sequence from Actaea racemosa with a high sequence similarity to enzymes from BAHD-superfamily was amplified (ArCAS) and expressed in E. coli cells. The encoded protein has a molecular mass of 50 kDa. Different hydroxycinnamoyl-CoA esters have been synthesised and piscidic acid was isolated from Quillaja saponaria extracts. Assays with p-coumaroyl-, caffeoyl-, sinapoyl- and feruloyl-CoA as acceptor substrates lead on to the formation of the respective cimicifugic acids. Optimal conditions for the enzyme assays were determined and kinetic assays were performed. The best accepted substrate was p-coumaroyl-CoA with a Km-value of 6.8 ± 2.3 µM. A reverse enzyme assay, using fukinolic acid and coenzyme A as substrates, showed the formation of fukiic acid by the enzyme. The isolation of fukiic acid was not possible, until now, so this result is a first evidence that fukiic acid is a substrate of ArCAS. The establishment of plant cell cultures from fresh plant material as well as seeds was not successful. Cell cultures would be important for expression analysis and further investigations on the biosynthesis of piscidic and fukiic acid. Investigations on the phylogenetic relationship of ArCAS with other BAHD-hydroxy-cinnamoyltransferases showed a close relationship to a spermidine hydroxycinnamoyltransferase from Malus domestica, as well as a malate hydroxycinnamoyltransferase from Trifolium pratense and a tetrahydroxyhexanedioic acid hydroxycinnamoyltransferase from Phaseolus vulgaris. However, ArCAS did not accept the substrates of these enzymes. Also substrates from other hydroxycinnamoyltransferase like p-hydroxyphenyllactic acid, quinic acid, shikimic acid or amino acids showed no product formation. ArCAS therefore has a high specificity for the formation of cimicifugic acids. Flowers, leaves and roots of Actaea racemosa were extracted and the total phenolic content determined using Folin-Ciocalteu reagent. The highest content could be found in leaves whereas the lowest was in the roots. Further investigations on these extracts revealed different distribution patterns of cimicifugic acids as well as different amounts of these acids in the different parts of the plant. ArCAS is the main enzyme for the biosynthesis of cimicifugic acid from (hydroxy)cinnamoyl-CoA esters and piscidic acid. The investigations presented in this thesis are a first step to elucidate the biosynthesis of cimicifugic acids in Actaea racemosa.