Lignans from Linum species of sections Syllinum and Linum.

The aryltetralin lignans 6-methoxypodophyllotoxin, 5'-demethoxy-6-methoxypodophyllotoxin as well as the corresponding 8'-epimers 6-methoxypicropodophyllin, and 5'-demethoxy-6-methoxypicropodophyllin were isolated from suspension cultures of Linum cariense, and 4'-demethyl-6-methoxypodophyllotoxin together with 6-methoxypodophyllotoxin from plants of L. tauricum, which both belong to section Syllinum of the genus Linum. Cell cultures of L. altaicum, L. austriacum ssp. euxinum and L. lewisii belonging to section Linum accumulate the naphthalene lignans justicidin B and isojusticidin B. The different lignans were identified by HPLC and spectroscopic methods.

Comparison of different extraction methods for the determination of podophyllotoxin and 6-methoxypodophyllotoxin in Linum species.

Three extraction methods for analysis of podophyllotoxin and its derivatives from Linum species were compared. No statistical difference on the percentage of recovery were found between the methods. The "glycosidase-method" showed the best result with respect to the accuracy studies; the "acetone-method" has an advantage compared to the other methods due to its capability to calculate the aglycone, lignan glycoside and total lignan. The content of podophyllotoxin and 6-methoxypodophyllotoxin in Linum mucronatum subsp. mucronatum Bertol, Linum arboreum L., and the endemic Turkey species of Linum flavum subsp. scabrinerve Davis were determined. This is the first report on the analysis of podophyllotoxin and 6-methoxy podophyllotoxin of natural collected Linum flavum subsp. scabrinerve and Linum arboreum.

Production of paclitaxel and baccatin III in a 20-L airlift bioreactor by a cell suspension of Taxus wallichiana.

A cell suspension culture of Taxus wallichiana (Himalayan Yew) was grown in shake flasks and a 20-L airlift bioreactor running for 28 days in a batch mode, and its capacity to accumulate paclitaxel and baccatin III was measured. When both culture types were in the highest productive state (from day 24 to day 28), there was a greater accumulation of paclitaxel and baccatin III in the bioreactor culture than in the shake flask culture (factor of 2.0 and 1.2, respectively). These increases in paclitaxel and baccatin III production cannot be related to the difference observed between the growth rates of both cultures, because when the bioreactor culture was at maximum productivity, its cell biomass, expressed in g L(-1) of dry weight, was similar to that obtained in the shake flask culture. It seems that these improvements were mainly due to adequate aeration and mixing of the culture in the bioreactor. The maximum yield observed for paclitaxel (20.84 mg x L(-1) day 24) and baccatin III (25.67 mg x L(-1) day 28) represents a productivity of 0.90 mg x L(-1) d(-1) and 0.93 mg x L(-1) x d(-1) respectively.

The production of cytotoxic lignans by plant cell cultures.

Cytotoxic lignans derived from podophyllotoxin are currently used in cancer chemotherapy. Podophyllotoxin for semi-synthetic derivatization is isolated from the rhizomes of Podophyllum plants growing wild, some of which are counted as endangered species. An alternative source for podophyllotoxin or related lignans may in future be cell cultures derived from different plant species, such as Podophyllum spp or Linum spp. These cell cultures were shown to accumulate considerable amounts of podophyllotoxin or 5-methoxypodophyllotoxin. Optimization of the cell cultivation regime might lead to a renewable source of cytotoxic lignans for medicinal uses. This Mini-Review summarizes the attempts to establish plant cell cultures for the production of podophyllotoxin and related lignans and their optimization towards high levels of these target compounds. It also summarizes the results of studies on the biosynthesis of podophyllotoxin and 5-methoxypodophyllotoxin.

Accumulation of lignans in suspension cultures of Linum mucronatum ssp. armenum (Bordz.) Davis.

For the first time callus and suspension cultures of Linum mucronatum ssp. annenum were initiated, grown in darkness at 25 degrees C and analyzed for lignans. 6-Methoxypodophyllotoxin was the main lignan besides smaller amounts of podophyllotoxin isolated and identified by chromatographic methods as well as by 1H NMR.

Deoxypodophyllotoxin 6-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum flavum involved in the biosynthesis of cytotoxic lignans.

Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrahydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degrees C. Deoxypodophyllotoxin is specifically accepted with an apparent Km of 20 microM and a saturation concentration of 200 microM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 microM NADPH; the apparent Km for this substrate is 36 microM.

High forskolin production in hairy roots of Coleus forskohlii.

Hairy roots of Coleus forskohlii were induced by infection with the Agrobacterium rhizogenes MAFF 03-01724 strain. Growth and forskolin production of two hairy root clones cultured in various liquid media were examined. Hairy root clone B9 grew well in woody plant liquid medium and showed a high forskolin yield (ca. 1.3 mg/ 100 ml flask) after 5 weeks of culture. The time course of growth and forskolin production of the clone B9 cultured in woody plant liquid medium was also examined. Rapid growth started at week 2 and continued until week 5. The highest forskolin yield (ca. 1.6 mg/100 ml flask) was obtained at week 5. Productivity was much higher than that previously reported.

Sesquiterpene lactone content in leaves of in vitro and field cultivated Arnica montana.

On the basis of GC and GC/MS analyses we report on the full qualitative and quantitative sesquiterpene lactone (STL) content of in vitro cultivated A. montana plantlets consisting of helenalin and 11alpha,13-dihydrohelenalin esters in approximately equal amounts. The accumulation of STL was shown to be correlated with tissue differentiation in the above-ground parts. The seasonal variation of STL content in leaves of A. montana cultivated in the proving field was investigated. Changes in the composition of the STL fraction were detected. While young plants accumulate mainly helenalin derivatives, the content of such compounds decreases to almost zero within about 6 weeks from the beginning of leaf formation while that of dihydrohelenalin type compounds increases at the same rate and remains constant for a longer period.

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.

Immobilization of digitoxin 12ß-hydroxylase, a cytochrome P-450-dependent enzyme from cell cultures of Digitalis lanata EHRH.

Attempts were made to immobilize digitoxin 12ß-hydroxylase, a membrane-bound, cytochrome P-450-dependent monooxygenase from cell cultures of Digitalis lanata. The optimum procedure was the entrapment of microsomes in 2% alginate by crosslinking the polysaccharide chains with CaCl2. After the immobilization of the enzyme about 70% of its activity was retained. The kinetic data such as the pH optimum and the optimum substrate concentrations were identical for the immobilized enzyme and freely suspended microsomes. Using ß-methyldigitoxin as a substrate enzyme activity could be observed for more than 20 h. A continuous flow system for immobilized digitoxin 12ß-hydroxylase is described.

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors.

Suspension cultures of Berberis wilsonae produce 4 berberine-type alkaloids: berberine, palmatine, columbamine and jatrorrhizine. In particular the formation of the phenolic alkaloids columbamine and jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. With higher aeration rates, berberine and jatrorrhizine yields can be increased considerably. Thus we reached an alkaloid yield of more than 3 g × 1(-1) with 50% dissolved oxygen tension in the medium. As far as we know this is one of the best results in fermenting of alkaloid-producing cell cultures.

Stability of biotransformation capacity in Digitalis lanata cell cultures after cryogenic storage.

Suspension cultures of Digitalis lanata were stored at the temperature of liquid nitrogen. After thawing, viable cultures were recovered which showed a growth rate equal to that of untreated cells. The capacity of Digitalis cells to transform ß-methyldigitoxin to ß-methyldigoxin remained unchanged after cryostorage. This was shown in 300-ml shake cultures as well as in 20-1 bioreactors. From these results it is clear that cryostorage is a suitable method for preserving cell lines with specific capacities to produce compounds of medical and industrial importance.