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.

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.

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.