Assessment of cytotoxic and genotoxic activity of alcohol extract of Polyscias filicifolia shoot, leaf, cell biomass of suspension culture and saponin fraction.

Some medicinal plants are the object of biotechnologists' special interest owing to their content of secondary metabolites, which have a strong pharmacological effect. Polyscias filicifolia is a plant known for long in traditional medicine of the Southeast Asia. Literature data suggest that it acts on the endocrine system, has adaptogenic and antiulcerative activity, shows bactericidal and insecticidal properties, restores the activity of the protein synthesis system in the conditions of long- and short-term anoxia, as well as reduces the effect of many mutagens in vitro. The purpose of the studies was to assess the cytotoxic and genotoxic effect of ethanol extracts from Polyscias filicifolia dry shoots and leaves obtained in vitro, as well as cell biomass from suspension culture. Saponin fraction from dried shoots was also tested. Initially, the cytotoxic effect was evaluated using the murine connective tissue cell line C3H/AN - L929. The genotoxic properties of the extracts were assessed using standard screening tests: the Ames test and the micronucleus test. Based on the obtained results it can be concluded that none of the extracts increases the number of revertants, both in tests with and without metabolic activation. The lack of in vitro genotoxic and mutagenic activity of tested shoot, dried leaf, cell biomass extracts, as well as the saponin fraction from dried shoots allows us to hope that Polyscias filicifolia could be used as a possible pharmaceutical raw material showing therapeutic properties.

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants.

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.

In vitro plant tissue cultures accumulate polyisoprenoid alcohols.

In vitro cultivated plant cells and tissues were found to synthesize polyisoprenoids. Taxus baccata suspension cell cultures accumulated polyisoprenoids of the same pattern as the parental tissue; methyl jasmonate or chitosan treatment almost doubled their content. All the root cultures studied accumulated dolichols as predominant polyisoprenoids. Roots of Ocimum sanctum grown in vitro accumulated approx. 2.5-fold higher amount of dolichols than the roots of soil-grown plants. Dolichols dominated over polyprenols in all Triticum sp. tissues studied.

Divergent pattern of polyisoprenoid alcohols in the tissues of Coluria geoides: a new electrospray ionization MS approach.

Polyisoprenoid alcohols of the plant Coluria geoides were isolated and analyzed by HPLC with UV detection to determine the nature of the polyprenol and dolichol mixture in the organs studied. In roots, a family of dolichols (Dol-15 to Dol-23, with Dol-16 dominating, where Dol-n is dolichol composed of n isoprene units) was accompanied by traces of polyprenols of similar chain lengths, whereas in hairy roots grown in vitro, identical patterns with a slightly broader chain-length range were found. Conversely, in leaves and seeds polyprenols were the dominant form, and their pattern was shifted toward longer chains (maximal content of Pren-19, where Pren-n is polyprenol composed of n isoprene units). Interestingly, the pattern of dolichols in seeds and leaves (in which Dol-17 dominated) was similar to that found in roots. Structures of the dolichols and polyprenols isolated were confirmed by the application of a new HPLC/electrospray ionization-MS method, which also offers a much higher sensitivity in detection of these compounds compared to a UV detector. The highest sensitivity was obtained when the [M + Na]+ ions of polyprenols and dolichols were recorded in the selected ion monitoring mode and a small amount of sodium acetate solution was added post-column to enhance the formation of these ions in an electrospray ion source.