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The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1 mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1 mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5 mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1 mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8 μg ml−1 for the HL-60, HeLa, and MCF-7 cells, respectively.

Enhanced taxane production was observed in a hybrid, two liquid phases containing, cultures of Taxus x media var. Hicksii hairy root carrying a taxadiene synthase transgene, supported with liquid perfluorodecalin (PFD) in degassed or aerated form. The hairy root cultures were elicited with methyl jasmonate (MJ, 100 lM) or coronatine (COR, 1 lM), and fed with sucrose and L-phenylalanine. The root growth was not stimulated by PFD addition, irrespective of the day of its application (day 0 and 14). However, in the cultures elicited with MJ and performed in the presence of PFD the final root biomass accumulation was higher than in cultures performed without PFD while the opposite effect was observed in cultures supplemented with COR. The highest paclitaxel content in root biomass was determined at the end of the cultures elicited with MJ and supplemented with PFD-degassed at day 0 or 14, 1,440.8 and 1,432.5 lg g -1 DW, respectively. The highest total (i.e. intracellular ? extracellular: both in aqueous and PFD phases) paclitaxel yield in flasks (149.15 lg flask -1 ) was noted after the application of PFD-degassed at day 14. The other taxane detected was baccatin III, only in the root biomass, with the highest content (76.9 lg g -1 DW) observed under COR treatment. Although COR stimulated paclitaxel production with less efficiency than MJ, it resulted in higher paclitaxel excretion to the liquid phases of culture medium and PFD.

The phytochemical investigation of the n-hexane extract from callus and cell suspension culture of Arnebia euchroma (Royle) Jonst. resulted in the isolation of nine isohexenylnaphthazarins: deoxyalkannin (1), alkannin (2), acetylalkannin (3), isobutyrylalkannin (4), β-hydroxyisovalerylalkannin (5), 2''-(S)-α-methylbutyrylalkannin (6), propionylalkannin (7), teracrylalkannin (8) and acetylshikonin (9). Their structures were determined by MS and NMR spectroscopy. Pigments 2–8 are isolated for the first time from Arnebia in vitro cultures, 4 and 7 are reported in the present work as novel metabolites within the Arnebia genus, while 9 is a known constituent of both natural roots and in vitro cultures of A. euchroma. Moreover, methyl jasmonate and 1-monoglyceryl olate, palmitate and stearate are reported for the first time within the Boraginaceae family. The antimicrobial and cytotoxic activities of all isolated pigment compounds were tested, revealing a very interesting profile.

Rhodiola (Crassulaceae) an arctic-alpine plant, is extensively used in traditional folk medicine in Asian and European countries. A number of investigations have demonstrated that Rhodiola preparations exhibit adaptogenic, neuroprotective, anti-tumour, cardioprotective, and anti-depressant effects. The main compounds responsible for these activities are believed to be salidroside, rosin and its derivatives which became the target of biotechnological investigations. This review summarizes the results of the diverse biotechnological approaches undertaken to enhance the production of salidroside, rosin and its derivatives in callus, cell suspension and organ in vitro cultures of selected Rhodiola species.