The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor.

The biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89 mg g-1 DW; equivalent to 63.3 mg L-1), taxodione (1.15 mg g-1 DW; equivalent to 17.4 mg L-1), 15-deoxy-fuerstione (2.15 mg g-1 DW; equivalent to 32.5 mg L-1), and 7-(2'-oxohexyl)-taxodione (0.076 mg g-1 DW; equivalent to 1.1 mg L-1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2'-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2'-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC50 = 0.05 µM with high selectivity, selectivity index (SI) = 38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1 µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC50 values ranging from 0.62 to 194.7 µM) and lower selectivity (SI value ranged from 0.4 to 5.0).

Antimicrobial and anti-biofilm properties of new taxodione derivative from hairy roots of Salvia austriaca.

The aim of the present report was to evaluate antimicrobial/anti-biofilm activity of 7-(2-oxohexyl)-taxodione, a novel taxodione derivative isolated from n-hexane extract of Salvia austriaca hairy roots. Antimicrobial assays showed that 7-(2-oxohexyl)-taxodione was at least 4 times more active than taxodione against methicillin-susceptible as well against methicillin-resistant staphylococci with MIC of 1.25-2.5 µgml(-1). This compound was less active against vancomycin-resistant enterococci (VRE), on the same level as taxodione (MIC ranged 10.0-20.0 µgml(-1)). The presence of 7-(2-oxohexyl)-taxodione in the culture medium (at MIC, ½ MIC or » MIC) decreased adhesion of staphylococci to abiotic surfaces, which in turn caused a reduction in biofilm formation during 24h, by approximately 25-30%. Also, the extent of established biofilm eradication was found to be significant, although it required an increased concentration of the compound. This is the first report on the antimicrobial activity of this, up to now not known compound, isolated from transformed roots of S. austriaca.

Genetic transformation of Salvia austriaca by Agrobacterium rhizogenes and diterpenoid isolation.

Hairy roots of Salvia austriaca Jacq. transformed with Agrobacterium rhizogenes strain A4 were obtained and transgenic status of the roots was confirmed by polymerase chain reaction (PCR) using rolB and rolC specific primers. The root cultures growing in half-strength Gamborg (1/2 B5) liquid medium supplemented with sucrose (30 g L(-1)) under light conditions (photoperiod: 16 h light/8 h dark) were examined for their ability to produce diterpenoids. From n-hexane extract the abietane-type diterpenoids royleanone, 15-deoxyfuerstione and taxodione were isolated and identified. This is the first report on the genetic transformation of S. austriaca.

Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: salvipisone as a potential anti-biofilm agent active against antibiotic resistant Staphylococci.

The antimicrobial activities of crude dichloromethane fractions from acetone extracts of Agrobacterium rhizogenes transformed roots and roots of field-grown plants of Salvia sclarea as well as four pure abietane diterpenoids isolated from the hairy root cultures were determined. The growth of Gram-positive bacteria (Staphylococcus aureus, S. epidermidis, Enterococcus faecalis) but not Gram-negative ones (Escherichia coli, Pseudomonas aeruginosa) or pathogenic fungi (Candida albicans) was inhibited by fractions tested at concentrations of 37.5-75.0 microgml(-1). Abietane diterpenoids: salvipisone, aethiopinone, 1-oxoaethiopinone and ferruginol were shown to be bacteriostatic as well as bacteriocidal for the cultures of S. aureus and S. epidermidis strains, regardless of their antibiotic susceptibility profile. This was demonstrated by using simultaneously the optical density measuring method and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction assay. The highest activity was shown by salvipisone which demonstrated also a very interesting activity when its effect on 24-h-old staphylococcal biofilm cells viability was examined. It limited the survival of biofilms formed by S. aureus as well as by S. epidermidis, putting this compound to the list of potential anti-biofilm agents, better than most of known antibiotics.

Thymol derivatives from hairy roots of Arnica montana.

Five known thymol derivatives were isolated from roots of Arnica montana transformed with Agrobacterium rhizogenes LBA 9402. The compounds were characterized by spectral methods. The pattern of thymol derivatives in light-grown hairy roots was slightly different from that in dark-grown ones. This is the first report on the presence of thymol derivatives in hairy roots of the plant.

Transformation of Catalpa ovata by Agrobacterium rhizogenes and phenylethanoid glycosides production in transformed root cultures.

Transformed root cultures of Catalpa ovata were established following shoots infection with four agropine strains of Agrobacterium rhizogenes. Frequency of root formation was dependent on the bacterial strain and the presence of acetosyringone in the incubation medium. It is the first report concerning the possibility of transforming Catalpa ovata by A. rhizogenes. Both transformed and untransformed root cultures of C. ovata were studied for their growth and phenylethanoid glycoside production. As with the roots of intact plants, cis- and trans-verbascoside as well as martynoside were produced in transformed and untransformed root cultures of C. ovata. In hairy roots, total (cis + trans) verbascoside production could be stimulated up to three-fold of that of roots of 6-month-old plants grown in a greenhouse, by using an appropriate root line cultured in liquid 1/2 B5 Gamborg medium containing indole-3-butyric acid (0.1 mg/l) in the dark but not light conditions. Transformed and untransformed root cultures of C. ovata were also found to have 10 times higher martynoside production than roots of intact plants.

Phenylpropanoid glycosides from Penstemon serrulatus.

Two new phenylpropanoid glycosides named cis-martynoside (1) and cis-leucosceptoside A (3) were recognized in cell suspension cultures of Penstemon serrulatus Menz. The structures of these compounds were determined on the basis of 1H NMR spectral data.

Studies on iridoids of tissue cultures of Penstemon serrulatus: isolation and their antiproliferative properties.

Penstemide and serrulatoloside as well as penstemide aglycone and serrulatoloside aglycone were isolated and identified in the calli and suspension cultures of Penstemon serrulatus. The influence of serrulatoloside, penstemide, and its aglycone on the spontaneous proliferation of mouse spleen lymphocytes or hepatoma cells in the Syrian hamster has been estimated in vitro. It has been found that these compounds produce a dose-dependent inhibition of [3H]-thymidine incorporation into the DNA of the examined cells.

[Analysis of phenolic acids in cultured cells and in Penstemon Serrulatus Menz]. / Zawartosc fenolokwasów w kulturach tkankowych i roslinach gruntowych Penstemon serrulatus Menz.

Qualitative and quantitative content of aromatic acids in callus tissues, cells in suspension cultures and intact plants of Penstemon serrulatus was determined by paper and gas chromatography. It has been observed that these acids occur in the investigated material mainly in bounded form. The largest quantities of phenolic acids were produced by callus tissues grown on Schenk and Hildebrandt's medium. The total content of aromatic acids found in the cells from suspension cultures was similar to the content of these compounds in intact plants, but twice lower than in callus tissue grown on the same medium.

Penstemide and serrulatoloside production in tissue cultures of Penstemon serrulatus Menz.

The contents of iridoid glycosides penstemide and serrulatoloside were determined with HPLC method in callus and cell cultures, plants regenerated with tissue culture method and in the intact plants of Penstemon serrulatus Menz.

Selection of penstemide and serrulatoloside producing clones in penstemon serrulatus by small-aggregate cloning.

A plating method is described for cultured cells of Penstemon serrulatus Menz. which allows effective cloning of aggregates consisting of less than 10 cells, on a simple synthetic medium. The obtained clones snowed high diversity with respect to their ability to produce penstemide, an ester iridoid of the Valeriana-type. Some of the clones produced higher quantities of this compound than the original cell line. The content of another iridoid glucoside of the same type, serrulatoloside, was also determined.