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In this study we verified our assumption that the genotoxicity of the effective anti-HIV drug 3'-azido-3'-dideoxythymidine (AZT) on human cells could be reduced by non-toxic concentrations of two antioxidants that occur frequently in nature (ursolic acid and lignin biopolymer). Cytotoxicity of these natural compounds, well-known by their antimutagenic effects, was evaluated by the trypan blue exclusion technique. Genotoxic activity of AZT was measured on the basis of AZT-induced single and double strand breaks to DNA in two histopathologically different types of human cells, hepatoma cells HepG2 and colonic cells Caco-2. Induction of DNA strand breaks was measured by the comet assay processed in parallel at pH > or = 13.0 (standard alkaline technique which enables to recognize single strand DNA breaks of different origin) and at pH = 9.0 (neutral technique which enables to recognize double strand DNA breaks). As the level of AZT-induced double strand DNA breaks was rather low, protective effects of the antioxidants tested were evaluated only against AZT-induced single strand DNA breaks by the standard alkaline comet assay. Our findings showed that 1 h pre-incubation of cells with ursolic acid or lignin preceding to 3 h treatment of cells with AZT (3 mg/ml) significantly decreased in both cell types the level of AZT-induced single strand DNA breaks. Pre-incubation of HepG2 or Caco-2 cells with a mixture of both natural antioxidants did not increase the effects of individual treatments. This study confirms that AZT is genotoxic toward both used cell types of human origin and that ursolic acid and biopolymer lignin can protect the cells studied against genotoxic effect of AZT.

Patuletin 7-O-beta-D-(6"-caffeoylglucoside) was isolated from the methanolic extract of the flowers of Anthemis tinctoria L. (Asteraceae). The isolated compound was identified by spectroscopic means and by comparison with authentic samples after enzymatic hydrolysis. A new flavonoid glycoside, tinctosid, from Anthemis tinctoria L. was isolated from a plant source for the first time.

Three diterpenoid quinones (royleanone- SAR 3, horminone- SAR 26, and acetyl horminone- SAR 43) isolated from the roots of Salvia officinalis L. were tested for their cytotoxic and DNA-damaging activity in human colon carcinoma cells Caco-2 and human hepatoma cells HepG2 cultured in vitro. Cytotoxicity was measured by the trypan blue exclusion technique and induction of apoptosis was evaluated by flow immunofluorocytometry after 30-300 min. exposure of HepG2 and Caco-2 cells to diterpenoid quinones and following 24 hr post-incubation in the culture medium. Induction of DNA breaks was measured after 60 min. exposure of cells to different concentrations of the compounds studied by the alkaline elution of DNA and by the Comet assay. Though all the quinones tested decreased the viability of the cells studied proportionally to the concentration and to the time of treatment (cytotoxicity= 30-60%), the increased level of apoptotic nuclei comparable to the level of apoptotic nuclei induced by a topoisomerase I inhibitor was proved only in HepG2 cells treated with 1x10(-4) mol/l SAR 26 or SAR 43. Either no or marginal increase of the level of apoptotic nuclei was observed in SAR 3-treated HepG2 cells and in SAR 3-, SAR 26- or SAR 43-treated Caco-2 cells. All compounds tested induced creation of DNA strand breaks in both cell types at concentrations >1x10(-7)-1x10(-6) mol/l. The occurrence of DNA strand breaks at different pH values as well as the kinetics of DNA breaks rejoining were evaluated only in colonic cells Caco-2. The Comet assay processed in parallel at pH 13.0 and pH 12.1 showed that strand breaks detected in SARs-treated colonic Caco-2 cells originated from alkali-labile sites, as induced DNA lesions were converted to DNA strand breaks only under strong alkaline conditions. The kinetics of DNA rejoining revealed that SARs-induced DNA breaks were repaired very slowly.

The homoisoflavonoid fraction obtained from the Muscari racemosum bulb ether extract (MRBEE) exhibited estrogenic activity by inducing proliferation of MCF7 cells in a dose-dependent manner. In the presence of estradiol MRBEE exhibited a dose-dependent antiestrogenic activity., (Copyright 2002 Elsevier Science B.V.)

The potential antimutagenic effect of the plant extract of Muscari racemosum bulbs, rich on 3-benzylidene-4-chromanones, was evaluated on three genetic model organisms. The mixture of three homoisoflavonoids was applied together with diagnostic mutagens in the Ames assay on four bacterial strains Salmonella typhimurium TA97, TA98, TA100, TA102, in the toxicity and mutagenicity/antimutagenicity assay on the yeast strain Saccharomyces cerevisiae D7, and in the simultaneous phytotoxicity and clastogenicity/anticlastogenicity assay on Vicia sativa (L.). The extract exerted antimutagenic and anticlastogenic effects due to the presence of homoisoflavonoids, which may be included in the group of natural antimutagens. This genotoxicological study suggests that homoisoflavonoids from M. racemosum (L.) owing to antimutagenic and anticlastogenic properties are of great pharmacological importance, and might be beneficial for prevention of cancer.

The reported investigation of the constituents of the petroleum ether extract of the root of Salvia officinalis L. confirmed the presence of the following diterpene quinones: 12.hydroxy-8, 12-abietadiene-11, 14-dione (royleanone), 7 alpha, 12-dihydroxy-8.12-abistadiene-11-14-dione (horminone) and 7 alpha-acetoxy-12-hydroxy-8, 12-abietadiene-11,14-dione (7-O-acetylhorminone). The compounds were identified on the basic of the interpretation of results of spectral analysis. Isolated royleanones show antimicrobial activity against gram-positive bacteria (Staphylococcus aureus). A study of abietate diterpenoids in the root of Salvia officinalis L. of Slovak provenance has been performed for the first time.