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In the present study, we examined cytotoxicity and extracellular and intracellular antiviral activity of frac­tionated extracts of wild and cultivated sage Salvia officinalis L. (Lamiaceae) in vitro using the WISH-VSV model system. Extracts were obtained by fractionating depigmented ethanol extracts of sage plants with supercritical CO2 at different pressures. Cytotoxicity was determined by examining cellular morphology in situ with the aid of a colorimetric micromethod and by cell staining with trypan blue. The fraction of distilled cultivated sage obtained at CO2 pressure of 300 bars and temperature of 60°C (149/3) was the most cytotoxic, with CTD10 44 μg/ml. That of non-distilled cultivated sage obtained at CO2 pressure of 500 bars and temperature of 100°C (144/5) was the least toxic (CTD10 199 μg/ml). Moreover, 144/5 had an antiviral effect at the intracellular level: when added 5 hours before VSV infection, it caused 100% reduction of CPE at concentrations of 99.5 and 199.0 μg/ml; when added after virus penetration had occurred, the same concentrations caused 35 and 60% reduction, respectively. The obtained results indicate that antiviral activity of 144/5 involves inhibition of the early steps of the virus infective cycle without a direct virucidal effect. Abbreviations: WISH - human amnion epithelial cells, VSV - vesicular stomatitis virus, HSV - herpes simplex virus, CPE - cytopathic effect, IS - selectivity index, TCID50 - tissue culture infective dose, CTD10 - 10% cytotoxic concentrations. U radu je ispitivana antiviralna aktivnost različito frakcionisanih ekstrakata divlje i gajene žalfije Salvia officinalis L. (Lamiaceae) u in vitro uslovima koristeći WISH-VSV model sistem. Ekstrakti su dobijeni frakcionisanjem depigmentisanog etanolnog biljnog ekstrakta pod različitim pritiskom CO2. Citotoksičnost je određivana praćenjem ćelijske morfologije in situ, i bojenjem ćelija sa tripan plavim. Frakcija gajene žalfije dobijena na CO2 pritisku od 300 bara i temperaturi od 60°C (149/3) je pokazala najveću citotoksičnost (CTD10 44 μg/ml). Frakcija nedestilisane gajene žalfije dobijena na CO2 pritisku od 500 bara i temperaturi od 100°C (144/5) je pokazala najmanju toksičnost (CTD10 199 μg/ml). Takođe, frakcija 144/5 je pokazala i antiviralni efekat na intracelularnom nivou: kada se ćelije tretiraju 5 sati pre VSV infekcije, redukcija CPE je bila 100% na koncentraciji od 99.5 i 199.0 μg/ml; kada se ćelije tretiraju posle ulaska virusa u ćeliju na istim koncentarcijama redukcija CPE iz­nosi 35% odnosno 60%. Dobijeni rezultati ukazuju da frakcija 144/5 ima antiviralnu aktivnost koja se ostvaruje krozinhibiciju ranih stupnjeva viralne infekcije bezdirektnog virucidalnog efekta.

Mutagenic and antimutagenic potential of essential oil (EO) of cultivated sage (S. officinalis L.) and its monoterpenes: thujone, 1,8-cineole, camphor and limonene against UVC-induced mutations was studied with Salmonella/microsome, E. coli WP2, E. coli K12 [Simić, D., Vuković-Gacić, B., Knezević-Vukcević, J., 1998. Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system. Mutat. Res. 402, 51-57] and S. cerevisiae D7 reversion assays. The toxicity of EO differed, depending on the strain used. The most sensitive were permeable strains TA100, TA102, E. coli K12 IB112 and non-permeable WP2. Mutagenic potential of EO and monoterpenes was not detected, with or without S9. EO reduced the number of UV-induced revertants in a concentration-dependent manner, reaching 50-70% of inhibition at the maximum non-toxic concentrations: 3 microl/plate (TA102), 5 microl/plate (WP2), 7.5 microl/plate (IB112), 30 microl/plate (E. coli K12 SY252) and 60 microl/plate (D7). The metabolic activation had no effect on antimutagenic potential of EO. Similar toxicity of monoterpenes was observed in TA100, E. coli SY252 and D7, with the exception of limonene (less toxic to D7). Reduction of UV-induced revertants by non-toxic concentrations of monoterpenes, tested with SY252 and D7, reached 40-50% at 15-20 microl/plate of thujone, 10 microl/plate of cineole and 1-10 microg/plate of camphor. Limonene showed antimutagenic effect only in D7. Our data recommend sage monoterpenes for further chemoprevention studies.

The inhibition of spontaneous and UV-induced mutations by essential oil (EO) of sage (Salvia officinalis L.) and its fractions F1-F5 containing different proportions of mono- and sesquiterpenes was studied with the Salmonella/microsome, E. coli K12, and S. cerevisiae D7 reversion assays. The EO, F1, and F2 exhibited antimutagenic potential against UV-induced mutations in all tests. Fractions F3 and F4 produced a toxic, mutagenic, or antimutagenic response, depend?ing on the test organism used. Reduction of spontaneous and UV-induced mutations by F5 was detected only in permeable strains of E. coli. The obtained results demonstrate antimutagenic activity of volatile sage terpenes and recommend them for further antimutagenesis and anticarcinogenesis studies.

Antibacterial activity of volatiles from sage against Gram-positive and Gram-negative bacteria from the ATCC collection was screened with the disk diffusion test. The essential oil and its fractions showed a significant antibacterial effect against S. aureus and B. subtilis. The minimum inhibitory concentrations were 1.25-2.5 ?L/mL for S. aureus and 0.15-2.5 ?L/mL for B. subtilis. The effect on S. aureus was bactericidal, while initial bactericidal effect on B. subtilis was impaired by the presence of a resistant fraction of the population, probably endospores. The results obtained with wild type and permeable strains of E. coli and S. typhimurium indicate that transport through the cell wall limits the antibacterial effect of sage volatiles.

The present study was carried out to investigate the antimutagenic activity of essential oil and crude extract of Phlomis fruticosa. This species belongs to the family Lamiaceae which includes a number of species with medical and pharmaceutical properties. The Escherichia coli K12 reversion assay for identifying antimutagens was used. The number of spontaneous and UV-induced Arg + revertants, as well as cell survival was monitored in the presence of non-toxic concentrations of the essential oil. The number of UV-induced revertants was slightly increased in the presence of essential oil. The ethanol extract demonstrated a significant effect on viable cells. Study on pharmacological and phytochemical activities as well as antimutagenetic potential against some chemical mutagens of the essential oils, total extract and their different fractions and constituents of Ph. fruticosa is in progress.

Escherichia coli K12 assay-system is designed in order to detect bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication. The assay is composed of four tests aimed at the detection of inhibition of spontaneous and induced mutations (Tests A and B) and the estimation whether the anti-mutagenic agent acts by increasing the fidelity of DNA replication (Test B), by inhibition of SOS error prone repair (Test C), or by favoring error-free recombinational repair (Test D). In Test A, repair proficient strain and its uvrA counterpart are used for detection of spontaneous and UV-induced mutations, while in Test B mismatch repair deficient strains ( mutH, mutS, mutL and uvrD ) are used for amplified detection of spontaneous mutations caused by replication errors. In Test C, repair proficient strain carrying sfiA::lacZ fusion is used for measuring the level of SOS induction by monitoring the level of β-galactosidase. In TestD, the strains carrying different recA alleles ( recA + , rcA730 and Δ recA ) are used for measuring intrachromosomal recombination between nonoverlapping deletions in duplicated lac operon, by monitoring Lac + recombinants. The assay-system is validated with model bioantimutagens and used for detection of anti-mutagenic potential of different terpenoid fractions from sage ( Salvia officinalis L.). Extract E1/3 of cultivated sage, distinguished from others by its high content of monoterpernoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Test B and C. In Test D, it enhances intrachromosomal recombination in untreated and UV-irradiated recA + and recA730 strains. The results suggest that the protective effect is due to stimulation of recombinational repair, similarly to coumarin. We speculate that monoterpenoids from sage enhance genetic recombination by intervening in a formation of RecA-DNA complex and channeling it into recombination reaction.

The effect of the recombinational deficiency on W-reactivation of UV-damaged phage lambda was explored. In this paper we show that W-reactivation is reduced by the recB21 and recF143 mutations after bleomycin (BM) and UV treatment. Combination of these mutations in the recB21recF143 double mutant blocks W-reactivation completely after BM induction, but leaves residual W-reactivation ability after UV-irradiation, which is abolished by the introduction of uvrB deficiency (delta(uvrB-chlA]. W-reactivation has been rendered constitutive in recB21C22sbcB15, but the efficiency of reactivation remained virtually constant over the range of BM and UV doses, indicating the role of the RecBC(D) enzyme in W-reactivation.

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.

The antimutagenic potential of Vitamin E due to its antioxidative properties was studied. The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action. The assay is composed of three tests. In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain. Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT). In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability. To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test. Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E. coli K12 assay-system as well as in the WP2 antimutagenicity test. This study confirms that mismatch repair is essential for repair of oxidative DNA damage. The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases. Moreover, it can reduce microsatellite instability. After further validation, the new E. coli K12 assay-system can be used to test the antimutagenic potential of antioxidants.

The results of a study of bioantimutagenesis, with emphasis on natural antimutagens from plant extracts with modulating effects on DNA repair in Escherichia coli bacteria are presented in this chapter. Comparative screening for spontaneous or induced mutagenesis, as well as expression of the SOS gene, sfiA was accomplished.

Activation of the RecA protein following UV-irradiation or bleomycin (BM) treatment was measured in rec mutants of E. coli by monitoring β-galactosidase activity. We provide evidence here that the defect in the recN mutant results in high constitutive and induced levels of activated RecA protein. In all rec mutants studied, with the exception of the recN mutant, induction of enzyme activity, following DNA-damaging treatments, was reduced relative to the wild type. The kinetics of induced sfiA expression indicates that the DNA-unwinding activity of the RecBCD enzyme plays a major role in SOS-signal formation. The RecF protein is not needed for BM induction in strains with a functional RecBCD pathway of recombination. However, a functional product of recF gene is implied in the formation of an efficient inducing signal after UV-irradiation, as well as in the additional processing of BM-induced lesions after exposure to the drug. A fully expressed RecF pathway of recombination does not provide a high level of activated RecA protein following DNA-damaging treatments.

The inhibition of cell division induced by bleomycin (BM) and UV irradiation in the set of rec mutants of E. coli K12 was studied. Data presented in this work indicate that BM treatment requires mainly the RecBC pathway for the induction of cell filamentation. In the rec B21 mutant cell filamentation is delayed and reduced compared to the wild type. Cell filamentation is BM-induced with similar kinetics in strains with a proficient RecBC recombination pathwa, ( rec + , rec F143 and rec N262), as well as is in the strain with a fully expressed RecF pathway ( rec B21 rec C22 sbc B15). Induction is completely abolished in the rec B21 rec F143 double mutant. On the other hand cell filamentation was induced similarly by UV irradiation in all strains with a functional rec F gene and in the strain with a fully operative RecF pathway, but it was delayed in the rec F143 and rec B21 rec F143 mutants.