SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

BACKGROUND: RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. PURPOSE: Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. METHODS: The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. RESULTS: Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. CONCLUSION: In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity.

Interaction between lichen secondary metabolites and antibiotics against clinical isolates methicillin-resistant Staphylococcus aureus strains.

The in vitro antimicrobial activities of five compounds isolated from lichens, collected in several Southern regions of Chile (including the Chilean Antarctic Territory), were evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, for the lichen compounds were evaluated. The MIC90 was ranging from 32 µg/ml for perlatolic acid to 128 µg/ml for α-collatolic acid. MBC90 was ranging from onefold up to twofold the MIC90 for each compound. A synergistic action was observed in combination with gentamicin, whilst antagonism was observed for some lichen compounds in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Iridoid glycosides from Alonsoa meridionalis.

A new iridoid glucoside has been isolated from the Chilean native Alonsoa meridionalis (L.f.) Kuntze. Its structure has been assigned as 6'-O-ß-d-glucopyranosyl-8-O-acetylharpagide (1) by using spectroscopic methods. Harpagoside (2), laterioside (3) and verbascoside (4) were also identified.

Henna through the centuries: a quick HPTLC analysis proposal to check henna identity

Henna leaves are the raw material of commercial body and hair dyes. According to historical and ethnobotanical information, henna was one of the first plants used for such purpose. However, differences can be observed between henna products by the origin of the raw material, the presence of other plants, or the addition of various contaminants that may cause allergies and permanent scarring. Nowadays henna is used everywhere but it lacks the necessary controls. We report a pharmacognostic study focused on quality control of henna's raw materials from different countries or based on other plants. The analytic approach based on High Performance Thin Layer Chromatography (HPTLC) was proposed as a reliable technique to evaluate natural products complex mixtures, as it is also the case of derived botanical marketed products.

Chemical, biological, morphoanatomical and antimicrobial study of Ocotea puchury-major Mart.

Ocotea puchury-major Mart. is a tree native to the Brazilian rain forest, where it is popularly known as puxurì. In local folk medicine the leaves are used for their sedative, gastroenteric and antireumatic properties. The morphoanatomical study determined those features useful in distinguishing this species from other closely related taxa. Chemical analysis was focused on the study of the volatile oil. Gas chromatography-mass spectrometry analyses indicated safrol as the main compound of the volatile oil (39%). The results confirm and authenticate the use of its leaves in folk medicine. Furthermore, safrol is economically important as the starting material for hemisynthesis of several products. The antimicrobial activity of the essential oil was studied which showed promising activity against environmental microorganisms as well as anti-inflammatory activity.

Antibacterial activity of selected metabolites from Chilean lichen species against methicillin-resistant staphylococci.

The in vitro antibacterial activities of eight compounds isolated from lichens, collected in several Southern regions of Chile (including Antarctica), were evaluated against methicillin-resistant clinical isolates strains of Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus warneri. The minimum inhibitory concentrations, calculated in microdilution, were ranging from 8 µg mL(-1) for sphaerophorin to 1024 µg mL(-1) for fumarprotocetraric acid. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

HPTLC fingerprint: a modern approach for the analytical determination of botanicals

Availability of rapid and reliable methods for detection of quality in plant raw materials and botanicals is urgently needed. The recent HPTLC instrumentation allows to obtain fingerprints useful to ascertain identity and composition. The results of direct application of HPTLC devices in selected cases, using the fingerprint approach, are here reported, considered and compared with other methods. HPTLC is proposed as an useful tool for analytical validation of the novel forms of natural products.

Cyclohexanones from Mimulus glabratus and M. luteus.

The phytochemical study of Mimulus glabratus A.Gray allowed the isolation of two cyclohexenones: the new compound 6-chlorohalleridone 1 and halleridone 2. Halleridone was also identified in Mimulus luteus L., together with dihydroalleridone 3, the naphtoquinone alpha-dunnione 4, ursolic acid and beta-sitosterol.

Monoterpene alkaloids from Argylia radiata.

Argylia radiata (L.) D. Don (Bignoniaceae) represents an important source of secondary metabolites, largely unexplored. The paper presents the isolation from the plant root of two new monoterpene alkaloids, 10-acetoxy-actinidine and 4-nor-7,8-dehydro-10-hydroxy-skytanthine, whose structures were elucidated by Mass spectrometry and 1H-NMR data.