Marcela Wilkens

Antimicrobial study of the resinous exudates and of diterpenoids and flavonoids isolated from some Chilean Pseudognaphalium (Asteraceae).

The antimicrobial properties of the resinous exudates from twigs and leaves of four Chilean species of Pseudognaphalium: P. viravira, P. robustum, P. heterotrichium and P. cheiranthifolium were tested against six gram negative bacteria and five gram positive bacteria. The extracts share similar antimicrobial activities against the gram positive bacteria. The antimicrobial activity correlated very well with the presence in the resinous exudates of ent-16-kauren-19-oic acid and to a lesser extent with the presence of ent-9(11),16-kauradien-19-oic. Introduction of an hydrophilic 3 beta-OH drastically reduced the antimicrobial activity of these compound. The activity was not correlated with the flavonoid content of those resinous exudates.

Antimicrobial study of the resinous exudate and of diterpenoids isolated from Eupatorium salvia (Asteraceae).

The antimicrobial properties of the resinous exudate from twigs and leaves of Eupatorium salvia were tested against five gram-negative and five gram-positive bacteria. Comparison of the antimicrobial activities of 7-hydroxy-8(17)-labden-15-oic acid (salvic acid) and of its acetate, both compounds isolated from the plant, with that of the crude extract suggested that the latter ester derivative was the major active component in the exudate. These results validate the vernacular medicinal use of Eupatorium salvia as an antiseptic agent.

Antibacterial Properties of 3 H-Spiro[1-benzofuran-2,1’-cyclohexane] Derivatives from Heliotropium filifolium

A re-examination of cuticular components of Heliotropium filifolium allowed the isolation of four new compounds: 3’-hydroxy-2’,2’,6’-trimethyl-3H-spiro[1-benzo-furan-2,1’-cyclohexane]-5-carboxylic acid (2), methyl 3’-acetyloxy-2’,2’,6’-trimethyl-3H-spiro[1-benzofuran-2,1’-cyclohexane]-5-carboxylate (3), methyl 3’-isopentanoyloxy-2’,2’,6’-trimethyl-3H-spiro[1-benzofuran-2,1’-cyclohexane]-5-carboxylate (4) and methyl 3’-benzoyloxy-2’,2’,6’-trimethyl-3H-spiro[1-benzofuran-2,1’-cyclohexane]-5-carboxylate (5). Compounds 2-5 were identified by their spectroscopic analogies with filifolinol (1), and their structures confirmed by chemical correlation with 1. The antimicrobial properties of the compounds were tested against Gram positive and Gram negative bacteria. Some of them proved to be active against Gram positive, but inactive against Gram negative bacteria. In searching for structure-activity relationships from the obtained MIC values, lipophilicity was shown to be an important variable.

ANTIBACTERIAL ACTIVITY OF COMPOUNDS ISOLATED OF THE RESINOUS EXUDATE FROM HELIOTROPIUM SINUATUM ON PHYTOPATHOGENIC BACTERIA

From the resinous exudates of Heliotropium sinuatum have been isolated and characterized two compounds, pentaeicosanol 1 and 1-hydroxy-3-doeicosanone 2. Those structures and previously isolated pure compounds have been tested in their antibacterial activity on phytopathogenic bacteria. Ketone 2 and the flavonoid hesperetine 10 showed antibacterial activity on Erwinia carotovora subsp. carotovora, while the flavonoid 3-O-methylgalangine 4 showed activity on Clavibacter michiganensis subsp. michiganensis

EXTERNAL FLAVONOIDS FROM HELIOTROPIUM MEGALANTHUM AND H. HUASCOENSE (BORAGINACEAE). CHEMOTAXONOMIC CONSIDERATIONS

Del extracto de compuestos superficiales obtenido con CH2Cl2de material fresco de especies de Heliotropium, se han aislado flavonoidesy derivados aromaticos geranilados. Entre los flavonoides, los tiposestructurales mas comunes corresponden a flavanonas y flavonolesy se han reportado solamente pocas flavonas. En este trabajo se informael aislamiento de varios flavonoides de los exudados de H. megalanthumyH. huascoense. Dos de los componentes aislados de H. huascoenseson flavonoides nuevos, que pertenecen a los grupos poco comunes de flavanonaspenta-O-substituidas y flavonoles hexa-O-substituidos. Estos resultadosmuestran una composicion de flavonoides externos en H. huascoense,diferente de las otras especies de Heliotropium estudiadas. Losresultados estan reafirmados por consideraciones morfologicas,en especial, la presencia o ausencia de tricomas glandulares en especiesde Heliotropium

Structure-Activity and Lipophilicity Relationships of Selected Antibacterial Natural Flavones and Flavanones of Chilean Flora

In this study, we tested eight naturally-occurring flavonoids—three flavanones and five flavones—for their possible antibacterial properties against four Gram-positive and four Gram-negative bacteria. Flavonoids are known for their antimicrobial properties, and due their structural diversity; these plant-derived compounds are a good model to study potential novel antibacterial mechanisms. The lipophilicity and the interaction of antibacterial compounds with the cell membrane define the success or failure to access its target. Therefore, through the determination of partition coefficients in a non-polar/aqueous phase, lipophilicity estimation and the quantification of the antibacterial activity of different flavonoids, flavanones, and flavones, a relationship between these parameters was assessed. Active flavonoids presented diffusion coefficients between 9.4 × 10−10 and 12.3 × 10−10 m2/s and lipophilicity range between 2.0 to 3.3. Active flavonoids against Gram-negative bacteria showed a narrower range of lipophilicity values, compared to active flavonoids against Gram-positive bacteria, which showed a wide range of lipophilicity and cell lysis. Galangin was the most active flavonoid, whose structural features are the presence of two hydroxyl groups located strategically on ring A and the absence of polar groups on ring B. Methylation of one hydroxyl group decreases the activity in 3-O-methylgalangin, and methylation of both hydroxyl groups caused inactivation, as shown for 3,7-O-dimethylgalangin. In conclusion, the amphipathic features of flavonoids play a crucial role in the antibacterial activity. In these compounds, hydrophilic and hydrophobic moieties must be present and could be predicted by lipophilicity analysis.

ANTIBACTERIAL ACTIVITY OF 13-EPI-SCLAREOL, A LABDANE TYPE DITERPENE ISOLATED FROM PSEUDOGNAPHALIUM HETEROTRICHIUM AND P. CHEIRANTHIFOLIUM (ASTERACEAE)

La actividad antibacteriana del 13-epi-esclareol, un diterpeno del grupo del labdano, aislado del exudado resinoso de las especies Pseudognaphalium heterotrichium y P. cheiranthifolium se estudio frente a cuatro bacterias Gram positivas y tres Gram negativas. La actividad antimicrobiana se determino usando tres metodos diferentes: medio solido, medio liquido y un micro-metodo en medio liquido. Adicionalmente se evaluo el efecto del compuesto sobre las bacterias en una curva de crecimiento y a traves de la viabilidad bacteriana. El 13-epi-esclareol presento actividad selectiva frente a las bacterias Gram positivas y a concentraciones de 30 µg/mL, produjo la lisis de Bacillus cereus y B. subtilis

Synergistic interactions between phenolic compounds identified in grape pomace extract with antibiotics of different classes against Staphylococcus aureus and Escherichia coli

Synergy could be an effective strategy to potentiate and recover antibiotics nowadays useless in clinical treatments against multi-resistant bacteria. In this study, synergic interactions between antibiotics and grape pomace extract that contains high concentration of phenolic compounds were evaluated by the checkerboard method in clinical isolates of Staphylococcus aureus and Escherichia coli. To define which component of the extract is responsible for the synergic effect, phenolic compounds were identified by RP-HPLC and their relative abundance was determined. Combinations of extract with pure compounds identified there in were also evaluated. Results showed that the grape pomace extract combined with representatives of different classes of antibiotics as β-lactam, quinolone, fluoroquinolone, tetracycline and amphenicol act in synergy in all S. aureus and E. coli strains tested with FICI values varying from 0.031 to 0.155. The minimal inhibitory concentration (MIC) was reduced 4 to 75 times. The most abundant phenolic compounds identified in the extract were quercetin, gallic acid, protocatechuic acid and luteolin with relative abundance of 26.3, 24.4, 16.7 and 11.4%, respectively. All combinations of the extract with the components also showed synergy with FICI values varying from 0.031 to 0.5 and MIC reductions of 4 to 125 times with both bacteria strains. The relative abundance of phenolic compounds has no correlation with the obtained synergic effect, suggesting that the mechanism by which the synergic effect occurs is by a multi-objective action. It was also shown that combinations of grape pomace extract with antibiotics are not toxic for the HeLa cell line at concentrations in which the synergistic effect was observed (47 μg/mL of extract and 0.6–375 μg/mL antibiotics). Therefore, these combinations are good candidates for testing in animal models in order to enhance the effect of antibiotics of different classes and thus restore the currently unused clinical antibiotics due to the phenomenon of resistance. Moreover, the use of grape pomace is a good and low-cost alternative for this purpose being a waste residue of the wine industry.

Enhanced Antibacterial Activity of Ent-Labdane Derivatives of Salvic Acid (7α-Hydroxy-8(17)-ent-Labden-15-Oic Acid): Effect of Lipophilicity and the Hydrogen Bonding Role in Bacterial Membrane Interaction

In the present study, the antibacterial activity of several ent-labdane derivatives of salvic acid (7α-hydroxy-8(17)-ent-labden-15-oic acid) was evaluated in vitro against the Gram-negative bacterium Escherichia coli and the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus. For all of the compounds, the antibacterial activity was expressed as the minimum inhibitory concentration (MIC) in liquid media and minimum inhibitory amount (MIA) in solid media. Structure activity relationships (SAR) were employed to correlate the effect of the calculated lipophilicity parameters (logPow) on the inhibitory activity. Employing a phospholipidic bilayer (POPG) as a bacterial membrane model, ent-labdane-membrane interactions were simulated utilizing docking studies. The results indicate that (i) the presence of a carboxylic acid in the C-15 position, which acted as a hydrogen-bond donor (HBD), was essential for the antibacterial activity of the ent-labdanes; (ii) an increase in the length of the acylated chain at the C-7 position improved the antibacterial activity until an optimum length of five carbon atoms was reached; (iii) an increase in the length of the acylated chain by more than five carbon atoms resulted in a dramatic decrease in activity, which completely disappeared in acyl chains of more than nine carbon atoms; and (iv) the structural factors described above, including one HBD at C-15 and a hexanoyloxi moiety at C-7, had a good fit to a specific lipophilic range and antibacterial activity. The lipophilicity parameter has a predictive characteristic feature on the antibacterial activity of this class of compounds, to be considered in the design of new biologically active molecules.

The diterpenoid ent-16-kauren-19-oic acid acts as an uncoupler of the bacterial respiratory chain.

ent-16-Kauren-19-oic acid is a bacteriolytic diterpenoid present in the resin of the medicinal plant Pseudognaphalium vira vira. The compound and its methyl ester showed strong activity against gram-positive bacteria, whereas the derivative 3beta-hydroxy-ent-kauren-19-oic acid was inactive against all assayed bacteria at the maximal concentration used (250 microg/mL). The bacteriolytic effect of ent-16-kauren-19-oic acid (5 microg/mL) was confirmed with cultures of Bacillus cereus and Staphylococcus aureus, whereas the methyl derivative (12 microg/mL) showed only a bacteriostatic effect. Both compounds stimulated oxygen consumption and proton conduction of whole cells, as reflected by an abrupt increase in the extracellular pH. These results indicate that ent-16-kauren-19-oic acid acts as a respiratory chain uncoupler, and that this function is strongly affected by minor structural substitutions, suggesting a tight activity-structure relationship. The ultimate effect of the uncoupling mechanism demonstrated by ent-16-kauren-19-oic acid is bacterial lysis. The disruption of the bacterial membrane integrity caused by the diterpenoid compound was determined using SYTOX Green stain and visualized by fluorescence microscopy.