Antibacterial effects of extracts obtained from plants of Argentina: Bioguided isolation of compounds from the anti-infectious medicinal plant Lepechinia meyenii.

ETHNOPHARMACOLOGICAL RELEVANCE: The mostly native species from Argentina are used in traditional medicine generally for the treatment of pain and inflammation, respiratory, gastro-intestinal and urinary disorders and as antiseptics. AIM OF THE STUDY: Since these ailments may be associated with bacterial infections and that it is necessary to discover alternative compounds with antibacterial activity, 69 extracts from these plants were screened for their activity against pathogenic bacteria. The most effective extract was then submitted to bioguided isolation to obtain the compounds responsible for this activity. MATERIALS AND METHODS: Extracts and fractions were screened using agar dilution, and compounds using microbroth dilution methods. A large panel of pathogenic bacteria was used, especially methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Bioguided fractionation was performed using successive chromatographic techniques, while the chemical structures of the isolated compounds were determined by nuclear magnetic resonance (NMR). Additionally, a series of derivatives of the most active compound were prepared in order to study the chemical features required for achieving the antibacterial effect. RESULTS: Lepechinia meyenii (Walp.) Epling (Lamiaceae) extract showed itself the most effective, with minimum inhibitory concentration (MIC) against Gram positive and negative bacteria ranging from 62.5 to 500 µg/mL, and showing better activity on MRSA than on MSSA. Activity-guided fractionation yielded the abietanes carnosol (1), rosmanol (2) and carnosic acid (3) as active principles, with MICs ranging from 15.6-31.2, 15.6-62.5 and 7.8-15.6 µg/mL, respectively against 15 MRSA strains, and 15.6-31.2, 31.2-62.5 and 7.8-15.6 µg/mL, respectively against 11 MSSA strains, maintaining higher activity against the resistant bacteria, as does the extract. In addition, Enterococcus faecalis was sensitive to 1-3 with MICs of 15.6-62.5 µg/mL. The structure activity analysis showed that 12-OH is necessary for remarkable activity, but methylation in C-20 significantly increased this, as observed with 20-methyl carnosate (5) displaying the greatest effect, even more so than 3, with MICs of 3.9 µg/mL against all the tested MRSA and 3.9-7.8 µg/mL against the MSSA. CONCLUSIONS: The results of this study contribute to validate the traditional antibacterial use of species native to Argentina, particularly of L. meyenii. The chemical structures of the compounds obtained may aid the design of antibacterial agents, especially those effective against MRSA.

Cytotoxic Activity of Extracts from Plants of Central Argentina on Sensitive and Multidrug-Resistant Leukemia Cells: Isolation of an Active Principle from Gaillardia megapotamica.

Plants are a significant reservoir of cytotoxic agents, including compounds with the ability to interfere with multidrug-resistant (MDR) cells. With the aim of finding promising candidates for chemotherapy, 91 native and naturalized plants collected from the central region of Argentina were screened for their cytotoxic effect toward sensitive and MDR P-glycoprotein (P-gp) overexpressing human leukemia cells by means of MTT assays. The ethanol extracts obtained from Aldama tucumanensis, Ambrosia elatior, Baccharis artemisioides, Baccharis coridifolia, Dimerostemma aspilioides, Gaillardia megapotamica, and Vernonanthura nudiflora presented outstanding antiproliferative activity at 50 µg/mL, with inhibitory values from 93 to 100%, when tested on the acute lymphoblastic leukemia (ALL) cell line CCRF-CEM and the resistant derivative CEM-ADR5000, while 70-90% inhibition was observed against the chronic myelogenous leukemia (CML) cell K562 and its corresponding resistant subline, Lucena 1. Subsequent investigation showed these extracts to possess marked cytotoxicity with IC50 values ranging from 0.37 to 29.44 µg/mL, with most of them being below 7 µg/mL and with ALL cells, including the drug-resistant phenotype, being the most affected. G. megapotamica extract found to be one of the most effective and bioguided fractionation yielded helenalin (1). The sesquiterpene lactone displayed IC50 values of 0.63, 0.19, 0.74, and 0.16 µg/mL against K562, CCRF-CEM, Lucena 1, and CEM/ADR5000, respectively. These results support the potential of these extracts as a source of compounds for treating sensitive and multidrug-resistant leukemia cells and support compound 1 as a lead for developing effective anticancer agents.

P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach.

This work examines the antitumor activity of an isomeric mixture (1), composed of the limonoids meliartenin and its interchangeable isomer 12-hydroxyamoorastatin. The results obtained showed that 1 displayed outstanding cytotoxic activity against CCRF-CEM, K562, A549 and HCT116 cells, with a highly selective effect on the latter, with an IC50 value of 0.2 µM. Based on this finding, HCT116 cells were selected to study the mechanism of action of 1. Cell cycle analysis revealed that 1 induced sustained arrest in the S-phase, which was followed by the triggering of apoptotic cell death and reduced clonogenic capacity. This cytotoxicity was seen to be preceded by the upregulation of the tumor suppressor p53 and its target effector p21. In addition, it was found that p53 expression was required for efficient cell death induction, and thus that the toxicity of 1 relies mainly on p53-dependent mechanisms. Taken together, these findings position 1 as a potent antitumor agent, with potential for the development of novel chemotherapeutic drugs based on the induction of S-phase arrest.

The Inhibitory Activity of Plants from Central Argentina on p-Hydroxyphenylpyruvate Dioxygenase. Isolation and Mechanism of Inhibition of a Flavanone from Flourensia oolepis.

The enzyme 4-hydroxyphenylpyruvate dioxygenase catalyzes the second step in the tyrosine degradation pathway. In mammals, this enzyme is the molecular target of drugs used for the treatment of metabolic disorders associated with defects in the tyrosine catabolism, mainly the fatal hereditary disease tyrosinemia type 1. This study evaluated the inhibitory effect of 91 extracts on 4-hydroxyphenylpyruvate dioxygenase from mostly native plants from central Argentina. Flourensia oolepis ethanol extract showed itself to be the most effective, and bioguided fractionation yielded pinocembrin (1) as its active principle. This flavanone, with an IC50 value of 73.1 µM and a KI of 13.7 µM, behaved as a reversible inhibitor of the enzyme and as a noncompetitive inhibitor. Molecular modeling studies confirmed the inhibitory potency of 1 and explained its activity by means of in silico determination of its binding mode in comparison to inhibitors of known activity, cocrystallized with 4-hydroxyphenylpyruvate dioxygenase. The main structural determinants that confer its potency are discussed. Analysis of the binding mode of the flavanone 1 with 4-hydroxyphenylpyruvate dioxygenase revealed the basis of the noncompetitive reversible mechanism of inhibition at the molecular level, which seems to be a common feature in this ubiquitous family of natural compounds. The resulting information may establish the basis for obtaining novel 4-hydroxyphenylpyruvate dioxygenase inhibitors for the treatment of tyrosinemia type 1 and other disorders associated with tyrosinase catabolism.

Antibacterial and Cytotoxic Activity of Compounds Isolated from Flourensia oolepis.

The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluated. Bioguided fractionation led to five flavonoids, identified as 2',4'-dihydroxychalcone (1), isoliquiritigenin (2), pinocembrin (3), 7-hydroxyflavanone (4), and 7,4'-dihydroxy-3'-methoxyflavanone (5). Compound 1 showed the highest antibacterial effect, with minimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250 µg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds 1-5 was determined by MTT assay on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR) phenotypes. Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC50 = 6.6-9.9 µM) and a lower effect against CML cells (IC50 = 27.5-30.0 µM). Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment, 1 induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites of F. oolepis extract, with 1 being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.

Fumigant toxicity of Citrus sinensis essential oil on Musca domestica L. adults in the absence and presence of a P450 inhibitor.

Essential oils (EOs) are potential tools for controlling Musca domestica L. In a fumigant assay, M. domestica adults treated with Citrus sinensis EO (LC50=3.9mg/dm(3)), with (4R)(+)-limonene (95.1%) being its main component, died within 15min or less. The terpenes absorbed by the flies and their metabolites, analyzed using SPME fiber, were (4R)(+)-limonene (LC50=6.2mg/dm(3)), α-pinene (LC50=11.5mg/dm(3)), ß-pinene (LC50=6.4mg/dm(3)), and two new components, carveol (LC50=1122mg/dm(3)) and carvone (LC50=19mg/dm(3)), in a proportion of 50, 6.2, 12.5, 6.3 and 25%, respectively. Carveol and carvone were formed by oxidation of (4R)(+)-limonene mediated by cytochrome P450, as was suggested by a fumigation assay on flies previously treated with piperonyl butoxide, a P450 inhibitor. In this experiment, an increase in the toxicity of the EO and (4R)(+)-limonene was observed, as well as a lower production of carveol and carvone.

Understanding the interactions between metabolites isolated from Achyrocline satureioides in relation to its antibacterial activity.

As part of our ongoing research on the antibacterial activity of Achyrocline satureioides, this study seeks to better understand the interactions between the metabolites isolated from this plant. For this purpose, the combined effect of 23-methyl-6-O-desmethylauricepyrone (1), quercetin (2) and 3-O-methylquercetin (3), obtained through bioguided fractionation from A. satureioides ethanol extract, was evaluated against Staphylococcus aureus and Escherichia coli. In first place, the antibacterial effect of the combination of flavonols 2 and 3 was assessed, as these showed individual effectiveness lower than or equal to that of the fraction from which they were obtained. When the flavonols were applied together at concentrations below their minimum inhibitory concentration (MIC) values, a synergistic effect (FICI<0.30) against S. aureus was observed. In addition, compounds 2 and 3 in combination reduced 1000 times the MIC of compound 1, showing a clear synergistic interaction (FICI<0.15) in treatments against the Gram (+) bacterium. The most active combination against E. coli showed an additive interaction (FICI<0.62) between the three assayed compounds 1-3. These results indicated the existence of concerted action between these metabolites, evidence of the importance of the synergistic interactions between the components of plant-derived extracts for the control of pathogenic bacteria.

Molecular response of Musca domestica L. to Mintostachys verticillata essential oil, (4R)+-pulegone and menthone.

Intense applications of synthetic insecticides for the control of adult Musca domestica have led to the insects developing resistance to most of them. In consequence, there is interest in new active ingredients as alternatives to conventional insecticides. Essential oils (EO) are potential tools for controlling M. domestica because of their effectiveness and their minimal environmental effects. In a fumigant assay, M. domestica adults treated with Minthostachys verticillata EO [LC(50)=0.5 mg/dm(3); majority components by SPME-GC: (4R)(+)-pulegone (67.5%), menthone (22.3%) and (4R)(+)-limonene (3.8%)], died within 15 min or less. The terpenes absorbed by the flies and their metabolites, analyzed using SPME fiber, were (4R)(+)-limonene (LC(50)=6.2 mg/dm(3)), menthone (LC(50)=1.9 mg/dm(3)), (4R)(+)-pulegone (LC(50)=1.7 mg/dm(3)) and a new component, menthofuran (LC(50)=0.3 mg/dm(3)), in a relative proportion of 12.4, 6.5, 35.9 and 44.2% respectively. Menthofuran was formed by oxidation of either (4R)(+)-pulegone or menthone mediated by cytochrome P450, as demonstrated by a fumigation assay on flies previously treated with piperonyl butoxide, a P450 inhibitor, which showed a decrease in toxicity of the EO, (4R)(+)-pulegone and of menthone, supporting the participation of the P450 oxidizing system in the formation of menthofuran. The enzymatic reaction of isolated fly microsomes with the EO or the (4R)(+)-pulegone produced menthofuran in both cases. Contrary to expectations, the insect detoxification system contributed to enhance the toxicity of the M. verticillata EO. Consequently, resistant strains overexpressing P450 genes will be more susceptible to either M. verticillata EO or (4R)(+)-pulegone and menthone.