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.

Inhibitory effects of compounds isolated from Lepechinia meyenii on tyrosinase.

To contribute enzymatic browning inhibitors to the food industry and also extend knowledge about the phytochemical profile of the anti-tyrosinase plant Lepechinia meyenii, its ethanol extract was subjected to bioguided fractionation. Three hydroxycinnamic acids, p-coumaric acid (1), caffeic acid (2) and rosmarinic acid (3), were isolated as mainly responsible for its activity. Compounds 1, 2 and 3 showed themselves highly effective for inhibiting tyrosinase with IC50 values of 0.30, 1.50 and 4.14 µM, respectively, for monophenolase activity and 0.62, 2.30 and 8.59 µM, respectively for diphenolase activity. This is the first report describing the isolation of the compounds causing the tyrosinase inhibitory activity of L. meyenii extract. The inhibitory kinetics of 1-3 using both L-tyrosine and L-DOPA as substrates was investigated and the results obtained were discussed at molecular level by docking analysis. The resulting compounds 1-3 and a phenolic-enriched fraction of the extract, 2.9-fold more active than the starting material, may be suitable as non-toxic and inexpensive alternatives for the control of deleterious enzymatic darkening.

Understanding Synergistic Toxicity of Terpenes as Insecticides: Contribution of Metabolic Detoxification in Musca domestica.

Essential oils, which are mixtures of terpenes, frequently show stronger insecticide activity, i.e., lower lethal dose 50 (LC50), than their most abundant terpenes. Synergy between terpenes provides a plausible explanation, but its demonstration has been elusive. In the present work, we look for an alternative explanation, by considering the influence of insect metabolic detoxification. Basically, we propose a model (metabolic model, MM) in which the LC50 of the major terpene in a mixture is expected to include a fraction that is detoxified by the insect, whereas a minor terpene would act unimpeded, showing a lower LC50 than when acting alone. In order to test this idea, we analyzed the effects of inhibiting the cytochrome P450 detoxification system with piperonyl butoxide (PBO), on the lethal concentration of terpenes as fumigants against Musca domestica. We found that, within a group of 10 terpenes [linalool, citronellal, (R)-α-pinene, 1,8-cineole, γ-terpinene, limonene, α-terpinene, (S)-ß-pinene, thymol and (R)-pulegone], seven showed the LC50PBO (the lethal concentration for PBO-treated flies) between 1.7 and 12.4 times lower than the corresponding LC50 when P450 was not inhibited. Only in one case, that of (R)-pulegone, was the LC50PBO greater than the LC50, while two terpenes [(S)-ß-pinene and thymol] showed no changes in toxicity. The increased activity of most terpenes (particularly linalool and citronellal) in PBO-treated flies supports our hypothesis that normally the LC50 includes a fraction of inactive compound, due to detoxification. Having previously determined that M. domestica preferentially oxidizes the most abundant terpene in a mixture, while terpenes in smaller proportions are poorly or not detoxified by the P450 system, we assessed whether the toxicity of minority terpenes in a mixture is similar to their activity under P450 inhibition. We chose suitable binary combinations in such a way that one terpene (in greater proportion) should be the target of P450 while the other (in smaller proportion) should intoxicate the fly with LC50PBO or similar. Combinations of 1,8-cineole-citronellal, 1,8-cineole-linalool, linalool-citronellal, (R)-pulegone-linalool, (R)-pulegone-1,8-cineole and (R)-pulegone-citronellal were assayed against M. domestica, and the LC50 of each mixture was determined and compared to values predicted by MM (considering the LC50PBO for minor component) or by the classical approach (LC50 for both components). The MM showed the best fit to the data, suggesting additive rather than synergistic effects, except for the combination of (R)-pulegone-citronellal that was clearly synergistic. Thus, the experimental data indicate that the insect preferentially oxidizes the major component in a mixture, while the terpene in lesser proportion acts as a toxicant, with higher toxicity than when it was assayed alone. These findings contribute to a deeper understanding of the higher toxicity of essential oils compared to their component terpenes and provide important information for the design of effective insecticides based on essential oils or terpenes.

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.

Tyrosinase inhibitory activity of a 6-isoprenoid-substituted flavanone isolated from Dalea elegans.

To aid the pharmaceutical and cosmetic industry in the development of alternatives to prevent melanin-related hyperpigmentation disorders, the plant Dalea elegans was submitted to fractionation with the aim of obtaining its anti-tyrosinase principle. Bioguided fractionation of D. elegans led to the isolation of 5,2',4'-trihydroxy-2″,2″-dimethylchromene-(6,7:5″,6″)-flavanone (1) as the active compound. This novel flavanone, named as dalenin, showed notable activity at inhibiting tyrosinase using l-tyrosine or l-DOPA as substrates with IC(50) values of 0.26 and 18.61 µM, respectively. This meant that the flavanone was 52 and 495 times more effective as a monophenolase inhibitor than hydroquinone and kojic acid, respectively. With l-DOPA as a substrate, compound 1 showed itself 59 times more effective at inhibiting the enzyme than hydroquinone and showed the same level of effectiveness as that of kojic acid. It was found that the flavanone behaved as a reversible inhibitor of the enzyme and that it was a mixed-I type or a non-competitive inhibitor with l-tyrosine or l-DOPA as substrates, respectively. Molecular modeling studies were conducted confirming the inhibitory potency of dalenin and showing that the 2',4'-dihydroxy substituents are important for the interaction with the enzyme. The results suggest that compound 1 has great potential to be further developed as a pharmaceutical and cosmetic agent for use in dermatological disorders associated with melanin.