Synthesis of a dehydrodieugenol B derivative as a lead compound for visceral leishmaniasis-mechanism of action and in vivo pharmacokinetic studies.

Leishmaniasis is a parasitic neglected tropical disease, affecting 12 million people. Available treatments present several limitations, with an increasing number of resistance cases. In the search for new chemotherapies, the natural product dehydrodieugenol B was used as a scaffold for the synthesis of a series of derivatives, resulting in the discovery of the promising analog [4-(4-(5-allyl-3-methoxy-2-((4-methoxybenzyl)oxy)phenoxy)-3-methoxybenzyl)morpholine, 1]. In this work, we investigated the effect of compound 1 on cell signaling in Leishmania (L.) infantum, culminating in cell death, as well as its immunomodulatory effect in the host cell. Additionally, we performed a pharmacokinetic profile study in an animal model. After treatment, compound 1 induced the alkalinization of acidocalcisomes and concomitant Ca2+ release in the parasite. These events may induce depolarization of the mitochondrial potential, with successive collapse of the bioenergetic system, leading to a reduction of ATP and reactive oxygen species (ROS) levels. The analysis of total proteins and protein profile by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) demonstrated that compound 1 also altered the parasite proteins after treatment. Transmission electron microscopy studies revealed ultrastructural damage to mitochondria; together, these data suggest that compound 1 may promote autophagic cell death. Additionally, compound 1 also induced an immunomodulatory effect in host cells, with a reduction of Th1 and Th2 cytokine response, characterizing an anti-inflammatory compound. The obtained pharmacokinetic profile in rats enhances the potential of the compound, with a mean plasma half-life (T1/2) of 21 h. These data reinforce the potential of compound 1 as a new lead for future efficacy studies.

Design, synthesis, docking studies and bioactivity evaluation of 1,2,3-triazole eugenol derivatives.

Aim: The design, synthesis, docking studies and evaluation of the in vitro antifungal and cytotoxic properties of eugenol (EUG) containing 1,2,3-triazole derivatives are reported. Most of the derivatives have not been reported.Materials & methods: The EUG derivatives were synthesized, molecular docked and tested for their antifungal activity.Results: The compounds showed potent antifungal activity against Trichophyton rubrum, associated with dermatophytosis. Compounds 2a and 2i exhibited promising results, with 2a being four-times more potent than EUG. The binding mode prediction was similar to itraconazole in the lanosterol-14-α-demethylase wild-type and G73E mutant binding sites. Additionally, the pharmacokinetic profile prediction suggests good gastrointestinal absorption and potential oral administration.Conclusion: Compound 2a is a promising antifungal agent against dermatophytosis caused by T. rubrum.

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Dehydrodieugenol isolated from Ocotea cymbarum induces cell death in human breast cancer cell lines by dysregulation of intracellular copper concentration.

In this work, two neolignans - dehydrodieugenol (1) and dehydrodieugenol B (2) - were isolated from leaves of Ocotea cymbarum (H. B. K.) Ness. (Lauraceae). When tested against two human breast cancer cell lines (MCF7 and MDA-MB-231), compound 1 was inactive (IC50 > 500 µM) whereas compound 2 displayed IC50 values of 169 and 174 µM, respectively. To evaluate, for the first time in the literature, the synergic cytotoxic effects of compounds 1 and 2 with ion Cu2+, both cell lines were incubated with equimolar solutions of these neolignans and Cu(ClO4)2·6H2O. Obtained results revealed no differences in cytotoxicity upon the co-administration of compound 2 and Cu2+. However, the combination of compound 1 and Cu2+ increases the cytotoxicity against MCF7 and MDA-MB-231 cells, with IC50 values of 165 and 204 µM, respectively. The activity of compound 1 and Cu2+ in MCF7 spheroids regarding the causes/effects considering the tumoral microenvironment were accessed using fluorescence staining and imaging by fluorescence microscopy. This analysis enabled the observation of a higher red filter fluorescence intensity in the quiescence zone and the necrotic core, indicating a greater presence of dead cells, suggesting that the combination permeates the spheroid. Finally, using ICP-MS analysis, the intracellular copper disbalance caused by mixing compound 1 and Cu2+ was determined quantitatively. The findings showcased a 50-fold surge in the concentration of Cu2+ compared with untreated cells (p > 0.0001) - 18.7 ng of Cu2+/mg of proteins and 0.37 ng of Cu2+/mg of protein, respectively. Conversely, the concentration of Cu2+ in cells treated with compound 1 was similar to values of the negative control group (0.29 ng of Cu2+/mg of protein). This alteration allowed us to infer that compound 1 combined with Cu2+ induces cell death through copper homeostasis dysregulation.

In Situ Preparation of Dehydrodieugenol-Loaded Silver Nanoparticles and their Antischistosomal Activity.

Schistosomiasis is a major neglected disease that imposes a substantial worldwide health burden, affecting approximately 250 million people globally. As praziquantel is the only available drug to treat schistosomiasis, there is a critical need to identify new anthelmintic compounds, particularly from natural sources. To enhance the activity of different natural products, one potential avenue involves its combination with silver nanoparticles (AgNP). Based on this approach, a one-step green method for the in situ preparation of dehydrodieugenol (DHDG) by oxidation coupling reaction using silver and natural eugenol is presented. AgNP formation was confirmed by UV-Vis spectroscopy due to the appearance of the surface plasmon resonance (SPR) band at 430 nm which is characteristic of silver nanoparticles. The nanoparticles were spherical with sizes in the range of 40 to 50 nm. Bioassays demonstrated that the silver nanoparticles loaded with DHDG exhibited significant anthelmintic activity against Schistosoma mansoni adult worms without toxicity to mammalian cells and an in vivo animal model (Caenorhabditis elegans), contributing to the development of new prototypes based on natural products for the treatment of schistosomiasis.

Incorporation of dehydrodieugenol, a neolignan isolated from Nectandra leucantha (Lauraceae), in lipid Langmuir monolayers as biomembrane models.

Dehydrodieugenol, a neolignan isolated from the Brazilian plant Nectandra leucantha (Lauraceae) with reported antiprotozoal and anticancer activity, was incorporated in Langmuir monolayers of selected lipids as cell membrane models, aiming to comprehend its action mechanism at the molecular level. The interaction of this compound with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG) was inferred through tensiometry, infrared spectroscopy, and Brewster angle microscopy. The interactions had different effects depending on the chemical nature of the lipid polar head, with expansion for DPPC monolayers, condensation for DPPE, and expansion (at low surface pressures) followed by the overlap of the isotherms (at high surface pressure values) for DPPS and DPPG. Effects caused by dehydrodieugenol in the negatively charged lipids were distinctive, which was also reflected in the hysteresis assays, surface potential-area isotherms, and rheological measurements. Infrared spectroscopy indicated that the drug interaction with the monolayer affects not only the polar groups, but also the acyl lipid chains for all lipids. These results pointed to the fact that the interaction of the drug with lipid monolayers at the air-water interface is modulated by the lipid composition, mainly considering the polar head of the lipids, as well as the hydrophobicity of the lipids and the drug. As negatively charged lipids pointed to distinctive interaction, we believe this can be related to the antiprotozoal and anticancer properties of the compound.

In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools

Background Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.(AU)

Evaluation of isoeugenol in inhibition of Staphylococcus aureus efflux pumps and their toxicity using Drosophila melanogaster model.

The Staphylococcus aureus bacteria is a pathogen considered opportunistic and that has been acquiring resistance to several classes of antibiotics, mainly due to the synthesis of efflux pumps, which are proteins that expel these drugs intracellularly, reducing their effectiveness. The objective of this study was to evaluate the ability of isoeugenol to inhibit S. aureus efflux pumps and to determine its toxicity against a eukaryotic model (Drosophila melanogaster). IS-58, K2068 and K4414 S. aureus strains were used in the study. Isoeugenol minimum inhibitory concentration (MIC) and antibiotic modulation were evaluated in efflux pump inhibitory tests as well as in ethidium bromide (EtBr) assays. Toxicity tests against D. melanogaster assessed mortality and negative geotaxis. Isoeugenol obtained a relevant MIC result and a synergism was observed when isoeugenol was associated with the antibiotics, mainly with ciprofloxacin. Isoeugenol was able to affect all three efflux pumps tested, especially in strain K4414. The mortality of D. melanogaster caused by isoeugenol administration started after 12 h of exposure, being volume dependent and having an LC50 of 81.69 µL/L. In the negative geotaxis test, a statistical difference was observed after 24h of exposure compared to the control, demonstrating that damage to the locomotor apparatus had occurred. Based on the results, isoeugenol is a putative efflux pump inhibitor, becoming an alternative in blocking these proteins, and demonstrated acute toxicity against D. melanogaster.

Variability of the Chemical Composition and Bioactivity between the Essential Oils Isolated from Male and Female Specimens of Hedyosmum racemosum (Ruiz & Pav.) G. Don.

Hedyosmum racemosum (Ruiz & Pav.) G. is a native species of Ecuador used in traditional medicine for treatment of rheumatism, bronchitis, cold, cough, asthma, bone pain, and stomach pain. In this study, fresh H. racemosum leaves of male and female specimens were collected and subjected to hydrodistillation for the extraction of the essential oil. The chemical composition of male and female essential oil was determined by gas chromatography-gas chromatography equipped with a flame ionization detector and coupled to a mass spectrometer using a non-polar and a polar chromatographic column. The antibacterial activity was assayed against five Gram-positive and two Gram-negative bacteria, and two dermatophytes fungi. The scavenging radical properties of the essential oil were evaluated by DPPH and ABTS assays. The chemical analysis allowed us to identify forty-three compounds that represent more than 98% of the total composition. In the non-polar and polar column, α-phellandrene was the principal constituent in male (28.24 and 25.90%) and female (26.47 and 23.90%) essential oil. Other main compounds were methyl chavicol, germacrene D, methyl eugenol, and α-pinene. Female essential oil presented a strong activity against Klebsiella pneumoniae (ATCC 9997) with an minimum inhibitory concentration (MIC) of 500 µg/mL and a scavenging capacity SC50 of 800 µg/mL.

Zuccagnia punctata Cav. Essential Oil into Poly(ε-caprolactone) Matrices as a Sustainable and Environmentally Friendly Strategy Biorepellent against Triatoma infestans (Klug) (Hemiptera, Reduviidae).

The main strategies against Triatoma infestans (primary vector responsible for the Chagas disease transmission) are the elimination or reduction of its abundance in homes through the application of insecticides or repellents with residual power, and environmental management through the improvement of housing. The use of plant-derived compounds as a source of therapeutic agents (i.e., essential oils from aromatic plants and their components) is a valuable alternative to conventional insecticides and repellents. Essential oil-based insect repellents are environmentally friendly and provide reliable personal protection against the bites of mosquitoes and other blood-sucking insects. This study investigates, for the first time to our knowledge, the potential repellent activity of Zuccagnia punctata essential oil (ZEO) and poly(ε-caprolactone) matrices loaded with ZEO (ZEOP) prepared by solvent casting. The analysis of its essential oil from aerial parts by GC-FID and GC-MS, MS allowed the identification of 25 constituents representing 99.5% of the composition. The main components of the oil were identified as (-)-5,6-dehydrocamphor (62.4%), alpha-pinene (9.1%), thuja-2, 4 (10)-diene (4.6%) and dihydroeugenol (4.5%). ZEOP matrices were homogeneous and opaque, with thickness of 800 ± 140 µm and encapsulation efficiency values above 98%. ZEO and ZEOP at the lowest dose (0.5% wt./wt., 96 h) showed a repellency of 33 and 73% respectively, while at the highest dose (1% wt./wt., 96 h) exhibited a repellent activity of 40 and 66 %, respectively. On the other hand, until 72 h, ZEO showed a strong repellent activity against T. infestans (88% repellency average; Class V) to both concentrations, compared with positive control N-N diethyl-3-methylbenzamide (DEET). The essential oils from the Andean flora have shown an excellent repellent activity, highlighting the repellent activity of Zuccagnia punctata. The effectiveness of ZEO was extended by its incorporation in polymeric systems and could have a potential home or peridomiciliary use, which might help prevent, or at least reduce, Chagas' disease transmission.

Enzymatic Synthesis of Eugenyl Acetate from Essential Oil of Clove Using Lipases in Liquid Formulation as Biocatalyst.

In this research, eugenyl acetate, a compound with flavoring, antioxidant, and antimicrobial properties, was obtained from essential oil of clove (Syzygium aromaticum) via liquid lipase-mediated acetylation. Clove essential oil was extracted by drag water vapor from dry flower buds and its physic-chemical characteristics were analyzed. For the enzymatic synthesis, an extensive evaluation of reaction parameters was accomplished through employment of distinct reaction temperatures, acetic anhydride to eugenol molar ratios, enzyme loads, and three different lipases (a lyophilized enzyme produced by solid-state fermentation of sunflower seed with Penicillium sumatrense microorganism and other two commercial lipases - Lipozyme TL 100L and CALB L). The product eugenyl acetate was confirmed by 1H-NMR, 13C-NMR Distortionless Enhancement by Polarization Transfer (DEPT 135), and Heteronuclear Multiple Bond Correlation (HMBC). Through optimized conditions (55 °C, acetic anhydride to eugenol molar ratio of 1:1, 10 wt% of Lipozyme TL 100L), 91.80% of conversion after 2 h was achieved to the eugenyl acetate production. With the results obtained, it was possible to conclude that the use of lipases in liquid formulation is a promising alternative for the synthesis of essential esters largely applied on food, cosmetic, and pharmaceutical industries.

In Silico Study Examining New Phenylpropanoids Targets with Antidepressant Activity.

BACKGROUND: Natural products, such as phenylpropanoids, which are found in essential oils derived from aromatic plants, have been explored during non-clinical psychopharmacology studies, to discover new molecules with relevant pharmacological activities in the central nervous system, especially antidepressant and anxiolytic activities. Major depressive disorder is a highly debilitating psychiatric disorder and is considered to be a disabling public health problem, worldwide, as a primary factor associated with suicide. Current clinically administered antidepressants have late-onset therapeutic actions, are associated with several side effects, and clinical studies have reported that some patients do not respond well to treatment or reach complete remission. OBJECTIVE: To review important new targets for antidepressant activity and to select phenylpropanoids with antidepressant activity, using Molegro Virtual Docker and Ossis Data Warris, and to verify substances with more promising antidepressant activity. RESULTS AND CONCLUSION: An in silico molecular modeling study, based on homology, was conducted to determine the three-dimensional structure of the 5-hydroxytryptamine 2A receptor (5- HT2AR), then molecular docking studies were performed and the predisposition for cytotoxicity risk among identified molecules was examined. A model for 5-HT2AR homology, with satisfactory results, was obtained indicating the good stereochemical quality of the model. The phenylpropanoid 4-allyl-2,6-dimethoxyphenol showed the lowest binding energy for 5-HT2AR, with results relevant to the L-arginine/nitric oxide (NO)/cGMP pathway, and showed no toxicity within the parameters of mutagenicity, carcinogenicity, reproductive system toxicity, and skin-tissue irritability, when evaluated in silico; therefore, this molecule can be considered promising for the investigation of antidepressant activity.

In vitro and in silico inhibitory effects of synthetic and natural eugenol derivatives against the NorA efflux pump in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium responsible for a number of diseases and has demonstrated resistance to conventional antibiotics. This study aimed to evaluate the antibacterial activity of eugenol and its derivatives allylbenzene, 4-allylanisole, isoeugenol and 4-allyl-2,6-dimethoxyphenol against the S. aureus NorA efflux pump (EP) in association with norfloxacin and ethidium bromide. The antibacterial activity of the compounds was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC). A reduction in the MIC of ethidium bromide (a substrate for several efflux pumps) or norfloxacin was used as a parameter of EP inhibition. Molecular modeling studies were used to predict the 3D structure and analyze the interaction of selected compounds with the binding pocket of the NorA efflux pump. Except for 4-allylanisole and allylbenzene, the compounds presented clinically effective antibacterial activity. When associated with norfloxacin against the SA 1199B strain, 4-allyl-2,6-dimethoxyphenol eugenol and isoeugenol caused significant reduction in the MIC of the antibiotic, demonstrating synergistic effects. Similar effects were observed when 4-allyl-2,6-dimethoxyphenol, allylbenzene and isoeugenol were associated with ethidium bromide. Together, these findings indicate a potential inhibition of the NorA pump by eugenol and its derivatives. This in vitro evidence was corroborated by docking results demonstrating favorable interactions between 4-allyl-2,6-dimetoxypheno and the NorA pump mediated by hydrogen bonds and hydrophobic interactions. In conclusion, eugenol derivatives have the potential to be used in antibacterial drug development in strains carrying the NorA efflux pump.

Cardiodepressive Effect of Eugenyl Acetate in Rodent Heart. / Efeito Cardiodepressor do Acetato de Eugenil em Coração de Roedor.

No presente trabalho investigou-se o efeito inotrópico do acetato de eugenil (AE), bem como sua ação sobre a corrente de Ca2+ do tipo L (ICa,L). Os experimentos de contratilidade foram realizados em átrio esquerdo isolado de cobaia exposto às concentrações crescentes da droga (1 a 5.000µM). O AE reduziu a força de contração atrial (IC50=558±24,06µM) de modo dependente de concentração. O efeito do AE sobre a ICa,L também foi avaliado em cardiomiócitos ventriculares isolados de camundongos, utilizando-se a técnica de "patch-clamp". O AE apresentou um efeito inibitório (IC50=1.337±221µM) sobre os canais de Ca2+ sensíveis à voltagem (CaV1.2). Em conclusão, o AE apesenta efeito cardiodepressor que se deve, pelo menos em parte, à diminuição da entrada de Ca2+ nos cardiomiócitos.

Antinociceptive Interaction and Pharmacokinetics of the Combination Treatments of Methyleugenol Plus Diclofenac or Ketorolac.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the main types of drugs used to treat pain, they have several adverse effects, and such effects can be reduced by combining two analgesic drugs. The aim of this study was to evaluate the nociceptive activity of methyleugenol combined with either diclofenac or ketorolac, and determine certain parameters of pharmacokinetics. For the isobolographic analysis, the experimental effective dose 30 (ED30) was calculated for the drugs applied individually. With these effective doses, the peak plasma concentration (Cmax) was found and the other parameters of pharmacokinetics were established. Methyleugenol plus diclofenac and methyleugenol plus ketorolac decreased licking behavior in a dose-dependent manner in phase II, with an efficacy of 32.9 ± 9.3 and 39.8 ± 9.6%, respectively. According to the isobolographic analysis, the experimental and theoretical ED30 values were similar for methyleugenol plus diclofenac, suggesting an additive effect, but significantly different for methyleugenol plus ketorolac (3.6 ± 0.5 vs. 7.7 ± 0.6 mg/kg, respectively), indicating a probable synergistic interaction. Regarding pharmacokinetics, the only parameter showing a significant difference was Cmax for the methyleugenol plus diclofenac combination. Even with this difference, the combinations studied may be advantageous for treating inflammatory pain, especially for the combination methyleugenol plus ketorolac.

Efeito Cardiodepressor do Acetato de Eugenil em Coração de Roedor / Cardiodepressive Effect of Eugenyl Acetate in Rodent Heart

Resumo No presente trabalho investigou-se o efeito inotrópico do acetato de eugenil (AE), bem como sua ação sobre a corrente de Ca2+ do tipo L (ICa,L). Os experimentos de contratilidade foram realizados em átrio esquerdo isolado de cobaia exposto às concentrações crescentes da droga (1 a 5.000μM). O AE reduziu a força de contração atrial (IC50=558±24,06μM) de modo dependente de concentração. O efeito do AE sobre a ICa,L também foi avaliado em cardiomiócitos ventriculares isolados de camundongos, utilizando-se a técnica de "patch-clamp". O AE apresentou um efeito inibitório (IC50=1.337±221μM) sobre os canais de Ca2+ sensíveis à voltagem (CaV1.2). Em conclusão, o AE apesenta efeito cardiodepressor que se deve, pelo menos em parte, à diminuição da entrada de Ca2+ nos cardiomiócitos.

Dehydrodieugenol improved lung inflammation in an asthma model by inhibiting the STAT3/SOCS3 and MAPK pathways.

BACKGROUND: Eugenol, a common phenylpropanoid derivative found in different plant species, has well-described anti-inflammatory effects associated with the development of occupational hypersensitive asthma. Dehydrodieugenol, a dimeric eugenol derivative, exhibits anti-inflammatory and antioxidant activities and can be found in the Brazilian plant species Nectandra leucantha (Lauraceae). The biological effects of dehydrodieugenol on lung inflammation remain unclear. PURPOSE: This study aimed to investigate the effects of eugenol and dehydrodieugenol isolated from N. leucantha in an experimental model of asthma. METHODS: In the present work, the toxic effects of eugenol and dehydrodieugenol on RAW 264.7 cells and their oxidant and inflammatory effects before lipopolysaccharide (LPS) exposure were tested. Then, male BALB/c mice were sensitized with ovalbumin through a 29-day protocol and treated with vehicle, eugenol, dehydrodieugenol or dexamethasone for eight days beginning on the 22nd day until the end of the protocol. Lung function; the inflammatory profile; and the protein expression of ERK1/2, JNK, p38, VAChT, STAT3, and SOCS3 in the lung were evaluated by immunoblotting. RESULTS: Eugenol and dehydrodieugenol were nontoxic to cells. Both compounds inhibited NO release and the gene expression of IL-1ß and IL-6 in LPS-stimulated RAW 264.7 cells. In OVA-sensitized animals, dehydrodieugenol reduced lung inflammatory cell numbers and the lung concentrations of IL-4, IL-13, IL-17, and IL-10. These anti-inflammatory effects were associated with inhibition of the JNK, p38 and ERK1/2, VAChT and STAT3/SOCS3 pathways. Moreover, treatment with dehydrodieugenol effectively attenuated airway hyperresponsiveness. CONCLUSION: The obtained data demonstrate, for the first time, that dehydrodieugenol was more effective than eugenol in counteracting allergic airway inflammation in mice, especially its inhibition of the JNK, p38 and ERK1/2, components of MAPK pathway. Therefore, dehydrodieugenol can be considered a prototype for the development of new and effective agents for the treatment of asthmatic patients.

Electrospray mass-spectrometry guided target isolation of neolignans from Nectandra leucantha (Lauraceae) by high performance- and spiral-coil countercurrent chromatography.

Nectandra leucantha (Lauraceae) is a tree indigenous to the tropical Atlantic forests of Brazil, one of the most biodiverse flora hotspots worldwide. This plant species contains high concentrations of neolignan and dehydrodieugenol derivatives that express significant in-vitro activities against various parasite strains. These activities are however responsible for severe tropical human infections, such as Leishmaniasis (Leishmania spp.) and Chagas disease (Trypanosoma cruzi), which have been classified by the World Health Organization (WHO) as Neglected Tropical Diseases (NTDs). In order to optimize the isolation process for these target metabolites, n-hexane extract of the leaves was separated by means of semi-preparative high performance countercurrent chromatography (HPCCC) and scale-up spiral-coil countercurrent chromatography (sp-CCC) systems. Several biphasic solvent mixtures were evaluated for their partitioning effects on neolignans, resulting in the selection of an optimized system n-hexane - ethylacetate - methanol - water (7:3:7:3, v/v/v/v). The chromatographic experiments on the HPCCC and sp-CCC were run in the head-to-tail mode with 500 mg and 16 g injections, respectively. For specific and multiple metabolite detection, the recovered CCC-fractions were off-line injected, in the sequence of recovery, to an electrospray mass-spectrometry (ESI-MS/MS) device. A projection of the single ion traces of the target compounds, in the positive ionization mode at a scan range of m/z 100-1500, located chromatographic areas where the co-elution effects occurred and pure target metabolites were present. Five major target neolignans were specifically detected, which enabled the accurate pooling of CCC-fractions for an optimum recovery of the metabolites. The direct comparison of the performance characteristics of the two CCC-devices, with very different mechanical designs was achieved by the conversion of the time axis into a partition ratio (KD) separation scale. As a result, the compound specific KD-elution values of the target neolignan were determined in high precision, while the comparison of the calculated separation factor (α) and resolution factor (RS) values revealed a superior separation performance for the HPCCC system. Also, the reproducibility of detected metabolites in the two CCC experiments was confirmed by small variations (ΔKD ±0.1). Neolignan target compounds with anti-parasite activities were successfully isolated in the 100 mg to 4 g range in a single lab-scale countercurrent chromatographic process step.

Semisynthetic eugenol derivatives as antifungal agents against dermatophytes of the genus Trichophyton.

PURPOSE: Eugenol, the main component of clove bud essential oil (Eugenia caryophyllus), has been linked to antimicrobial, anti-inflammatory, insecticidal and immunomodulatory properties. The purpose of this study was to evaluate the antifungal and cytotoxic activity of eugenol, the essential oil of Eugenia caryophyllus, and some semisynthetic derivatives of eugenol against dermatophytes of the genus Trichophyton. METHODOLOGY: We evaluated the antifungal effect of the compounds, determining the minimum inhibitory concentrations (MICs) by the microdilution method and the minimum fungicidal concentrations by cultures from the inhibitions. Additionally, the inhibition of the radial growth of the mycelium of the dermatophyte fungi was tested by poisoned substrate. Cytotoxicity was measured by the colorimetric method on Vero cells. RESULTS: All of the eugenol compounds tested exhibited antifungal properties, showing MICs of 62.5-500 µg ml-1 , determined within three dermatophyte species: Trichophyton rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans. Among these derivatives, methyl isoeugenol, at concentrations of 300 and 100 µg ml-1, was found to completely inhibit (100 %) radial growth of the mycelium of all three species after 20 days of treatment. Additionally, phenotypic variations related to the decrease in pigment production of T. rubrum were observed after treatment with O-ethyl and O-butyl isoeugenol derivatives. Meanwhile, all of the tested (iso)eugenol molecules exhibited moderate toxicity in Vero cells [50 % cytotoxic concentration (the concentration required for a 50 % reduction in cell viability; CC50): 54.06-265.18 µg ml-1 ). CONCLUSION: The results suggest that the semisynthetic eugenol derivatives (SEDs) show promising antifungal activity and selectivity against dermatophyte fungi.

Antifungal Activity of Eugenol Derivatives against Botrytis Cinerea.

Botrytis cinerea is a worldwide spread fungus that causes the grey mold disease, which is considered the most important factor in postharvest losses in fresh fruit crops. Consequently, the control of gray mold is a matter of current and relevant interest for agricultural industries. In this work, a series of phenylpropanoids derived from eugenol were synthesized and characterized. Their effects on the mycelial growth of a virulent and multi-resistant isolate of B. cinerea (PN2) have been evaluated and IC50 values for the most active compounds range between 31⁻95 ppm. The antifungal activity exhibited by these compounds is strongly related to their chemical structure, i.e., increasing activity has been obtained by isomerization of the double bond or introduction of a nitro group on the aromatic ring. Based on the relationship between the fungicide activities and chemical structure, a mechanism of action is proposed. Finally, the activity of these compounds is higher than that reported for the commercial fungicide BC-1000 that is currently employed to combat this disease. Thus, our results suggest that these compounds are potential candidates to be used in the design of new and effective control with inspired natural compounds of this pathogen.

Myroxylon pereirae resin (balsam of Peru) - A critical review of the literature and assessment of the significance of positive patch test reactions and the usefulness of restrictive diets.

In this article, contact allergy to Myroxylon pereirae resin (MP) (balsam of Peru) is reviewed. The topics presented include the uses, the chemical composition, the frequency of sensitization, the relevance of positive reactions, the MP-containing products causing allergic contact dermatitis, co-reactivity with other fragrance and non-fragrance materials, the sensitizers, the usefulness of MP as a "marker" of fragrance allergy, and the effectiveness of, and indications for, "balsam-restrictive" diets. Sensitization to MP occurs in 4% to 8% of patients routinely tested for suspected contact dermatitis. There are few adequate data on relevance. Topical pharmaceuticals were formerly, but are not today, important sources of sensitization. Cosmetics and foods or drinks are hardly ever products responsible for sensitization to MP. Positive patch test reactions in the large majority probably result from previous sensitization to MP constituents because of their presence in fragrances and fragranced products, MP thereby acting as marker (or "indicator") of fragrance allergy. However, fragrance mix I is a more sensitive marker, and the added diagnostic value of testing with MP is unknown. The allergenic ingredients of MP include isoeugenol, eugenol and cinnamyl alcohol, but there are other-largely unknown-chemicals that are responsible for contact allergy. Suggestions are given for further research to address questions thus far unanswered and to improve patient care.