Inclusion of Ethyl Acetoacetate Bearing 7-Hydroxycoumarin Dye by ß-Cyclodextrin and its Cooperative Assembly with Mercury(II) Ions: Spectroscopic and Molecular Modeling Studies.

The inclusion of the fluorescent organic dye, ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-oxopropanoate (1) by the host ß-cyclodextrin (ß-CD), and its response toward mercuric ions (Hg2+ ), was studied by UV/Vis, fluorescence, and 1 H NMR spectroscopic analyses, mass spectrometry and molecular modeling studies. 1 H NMR measurements together with molecular modeling studies for dye 1 demonstrate that it exhibits two tautomeric forms (keto and enol); however, when the dye is included into the ß-CD cavity, the enol form predominates. Moreover, by using spectroscopic and spectrometry techniques, a 1:1 stoichiometry was determined for the complexes formed between dye 1 (enol form) and ß-CD, with a binding constant (Kb1 =1.8×104 m-1 ) and for the dye 1 (keto form)-Hg2+ (Kb2 =2.3×103 m-1 ). Interestingly, in the presence of 1-ß-CD complex and mercuric ions, a ternary supramolecular system (Hg-1-ß-CD complex) was established, with a 1:1:1 stoichiometry and a Kb3 value of 4.3×103 m-1 , with the keto form of the dye being the only one present in this assembly. The three-component system provides a starting point for the development of novel and directed supramolecular assemblies.

Absence of genotoxic effects of the coumarin derivative 4-methylesculetin in vivo and its potential chemoprevention against doxorubicin-induced DNA damage.

4-Methylesculetin (4-ME) is a synthetic derivative of coumarin that displays a potent reactive oxygen species (ROS) scavenger and metal chelating agent and therefore has been produced to help reduce the risk of human disease. The main objective of this study was to investigate the in vivo genotoxicity of 4-ME and initially to verify its potential antigenotoxicity on doxorubicin (DXR)-induced DNA damage. Different doses of 4-ME (500, 1000 and 2000 mg kg(-1) body weight) were administered by gavage only or with a simultaneous intraperitoneal (i.p.) injection of DXR (80 mg kg(-1)). The following endpoints were analyzed: DNA damage in peripheral blood, liver, bone marrow, brain and testicle cells according to an alkaline (pH > 13) comet assay and micronucleus induction in bone marrow cells. Cytotoxicity was assessed by scoring polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). No differences were observed between the negative control and the groups treated with a 4-ME dose for any of the endpoints analyzed, indicating that it lacks genotoxic and cytotoxic effects. Moreover, 4-ME demonstrated protective effects against DXR-induced DNA damage at all tested doses and in all analyzed cell types, which ranged from 34.1% to 93.3% in the comet assay and 54.4% to 65.9% in the micronucleus test.

Evaluation of the antioxidant capacity of synthesized coumarins.

Coumarins are secondary metabolites that are widely distributed within the plant kingdom, some of which have been extensively studied for their antioxidant properties. The antioxidant activity of coumarins assayed in the present study was measured by different methods, namely the 1,1-diphenyl-2-picryl-hydrazyl (DPPH(•)) method, cyclic voltammetry and the antioxidant capacity against peroxyl radicals (ACAP) method. The 7,8-dihydroxy-4-methylcoumarin (LaSOM 78), 5-carboxy-7,8-dihydroxy-4-methylcoumarin (LaSOM 79), and 6,7-dihydroxycoumarin (Esculetin) compounds proved to be the most active, showing the highest capacity to deplete the DPPH radicals, the highest antioxidant capacity against peroxyl radicals, and the lowest values of potential oxidation.

Suppression of TNBS-induced colitis in rats by 4-methylesculetin, a natural coumarin: comparison with prednisolone and sulphasalazine.

The aim of the present study was to compare the effects of the 4-methylesculetin with those produced by prednisolone and sulphasalazine and to elucidate the mechanisms involved in its action. Colitis was induced in rat by instillation of trinitrobenzenesulphonic acid (TNBS). The colon damage was evaluated using macroscopic, microscopic and biochemical analysis. In addition, in vitro studies were performed to evaluate cytokine production in cell cultures using the murine macrophage cell line RAW264.7, mouse splenocytes and the human colonic epithelial cell line Caco-2. 4-Methylesculetin produced a reduction of the macroscopic damage score and the recovery of the intestinal cytoarchitecture. These effects were associated with a prevention of the GSH depletion and an inhibition in AP activity. After colitis relapse, 4-methylesculetin improved the colonic inflammatory status as evidenced by histological findings, with a reduction in apoptosis, as well as biochemically by inhibition of colonic myeloperoxidase, alkaline phosphatase and metalloproteinase 9 activities. Paired with this inhibitive activity, there was a decrease in malondialdehyde content and in IL-1ß levels. In vitro assays revealed that 4-methylesculetin promoted an inhibition in IL-1ß, IL-8, IL-2 and IFN-γ production in cell cultures. In conclusion, 4-methylesculetin showed similar efficacy to that obtained with either prednisolone or sulphasalazine, both in the acute phase of colitis as well as following a curative protocol. The intestinal anti-inflammatory activity by 4-methylesculetin is likely related to its ability in reduce colonic oxidative stress and inhibit pro-inflammatory cytokine production.

In vitro antifungal activity of coumarin extracted from Loeselia mexicana Brand.

The bis-coumarin daphnoretin and its monomeric precursors scopoletin and umbelliferone were isolated for the first time from the aerial part of Loeselia mexicana Brand (a vegetal species used in Mexican traditional medicine) using chromatographic techniques. The structures of these compounds were determined by (1) H and (13) C NMR analyses. These coumarins were evaluated for in vitro antifungal activity. The three compounds tested showed significant antifungal activity.

Mechanisms involved in the antinociceptive effects of 7-hydroxycoumarin.

7-Hydroxycoumarin (umbelliferone, 1), the main metabolite of coumarin, has been reported to produce potent antinociceptive effects in animal models of pain. However, the biochemical events involved in antinociception mediated by 1 are currently not well understood. In the present study, the mechanisms by which 1 exerts its pharmacological actions were investigated. Acute pretreatment of mice with 1 produced a long-lasting antinociceptive effect against complete Freund's adjuvant (CFA)-induced hyperalgesia. The subchronic administration of 1 inhibited CFA-induced hyperalgesia and paw edema, while it did not cause any apparent toxicity. Another set of experiments showed that 1 inhibited carrageenan-induced mechanical hyperalgesia, but not mechanical hyperalgesia induced by prostaglandin E(2) (PGE(2)), suggesting that it acts upstream of PGE(2.) Treatment with 1 was able to prevent the plantar tissue neutrophil influx induced by local inflammatory stimuli. In addition, 1 exhibited inhibitory effects on the release of hyperalgesic cytokines (TNF-α and IL-1ß) and the production of PGE(2), a directly acting hyperalgesic mediator. The present results suggest that the antinociceptive effect of 1 is correlated with the inhibition of neutrophil migration, cytokine release, and PGE(2) production and are supportive of the further investigation of the therapeutic potential of 1 to control inflammatory pain.

Determination of umckalin in commercial tincture and phytopreparations containing Pelargonium sidoides by HPLC: comparison of sample preparation procedures.

Roots of Pelargonium sidoides D.C. are used for the production of phytomedicines. Current quality control of phytopreparations containing P. sidoides extracts has been made in terms of total phenolics content. In this work we describe the development and validation of an HPLC method for the analysis of P. sidoides tincture and commercial syrup phytopreparations using umckalin (7-hydroxy-5,6-dimethoxycoumarin) as chemical marker. Two sample preparation procedures, liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were also developed and compared. The samples were analyzed by RP-HPLC and the two methods were then validated and compared. The repeatability of the two procedures showed coefficients of variation (CV) of 1.2% for SPE procedure, and 1.3% for LLE. Recovery for both methods was higher than 95.2%. The linearity showed correlation coefficients better than 0.999 for both methods. The detection and quantification limit were 0.0098 and 0.0298microgmL(-1), respectively. The validated procedure was then used for the analysis of tincture and five batches of two commercial phytopreparations containing P. sidoides tincture.

Intestinal anti-inflammatory activity of esculetin and 4-methylesculetin in the trinitrobenzenesulphonic acid model of rat colitis.

Coumarins comprise a broad class of phenolic compounds that influences the formation and scavenging of reactive oxygen species and the processes involving free radical-mediated injury. In light of the antioxidant and anti-inflammatory properties of esculetin and 4-methylesculetin, the aim of this study was to investigate the effects of these compounds in an experimental model of rat colitis induced by trinitrobenzenesulphonic acid (TNBS). For this purpose, macroscopic (diarrhoea, extension of lesion, colonic weight/length ratio and damage score) and biochemical parameters (myeloperoxidase, alkaline phosphatase and glutathione) were evaluated. Our results reveal that these compounds, particularly 4-methylesculetin, may be effective for the treatment of intestinal inflammatory bowel disease. In the acute colitis model, esculetin promoted a reduction in the extension of the lesion accompanied by a reduction in the incidence of diarrhoea and restoration of the glutathione content. Similar effects were produced by the administration of 4-methylesculetin, which also inhibited the myeloperoxidase and alkaline phosphatase activities in the acute intestinal inflammatory process and in the model of colitis relapse. The effect of the esculetin and 4-methylesculetin on the inflammatory process may be related to their antioxidant and anti-inflammatory properties, as observed in this study. The evidence for better effects of 4-methylesculetin in comparison to those demonstrated by esculetin in both experimental settings could be attributed to the presence of the methyl group at C-4 of 4-methylesculetin.

Effects of umbelliferone in a murine model of allergic airway inflammation.

The therapeutic effects of umbelliferone (30, 60 and 90 mg/kg), a coumarin isolated from Typha domingensis (Typhaceae) were investigated in a mouse model of bronchial asthma. BALB/c mice were immunized and challenged by nasal administration of ovalbumin. Treatment with umbelliferone (60 and 90 mg/kg) caused a marked reduction of cellularity and eosinophil numbers in bronchoalveolar lavage fluids from asthmatic mice. In addition, a decrease in mucus production and lung inflammation were observed in mice treated with umbelliferone. A reduction of IL-4, IL-5, and IL-13, but not of IFN-gamma, was found in bronchoalveolar lavage fluids of mice treated with umbelliferone, similar to that observed with dexamethasone. The levels of ovalbumin-specific IgE were not significantly altered after treatment with umbelliferone. In conclusion, our results demonstrate that umbelliferone attenuates the alteration characteristics of allergic airway inflammation. The investigation of the mechanisms of action of this molecule may contribute for the development of new drugs for the treatment of asthma.

A rapid TLC autographic method for the detection of glucosidase inhibitors.

A new bioautographic assay suitable for the localisation of beta-glucosidase inhibitors present in a complex matrix is described. Enzyme activity was detected using esculin as the substrate to produce esculetin, which reacts with ferric ion to form a brown complex.