Monica Dal Sasso

Anti-Inflammatory Activity of Thymol: Inhibitory Effect on the Release of Human Neutrophil Elastase

Elastase, a serine proteinase released by activated human neutrophils, can degrade a wide variety of biomacromolecules including elastin, and is considered a marker of inflammatory diseases. As the logical strategy to protect tissue is to inhibit excessive elastase activity, experimental and clinical researches have concentrated on trying to find efficient elastase inhibitors. As thymol, one of the major components of thyme oil with a phenolic structure, has been credited with a series of pharmacological properties, that include antimicrobial and antioxidant effects, the aim of this study was to explore whether it can also interfere with the release of elastase by human neutrophils stimulated with the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). After the neutrophils were incubated with increasing amounts of thymol (2.5, 5, 10, 20 µg/ml), elastase release was initiated by fMLP and measured using MeO-Suc-Ala-Ala-Pro-Val-MCA. The results showed that thymol inhibited fMLP-induced elastase release in a concentration-dependent manner, with the effects of 10 and 20 µg/ml being statistically significant. The behavior of cytosolic calcium mobilization revealed by fura-2 closely resembled that of elastase, thus suggesting that they may be related. The hydrophobic nature of thymol means that it can approach ion channel proteins through the lipid phase of the membrane, alter the local environment of calcium channels and thus inhibit capacitative calcium entry. In brief, thymol inactivates calcium channels machinery, thus triggering a corresponding reduction in elastase. The antibacterial and antimycotic activity of thymol is already well known, but our findings that it inhibits elastase extend our knowledge of the anti-inflammatory activity of this interesting molecule that is already credited with antioxidant activity. These two latter characteristics make thymol a molecule that can have helpful effects in controlling the inflammatory processes present in many infections.

Thymol inhibits Candida albicans biofilm formation and mature biofilm

Candida albicans has a high propensity to develop biofilms that are resistant to traditional antifungal agents. Thymol is credited with a series of pharmacological properties including antimicrobial and antifungal effects. As C. albicans biofilms are known to be important factors underlying its virulence and pathogenicity, the aim of this study was to investigate whether thymol can interfere with biofilm formation as well as acting on mature biofilms. Tests of C. albicans strains ATCC 3153A and ATCC MYA 2876 showed that thymol interferes with the starting phases of biofilm production as well as with mature C. albicans biofilms. The metabolic activity of sessile cells was reduced by >90% at twice the minimum inhibitory concentration of planktonic cells. As biofilm is a multifactorial phenomenon, the multiple mechanisms of thymol (terpenes) could act on different steps in the evolution of mature biofilm.

Antioxidant Potential of Thymol Determined by Chemiluminescence Inhibition in Human Neutrophils and Cell-Free Systems

Thyme essential oil and thymol have antimicrobial, antifungal and antioxidant activities. Their antioxidant activity has been studied almost exclusively by means of chemical testing in order to be able to use it for food preservation purposes. The aim of this luminol amplified chemiluminescence (LACL) study was to investigate whether thymol can interfere with the production of reactive oxygen species, nitric oxide and the nitric oxide-derived peroxynitrite released by human neutrophils after activation by fMLP and PMA with and without the addition of the L-arginine (L-Arg) nitric oxide donor to the medium. The lowest thymol concentration that was still active in reducing LACL was 2.73 µg/ml, and there was a progressive linear inhibition of LACL from this concentration to 21.87 µg/ml, the highest thymol concentration investigated. This was also observed in the case of both fMLP and PMA stimulation with or without L-Arg. In cell-free systems using H2O2/HOCl– and SIN-1 as radical producers, a significant scavenging activity of thymol was present already at 0.08 and 0.68 µg/ml respectively, and these are very low concentrations. These findings can be related to the phenolic structure of thymol, because phenolic compounds have redox properties and play an important role in adsorbing and neutralizing free radicals and peroxynitrite, and in decomposing peroxides. Our findings in human neutrophils are pharmacologically relevant as they imply that thymol is a potential antioxidant and anti-inflammatory agent in human cells.

Thermal Treatment of Eggplant (Solanum melongena L.) Increases the Antioxidant Content and the Inhibitory Effect on Human Neutrophil Burst

The aim of this study was to compare the amount and activity of phytonutrients in raw, grilled, and boiled eggplant fruit using chemical measures and a biological assay of oxidative bursts in human neutrophils. The thermally treated samples showed various changes in their chemical composition (dry matter, soluble solids, acidity, and the amount of alcohol insoluble substances) due to the cooking processes and were much richer in the main phenolic compounds such as chlorogenic and caffeic acids, which are known to be antioxidants. Consequently, their free radical scavenging activity was significantly higher, especially that of superoxide anion. The biological assay of oxidative bursts from human neutrophils in the presence of N-formyl-methionyl-leucyl-phenylalanine confirmed the greater activity of extracts of the cooked eggplants with respect to raw eggplants. Successive extract dilutions showed a significant activity up to 1.25 microg/mL after cooking, while raw fruits resulted in an activity up to 10.00 microg/mL. These results showed that the thermal treatment commonly used before consumption can increase the content and biological activity of antioxidant compounds of eggplants.

Antioxidant Effect of Sulphurous Thermal Water on Human Neutrophil Bursts: Chemiluminescence Evaluation

Background: The activities of the HS (sulfhydryl or thiolic) group in the cysteine of glutathione or various low-weight soluble molecules (thiolic drugs), such as N-acethylcysteine, mesna, thiopronine and dithiotreitol or stepronine and erdosteine (prodrugs), include its antioxidant activity in the airways during the release of reactive oxygen or nitrogen species (ROS, RNS) by polymorphonuclear neutrophils (PMNs) activated in response to exogenous or endogenous stimuli. Objective: In addition to being administered by means of thiolic molecules, the HS group can also be given by means of the inhalation of sulphurous thermal water. The aim of this study was to investigate the effect of sulphurous thermal water on the release of ROS and RNS during the bursts of human PMNs. Methods: The luminol-amplified chemiluminescence methodology was used to investigate the ROS and RNS released by PMNs stimulated with N-formyl-methionyl-leucyl-phenylalanine and phorbol-12-myristate-13-acetate, before and after incubation with sulphurous water. Effects on cell-free systems were also investigated. Results: The water significantly reduced the luminol-amplified chemiluminescence of N-formyl-methionyl-leucyl-phenylalanine- andphorbol-12-myristate-13-acetate-activated PMNs on average from 0.94 to 15.5 µg/ml of HS, even after the addition of L-arginine, a nitric oxide (NO) donor. Similar findings have also been obtained in a cell-free system, thus confirming the importance of the presence of the HS group (reductive activity). Conclusions: The positive effects of the activity of sulphurous thermal waters has been partially based on the patients’ subjective sense of wellbeing and partially on not always easy to quantify symptomatic (or general) clinical improvements. Our findings indicate that, in addition to their known mucolytic activity and trophic effects on respiratory mucosa, the HS groups present in the sulphurous thermal water of this spring also have antioxidant activity that contributes to the therapeutic effects of the water in upper and lower airway inflammatory diseases.

Inhibitory activity of thymol on native and mature Gardnerella vaginalis biofilms: in vitro study

Bacterial vaginosis (BV) is the most frequent diagnosis made in women with lower genital tract symptoms. It has recently been observed that 90 % of subjects with BV show the growth of bacteria in the form of biofilms as against only 10% without BV, and that Gardnerella vaginalis was the predominant species. The propensity of G. vaginalis to form biofilm is clinically relevant because this form of growth allows it to tolerate higher concentrations of certain antibiotics, thus increasing the possibilty of recurrent BV even after apparently curative therapy. The aim of this study was to investigate whether thymol (CAS 89-83-8), a molecule present in thyme essential oil, that is credited with having a series of pharmacological properties including antimicrobial and antifungal effects, can interfere with newly formed and mature G. vaginalis biofilms. The ability of G. vaginalis ATCC 49145 and two G. vaginalis strains isolated from human BV to form biofilm in flat-bottomed 96-well microtitre plates was verified, and the effects of thymol concentrations ranging from 1 to 1/16 MIC (minimum inhibitory concentration) on preformed and mature biofilms was investigated by means of spectrophotometric analysis, Nomarski interference contrast microscopy, and fluorescence microscopy with live-dead cell visualisation (SYTO 9 and propidium iodide). Native biofilm was inhibited by concentrations ranging from 1 MIC to 1/8 MIC (32.77% +/- 2.37 to 11.39% +/- 1.46), and mature biofilm was inhibited by concentrations ranging from 1 MIC to 1/4 MIC (26.18% +/- 1.36 to 13.20% +/- 1.44). Nomarski interference contrast and fluorescence microscopy visually confirmed these findings. As biofilm is a multi-factorial phenomenon, the multiple mechanisms of thymol may act on different steps in the evolution of mature biofilm.

Thymol: Inhibitory Activity on Escherichia coli and Staphylococcus aureus Adhesion to Human Vaginal Cells

Abstract Adhesion is an important starting event in the pathogenesis of bacterial infection because the microorganisms must first adhere to host tissue in order to multiply and create a colony or colonies before specific symptoms allow the disease process to be detected. This is particularly true in the case of female urogenital infections, including urinary tract infections, bacterial vaginosis and vaginitis. Thymol is a component of thyme essential oil, which has been reported to possess interesting antimicrobial effects on various microorganisms; however, its ability to interact with the adhesion of bacteria (an important determinant of bacterial virulence) has not been investigated. The aim of this study was to assess whether thymol interferes with the adhesion of Escherichia coli and Staphylococcus aureus to human vaginal epithelial cells. The adhesiveness of three strains of E. coli to vaginal cells was significantly reduced at thymol concentrations ranging from 1/2 MIC to 1/32 MIC, and in three strains of S. aureus at concentrations ranging from 1/2 MIC to 1/16 MIC. Sub-MICs down to 1/8 MIC also significantly reduced the hemagglutination of E. coli, which is correlated with fimbriation and thus provides a clue relating to the interference of thymol, a phenolic structure with an hydroxyl group, on the physicochemical characteristics of the outer surface of bacteria. This is of interest for the strategy of protecting against vaginitis or vaginosis using drugs other than antibiotics.

A combination of budesonide and the SH-metabolite I of erdosteine acts synergistically in reducing chemiluminescence during human neutrophil respiratory burst

Activated neutrophils can release superoxide anion and nitric oxide (NO), which subsequently combine with each other to yield peroxynitrite anions, powerful and harmful oxidants that preferentially mediate the oxidation of the thiol groups in proteins and non-protein molecules. These oxidants play a direct role in the inflammatory process in chronic obstructive pulmonary disease and asthma by increasing the number of neutrophils and macrophages that induce a self-sustaining phlogogenic loop. Budesonide (BUD) and erdosteine (a muco-active drug which, after metabolization, produces an active metabolite (Met I) with a sulfhydryl group) are both active in reducing the release of superoxide anion, NO and peroxynitrite, and can be administered to patients with respiratory diseases. The aim of this study was to investigate the possible synergistic in vitro effect of BUD and Met I on chemiluminescence generation during fMLP-stimulated respiratory bursts of human neutrophils with the NO donor L-arginine, added to the incubating medium. The investigated BUD concentrations ranged from 6 × 10–8 to 1 × 10–6 mol/l in logarithmic scale and a significant and progressive reduction in luminol-amplified chemiluminescence (LACL) was observed at concentrations ranging from 2.5 × 10–7 to 1 × 10–6 mol/l. The investigated concentrations of Met I varied from 0.62 to 10 µg/ml. No significant changes were observed at 0.62, 1.25, and 2.5 µg/ml, but a significant decrease in LACL was observed at 5 and 10 µg/ml. When the two drugs were combined, there was a greater significant decrease in LACL versus the single drugs with the combinations of BUD 1 × 10–6 mol/l plus Met I 10 µg/ml, BUD 5 × 10–7 mol/l plus Met I 5 µg/ml, BUD 2.5 × 10–7 mol/l plus Met I 2.5 µg/ml, and BUD 1.25 × 10–7 mol/l plus Met I 1.25 µg/ml. A further interesting finding was that the combination of BUD 2.5 × 10–7 mol/l plus Met I 2.5 µg/ml and BUD 1.25 × 10–7 mol/l plus Met I 1.25 µg/ml significantly decreased LACL, whereas the single concentrations had no significant effect, thus indicating the possibility of extending the duration of the effect. Our findings indicate a synergistic antioxidant effect when BUD and Met I are given together, which is of interest for counteracting the airway phlogosis involved in many respiratory diseases.

Effects of sulphurous water on human neutrophil elastase release

Background: Molecules bearing a sulphide (HS) group, such as glutathione, play a fundamental role in the defensive system of human airways, as shown by the fact that the lining fluid covering the epithelia of the respiratory tract contains very high concentrations of glutathione: the lungs and nose, respectively, contain about 140 and 40 times the concentrations found in plasma. Consequently, various low-weight soluble molecules bearing an HS group (including N-acetylcysteine, mesna and thiopronine, and prodrugs such as stepronine and erdosteine) have been used for therapeutic purposes. HS groups can also be therapeutically administered by means of sulphurous thermal water containing HS groups. The aim of this study was to investigate the direct activity of such water on the release of elastase by activated human neutrophils. Method: After the neutrophils were incubated with increasing amounts of sulphurous water or the HS/hydrogen sulphide donor sodium hydrosulphide (NaHS), elastase release was initiated by N-formyl-methionyl-leucyl-phenylalanine and measured by means of spectrofluorimetry using methylsuccinylalanylprolylvalyl-methylcoumarin amide as the fluorogenic substrate. To verify the presence of direct action on elastase we determined the diameter of the area of elastinolysis on elastine-agarose gel plates. Results: The sulphurous water significantly inhibited elastase release at HS concentrations ranging from 4.5 to 18 μg/ml, as assayed using the iodometric method; in the case of NaHS, the inhibition was significant at HS concentrations ranging from 2.2 to 18 μg/ml. The concentration-effect regression lines of both were parallel and neither showed any direct elastolytic activity. Conclusions: Previous claims concerning the activity of sulphurous water have been based on the patients’ subjective sense of wellbeing and on symptomatic (or general) clinical improvements that are not easy to define or quantify exactly. Our findings indicate that, in addition to its known mucolytic and antioxidant activity, sulphurous water also has an anti-elastase activity that may help to control the inflammatory processes of upper and lower airway diseases.

Budesonide reduces superoxide and peroxynitrite anion chemiluminescence during human neutrophil bursts

Many lung disorders are characterized by airway inflammation involving the recruitment of inflammatory cells, and leading to the release of oxidant and inflammatory mediators. The overproduction of superoxide anion (O2–DELETE) and nitric oxide (NO) during the respiratory bursts of neutrophils leads to production of peroxynitrite, a highly damaging oxidant with an important role in the inflammatory loop causing airway hyper-reactivity in respiratory diseases like asthma. The aim of this study was to investigate in vitro the effects of a 1-hour incubation with budesonide at 2.5 × 10–7, 5 × 10–7, 1 × 10–6, 2 × 10–6 and 4 × 10–6 mol/l on O2–DELETE, NO, and peroxynitrite production during the respiratory burst of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10–7 mol/l) or phorbol 12-myristate 13-acetate (PMA, 2 × 10–6 mol/l), as documented by luminol-amplified chemiluminescence (LACL). In absence of L-arginine, budesonide (5 × 10–7 to 4 × 10–6 mol/l) dose-dependently reduced both fMLP- and PMA-induced LACL (18.3–50.6%). In the presence of L-arginine (100 µg/ml), a NO donor increasing peroxynitrite production, LACL increased 3–5 times compared with baseline, but budesonide dose-dependently reduced LACL (25.5–59.6%). Mifepristone (4 × 10–6 mol/l), a glucocorticoid receptor antagonist, inhibited the effect of budesonide on LACL, thus confirming that budesonide reacts with glucocorticoid receptors to exert an antioxidant activity. These results suggest that budesonide target rapidly human neutrophils leading to a fast reduction in both NO and peroxynitrite production, and are consistent with decrease in exhaled NO levels after treatment with budesonide in patients with asthma.