Pier Carlo Braga

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.

Effect of aspirin on serotonin and met-enkephalin in brain: correlation with the antinociceptive activity of the drug

Intravenous administration of acetyl salicylate of lysine, a soluble salt of aspirin, reduced in rats the firing discharge of thalamic neurones, evoked by noxious stimuli. Concomitantly, concentrations of 5-hydroxyindole acetic acid increased, while those of met-enkephalin-like immuno-reactive derivatives were decreased in several areas of the brain. Similar electrophysiological and biochemical responses were obtained by administering tryptophan or 5-hydroxytryptophan plus carbidopa. The effect of aspirin on the evoked firing of the thalamic neurones was counteracted by pretreating the animals with metergoline. On the other hand, naloxone did not antagonize the inhibitory effect of aspirin and 5-hydroxytryptophan on pain-induced neuronal excitation. These data indicate that a serotonin-, but not a naloxone-sensitive opiate mechanism, may be relevant for aspirin-mediated antinociception.

Calcitonin and its antinociceptive activity: Animal and human investigations 1975–1992

Calcitonin (CT) is a polypeptide hormone produced in the thyroid gland that regulates, blood calcium levels and bone calcium metabolism.The unexpected finding of binding sites for calcitonin in several areas of the brain oriented attention to activities of CT in the central nervous system and also to its antinociceptive action.The first report of this last effect was in 1975, and the many different experimental and clinical data on this topic reported since then are reviewed here.The heterogenous findings have been organized according to the logical classification of animal and human studies. For each of these headings, subheadings such as acute and chronic pain, different kinds of administration and different procedures used to record the results, are considered.The several proposed mechanisms of action, involving serotoninergic, catecholaminergic, Ca2+ fluxes, protein phosphorylation, β-endorphin production, cyclooxygenase inhibition and histamine interference are also reviewed.Calcitonin, neurotensin, substance P, VIP and, recently, CGRP are some of the non-opioid peptides that have been reported to interfere with pain and that open up a new, alternative way of investigating antinociceptive drugs different than opioid or opioid-like agents.An examination of the state-of-investigation of calcitonins antinociceptive activity in the last 17 years shows that many experimental studies indicate the existence of this effect, including studies in humans, and this opens up perspectives for therapy with a new class of antinociceptive agents.

Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.

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.

Influence of Age on Oxidative Bursts (Chemiluminescence) of Polymorphonuclear Neutrophil Leukocytes

The release of reactive oxygen species (ROS) during neutrophil oxidative bursts is the last of a sequence of different steps leading to the neutralization of pathogen microorganisms. Using luminol-amplified chemiluminescence (LACL), the oxidative burst activity of neutrophils in elderly people (≥75 years) was compared with that in younger controls (39 years on average) after activation with both particulate (Candida albicans) and soluble (formyl-methionyl-leucyl-phenylalanine; fMLP) stimulants. After Candida stimulation, a reduction in LACL was observed in the elderly subjects in comparison with the controls, but the difference did not reach statistical significance. After fMLP stimulation, the reduction in LACL was significant, thus suggesting that the Candida pathway of chemiluminescence production seems to be less affected than the fMLP pathway. This finding raises questions concerning the complex differences in the pathways of cell killing and ROS generation, and their efficacy in the elderly. Various possible explanations are discussed, all of which need further investigation.

Human pharmacokinetics and distribution in various tissues of ceftriaxone

Multiple-dose pharmacokinetics of ceftriaxone were investigated in 7 patients with bronchopneumonia using an intramuscular regimen of 1 g given every 24 h for 7 days. Serum, sputum, and urine samples were collected serially following the first dose (day 1) and last dose (day 7). Mean peak serum concentrations of ceftriaxone occurred at 2 h on both days and were 67.8 and 75.1 micrograms/ml, respectively, on day 1 and day 7. Ceftriaxone had a half-life of 6.9 h on day 1 and 7.4 h on day 7. The half-life of ceftriaxone in sputum was 5.9 and 6.6 h, respectively, on days 1 and 7. Approximately 50% of the dose of ceftriaxone was recovered in the urine within 24 h on day 1, 60% on day 7. Tissue distribution of ceftriaxone was determined in 103 patients following intramuscular administration of a single 1-gram dose at different times up to 24 h prior to surgery. High concentrations of ceftriaxone were found in lung, tonsil, middle ear mucosa, and nasal mucosa, and therapeutic levels of ceftriaxone persisted for 24 h after administration.

Evaluation of the Immunostimulating Activity of Erythromycin in Man

The effects of erythromycin on the immune system have been studied in healthy volunteers and patients suffering from chronic bronchopneumonial diseases, by means of the following assays: phagocytosis, natural killer activity and superoxide anion production. The tests were performed before and after oral administration of 1 g of erythromycin. The findings suggest that erythromycin enhances phagocytosis by means of increasing ingestion of microorganisms, superoxide anion (O2-) production as well as natural killer activity. Under the experimental conditions described these effects appear 4-6 h after drug intake and reach their maximum around the 8th hour.