244 Original Article
Diclofenac-Choline Antioxidant Activity Investigated by means of Luminol Amplified Chemiluminescence of Human Neutrophil Bursts and Electron Paramagnetic Resonance Spectroscopy Authors
P. C. Braga1, N. Lattuada1, V. Greco1, V. Sibilia1, M. Falchi1, T. Bianchi2, M. Dal Sasso1
Affiliations
1
2
Key words ▶ diclofenac-choline ● ▶ electron paramagnetic ● resonance (EPR) ▶ human neutrophils ● ▶ chemiluminescence ● ▶ antioxidant activity ●
Abstract
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1377002 Published online: June 11, 2014 Drug Res 2015; 65: 244–251 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Prof. P. C. Braga Department of Medical Biotechnology and Translational Medicine School of Medicine Via Vanvitelli 32 20129 Milano Italy Tel.: + 39/02/50316 990 [email protected]
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A new diclofenac salt called diclofenac-choline (DC) has recently been proposed for the symptomatic treatment of oropharyngeal inflammatory processes and pain because its greater water solubility allows the use of high concentrations, which are useful when the contact time between the drug and the oropharyngeal mucosa is brief, as in the case of mouthwashes or spray formulations. The antioxidant activity of DC has not yet been investigated, and so the aim was to use luminol-amplified-chemiluminescence (LACL) to verify whether various concentrations of DC (1.48, 0.74 and 0.37 mg/mL for incubation times of 2, 4 and 8 min) interfere with oxygen and nitrogen radicals during the course of human neutrophils respiratory bursts; electron paramagnetic resonance (EPR) spectroscopy was
Introduction
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The inflammatory process generally has a localised protective function and is induced by a variety of stimuli, including micro-organisms, free radicals or oxidants, injured tissue, airborne pollutants, and allergens. The mechanism of inflammation is very complex and involves many different types of molecules and metabolic pathways [1, 2]. Inflammatory processes cause various diseases depending on the inflamed tissue or organ involved, but they all share the activation of a “stress signalling pathway” and the concomitant production of inflammatory cytokines [3, 4]. The chemotactic signals generated at the site of inflammation lead to the rapid and often massive recruitment and activation of polymorphonuclear neutrophils (PMNs), which are themselves major cell mediators of inflammation as they contribute to generating a “phlogogenic loop” as a result of their ability to generate inflammation
Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
used to investigate its direct antiradical (scavenger) activity. The EPR findings showed that DC has concentration-dependent scavenging activity against the ABTS, the DPPH, and the hydroxyl radicals, but no activity on superoxide anion, as has been previously reported in the case of other NSAIDs. LACL revealed an inhibitory effect that was statistically significant after only 2 min of incubation, and similar after 4 and 8 min. The effects on the peroxynitrite radical paralleled those observed in the previous test. High concentrations and short incubation times showed that there is no interference on PMN viability, and so the inhibitory findings must be attributed to the effect of the drug. The anti-inflammatory effects of DC cannot be attributed solely to the inhibition of prostaglandin synthesis, but its effects on free radicals and neutrophil bursts suggest that they may contribute to its final therapeutic effect.
by releasing reactive oxygen and nitrogen species (ROS/RNS), lysosomal enzymes, histaminereleasing factor, chemoattractant such as IL-8, and cytokines such as IL-1βb and TNFα [5, 6]. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of various acute and chronic pain and inflammatory conditions because of their anti-inflammatory, analgesic and antipyretic properties. Inflammatory processes in the oropharyngeal cavity can be sustained by factors such as acute pharyngitis and laryngitis, gingivitis, dental surgery, etc., and these are also frequently treated with NSAIDs [7–9]. The most frequently prescribed NSAID for the treatment of inflammation-related conditions is diclofenac, a phenylacetic acid derivative (2-[2, 6]–dichloranilinophenylaceticacid]) [10, 11], which is available in oral, intravenous, suppository and topical (transdermal patch or gel) formulations. However, the most frequently used medications for the symptomatic treatment
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received 11.11.2013 accepted 03.05.2014
Department of Medical Biotechnology and Translational Medicine, School of Medicine, University of Milan, Milan, Italy AVIS Comunale di Milano, Ospedale Niguarda, Milano, Italy
Original Article
Materials and Methods
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Fenton reaction model with the EPR detection of HO•
The first series of tests used the spin trapping method [26], which is based on the rapid reaction of many radicals with certain chemical acceptor molecules (spin trapping agents) to produce more stable secondary radicals. The diamagnetic spin trap nitrone DMPO (5,5-dimethyl-1-pyrrolidine-N-oxide) (Sigma Chemicals Co., St Louis, MO, USA) was added to the reaction mixture to produce the relatively long-lived free radical product DMPO-OH, which can be easily investigated by EPR. The ROS scavenging activity of various concentrations (1.48, 0.74 and 0.37 mg/mL) of diclofenac-choline (Di Schiena Holos, Robecco sul Naviglio, Milan, Italy) was evaluated by assessing its ability to scavenge the most potent active oxygen species HO• [27]. To obtain the Fenton reaction, the final concentrations were: FeSO4 • 7 H2O 0.31 mM/L (Sigma), DTPA 0.34 mM/L (Sigma), H2O2 0.31 mM/L (Sigma) and DMPO 0.78 mM/L (Sigma). The Fenton reaction was initiated by mixing the Fe-DTPA solution with DC or distilled water (control) and then adding the H2O2 solution. The hydroxyl radical generated by a standard Fenton reaction was trapped using DMPO in accordance with a slightly modified version of a previously described method [26, 28]. The solutions were carefully mixed in a glass tube and then placed in a 100 μL capillary tube for EPR analysis. The EPR spectra were recorded after exactly 1 min, and the resulting DMPO-OH (consisting of a quartet of resonances with 1:2:2:1 relative intensities) was
detected using an X-band EPR spectrometer Miniscope MS 200 (Magnettech, Berlin Germany), whose parameters were: field modulation 100 KHz, modulation amplitude 2000 mG, field constant 60 s, centre field 3 349.39 G, sweep width 99.70 G, X-band frequency 9.64 GHz, attenuation 7, and gain 100. The percentage HO• scavenging activity of the assayed solution was calculated using the formula: 100 • (h0 − hX)/h0 [ %], where ho and hx are the relative heights of the highest resonance signal (mm) of the DMPO-OH adduct spectra in a reaction mixtures without and with DC.
KO2 in crown-ether as a source of O2 − •
In the second series of tests, the EPR analysis was based on the spin trapping of O2 − • generated by potassium superoxide (KO2) in DMSO, with the addition of 18-crown-6-ether to complex K + , conditions under which a DMPO-OOH adduct has been observed [29]. A typical reaction mixture contained 7.29 mM/L KO2 (Sigma), 9.01 mM/L crown-ether (Sigma) in DMSO, 14.29 mM/L DMPO (Sigma) and the previously used concentrations of DC. The reaction mixture was stirred and transferred into a 100 μL capillary tube for EPR analysis, and the EPR spectra were recorded after exactly 30 s. The resulting DMPO-OOH was detected using an X-band EPR spectrometer Miniscope MS 200 (Magnettech, Berlin Germany), whose parameters were: field modulation 100 KHz, modulation amplitude 2500 mG, field constant 120 s, centre field 3349.39 G, sweep width 147.76 G, X-band frequency 9.64 GHz, attenuation 7, and gain 800. The intensity of EPR was calculated using the formula: 100 • (h0 −hX)/h0 [ %], where ho and hx are the relative heights of the highest resonance signal (mm) of the DMPO-OOH adduct spectra in a reaction mixture without and with DC.
DPPH electron paramagnetic resonance (EPR) spectrometry
The radical scavenging activity of DC was investigated by means of electronic paramagnetic resonance spectrometry, a standard methods for detecting free radicals that uses the stable free-radical 2, 2-diphenyl-1-picryhydrazyl (DPPH•)(Sigma). DPPH• is reduced when it reacts with an antioxidant compound donating hydrogen, and this reduction is recorded on the basis of the corresponding inhibition of the EPR spectrum [30]. The EPR analysis was made using a Magnettech MS200 EPR spectrometer (Magnettech, Berlin, Germany). A blank probe was obtained by mixing 25 μL of a 30 μM ethanol solution of DPPH• and 225 μL ethanol. The concentration range of the investigated DC was 1.48, 0.74 and 0.37 mg/mL. The EPR signals were recorded 5 min after the start of the reaction under the following conditions: field modulation 100 KHz, modulation amplitude 0.226 G, field constant 40.96 ms, conversion time 671.089 ms, centre field 3 440,00 G, sweep with 100.00 G, X-band frequency 9.64 GHz, power 20 mV, temperature 24 °C. The scavenging activity of DC was defined as 100 • (h0 −hX)/h0 [ %], where h0 is the height of the third peak in the EPR spectrum of DPPH• free radicals in the blank, and hX the height of the third peak in the EPR spectrum of the DPPH• free radicals in the test sample.
Scavenging of ABTS● +
The free radical scavenging capacity of the same DC concentrations was studied using a radical cation decolorisation assay, based on the reduction of the ABTS● + radical. We used the method of Re et al. [31]. Briefly, ABTS (Sigma) was dissolved in deionised water to a concentration of 7 mM, and the ABTS radiBraga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
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of local oropharyngeal inflammation and pain are mouthwashes, sprays and gargles, whose advantages over other formulations include fewer side effects and drug interactions, and particularly their greater contact and activity at the oropharyngeal site where the main inflammatory mediators are released. The main mechanism of action explaining the activity of NSAIDs is the inhibition of prostaglandin synthesis, however it has been reported that some NSAIDs can also scavenge free radicals [12– 16] and inhibit the respiratory bursts of human neutrophils and the related release of ROS/RNS [17–20], which indicates the presence of an important additional mechanism that needs to be taken into account and investigated when making a pharmacological evaluation. Diclofenac-choline (DC), (obtained by a new salification process of diclofenac) has recently been proposed in the spray formulation for the symptomatic treatment of oropharyngeal inflammatory conditions and pain. The antioxidant activity of DC has not yet been investigated, and the aim of this study was to use luminol amplified chemiluminescence (LACL) to verify whether the new salt formulation interferes with ROS/ RNS during the course of human PMN respiratory bursts. LACL has been widely used to detect the PMN production of ROS/RNS under various conditions [21–23]. In order to yield light, luminol has to undergo 2-electron oxidation and form an unstable endoperoxide, which decomposes to an excited state (3-aminophthalic acid), and then relaxes to the ground state by emitting photons [24, 25] that are amplified by the phototube of a luminometer. The salt’s direct antiradical (scavenger) activity was investigated by means of electron paramagnetic resonance (EPR) spectroscopy, which is the only means of providing direct evidence of the presence of a free radical (a paramagnetic species containing an unpaired electron that can be physically detected by EPR) [26] and the ability of other molecules to scavenge it.
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246 Original Article
Human PMN harvesting
Peripheral venous blood was obtained from healthy adult donors by the AVIS Blood Donor Department of Niguarda Hospital (Milan, Italy), the Ethics Committee of which approved the study. Blood (5 mL) was stratified on 3 mL of a Polymorphprep cell separation medium (Axis-Shield PocAs, Oslo, Norway), and the PMNs were separated by means of density gradient centrifugation. After centrifugation, the upper mononuclear cell band was discarded, and the lower PMN band was washed in RPMI 1640 medium containing glutamine (Sigma). When necessary, any residual erythrocytes in the granulocyte preparation were lysed using a 0.15 mol/L NH4Cl solution (pH 7.4). After the aggregates had been disrupted by being passed through a needle with an internal diameter of 150 µm, the PMNs were collected, washed in HBSS, and tested for viability by means of Trypan blue exclusion. The number of cells in the final cell suspension used for each test was adjusted by counting in a Burker chamber (interference contrast microscopy).
Measurement of oxidative burst responses by means of LACL
PMN oxidative bursts are associated with the generation of ROS such as superoxide anion, hydrogen peroxide, oxygen radical, hydroxyl radical and hypochlorous acid. As luminol degradation by ROS is associated with luminescence, the inclusion of luminol in the reaction medium provides a sensitive means of detecting PMN respiratory bursts. LACL was investigated using the soluble stimulant phorbol 12 myristate 13 acetate (PMA), which is frequently used to stimulate PMN respiratory bursts and directly activates intracellular protein kinase (PKC). The measurements were made using a slightly modified version of the procedure described by Briheim and Dahlgren [22]. Briefly, 0.1 mL of a PMN suspension (1 × 106 cells/mL) plus 0.2 mL of 2 × 10–5 mol/L of luminol (Sigma) were put into a 3 mL flat-bottomed polystyrene vial. The vial was placed in the light-proof chamber of a Luminometer 1250 (Bio Orbit, Turku, Finland), and the carousel was rotated to bring the sample in line with the photomultiplier tube in order to record background activity. A volume of 0.1 mL PMA 2.6 × 10 − 6 mol/L was added to HBSS to reach a final volume of 1 mL. The resulting light output was continuously recorded in millivolts on a chart recorder, and simultaneously by means of a digital printout set to record intervals of 10 s. All of the constituents of the mixture were kept at 37 °C during the reaction by passing water from a thermostatically controlled circulation system through a polished hollow metal sample holder. No mixing took place during the recordings. The gain control was set to give a recording of 10 mV for a built-in standard. A background subtraction control zeroed the instrument before the addition of Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
PMA. The LACL response patterns were identified by calculating the peak values (mV) and the times to peak values (min, sec). The effects of DC was evaluated at concentrations of 1.48, 0.74, and 0.37 mg/mL, and incubation times of 2, 4 and 8 min at 37 °C. A second series of tests were performed in the same way, but with L-arginine (L-Arg) 170 µg/mL (Sigma) added to the medium incubating the PMNs as a NO donor, in order to be able to read NO-derived peroxynitrite LACL. In these tests, the effects of DC were evaluated at the same concentrations and incubation times as in the first series.
Viability tests
Topical agents frequently require the use of high drug concentrations in order to exert their specific biochemical effects. We therefore verified whether high DC concentrations affect cell viability, which would explain the salt’s inhibitory activity. Buccal cell viability was tested after short-term incubations with high DC concentrations using the Trypan blue exclusion test, and by measuring lactate dehydrogenase (LDH) in the supernatant of PMNs incubated with the medium and the drug under the same conditions.
Buccal cell collection
After being informed of the nature of the study and giving their informed consent, healthy, non-smoking volunteers laboratory personnel were first asked to rinse their mouths thoroughly with filtered tap water, after which exfoliated buccal cells were obtained by scraping the inside of both cheeks with a plastic stick, and rinsed into a tube with saline. The cells were incubated with the various DC concentrations for 2, 4 and 8 min, and their viability was measured by means of Trypan blue dye exclusion (100 µL of cell suspension with 100 µL of 0.2 % Trypan blue solution). The excluded cells were counted using a hemacytometer under a microscope with a Nomarsky interference contrast setting. 100 cells were counted in each preparation, and the cell viability was calculated as the ratio between the living and total number of counted cells.
LDH activity assay
Neutrophil viability was assessed by determining the activity of released dehydrogenase (LDH) after the cells had been incubated with various concentrations of DC for 2, 4 and 8 min using a Cytotox-ONE assay kit (Promega, Madison, USA) in accordance with the manufacturer’s instructions.
Statistical analysis
4 assays of each concentration were made for each test, and the statistical significance of the differences was calculated by means of one-way ANOVA followed by multiple paired comparisons using Dunnett’s test. The differences were considered statistically significant when the p-value was ≤ 0.05. The Trypan blue buccal cell data were statistically analysed using Wilcoxon’s test.
Results
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The antioxidant activity of this new diclofenac salt was assessed in 2 steps. The aim of the first was to confirm that the presence of choline did not chemically interfere with the activity of diclofenac (the EPR study).
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cal cation (ABTS● + ) was produced by reacting the ABTS solution (1 mL) with 2.45 mM potassium persulfate (10 µL) (Sigma) and leaving the mixture (stock solution) in the dark at room temperature for 12–16 h to give a dark blue solution. For this study, the ABTS● + solution was diluted in deionised water to an absorbance of 0.700 ( ± 0.020) at 734 nm, and an appropriate solvent blank reading was made (AB). An aliquot of the test sample (100 µL) was mixed with the ABTS● + solution (900 µL) in a 1 mL cuvette, and its adsorbance was recorded for 5 min (AE). All of the solutions were used on the day of preparation, and all of the determinations were carried out in duplicate. The percentage inhibition of ABTS● + was calculated using the formula: %inhibition = [(AB − AE)/AB] × 100.
Original Article
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Fig. 1 Panel a Percentage quenching effect of various amounts of diclofenac-choline using DMPO to trap the HO• radical (** = p ≤ 0.01). Panel b Examples of EPR spectra: A = control; B–D = effects of concentrations ranging from 1.48 mg/mL to 0.37 mg/mL.
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Fig. 2 Panel a Percentage quenching effect of various amounts of diclofenac-choline using DMPO to trap the O2 − • radical. Panel b Examples of EPR spectra: A = control; B–D = effects of concentrations ranging from 1.48 mg/mL to 0.37 mg/mL.
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The hydroxyl radical scavenging activity of the various DC concentrations is shown in ● ▶ Fig. 1 (Panel a). The quartet resonance ▶ Fig. 1, Panel b) was significantly reduced by DC of DMPO-OH ( ● concentrations ranging from 1.48 mg/mL to 0.37 mg/mL (from ▶ Fig. 1). ● ▶ Fig. 2, panel b, 94.57 ± 0.51 % to 76.56 ± 1.36 %) ( ● shows representative spectra of DMPO-OOH (from KO2 + crownether). DC was not active in scavenging superoxide anion at con▶ Fig. 2, centrations ranging from 1.48 mg/mL to 0.37 mg/mL ( ● panel a). The EPR spectra of DPPH• free radicals in the blank were characterised by the 5 lines of relative intensities 1:2:3:2:1 ▶ Fig. 3, panel b). The various concentrations of DC induced a ( ● significant decrease in the intensity of the lines of the DPPH• spectrum from the concentration of 1.48 mg/mL to 0.74 mg/mL ▶ Fig. 3, panel a), with a reduction from 19.84 ± 0.76 % to ( ● 2.89 ± 0.78 % respectively. ● ▶ Fig. 4 shows the effect of DC on ABTS● + radicals, which are very sensitive. All of the DC concentrations had > 90 % inhibitory activity.
In general, all of these findings indicated the presence of scavenging activity, whose relative intensity varied depending on the type of radical. After confirming that DC possesses anti-radical activity, the second step was to investigate whether this activity was maintained when challenged with human neutrophils. Microscopic examination of the human neutrophil suspensions showed that the population of neutrophils was always ≥ 95 %, and viability always ≥ 94 %. None of the DC concentrations used in the PMA tests affected PMN viability. ● ▶ Fig. 5 shows the comparative effects of the various concentrations on the LACL of PMA-induced PMN respiratory bursts after different incubation times. The lowest concentration that still had significant antioxidant activity was 0.37 mg/mL and, from this concentration to 1.48 mg/mL (the highest investigated concentration), there was a similarly significant concentration-dependent inhibition of peak Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
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% inhibition
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248 Original Article
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Fig. 3 Panel a Percentage quenching effect of various amounts of diclofenac-choline on the DPPH radical (** = p ≤ 0.01). Panel b Examples of EPR spectra: A = control; B–D = effects of concentrations ranging from 1.48 mg/mL to 0.37 mg/mL.
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Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
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Fig. 4 Effects of the inhibitory activity of various concentrations of diclofenac-choline on the ABTS radical (** = p ≤ 0.01).
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▶ Fig. 5). chemiluminescence at the different incubation times ( ● The times to peak chemiluminescence generally overlapped at the various concentrations, and were not significantly different from those of the controls. LACL can be used to investigate not only dynamic ROS generation, but also the generation of NO. The increase in chemiluminescence during the stimulation of PMNs is generally attributed to ROS, but the addition of L-Arg rapidly leads to a peak chemiluminescence that is many times greater than that reached without it because L-Arg (a substrate for NO synthetase) gener-
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Fig. 5 Effects of various of diclofenac-choline concentrations and incubation times (2, 4 and 8 min) on the LACL of the PMN respiratory bursts induced by PMA (** = p ≤ 0.01).
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Fig. 6 Effects of various of diclofenac-choline concentrations and incubation times (2, 4 and 8 min) on the LACL of the PMN respiratory bursts induced by PMA after L-Arg incubation (** = p ≤ 0.01).
ates NO in bursts, which then combines with O2 − • to yield peroxynitrite anions as a by-product of PMN activation. These anions oxidise luminol and rapidly create very bright chemiluminescence, which is consistent with our findings. When L-Arg was added to the reaction medium as a NO donor, baseline LACL increased approximately 3–4 times. The inhibiting behaviour of the same concentrations of DC was confirmed under these new conditions, and parallelled those previously obtained without the addition of L-Arg. The lowest concentration that significantly reduced LACL was 0.37 mg/mL and, from that concentration to 1.48 mg/mL, the concentration-dependent inhibition of peak ▶ Fig. 6). The time to peak chemiluminescence was significant ( ● LACL was not significantly different from that of the control.● ▶ Fig. 7 shows the findings concerning the viability of PMNs when incubated with the various DC concentrations for different times. High concentrations did not influence cell viability or reduce the number of respiratory bursts. This was confirmed by the Trypan blue exclusion test on exfoliated buccal cells. Generally, a relative low viability was expected for these type of terminally differentiated cell populations with high reneval rate. However, even under this situation the viability of buccal cells
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Fig. 7 Effects of diclofenac-choline on the viability of human PMN cells. LDH measured in the supernatant of PMN cells after incubation with various concentrations of diclofenac-choline for different times (** = p ≤ 0.01).
was unaffected by high DC concentrations for brief times of ▶ Fig. 8). incubation ( ●
Discussion
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The production of excess free radicals at the site of inflammation plays a key role in the progression of inflammatory processes, and so controlling radicals is a potential target of NSAIDs. In addition to its well-known anti-inflammatory effects, recent findings have shown that diclofenac sodium protects human erythrocytes against hemolysis by peroxyl radicals [15], bleaches the ABTS radical cation and reduces radicals in the oxygen cation absorbance capacity assay [16], thus suggesting that it has scavenging activity. Our DC data confirmed its concentration-dependent scavenging activity against the ABTS and DPPH radicals, and the hydroxyl radical generated by the Fenton reaction, but it had no activity against superoxide anion investigated by EPR. This has also been reported in the case of other NSAIDs such as nimesulide [14], ketorolac, tolmetin and oxaprozin [13] and diclofenac itself in Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
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erally concluded that NSAIDs and diclofenac have an inhibitory effect [17–20]. However, these data have been obtained using relatively small concentrations and long incubation times of 30 or 60 min neither of which apply to the frequent short time associated with the practical use of NSAID-containing mouthwashes or sprays. NSAIDs are generally lipophilic and this facilitates their penetration into tissues and with reference to oral or gingival tissues it has been reported a local inhibition of PGE2 levels within 1 h [34, 35]. Diclofenac seems to be quickly absorbed by the oral mucosa and particularly by human neutrophil with peak levels reached after 2 min [36], but no data have been published concerning its activity after this short period of contact, which is similar to that associated with the use of mouthwashes or sprays. In order to investigate the activity of the new diclofenac salt we used stricter conditions of high concentrations (up to 1.48 mg/mL) and shorter incubation times of 2, 4, and 8 min and found that the inhibitory concentration-effect ratio was statistically significant after only 2 min of incubation, and remained so after 4 and 8 min. Furthermore, the addition of L-Arg (which generates the peroxynitrite radical) led to a statistically significant concentration-effect reduction in the LACL of stimulated neutrophils that paralleled the findings of the previous tests. As the high concentrations and short incubation times did not interfere with PMN viability the inhibitory findings must be attributed to the effect of the drug. Partsch et al. [37] obtained similar results using diclofenac alone at 10 − 2 M. On the basis of our findings, it seems that the anti-inflammatory effects of DC cannot be attributed solely to the inhibition of prostaglandin synthesis because it is likely that its action on free radicals and neutrophil bursts also contributes to its final therapeutic effect [17].
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Fig. 8 Effects of various diclofenac-choline concentrations and incubation times on the viability of human buccal cells revealed by the Trypan blue exclusion test (** = p ≤ 0.01).
the xanthine-xanthine oxidase system [15], although differences in NSAID structure activity relationships, the method of generation of the assayed radicals, and the measurement systems used must all be taken into account when making a final interpretation [13]. The lack of superoxide anion scavenging activity is due to the particular molecular structure of DC, which does not have the classical hydrogen-donating moieties typical of the superoxide anion scavenging polyphenols [32]. DC is a carboxylic acid and the only part of it that could play a role in superoxide scavenging would be the carboxyl function; however, previous studies have found that natural carboxylic acids have little effect on superoxide [33], and this is further confirmed by our data. With reference to local oropharyngeal inflammation and pain, topical formulation of NSAIDs such as flurbiprofen gel, ketorolac rinse or diclofenac itself have been found to lead to useful clinical results. Activated PMNs are present during oropharyngeal inflammation and various authors have challenged NSAIDs and diclofenac with neutrophil respiratory bursts releasing ROS/RNS, and gen-
Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
▼
We would like to thank Di Schiena Holos (Robecco sul Naviglio, Milan, Italy) for the kind gift of diclofenac-choline.
Declaration of Interest
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The authors declare that they have no conflict of interest.
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250 Original Article
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23 Robinson PD, Wakefield D, Breit SN et al. Chemiluminescence response to pathogenic organisms: normal human polymorphonuclear leukocytes. Infect Immun 1984; 43: 744–752 24 Allen RC. Phagocytic oxygenation activities and chemiluminescence; a kinetic approach to analysis. Methods Enzymol 1986; 133: 449–493 25 Merenyi G, Lind J, Eriksen TE. Luminol chemiluminescence: chemistry, excitation, emitter. J Biolum Chemilum 1990; 5: 53–56 26 Kopani M, Celec P, Danisovic L et al. Oxidative stress and electron spin resonance. Clin Chim Acta 2006; 364: 61–66 27 Falchi M, Bertelli A, Lo Scalzo R et al. Comparison of cardioprotective abilities between theflesh and skin of grapes. J Agric Food Chem 2006; 54: 6613–6622 28 Valavanidis A, Nisiotu C, Papageorgiou Y et al. Comparison of the radical scavenging potential of polar and lipid fractions of olive oil and other vegetable oils under normal conditions and after thermal treatments. J Agr Food Chem 2004; 52: 2358–2365 29 Reszka K, Chignell CF. Spin trapping of the superoxide radical in aprotic solvents. Free Rad Res Commun 1991; 14: 97–106 30 Braga PC, Dal Sasso M, Culici M et al. Antioxidant activity of Calendula officinalis extract: inhibitory effects on chemiluminescence of human neutrphil bursts and electron paramagnetic resonance (EPR) spectroscopy. Pharmacology 2009; 83: 348–355 31 Re R, Pellegrini N, Proteggente A et al. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Rad Biol Med 1999; 26: 1231–1237 32 Deeble DJ, Parson BJ, Phillips CO et al. Superoxide reactions in aqueous solutions of pyrogallol and N-proyl gallate: the involvement of phenoxyl radicals. A pulse radiolysis study. Int J Rad Biol 1988; 54: 179–193 33 Bielski BHJ, Ritcher HW. A study of the superoxide radical chemistry by stopped-flow radiolysis and radiation induced oxygen comsumption. J Amer Chem Soc 1977; 99: 3019–3023 34 Salvi GE, Williams RC, Offenbacher S. Nonsteroidal anti-inflammatory drugs as adjuncts in the management of periodontal diseases and peri-implantis. Curr Opin Periodontol 1997; 4: 51–58 35 Williams RC, Jeffcoat MK, Howell TH et al. Topical flurbiprofen treatment of periodontitis in beagles. J Periodontal Res 1988; 23: 166–169 36 Perianin A, Giroud JP, Hakim J. Stimulation of human polymorphonuclear leukocytes potentiates the uptake of diclofenac and the inhibition of chemotaxis. Biochem Pharmacol 1990; 40: 2039–2045 37 Partsh G, Schwarzer C, Eberl R. The effects of ibuprofen and diclofenac on the chemotaxis and adenosine triphosphate level of polymorphonuclear cells in vitro. J Rheumatol 1990; 17: 583–588
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Original Article
Diclofenac-Choline Antioxidant Activity Investigated by means of Luminol Amplified Chemiluminescence of Human Neutrophil Bursts and Electron Paramagnetic Resonance Spectroscopy Authors
P. C. Braga1, N. Lattuada1, V. Greco1, V. Sibilia1, M. Falchi1, T. Bianchi2, M. Dal Sasso1
Affiliations
1
2
Key words ▶ diclofenac-choline ● ▶ electron paramagnetic ● resonance (EPR) ▶ human neutrophils ● ▶ chemiluminescence ● ▶ antioxidant activity ●
Abstract
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1377002 Published online: June 11, 2014 Drug Res 2015; 65: 244–251 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Prof. P. C. Braga Department of Medical Biotechnology and Translational Medicine School of Medicine Via Vanvitelli 32 20129 Milano Italy Tel.: + 39/02/50316 990 [email protected]
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A new diclofenac salt called diclofenac-choline (DC) has recently been proposed for the symptomatic treatment of oropharyngeal inflammatory processes and pain because its greater water solubility allows the use of high concentrations, which are useful when the contact time between the drug and the oropharyngeal mucosa is brief, as in the case of mouthwashes or spray formulations. The antioxidant activity of DC has not yet been investigated, and so the aim was to use luminol-amplified-chemiluminescence (LACL) to verify whether various concentrations of DC (1.48, 0.74 and 0.37 mg/mL for incubation times of 2, 4 and 8 min) interfere with oxygen and nitrogen radicals during the course of human neutrophils respiratory bursts; electron paramagnetic resonance (EPR) spectroscopy was
Introduction
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The inflammatory process generally has a localised protective function and is induced by a variety of stimuli, including micro-organisms, free radicals or oxidants, injured tissue, airborne pollutants, and allergens. The mechanism of inflammation is very complex and involves many different types of molecules and metabolic pathways [1, 2]. Inflammatory processes cause various diseases depending on the inflamed tissue or organ involved, but they all share the activation of a “stress signalling pathway” and the concomitant production of inflammatory cytokines [3, 4]. The chemotactic signals generated at the site of inflammation lead to the rapid and often massive recruitment and activation of polymorphonuclear neutrophils (PMNs), which are themselves major cell mediators of inflammation as they contribute to generating a “phlogogenic loop” as a result of their ability to generate inflammation
Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
used to investigate its direct antiradical (scavenger) activity. The EPR findings showed that DC has concentration-dependent scavenging activity against the ABTS, the DPPH, and the hydroxyl radicals, but no activity on superoxide anion, as has been previously reported in the case of other NSAIDs. LACL revealed an inhibitory effect that was statistically significant after only 2 min of incubation, and similar after 4 and 8 min. The effects on the peroxynitrite radical paralleled those observed in the previous test. High concentrations and short incubation times showed that there is no interference on PMN viability, and so the inhibitory findings must be attributed to the effect of the drug. The anti-inflammatory effects of DC cannot be attributed solely to the inhibition of prostaglandin synthesis, but its effects on free radicals and neutrophil bursts suggest that they may contribute to its final therapeutic effect.
by releasing reactive oxygen and nitrogen species (ROS/RNS), lysosomal enzymes, histaminereleasing factor, chemoattractant such as IL-8, and cytokines such as IL-1βb and TNFα [5, 6]. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of various acute and chronic pain and inflammatory conditions because of their anti-inflammatory, analgesic and antipyretic properties. Inflammatory processes in the oropharyngeal cavity can be sustained by factors such as acute pharyngitis and laryngitis, gingivitis, dental surgery, etc., and these are also frequently treated with NSAIDs [7–9]. The most frequently prescribed NSAID for the treatment of inflammation-related conditions is diclofenac, a phenylacetic acid derivative (2-[2, 6]–dichloranilinophenylaceticacid]) [10, 11], which is available in oral, intravenous, suppository and topical (transdermal patch or gel) formulations. However, the most frequently used medications for the symptomatic treatment
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received 11.11.2013 accepted 03.05.2014
Department of Medical Biotechnology and Translational Medicine, School of Medicine, University of Milan, Milan, Italy AVIS Comunale di Milano, Ospedale Niguarda, Milano, Italy
Original Article
Materials and Methods
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Fenton reaction model with the EPR detection of HO•
The first series of tests used the spin trapping method [26], which is based on the rapid reaction of many radicals with certain chemical acceptor molecules (spin trapping agents) to produce more stable secondary radicals. The diamagnetic spin trap nitrone DMPO (5,5-dimethyl-1-pyrrolidine-N-oxide) (Sigma Chemicals Co., St Louis, MO, USA) was added to the reaction mixture to produce the relatively long-lived free radical product DMPO-OH, which can be easily investigated by EPR. The ROS scavenging activity of various concentrations (1.48, 0.74 and 0.37 mg/mL) of diclofenac-choline (Di Schiena Holos, Robecco sul Naviglio, Milan, Italy) was evaluated by assessing its ability to scavenge the most potent active oxygen species HO• [27]. To obtain the Fenton reaction, the final concentrations were: FeSO4 • 7 H2O 0.31 mM/L (Sigma), DTPA 0.34 mM/L (Sigma), H2O2 0.31 mM/L (Sigma) and DMPO 0.78 mM/L (Sigma). The Fenton reaction was initiated by mixing the Fe-DTPA solution with DC or distilled water (control) and then adding the H2O2 solution. The hydroxyl radical generated by a standard Fenton reaction was trapped using DMPO in accordance with a slightly modified version of a previously described method [26, 28]. The solutions were carefully mixed in a glass tube and then placed in a 100 μL capillary tube for EPR analysis. The EPR spectra were recorded after exactly 1 min, and the resulting DMPO-OH (consisting of a quartet of resonances with 1:2:2:1 relative intensities) was
detected using an X-band EPR spectrometer Miniscope MS 200 (Magnettech, Berlin Germany), whose parameters were: field modulation 100 KHz, modulation amplitude 2000 mG, field constant 60 s, centre field 3 349.39 G, sweep width 99.70 G, X-band frequency 9.64 GHz, attenuation 7, and gain 100. The percentage HO• scavenging activity of the assayed solution was calculated using the formula: 100 • (h0 − hX)/h0 [ %], where ho and hx are the relative heights of the highest resonance signal (mm) of the DMPO-OH adduct spectra in a reaction mixtures without and with DC.
KO2 in crown-ether as a source of O2 − •
In the second series of tests, the EPR analysis was based on the spin trapping of O2 − • generated by potassium superoxide (KO2) in DMSO, with the addition of 18-crown-6-ether to complex K + , conditions under which a DMPO-OOH adduct has been observed [29]. A typical reaction mixture contained 7.29 mM/L KO2 (Sigma), 9.01 mM/L crown-ether (Sigma) in DMSO, 14.29 mM/L DMPO (Sigma) and the previously used concentrations of DC. The reaction mixture was stirred and transferred into a 100 μL capillary tube for EPR analysis, and the EPR spectra were recorded after exactly 30 s. The resulting DMPO-OOH was detected using an X-band EPR spectrometer Miniscope MS 200 (Magnettech, Berlin Germany), whose parameters were: field modulation 100 KHz, modulation amplitude 2500 mG, field constant 120 s, centre field 3349.39 G, sweep width 147.76 G, X-band frequency 9.64 GHz, attenuation 7, and gain 800. The intensity of EPR was calculated using the formula: 100 • (h0 −hX)/h0 [ %], where ho and hx are the relative heights of the highest resonance signal (mm) of the DMPO-OOH adduct spectra in a reaction mixture without and with DC.
DPPH electron paramagnetic resonance (EPR) spectrometry
The radical scavenging activity of DC was investigated by means of electronic paramagnetic resonance spectrometry, a standard methods for detecting free radicals that uses the stable free-radical 2, 2-diphenyl-1-picryhydrazyl (DPPH•)(Sigma). DPPH• is reduced when it reacts with an antioxidant compound donating hydrogen, and this reduction is recorded on the basis of the corresponding inhibition of the EPR spectrum [30]. The EPR analysis was made using a Magnettech MS200 EPR spectrometer (Magnettech, Berlin, Germany). A blank probe was obtained by mixing 25 μL of a 30 μM ethanol solution of DPPH• and 225 μL ethanol. The concentration range of the investigated DC was 1.48, 0.74 and 0.37 mg/mL. The EPR signals were recorded 5 min after the start of the reaction under the following conditions: field modulation 100 KHz, modulation amplitude 0.226 G, field constant 40.96 ms, conversion time 671.089 ms, centre field 3 440,00 G, sweep with 100.00 G, X-band frequency 9.64 GHz, power 20 mV, temperature 24 °C. The scavenging activity of DC was defined as 100 • (h0 −hX)/h0 [ %], where h0 is the height of the third peak in the EPR spectrum of DPPH• free radicals in the blank, and hX the height of the third peak in the EPR spectrum of the DPPH• free radicals in the test sample.
Scavenging of ABTS● +
The free radical scavenging capacity of the same DC concentrations was studied using a radical cation decolorisation assay, based on the reduction of the ABTS● + radical. We used the method of Re et al. [31]. Briefly, ABTS (Sigma) was dissolved in deionised water to a concentration of 7 mM, and the ABTS radiBraga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
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of local oropharyngeal inflammation and pain are mouthwashes, sprays and gargles, whose advantages over other formulations include fewer side effects and drug interactions, and particularly their greater contact and activity at the oropharyngeal site where the main inflammatory mediators are released. The main mechanism of action explaining the activity of NSAIDs is the inhibition of prostaglandin synthesis, however it has been reported that some NSAIDs can also scavenge free radicals [12– 16] and inhibit the respiratory bursts of human neutrophils and the related release of ROS/RNS [17–20], which indicates the presence of an important additional mechanism that needs to be taken into account and investigated when making a pharmacological evaluation. Diclofenac-choline (DC), (obtained by a new salification process of diclofenac) has recently been proposed in the spray formulation for the symptomatic treatment of oropharyngeal inflammatory conditions and pain. The antioxidant activity of DC has not yet been investigated, and the aim of this study was to use luminol amplified chemiluminescence (LACL) to verify whether the new salt formulation interferes with ROS/ RNS during the course of human PMN respiratory bursts. LACL has been widely used to detect the PMN production of ROS/RNS under various conditions [21–23]. In order to yield light, luminol has to undergo 2-electron oxidation and form an unstable endoperoxide, which decomposes to an excited state (3-aminophthalic acid), and then relaxes to the ground state by emitting photons [24, 25] that are amplified by the phototube of a luminometer. The salt’s direct antiradical (scavenger) activity was investigated by means of electron paramagnetic resonance (EPR) spectroscopy, which is the only means of providing direct evidence of the presence of a free radical (a paramagnetic species containing an unpaired electron that can be physically detected by EPR) [26] and the ability of other molecules to scavenge it.
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Human PMN harvesting
Peripheral venous blood was obtained from healthy adult donors by the AVIS Blood Donor Department of Niguarda Hospital (Milan, Italy), the Ethics Committee of which approved the study. Blood (5 mL) was stratified on 3 mL of a Polymorphprep cell separation medium (Axis-Shield PocAs, Oslo, Norway), and the PMNs were separated by means of density gradient centrifugation. After centrifugation, the upper mononuclear cell band was discarded, and the lower PMN band was washed in RPMI 1640 medium containing glutamine (Sigma). When necessary, any residual erythrocytes in the granulocyte preparation were lysed using a 0.15 mol/L NH4Cl solution (pH 7.4). After the aggregates had been disrupted by being passed through a needle with an internal diameter of 150 µm, the PMNs were collected, washed in HBSS, and tested for viability by means of Trypan blue exclusion. The number of cells in the final cell suspension used for each test was adjusted by counting in a Burker chamber (interference contrast microscopy).
Measurement of oxidative burst responses by means of LACL
PMN oxidative bursts are associated with the generation of ROS such as superoxide anion, hydrogen peroxide, oxygen radical, hydroxyl radical and hypochlorous acid. As luminol degradation by ROS is associated with luminescence, the inclusion of luminol in the reaction medium provides a sensitive means of detecting PMN respiratory bursts. LACL was investigated using the soluble stimulant phorbol 12 myristate 13 acetate (PMA), which is frequently used to stimulate PMN respiratory bursts and directly activates intracellular protein kinase (PKC). The measurements were made using a slightly modified version of the procedure described by Briheim and Dahlgren [22]. Briefly, 0.1 mL of a PMN suspension (1 × 106 cells/mL) plus 0.2 mL of 2 × 10–5 mol/L of luminol (Sigma) were put into a 3 mL flat-bottomed polystyrene vial. The vial was placed in the light-proof chamber of a Luminometer 1250 (Bio Orbit, Turku, Finland), and the carousel was rotated to bring the sample in line with the photomultiplier tube in order to record background activity. A volume of 0.1 mL PMA 2.6 × 10 − 6 mol/L was added to HBSS to reach a final volume of 1 mL. The resulting light output was continuously recorded in millivolts on a chart recorder, and simultaneously by means of a digital printout set to record intervals of 10 s. All of the constituents of the mixture were kept at 37 °C during the reaction by passing water from a thermostatically controlled circulation system through a polished hollow metal sample holder. No mixing took place during the recordings. The gain control was set to give a recording of 10 mV for a built-in standard. A background subtraction control zeroed the instrument before the addition of Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
PMA. The LACL response patterns were identified by calculating the peak values (mV) and the times to peak values (min, sec). The effects of DC was evaluated at concentrations of 1.48, 0.74, and 0.37 mg/mL, and incubation times of 2, 4 and 8 min at 37 °C. A second series of tests were performed in the same way, but with L-arginine (L-Arg) 170 µg/mL (Sigma) added to the medium incubating the PMNs as a NO donor, in order to be able to read NO-derived peroxynitrite LACL. In these tests, the effects of DC were evaluated at the same concentrations and incubation times as in the first series.
Viability tests
Topical agents frequently require the use of high drug concentrations in order to exert their specific biochemical effects. We therefore verified whether high DC concentrations affect cell viability, which would explain the salt’s inhibitory activity. Buccal cell viability was tested after short-term incubations with high DC concentrations using the Trypan blue exclusion test, and by measuring lactate dehydrogenase (LDH) in the supernatant of PMNs incubated with the medium and the drug under the same conditions.
Buccal cell collection
After being informed of the nature of the study and giving their informed consent, healthy, non-smoking volunteers laboratory personnel were first asked to rinse their mouths thoroughly with filtered tap water, after which exfoliated buccal cells were obtained by scraping the inside of both cheeks with a plastic stick, and rinsed into a tube with saline. The cells were incubated with the various DC concentrations for 2, 4 and 8 min, and their viability was measured by means of Trypan blue dye exclusion (100 µL of cell suspension with 100 µL of 0.2 % Trypan blue solution). The excluded cells were counted using a hemacytometer under a microscope with a Nomarsky interference contrast setting. 100 cells were counted in each preparation, and the cell viability was calculated as the ratio between the living and total number of counted cells.
LDH activity assay
Neutrophil viability was assessed by determining the activity of released dehydrogenase (LDH) after the cells had been incubated with various concentrations of DC for 2, 4 and 8 min using a Cytotox-ONE assay kit (Promega, Madison, USA) in accordance with the manufacturer’s instructions.
Statistical analysis
4 assays of each concentration were made for each test, and the statistical significance of the differences was calculated by means of one-way ANOVA followed by multiple paired comparisons using Dunnett’s test. The differences were considered statistically significant when the p-value was ≤ 0.05. The Trypan blue buccal cell data were statistically analysed using Wilcoxon’s test.
Results
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The antioxidant activity of this new diclofenac salt was assessed in 2 steps. The aim of the first was to confirm that the presence of choline did not chemically interfere with the activity of diclofenac (the EPR study).
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cal cation (ABTS● + ) was produced by reacting the ABTS solution (1 mL) with 2.45 mM potassium persulfate (10 µL) (Sigma) and leaving the mixture (stock solution) in the dark at room temperature for 12–16 h to give a dark blue solution. For this study, the ABTS● + solution was diluted in deionised water to an absorbance of 0.700 ( ± 0.020) at 734 nm, and an appropriate solvent blank reading was made (AB). An aliquot of the test sample (100 µL) was mixed with the ABTS● + solution (900 µL) in a 1 mL cuvette, and its adsorbance was recorded for 5 min (AE). All of the solutions were used on the day of preparation, and all of the determinations were carried out in duplicate. The percentage inhibition of ABTS● + was calculated using the formula: %inhibition = [(AB − AE)/AB] × 100.
Original Article
a
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Fig. 1 Panel a Percentage quenching effect of various amounts of diclofenac-choline using DMPO to trap the HO• radical (** = p ≤ 0.01). Panel b Examples of EPR spectra: A = control; B–D = effects of concentrations ranging from 1.48 mg/mL to 0.37 mg/mL.
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The hydroxyl radical scavenging activity of the various DC concentrations is shown in ● ▶ Fig. 1 (Panel a). The quartet resonance ▶ Fig. 1, Panel b) was significantly reduced by DC of DMPO-OH ( ● concentrations ranging from 1.48 mg/mL to 0.37 mg/mL (from ▶ Fig. 1). ● ▶ Fig. 2, panel b, 94.57 ± 0.51 % to 76.56 ± 1.36 %) ( ● shows representative spectra of DMPO-OOH (from KO2 + crownether). DC was not active in scavenging superoxide anion at con▶ Fig. 2, centrations ranging from 1.48 mg/mL to 0.37 mg/mL ( ● panel a). The EPR spectra of DPPH• free radicals in the blank were characterised by the 5 lines of relative intensities 1:2:3:2:1 ▶ Fig. 3, panel b). The various concentrations of DC induced a ( ● significant decrease in the intensity of the lines of the DPPH• spectrum from the concentration of 1.48 mg/mL to 0.74 mg/mL ▶ Fig. 3, panel a), with a reduction from 19.84 ± 0.76 % to ( ● 2.89 ± 0.78 % respectively. ● ▶ Fig. 4 shows the effect of DC on ABTS● + radicals, which are very sensitive. All of the DC concentrations had > 90 % inhibitory activity.
In general, all of these findings indicated the presence of scavenging activity, whose relative intensity varied depending on the type of radical. After confirming that DC possesses anti-radical activity, the second step was to investigate whether this activity was maintained when challenged with human neutrophils. Microscopic examination of the human neutrophil suspensions showed that the population of neutrophils was always ≥ 95 %, and viability always ≥ 94 %. None of the DC concentrations used in the PMA tests affected PMN viability. ● ▶ Fig. 5 shows the comparative effects of the various concentrations on the LACL of PMA-induced PMN respiratory bursts after different incubation times. The lowest concentration that still had significant antioxidant activity was 0.37 mg/mL and, from this concentration to 1.48 mg/mL (the highest investigated concentration), there was a similarly significant concentration-dependent inhibition of peak Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
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Fig. 3 Panel a Percentage quenching effect of various amounts of diclofenac-choline on the DPPH radical (** = p ≤ 0.01). Panel b Examples of EPR spectra: A = control; B–D = effects of concentrations ranging from 1.48 mg/mL to 0.37 mg/mL.
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Fig. 4 Effects of the inhibitory activity of various concentrations of diclofenac-choline on the ABTS radical (** = p ≤ 0.01).
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▶ Fig. 5). chemiluminescence at the different incubation times ( ● The times to peak chemiluminescence generally overlapped at the various concentrations, and were not significantly different from those of the controls. LACL can be used to investigate not only dynamic ROS generation, but also the generation of NO. The increase in chemiluminescence during the stimulation of PMNs is generally attributed to ROS, but the addition of L-Arg rapidly leads to a peak chemiluminescence that is many times greater than that reached without it because L-Arg (a substrate for NO synthetase) gener-
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ates NO in bursts, which then combines with O2 − • to yield peroxynitrite anions as a by-product of PMN activation. These anions oxidise luminol and rapidly create very bright chemiluminescence, which is consistent with our findings. When L-Arg was added to the reaction medium as a NO donor, baseline LACL increased approximately 3–4 times. The inhibiting behaviour of the same concentrations of DC was confirmed under these new conditions, and parallelled those previously obtained without the addition of L-Arg. The lowest concentration that significantly reduced LACL was 0.37 mg/mL and, from that concentration to 1.48 mg/mL, the concentration-dependent inhibition of peak ▶ Fig. 6). The time to peak chemiluminescence was significant ( ● LACL was not significantly different from that of the control.● ▶ Fig. 7 shows the findings concerning the viability of PMNs when incubated with the various DC concentrations for different times. High concentrations did not influence cell viability or reduce the number of respiratory bursts. This was confirmed by the Trypan blue exclusion test on exfoliated buccal cells. Generally, a relative low viability was expected for these type of terminally differentiated cell populations with high reneval rate. However, even under this situation the viability of buccal cells
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was unaffected by high DC concentrations for brief times of ▶ Fig. 8). incubation ( ●
Discussion
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The production of excess free radicals at the site of inflammation plays a key role in the progression of inflammatory processes, and so controlling radicals is a potential target of NSAIDs. In addition to its well-known anti-inflammatory effects, recent findings have shown that diclofenac sodium protects human erythrocytes against hemolysis by peroxyl radicals [15], bleaches the ABTS radical cation and reduces radicals in the oxygen cation absorbance capacity assay [16], thus suggesting that it has scavenging activity. Our DC data confirmed its concentration-dependent scavenging activity against the ABTS and DPPH radicals, and the hydroxyl radical generated by the Fenton reaction, but it had no activity against superoxide anion investigated by EPR. This has also been reported in the case of other NSAIDs such as nimesulide [14], ketorolac, tolmetin and oxaprozin [13] and diclofenac itself in Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
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80 60 2 min
40 20 0
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erally concluded that NSAIDs and diclofenac have an inhibitory effect [17–20]. However, these data have been obtained using relatively small concentrations and long incubation times of 30 or 60 min neither of which apply to the frequent short time associated with the practical use of NSAID-containing mouthwashes or sprays. NSAIDs are generally lipophilic and this facilitates their penetration into tissues and with reference to oral or gingival tissues it has been reported a local inhibition of PGE2 levels within 1 h [34, 35]. Diclofenac seems to be quickly absorbed by the oral mucosa and particularly by human neutrophil with peak levels reached after 2 min [36], but no data have been published concerning its activity after this short period of contact, which is similar to that associated with the use of mouthwashes or sprays. In order to investigate the activity of the new diclofenac salt we used stricter conditions of high concentrations (up to 1.48 mg/mL) and shorter incubation times of 2, 4, and 8 min and found that the inhibitory concentration-effect ratio was statistically significant after only 2 min of incubation, and remained so after 4 and 8 min. Furthermore, the addition of L-Arg (which generates the peroxynitrite radical) led to a statistically significant concentration-effect reduction in the LACL of stimulated neutrophils that paralleled the findings of the previous tests. As the high concentrations and short incubation times did not interfere with PMN viability the inhibitory findings must be attributed to the effect of the drug. Partsch et al. [37] obtained similar results using diclofenac alone at 10 − 2 M. On the basis of our findings, it seems that the anti-inflammatory effects of DC cannot be attributed solely to the inhibition of prostaglandin synthesis because it is likely that its action on free radicals and neutrophil bursts also contributes to its final therapeutic effect [17].
20 0
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0.74
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Fig. 8 Effects of various diclofenac-choline concentrations and incubation times on the viability of human buccal cells revealed by the Trypan blue exclusion test (** = p ≤ 0.01).
the xanthine-xanthine oxidase system [15], although differences in NSAID structure activity relationships, the method of generation of the assayed radicals, and the measurement systems used must all be taken into account when making a final interpretation [13]. The lack of superoxide anion scavenging activity is due to the particular molecular structure of DC, which does not have the classical hydrogen-donating moieties typical of the superoxide anion scavenging polyphenols [32]. DC is a carboxylic acid and the only part of it that could play a role in superoxide scavenging would be the carboxyl function; however, previous studies have found that natural carboxylic acids have little effect on superoxide [33], and this is further confirmed by our data. With reference to local oropharyngeal inflammation and pain, topical formulation of NSAIDs such as flurbiprofen gel, ketorolac rinse or diclofenac itself have been found to lead to useful clinical results. Activated PMNs are present during oropharyngeal inflammation and various authors have challenged NSAIDs and diclofenac with neutrophil respiratory bursts releasing ROS/RNS, and gen-
Braga PC et al. Antiox Activity Of Diclofenac-Choline … Drug Res 2015; 65: 244–251
▼
We would like to thank Di Schiena Holos (Robecco sul Naviglio, Milan, Italy) for the kind gift of diclofenac-choline.
Declaration of Interest
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The authors declare that they have no conflict of interest.
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