2. Oxidation processes

At the same time, many neuroinflammatory mediators, including oxidative agents such as reactive oxygen species (ROS), were found to be upregulated in neurodegenerative disorders (ND) that affect human brain areas [4, 5]. This fact immediately allows the proposal of some kind of cause-effect link between the presence of ROS, oxidation processes, neuroinflammation, and

Oxidative stress is a process that occurs in early stages of ND and is considered an identifier mark for their detection as could be evaluated by DNA, RNA, lipids, and protein oxidation levels [6–8]. Simultaneously, several studies have observed an inverse correspondence between prolonged NSAID administration and the development of some ND in humans, (for review, see Ref. [9]). So, it is now accepted that NSAIDs could play a protective role on many ND and one of the reasons of the great interest for getting more insight into the elucidation of the pathways and mechanisms of the oxidative processes in which several NSAIDs and different

The present chapter will analyze the results presented in two relatively recent papers that have been dedicated to evaluate the possible action of some NSAIDs as protectors against ROSmediated oxidation/deterioration of biological targets [10, 11]. Those research works are focused on NSAIDs from different chemical structure classes, one salicylic acid derivative, diflunisal (DFN), an indolic acid derivative, indomethacin (IMT) (Figure 1) and the enolic acid derivatives, oxicams, represented by meloxicam (MEL), tenoxicam (TEN) and piroxicam (PIR)

Figure 1. Chemical structures of a: 2′,4′-difluoro-4-hydroxyphenyl-3-carboxylic acid, diflunisal (DFN) and b: 2-{1′-[(4-

Figure 2. Chemical structures of a: [4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3-carboxamide 1,1 dioxide], meloxicam (MEL), b: [4-hydroxy-2-methyl-N-(pyridin-2-yl)-2H-thieno(2,3-e)-1,2 thiazine-3-carboxamide 1,1 dioxide], tenoxicam (TEN) and c: [4-hydroxy-2-methyl-N-(pyridin-2-yl)-2H-1,2-benzothiazine-3-carboxamide 1,1-diox-

chlorophenyl)carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl} acetic acid, indomethacin (IMT).

ND pathogenesis [4, 5].

246 Pain Relief - From Analgesics to Alternative Therapies

ROS take part.

(Figure 2).

ide], piroxicam (PIR).

Many compounds in the presence of oxygen and any electron donor can generate different ROS—by energy and/or electron transfer processes—like singlet molecular oxygen, O2ð 1 ΔgÞ, superoxide radical anion (O•<sup>−</sup> <sup>2</sup> ) or hydrogen peroxide (H2O2) among others. An interesting example of those compounds is vitamin B2, riboflavin (Rf), a naturally occurring endogenous compound of singular importance, present in practically all living organisms. Rf absorbs energy in the wavelength range of visible light, being a well-known photosensitizer for oxidative processes [12, 13]. Upon selective absorption of energy, Rf is promoted from its ground state to electronically excited singlet state (<sup>1</sup> Rf� ) (Eq. (1)).

$$\mathbf{Rf} + h\boldsymbol{\nu} \to \,^1\mathbf{Rf}^\* \tag{1}$$

The generated <sup>1</sup> Rf � can decay to the original ground state or produce the electronically excited triplet state (<sup>3</sup> Rf� ) (Eq. (2)).

$$\text{^1Rf^\*} \rightarrow \text{^3Rf^\*} \tag{2}$$

The <sup>3</sup> Rf� may react with the ground state oxygen (O2ð<sup>3</sup>Σ<sup>−</sup> <sup>g</sup>Þ) (Eq. (3)) to form superoxide radical anion (O•<sup>−</sup> <sup>2</sup> ), with a very low quantum yield (0.009) (Krishna, 1991).

$$\rm{^3Rf^\*(^3\Sigma\_g^-)} \to \rm{Rf^{\*+}} + \rm{O\_2^{\*-}} \tag{3}$$

In living organisms, a great number of biomolecules essential to life such as DNA, RNA, lipids, and proteins, can be oxidized by the generated ROS producing oxidative stress [6–8, 14]. Among other substrates, NSAIDs are compounds that can be oxidized in the presence of Rfgenerated ROS and as shown can act as quenchers of electronically excited states of Rf (Eqs. (4) and (5)).

$$\text{R}^{1}\text{R}\text{f}^{\*} + \text{NSAIDs} \rightarrow \text{Rf} + \text{NSAIDs} \text{ or } \text{P} (4) \text{ rate constant } ^{1}\text{k}\_{\text{q}} \tag{4}$$

$$\text{s}^{\text{3R}}\\\text{Rf}^{\text{\*}} + \text{NSAIDs} \rightarrow \text{Rf}^{\text{\*-}} + \text{NSAIDs}^{\text{\*+}} \text{ rate constant}^{\text{3}}\\k\_{\text{q}} \tag{5}$$

The protonation of Rf•<sup>−</sup> at neutral pH can generate the species RfH• (pKa = 8.3), (Eq. (6)).

$$\text{Rf}^{\bullet-} + \text{H}^+ \rightleftharpoons \text{RfH}^\bullet \tag{6}$$

Its bimolecular decay through a disproportionation reaction can yield the ground state of the vitamin and fully reduced Rf (Eq. (7)).

$$\text{2RfH}^{\bullet} \rightarrow \text{Rf} + \text{RfH}\_{2} \tag{7}$$

The last product, in the presence of ground state oxygen, is reoxidized to Rf radical and superoxide radical anion (O•<sup>−</sup> <sup>2</sup> ) (Eq. (8)).

$$\text{RfH}\_2 + \text{O}\_2(^{3}\text{L}^{-}\_{\text{g}}) \rightarrow \text{RfH}\_2^{\bullet+} + \text{O}\_2^{\bullet-} \text{ rate constant } k\_8 \tag{8}$$

The electron transfer process, in Eq. (8) is relevant as a source of H2O2 (Eq. (9)), another important already-mentioned ROS.

$$\text{RfH}\_2^{\bullet+} + \text{O}\_2^{\bullet-} \rightarrow \text{Rf} + \text{H}\_2\text{O}\_2 \tag{9}$$

In parallel, the generated O•<sup>−</sup> <sup>2</sup> can chemically react with a substrate, according to Eqs. (10) and (11), respectively, illustrates the processes that occur with NSAIDs.

$$\text{CO}\_2^{\bullet -} \text{NSAIDs} \to \text{P}(10) \quad \text{rate constant } k\_{10} \tag{10}$$

$$\rm H\_2O\_2 + NSAIDs \to P(11) \tag{11}$$

Another possible pathway for <sup>3</sup> Rf� is the energy transfer reaction with O2ð<sup>3</sup>Σ<sup>−</sup> <sup>g</sup>Þ which generates O2ð 1 ΔgÞ, with reported quantum yield of 0.49 in water [15] (Eq. (12)).

$$\rm{^3Rf^\*} + \rm{O\_2}(^3\Sigma\_{\rm g}^-) \rightarrow \rm{Rf} + \rm{O\_2}(^1\Delta\_{\rm g}) \text{ rate constant } k\_{\rm ET} \tag{12}$$

The O2ð 1 ΔgÞ formed may be physically quenched either by the solvent (Eq. (13)).

$$\mathbf{O}\_2(^1\Delta\_{\mathfrak{g}}) \to \mathbf{O}\_2(^3\Sigma\_{\mathfrak{g}}^-) \tag{13}$$

or by a substrate, as happens in the presence of NSAIDs (Eq. (14)).

$$\text{O}\_2(\text{}^1\text{A}\_{\text{g}}) + \text{NSAIDs} \rightarrow \text{O}\_2(\text{}^3\text{E}\_{\text{g}}^-) + \text{NSAIDs} \text{ rate constant } k\_{\text{q}} \tag{14}$$

Finally, Eq. (15) represents the main pathway of substrate disappearance in O2ð 1 ΔgÞ mediated processes.

$$\text{O}\_2(\:^1\text{A}\_\%) + \text{NSAIDs} \rightarrow \text{P}(\text{15}) \text{ rate constant } k\_\text{r} \tag{15}$$

k<sup>t</sup> being the overall rate constant for physical plus chemical quenching processes (Eq. (16)).

$$k\_{\mathbf{t}} = k\_{\mathbf{r}} + k\_{\mathbf{q}} \tag{16}$$

In order to get more insight into the behavior of NSAIDs toward Rf-generated ROS several in vitro experiments were performed.

#### 2.1. Stationary photolysis: riboflavin-photosensitization

In complex biological structures, Rf and NSAIDs may occupy the same locations. Kinetic and mechanistic aspects of their mutual interaction constitute the crucial information for understanding the behavior of NSAIDs toward Rf-generated ROS and the potential in vivo consequences.

Using a home-made photolyzer, aerated neutral aqueous solutions of each of the following NSAIDs DFN, IMT, MEL, TEN, and PIR, were irradiated with the light of a 150W quartzhalogen lamp, in the presence of Rf as a sensitizer. All the NSAIDs used as substrates are transparent to visible light. Nevertheless, in order to assure that they do not absorb any incident radiation, a cut-off filter at 400 nm was employed. The processes were followed by the absorption spectra using a diode array spectrophotometer (Hewlett Packard 8452A). The light irradiation induced changes in the absorption spectra of the mixtures 0.05 mM DFN + 0.04 Rf (Figure 3), 0.05 mM IMT + 0.04 mM Rf (Figure 3, inset A) and 0.05 mM MEL + 0.04 mM Rf (Figure 4). The processes could be monitored from the absorbance decay at the respective absorption maxima for each substrate. In this way, the rates of sensitized photoxygenation for each NSAID were determined.

RfH2 þ O2ð

important already-mentioned ROS.

248 Pain Relief - From Analgesics to Alternative Therapies

In parallel, the generated O•<sup>−</sup>

Another possible pathway for <sup>3</sup>

3

O2ð

in vitro experiments were performed.

ates O2ð 1

The O2ð 1

processes.

quences.

3 Σ−

RfH•<sup>þ</sup>

(11), respectively, illustrates the processes that occur with NSAIDs.

3 Σ−

or by a substrate, as happens in the presence of NSAIDs (Eq. (14)).

<sup>1</sup> <sup>Δ</sup>gÞ þ NSAIDs ! O2<sup>ð</sup>

O2ð 1

2.1. Stationary photolysis: riboflavin-photosensitization

O•<sup>−</sup>

Rf� þ O2ð

<sup>g</sup>Þ ! RfH•<sup>þ</sup>

<sup>2</sup> <sup>þ</sup> <sup>O</sup>•<sup>−</sup>

ΔgÞ, with reported quantum yield of 0.49 in water [15] (Eq. (12)).

O2ð 1

Finally, Eq. (15) represents the main pathway of substrate disappearance in O2ð

<sup>g</sup>Þ ! Rf þ O2ð

ΔgÞ formed may be physically quenched either by the solvent (Eq. (13)).

3 Σ−

k<sup>t</sup> being the overall rate constant for physical plus chemical quenching processes (Eq. (16)).

In order to get more insight into the behavior of NSAIDs toward Rf-generated ROS several

In complex biological structures, Rf and NSAIDs may occupy the same locations. Kinetic and mechanistic aspects of their mutual interaction constitute the crucial information for understanding the behavior of NSAIDs toward Rf-generated ROS and the potential in vivo conse-

ΔgÞ ! O2ð

<sup>2</sup> <sup>þ</sup> <sup>O</sup>•<sup>−</sup>

The electron transfer process, in Eq. (8) is relevant as a source of H2O2 (Eq. (9)), another

<sup>2</sup> rate constant k<sup>8</sup> (8)

<sup>g</sup>Þ which gener-

<sup>2</sup> ! Rf þ H2O2 (9)

ΔgÞ rate constant kET (12)

<sup>g</sup>Þ (13)

1

ΔgÞ mediated

<sup>g</sup>Þ þ NSAIDs rate constant k<sup>q</sup> (14)

k<sup>t</sup> ¼ k<sup>r</sup> þ k<sup>q</sup> (16)

ΔgÞ þ NSAIDs ! Pð15Þ rate constant k<sup>r</sup> (15)

<sup>2</sup> can chemically react with a substrate, according to Eqs. (10) and

<sup>2</sup> NSAIDs ! Pð10Þ rate constant k<sup>10</sup> (10)

Rf� is the energy transfer reaction with O2ð<sup>3</sup>Σ<sup>−</sup>

1

3 Σ−

H2O2 þ NSAIDs ! Pð11Þ (11)

In parallel experiments, using a specific oxygen electrode (Orion 97-08) the oxygen concentration was measured during irradiation of the same mixtures in aqueous solutions in a closed Pyrex cell [10]. Under these conditions, all the NSAIDs under study showed oxygen consumption. Regarding the oxicams family, TEN and PIR presented the lowest rate of oxygen consumption. It was a little bit higher for MEL (Figure 4, inset B). In the corresponding set for DFN and IMT, the rate of oxygen uptake was significantly higher for the latter (Figure 3, inset B).

Figure 3. Changes in UV-vis absorption spectra of a pH 7 aqueous solution of 0.05 mM DFN plus 0.04 mM Rf upon photoirradiation taken vs. a 0.04 mM Rf aqueous solution (spectrum a). Cut-off 400 nm interference filter, under airsaturated conditions. Numbers on the spectra represent photoirradiation time in seconds. (Inset A) Changes in UV-vis absorption spectrum of a pH 7 aqueous solution of 0.05 mM IMT plus 0.04 mM Rf upon photoirradiation taken vs. a 0.04 mM Rf aqueous solution (spectrum b). Cut-off 400 nm interference filter, under air-saturated conditions. Numbers on the spectra represent photoirradiation time in seconds. (Inset B) Oxygen consumption vs. photoirradiation time in pH 7 aerated aqueous solutions for the systems: a: Rf (A446 = 0.46) plus DFN (0.4 mM); b: Rf (A446 = 0.46) plus ITM (0.4 mM). Reprinted from Purpora et al. [10], © (2013), with permission from The American Society of Photobiology, a Wiley Company, John Wiley & Sons, Inc.

Figure 4. Changes in UV-vis absorption spectra of aqueous solution of 0.05 mM MEL plus 0.05 mM Rf upon photoirradiation taken vs. 0.05 mM Rf aqueous solution (spectrum a). Cut-off 450 nm interference filter, under airsaturated conditions. Numbers on the spectra represent photoirradiation time in minutes. (Inset A) Changes in UV-vis absorption spectra of aqueous solution of 0.05 mM MEL plus 0.05 mM RB upon photoirradiation taken vs. 0.05 mM RB aqueous solution (spectrum b). Cut-off 450 nm interference filter, under air-saturated conditions. Numbers on the spectra represent photoirradiation time in minutes. (Inset B) Oxygen consumption vs. photoirradiation time under air saturated conditions for the systems: a: Rf 0.05 mM plus TEN (0.5 mM) in MeOH-H2O (buffer pH 7) 1:1 v/v; b: Rf 0.05 mM plus 0.5 mM PIR in MeOH-H2O (buffer pH 7) 1:1 v/v; c: Rf 0.05 mM plus 0.5 mM MEL in aqueous buffer pH 7. λirr > 480 nm, cutoff filter. Reprinted from Ferrari et al. [11], © (2015), with permission from Elsevier B.V.

From all these preliminary findings, we assume that the transformations in NSAIDs can be attributed to interactions with electronically excited states of Rf with the possible participation of photogenerated ROS.

#### 2.1.1. Kinetics and mechanism

The xantenic dye Rose Bengal (RB) is one of the most frequently employed photosensitizers that exclusively generate O2ð 1 ΔgÞ, with a quantum yield of 0.7 in aqueous media [15, 16]. So, experiments performed in the presence of RB involved possible O2ð 1 ΔgÞ-mediated oxidation of NSAIDs. In this case, eventual interferences of other ROS that could be generated by Rf were avoided. Comparing the rates of substrate consumption by Rf – photosensitization with those in the presence of RB it was possible to elucidate the relevance of O2ð 1 ΔgÞ in relation to other ROS also generated by Rf.

The combination of stationary and time-resolved experiments unambiguously demonstrates the participation of O2ð 1 ΔgÞ in NSAIDs' photooxidation processes. Using time-resolved phosphorescence detection (TRPD) [17], the overall quenching rate constant of O2ð 1 ΔgÞ by NSAIDs, Interaction of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) with Reactive Oxygen Species (ROS)... http://dx.doi.org/10.5772/66478 251


(a) In MeOH; (b) in D2O, pH 7; (c) in MeOH-D2O (pD 7) 1:1 v/v; (d) in dioxane-water (molar fraction of water = 0.91) Source: [18]; (e) in MeCN Source: [19].

Table 1. Values for the rate constants for the interactions of each NSAID by quenching with electronically excited singletð 1 kqÞ, and triplet (<sup>3</sup>kq) of riboflavin; overall rate constants <sup>ð</sup>kt<sup>Þ</sup> and reactive <sup>ð</sup>kr<sup>Þ</sup> for the interaction of <sup>O</sup>2<sup>ð</sup> 1 ΔgÞ with each NSAID.


Source: [10, 11].

From all these preliminary findings, we assume that the transformations in NSAIDs can be attributed to interactions with electronically excited states of Rf with the possible participation

Figure 4. Changes in UV-vis absorption spectra of aqueous solution of 0.05 mM MEL plus 0.05 mM Rf upon photoirradiation taken vs. 0.05 mM Rf aqueous solution (spectrum a). Cut-off 450 nm interference filter, under airsaturated conditions. Numbers on the spectra represent photoirradiation time in minutes. (Inset A) Changes in UV-vis absorption spectra of aqueous solution of 0.05 mM MEL plus 0.05 mM RB upon photoirradiation taken vs. 0.05 mM RB aqueous solution (spectrum b). Cut-off 450 nm interference filter, under air-saturated conditions. Numbers on the spectra represent photoirradiation time in minutes. (Inset B) Oxygen consumption vs. photoirradiation time under air saturated conditions for the systems: a: Rf 0.05 mM plus TEN (0.5 mM) in MeOH-H2O (buffer pH 7) 1:1 v/v; b: Rf 0.05 mM plus 0.5 mM PIR in MeOH-H2O (buffer pH 7) 1:1 v/v; c: Rf 0.05 mM plus 0.5 mM MEL in aqueous buffer pH 7. λirr > 480 nm, cut-

The xantenic dye Rose Bengal (RB) is one of the most frequently employed photosensitizers

NSAIDs. In this case, eventual interferences of other ROS that could be generated by Rf were avoided. Comparing the rates of substrate consumption by Rf – photosensitization with those

The combination of stationary and time-resolved experiments unambiguously demonstrates

ΔgÞ, with a quantum yield of 0.7 in aqueous media [15, 16]. So,

ΔgÞ in NSAIDs' photooxidation processes. Using time-resolved phos-

1

1

ΔgÞ-mediated oxidation of

ΔgÞ in relation to other

ΔgÞ by NSAIDs,

1

of photogenerated ROS.

2.1.1. Kinetics and mechanism

250 Pain Relief - From Analgesics to Alternative Therapies

that exclusively generate O2ð

ROS also generated by Rf.

the participation of O2ð

1

1

experiments performed in the presence of RB involved possible O2ð

off filter. Reprinted from Ferrari et al. [11], © (2015), with permission from Elsevier B.V.

in the presence of RB it was possible to elucidate the relevance of O2ð

phorescence detection (TRPD) [17], the overall quenching rate constant of O2ð

Table 2. Values for the ratio of the reactive and overall rates kr=kt, and relative rates of each NSAID consumption upon Rf (RRRf) and RB (RRRB) photosensitization.

k<sup>t</sup> (Table 1) was determined. Hence, k<sup>t</sup> in the order of 107 M<sup>−</sup><sup>1</sup> s<sup>−</sup><sup>1</sup> allows the consideration of these substrates as good quenchers of O2ð 1 ΔgÞ. The kr=k<sup>t</sup> ratio accounts for the fraction of the overall quenching of O2ð 1 ΔgÞ that produces chemical transformation in the substrate (Table 2). Low kr=k<sup>t</sup> values denote that O2ð 1 ΔgÞ removal will proceed without a significant loss of the present NSAIDs, which act as a scavenger [18, 19].

#### 2.2. Interaction of NSAIDs with photogenerated ROS

Some compounds that are specific ROS quenchers have been used to elucidate which species are effectively involved in a given oxidative event [20, 21]. Catalase from bovine liver (CAT) reacts with H2O2, so the photodegradation via process in Eq. (11) is inhibited due to the process represented by Eq. (17).

$$\text{2H}\_2\text{O}\_2 + \text{CAT} \rightarrow 2\text{H}\_2\text{O} + \text{O}\_2(^{3}\text{L}^{-}\_{\text{g}}) \tag{17}$$

The enzyme superoxide dismutase from bovine erythrocytes (SOD) dismutates the species O•<sup>−</sup> <sup>2</sup> , as shown by Eq. (18).

$$2\text{O}\_2^- + 2\text{H}^+ + \text{SOD} \rightarrow \text{O}\_2(^3\text{E}\_{\text{g}}^-) + \text{H}\_2\text{O}\_2\tag{18}$$

Meanwhile, sodium azide (NaN3) is a known physical quencher of O2ð 1 ΔgÞ, with a reported rate constant k<sup>q</sup> of 4.5 · 108 M−<sup>1</sup> s <sup>−</sup><sup>1</sup> in water at pH 7 (Eq. (14) with NaN3 instead of NSAIDs) [22]. Several oxygen consumption experiments of NSAIDs upon Rf-photosensitization were performed adding each of these specific ROS interceptors. With DFN or IMT solutions different extent of decrease in the rates of oxygen consumption were observed upon using any of these three quenchers. This fact confirms a significant participation of O2ð 1 ΔgÞ in the degradation of

Figure 5. Bar diagrams for the relative rates of oxygen consumption with aqueous solutions pH 7, 0.5 mM IMT plus Rf (A445 = 0.5) as function of photoirradiation time (cut-off 400 nm): ITM: alone; IMT + CAT: in the presence of 1µg mL−<sup>1</sup> CAT; IMT + SOD: in the presence of 1µg mL−<sup>1</sup> SOD; IMT + NaN3: in the presence of 1 mM NaN3. Reprinted from Purpora et al. [10], © (2013), with permission from The American Society of Photobiology, a Wiley Company, John Wiley & Sons, Inc.

Figure 6. Bar diagrams for the relative rates of oxygen consumption upon photoirradiation as function of photoirration time (cut-off filter 400 nm) in the presence of Rf 0.05 mM with the following solutions: 0.5 mM PIR in MeOH-H2O (buffer pH 7) 1:1 v/v plus 1 µg mL−<sup>1</sup> SOD; 0.5 mM PIR in MeOH-H2O (buffer pH 7) 1:1 v/v; 0.5 mM TEN in MeOH-H2O (buffer pH 7) 1:1 v/v plus 1 µg mL−<sup>1</sup> SOD; 0.5 mM TEN in MeOH-H2O (buffer pH 7) 1:1 v/v; 0.5 mM MEL plus 1 µg mL<sup>−</sup><sup>1</sup> SOD in aqueous buffer pH 7; 0.5 mM MEL in aqueous buffer pH 7. Reprinted from Ferrari et al. [11], © (2015), with permission from Elsevier B.V.

the analgesics DFN and IMT, in which also O•<sup>−</sup> <sup>2</sup> and H2O2 take part. Bar diagram of the relative rates illustrates the results obtained with IMT solutions in the presence of each specific quencher (Figure 5); DFN solutions presented similar qualitative results.

2O�<sup>−</sup>

s

constant k<sup>q</sup> of 4.5 · 108 M−<sup>1</sup>

252 Pain Relief - From Analgesics to Alternative Therapies

from Elsevier B.V.

Meanwhile, sodium azide (NaN3) is a known physical quencher of O2ð

three quenchers. This fact confirms a significant participation of O2ð

<sup>2</sup> þ 2H<sup>þ</sup> þ SOD ! O2ð

Several oxygen consumption experiments of NSAIDs upon Rf-photosensitization were performed adding each of these specific ROS interceptors. With DFN or IMT solutions different extent of decrease in the rates of oxygen consumption were observed upon using any of these

Figure 5. Bar diagrams for the relative rates of oxygen consumption with aqueous solutions pH 7, 0.5 mM IMT plus Rf (A445 = 0.5) as function of photoirradiation time (cut-off 400 nm): ITM: alone; IMT + CAT: in the presence of 1µg mL−<sup>1</sup> CAT; IMT + SOD: in the presence of 1µg mL−<sup>1</sup> SOD; IMT + NaN3: in the presence of 1 mM NaN3. Reprinted from Purpora et al. [10], © (2013), with permission from The American Society of Photobiology, a Wiley Company, John Wiley & Sons, Inc.

Figure 6. Bar diagrams for the relative rates of oxygen consumption upon photoirradiation as function of photoirration time (cut-off filter 400 nm) in the presence of Rf 0.05 mM with the following solutions: 0.5 mM PIR in MeOH-H2O (buffer pH 7) 1:1 v/v plus 1 µg mL−<sup>1</sup> SOD; 0.5 mM PIR in MeOH-H2O (buffer pH 7) 1:1 v/v; 0.5 mM TEN in MeOH-H2O (buffer pH 7) 1:1 v/v plus 1 µg mL−<sup>1</sup> SOD; 0.5 mM TEN in MeOH-H2O (buffer pH 7) 1:1 v/v; 0.5 mM MEL plus 1 µg mL<sup>−</sup><sup>1</sup> SOD in aqueous buffer pH 7; 0.5 mM MEL in aqueous buffer pH 7. Reprinted from Ferrari et al. [11], © (2015), with permission

3 Σ−

<sup>g</sup>Þ þ H2O2 (18)

ΔgÞ, with a reported rate

ΔgÞ in the degradation of

1

1

<sup>−</sup><sup>1</sup> in water at pH 7 (Eq. (14) with NaN3 instead of NSAIDs) [22].

Similar experiments were performed using solutions 0.5 mM of the three oxicams and NaN<sup>3</sup> NaN3 or SOD. The participation of O2ð 1 ΔgÞ in the oxidation processes of these NSAIDs was revealed by the lower rates of oxygen uptake (Figure 6). As in the previous cases, for MEL the presence of SOD produced a decrease in the rates of oxygen consumption. Meanwhile for TEN and PIR it was the other way around. This fact can be due to the participation of O•<sup>−</sup> <sup>2</sup> with different mechanistic roles. The regeneration of O2ð<sup>3</sup>Σ<sup>−</sup> <sup>g</sup><sup>Þ</sup> (Eq. (18)) at expenses of O•<sup>−</sup> <sup>2</sup> increases the O2ð 1 ΔgÞ leading to the detected rates increased.
