*Grape Pomace Polyphenols*

From the chemical structure point of view, polyphenols are compounds made from several hydroxyl groups attached to an aromatic ring. These molecules can vary from simple to complex structures [69]. Also, these compounds are synthesized in plants and exist as glycosides, which are further formed from the basic polyphenol structure and glycosylic radical (sugar fragment) [70]. GP polyphenols are divided into two classes: flavonoids and non-flavonoids (Figure 1). Grape pomace flavonoids are further subdivided into flavonols, anthocyanins, flavanols, proanthocyanidins, and anthocyanidins, while GP non-flavonoids in stilbenes and phenolic acids [71,72].

The main structure of a flavonoid is made up of two phenyl radicals (rings A and B) linked to a heterocyclic ring, which contains an atom of oxygen (ring C). Based on the oxidation state and hydroxyl radicals' distribution pattern on the heterocyclic ring, flavonoids are further divided into the classes mentioned before [69,70] (Figure 2).

**Figure 2.** Flavonoid generic structure, structures of the main grape pomace polyphenols classes, as well as the most representative compounds.

Flavonols structure presents a C2−C3 double bond with a hydroxyl radical at the C3 position and ring B coupled to the C2 position. Flavanols structure presents with a hydroxyl radical situated at the C3 position, and their ring B is attached to the C2 position [70,73,74] (Figure 2). Proanthocyanidins, also called procyanidins or condensed tannins, are formed from subunits of flavanols that bond together in dimers (usually referred to as B-series—B1, B2, B3, B4, and B5) or trimers (known as C-series—C1, and C2). This series of procyanidins are being found in grape skins and seeds [75] (Figure 2). Anthocyanins present double bonds in the heterocyclic ring, their aromatic ring B being bonded to the C2 position. They represent the glycosylated form of anthocyanidins (aglycone) which result from the bond between the hydroxyl group at C3 and the sugar fragment and are the most abundant polyphenols in the peel of red grapes [70,74] (Figure 2). It is known that anthocyanins are only found in red GP because they act as a natural colorant, giving the red grape specific color. High contents of anthocyanins are found in red GP also because of the red grape skin thickness as compared to the white ones [76,77].

Compared to flavonoids, non-flavonoid polyphenols have one aromatic ring as a basic structure. Non-flavonoid molecules found in GP are phenolic acids, and stilbenes [69,70,78]. Phenolic acids are further divided into hydroxybenzoic and hydroxycinnamic acids. As representants of hydroxybenzoic acids, there are gallic, p-hydroxybenzoic, and syringic acids, and for the hydroxycinnamic acids, caffeic, p-coumaric, ferulic and synaptic acids are the most found in GP [69,70,78,79]. Stilbenes are formed from two aromatic rings bounded through the ethylene radical. The most known and studied stilbene is resveratrol. Besides GP, stilbenes are reported to be found also in grapes, and wine [69,70,78] (Figure 2).

The polyphenolic composition from different assortments of GP may differ based on the grape cultivar, type of soil, weather, geographical location, and winemaking process [76,80]. The content of polyphenols found in GP differ from study to study; some suggest that red GP possess the highest content of polyphenols, and other suggest otherwise, but the principal idea is that no matter which GP is analyzed, all of them possess high quantities of polyphenols. For instance, Kammerer et al. 2004 studied the polyphenol composition of 14 different red and white GP [79]. The study did not find significant differences in composition between red and white varieties except for the presence of anthocyanins found in red ones [79].

#### **5. Grape Pomace Polyphenols Benefic Actions**

Grape pomace polyphenols research studies have grown in the last decades, given their potential benefic effects on promoting human health. Some of their benefic actions are observed in oxidative stress and inflammation aiming at homeostasis restoration. Regarding the antioxidant effect, polyphenols can modulate the endogenous pathway responsible for combating oxidative stress. These effects can be achieved by polyphenols capacity to activate the nuclear factor E2 and to up-regulate superoxide dismutase, catalase, glutathione, glutathione peroxidase, and heme-oxidase 1 [81,82] or their capacity to scavenge and chelate reactive oxygen species involved in ROS production [83] (Figure 3). In inflammation, polyphenols are reported to inhibit the mitogen-activated kinase pathway, Nf-kB, anddown-regulate cytokines and chemokines [81,82]. Polyphenols also inhibit cyclooxygenase and lipoxygenase, which are involved in the arachidonic acid signaling pathway, being responsible for synthesizing prostaglandin, thromboxane A2, and leukotrienes which further increase inflammatory response [73,74,82] (Figure 3).

**Figure 3.** Proposed antioxidant and anti-inflammatory actions of polyphenols from grape pomace.

Further, there are presented the *in vitro* and *in vivo* beneficial effects of different red and white GP in oxidative stress and inflammatory conditions.

#### *5.1. In Vitro Beneficial Actions of Grape Pomace in Oxidative Stress and Inflammation*

The *in vitro* studies, as presented in Table 1, can offer the possibility to investigate and identify the diversity of related diseases in which GP exerts the optimum antioxidant and anti-inflammatory effects.

The *in vitro* beneficial action of GP was studied by Goutzourelas et al. (2015) [84]. They investigated an extract of red GP on muscle and endothelial cells using non-cytotoxic doses to check the effect of GP polyphenols extract on cells' antioxidant enzymes [84]. The red GP extract was investigated as a mixture of compounds that contained phenolic acids (caftaric acid, gallic acid), anthocyanins, flavanols (epicatechin and catechin), flavonols (quercetin), and anthocyanidins. It was observed that GP treatment increased Glutathione S-transferase (GST) and GSH levels in both cell lines. CAT levels were decreased in endothelial cells, while in muscle cells it showed no significant differences. SOD and HO-1 presented no differences in any population. An explanation for these inconstant findings, in which some of the antioxidant enzymes are not modified, is the ability of GP to enhance other antioxidant systems (GSC, GSH) [84] (Table 1). Another *in vitro* study, of Pop et al. (2022), investigated the antioxidant effect of red GP (mixture of Pinot Noir, Cabernet Sauvignon, Fetească Neagră, and Mamaia cultivars) and white GP (mixture of Sauvignon Blanc and Muscat Ottonel cultivars) added to a mouthwash on both H2O2 exposed and non-exposed fibroblast cells [90]. They observed that both red grape pomace (RGP) and white grape pomace (WGP) decreased ROS levels in a dose-dependent matter (100 < 200 < 300 μg/mL). Similar to the non-exposed condition, in the presence of H2O2, red GP and white GP led to a significant decrease in ROS levels, the only difference being that while red GP effect was dose-dependent, and white GP produced a non-dependent action [90]. Moreover, they also studied the anti-inflammatory effects of these extracts on lipopolysaccharides (LPS) induced inflammation in cells [90]. It was observed that while in the case of white GP a dose of 100 μg/mL was sufficient to induce a significant reduction of interleukin (IL) -8 levels, for red GP was necessary a higher dose of 200 μg/mL. At the dose of 300 μg/mL, both extracts significantly reduced IL-8 levels, but not even the highest dose did significantly

reduce the levels of IL-6. In the case of IL-1β, the lowest dose, 100 μg/mL, reduced its level to a similar one found in the non-exposed cells, while the doses of 200 and 300 μg/mL reduced, even more, the levels of IL-1β [90].

**Table 1.** *In vitro* beneficial actions of grape pomace in oxidative stress and inflammation.


Abbreviations: CAT—catalase; CGRP—calcitonin gene-related peptide; COX 2—cyclooxygenase 2; GR glutathione reductase; GS—glutathione syntase; GCLC—glutamate-cysteine ligase catalytic subunit; GPx1 glutathione peroxidase 1; GSH—glutathione; GSSG—glutathione disulfide; HO-1—heme oxygenase 1; MDA malondialdehyde; NF-*κ*B—nuclear factor kappa-light-chain-enhancer of activated B cells; NF-*κ*B p65—nuclear factor kappa-light-chain-enhancer of activated B cells transcription factor; NOX4—nicotinamide adenine dinucleotide phosphate oxidase 4; NQO1 —nicotinamide adenine dinucleotide plus hydrogen quinone oxidoreductase 1 mRNA Nrf2—messenger ribonucleic acid nuclear factor erythroid 2-related factor 2; p38-MAPK—p38mitogen-activated protein kinase PGE2—prostaglandin E2; pAkt—phosphorylated protein kinase B; pI*κ*Bα—phosphorylated inhibitor of kappa B; pIKK—phosphorylated I*κ*B kinase; pNrf2—phosphorylated Nrf2 ROS—reactive oxygen species; SIRT1—sirtuin 1; SOD2—superoxide dismutase 2; TBARS—thiobarbituric acid reactive substances; TNF-α—tumor necrosis factor alpha.

Marzulli et al. (2018), treated mononuclear cells with phorbol 12-myristate 13-acetate (PMA) to activate inflammation, and with different GPs (red Negroamaro cultivar or white Koshu cultivar) extracts (water, ethanol), to observe their immunomodulatory effects [91]. In terms of cytokine release, all GP fractions and extracts increased anti-inflammatory (IL-10) and pro-inflammatory (IL-12, IL-1β, IL-6, tumor necrosis factor-alpha (TNF-α)) cytokines. The water extracts of both GPs managed to increase T regulatory cells and forkhead box P3 (FoxP3) protein, which is responsible for the genes activity control that are involved in the immune system regulation. Another benefic effect of GPs extracts is FoxP3 increase which is a marker with a role in stabilizing the T regulatory cells' function. All extracts lowered the release of granzyme (GrB) compared to PMA treated group [91]. GrB

is an enzyme secreted by cytolytic T cells with role in cell necrosis leading to harmful effects on homeostasis [91]. Regarding intracellular cytokines, the water extract of red Negroamaro GP increased TNF-α and IL-10 content in monocytes, while the red Negroamaro GP ethanol extract increased IL-12 and IL-10 levels in lymphocytes. Further, the white Koshu GP water extract increased monocyte levels of IL-10 and IL-12, while the white Koshu GP ethanol extract increased lymphocyte levels of TNF-α and IL-10. IL-10 was increased by both water or ethanolic, red or white GP extracts and as underlined by authors [91], the release of IL-10 by T cells and monocytes is a key step in maintaining the immune homeostasis. In conclusion, GPs extracts could induce immune homeostasis through the anti-inflammatory IL-10 secretion which counterbalances the pro-inflammatory cytokines (IL-12 and TNFα) [91]. Another study that reinforces the anti-inflammatory effects of RGP from *Vitis vinifera* L. cv. Montepulciano d'Abruzzo on LPS-stimulated macrophages is that of Mollica et al. (2021). They observed that the extract significantly inhibited the release of cytokines (IL-6, TNF-α, and IL-1β), the maximal inhibitory action being at the dose of 100 μg/mL [92].

The possible potential impact of GP extracts on *in vitro* calcitonin gene-related peptide (CGRP) secretion was investigated as a potential mechanism to influence migraine [85]. The treatment of CA 77 cells with different red GP extracts showed a significant decrease in CGRP levels. CGRP is a gene that represents a key mediator of migraine-induced inflammation [85]. The results suggest that GP extracts had anti-inflammatory effect preventing the release of CGRP in migraine [8885].

White GP extract and a mixture of red and white GP extract, in different concentrations (100, 200, 500 μg/mL dry extract *w*/*v*), were added to Caco-2 cells after treatment with an inflammation inducer (IL-1β) to observe the effects on IL-8 secretion and NF-*κ*B expression [93]. Grape pomaces were hydrolyzed enzymatically to determine if anti-inflammatory effects would be augmented. Both white and red GP contained quercetin, catechin, resveratrol, gallic and caffeic acids, trans-resveratrol, rutin, and procyanidin B2 [93]. All GP fractions (100, 200 μg/mL dry extract *w*/*v*) with or without enzymatic transformation decreased ROS levels, while treatment with GP extracts in higher concentration (500 μg/mL dry extract *w*/*v*) showed a considerable increase in ROS levels. Furthermore, NF-*κ*B expression and prostaglandin E2 (PGE2) levels were significantly reduced in all fractions. At the same time, IL-8 secretion revealed a more substantial drop in enzymatically treated fractions of mixed GP, presenting beneficial effects of enzyme hydrolysis. The mixed GP had a more potent anti-inflammatory effect due to the high content of anthocyanins found in red GP [89].

Concerning the benefic antioxidant and anti-inflammatory GP actions, the literature presents a large variety of experimental settings that can be considered for future *in vivo* research. Also, we can observe that there is still space for other hypotheses, for both red and white GPs, but especially for the white ones which were much less investigated.

Further, in the next step the GP effects *in vivo* studies were analyzed. The *in vivo* studies usually use rodents to induce different models of inflammation, but fish and lamb were also introduced.

### *5.2. In Vivo Beneficial Actions of Grape Pomace in Oxidative Stress and Inflammation*

The effect of GP extracts on the pathophysiology of oxidative stress and inflammation in various types of diseases can be well documented using different *in vivo* experimental models. These types of studies are very important in deciding whether the GP can be further used in safe conditions in human clinical trials.

The antioxidant and anti-inflammatory effects of both fresh and fermented GP extracts (*Vitis vinifera* L. cultivars, Fetească neagră, and Pinot noir, from Romania) were investigated using and a rat model of induced inflammation by turpentine oil [94]. The administration of turpentine oil increased the total oxidative status, oxidative stress index and reduced total antioxidant reactivity [94]. Treatment with GP decreased total oxidative status and oxidative stress index in a dose-dependent manner, but total antioxidant reactivity was not modified. All GP's fractions significantly reduced malondialdehyde (MDA) levels. Total thiols were considerably lessened by turpentine, but GP managed to increase them in a concentration-dependent way. The same results were observed in the case of NOx production. 3NT was also increased by turpentine, but GP varieties decreased the levels. Due to higher phenolic content, the fresh extract showed a higher antioxidant effect. MDA is a lipid peroxidation waste product with hazardous potential for normal homeostasis. Thiols, under oxidative stress, manage to form disulphide bonds between them to reduce oxidative stress. NO presents a dual effect based on its concentrations. Small doses possess an antioxidant effect, while high doses can cause an increase in oxidative stress through the synthesis of new and stronger radicals. 3NT is a waste product resulting from tyrosine nitration induced by reactive nitrogen species [94]. The authors concluded that GP extracts could be used considered a potential agent in nutraceuticals formulation.

An interesting study that evaluates the effects of red GP flour dietary inclusion on growth, anti-inflammatory, antioxidant, innate-adaptive immunity, and on immune genes expression was performed on *Labeo rohita* fish against *Flavobacterium columnaris* induced infection [95]. Treatment with 200 and 300 mg GP flour showed a significant increase in GSH, SOD, and GPx activities as compared to regular diet or 100 mg GP supplementation, in both infected and uninfected groups. Regarding GP action on innate-adaptive immune activity, higher doses of GP (200, 300 mg) increased phagocytosis, alternative-complement pathway activity, raised IgM levels, and serum lysozyme (Lyz) activity when compared to regular diet or 100 mg GP supplementation in infected or uninfected group. In terms of immune-related genes, Lyz, (β-2 microglobulin) β-2M, 3rd component complement (CC3), and immunoglobulin M (IgM) gene expression pointed out a significant growth in infected fish with 200, 300 mg GP supplementation compared to other groups. However, the uninfected group treated with the same doses of GP showed higher gene expression than the infected group. Antioxidant related-genes were measured, and SOD, GPx, nuclear factor erythroid 2-related factor 2 (Nrf2), and (natural killer-cell enhancing factor β) NKEFβ were remarkably higher in all groups treated with raised doses of GP compared to 100 mg GP diet or regular diet in infected or uninfected groups. Furthermore, uninfected groups treated with high doses of GP showed a more significant increase in SOD and GPx expression levels. As for pro-inflammatory-related genes, IL-1β and TNF-α were not modified in any group. Hepcidin and toll-like receptor-22 (TLR22) expression were increased in infected and uninfected groups treated with a high dose of GP [95].

Therefore, in Rajkovi´c et al. (2022), GP was given to piglets to assess their positive effects on the animal organism without antibiotics side effects [96]. During the experiment tissue samplings (liver, jejunum, ileum) were collected on days 27/28 and 55/56, while blood samples were taken on days 6, days 27/28, and 55/56. Regarding antioxidant enzymes, GPx (GPx1-liver, GPx-2 jejunum, and ileum) wasn't different between diets, but the enzyme activity was significantly increased on days 55/56 compared to 27/28 [96]. About, SOD and Manganese Superoxide Dismutase (Mn-SOD) enzymes, there weren't any differences between diets in jejunum, ileum, and liver, but there was an increase between sampling dates, in days 55/56 compared to 27/28 in the liver. The copper superoxide dismutase system (Cu-SOD or SOD1) presented no distinction between any sampling days in the liver or ileum. CAT activity wasn't affected by any of the diets in the jejunum and liver, but there were differences between sampling days in the liver and ileum. TBARS concentrations weren't affected by diets in any organs, only in the jejunum between sampling days (decreased levels on days 55/56 compared to 27/28). GPx2 and SOD1 gene expression were modified at the jejunum level (decreased in days 55/56 compared to 27/28), while CAT expression presented the same results at the ileum level. In the liver, the authors have observed differences between samples for SOD1, CAT, and GPx1 in the liver, with a higher expression on days 27/28 compared to 55/56. In terms of inflammation, pig major acute phase-protein serum levels presented a decrease on days 55/56 and 27/28 compared to day 6 [96]. MDA serum levels decreased through sampling days while for SOD different fluctuations were noticed, without showing any significant values on day 55/56 versus other time points. As a speculative explanation for the variation of antioxidant enzymes

decreasing it can be stated that the systemic presence of antioxidant substances can lead to a decreasing need for endogenous antioxidant enzymes production [96].

Another important direction in GP research is to check whether it is suitable to be used as an adjuvant treatment in different pathologies to reduce conventional drugs side effects. Thus, in Mossa et al. (2015) study, cypermethrin was given to female rats to observe toxic effects on the liver and kidneys, and white GP was added to check whether it can counter these toxic effects [97]. The assessment of kidneys and liver biomarkers showed a dose-dependent fall in liver enzymes: aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), and alkaline phosphatase (ALP), and a decrease kidneys urea nitrogen and creatine. Also, total proteins and albumin revealed a significant increase in GP treated group. The histological analysis pointed out significant changes due to inflammatory infiltrate in cypermethrin groups, while the GP supplemented group had regressed values for all biomarkers. Similar results were also observed in histological studies of kidneys samples. This may be due to the antioxidant effects of the white GP [97]. This study offers important evidence regarding the use of GP extract with hepatorenal protective activity and encourages future studies to investigate whether it can be used to reduce other drugs adverse reactions.

So far, the existing studies on GP suggest that through its anti-inflammatory and antioxidant effects, GP can be considered a potent agent that can contribute to the restoration of homeostasis to control levels or that can reduce different drug side effects (Table 2).

**Grape Pomace Models Polyphenols Content Antioxidant and Anti-Inflammatory Effects References** *Red grape pomace variety* Tempranillo variety (Burgos, Spain) Spontaneously hypertensive rats Proanthocyanidin, anthocyanins, quercetin -TAC increased; -decreased lipid peroxidation and carbonyl groups; -increased NO levels -increased HO-1, SOD2, eNOS gene expression; [98] Alicante and Pinot varieties (France) polyphenol-enriched Alicante Dextran Sulfate Sodium-Induced colitis in Wistar male Anthocyanins -improved histological score; -decreased MPO activity; -increased SOD activity in polyphenol-enriched Alicante; -decreased IL-1*α*, IL-6 IFN-*γ* levels; -decreased IL-1β levels in Alicante and Pinot; -decreased IL6, ICAM-1, MMP-9 gene expression; -decreased IL-1β, iNOS gene expression in Alicante and Pinot; -decreased TNF*α*, NF*κ*B p65, COX2 gene expression in polyphenol-enriched Alicante; [99] Malbec variety (Gualtallary, Mendoza, Argentina) High fructose diet-induced Metabolic syndrome in Wistar rats Quercetin, epicatechin, catechin, trans-resveratrol, ferulic, gallic, caffeic, syringic, p-coumaric acids -reduced CRP levels; -reduced NADPH oxidase activity; -increased adiponectin; -reduced resistin; -increased insulin sensitivity; [100] Dimrit grapes variety 96 laying Hens given different GP concentrations Catechin, Epicatechin, Gallocatechin, Epigallocatechin, Phenolic acids, Gallic acid, Caffeic acid, p-cumaric acid -decreased plasma MDA levels; -decreased egg yolk MDA levels; [101] Red wine grape pomace 18 crossbreed lambs given different GP diets (5%, 10%) - -increased TAOC and SOD, GPX activity in longissimus dorsi muscle; -decreased ROS and MDA levels in longissimus dorsi muscle; [102] Red wine grape pomace 24 crossbreed ram lambs under pen conditions given GP diets (5%, 10%) - -decreased MDA and ROS levels in lamb testes; -increased CAT, SOD, GPx4 activity in lamb testes; -increased TAOC (GP 10%); -increased SOD, GPx4 mRNA expression (GP 10%); -increased CAT, SOD, GPx4 protein abundance; [103] Tempranillo variety Wistar rats given high-fat diet - -decreased IL-1β and TNF-α levels; -increased FRAP plasma and liver levels; -increased liver GSH/GSSG ratio; -decreased plasma and liver MDA and carbonyl groups levels; -decreased 8-hydroxydeoxyguanosine plasma levels; [104]

**Table 2.** *In vivo* beneficial actions of grape pomace in oxidative stress and inflammation.



Abbreviations: AOPP—advanced oxidation protein product; CARB—protein carbonyls; CAT—catalase; COX 2 cyclooxygenase 2; CRP—C-reactive protein; DPPH—2,2-diphenyl-1-picrylhydrazyl; FRAP—ferric ion antioxidant reducing power; GP—grape pomace; GPx—glutathione peroxidase; G-GCS—G-synthase glutamyl cysteine; GSH—glutathione; GSSG—glutathione disulfide; GST—glutathione-s-transferase; HO-1—heme oxygenase 1; ICAM-1—Intercellular Adhesion Molecule 1; IFN-G—interferon gamma; MDA—malondialdehyde; MMP-9 matrix metalloproteinase 9; MPO—myeloperoxidase activity; NADPH—nicotinamide adenine dinucleotide phosphate; NF-*κ*B p65—nuclear factor kappa-light-chain-enhancer of activated B cells transcription factor; eNOSendothelial nitric oxide synthase; iNOS—inducible nitric oxide synthase; NO—nitric oxide; oxLDL- Oxidized lowdensity lipoprotein; PAI-1—Plasminogen activator inhibitor-1; ROS—reactive oxygen species; SOD—superoxide dismutase; TAC—total antioxidant capacity; TAOC—total antioxidant capacity; TAS—total antioxidant status; TBARS—thiobarbituric acid reactive substances; TNF-α—tumor necrosis factor alpha.

#### **6. Non-Steroidal Anti-Inflammatory Drugs**

Non-steroidal anti-inflammatory drugs (NSAIDs) represent a group of chemically distinct compounds that act through the same mechanism producing a reversible inhibition of cyclooxygenase (COX) 1 and 2 isoenzymes, enzymes that produce prostaglandins (PGs). The main representatives of the group of NSAIDs that act non-selectively on COX-1 and COX-2 are aspirin, ibuprofen, diclofenac, indomethacin, naproxen, ketorolac, piroxicam and meloxicam [119]. The main difference between these two isoenzymes is represented by the fact that while COX-1 is a constitutive enzyme which produce regularly PGs that have a protective role mainly on the stomach and kidney, COX-2 expression is induced by inflammatory stress and PGs resulted from this pathway lead to the swelling and pain associated with inflammation [120]. The ability of NSAIDs to reduce pain and swelling associated with several inflammatory diseases and the fact that NSAIDs are over-the-counter drugs, contributed to their large-scale use, being of the most sold drugs worldwide [121]. Besides these beneficial effects, NSAIDs have multiple adverse reactions, mainly related to COX-1 inhibition. Thus, the most common adverse reactions are the gastrointestinal ones. Long-term usage of NSAIDs could lead especially to gastric ulcer, but can also alter renal function and sodium exchange and could lead to hypertension and/or renal failure. On the cardiovascular system, COX inhibition could lead to heart failure exacerbation. On hepatic activity, COXs function alteration could lead to acute liver injury [121]. Hypersensitivity reactions are also described and manifest clinically by NSAIDs-exacerbated respiratory diseases (rhinosinusitis, bronchial asthma, pneumonitis), NSAIDs-exacerbated cutaneous diseases (urticaria, photo-contact dermatitis, angioedema), NSAIDs-induced diseases (nephritis, aseptic meningitis) and anaphylaxis [122–125]. Because of these multiple and heterogenous adverse effects associated with COX-1 inhibition, pharmacology researchers discovered other compounds from the class of NSAIDs, celecoxib, which selectively inhibits COX-2. However, although studies have shown a reduced adverse effect on the gastrointestinal system, namely a lower risk of gastric ulcer, this compound more frequently leads to acute coronary syndromes [121]. Besides the anti-inflammatory activity of

NSAIDs, another area of interest was represented by their antioxidant activity. In this way, Costa et al. (2006) focused on the antioxidant activity via the *in vitro* scavenging ability of ROS and reactive nitrogen species (RNS) of ibuprofen, flurbiprofen, fenbufren, fenoprofen, naproxen, ketoprofen and indoprofen [126]. They observed that the greatest scavenging activity of ROS for O2<sup>−</sup> was equal for fenbufen, flurbiprofen, indoprofen, ketoprofen, for H2O2 was equal for ketoprofen, indoprofen, fenbufen and for HO was equal for fenoprofen and ibuprofen. In the case of RNS, for NO the greatest scavenging activity was that of indoprofen, and for ONOO− was indoprofen [126].

However, in the *in vivo* studies no antioxidant activity was observed, but, on the contrary, a prooxidant effect was described. One example is represented by the study of Nawaz et al. (2021) who observed the effect of NSAIDs treatment on antioxidant status and oxidative stress in patients with rheumatoid arthritis [127]. They highlighted that the group of patients with rheumatoid arthritis under the treatment with NSAIDs, compared with the other three groups (control group, patients without rheumatoid arthritis and under the treatment with NSAIDs, patients with rheumatoid arthritis who did not take NSAIDs), showed the highest oxidative stress and the lowest free radical scavenging ability [127].

Taking into consideration all of the above, namely the multitude of adverse reactions associated with NSAIDs treatment, it is necessary to find a potent substitute for these drugs, one that has strong anti-inflammatory effects with as few adverse reactions as possible.
