**7. Comparative Effect of Polyphenols Found in Grape Pomace Versus Non-Steroidal Anti-Inflammatory Drugs in Oxidative Stress and Inflammation**

Several diseases use NSAIDs to decrease vasodilatation, cell adhesion to vascular endothelium, cells migration to inflammation site, cytokine synthesis, and further tissue damage. However, as we already mention before, NSAIDs also possess several adverse reactions (gastrointestinal bleeding, hepatic, renal toxicity), which can cause a limitation in use for more extended periods. This led to studying different natural compounds to compare their effects with different NSAIDs (Table 3).

Unfortunately, we were unable to find studies in which the anti-inflammatory and antioxidant effects of GP whole extract were compared with those induced by NSAIDs, but only of several other compounds extracted from GP, such as resveratrol, flavonol (quercetin) and hydroxybenzoic acid (gallic acid). Therefore, this section will examine the differences between *in vitro* and *in vivo* effects of NSAIDs and different phenolic compounds that are also found in GP.

Thus, Zhang et al. (2021), subjected rat glial cells to LPS induced-inflammation and treated them with different doses of resveratrol (0.1–20 μM) or ibuprofen to see the differences [136]. Resveratrol showed a higher decrease in TNF-α (0.1–20 μM) and ROS production (20 μM) compared to ibuprofen [136]. In another study, horse immune cells were cultured with resveratrol or different NSAIDs (flunixin meglumine or phenylbutazone) to observe the effects on pro-inflammatory cytokine production. Resveratrol managed to lower interferon (IFN)-Gand TNF-α levels, similar to of NSAIDs [137].

As for*in vivo* studies, resveratrol patches were given to rats injected with carrageenan [138]. Diclofenac gel was used to compare the effects of the two substances. Resveratrol and diclofenac decreased paw swelling, but resveratrol caused a much longer inflammation reduction when compared to diclofenac. Also, the resveratrol group presented the lowest paw swelling compared to the diclofenac and control groups [138].

Another polyphenol found in GP and recognized for its anti-inflammatory and antioxidant effects is represented by quercetin, which is a flavonol. In Wei et al. study (2019), osteoarthritis was surgically induced in the left knee in rats, that were also treated with quercetin or celecoxib (CXB) as a reference drug [139]. In terms of antioxidant effects, quercetin and CXB groups managed to increase SOD serum and synovial fluid levels compared to the untreated group. Metalloproteinases (MMPs) are a family of enzymes responsible for cartilage degradation and osteoarthritis appearance [139]. Tissue inhibitor metalloproteinase (TIMP) is accountable for cartilage repair through the downregulation

of MMPs [139]. In this study, quercetin and CXB groups decreased MMP-13 in serum, synovial fluid, and synovium, while TIMP-1 levels increased in these groups compared to the untreated osteoarthritis group [139]. In this work, quercetin presented comparable effects with celecoxib, both having protective effects [139]. Heydari Nasrabadi et al. (2022) also analyzed the effects of quercetin in monosodium iodoacetate-induced osteoarthritis in rats injection [140]. In this experiment, ibuprofen was used as a reference drug. In histopathological studies of the rats' knees, the inflammatory cells were significantly decreased in the treated groups (quercetin and ibuprofen) compared to the untreated group. In addition, MMP-3 and MMP-13 were measured, and the results showed that the quercetin group reduced, even more, the levels of MMPs compared to the ibuprofen group [140].

**Table 3.** Grape pomace polyphenols versus non-steroidal anti-inflammatory drugs in oxidative stress and inflammation.


Abbreviations: COX2—cyclooxygenase 2; iNOS—inducible nitric oxide synthase; MCP-1—monocyte chemoattractant protein-1; MMP-13—mettaloproteinase 13; NF-*κ*B p65—nuclear factor kappa-light-chain-enhancer of activated B cells transcription factor; NOx—serum total nitrate/nitrite; PGE2—prostaglandin E2; RES—resveratrol; TBARS—thiobarbituric acid reactive substances; TNF-α—tumor necrosis factor alpha.

When taking into consideration the oxidative stress induced by the treatment with NSAIDs, other researchers focused on finding a substance that could limit this pro-oxidative side effect. In this matter, a great phenolic compound found in high quantities in GP, gallic acid, has proven to be a potent adjuvant in this case. Thereby, Moradi et al. (2021) evaluated if gallic acids taken alongside diclofenac had an impact on the oxidative status of 30 Wistar rats divided into 5 groups [141]. They observed that while only under diclofenac treatment there was a significant increase in urea, AST, ALT, creatinine, uric acid serum levels, and in IL-1β expression, in the group which received gallic acid after diclofenac there were registered reduced levels of these biochemical parameters and gene expression [141]. Moreover, the treatment with gallic acid alongside diclofenac led to a decrease in nitrate content, serum and renal MDA levels, and protein carbonyl level. Furthermore, on antioxidant enzymes activity, treatment with diclofenac and gallic acid led to an increase in renal SOD and CAT activities and renal GSH levels. The histopathological examination of the kidney highlighted that the group which received both diclofenac and gallic acid presented a reduced lymphocytic cell infiltration, focal hemorrhage, and vacuolar degeneration of tubular epithelial cells in comparison with the group which received only diclofenac [141]. Another study that reinforces these observations is that of Esmaeilzadeh et al. (2020) who investigated the hepatoprotective activity of gallic acid in diclofenac-induced liver toxicity in 30 Wistar rats [142]. They observed that while diclofenac led to an increase in GOT, GPT, ALP, and total bilirubin levels, gallic acid reduced the levels of these parameters. Moreover, at a dose of 100 mg/kg gallic acid, there was observed a significantly decreased compared with a dose of 50 mg/kg gallic acid [142]. The same difference between these two doses was also observed in the increase of plasma antioxidant capacity, hepatic SOD, GPx, GSH, and CAT activities and in the decrease of nitrite content, protein carbonyl levels, serum, and liver MDA levels compared with the group which received only diclofenac. The histopathological examination of the liver, both doses of gallic acid led to a significantly reduced in lymphocytic cell infiltration and liver degeneration [142].

These *in vivo* studies also suggest that GP is a valuable candidate that could be used as a potential therapeutic agent capable of reducing oxidative stress and inflammation and also as an adjuvant treatment in the attempt to reduce the side effects.

### **8. Conclusions**

GP extracts due to their rich content in flavonols, anthocyanins, anthocyanidins, flavanols, proanthocyanidins, stilbenes, and phenolic acids are offering new perspectives as possible therapeutic agents. In vivo and *in vitro* studies showed promising results for both GP whole extracts and different types of polyphenols that are contained in it, in reducing oxidative stress and inflammatory markers in different models of chronic inflammation.

The first evidence in the direction of this possible therapeutic use was represented by in vitro studies. In these works, the antioxidant effects were demonstrated especially by the decrease in ROS, MDA, and TBARS levels and by the increase in GSH levels. The anti-inflammatory effects were given by the inhibition of some inflammatory pathways such as NF-kB and PGE2, an inhibition that led to a decrease in levels of inflammatory markers such as IL-8. These antioxidant and anti-inflammatory effects are also validated in *in vivo* studies. In terms of antioxidant activity, in addition to the effects already observed in the *in vitro* studies, an increase in CAT, SOD, and GPx4 levels and stimulation of eNOS gene expression were also seen. Similarly, the *in vivo* studies brought additional data regarding the anti-inflammatory activity, observing an inhibition of the release of several inflammatory markers such as IL-1*α*, IL-1β, IL-6, IFN-*γ*, TNF-α, and CRP. Anyway, it is still necessary to implement this research in clinical trials to be able to conclude the possible use of GP as an antioxidant and anti-inflammatory therapeutical agent. Further, most of the studies have shown that GP extracts are more effective than a single polyphenol, possibly because of polyphenols synergistic action that interferes with more than one pathophysiological mechanism. Compared to the antioxidant and anti-inflammatory effects of NSAIDs, the literature currently offers only the effects of single polyphenols, such as

resveratrol, quercetin, and gallic acid. However, single polyphenols possess similar antiinflammatory and antioxidant effects as the classical NSAIDs. Thereby, further comparative studies are needed to investigate if the entire complex of polyphenols that exists in GP may induce a similar or a better effect. The question that rises from these studies is whether the best results can be obtained when single isolated phenolic compounds with a known concentration are used or whether a standardized mixture of these compounds is used? If this hypothesis is validated, in the future GP could become an add-on therapeutic measure that could be used for better control of chronic inflammation than monotherapy with NSAIDs.

**Author Contributions:** Conceptualization, V.S.C., S, .O.M., I.C.B. and R.M.P.; methodology, S, .O.M., D.C.M. and A.M.L.; software A.M.L.; validation, V.S.C., I.C.B. and A.D.B.; formal analysis, V.S.C. and I.C.B.; investigation, S, .O.M., D.C.M. and A.M.L.; resources, A.D.B.; data curation, V.S.C.; writing original draft preparation, V.S.C., S, .O.M. and R.M.P.; writing—review and editing, R.M.P. and A.D.B.; visualization, I.C.B.; supervision, R.M.P.; project administration, V.S.C.; funding acquisition, I.C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
