3.1.4. Pharmacological Studies

Flavonoids, phytochemicals present in di fferent *T. esculenta* parts have pharmacological properties that are the focus of several studies, in which, among others, the anti-inflammatory, antioxidant and anti-tumor properties associated with these compounds are described. A study analyzed the anti-inflammatory activity of some flavonoids with a focus on the possible regulatory role in the activation of NLRP3 inflammasome. The mechanism proposed for inhibiting activation by flavonoids was based on the regulation of the expression of inflammasome components such as the amino-terminal pyrin domain (PYD), which interacts with the ASC pyrin domain (caspase

recruitment domain) to initiate the assembly of the inflammasome; the central nucleotide binding and oligomerization domain (NACHT), which has the ATPase activity necessary for NLRP3 oligomerization after activation; and C-terminal leucine-rich repeat (LRR) domain, whose function has not ye<sup>t</sup> been identified. These changes can prevent its assembly and lead to inhibition of caspase-1 activation and, consequently, maturation and secretion of pro-inflammatory cytokines [41,42].

Luteolin (**6**), a flavonoid found in *T. esculenta* seeds, has anti-inflammatory activity attributed to its ability to reduce the generation of reactive oxygen species (ROS) and inhibit the activation of NLRP3 inflammasome. Previous studies have described some Pattern-Recognition Receptors (PRR) located in the cytoplasm of cells, such as dendritic cells and macrophages, which are also involved in the induction of inflammatory responses [43]. Among these receivers, some belong to the family of NOD-like receptors (NRLs). NLRs are a large family of intracellular PRRs with similar structure [44,45]. Among the various types of NLRs, NLRP3 is recognized for responding to various stimuli, being responsible for the inflammasome activation (NLRP3 inflammasome), involved in the recruitment and activation of caspase-1 (pro-caspase 1) in association with the ASC adapter protein [46]. The role of activated caspase-1 is crucial for the conversion of pro-interleukin 1 beta (pro-IL-1β) and pro-interleukin 18 (pro-IL-18) into their mature and biologically active forms [47]. Thus, luteolin (**6**) is able to reduce the expression of interleukin-1 beta (IL-1β), a cytokine with a primary role in the inflammatory response, and interleukin-8 (IL-8), a chemokine that stimulates migration of immune cells. Another anti-inflammatory flavonoid found in *T. esculenta* seeds is rutin (**10**), which promotes the inhibition of the NLRP3 inflammasome activation through the negative regulation of the NLRP3, ASC and caspase-1 expression and reduced production of IL-1β and interleukin-18 (IL-18), which is also known as an interferon-γ inducing factor (IFN-γ) [41,48].

A previous study revealed the immunoregulatory properties of naringenin (**7**), a flavonoid also found in *T. esculenta* seeds. This compound promotes the inhibition of nuclear transcription factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and reduces the production of tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6), a pro-inflammatory cytokine [49].

Jhang et. al. [50] analyzed the therapeutic potential of catechin (**3**) and gallic acid (**5**), both found in *T. esculenta* fruits and possible anti-inflammatory properties were found. After subcutaneous injection in mice, whose inflammation was stimulated by the administration of monosodium urate (MSU), significant reduction in the production and secretion of IL-1β and IL-6 was observed. The secretion of IL-1β was modulated through two pathways that include the NF-κB pathway, which provides pro-IL-1β and the NLRP3 inflammasome pathway, which promotes the release of IL-1β from pro-IL-1β. The study demonstrated that catechin (**3**) and gallic acid (**5**) have potent activity to eliminate superoxide anions, consequently inhibiting MSU-induced IL-1β secretion and NLRP3 inflammasome activation. Catechin (**3**) also regulated the oxidative stress status in mitochondria through positive regulation of thioredoxin (TRX) and deglycase protein DJ-1 (DJ-1) and these e ffects prevented mitochondrial damage caused by MSU attack. These results sugges<sup>t</sup> that catechin intake has potential to prevent acute gou<sup>t</sup> attacks. In addition, researchers have demonstrated the role of these compounds on the intracellular calcium concentration, which is high during the inflammatory process, with significant reduction in calcium concentration by catechin (**3**), but not by gallic acid (**5**) (Figure 1a,b).

**Figure 1.** Molecular mechanism of flavonoids in inflammasome regulation: (**a**) Studies sugges<sup>t</sup> that oxidative stress is an important mediator of monosodiumurate (MSU) induced inflammation [51]. The formation of reactive oxygen species (ROS) induces nuclear translocation of Nuclear Factor-kappa B (NF-kB) via phosphorylation by IkB kinase, which binds to target DNA that regulates Pro-IL-1β and Pro-IL-8 gene expression. In addition, ROS dissociates the thioredoxin (TRX) and thioredoxin interaction protein (TXNIP) conjugation [52], and released TXNIP further recruits and binds to NLRP3 inflammasome, leading to the release of IL-1β [53] and IL-8. NLRP3 inflammasome consists of NLRP3, caspase recruitment domain (ASC), and pro-caspase-1. Mitochondrial ROS (MtROS) is also associated with NLRP3 inflammasome activation [53]. In the process of NLRP3 inflammasome activation, activated caspase-1 transforms pro-IL-1b and pro-IL-18 into mature IL-1b and IL-18, resulting in the release of inflammatory cytokines; (**b**) Flavonoid uptake occurs either via passive diffusion through the cell membrane, or through membrane bound transport proteins. Cut circles indicate different points of flavonoid action, inhibiting the process of inflammasome formation with subsequent inhibition of inflammatory events [54]. Phenolic compounds block the inflammatory process by inhibiting ROS formation, thereby reducing the formation of pro-inflammatory cytokines. The nature and position of substituents in relation to the hydroxyl group affect the activity of polyphenols. The easily ionizable carboxylic group contributes to the efficient hydrogen donation tendency of phenolic acids [55]. Gallic acid has high antioxidant activity rate. This is due to a beneficial influence of carboxylate on the antioxidant activity of phenolic acids [56]. The tricyclic structure of flavonoids, such as catechin, determines their antioxidant effect. Phenolic quinoid tautomerism and the localization of electrons over the aromatic system eliminate reactive oxygen species. These aromatic rings directly neutralize free radicals and increase antioxidant defense [57]; (**c**) DAMPs/PAMPs bind to their receptor on the cell membrane and activate a signaling cascade. As a consequence, activation and formation of NRLP3 inflammasome occur, where the formation of active caspase-1 catalyzes the cleavage and secretion of mature IL-1β and IL-18, leading to propagate inflammation [54]. ASC, caspase recruitment domain; C-JUN/JNK, c-Jun N-terminal Kinase; CARD, caspase recruitment domain; DAMPs, damage-associated molecular patterns; IκB, inhibitor of κB; IKKα, IkBkinase α; IKKβ, IkBkinase β; IL-1β, Interleukin 1-beta; IL-8, Interleukin 8; LRR, leucine-rich repeats; MAP3Ks, mitogen-activated protein 3 kinases; MEK, mitogen-activated protein kinase; MKK4, mitogen-activated protein kinasekinase 4; MKK7, mitogen-activated protein kinase kinase 7; MtROS, Mitochondrial ROS; MSU, monosodiumurate; NACHT, central nucleotide-binding and oligomerization domain; NEMO, NF-kappa-B essential modulator; NF-KB, Nuclear Factor-kappa B; p38a, p38 kinase α; p50, NF-KB, Nuclear Factor-kappa B 1 (NF-KB1); p65, RelA; PAMPs, pathogen-associated molecular patterns; PYD, pyrin domain; ROS, reactive oxygen species; TXNIP, thioredoxin interaction protein; TRX, thioredoxin; TXNIP, thioredoxin interaction protein.

For the inflammasome activation, danger signals such as Damage-Associated Molecular Patterns (DAMPs) or Pathogen-Associated Molecular Patterns (PAMPs) bind to PRRs on the cell membrane and trigger the activation of a signaling cascade, which includes the activation of NF-κB, MAPK and MAPK activating protein kinase (MEK), leading to activation and assembly of the inflammasome, a protein complex where active caspase-1 catalyzes the cleavage and secretion of IL-1β and IL-18, important pro- inflammatory signaling gents. In this context, flavonoids cross the membrane through passive or facilitated di ffusion using membrane-bound transport proteins. In the cell, di fferent flavonoids act in the same pathway or in di fferent pathways, blocking the formation of inflammasome and consequently inhibiting the inflammatory process (Figure 1c) [54].

The flavonoid quercetin (**2**), found in *T. esculenta* fruits, has anti-dyslipidemic activity, which is also associated with inflammation, since the accumulation of lipids is a factor that contributes to the inflammatory response and inflammasome formation. Quercetin (**2**) acts by suppressing the NLRP3 expression, inhibiting caspase-1 and the production of IL-1β, also reducing the levels of lipids, more specifically triacylglycerols [58].

In addition, quercetin (**2**) also has antioxidant activity, which is mainly mediated by its e ffects on glutathione (GSH), enzyme activity, signal transduction pathways and ROS. Increase in GSH levels in rats was observed after quercetin administration (**2**), which increases the antioxidant capacity of these animals, since GSH acts as hydrogen donor in the reaction of conversion of hydrogen peroxide into water, catalyzed by superoxide dismutase (SOD), reducing its toxicity. Quercetin (**2**) also acts by increasing the expression of endogenous antioxidant enzymes, including catalase (CAT) and glutathione peroxidase (GPx). In signal transduction pathways, quercetin (**2**) acts in the regulation of kinase protein activated by AMP (AMPK) and MAPK, stimulated by ROS, promoting antioxidant defense and maintaining the oxidative balance, since ROS lead to the activation of several pro-inflammatory and apoptotic signaling events mediated by p53, a cell cycle regulatory protein (Figure 2). Also, quercetin (**2**) inhibits the p38MAPK/inducible nitric oxide synthase (iNOS) signaling pathways, negative regulation of NF-κB levels and positive regulation of SOD activity to promote antioxidant activity [59].

**Figure 2.** Antioxidant effect of quercetin on enzyme activity, signal transduction pathways and reactive oxygen species (ROS). Several conditions and environmental factors can increase ROS production. Besides, the mitochondrial electron transport chain is an important source of intracellular ROS generation. Flavonoid uptake occurs either via passive diffusion through cell membrane, or through membrane bound transport proteins [54]. After entering the cell, quercetin acts through the regulation of the enzyme-mediated antioxidant defense system and the non-enzymatic antioxidant defense system. Nuclear factor erythroid 2–related factor 2 (NRF2), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase (MAPK) pathways induced by ROS to promote the antioxidant defense system and maintain oxidative balance can also be regulated by phenolic compounds such as quercetin [59]. Through the neutralizing effect of ROS, quercetin can develop important anti-inflammatory effect due to inhibition of the Nuclear Factor-kappa B (NF-KB) pathway, preventing the activation of NRLP3 inflammasome (shown in Figure 1B). Through the p53 pathway, ROS induce apoptotic events. Therefore, quercetin can prevent apoptosis induced by excess ROS. In addition, it enhances the production of Apurinic/apyrimidinic Endonuclease 1/ Redox Effector Factor 1 (APE1/Ref1), activation of various signaling events and the NF-E2-related factor (NRF2)-mediated activation of genes, containing antioxidant response elements (ARE) and NF-κB [60–64]. AMPK, AMP-activated protein kinase; AP-1, activator protein 1; APE1, Apurinic/apyrimidinic endonuclease 1; ARE, antioxidant response element; Bax, BCL2 Associated X; CAT, catalase; CREB, cAMP-response element binding protein; EGR1, Early Growth Response 1; ERK, Extracellular signal-regulated kinase; GSH, glutathione; GSHPx, Glutathione peroxidase; IκB, κB inhibitor; JNK, c-Jun N-terminal Kinase; KEAP1, Kelch-like ECH-associated protein 1; Maf, musculoaponeurotic fibrosarcoma; MAPK, mitogen-activated protein kinase; MtROS, Mitochondrial ROS; NF-KB, Nuclear Factor-kappa B; Nrf2, nuclear factor erythroid 2–related factor 2; PDGFR, Platelet-derived growth factor receptors; PI3K, phosphatidylinositol-3-kinase; Ref-1, redox effector factor 1; ROS, reactive oxygen species; SOD, Superoxide dismutase.

*p*-Coumaric acid (**8**), identified in *T. esculenta* fruits, presents a phenyl hydroxyl group in its molecular structure, capable of promoting the neutralization of free radicals such as superoxide anion 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH) and hydrogen peroxide (H2O2). This antioxidant property is intensified after conjugation with quinic acid (**9**), also found in *T. esculenta* fruits. In addition, *p*-coumaric acid (**8**) also has antimicrobial activity tested against three Gram-positive bacteria (*Streptococcus pneumonia, Staphylococcus aureus* and *Bacillus subtilis*) and three Gram-negative bacteria (*Escherichia coli*, *Shigelladys enteriae* and *Salmonella typhimurium*), by increasing the permeability of the bacterial membrane and binding to the phosphate anion of the DNA (deoxyribonucleic acid), altering the processes of bacterial transcription and replication [65].

In a previous study, researchers analyzed the antioxidant properties of *T. esculenta* fruits using two tests, the scavenging of DPPH radicals and the iron reduction capacity. Antioxidant activity was detected in the seed extract, in which naringenin (**7**), luteolin (**6**) and rutin (**10**) flavonoids were identified and also for pulp extracts, where phenolic compounds such as gallic acid (**5**), *p*-coumaric acid (**8**), rutin (**10**), catechin (**3**), epicatechin (**4**) were also found, as well the cyclitol quinic acid (**9**), to which antioxidant activity can be attributed [33].

Flavonoids mirecetin (**1**) and quercetin (**2**), also found in *T. esculenta* fruits, have significant antiproliferative activity, suggesting a chemopreventive and anti-tumor potential that should be investigated in the future [32]. [35] reported two other properties of *T. esculenta* phytochemicals, diuretic and antihypertensive. Studies have shown that the hydroalcoholic extract obtained from *T. esculenta* leaves and stem promotes significant increase in urinary volume, without changing urine pH and density, indicating a diuretic effect, and significant increase in renal potassium elimination, which are properties related to phenolic acids and flavonoids found in extracts.

Pinheiro et al. [66] analyzed the possible antifungal activity of lectin extracted from *T. esculenta* seeds. This activity was tested on *Microsporum canis*, a filamentous keratinophilic fungus that causes infections in skin, hair and nails in humans and animals. The results show the ability of lectin to inhibit the growth of the fungus, which may be associated with the interaction of lectin with carbohydrates on the surface of microorganism such as d-mannose and *N*-acetyl-glucosamine, since the addition of these carbohydrates caused inhibition of the antifungal effect, probably due to competition for the interaction of fungal carbohydrates with lectin. The ability of lectin to inhibit the adherence of microorganisms and exert antimicrobial effects was analyzed in another study, which tested such properties on bacteria *Streptococcus mutans* UA159, *Streptococcus sobrinus* 6715, *Streptococcus sanguinis* ATCC10556, *Streptococcus mitis* ATCC903 and *Streptococcus oralis* PB182. The results indicate that lectin was not able to inhibit the growth of bacteria at any of the applied concentrations and also did not inhibit the adherence of microorganisms, that is, it does not present antimicrobial activity and does not inhibit biofilm formation [67].
