Gluten-Free White Quinoa Flour Exhibits Antioxidant and Anti-Inflammatory Activity

Abstract

Gluten-free foods may help address oxidative stress and inflammation linked to gluten-related disorders. This study characterized the phytochemical profile of a 70% ethanolic extract from commercial white quinoa (Chenopodium quinoa Willd.) flour (Peru) and evaluated its antioxidant and anti-inflammatory activity in vitro and in vivo in a rat model of acute inflammation. Total polyphenols and flavonoids were quantified spectrophotometrically, while individual phenolics were profiled by HPLC-DAD-ESI-MS. Antioxidant capacity was assessed in vitro using DPPH, FRAP, H₂O₂, and nitric oxide (NO) scavenging assays. For in vivo testing, male Wistar rats received for 10 days quinoa extract (100%—1 g/mL, 50–0.5 g/mL, or 25–0.25 g/mL) either therapeutically (after turpentine-induced inflammation) or prophylactically (before induction), with diclofenac and Trolox as reference controls; systemic oxidative stress (TOS, TAC, OSI, AOPP, MDA, NO, 3-NT, total thiols) and inflammatory mediators (NF-κB p65, IL-1β, IL-18, caspase-1, IL-10) were measured by spectrophotometry/ELISA and explored multivariately by PCA. Quinoa extract contained measurable phenolic and flavonoid levels (TPC 1.25 mg GAE/g d.w.; TFC 68.5 mg QE/100 g d.w.) and was dominated by flavonoid glycosides and hydroxybenzoic acids. It showed strong radical-scavenging/reducing activity in vitro. In vivo, the extract dose-dependently attenuated turpentine-induced nitro-oxidative stress and reduced key pro-inflammatory markers (notably NF-κB, IL-1β, IL-18, and caspase-1), in several endpoints matching or exceeding diclofenac/Trolox effects, while IL-10 was largely unchanged. These findings support white quinoa flour extract as a phytochemical-rich, gluten-free ingredient with promising antioxidant and anti-inflammatory potential, warranting further translational investigation.

1. Introduction

Celiac disease is a chronic autoimmune enteropathy in which dietary gluten triggers intestinal inflammation in genetically susceptible individuals [1]. Recent reports indicate that the incidence of celiac disease and gluten intolerance is increasing worldwide [2], reflecting a multifactorial interplay between genetic risk and environmental exposures [3]. Gliadin peptides can damage the intestinal mucosa through immunomodulatory effects and by amplifying oxidative and inflammatory pathways. Clinically, gastrointestinal manifestations may be accompanied by extra-intestinal symptoms, likely because intestinal inflammation increases antigen exposure, autoantibody production, and systemic immune activation [4,5]. A strict gluten-free diet remains the cornerstone of management and is usually associated with clinical improvement [4,5]. In addition, diets enriched in antioxidant micronutrients and phytochemicals (e.g., vitamins C and E, polyphenols, and carotenoids) may help modulate oxidative stress and inflammatory mediator production, supporting intestinal barrier integrity [6].

Quinoa (Chenopodium quinoa Willd.) has attracted considerable attention from researchers and consumers because it fits well within gluten-free dietary patterns [7]. Originating in the Andean region of South America, quinoa has been cultivated for millennia and is now grown across North America, Europe, and Asia as its popularity has expanded [8]. Although it is botanically a pseudocereal, quinoa is used similarly to grains and provides a dense nutrient profile [8]. Quinoa flour can help meet the needs of gluten avoidance while also improving overall dietary quality, in part because quinoa provides a complete protein with all nine essential amino acids [2]. It is also described as having a low glycemic index and is valued in plant-based diets [9]. Beyond macronutrients, quinoa contains a broad range of bioactive constituents (saponins, phenolics, flavonoids, betalains, and phytosterols) and is a source of fiber and minerals such as magnesium and phosphorus [10]. Quinoa has even been highlighted for its suitability in constrained diets, including those considered for space missions [2]. These features contribute to quinoa’s reputation as a functional food and support continued interest in its potential health benefits [9,10].

Quinoa-derived metabolites have been associated with multiple biological effects, including antioxidant, cytotoxic, antidiabetic, and anti-inflammatory activities [8,9,10]. To our knowledge, there is no in vivo study analyzing the mechanisms of the antioxidant and anti-inflammatory activities of the whole-grain quinoa flour extract. A structure–activity relationship study of the main flavonoid aglycones from quinoa (kaempferol and quercetin) found their antioxidant activity and confirmed in vitro antioxidant effects. Only quinoa bran extract’s anti-oxidant and anti-inflammatory effects have been verified by in vivo studies, and the anti-inflammatory mechanism was NFkB pathway inhibition [9].

Therefore, the first objective of the present study was to analyze in vivo the antioxidant and anti-inflammatory effects and mechanisms of whole-grain quinoa flour ethanol extract through an experiment on turpentine-induced acute inflammation in rats. The second objective was to perform a correlation analysis between the oxidative stress and inflammation biomarkers. The third objective was to associate the phytochemical profile of a white quinoa flour ethanolic extract with the antioxidant and anti-inflammatory activities.

2. Results

2.1. Phytochemical Analysis

The quinoa flour ethanolic extract contained 1.25 ± 0.02 mg GAE/g d.w. (TPC) and 68.5 ± 0.52 mg QE/100 g d.w. (TFC).

HPLC analysis indicated that the phenolic profile of the quinoa extract was dominated by flavonoid derivatives (approximately 70% of the quantified phenolics), whereas phenolic acids accounted for about 30%. The main constituents were kaempferol-rhamnosyl-rhamnosyl-glucoside (peak 8; ~28% of total quantified phenolics), 2,3-dihydroxybenzoic acid (peak 2; ~20%), and quercetin-rhamnosyl-rhamnosyl-glucoside (peak 6; ~13%). Quercetin-xylosyl-glucoside (peak 9) was also detected at a relatively high level (~12%). Details are shown in Figure 1 and

Table 1. HPLC-DAD-ESI-MS profile of phenolic compounds in white quinoa flour ethanolic extract.

Peak No.	Rt (min)	UVλmax (nm)	[M+H]+ (m/z)	Compound	Concentration (μg/mL)
1	2.75	280	139	2-Hydroxybenzoic acid *	28.15 ± 1.66
2	3.06	275	155	2,3-Dihydroxybenzoic acid *	162.14 ± 0.74
3	9.35	280	155	Protocatechuic acid *	23.32 ± 0.10
4	10.32	280	155	2,4-Dihydroxybenzoic acid *	16.99 ± 0.46
5	13.13	290	169	Vanillic acid *	11.34 ± 0.04
6	13.87	360, 255	757, 303	Quercetin-rhamnosyl-rhamnosyl-glucoside **	104.80 ± 1.10
7	14.23	360, 255	743, 303	Quercetin-xylosyl-rhamnosyl-glucoside **	63.78 ± 0.58
8	14.51	350, 260	741, 287	Kaempferol-rhamnosyl-rhamnosyl-glucoside **	225.81 ± 1.13
9	14.95	360, 255	597, 303	Quercetin-xylosyl-glucoside **	97.39 ± 1.44
10	15.49	350, 250	481, 319	Myricetin-glucoside **	73.37 ± 0.66
				Total phenolics	813.10 ± 3.13

* Compounds belonging to hydroxybenzoic acids subclass; ** compounds belonging to flavonols subclass.

.

2.2. In Vitro Antioxidant Capacity Testing

The white quinoa flour ethanolic extract showed notable in vitro antioxidant and radical-scavenging activity (

Table 2. Antioxidant and anti-inflammatory activity in vitro of white quinoa flour ethanolic extract.

	DPPH μg TE/g d.w.	FRAP μg TE/g d.w.	H2O2 Scavenging Activity mg TE/g d.w.	NO Scavenging Activity μM QE/mL/g d.w.
Extract	265.31	277.75	155.10	232.47
TROLOX	11.2	19.97	24.23	-
Quercetin	-	-	-	20.58

DPPH = 2,2-diphenyl-1-picrylhydrazyl radical scavenging; H₂O₂ = hydrogen peroxide scavenging activity; FRAP = ferric-reducing antioxidant power; NO = nitric oxide scavenging activity; TE = Trolox equivalents; QE = quercetin equivalents.

). Compared with Trolox, the extract exhibited higher DPPH scavenging, H₂O₂ scavenging, and ferric-reducing power (p < 0.01). NO scavenging was also significantly higher when compared with quercetin (p < 0.01).

2.3. In Vivo Antioxidant and Anti-Inflammatory Activity

2.3.1. Therapeutic Plan Effects

Induction of acute inflammation by intramuscular turpentine oil produced pronounced oxidative stress in the inflammation (INFL) group compared with controls. Total oxidative status (TOS), oxidative stress index (OSI), advanced oxidation protein products (AOPP), malondialdehyde (MDA), nitrites (NO), and 3-nitrotyrosine (3-NT) increased significantly (p < 0.001), whereas total antioxidant capacity (TAC) (p < 0.001) and total thiols (SH) (p < 0.01) decreased. Diclofenac and Trolox reduced TOS, OSI, AOPP, MDA, NO, and 3-NT (p < 0.001) and increased TAC and SH (p < 0.01) (Figure 2).

Therapeutic administration of the white quinoa flour ethanolic extract attenuated nitro-oxidative stress by decreasing NO synthesis and markers of protein and lipid oxidation (p < 0.001), while increasing antioxidant capacity (TAC) and thiols (SH) (p < 0.01). For TOS, OSI, AOPP, and NO, the inhibitory effects were stronger than those observed with diclofenac or Trolox. For TAC, only Q100 produced a larger increase than diclofenac or Trolox. SH increased more in the Trolox group than in the quinoa extract group (Figure 2).

NF-κB, IL-1β, IL-18, and caspase-1 were markedly elevated in the INFL group versus controls (p < 0.001), and diclofenac and Trolox reduced these parameters (p < 0.01). Therapeutic treatment with the white quinoa flour ethanolic extract lowered these inflammatory mediators in a concentration-dependent manner, with Q100 showing the strongest anti-inflammatory effect. For caspase-1 and IL-18, the quinoa extract produced greater inhibition than diclofenac or Trolox. IL-10 was not influenced by inflammation or by the tested treatments (Figure 3).

PCA was performed to examine the multivariate relationships among oxidative stress and inflammatory biomarkers. Three principal components were retained, explaining 75.9% of the total variance (PC1: 54.4%, PC2: 12.9%, PC3: 8.6%). Communalities after extraction were generally high, indicating adequate representation of the variables within the reduced component space. The rotated solution showed that PC1 was characterized by strong positive loadings for NF-κB, caspase activity, TOS/OSI, MDA, IL-1β, IL-18, NO, and 3-NT, together with negative loadings for SH and TAC, defining a dominant axis of oxidative stress and inflammatory burden inversely associated with antioxidant capacity. PC2 was primarily associated with AOPP, NO, and SH, reflecting variability related to protein oxidative modification and nitric oxide-dependent processes. PC3 was mainly defined by IL-10, identifying an independent regulatory inflammatory dimension. Correlations between components were moderate to low, indicating that these components represent related but distinct biological patterns. Group-specific correlation circles further illustrated a progressive redistribution of biomarkers from the inflamed profile toward lower oxidative-inflammatory load with increasing concentration of the extract (Figure 4).

2.3.2. Prophylactic Plan Effects

Intramuscular turpentine oil induced acute inflammation and pronounced oxidative stress, increasing TOS, OSI, AOPP, MDA, NO, and 3-NT (p < 0.001) and decreasing TAC (p < 0.001) and SH (p < 0.01). Trolox pretreatment efficiently limited the rise in TOS, OSI, AOPP, MDA, NO, and 3-NT (p < 0.001) and attenuated the reductions in TAC and SH (p < 0.01) (Figure 5).

In the prophylactic setting, pretreatment with the white quinoa flour ethanolic extract mitigated nitro-oxidative stress after turpentine oil administration. Oxidant markers (TOS, OSI, AOPP, MDA, NO, and 3-NT) showed smaller increases (p < 0.001), accompanied by higher TAC and SH (p < 0.01). Only 3-NT and SH were more strongly influenced by Trolox prophylaxis; for the remaining oxidative stress markers, the quinoa extract produced a more pronounced protective effect.

NF-κB, IL-1β, IL-18, and caspase-1 were markedly increased in the INFL group versus controls (p < 0.001), and Trolox reduced these parameters (p < 0.01). In the prophylactic design, both Trolox and the white quinoa flour ethanolic extract prevented the increase in NF-κB, IL-1β, IL-18, and caspase-1 in a concentration-dependent manner, with higher extract concentrations showing stronger inhibition. IL-10 was not significantly affected by inflammation; however, Q50 and Q25 increased this cytokine (p < 0.05) (Figure 6).

Principal component analysis (PCA) was performed to investigate the multivariate relationships among oxidative stress and inflammatory biomarkers in the prophylactic setting. Based on eigenvalues > 1 and inspection of the scree plot, three principal components were retained, explaining 81.5% of the total variance (PC1: 51.8%, PC2: 19.7%, PC3: 10.0%). Communalities after extraction were generally high (0.52–0.97), indicating that most variables were well represented within the reduced component space. The oblimin-rotated solution revealed that PC1 was characterized by strong positive loadings of 3-NT, NFκB, caspase activity, IL-1β, IL-18, MDA, NO, and OSI/TOS, together with negative loadings for SH and TAC, defining a dominant axis integrating oxidative stress burden and inflammatory signaling inversely related to antioxidant capacity. PC2 was primarily associated with NO, AOPP, and SH, reflecting variability related to protein oxidative modification and nitric oxide-dependent processes. PC3 was mainly defined by IL-10, identifying an independent regulatory inflammatory dimension. Correlations among components were low to moderate, confirming that these components represent related but distinct biological patterns. This analysis demonstrates a clear separation between inflamed and prophylactically treated groups and reveals a dose-dependent redistribution of oxidative and inflammatory biomarkers across the principal component space (Figure 7).

3. Discussions

Overall, the present work characterizes the polyphenol profile of white quinoa flour and links this composition to measurable antioxidant and anti-inflammatory effects.

Phenolic compounds are plant secondary metabolites that contribute to health-promoting properties. They can influence carbohydrate and lipid metabolism and have been associated with antioxidant, anti-inflammatory, and antiproliferative activities, which may support the prevention of non-communicable diseases. Reported phenolic levels in quinoa vary widely, largely because genotype, cultivation conditions, and post-harvest processing affect composition [11]. In this study, we quantified phenolics in quinoa flour extract using both spectrophotometric assays and chromatographic profiling.

Total phenolic content is commonly estimated using the Folin–Ciocalteu assay [12,13]. The TPC of our Peruvian sample (1.25 mg GAE/g d.w.) was lower than values reported for other Peruvian samples (5 mg GAE/g d.w.) and for products from the USA market (4.7 mg GAE/g d.w.), the Korean market (3.84 mg GAE/g d.w.) [14], and the Italian market (1.8 mg GAE/g d.w.) [7]. Higher TPC is typically observed in pigmented varieties compared with white ones, and milling degree can also reduce phenolics because these compounds are concentrated in the outer grain layers [7].

Flavonoids represent a major subgroup of phenolics and are often reported as total flavonoid content (TFC) in quercetin equivalents. Flavonoid classes include flavonols, flavan-3-ols, flavones, isoflavones, flavanones, anthocyanidins, and chalcones, many of which exhibit antioxidant activity. Quantifying TFC helps estimate how quinoa may contribute to the dietary intake of functional phytochemicals. In the literature, TFC values for quinoa have been reported at 2.18 mg QE/100 g d.w. for other Peruvian samples, 1.76 mg QE/100 g d.w. for USA samples, and 1.60 mg QE/100 g d.w. for Korean samples (Lee & Sim, 2018), which are lower than the TFC measured here (68.5 mg QE/100 g d.w.) [14]. Processing can increase measured TFC values [7].

The influence of genotype, agroecological factors, and crop management on quinoa bioactive composition remains insufficiently explored [10], and extraction protocols can substantially affect recovery yields [9]. Our HPLC-DAD-ESI-MS analysis identified two dominant phenolic groups in the white quinoa ethanolic extract—phenolic acids and flavonoids—which together represent the most abundant dietary phenolics [7]. Phenolic acids can act as antioxidants via radical scavenging and metal chelation, thereby limiting oxidative stress and contributing to chronic disease prevention [8,9]. In our profile, 2,3-dihydroxybenzoic acid was the most abundant phenolic acid and is reported to rank among the stronger antioxidants within hydroxybenzoic acids [15]. Flavonoids contribute through multiple mechanisms, including direct ROS scavenging, inhibition of oxidase-derived superoxide, induction of antioxidant enzymes, and trace metal chelation [16]. Several flavonol glycosides were detected (e.g., kaempferol-3-O-rhamnosyl-rhamnosyl-glucoside, quercetin-rhamnosyl-rhamnosyl-glucoside [17], and quercetin-xylosyl-glucoside [18]. Although glycosylation can lower the intrinsic antioxidant activity compared with the corresponding aglycones [19], dietary glycosides may be hydrolyzed by gut microbiota in vivo, releasing aglycones that enhance antioxidant defenses [20]. Taken together, our phytochemical data support white quinoa ethanolic extract as a relevant source of natural antioxidants.

Because oxidative stress contributes to the pathogenesis of many disorders, we further assessed the antioxidant potential of the quinoa extract. Using DPPH, FRAP, H₂O₂, and NO assays, the white quinoa flour ethanolic extract demonstrated strong in vitro activity, exceeding the reference antioxidant TX in these tests. This performance is consistent with the polyphenol-rich composition of the extract and likely reflects synergistic effects among its constituents [11].

Although quinoa phytochemistry has been widely described, additional work is needed to clarify the mechanisms underlying its antioxidant and anti-inflammatory effects and to inform evidence-based recommendations for specific health contexts.

Oxidative stress has been implicated in a broad range of disorders, including cancer, Alzheimer’s disease, atherosclerosis, and depression. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are continuously generated during metabolism, mitochondrial activity, and immune responses. ROS include free radicals such as superoxide (O₂•⁻) and hydroxyl radical (HO•), as well as non-radical oxidants such as hydrogen peroxide (H₂O₂); additional oxidants include lipid and protein peroxides and oxidized nucleic acids. RNS include nitric oxide and peroxynitrite [21]. Beyond causing damage at high levels, certain ROS also participate in redox signaling, so disruptions in their steady-state levels can alter cellular signaling pathways [11]. Organisms counterbalance oxidant production through low-molecular-weight antioxidants (e.g., tocopherols, ascorbate, uric acid, melatonin) and antioxidant enzymes; oxidative stress may develop when oxidant generation increases and/or antioxidant defenses decline. For this reason, oxidative stress is typically evaluated using a panel of markers that reflect oxidatively modified biomolecules and changes in enzymatic or non-enzymatic antioxidant capacity [21].

There is a strong interest in identifying safe, plant-derived compounds that can counteract oxidative stress. Dietary antioxidants can help neutralize endogenously generated free radicals and support the body’s antioxidant defenses, potentially lowering the risk of oxidative stress-related metabolic and chronic diseases [9]. Whole grains are rich in phytochemicals and may help limit oxidative stress-driven inflammation [22]. In our model, quinoa flour ethanolic extract reduced total oxidative status (TOS) and oxidative stress index (OSI) and increased total antioxidant capacity (TAC) in vivo. These effects likely reflect synergism among quinoa phenolics and flavonoids [23]. To better interpret the mechanisms, we also assessed additional biomarkers that capture oxidative damage and adaptive antioxidant responses [24].

Advanced oxidation protein products (AOPPs) are generated when proteins—particularly albumin—undergo oxidative modification. Their accumulation can exacerbate cellular dysfunction and inflammation and is associated with disturbed redox homeostasis [25]. In both therapeutic and prophylactic designs, white quinoa extract reduced AOPP levels; in the therapeutic plan, the reduction was stronger than that observed with diclofenac or Trolox. Quinoa’s antioxidant constituents, including polyphenols, flavonoids, and vitamin E, may limit the oxidative reactions that contribute to AOPP formation [26]. Additionally, quinoa’s balanced amino acid profile may support protein turnover and recovery under oxidative conditions. Further studies should clarify the pathways through which quinoa influences AOPPs.

Malondialdehyde (MDA), a lipid peroxidation byproduct, is widely used as an indicator of oxidative damage. Elevated MDA has been linked to cardiovascular and neurodegenerative diseases, aging, and cancer [21]. Prior studies reported that quinoa consumption can reduce oxidative stress and lower plasma MDA [11,27]. Consistent with these observations, quinoa flour ethanolic extract reduced MDA in both the therapeutic and prophylactic plans, with an effect comparable to Trolox. This suggests that quinoa-derived antioxidants can limit lipid peroxidation and may support preventive nutritional strategies in conditions associated with increased MDA.

Nitric oxide (NO) is a signaling molecule produced from L-arginine by nitric oxide synthases (NOS) and participates in multiple physiological processes [28]. During inflammation, inducible NOS (iNOS) in activated immune cells can generate excessive NO, contributing to tissue injury; thus, NO is commonly used as an inflammation-related marker and therapeutic target. Diet and metabolic factors can influence NO biology [28]. Quinoa provides nutrients relevant to vascular and redox homeostasis, including magnesium (an enzymatic cofactor), arginine (NO precursor), potassium, and antioxidant phytochemicals. In our study, quinoa flour ethanolic extract reduced inflammation-triggered NO synthesis. This aligns with reports that quinoa components can inhibit NO production in LPS-stimulated macrophages [29] and that quinoa seed extracts may indirectly inhibit iNOS and lower NO levels [30]. Therapeutic administration produced stronger NO inhibition than diclofenac or Trolox, whereas prophylactic administration showed efficacy comparable to Trolox.

3-Nitrotyrosine (3-NT) forms when tyrosine residues are nitrated under nitro-oxidative conditions, often involving interactions between NO and ROS. It is used as a marker of inflammation-associated oxidative damage and has been reported to increase in cardiovascular, neurodegenerative, and chronic inflammatory diseases [24]. Measuring 3-NT in tissues or blood can therefore provide information on nitro-oxidative injury [31]. In our experiment, quinoa flour ethanolic extract reduced 3-NT, consistent with its overall lowering effect on oxidation and NO synthesis.

Total antioxidant capacity (TAC) offers a rapid estimate of overall antioxidant ‘power’, even though it does not identify the specific antioxidants present. In plasma, uric acid contributes substantially to TAC, and protein thiols (SH) represent another major component; SH is particularly sensitive to oxidative stress [32]. In our study, quinoa extract increased SH significantly, although the magnitude was smaller than the increase observed with Trolox. These findings support the view that quinoa extract can act as an exogenous antioxidant in this model.

Oxidative stress and inflammation are closely connected: oxidative injury can initiate inflammatory signaling, and inflammation can further amplify oxidative stress. The recruitment and activation of immune cells is coordinated by mediators such as NF-κB, the NLRP3 inflammasome, IL-1β, IL-18, and IL-10 [33]. While acute inflammation is protective, chronic inflammation contributes to cardiometabolic disease, autoimmunity, and cancer. Dietary patterns are increasingly recognized as modulators of inflammatory tone, and quinoa is of interest due to its bioactive profile. Most anti-inflammatory studies have focused on quinoa seeds [9]. Quinoa’s phytochemicals, particularly flavonoids, may reduce inflammation by influencing eicosanoid pathways and by inhibiting iNOS induction and expression [6,8,16]. In addition, quinoa whole flour supplies omega-3 fatty acids in small amounts, a complete amino acid profile, dietary fiber, minerals, and antioxidants, all of which may contribute to anti-inflammatory effects [23].

NF-κB is generally retained in the cytoplasm by inhibitory IκB proteins. Upon activation by cytokines, pathogens, or oxidative stress, IκBs are degraded and NF-κB translocates to the nucleus, where it drives the expression of genes involved in immune responses, inflammation, and cell survival [16]. Nutrition can modulate NF-κB signaling, particularly through antioxidant and anti-inflammatory dietary components. Several whole-grain quinoa constituents—including peptides, rutin, fatty acids, anthocyanins, and tocopherols—have been reported to inhibit NF-κB activation [9]. In our study, quinoa flour ethanolic extract rich in polyphenols reduced NF-κB levels too. Flavonoid glycosides/aglycones, such as quercetin and kaempferol from the quinoa flour extract, were found to inhibit NLRP3 inflammasome pro-inflammatory cytokines transcription by inhibiting NF-κB activation [34,35]. However, due to the low bioavailability of the flavonoids after oral administration, the effects may not be significant. In the therapeutic plan, the effect was comparable to diclofenac and Trolox, whereas prophylactic administration produced a smaller inhibition. These findings suggest that quinoa extract may attenuate inflammatory responses, at least in part, through modulation of the NF-κB pathway.

The potential synergism among the extract’s constituents and those from the whole quinoa grain flour warrants further investigation.

The nutrient and phytochemical composition of quinoa also makes it relevant in the context of inflammasome biology. Inflammasomes are intracellular protein complexes that coordinate innate immune responses; however, excessive activation can drive pathological inflammation in disorders such as autoimmune disease, type 2 diabetes, neurodegeneration, and atherosclerosis. The NLRP3 inflammasome is particularly important because it responds to diverse pathogen- and damage-associated signals, ROS, and crystalline particles. Recent studies suggest that quinoa extracts can activate antioxidant defenses (e.g., Nrf2 signaling) while suppressing NF-κB/NLRP3 inflammasome pathways [36], and that quinoa bioactives may inhibit inflammasome activation by limiting ROS and downstream cytokine release [37].

Caspase-1 is activated downstream of inflammasomes such as NLRP3. Once active, it cleaves pro-IL-1β and pro-IL-18 into mature cytokines and can promote pyroptotic cell death [38]. A rat study using sprouted black quinoa extract in a heat-stress liver injury model reported dose-dependent reductions in caspase-1 [36]. In the present study, quinoa flour ethanolic extract inhibited caspase-1 in both therapeutic and prophylactic designs, with stronger inhibition than Trolox or diclofenac. This is consistent with the extract’s overall reduction in oxidative stress and NF-κB signaling. Reduced caspase-1 activity would be expected to decrease activation of pro-IL-1β and pro-IL-18 [39].

IL-1β is a key pro-inflammatory cytokine produced by activated macrophages and monocytes during infection or tissue injury. While it is important for host defense, excessive IL-1β contributes to inflammatory pathology, including rheumatoid arthritis, Alzheimer’s disease, and sepsis [40]. Direct evidence on quinoa and IL-1β remains limited, but in our experiment, IL-1β decreased after treatment with quinoa flour ethanolic extract, with an effect comparable to Trolox. This may reflect multiple actions, including reduced oxidative stress (a trigger for IL-1β activation) and potential gut-mediated immunomodulation via dietary fiber effects on microbiota and barrier function [41].

IL-18 signals through its receptor (IL18R) and can activate NF-κB, promoting the production of other pro-inflammatory cytokines and amplifying tissue injury [42]. At the same time, IL-18 can have context-dependent roles and has been described as a ‘double-edged sword’ cytokine that may either exacerbate inflammation or support immune homeostasis [43]. Elevated IL-18 is implicated in autoimmunity, infection, and cancer [41], and the concept of IL-18-opathies underscores interest in therapeutic IL-18 blockade [43]. Diets rich in antioxidants may help reduce chronic inflammatory tone and rebalance cytokines such as IL-18 [36]. In our study, quinoa flour ethanolic extract reduced IL-18 in both designs, with an effect comparable to Trolox and stronger than diclofenac. Together with the reduction in mature IL-1β/IL-18, these changes may help interrupt ROS–NF-κB positive feedback loops that sustain inflammation [43].

IL-10 is a cytokine that targets both innate and adaptive immune responses, having opposite effects depending on the condition. It has an immunosuppressive activity in order to reduce tissue damage caused by excess and uncontrolled inflammatory responses. In certain conditions, like tumors, IL-10 has immunostimulatory activity [44]. In turpentine-induced inflammation with quinoa flour ethanol extract treatments, prophylactic or therapeutic, IL-10 did not register significant changes. In the prophylactic plan, inflammation lasted only 24 h, and in many acute inflammation models, IL-10 protein peaked later than IL-1β, commonly in the 6–24 h window [45]. In the therapeutic plan, the antioxidant and anti-inflammatory effect of the extract may have reduced IL-10 synthesis stimulation.

PCA provided complementary insight by integrating oxidative stress indices and inflammatory mediators into a combined oxidative-inflammatory pattern. In the score plot, treated samples separated from controls along components heavily weighted on both oxidative damage markers and inflammatory biomarkers, forming an oxidative–inflammatory axis that supports the concept that oxidative stress and inflammation reinforce one another [46]. Quinoa flour ethanolic extract shifted this cluster toward the control profile, indicating concurrent normalization of oxidative and inflammatory pathways. This combined oxidative stress-inflammation burden may contribute to disease severity [47]. Overall, our PCA results are consistent with a dual mechanism of action for the quinoa extract across redox and inflammatory signaling.

Our study presents some limitations. The small sample size limits the statistical power. The use of a single experimental model of acute inflammation does not capture the complexity of chronic or multifactorial inflammatory diseases. The variability in phytochemical composition due to genetic, environmental, and seasonal factors can be attributed to the extraction method not being addressed. Another important limitation is that safety/toxicology evaluation was not included in the present work. Future studies should aim to address these limitations and expand the translational potential of quinoa flour ethanol extract.

4. Materials and Methods

4.1. Chemicals

All analytical-grade reagents were obtained from Merck (Darmstadt, Germany) or Sigma-Aldrich (Munich, Germany) unless otherwise specified. The following compounds were used for spectrophotometric and antioxidant assays: Folin–Ciocâlteu reagent, sodium carbonate, sodium acetate, aluminum chloride, methanol, acetic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Griess–Ilosvay reagent, sodium nitroprusside, phosphate-buffered saline (PBS), N-(1-naphthyl)ethylenediamine dihydrochloride (NEDD), sulfanilic acid, hydrogen peroxide (H₂O₂), 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ), acetate buffer, ferric chloride, xylenol orange, o-dianisidine dihydrochloride (3,3′-dimethoxybenzidine), thiobarbituric acid, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), butylated hydroxytoluene (BHT), 1,1,3,3-tetrahydroxypropane, vanadium(III) chloride (VCl₃), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).

HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). Ultrapure water was obtained using a Direct-Q UV purification system from Millipore (Burlington, MA, USA).

Reference standards used for chromatographic identification and quantification included gallic acid (>99%, HPLC grade), quercetin (>99.8%, HPLC grade), chlorogenic acid (>95%, HPLC grade), luteolin (>99%, HPLC grade), and rutin (>94%, HPLC grade), all supplied by Sigma (St. Louis, MO, USA).

Commercial ELISA kits were employed for the determination of rat 3-nitrotyrosine (3-NT; EU2560), NF-κB p65 (ER1187), IL-1β (ER1094), IL-18 (ER1094), and caspase-1 (E-EL-R0371). Kits for 3-NT, NF-κB p65, IL-1β, and IL-18 were purchased from FineTest (Wuhan, Hubei, China), while the caspase-1 kit was obtained from Elabscience Innovation Bionovation Inc. (Houston, TX, USA).

4.2. Plant Material Collection and Quinoa Flour Extraction

Commercial white organic quinoa flour from Peru was purchased from AGRO DELIVERY SRL, Chitila, Romania, and stored at room temperature until analysis. It was extracted with 70% ethanol using a modified Squibb cold repercolation procedure performed at room temperature. Quinoa flour was loaded into three percolators: in the first percolator 150 g, in the second percolator 90 g, and in the third percolator 60 g. Then quinoa flour was soaked with 150 mL 70% ethanol. After two days, the three percolated fractions (60 mL, 90 mL, and 120 mL) were collected and mixed. The quinoa flour extract had a concentration of 1:1 g quinoa flour/mL extract (w:v) in 30% ethanol. Extract was stored at 4 °C until analysis [48].

4.3. Phytochemical Analysis

4.3.1. Total Polyphenol Content

Total polyphenol content (TPC) was measured using a modified Folin–Ciocalteu assay. The quinoa flour extract was diluted 1:25, mixed with Folin–Ciocalteu reagent and distilled water, and sodium carbonate solution. After 30 min of incubation in the dark, quinoa flour extract absorbance was read at 760 nm. TPC was expressed as mg gallic acid equivalents per g dry weight (mg GAE/g d.w.) by using a gallic acid calibration curve (R² = 0.9992; 0.002–0.16 mg/mL) [48].

4.3.2. Total Flavonoid Content

Total flavonoid content (TFC) was determined by the AlCl₃ colorimetric method. Quinoa flour extract was mixed with methanol, followed by the addition of AlCl_3, potassium acetate (1 M) and dH₂O. After 15 min of incubation at room temperature, absorbance was recorded at 510 nm. TFC was calculated using a quercetin calibration curve (R² = 0.997; 0.004–0.2 mg/mL) and expressed as mg quercetin equivalents per 100 g dry weight (mg QE/100 g d.w.) [48].

4.3.3. HPLC-ESI MS Analysis

The quinoa ethanolic extract was characterized by high-performance Liquid Chromatography Coupled with Electrospray Ionization Mass Spectrometry (HPLC-DAD MS) (Agilent 1200 HPLC with DAD; Agilent 6110 MS). An Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) was used for separation at room temperature. The mobile phases were: mobile phase A—0.1% acetic acid in water with acetonitrile (99:1, v/v); mobile phase B—0.1% acetic acid in acetonitrile (v/v). The gradient was as follows: 95% A (0–2 min), 95–60% A (2–18 min), 60–10% A (18–20 min), 10% A (20–24 min), then return to 95% A in 1 min and hold for 5 min. The flow rate was 0.5 mL/min. DAD chromatograms were recorded at 280 nm (phenolic acids) and 340 nm (flavonoids). MS detection used ESI in positive mode (350 °C, 3000 V capillary voltage, nitrogen 8 L/min) with scanning from 100 to 1000 m/z. Compounds were tentatively identified using UV–Vis spectra, mass spectra, retention times, co-chromatography with available standards, and literature data. Prior to injection, the lyophilized quinoa flour extract was dissolved in MeOH. Calibration curves of standards dissolved in methanol were used for polyphenols quantification: luteolin for flavones (R² = 0.9972; LOD 0.26 μg/mL; LOQ 0.95 μg/mL), chlorogenic acid for phenolic acids (R² = 0.9937; LOD 0.41 μg/mL; LOQ 1.64 μg/mL), and rutin for flavonols (R² = 0.9981; LOD 0.21 μg/mL; LOQ 0.84 μg/mL) [48].

4.4. In Vitro Antioxidant Activity Analysis

4.4.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Capacity

The DPPH radical scavenging activity of the quinoa flour extract was assessed using a standard protocol. Quinoa extract was mixed with DPPH solution in methanol and after 30 min of incubation at room temperature in the dark, absorbance was measured at 517 nm. Antioxidant activity was calculated as AA% = (1 − A sample/A control) × 100. Results were expressed as μg Trolox equivalents per g dry weight (μg TE/g d.w.) by using a Trolox calibration curve (R² = 0.986; 0.5–5 μg/mL) [48].

4.4.2. Ferric Reducing Antioxidant Power Assay

Ferric reducing antioxidant power (FRAP) was measured as described previously [48]. Quinoa flour ethanolic extract was mixed with FRAP reagent and mixed thoroughly. After 30 min of incubation at room temperature, absorbance was read at 593 nm. Results were reported as microgram Trolox equivalents per gram dry weight plant material (μg TE/g d.w.) by using a Trolox standard curve (R² = 0.786; 50–500 μg).

4.4.3. Hydrogen Peroxide Scavenging Activity

Hydrogen peroxide scavenging activity (H₂O₂) was evaluated following a published method. Quinoa flour extract was added to an H₂O₂ solution, and after 10 min incubation at room temperature, absorbance was measured at 230 nm against phosphate buffer. Scavenging activity was calculated as (1 − A sample/A control) × 100. Results were reported as IC50, and converted to mg Trolox equivalents per g dry weight plant material (mg TE/g d.w.) by using a standard Trolox curve (R² = 0.998; 250–1000 μg/mL) [48].

4.4.4. The Nitric Oxide Radical Scavenging Assay

For the nitric oxide scavenging assay (NO), sodium nitroprusside (SNP) was used as an NO donor. Quinoa flour ethanol extract was mixed with SNP solution (2 mL SNP + 0.5 mL PBS, pH 7.4) and incubated at 25 °C for 2.5 h. Then sulphanilic acid and N-(1-naphthyl) ethylenediamine dihydrochloride were added. Samples were vortexed and incubated in the dark for 30 min, and absorbance was measured at 546 nm. Inhibition was calculated as (1 − A sample/A blank) × 100. Results were expressed as IC50, and converted into mg quercetin equivalents per g dry weight plant material (μM QE/g d.w.) by using a quercetin standard curve (R² = 0.997; 5–50 μM) [48].

All assays were performed in triplicate. Spectrophotometric measurements were performed with a UV-Vis spectrophotometer (Jasco V-350, Jasco International Co., Ltd., Tokyo, Japan).

4.5. In Vivo Experimental Design

4.5.1. Experimental Protocol

Adult male Wistar rats (200–250 g) were obtained from the Establishment for Breeding and Use of Laboratory Animals, “Iuliu Hațieganu” University of Medicine and Pharmacy (Cluj-Napoca, Romania). Animals were housed under standard conditions (25 ± 1 °C; 55 ± 5% relative humidity; 12 h light/dark cycle) with ad libitum access to standard chow and water. After 7 days of acclimatization, rats were labeled and randomly divided into 11 groups (n = 9). The SHAM group served as a healthy control without treatment. Acute inflammation was induced by intramuscular turpentine oil (6 mL/kg b.w.). Two treatment plans administered orally by gavage (1 mL/rat/day) were used. The stock extract concentration was 1000 mg quinoa flour per mL extract. The extract was tested at three pre-established concentrations: the stock extract 1000 mg/mL, abbreviated as Q100; the extract diluted with distilled water 1:1 (v/v) to obtain 500 mg/mL and abbreviated as Q50; the extract diluted with distilled water 1:4 (v/v) to obtain 250 mg/mL and abbreviated as Q25 [36]. All treatments were administered by oral gavage in a volume of 1 mL/rat/day and prepared fresh each day.

In the therapeutic plan, inflammation was induced on the first day, followed by the 10th days treatment. Six different groups of rats were treated as follows: INFL group received tap water (1 mL/rat/day); DICLO group received diclofenac (10 mg/kg b.w./day); TX group received Trolox (50 mg/kg b.w./day); INFL/Q100 group received Q100 (1000 mg/kg b.w.), INFL/Q50 group received Q50 (500 mg/b.w.), and INFL/Q25 group received Q25 (250 mg/kg b.w.). In the prophylactic plan, after the 10th day of treatment with Q100 (1000 g/kg b.w.), Q50 (500 mg/kg b.w.), and Q25 (250 mg/kg b.w.), inflammation was induced on day 11. On the 12th day, general anesthesia was induced (ketamine 60 mg/kg b.w. plus xylazine 15 mg/kg b.w.) [46], blood samples were drawn by retro-orbital puncture, and serum was stored at −80 °C until analysis.

The project was approved by the Veterinary Sanitary Direction and Food Safety Cluj-Napoca (No. 372/04.07.2023). All the procedures comply with Directive 2010/63/EU, and Romanian national law 43/2014 for the protection of animals used for scientific purposes.

4.5.2. Oxidative Stress Biomarkers Assessment

Total Oxidative Status (TOS)

Total oxidative status (TOS) was quantified based on the oxidation of ferrous ions to ferric ions in an acidic environment in the presence of reactive oxygen species. Absorbance was measured at 560 nm and results were expressed as μmol H₂O₂ equivalents/L (μM H₂O₂ equiv./L) [49].

Total Antioxidant Capacity (TAC)

Total antioxidant capacity (TAC) was assessed by measuring suppression of hydroxyl radical generation in a Fenton-type reaction by antioxidants present in serum. Absorbance was recorded at 444 nm and results were reported as mmol Trolox equivalents/L (mM TE/L) [50].

Oxidative Stress Index (OSI)

Oxidative stress index (OSI) provides an integrated estimate of oxidative stress burden, and it is calculated as the ratio between TOS and TAC [51].

Advanced Oxidation Protein Products (AOPP)

Advanced oxidation protein products (AOPP) are a biomarker of protein oxidation [52]. Serum samples and chloramine-T (blank) were diluted to 10% in PBS, potassium iodide and glacial acetic acid were added, and absorbance was measured at 340 nm. AOPP concentrations were expressed as μM chloramine-T equivalents/L by using a chloramine-T hydrate (Sigma Ltd., St. Louis, MO, USA) standard curve (R² = 0.9992; 0–100 μM).

Malondialdehyde (MDA)

Malondialdehyde (MDA) is a biomarker of lipid peroxidation. It was measured by using a thiobarbituric acid-based method [53]. Serum sample was mixed with 40% trichloroacetic acid and 0.67% thiobarbituric acid. Then the mixture was heated for 30 min in a boiling water bath, then cooled in ice, and centrifuged for 5 min at 3461 g. MDA concentration absorbance was measured at 532 nm, and results were expressed as nM/mL serum.

Nitric Oxide Synthesis (NO)

Nitric oxide production was estimated by determining total nitrites and nitrates (NOx) using the Griess reaction. First, serum proteins were removed by extraction with methanol/diethyl ether (3:1, v/v). Then vanadium (III) chloride was added in order to reduce nitrates to nitrites. Sample absorbance was measured at 540 nm, and results were expressed as nitrite μM/L by using a standard curve (R² = 0.9992; 1–200 μM) [54].

3-Nitrotyrosine (3NT)

3-Nitrotyrosine (3NT), a marker of peroxynitrite-mediated oxidative damage, was quantified using an ELISA kit (E-EL-0040) (Elabscience Biotechnology Inc., Houston, TX, USA) according to the manufacturer’s protocol. Results were expressed as ng/mL [48].

Total Thiols (SH)

Total thiols (SH) were determined with modified Ellman’s reagent by adding formaldehyde solution [55]. Absorbance was at 412 nm, and SH concentrations were expressed as mM glutathione equivalents per mL (mM GSH/mL).

A UV-Vis spectrophotometer (Jasco V-350, Jasco International Co., Ltd., Tokyo, Japan) was used to read all biochemical oxidative stress tests.

4.5.3. Inflammatory Biomarkers Assessment

Systemic inflammatory response was evaluated by ELISA quantification of NF-κB-p65 (E-EL-RO674), IL-1β (E-EL-0012), IL-18 (E-EL-R0567), caspase-1 (E-EL-R0371), and IL-10 (E-EL-R0016). Assays were performed according to manufacturers’ instructions and results were reported as pg/mL. ELISA procedures used a Biotek Microplate 50 TS washer and an 800 TS microplate reader (Agilent Technologies Inc., Santa Clara, CA, USA).

4.6. Statistical Analysis

Study results were presented as mean ± standard deviation (SD) for normally distributed variables. Group comparison was performed with a one-way ANOVA test, followed by a post hoc Holm–Bonferroni-adjusted pairwise test. Principal component analysis (PCA) was used to examine correlations among the parameters. Statistical significance was set at p < 0.05. Data were analyzed using SPSS v26.0 (SPSS Inc., Chicago, IL, USA) and R v.5.1 (R Foundation for Statistical Computing, Vienna, Austria).

5. Conclusions

In conclusion, this study investigated both prophylactic and therapeutic effects of quinoa flour ethanolic extract on inflammation and inflammation-associated oxidative stress in an acute experimental model. The results indicate that the extract can reduce oxidative damage while enhancing antioxidant defenses (lower ROS-related markers and higher antioxidant capacity). In parallel, the extract attenuated inflammatory signaling, as reflected by decreases in NF-κB, caspase-1, IL-1β, and IL-18. These effects are likely driven by the combined action of multiple functional constituents (flavonoids, polyphenols, and other nutrients). Collectively, the findings support further exploration of quinoa flour ethanolic extract as a dietary adjunct for prevention or support in conditions linked to oxidative stress and inflammation, including in oncology-related contexts. Additional studies are needed to define effective doses, mechanisms, and translational strategies for dietary interventions based on quinoa-derived products.