Antioxidant Potential of Myrciaria tenella Fruit Extracts: In Vitro and In Vivo Protection Against Oxidative Stress

Abstract

Myrciaria tenella (cambuí) is a native Brazilian fruit traditionally recognized for its sensory attributes and medicinal properties, including antidiabetic, anti-inflammatory, antimicrobial, and gastroprotective activities. This study evaluated the antioxidant activity of unripe and ripe M. tenella fruits using in vitro and in vivo experimental models. Four extracts were prepared: aqueous unripe (VA), aqueous ripe (MA), hydroethanolic unripe (VE), and hydroethanolic ripe (ME). Antioxidant activity was assessed through biochemical assays and cellular models using NIH/3T3 fibroblasts and RAW 264.7 macrophages. In RAW 264.7 cells, oxidative stress modulation was investigated using hydrogen peroxide-induced stress and lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production. In NHI/3T3 cells, wound healing, copper sulphate (CuSO₄)-induced oxidative stress, intracellular reactive oxygen species (ROS) generation, and nuclear morphology following DAPI staining were evaluated. Total phenolic content was quantified using the Folin–Ciocalteu method and analyzed by HPLC-DAD. In vivo toxicity and antioxidant protection were assessed using Tenebrio molitor larvae exposed to CuSO₄-induced oxidative stress. Among the extracts, VE exhibited the highest metal-chelating activity (Cu: 78.6%; Fe: 37.7%) and total phenolic content (50.64 mg GAE/mg). HPLC-DAD analysis identified gallic acid in all extracts, kaempferol derivatives in hydroethanolic extracts (VE and ME), and catechin derivatives in aqueous extracts (VA and MA). All extracts were non-cytotoxic and demonstrated protective effects against oxidative stress in vitro. In vivo assays confirmed the absence of toxicity and significant antioxidant protection, with VE resulting in 80% larval survival.

1. Introduction

Cells are constantly exposed to oxidative stress resulting from metabolic reactions, which increase intracellular levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS). At moderate levels, ROS and RNS are essential for physiological processes, serving as signaling molecules in various metabolic pathways. Additionally, endogenous antioxidant enzymes play an important role in maintaining redox homeostasis [1].

Oxidative stress occurs when excessive ROS and/or RNS disrupt the cellular balance, leading to damage to critical biomolecules such as DNA, proteins, and lipids [2]. This imbalance has been associated with several pathological conditions, including cardiovascular disorders [3], kidney diseases [4], aging [5], neurological disorders [3], diabetes [6], inflammation [7], and cancer [8].

Medicinal plants have long played an important role in folk medicine, providing alternative therapeutic approaches for various health conditions. Studies involving plant extracts have demonstrated therapeutic potential in the management of diabetes, cancer, inflammation, microbial infections, and oxidative stress [9]. These beneficial effects are mainly attributed to bioactive phytochemicals, including phenolic compounds, terpenes, alkaloids, saponins, and quinones [10,11]. Understanding the mechanisms of action of these components is important for their application in disease prevention, treatment strategies, and as nutritional supplements [12].

The antioxidant potential of phenolic compounds, particularly flavonoids (such as kaempferol and catechins) and phenolic acids (e.g., gallic acid) is well documented. These compounds exert antioxidant effects through mechanisms including enzyme regulation, protection against superoxide anion, and the reduction in nitric oxide and hydroxyl radicals [13].

Fruits are also important sources of bioactive compounds with antioxidant properties [14]. They are commonly consumed in various forms, such as juices, jellies, and fermented beverages. The antioxidant activity of fruits is frequently correlated with their phenolic content [15]. During ripening, fruits undergo significant chemical changes that affect their phytoconstituent profiles [14,16]. Typically, unripe fruits contain higher concentrations of phenolic compounds, such as tannins, which may provide protective effects against predators. In contrast, ripe fruits generally present higher levels of sugars, carotenoids, and anthocyanins, enhancing their flavor and appeal [17,18].

Myrciaria tenella (DC.) O. Berg, a shrub native to the Atlantic Forest biome, produces edible, spherical fruits ranging from red to dark purple and measuring approximately 8–10 mm in diameter. The sweet pulp of the fruit is widely consumed by both birds and humans [19].

Studies on the Myrciaria genus, particularly those focusing on its leaves and essential oils, have revealed high levels of phenolic compounds, tannins, and terpenoids. These phytoconstituents exhibit a wide range of pharmacological activities, including antioxidants, antidiabetic, antiproliferative, anti-inflammatory, antihypertensive, antimicrobial, antifungal, and gastroprotective effects, reinforcing the functional potential of the species [10,20,21,22,23].

Previous research conducted by our group on Myrciaria tenella leaf extracts identified tannins, saponins, and ursolic acid [23]. These studies demonstrated antioxidant activity via biochemical assays, as well as antitumor effects against human cervical adenocarcinoma (HeLa) and gastric adenocarcinoma (AGS) cell lines. In addition, in vivo assays using Caenorhabditis elegans revealed that ethanolic extracts were able to reduce basal ROS levels.

No information on the centesimal composition or active compounds of fruits from this species has been reported in the literature. Nevertheless, a study on lyophilized Myrciaria floribunda fruit reported lower protein and lipid contents compared with other fruits from the same family, such as Psidium guajava (13.28 g protein·100 g⁻¹ dw and 4.95 g lipids·100 g⁻¹ dw) and Syzygium cumini (8.57 g protein·100 g⁻¹ dw and 4.29 g lipids·100 g⁻¹ dw) [24].

Regarding the chemical composition and bioactivities of Myrciaria fruits, including Myrciaria floribunda, HPLC analysis of free sugars revealed the presence of fructose, glucose, and sucrose at levels comparable to those reported for Syzygium cumini (18.6 g·100 g⁻¹ dw fructose and 21.0 g·100 g⁻¹ dw glucose) [25].

Despite these findings, studies specifically addressing the antioxidant potential of M. tenella fruits remain limited. Therefore, the present study aimed to investigate the antioxidant properties of aqueous and hydroethanolic extracts obtained from M. tenella fruits at two different maturation stages: unripe and ripe.

The antioxidant activity of these extracts was evaluated using multiple in vitro assays, including biochemical assays and cell-based analyses done on RAW 264.7 and NIH/3T3 cell lines. Following promising results, the extracts were further assessed using the invertebrate model Tenebrio molitor.

Phytochemical analysis using HPLC-DAD suggests the presence of gallic acid in all extracts, kaempferol derivative in hydroethanolic extracts, and catechin derivative in aqueous extracts.

The suggested phenolic phytoconstituents likely contribute to the antioxidant properties observed in vitro. Furthermore, assays employing the in vivo Tenebrio molitor model supported the antioxidant profile of the extracts, as evidenced by increased survival rates and reduced melanization following oxidative stress.

Overall, the results indicated that M. tenella fruit extracts, particularly those obtained from unripe fruits, exhibited significant potential as natural agents for mitigating oxidative stress-related conditions. These findings highlight their possible applications in the development of nutraceuticals, pharmaceuticals, and dietary supplements aimed at promoting health and reducing oxidative damage.

2. Results

2.1. In Vitro Antioxidant Profile of Myrciaria tenella Fruit Extracts

This study evaluated the antioxidant potential of unripe and ripe Myrciaria tenella fruits. Aqueous extracts were designated as VA (unripe) and MA (ripe), and the hydroethanolic extracts were VE (unripe) and ME (ripe). Antioxidant activity was assessed using five different assays: Total Antioxidant Capacity (TAC), Reducing Power, DPPH Radical Scavenging, and metal chelation assays for iron and copper. All experiments were performed at a concentration of 100 µg/mL.

The TAC assay demonstrated that unripe extracts (VA and VE) exhibited higher antioxidant activity than ripe extracts (MA and ME), with values of 168 and 164 mg ascorbic acid equivalents per mg of sample, respectively. In contrast, ripe extracts presented lower values, with 124 mg for MA and 127 mg for ME (Figure 1a).

Figure 1. Antioxidant activity of Myrciaria tenella fruit extracts evaluated by biochemical assays. The x-axis represents the extracts VA, VE, MA, and ME, while the y-axis shows the estimated antioxidant activity of the different M. tenella extracts at a concentration of 100 µg/mL. (a) Total Antioxidant Capacity (TAC) expressed in µgEqAA/mg extract; (b) Reducing power (%); (c) DPPH radical scavenging (%); (d) Metal chelating activity: Copper chelation (%); (e) Metal chelating activity: Iron chelation (%). VA: aqueous unripe extract of M. tenella fruits; VE: hydroethanol unripe extract; MA: aqueous ripe extract; ME: hydroethanol ripe extract. The assays were performed in duplicate, with each extract tested in triplicate. Data were analyzed using ANOVA followed by Tukey’s test (p ≤ 0.05), different letters (a–d) indicate statistically significant differences.

Similarly, in the Reducing Power assay, unripe extracts showed greater activity, with VE reaching 124.6% and VA 64.16%. Among the ripe extracts, MA and ME displayed comparable reducing capacity, with values of 43.16% and 30%, respectively (Figure 1b). DPPH radical scavenging activity was comparable for VA, VE, and ME (approximately 87%), whereas MA exhibited lower activity (63.2%) (Figure 1c).

In the copper ion chelation assay, all the extracts demonstrated chelating activity (Figure 1e).

Overall, the results obtained with these five biochemistry assays indicate that M. tenella fruit extracts exhibit antioxidant activity through various mechanisms, including electron donation (TAC and Reducing Power assays), free radical scavenging (DPPH assay), and metal chelation (iron and copper assays). In addition, extracts derived from unripe fruits generally demonstrated greater antioxidant potential than those obtained from ripe fruits.

2.2. Antioxidant Activity of M. tenella Fruit Extracts on RAW 264.7 Macrophage Cells

Initially, the cytotoxic potential of M. tenella extracts was evaluated at three different concentrations: 50, 100, and 250 µg/mL. The results demonstrated that none of the extracts exhibited cytotoxicity effect at any of the tested concentrations (Figure 2a). At the highest concentration of 250 µg/mL, all extracts maintained cell viability, with MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) reduction values of 93% (VA), 117% (VE), 101% (MA), and 104% (ME). Based on these findings, the concentration of 100 µg/mL was selected for subsequent assays.

Figure 2. In vitro assays performed on RAW 264.7 macrophage cells. (a) cytotoxic evaluation of M. tenella fruit extracts at concentrations of 50, 100, and 250 µg/mL. The y-axis represents the percentage of MTT reduction, and the x-axis corresponds to the different M. tenella extracts at the indicated concentrations. (b) Effects of extracts on cell viability following oxidative stress induced by H₂O₂ stressor. The y-axis indicates the percentage of MTT reduction, and the x-axis represents the different extracts tested at 100 µg/mL. NC (negative control) corresponds to macrophages cultured in medium only, while PC (positive control) represents macrophages treated with the H₂O₂ oxidative stressor. (c) Percentage of nitric oxide (NO) production after treatment with M. tenella extracts. The y-axis represents NO production (%), and the x-axis corresponds to treatments with M. tenella extracts at 100 µg/mL in combination with the LPS (1 µg/mL) stimulus. NC represents macrophages cultured in medium only, and PC represents macrophages treated with LPS, with PC set as 100% NO production. VA: aqueous unripe fruit extract; VE: hydroethanolic unripe fruit extract; MA: aqueous ripe fruit extract; ME: hydroethanolic ripe fruit extract. In panels (b,c), different letters (a–d) indicate statistically significant differences between extracts and NC compared with PC, according to Tukey’s test (p < 0.0001). In panel (a), letters indicate statistically significant differences among extracts, as determined by Tukey’s test (p < 0.01).

The antioxidant potential of the extracts was subsequently evaluated using H₂O₂ as an oxidative stressor in RAW 264.7 macrophages (Figure 2b). The results showed that all M. tenella extracts had similar MTT reduction percentages: 92% (VA), 89.89% (VE), and 74.54% (MA). In contrast, ME extract exhibited a higher MTT reduction value of 124.82% compared with the positive control (PC). These findings suggest that both unripe and ripe M. tenella extracts display antioxidant potential under oxidative stress conditions.

To further investigate the antioxidant activity, the anti-inflammatory effects of the extracts were assessed by inducing an inflammatory response with lipopolysaccharide (LPS), followed by measurement of nitric oxide (NO) (Figure 2c). RAW 264.7 macrophages were treated with M. tenella extracts at a concentration of 100 µg/mL and subsequently stimulated with LPS. The negative control (NC), consisting of cell cultured in Dullbecco´s Modified Eagle Medium (DMEM), exhibited 11.74% NO production relative to the positive control (PC), which was set as 100%. Treatment with M. tenella extracts significantly reduced NO production compared with the PC, with inhibition values of 29.7% (VA), 31% (VE), 39% (MA), and 20% (ME).

2.3. Effect on Cell Migration of M. tenella Extracts Using the NIH/3T3 Cell Line by Wound Healing Assay

Considering M. tenella fruit extracts exhibit antioxidant and anti-inflammatory effects in RAW 264.7 macrophage—processes known to play an important role in tissue repair—their influence on cell migration was subsequently investigated using a wound healing assay.

As this experiment was done using the NHI/3T3 fibroblast cell line, the cytotoxic potential of extracts was initially evaluated at concentrations of 50, 100, and 250 µg/mL by MTT reduction (Figure 3a). The results demonstrated that all the unripe (VA and VE) and ripe (MA and ME) extracts showed similar MTT reduction value and did not exhibit any cytotoxic effects. Based on this result, the concentration of 100 µg/mL was selected for subsequent assays. MTT reduction observed for VA, VE, MA, and ME extracts were 118%, 132%, 120%, and 95%, respectively, indicating high cell viability in all treatments.

Figure 3. Effect of M. tenella fruit extracts on cell migration assessed by the wound healing assay. (a) MTT reduction in NIH/3T3 cells treated for 24 h with M. tenella fruit extracts. (b) Bar graph showing the effects of control and M. tenella extracts on wound closure, with comparisons made relative to the initial time point (0 h). (c) Line graph illustrating wound closure progression over time (0, 12, and 24 h) for control and extract-treated groups. (d) Representative microscopic images of the wound healing assay at 0, 12, and 24 h following treatment with VA, VE, MA, ME, and the control group. Measurements were performed in intermediate regions of the scratch. In the control group, no extract was added. VA corresponds to the aqueous unripe fruit extract; VE to the hydroethanolic unripe fruit extract; MA to the aqueous ripe fruit extract; and ME to the hydroethanolic ripe fruit extract. (ns) indicates no statistically significant difference. Different letters (a–d) indicate statistically significant differences relative to the respective 0 h time point, while **** indicate statistically significant differences between extract-treated groups and the control, according to Tukey’s test (p < 0.0001).

Cell migration was then assessed using the wounding healing assay by measuring the distance of closure. As shown in Figure 3b,c, the NHI/3T3 cells were treated with the extracts at 100 µg/mL, and the migration was evaluated by comparing the remaining wound area with the control at 12 and 24 h. At 12 h, all treatments exhibited migration rates similar to the control group. However, after 24 h, the control group showed approximately 33% wound closure.

Treatment with VA, VE, and ME extracts resulted in significantly enhanced migration, with scratch closure values of approximately 56%, 48%, and 46%, respectively (Figure 3b–d). In contrast, MA treatment resulted in 35% wound closure, comparable to the control condition (Figure 3b–d). These findings suggest that M. tenella fruit extracts contain bioactive compounds capable of stimulating fibroblast migration in the NIH/3T3 cell line. Based on the observed activity, the effectiveness of the treatments can be ranked as follows: (VA = VE = ME) > (MA = C).

2.4. Effect of M. tenella Extracts on NIH/3T3 Fibroblast Cells Exposed to Oxidative Stress (CuSO₄ 25 μM + 1 mM Ascorbate)

2.4.1. Reduction in MTT and ROS-DCFH in NIH/3T3 Fibroblast Cells

Based on the results described above, which demonstrated the absence of cytotoxic effects, the protective potential of M. tenella fruit extracts under oxidative stress conditions was subsequently evaluated. All extracts exhibited protective activity in the NIH/3T3 cell line against oxidative stress induced by 25 μM CuSO₄ combined with 1 mM ascorbate (Figure 4). This protective effect was evidenced by MTT reduction values of approximately 92%, compared with 64% observed in the positive control (PC) following oxidative stress treatment (Figure 4a).

Figure 4. Protective effects of M. tenella fruit extracts against copper-induced oxidative stress. (a) Percentage of MTT reduction in NIH/3T3 fibroblasts exposed to oxidative stress induced by 25 µM CuSO₄ combined with 1 mM ascorbate and treated with M. tenella fruit extracts. VA corresponds to the aqueous unripe fruit extract; VE to the hydroethanolic unripe fruit extract; MA to the aqueous ripe fruit extract; and ME to the hydroethanolic ripe fruit extract. NC represents the negative control (culture medium only), while PC represents the positive control (culture medium supplemented with 25 µM CuSO₄ and 1 mM ascorbate). Statistical differences between treatments were analyzed using Tukey’s test (p < 0.0001) in comparison with the PC group, as indicated by different letters (a, b). (b) Relative fluorescence intensity (%) used to assess intracellular reactive oxygen species (ROS) levels by the DCFH-DA assay. NIH/3T3 fibroblasts were treated with M. tenella fruit extracts, and fluorescence was quantified using a microplate reader following 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Statistical differences between treatments were analyzed using Tukey’s test (p < 0.0001) compared with the PC group, as indicated by different letters (a, b). (c) Representative fluorescence microscopy images of NIH/3T3 cells treated with M. tenella fruit extracts. Green fluorescence intensity corresponds to intracellular ROS production, with a marked reduction observed in extract-treated groups relative to the positive control.

To further investigate the antioxidant mechanism, the same oxidative stress protocol was applied to evaluate the ability of the extracts to reduce intracellular reactive oxygen species (ROS). The DCFH-DA was employed for this purpose. DCFH-DA is a fluorogenic probe that diffuses into cells and is deacetylated by intracellular esterases to form 2′,7′-dihydrodichlorofluorescein (DCFH), which is subsequently oxidized by ROS to the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF). Fluorescence was detected by spectrofluorometry at excitation and emission wavelengths of 480 and 530 nm, respectively.

Quantification of fluorescence using the DCFH probe revealed a marked reduction in ROS levels compared with the negative control (55%) (Figure 4b). Specifically, treatment with M. tenella extracts resulted in ROS values of 38% (VA), 43% (VE), and 28% (MA and ME) (Figure 4b).

In addition, green fluorescence imaging was performed, as shown in Figure 4c, in which fluorescence intensity corresponds to intracellular ROS production. Strong fluorescence was observed in the positive control (100%), whereas a pronounced reduction in fluorescence intensity was detected in cells treated with M. tenella extracts. All values are expressed relative to the positive control (PC).

2.4.2. DAPI Staining in NIH/3T3 Fibroblast Cells

To evaluate the protective effect of fruit extracts against oxidative stress-induced damage to nuclear DNA, NIH/3T3 fibroblast cells exposed to 25 μM CuSO₄ combined with 1 mM ascorbate and treated with the different extracts were stained with DAPI, a DNA-specific fluorescent marker widely used for the assessment of nuclear morphology. Because the plasma membrane of apoptotic cells becomes compromised, increased amounts of DAPI can enter the cell, resulting in more intense blue fluorescence. Moreover, characteristic nuclear alterations associated with apoptosis, such as chromatin condensation and nuclear fragmentation, facilitate the visual identification of apoptotic cells following DAPI staining cells stained with DAPI.

The effects of M. tenella fruit extracts on nuclear integrity were evaluated by comparison with the positive control (PC). The results demonstrated that all extracts were able to preserve a high number of cells with intact nuclei, exceeding the values observed in the negative control (NC; 116 cells). In contrast, the PC group exhibited only 54 cells with normal nuclear morphology, indicating substantial damage induced by oxidative stress (Figure 5).

Figure 5. NIH/3T3 fibroblast cell line stained with DAPI following oxidative stress induction and treatment with M. tenella fruit extracts. (a) Absolute number of cells exhibiting intact nuclei after treatment with M. tenella fruit extracts and exposure to oxidative stress induced by 25 µM CuSO₄ combined with 1 mM ascorbate. Different letters (a, b) indicate statistically significant differences between treatments, as determined by Tukey’s test (p < 0.0001) compared with the positive control (PC). (b) Representative fluorescence microscopy images showing NIH/3T3 cells subjected to oxidative stress and treated with M. tenella fruit extracts. The orange arrow in the PC group indicates a plaque formed by the accumulation of DAPI-stained nuclear material. VA: aqueous unripe extract; VE: hydroethanolic unripe extract; MA: aqueous ripe extract; ME: hydroethanolic ripe extract. NC: negative control (untreated); PC: positive control (25 µM CuSO₄ + 1 mM ascorbate).

In the PC group, the presence of apoptotic plaques formed by exposed nuclear material (red arrow) was observed, reflecting severe nuclear damage. By comparison, the NC group displayed less intense nuclear fluorescence, consistent with preserved basal nuclear integrity. Treatment with M. tenella fruit extracts resulted in cell counts comparable to the NC, with the following values: VA, 143 cells; VE, 119 cells; MA, 132 cells; and ME, 106 cells. The altered nuclear morphology observed in the stressed group was associated with DNA damage and the initiation of apoptotic cell death (Figure 5b).

Taken together, these images demonstrate a higher number of intact nuclei in extract-treated groups compared with the PC, suggesting that M. tenella fruit extracts exert a protective effect against oxidative stress-induced nuclear damage.

2.5. Phytochemical Composition of M. tenella Fruit Extracts

2.5.1. Chemical Composition of the Extracts

With respect to the chemical composition of bioactive metabolites, the unripe hydroethanolic extract (VE) exhibited the highest total phenolic content (50.64 mg GAE/mg of extract), followed by the unripe aqueous extract (VA), which presented 32.52 mg GAE/mg extract. In contrast, the hydroethanolic ripe (ME) and aqueous ripe (MA) extracts showed the lower phenolic levels, with values of 22.68 and 12.36 mg GAE/mg of extract, respectively.

2.5.2. HPLC-DAD

These extracts were further analyzed by high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) to tentatively identify the major biomolecules present (Figure 6 and Supplementary Figure S2). Retention times and UV-Vis spectra were evaluated, and the detected peaks were compared with previously isolated standard compounds from laboratory libraries.

Figure 6. Chromatographic profiles of Myrciaria tenella fruit extracts obtained by HPLC-DAD analysis. The unripe hydroethanolic (VE) is shown in a chromatogram processed at l = 352 nm. In this chromatogram, peak 1 corresponds to gallic acid, while peak 2 corresponds to a kaempferol derivative. The aqueous ripe extract (MA) profile is shown in a chromatogram processed at l = 280 nm, in which peak 1 corresponds to gallic acid and peak 3 corresponds to a catechin derivative. Partial molecular structures of the tentatively identified compounds are illustrated alongside their respective UV-Vis spectra. In all chromatograms, the x-axis represents retention time (min), and the y-axis indicates peak intensity (mAU).

In both the unripe hydroethanolic (VE) and ripe hydroethanolic (ME) extracts, gallic acid (peak 1, tr = 1.9 min, phenolic acid) and a kaempferol derivative (peak 2, tr = 18.0 min, flavonol) were identified. Although the same compounds were detected in both extracts, the kaempferol derivative was present at a higher relative abundance in the ME extract compared with VE.

By contrast, the aqueous unripe (VA) and aqueous ripe (MA) extracts contained gallic acid (peak 1, tr = 1.9 min, phenolic acid) and a catechin derivative (peak 3, tr = 2.8 min, flavanol) (Figure 6 and Supplementary Figure S2). In this case, catechin derivative was observed at higher levels in the MA extract, approximately twice that detected in the VA.

These results indicate that ripe fruit extracts contain higher levels of phytoconstituents than unripe extracts. Gallic acid was the predominant compound in all four extracts, while kaempferol derivatives were detected exclusively in hydroethanolic extracts, and catechin derivatives in aqueous extracts. The differences observed among the extracts may be attributed to the extraction method, which influenced the relative abundance of these compounds (Figure 6). Nevertheless, additional analyses are required to fully characterize other constituents present in the extracts.

2.6. Toxicological and Protective Effects of M. tenella Fruit Extracts on Tenebrio molitor

Based on the previous results demonstrating that M. tenella fruit extracts were not cytotoxic and were able to protect cells against oxidative stress, their effects were further evaluated using an in vivo invertebrate model, Tenebrio molitor. The first parameter assessed was the potential toxicity of the extracts in this organism.

T. molitor larvae were inoculated with VA, VE, MA, and ME extracts at two different concentrations (100 and 250 µg/mL), and survival curves were generated over a 10-day period (Figure 7a,b). During this time, survival rates remained comparable to those observed in the phosphate-buffered saline (PBS) control group, which exhibited 100% survival. At a concentration of 100 µg/mL, the VE extract resulted in a larval survival rate of 90%, while the remaining extracts showed survival rates of 86.7% (MA) and 83.3% (VA and ME). At 250 µg/mL, similar results were observed, with MA exhibiting a survival rate of 90%, and VE, VA, and ME presenting survival rates of 86.7% and 83.3%, respectively.

Figure 7. Oxidative protective effects of VA, VE, MA, and ME extracts in T. molitor larvae. (a) Effect of extracts at 100 µg/mL on larvae survival. (b) Effects of extracts at 250 µg/mL on larval survival. For panels (a,b), larvae (n = 30 per group) were inoculated with extracts at concentrations of 100 and 250 µg/mL, and survival was monitored over a 10-day period. The negative control group consisted of larvae inoculated with phosphate-buffered saline (PBS) only. (c) Effects of extracts on the survival of T. molitor larvae exposed to CuSO₄. Survival was evaluated over 15 days. The negative control (NC) consisted of larvae receiving two PBS inoculations, while the positive control (PC) consisted of larvae inoculated with 25 µM CuSO₄ followed by PBS. (d) Effects of extracts on melanization in T. molitor larvae evaluated 15 days after inoculation. Values are expressed as mean ± standard deviation. Asterisks indicate statistically significant differences compared with the PC group, as determined by Dunnett’s test (* p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Different letters (a–d) indicate statistically significant differences among extracts, according to Tukey’s test (p < 0.001).

Altogether, these results indicate that M. tenella fruit extracts did not exhibit toxic effects in the T. molitor model over the 10-day experimental period.

Based on these results, the protective effects of the extracts against oxidative stress induced by CuSO₄ were subsequently evaluated (Figure 7c). The positive control (PC) exhibited a survival rate of 60%. Similar survival rates were observed in larvae treated with VA and ME extracts, with values of 63.3% and 60%, respectively (Figure 7c). In contrast, treatment with VE or MA extracts at a concentration of 100 μg/mL resulted in higher survival rates of 80% and 73.3%, respectively (Figure 7c), indicating enhanced protection against oxidative stress.

Another parameter assessed was the degree of melanization, used as an indicator of oxidative stress response in T. molitor larvae (Figure 7d). A higher optical density (OD) value was observed in larvae subjected to CuSO₄-induced stress (PC: 1.270) compared with the negative control (NC: 0.392). When control values were used as reference, all extract-treated groups exhibited a statistically significant reduction in melanization. Among the extracts, VE (OD = 0.432) and MA (OD = 0.438) presented values comparable to the negative control, further supporting their protective effects against oxidative stress. The ME (OD = 0.566) and VA (OD = 0.865) extracts also reduced melanization relative to the CuSO₄-stressed positive control.

The combined in vitro and in vivo results indicate antioxidant protective effects of M. tenella fruit extracts, enabling comparisons between unripe and ripe fruits as well as between aqueous and hydroethanolic extraction methods.

3. Discussion

Several endogenous and environmental stressors can induce metabolic alterations that lead to oxidative imbalance, resulting in cellular damage and functional impairments associated with aging, chronic inflammation, asthma, neurodegenerative and cardiovascular diseases, and cancer [3]. These effects have intensified scientific interest in the identification of bioactive molecules with antioxidant properties capable of restoring redox homeostasis and contributing to improved nutrition, supplementation strategies, and the prevention or treatment of diseases related to excessive oxidative stress [26].

Plants represent an important source of bioactive compounds, commonly consumed as juices, teas, and tinctures, prompting research to understand phytoconstituents [8]. Investigations involving plant extracts may support their application in dietary supplements, nutraceuticals, and pharmaceutical products [27]. As life expectancy increases, phytochemical antioxidants have gained attention due to their potential health-promoting and therapeutic effects [7]. Nevertheless, the biological potential of many phytochemicals remains insufficiently explored, motivating ongoing investigations by both industry and academia [9]. Regular consumption of fruits and their phytoconstituents has been associated with multiple health benefits, including anti-aging, cardiovascular protection, and reduced risk of neurodegenerative disease [5].

The phenolic content responsible for antioxidant activity in fruits may vary throughout the ripening, depending on the species, with reports indicating either decreases [17], or increases in phenolic levels [14,16]. The growing interest in understanding the biological functions and cellular interactions of phenolic compounds has also contributed to the expansion of nutraceutical research, which provides health benefits but does not replace conventional pharmacological treatments. For instance, tannins present in grapes and cherries have been shown to reduce inflammatory cytokine production [28], while flavonoids such as quercetin and kaempferol, commonly found in dark-colored fruits and green leaves, exhibit cardioprotective and antihypertensive effects [29].

In the present study, the antioxidant and anti-inflammatory properties of extracts obtained from unripe and ripe Myrciaria tenella fruits, whose fresh juice is traditionally consumed, were investigated. The aqueous and hydroethanolic extraction methods employed are simple and reproducible, facilitating both the identification of bioactive compounds and the evaluation of their biological activities. A major difference observed between unripe and ripe fruits was the total phenolic content, which was nearly twice as high in unripe fruits. These variations are likely associated with the ripening process, which promotes quantitative and qualitative changes in phytoconstituent composition and, consequently, biological activity [30].

Differences in phytoconstituent profiles were also influenced by the extraction method. Catechin derivatives (flavan-3-ols) were identified in aqueous extracts, whereas kaempferol derivatives (flavonols) were detected in hydroethanolic extracts. Gallic acid, a phenolic acid, was present in extracts obtained using both methods. Consistent with these findings, Martins et al. [31] identified p-coumaric acid, ellagic acid, epicatechin, rutin, quercetin, and catechin in M. tenella leaves, while Rybka et al. [15] reported M. tenella extracts as richt sources of phenolic compounds with high antioxidant potential.

Research on Myrciaria tenella fruits (also referred to as Eugenia tenella) remains limited; however, studies involving other species from this botanical family—such as Eugenia uniflora (pitanga), Plinia cauliflora (jabuticaba), Myrciaria dubia (camu-camu), and Eugenia stipitata (araçá-boi)—have reported the presence of diverse bioactive compounds, including anthocyanins, flavonoids, terpenes, phenolic acids, and carotenoids [12,32,33]. Fruits from the Myrciaria genus are also recognized as important sources of essential minerals, vitamins, phenolic compounds, carotenoids, sugars, and dietary fiber [34,35].

Flavonoids constitute a major class of phenolic compounds whose antioxidant activity depends on structural characteristics, such as the number and position of hydroxyl groups, electron transfer capacity, hydrogen or electron donation, and the ability to chelate metal ions, including iron and copper [36]. The biochemical assays performed in the present study demonstrated the antioxidant potential of all four M. tenella fruit extracts. Notably, antioxidant activity observed in fruit extracts was markedly higher than that previously reported for leaf extracts. For instance, TAC values ranged from 127 to 168 µg EqAA/g in fruit extracts, whereas leaf extracts exhibited values between 13 and 50 µg EqAA/g [23].

Furthermore, copper chelation activity observed in this study was approximately threefold higher than that reported for M. dubia extracts [32], suggesting a strong metal-chelating capacity of M. tenella fruits. Likewise, the antioxidant activity of M. tenella fruit extracts—assessed through free radical scavenging and electron donation—was 10- to 40-fold higher than that reported for M. vexator [35].

The antioxidant potential of M. tenella fruit extracts was further investigated using hydrogen peroxide (H₂O₂)- and copper sulfate-induced oxidative stress models in RAW 264.7 macrophages and NIH/3T3 fibroblasts, respectively. Hydrogen peroxide is a reactive oxygen species capable of disrupting cellular redox homeostasis and is therefore widely used to evaluate cellular oxidative responses and antioxidant agents [37]. In the present study, M. tenella fruit extracts exhibited no cytotoxic effects under the experimental conditions tested.

Comparable findings have been reported for other species of the genus. Carmo et al. [34] observed no cytotoxicity for M. dubia extracts, while Ribeiro et al. [23] and Lima et al. [38] reported similar results for leaf extracts from M. tenella and M. ferruginea, respectively, in 3T3 murine fibroblasts. Additionally, treatment of RAW 264.7 cells with M. tenella fruit extracts followed by H₂O₂ exposure resulted in increased cell viability, indicating that phenolic compounds present in these extracts may mitigate oxidative stress-induced damage. This observation is consistent with findings reported by Su et al. [33], who demonstrated that flavonoids from another Myrciaria species enhanced RAW 264.7 cell survival under oxidative stress conditions. Likewise, Lin et al. [39] reported that low concentrations of curcumin protected cells from oxidative injury, while Zhuang et al. [40] demonstrated that phenolic compounds derived from rambutan (Nephelium lappaceum) bark effectively protected HepG2 cells against oxidative damage.

Copper sulfate is a well-established inducer of oxidative stress, as it promotes increased reactive oxygen species (ROS) production, reduces endogenous antioxidant defenses, enhances mitochondrial membrane permeability, and ultimately triggers cell death. Due to their high sensitivity to copper ionophores, mitochondria undergo metabolic disruption when exposed to excessive copper levels [41]. In addition, the copper redox cycle leads to the formation of hydroxyl radicals, which are involved in enzymatic processes such as energy metabolism, respiration, and DNA synthesis [42]. Under pathological conditions, however, copper acts as a potent stressor by generating H₂O₂ and hydroxyl radicals, resulting in lipid peroxidation, DNA damage, and impairment of other cellular components. These alterations may promote inflammation, autophagy, and cellular dysfunction, potentially contributing to senescence, organ failure, and disease development [43].

Tsvetkov et al. [44] described the process of lipoylation, whereby copper binds to mitochondrial proteins and induces a toxic state that promotes cell death. Copper-induced ROS generation is further intensified by physiological reducing agents such as ascorbate, which facilitates the conversion of Cu²⁺ to Cu⁺—the redox-active form involved in ROS-producing reactions [45]. Excess intracellular copper accumulation has been associated with several pathological conditions, including Wilson’s disease, liver cirrhosis, neurodegenerative disorders, anemia, cardiovascular diseases, cancer, and diabetes mellitus [6,46,47,48].

The protective effects of Myrciaria tenella fruit extracts under copper-induced oxidative stress were evaluated using NIH/3T3 fibroblast cells. Under the experimental conditions tested, these extracts exhibited no cytotoxic effects. ROS levels measured using the DCFH-DA probe revealed markedly reduced fluorescence intensity (below 44%) in extract-treated cells, suggesting effective mitigation of oxidative stress. These findings are consistent with those reported by Bittner Fialová et al. [49], who demonstrated that Mentha rhizome extracts inhibited ROS formation in NIH/3T3 cells exposed to H₂O₂.

In contrast, Inthi et al. [50] reported that Houttuynia cordata extracts increased ROS production in breast cancer cells, leading to cell death. Similarly, Dhivya et al. [51] observed time-dependent ROS generation in copper-treated breast cancer cells, reinforcing the context-dependent nature of phytochemical activity.

Cell viability assays based on metabolic activity and nuclear integrity demonstrated that M. tenella fruit extracts preserved cell number and morphology under copper-induced stress, in contrast to the positive control, which exhibited reduced viability [52]. Nuclear staining further revealed mild apoptotic features, including chromatin condensation and fragmentation [53]. Comparable observations were reported by Gülnar et al. [54], who demonstrated that phenolic-rich plant extracts induced apoptosis in 3T3-L1 preadipocytes, as evidenced by increased chromatin condensation following DAPI staining.

The scratch assay is widely used to simulate wound healing in vitro, allowing the evaluation of cellular migration mechanisms and the activity of potential wound-healing agents [55]. In the present study, M. tenella fruit extracts promoted enhanced wound closure, with migration rates of 52% for VE, 47% for VA, and 45% for ME, compared with 29% observed in the control group. Similar findings were reported by Teplicki et al. [56], who demonstrated that Aloe vera treatment increased fibroblast migration by approximately 29%.

Conversely, Derici et al. [57] reported Bolanthus. spergulifolius extracts delayed wound healing in 3T3-L1 adipocytes. Supporting our results, Vaid et al. [58] showed that Aloe vera extract stimulated fibroblast migration by 29% compared to the negative control (17%) within 24 h, suggesting its potential use in wound treatment.

In contrast, Derici et al. [57] reported delayed wound healing following treatment with Bolanthus spergulifolius extracts in 3T3-L1 adipocytes, indicating that phytochemical effects on cell migration may vary depending on plant species, extract composition, and experimental conditions. Supporting the results observed in the present study, Vaid et al. [58] demonstrated that Aloe vera extract stimulated fibroblast migration by 29% relative to the negative control (17%) within 24 h, suggesting its potential application in wound repair processes. In addition, their MTT assay confirmed increased cell viability and cytoprotective effects following peroxide-induced oxidative stress, while the wound-healing assay revealed enhanced cell migration after 20 h of injury.

In parallel, the anti-inflammatory potential of M. tenella fruit extracts was evaluated in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Treatment with the extracts resulted in a reduction in nitric oxide (NO) production of approximately 30% for VA and VE, 39% for MA, and 21% for ME. Comparable results were reported by Gonçalves et al. [59], who demonstrated that M. tenella leaf extracts reduced NO production by 44% without exhibiting cytotoxic effects, as confirmed by MTT assays. Similarly, Pereira et al. [20] observed that extracts from Myrciaria glazioviana preserved RAW 264.7 macrophage viability.

Collectively, these findings indicate that M. tenella fruit extracts are capable of attenuating H₂O₂- and copper-induced oxidative stress, preserving fibroblast viability, limiting excessive ROS accumulation, and promoting cell migration. These protective effects are likely associated with the presence of antioxidant phenolic compounds, highlighting the potential therapeutic relevance of M. tenella extracts in conditions related to oxidative stress and impaired tissue repair.

Although in vitro assays provide valuable insights into cellular mechanisms, they do not fully reflect the complexity of oxidative stress responses at the organismal level. Therefore, to further validate the biological relevance of the observed antioxidant effects, the protective potential of M. tenella fruit extracts was subsequently evaluated using an in vivo model, Tenebrio molitor. Invertebrate organisms represent valuable alternatives to vertebrate models due to their ease of handling, short life cycle, and low maintenance costs. Accordingly, T. molitor (yellow mealworm) has been increasingly employed as a practical experimental model [60]. Its widespread use is attributed to its manageable size, well-characterized immune response, capacity to control pathogens, and additional biotechnological relevance, including its potential as a polymer-degrading organism [61].

Given the copper-chelating capacity of M. tenella fruit extracts and their ability to preserve NIH/3T3 cell viability under CuSO₄-induced stress, these extracts were also investigated in the T. molitor in vivo model. Survival assays demonstrated that the extracts were non-toxic and effectively mitigated oxidative stress, maintaining elevated survival rates, particularly for VE (80%) and MA (73%) treatments.

Comparable studies conducted by Cordeiro et al. [62] also employed T. molitor to assess extract toxicity and antioxidant potential, reporting protection against CuSO₄-induced oxidative damage. Their findings support the role of phenolic compounds in protecting invertebrate models from oxidative stress. In addition, Naccarato et al. [63] reported significant alterations in melanization responses in T. molitor following exposure to herbicides and fertilizers, reinforcing the relevance of this model for oxidative stress assessment.

Considering the phytoconstituents tentatively identified by HPLC in M. tenella fruit extracts, gallic acid has been associated with several biological activities, including the reduction in inflammatory mediators and the upregulation of antioxidant enzyme expression in RAW 264.7 macrophages [64]. Furthermore, gallic acid has been shown to attenuate liver damage in iron-overloaded mice by decreasing malondialdehyde levels and increasing superoxide dismutase (SOD) and glutathione (GSH) expression [65]. This compound also reduces β-galactosidase activity and oxidative stress markers in rat embryonic fibroblasts, suggesting a role in the regulation of cellular senescence and metabolic dysfunction through antioxidant mechanisms [66]. In agreement, Variya et al. [67] demonstrated that gallic acid derived from Emblica officinalis fruit juice improves insulin sensitivity and glucose homeostasis in adipocytes.

In addition to gallic acid, other phenolic compounds identified in M. tenella fruit extracts may also contribute to the biological activities observed in this study, particularly flavonoids such as kaempferol.

Kaempferol, a widely studied flavonoid, has been associated with therapeutic applications in cancer, cardiovascular diseases, metabolic disorders, and neurological conditions. Its biological activity involves the regulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a central transcription factor in redox homeostasis, as well as the modulation of inflammatory signaling pathways, including JNK, MAPKs, ERK, and PI3K/Akt. These mechanisms contribute to its reported anti-inflammatory, antimicrobial, and endothelial-protective properties [37,68,69]. Kaempferol has been identified in seeds of Myrciaria species, including Eugenia uniflora L. (pitanga) [70].

Catechins are abundant in Camellia sinensis (green tea) and exhibit pronounced antioxidant activity. These compounds scavenge reactive oxygen species by inhibiting pro-oxidant enzymes and activating endogenous antioxidant systems, such as glutathione S-transferase and superoxide dismutase. Moreover, catechins function as metal chelators by binding excess iron and copper through their gallate groups, thereby contributing to the regulation of metal homeostasis [71].

The present findings emphasize the relevance of the tested M. tenella fruit extracts, which contain pharmacologically active phenolic compounds closely associated with the biological effects observed throughout the study. These constituents displayed antioxidant activity across multiple experimental models, suggesting their potential contribution to protective mechanisms against oxidative stress. The occurrence of such compounds in M. tenella fruits supports further investigation of their biotechnological applications, particularly regarding the use of unripe fruit extracts in pharmaceutical, nutraceutical, and dietary supplement development. Notably, the VE extract—likely due to its higher phenolic content—exhibited consistent antioxidant effects in both in vitro and in vivo assays.

4. Materials and Methods

4.1. Reagents

Potassium ferricyanide, Iron (II) sulfate, trichloroacetic acid, Folin–Ciocalteu reagent, and sulfuric acid were purchased from Merck (Darmstadt, Germany). Ethylenediaminetetraacetic acid (EDTA), gallic acid, ascorbic acid, quercetin, methionine, pyrocatechol violet, riboflavin, and ammonium molybdate were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). DPPH (2,2-diphenyl-1-picrylhydrazyl) was purchased from Fluka (Seelze, Germany). Cupric sulfate was obtained from Chemical Kinetics. Penicillin and streptomycin were obtained from Gibco (Fort Worth, TX, USA). Nitro Blue Tetrazolium (NBT), monosaccharides, methionine, and ammonium molybdate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Dulbecco’s Modified Eagle Medium (DMEM), L-glutamine, sodium bicarbonate, sodium pyruvate, and phosphate-buffered saline (PBS) were purchased from Invitrogen Corporation (Burlington, ON, Canada). Trypsin was obtained from Vitrocell (Campinas, SP, Brazil), and fetal bovine serum (FBS) was purchased from Cultilab (Campinas, SP, Brazil).

Mouse embryonic fibroblasts (NIH/3T3, ATCC^® CRL-1658^TM) and mouse macrophage cells (RAW 264.7, ATCC^® TIB-71^TM) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). All other solvents and chemicals used were of analytical grade.

4.2. Plant Material

The fruits of Myrciaria tenella were collected in March 2020 in the Rio do Fogo (5°16′38″ S, 35°23′06″ W), Rio Grande do Norte, Brazil. The region (Supplementary Figure S1) presents a tropical climate, with a rainy season between March and August and an average temperature of around 28.5 °C.

The plant was identified by Dr. Leonardo de Melo Versieux (Universidade Federal do Rio Grande do Norte-UFRN), and a voucher specimen was deposited under number 20383. This study was registered under SISBIO (77313-1) and SISGEN (A39FD4C).

4.3. Preparation of Fruit Extracts

Myrciaria tenella fruits at two developmental stages, unripe and ripe, were used to prepare aqueous and hydroethanolic extracts. Unripe fruits were characterized by green, yellow, or light red coloration, whereas ripe fruits displayed red to purple coloration. The extracts were designated as follows: VA (aqueous unripe), VE (hydroethanolic unripe), MA (aqueous ripe), and ME (hydroethanolic ripe).

For each extraction, 100 g of fruits were washed, crushed, and macerated with 1000 mL of the extraction solvent—distilled water for aqueous extracts or 70% ethanol for hydroethanolic extracts—at a 1:10 w/v ratio (plant material: solvent). Maceration was performed under continuous agitation for 24 h at 26 °C, using flasks wrapped with aluminum foil to protect from light exposure.

The extracts were filtered through Whatman No. 1 paper, and solid residues were discarded. Solvent removal was carried out using a rotary evaporator at 40 °C until the volume was reduced to approximately 25 mL. The concentrated extracts were transferred to conical tubes and freeze-dried using a lyophilizer. The resulting dried extracts were weighed, resuspended in distilled water at a concentration of 100 mg/mL, and stored at −20 °C until further use.

4.4. In Vitro Antioxidant Activity

Different assays were employed to evaluate the antioxidant activity of the extracts, including total antioxidant capacity (TAC), DPPH radical scavenging, reducing power, and copper and iron chelation [72].

4.4.1. Total Antioxidant Capacity (TAC)

This colorimetric assay evaluates the ability of the extracts to reduce molybdenum + 6 to molybdenum + 5, forming a green phosphate/Mo + 5 complex at acid pH [73]. Extract samples (10 mg/mL) were mixed with reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, 4 mM ammonium molybdate, reaching a final volume of 1 mL. The mixtures were incubated at 100 °C for 90 min. After cooling to a room temperature, absorbance was measured at 695 nm using a spectrophotometer (Biotek Epoch Microplate, Santa Clara, CA, USA).

Total antioxidant capacity (TAC) was expressed as ascorbic acid equivalents per mg of sample (µg EqAA/mg), based on a calibration curve constructed with ascorbic acid (Sigma-Aldrich Co., St. Louis, MO, USA).

Reducing power, DPPH radical scavenging, copper, and iron chelation assays were performed at 50, 100, and 250 µg/mL to determine the lowest effective concentration.

4.4.2. Reducing Power Assay

Samples were incubated with a solution containing phosphate buffer (0.2 M, pH 6.6) and potassium ferricyanide (1%) at 50 °C for 20 min, with a final volume of 4 mL. Potassium ferricyanide (Fe³⁺) was reduced to potassium ferrocyanide (Fe²⁺), which in the presence of iron chloride formed a greenish blue color known as Prussian blue {Fe₄[Fe(CN)₆]₃}. The reaction was completed by adding the TCA solution (10%) and then it was homogenized with distilled water and ferric chloride (0.1%). The absorbance was read at 700 nm at a spectrophotometer (Biotek Epoch Microplate, Santa Clara, CA, USA). The result was expressed as the relatively reduced capacity of samples (%), considering the activity of the reducing capacity of ascorbic acid at 0 as 100%, and the activity of the reducing capacity of ascorbic acid at 0.1 mg/mL.

4.4.3. DPPH Radical Scavenging

This assay was performed to evaluate the hydrogen-donating ability of the extracts to neutralize the stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl). In a 96-well plate, 100 µL of each sample at the evaluated concentration was mixed with a 150 µM DPPH solution prepared in ethanol. The control consisted of DPPH solution without the extract.

The reactions were incubated in the dark at room temperature for 30 min, according to the method described by Shimada et al. [74].

Absorbance was measured at 517 nm using a microplate spectrophotometer (Biotek Epoch, Santa Clara, CA, USA).

Radical scavenging activity was expressed as percentage (%) and calculated using the following equation:

$$\mathrm{DPPH}\mathrm{scavenging}\mathrm{activity}(\%)=[1-\left({\displaystyle \frac{A_{\mathrm{sample}}}{A_{\mathrm{control}}}}\right)]\times 100$$

where A_sample represents the absorbance of the extract-treated sample and A_control represents the absorbance of the DPPH solution alone.

4.4.4. Copper Chelation

The copper chelation capacity of the extracts was evaluated using pyrocatechol violet as an indicator. The reaction mixture consisted of pyrocatechol violet (4 mM) and copper sulfate pentahydrate (50 µg/mL) prepared in acetate buffer. After homogenization, the chelation of Cu²⁺ ions by the extracts was assessed by measuring absorbance at 632 nm using a microplate spectrophotometer (BioTek Epoch, Santa Clara, CA, USA).

Copper chelation activity was calculated according to the equation:

$$\mathrm{Copper}\mathrm{chelation}(\%)=[1-\left({\displaystyle \frac{A_{\mathrm{sample}}}{A_{\mathrm{blank}}}}\right)]\times 100$$

where A_sample corresponds to the absorbance in the presence of extract and A_blank corresponds to the reaction mixture without extract.

4.4.5. Iron Chelation

Iron chelating activity was determined using the ferrozine assay, as described by Decker and Welch [75]. The reaction mixture contained the extract, 0.05 mL of FeCl₂ solution (2 mM), and 0.2 mL of ferrozine solution (5 mM), adjusted to a final volume of 1 mL. The mixture was incubated at room temperature for 10 min.

Absorbance was measured at 562 nm using a microplate spectrophotometer (BioTek Epoch, Santa Clara, CA, USA) and compared with the blank solution.

Iron chelation activity was calculated using the following equation:

$$\mathrm{Iron}\mathrm{chelation}(\%)=[1-\left({\displaystyle \frac{A_{\mathrm{sample}}}{A_{\mathrm{blank}}}}\right)]\times 100$$

4.5. Cell Lines and Cell Culture

The cell lines used in this study were murine macrophages/monocytes (RAW 264.7, ATCC TIB-71) and NIH/3T3 fibroblasts (ATCC CRL-1658). Both cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

4.5.1. MTT Reduction Assay

The MTT assay was performed to evaluate the cytotoxic effects of the extracts on RAW 264.7 macrophages (ATCC^® TIB-71^TM) and NIH/3T3 fibroblast cells (ATCC^® CRL-1658^TM), as previously described [76].

Cells were seeded in 96-well plates at a density of 1.0 × 10⁴ cells/well in DMEM supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. After 24 h, the medium was replaced with DMEM without FBS, followed by an additional 24 h incubation period.

Subsequently, DMEM supplemented with 10% FBS containing the extracts at concentrations of 50, 100, or 250 µg/mL was added to the cells. After 24 h of treatment, MTT solution (1 mg/mL) was added to each well, and the plates were incubated for 4 h at 37 °C.

Formazan crystals were solubilized with absolute ethanol, and absorbance was measured at 570 nm using a microplate reader (BioTek Epoch, Santa Clara, CA, USA). The negative control (NC) consisted of cells cultured in DMEM with FBS without extract treatment and was considered as 100% cell viability.

MTT reduction was calculated using the following equation:

$$\mathrm{MTT}\mathrm{reduction}(\%)=\left({\displaystyle \frac{A_{\mathrm{sample}}}{A_{\mathrm{negative}\mathrm{control}}}}\right)\times 100$$

4.5.2. In Vivo Oxidative Stress Assay with H₂O₂ in RAW 264.7 Macrophages

To evaluate the antioxidant potential of the extracts under oxidative stress conditions, RAW 264.7 macrophages were exposed to hydrogen peroxide (H₂O₂) (Merck, Darmstadt, Germany).

Initially, five concentrations of H₂O₂ (0.1, 0.3, 0.5, 0.7, and 1.0 mM) were tested to determine the inhibitory concentration that reduced cell viability by approximately 50% (IC₅₀). Based on this analysis, 0.3 mM H₂O₂ was selected for subsequent assays.

Cells were seeded in 96-well plates at a density of 10 × 10⁴ cells/well in DMEM supplemented with 10% FBS and incubated for 24 h at 37 °C with 5% CO₂. The medium was then replaced with DMEM without FBS for an additional 24 h.

Cells were subsequently treated with the extracts at 100 µg/mL in the presence of 0.3 mM H₂O₂ for 2 h. After treatment, the medium was replaced with DMEM containing 10% FBS, and cells were incubated for another 24 h.

Cell viability was assessed using the MTT assay as described in Section 4.5.1. Absorbance was measured at 570 nm, and MTT reduction was calculated using the following equation:

$$\mathrm{MTT}\mathrm{reduction}(\%)=\left({\displaystyle \frac{A_{\mathrm{sample}}}{A_{\mathrm{control}}}}\right)\times 100$$

4.5.3. NO Reduction Assay in RAW 264.7 Macrophages

Nitric oxide (NO) production was evaluated as an indicator of inflammatory response in RAW 264.7 macrophages. Lipopolysaccharide (LPS) from bacteria (Sigma-Aldrich Co., St. Louis, MO, USA) was used to induce inflammation.

Cells were seeded in 24-well plates at a density of 3.0 × 10⁵ cells/well in DMEM supplemented with 10% FBS and incubated for 24 h at 37 °C with 5% CO₂. The medium was then replaced with DMEM without FBS for an additional 24 h.

Subsequently, cells were treated with M. tenella extracts (100 µg/mL) in the presence of LPS (1 µg/mL). The negative control consisted of untreated cells, while the positive control consisted of cells treated with LPS only.

After 24 h of incubation, 50 µL of culture supernatant was mixed with 50 µL of Griess reagent (Sigma-Aldrich Co., St. Louis, MO, USA), containing 5% sulfanilamide and 0.5% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid. The mixture was incubated for 10 min at 25 °C.

Absorbance was measured at 540 nm using a microplate reader (BioTek Epoch, Santa Clara, CA, USA). Nitrite concentration was determined using a sodium nitrite standard curve.

Nitric oxide production was expressed as a percentage relative to the positive control, according to the equation:

$$\mathrm{NO}\mathrm{production}(\%)=\left({\displaystyle \frac{\mu M_{\mathrm{nitrite}\left(\mathrm{sample}\right)}}{\mu M_{\mathrm{nitrite}\left(\mathrm{positive}\mathrm{control}\right)}}}\right)\times 100$$

4.5.4. NIH/3T3 Fibroblast Cells Migration Assay (Wound Healing)

The effect of M. tenella fruit extracts on the migratory behavior of NIH/3T3 cells (ATCC CRL-1658) was evaluated using the wound healing assay, as previously described [55].

NIH/3T3 cells were seeded in 24-well plates at a density of 1 × 10⁶ cells/well in DMEM supplemented with 10% fetal bovine serum (FBS). After 24 h, a confluent monolayer was formed. The medium was removed, and a linear scratch was generated in the cell monolayer using a sterile 200 µL pipette tip. The wells were gently washed with phosphate-buffered saline (PBS) to remove detached cells.

The extracts (100 µg/mL) were added in DMEM containing 10% FBS. Images of the wound area were captured at 0, 12, and 24 h using a Nikon Eclipse Ti microscope equipped with a Moticam 5+ camera and a 40× objective.

Wound width was measured using NIS-Elements AR (advanced research) 4.0 software by determining the shortest distance between the opposing edges of the scratch [77]. Cell migration was expressed as the percentage of wound closure relative to the initial wound area (0 h).

4.5.5. In Vivo Oxidative Stress Assay Using CuSO₄ and Ascorbate in NIH/3T3 Fibroblasts Cells

The NIH/3T3 fibroblast cell line was used to evaluate the antioxidant activity of the extracts under copper-induced oxidative stress conditions. Oxidative stress was induced using copper sulfate (CuSO₄) in the presence of ascorbate [78].

Initially, the IC₅₀ of CuSO₄ in the presence of 1 mM ascorbate was determined by testing CuSO₄ concentrations ranging from 1 to 30 µM. Based on these results, 25 µM CuSO₄ was selected, as it reduced cell viability by approximately 50%.

For the assay, NIH/3T3 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well in DMEM supplemented with 10% FBS and incubated at 37 °C under 5% CO₂ for 24 h. The medium was then replaced with DMEM without FBS and incubated for an additional 24 h.

Subsequently, extracts (100 µg/mL) and CuSO₄ (25 µM) were added, followed by the addition of ascorbate (1 mM) after 15 min. Cells were incubated for 45 min at 37 °C under 5% CO₂. The medium was then replaced with DMEM containing 10% FBS, and cells were incubated for 24 h.

Cell viability was assessed using the MTT reduction assay. After incubation with MTT for 4 h, formazan crystals were dissolved in absolute ethanol and shaken for 15 min. Absorbance was measured at 570 nm using a microplate reader (BioTek Epoch, Santa Clara, CA, USA). Negative and positive controls were included to evaluate the effects of CuSO₄ and ascorbate.

4.5.6. Assessment of Intracellular ROS Generation Using the DCFH-DA Fluorescent Probe

Intracellular reactive oxygen species (ROS) levels were determined using the fluorescent probe DCFH-DA, as previously described [79].

NIH/3T3 cells were seeded in 24-well plates at a density of 2 × 10⁵ cells/well in 500 µL of DMEM supplemented with 10% FBS and incubated at 37 °C with 5% CO₂ for 24 h.

Cells were then treated with the extracts (100 µg/mL) and CuSO₄ (25 µM), followed by the addition of ascorbate (1 mM) after 15 min. After 45 min of incubation, the medium was replaced with 10 µM DCFH-DA solution (prepared from a stock solution of 4.85 mg DCFH-DA in 1 mL DMSO) and incubated for 2 h. Cells were washed twice with DMEM and once with PBS. Subsequently, 100 µL of trypsin (2.5 g/L) was added for 8 min to detach the cells, which were then transferred to 1.5 mL microtubes. A 0.1 M sodium citrate solution was added, and samples were sonicated three times for 5 s.

The suspensions were centrifuged at 26,000× g for 10 min at 4 °C. The supernatant (200 µL) was transferred to a black 96-well plate, and fluorescence was measured using a GloMax^® Multi Detection System (Promega, Madison, WI, USA) with excitation at 485 nm and emission at 530 nm.

4.5.7. Nucleus Analysis After DAPI Labeling

DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent dye widely used for nuclear staining, allowing visualization of nuclear morphology and identification of chromatin alterations under fluorescence microscopy. DAPI preferentially binds to AT-rich regions of DNA and emits blue fluorescence under UV excitation [80].

To evaluate nuclear alterations induced by CuSO₄- and ascorbate-mediated oxidative stress, NIH/3T3 cells were subjected to DAPI staining. Cells were seeded at a density of 2 × 10⁵ cells/well in 24-well plates containing 500 µL DMEM and incubated at 37 °C with 5% CO₂ for 24 h. The medium was replaced with DMEM without FBS and incubated for an additional 24 h.

Extracts (100 µg/mL) were added, followed by CuSO₄ (25 µM) and ascorbate (1 mM) for 45 min. After treatment, the medium was replaced with DMEM supplemented with 10% FBS, and cells were incubated for 24 h.

Cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 30 min, and stained with DAPI (1 µg/mL) for 30 min. After washing with PBS, cells were visualized using a fluorescence microscope (TE-Eclipse 300, Nikon, Yuko, Japan).

4.6. Chemical Composition of M. tenella Fruit Extracts

Total Phenolic Content (TPC)

The quantification of the total content of phenolic compounds was carried out by the colorimetric method using the Folin–Ciocalteu (Sigma-Aldrich Co., St. Louis, MO, USA) reagent and gallic acid as a reference standard for the calibration curve [72,81]. The results were expressed as gallic acid equivalents/mg of extract.

4.7. High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD)

HPLC-DAD analysis was performed according to Melo et al. [82] using an Agilent 1260 system equipped with a Zorbax C18 column (150 mm × 4.6 mm × 3.5 µm), maintained at 45 °C.

A gradient elution was carried out with 0.1% acetic acid (AcOH) and acetonitrile (CH₃CN) as the mobile phases. The gradient programs consisted of 10% CH₃CN (0–6 min), increased to 15% (6–7 min), followed by 50% (22–32 min), and reaching 100% (32–42 min), which was maintained isocratically for an additional 8 min.

The flow rate was set at 1.0 mL/min, and 10 µL of each extract solution (2 mg/mL) was injected. Chromatograms were recorded at 280 nm and 352 nm. Compound identification was based on comparisons of retention times and UV-visible spectra with previous isolated reference compounds from the Phytochemistry Laboratory (University of São Paulo).

4.8. In Vivo Antioxidant Capacity Using Tenebrio molitor as an In Vivo Model

4.8.1. Tenebrio molitor Maintenance

Tenebrio molitor larvae were maintained in plastic containers (30 × 45 × 15 cm) at room temperature (25–29 °C), in the absence of light. To optimize colony development, insects were separated according to life cycle stage (larvae, pupa, and adult beetles).

The feeding substrate consisted of 36.36% wheat bran, 18.18% wheat germ, 18.18% wheat flour, 16.36% oat flakes, 10.9% soybean extract, and 0.04% chloramphenicol (Patrícia Canteri de Souza, personal communication).

4.8.2. Action of the Extracts on Tenebrio molitor Survival

An in vivo assay was conducted to evaluate the potential toxicity of the extracts in invertebrate organisms. Larvae weighing 100–150 mg were randomly assigned to experimental groups of 10 individuals, with three biological replicates, totaling 30 larvae per treatment.

Larvae were placed in Petri dishes at room temperature and anesthetized on ice. Extracts at concentrations of 100 µg/mL or 250 µg/mL were injected (5 µL) into the ventral hemocoel between the second and third abdominal segments using a Hamilton microsyringe (701 N, 26 gauge, 10 µL). Negative control groups received phosphate-buffered saline (PBS: NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 10 mM, KH₂PO₄ 1.76 mM; pH 7.4), while positive controls received only the oxidative stressor. Larval survival was monitored every 24 h for 10 days. Death was defined by the absence of response to mechanical stimulation and the presence of cuticular melanization.

4.8.3. Effect of Extracts on Tenebrio molitor Survival Under CuSO₄-Induced Oxidative Stress

This assay was performed according to Cordeiro et al. [62]. T. molitor larvae (100–150 mg) were randomly divided into groups of 10 individuals, with three biological replicates per treatment.

Larvae were anesthetized on ice and injected with 5 µL of 0.25% CuSO₄ into the ventral hemocoel between the second and third abdominal segments to induce oxidative stress. After 1 h, 5 µL of extract solution (100 µg/mL) was administered. Positive controls received CuSO₄ followed by PBS, whereas negative controls received two PBS injections. Survival was monitored every 24 h for 15 days. Death was determined by lack of movement upon mechanical stimulation and visible melanization.

4.8.4. Melanization Analysis in Tenebrio molitor Under CuSO₄-Induced Oxidative Stress

Melanization was quantified according to Jorjão et al. [83], with minor modifications. Biological triplicates were performed using five larvae per group, totaling 15 larvae per treatment.

Larvae from the oxidative stress assay (Section 4.8.3) were anesthetized on ice, and an incision was made in the head region for hemolymph collection. Samples were homogenized in PBS at a 1:100 ratio (larvae/µL) and centrifuged at 21,913× g for 15 min at 4 °C. Melanization was quantified by measuring absorbance at 405 nm using a microplate reader (BioTek Epoch, Santa Clara, CA, USA). Results were expressed as optical density (OD).

4.9. Language Editing

To improve the clarity, coherence, and flow of the English language in the manuscript, the authors utilized ChatGPT version 4.0. This model was developed by OpenAI (San Francisco, CA, USA). The use of this tool was limited to linguistic revision and did not influence the scientific content, data interpretation, or conclusions of the study.

The final language verification was done by Nathalia Maira Cabral de Medeiros.

5. Conclusions

This study highlights the biological potential of Myrciaria tenella fruit extracts, particularly the unripe hydroethanolic extract (VE), which presented the most prominent antioxidant activity. The observed effects are associated with the presence of pharmacologically active phytoconstituents, especially phenolic compounds, as illustrated in Figure 8. Both in vitro and in vivo assays demonstrated that the extracts effectively protected cells against oxidative stress induced by H₂O₂ and CuSO₄, reduced nitric oxide production in LPS-stimulated macrophages, and enhanced the survival of Tenebrio molitor larvae under CuSO₄-induced stress. Phytochemical analyses identified phenolic acids and flavonoids, including gallic acid, catechin derivatives, and kaempferol derivatives, which likely contribute to the antioxidant responses observed (Figure 8). Although these findings support the therapeutic relevance of M. tenella fruit extracts, further studies are required to expand phytochemical characterization and to evaluate their biological effects in animal models. Considering the heterogeneous composition of these extracts and the potential synergistic interactions among their constituents, comprehensive toxicological and pharmacological investigations are essential to ensure safety and efficacy. Overall, these results reinforce the potential of M. tenella fruits as promising natural sources of antioxidants, supporting their future exploration as supplements, nutraceuticals, or pharmacotherapeutic agents.

Figure 8. Bioactive compounds identified by HPLC-DAD in Myrciaria tenella fruit extracts and their potential biological activities inferred from the assays performed in this study. The ↑ corresponds an increase and ↓ corresponds to a decrease.