In Vitro Bioactivity of a Supercritical CO Naringenin-Rich Extract on Diabetes-Related Metabolic Disturbances.

1. Introduction
Lippia graveolens, also known as Mexican oregano, is a Verbenaceae shrub widely distributed throughout Mexico. It is used in Mexican traditional medicine as an analgesic, to treat inflammation, as a remedy for diarrhea, for menstrual disorders, as an antispasmodic, and for diabetes, as well as a spice in traditional cuisine [1]. Some reports from plants of the Lippia genus indicate the ability to reduce blood glucose levels in rats [2] and to improve insulin regulation [3]. Most studies on Lippia graveolens (LG) focus on its lipophilic extracts, which are rich in essential oils. However, LG also has polar compounds, including phenolic compounds (PCs), especially flavonoids, such as cirsimaritin, quercetin, phloridzin, luteolin, and naringenin [4].
Various health benefits have been attributed to these PCs. For instance, quercetin has shown an effect on glucose uptake in human hepatic (HepG2) cells in an insulin resistance model similar to metformin [5], and cirsimaritin has shown an impact against metastasis in human breast cancer (MDA-MB-231) cells [6]. Moreover, naringenin (4′,5,7-trihydroxyflavanone), a flavonoid commonly found in herbs and citrus fruits, has been reported to exhibit biological activity against oxidative stress, inflammation, and diabetes [4,7]. Naringenin has also shown potential to alleviate insulin resistance [8] and reduce inflammation by inhibiting NO production [9].
Insulin resistance is a physiological state in which tissues do not respond to normal insulin levels and is associated with several conditions, including type 2 diabetes, atherosclerosis, and metabolic syndrome [10]. The mechanism underlying insulin resistance remains unclear; however, oxidative stress and inflammation are closely associated with it. The hyperglycemic state characteristic of diabetes promotes the overproduction of oxidative species (ROS) in β-cells via the mitochondrial respiratory chain, inducing DNA damage, impairing function, increasing stress, and decreasing insulin sensitivity. Hyperglycemia also leads to low-grade chronic inflammation, characterized by elevated levels of pro-inflammatory cytokines and markers, including tumor necrosis factor α (TNF-α), interleukin (IL)-6, and C-reactive protein (CRP), which interfere with regular insulin signaling [11,12,13].
One of the first challenges in characterizing potential functional ingredients is determining their bioaccessibility. To exert their bioactivities, the compounds must be bioaccessible, reaching the intestine and being released from the food matrix for absorption [14]. The bioaccessibility of flavonoids is linked to their chemical structure, as these compounds undergo degradation or transformation in response to pH changes and enzymatic activity during digestion [15,16,17]. Likewise, the extraction method affects the structural stability of the extracted molecules and the overall phytochemical profile obtained, which, in turn, influences bioactivity.
Traditional methods for extracting flavonoids often use organic solvents, which can be harmful to health and the environment. Modern extraction technologies, such as supercritical carbon dioxide (CO2) extraction, offer a safer alternative. This technique leverages supercritical CO2’s unique properties—its ability to diffuse like a gas and extract like a liquid—to enhance extraction efficiency. The extraction power can be adjusted by varying temperature, pressure, cosolvent proportion, and flow rate [18]
In our previous study, we optimized the extraction of naringenin from LG using supercritical CO2. We observed that simulated gastrointestinal digestion decreased the antioxidant capacity (as evaluated by in vitro chemical assays) of the extract [19]. However, these evaluations do not correlate with the complexity of biological systems, limiting the ability to predict the biological potential of these compounds for human health.
On the other hand, the extraction method has been shown to impact the phytochemical profile significantly and, thus, the biological performance of an extract. Therefore, it is essential to assess whether supercritical fluid extraction not only improves yield but also produces an extract with better biological properties than those obtained through conventional methods. Hence, the present study aimed to compare the antioxidant, anti-inflammatory, and antidiabetic effects of naringenin-rich extracts obtained from LG by two different extraction methods, CO2 supercritical extraction and conventional solid-liquid extraction, both subjected to simulated gastrointestinal digestion.

2. Results and Discussion

2.1. Flavonoid Content and the Digestion Effect of the Extracts
The flavonoids identified in the crude extracts and intestinal phases were naringenin, cirsimaritin, phloridzin, and quercetin (Figure S1). Naringenin was the major flavonoid in both the supercritical and methanolic extracts. In the supercritical extract, the subsequent flavonoids in order of abundance relative to the sum of the flavonoids tested were cirsimaritin (7.7%), quercetin (5.6%), phloridzin (1.4%), apigenin (0.86%), and luteolin (0.41%). In contrast, in the methanolic extract, the order was phloridzin (17.4%), cirsimaritin (11%), quercetin (8.8%), luteolin (2.22%), and apigenin (1.47%). However, simulated gastrointestinal digestion significantly reduced flavonoid content. The bioaccessibility for the supercritical extract was as follows: phloridzin 0%, quercetin 0.02%, luteolin 1.3%, apigenin 4.8%, naringenin 0.9%, and cirsimaritin 1.01%. In the methanolic extract, the bioaccessibility was phloridzin 7%, quercetin 0.02%, luteolin 0.19%, apigenin 0.4%, naringenin 1.3%, and cirsimaritin 0.8%.
The low bioaccessibility of these compounds is attributed to the poor stability of flavonoids during the simulated gastrointestinal digestion, especially in the intestinal section, where the pH tends to be neutral to alkaline. At this pH, reports have shown that flavonoid content decreases [20,21] due to oxidation, dihydroxylation, and polymerization reactions [22,23]. Notably, the higher bioaccessibility of phloridzin in the methanolic extract is attributed to its higher quantity and the presence of a sugar substituent, which confer greater stability compared to the aglycones [24,25]. In contrast, in the supercritical extract, the loss of quercetin (compared with the other flavonoids) is due to the low solubility of this flavonol at intestinal pH [26], as it is found in a protonated form (pKa = 7.58).
Regarding the extraction methods, methanolic extraction was selected as a conventional method due to its widespread use for extracting phenolic compounds from oregano species [27]. This allowed us to compare this method with the supercritical CO2 extraction, which provided a different flavonoid profile and content. The extraction conditions can explain the differences in the flavonoid profile and content. For instance, the supercritical extraction conditions used were appropriate for enhancing the yields of naringenin and other flavonoids with similar polarity. In contrast, methanolic extraction was not targeted to a specific compound, resulting in the extraction of more polar compounds, such as phloridzin. Despite this, naringenin remained the most abundant compound in both extracts.
A similar flavonoid profile has been reported in previous studies of LG with various extraction methods [3,28]. In contrast, the high naringenin content in our study is attributed to the supercritical extraction conditions (pressure of 168 bar, temperature of 58.4 °C, and cosolvent proportion of 12.46%), which enhance the extraction of this flavanone. For instance, increasing the temperature increases vapor pressure and, along with the addition of ethanol, improves the solubility for the extraction of naringenin and compounds with similar polarity [19].

2.2. Cell Viability
The viability of Caco-2 cells was assessed after exposure to crude extracts and their intestinal phases at concentrations ranging from 100 to 800 µg/mL. Cell viability remained above 80% at almost all concentrations, except at 800 µg/mL, where a significant decrease was observed (Figure 1). On the other hand, the viability of RAW 264.7 and HepG2 cells was assessed in the same way as the Caco-2 cells. In these assays, cell viability remained above 80% (Figures S4 and S5). However, a concentration of 50 µg/mL was used to ensure better viability in both assays.

2.3. Cellular Antioxidant Activity
The CAA of the crude extracts and intestinal phases is shown in Figure 2. No significant differences were observed between the supercritical and methanolic extracts. However, the simulated gastrointestinal process significantly reduced CAA for both extracts, with no notable differences between the intestinal phases. Despite the decrease, CAA remained above 79% for both extracts.
In contrast to chemical antioxidant assays, assessing the antioxidant capacity in cell models provides more realistic physiological conditions, including pH, temperature, and cell metabolism [29]. During gastrointestinal digestion, PCs are degraded by enzymes and pH changes [30], thereby reducing the antioxidant activity of the extracts. A similar decrease in CAA has been reported for naringenin and other flavonoids, such as quercetin and luteolin, following simulated gastrointestinal digestion [17].
Although the flavonoid profiles of the supercritical and methanolic extracts differ (Figure S1), no significant differences in CAA were observed between the extracts or their intestinal phases (SIP vs. MIP). This suggests that variations in the PC profile do not substantially affect antioxidant capacity, as these compounds act similarly in counteracting reactive oxygen species (ROS). In this sense, the extracts and intestinal phases contain naringenin, cirsimaritin, quercetin, phloridzin, apigenin, and luteolin in different quantities, which are hydroxylated in the range of two to five hydroxyl groups. This is important as flavonoids are known to exert their antioxidant capacity by donating hydrogen atoms from their hydroxyl groups [31,32]. Although naringenin’s antioxidant capacity is lower compared to other compounds like quercetin, due to the lack of the C2–C3 double bond and catechol moiety in the B ring [33], its high concentration in the extracts and intestinal phases, along with its potential synergy with other compounds such as quercetin [34], might have contributed to the observed CAA. Nevertheless, other phenolic compounds not identified in the extract could also contribute to the synergistic effect, and their identification and quantification should be considered in future studies.
The gastrointestinal digestion caused a significant reduction in antioxidant capacity across all assays. However, in the present study, the decrease in CAA was less pronounced than in the chemical assays previously used (TEAC and ORAC) [19]. In this regard, the TEAC assay measures an antioxidant’s ability to reduce the ABTS•+ radical. It works mainly via a single-electron transfer (SET) mechanism [35], while the ORAC assay evaluates the antioxidant’s capacity to stop a peroxyl radical chain reaction initiated by AAPH, and the mechanism is based on hydrogen atom transfer (HAT) [31]. On the other hand, the CAA assay involves cellular metabolism, such as the absorption, distribution, and metabolism of antioxidants in a living organism. This method is based on the ability of the antioxidant to protect a fluorescent probe against peroxyl radicals generated by AAPH in cells under physiological conditions [36]. In this regard, several reports indicate that the antioxidant activity of extracts is better reflected by the CAA.
Additionally, the gastrointestinal process transformed the compounds into structures that retain antioxidant activity similar to their parent compounds (Figure S2). For example, flavanones can be converted into chalcones [37]. In addition, compared to a study by Gutiérrez-Grijalva et al. [38], an LG extract (80% methanol) showed no difference between the intestinal phase (IP) and the crude extract. This outcome can be attributed to the polarity of the extract, which favored the extraction of glycosylated flavonoids, compounds that are generally more bioaccessible [17].

2.4. Nitric Oxide Production
As shown in Figure 3, NO production in stimulated RAW 264.7 macrophages was significantly reduced by 10.8% with the supercritical extract and by 22% with the methanolic extract. Following simulated digestion, the SIP showed greater NO reduction (38.1%) than the supercritical crude extract. In contrast, the MIP still had the capacity to reduce the NO production (15.8%), although it was less effective than the crude methanolic extract.
Flavonoids are well known for their ability to reduce NO production through various mechanisms, which depend on their chemical structure. For instance, naringenin, the most abundant compound in the SIP and MIP, has been shown to downregulate inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages [9]. This reduction is linked to the inhibition of the NF-κB pathway, achieved by preventing the degradation of IκBα, a natural inhibitor of NF-κB, thereby blocking pathway activation [39].
The methoxylated flavonoid cirsimaritin has been reported to act similarly to naringenin in reducing iNOS expression through blocking IκBα degradation. Besides, a decrease in Akt phosphorylation has been observed [40], a serine/threonine kinase that phosphorylates IKKα/β and subsequently degrades IκBα [41]. Although studies indicate that phloridzin does not inhibit NO production, its aglycone, phloretin, has been shown to reduce iNOS expression by inhibiting NF-κB p65 nuclear translocation [42]. We hypothesize that during the gastrointestinal process, phloridzin undergoes hydrolysis, yielding phloretin [25]. Moreover, luteolin suppresses NO production by inhibiting NF-κB and the activator protein 1 (AP-1) pathways, reducing p65 and c-Jun, respectively [43]. Reports indicate that quercetin can decrease iNOS by suppressing Iκ-B phosphorylation, thus reducing the liberation of NF-κB from the NF-κB/Iκ-B complex [44]. At the same time, apigenin inhibits iNOS expression by suppression of mitogen-activated protein kinases (MAPKs) phosphorylation (ERK and JNK) [45].
The decrease in NO production by the supercritical intestinal phase could be related to the presence of the flavonoids. In this sense, naringenin, cirsimaritin, apigenin, and luteolin, due to their reported ability to inhibit NF-κB activation. Although the concentration of flavonoids in the supercritical intestinal phase was lower compared to the crude supercritical extract, the NO production was significantly decreased. This increase in potency despite lower concentrations can be attributed to the transformation of compounds into more bioactive metabolites [46]. Nevertheless, the presence of other phenolic compounds in the extract could also be affecting NO production regulation and should be analyzed in future studies.

2.5. Glucose Uptake
The glucose uptake in HepG2 cells is shown in Figure 4. The results indicated that MIP and SIP increase glucose uptake compared with the insulin-resistant control, with no significant difference between them. The enhanced glucose uptake could be attributed to several factors. For instance, naringenin, found in high concentrations in the supercritical extract, is a potential insulin receptor activator, promoting its autophosphorylation and initiating the insulin signaling pathway [47]. Also, this flavanone could inhibit the protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signaling [8]. It can induce Akt activation and promote 5′AMP-activated protein kinase (AMPK) phosphorylation [48], an enzyme that controls glucose production by inhibiting gluconeogenic gene expression and increasing the GLUT2 expression [49]. Furthermore, a study in diabetic rats also showed increased AMPK and GLUT2 protein expression in the liver when cirsimaritin was administered [50]. Moreover, phloridzin induces activation of IRS2 and subsequently of the PI3K/Akt pathway, ultimately activating the proteins glycogen synthase kinase-3β (GSK3β) and forkhead box protein O1 (FOXO1), which are negative regulators of gluconeogenesis and glycogenolysis enzymes and positive regulators of glycogen synthesis [51]. In addition, luteolin and apigenin downregulate gluconeogenic and lipogenic gene expression, thereby reducing hepatic glucose production [52].
These results agree with other reports of glucose uptake in insulin-resistant HepG2 cells. For instance, pretreatment with flavonoids, such as quercetin, increased glucose uptake to a level similar to that of the drug metformin. In contrast, the glucose uptake effects of luteolin, luteolin-7-O-glucoside, kaempferol, and apigenin were better than those of metformin but did not reach levels observed in healthy cells [53]. Similarly, baicalein, isorhamnetin-3-O-rutinoside, apigenin-7-O-glucoside, kaempferol-7-O-β-glucoside, and cyanidin-3-O-glucoside at a concentration of 60 µM improved glucose uptake compared to the insulin resistance control, but did not reach the levels of the rosiglitazone control [54]. Additionally, other extracts rich in phenolic compounds, such as fermented and non-fermented chili pepper extracts, containing kaempferol-3-O-rutinoside, caffeic acid, kaempferol-3-O-glucoside, luteolin, and apigenin, improved glucose uptake in the insulin resistance model. They did not reach healthy cells or rosiglitazone controls [55].
In this case, the bioactivity of both extracts obtained by supercritical and methanolic extraction was statistically the same. In this context, some flavonoids exhibit a synergistic effect, thereby exerting bioactivity beyond their individual effects [56]. So, our observations could be attributed to a possible synergistic effect between the flavonoids in the extracts. In addition, a synergistic effect with other compounds not identified in the extracts is a possibility that should not be discarded.

3. Materials and Methods

3.1. Plant Material
Wild oregano (Lippia graveolens) was obtained in Santa Gertrudis, Durango, Mexico (N 23°32′43.8″ W 104°22′20.8″). The identification was carried out at the Herbarium of the School of Agriculture at the Universidad Autónoma de Sinaloa, under catalog number FA-UAS-017005. The aerial parts, including flowers, leaves, and small stems, were dried using a Food Dehydrator Parallax Hyperware (Excalibur, Sacramento, CA, USA) at 40 °C for 24 h, and then ground into a powder. The resulting powder was stored at −20 °C until further analysis.

3.2. Supercritical CO2 Extraction
The supercritical extraction process was carried out according to Picos-Salas et al. [19] using an MV-10 ASFE system (Waters Corporation, Milford, MA, USA). Briefly, 2.5 g of ground oregano was placed in an extraction vessel. The extraction was performed at 166 bar, 58.4 °C, 12.46% v/v of ethanol (cosolvent, 99.9%), total flow of 5 mL/min (CO2 + ethanol), 30 min of static extraction (supercritical CO2 entering the vessel without going out), and 45 min of dynamic extraction (supercritical CO2 passing through the vessel). These extraction conditions provide a high yield of naringenin from oregano using low pressure, a low cosolvent proportion, and a moderate temperature. The obtained extract was dried in a vacuum concentrator, resuspended in ethanol, and stored at −20 °C. The extraction procedure was repeated three times (n = 3).

3.3. Preparation of Conventional Methanolic Extract
To serve as a high-yield reference for bioactivity comparison, a conventional methanolic extraction was performed according to the method reported by Picos-Salas et al. [19]. Briefly, 0.1 g of oregano was macerated in 10 mL of methanol and shaken for 2 h in a Rotator HAG (FINEPCR, Gunpo, Republic of Korea). Then, the mixture was centrifuged for 15 min at 11,627× g and 4 °C in a Z 36 HK centrifuge (HERMLE, Franklin, WI, USA). The supernatant was collected, dried using a rotary evaporator R-300 (Buchi, Flawil, Switzerland), and resuspended in methanol. The extraction was completed in triplicate (n = 3).

3.4. Simulated Digestion
The simulated digestion process was performed according to the INFOGEST protocol [57], which simulates conditions in the mouth, stomach, and small intestine (Table S1). First, 1 mL of either the supercritical or methanolic extract was mixed with oral solution (0.8 mL), 0.3 M CaCl2 (5 µL), 75 U/mL amylase (0.1 mL), and distilled water (0.095 mL), then incubated at 37 °C for 2 min in an oscillator. Later, the gastric solution (1.6 mL) was added, and the pH was adjusted to 3. Additionally, 0.3 M CaCl2 (1 µL), 2000 U/mL pepsin (0.1 mL), 60 U/mL lipase (0.1 mL), and distilled water (0.199 mL) were added, with the pH readjusted to 3. The resulting mixture was incubated at 37 °C for 2 h with oscillation. For the intestinal phase, the pH was adjusted to 7, and the intestinal solution was added along with 0.3 M CaCl2 (8 µL), 100 U/mL pancreatin (1 mL), and distilled water (0.792 mL). The final solution was incubated at 37 °C for 2 h with oscillation. After the in vitro gastrointestinal process, methanol was added to the final mixture at a 1:1 v/v ratio to precipitate proteins, and the solution was refrigerated at −20 °C for 20 min. Afterward, it was centrifuged at 11,627× g for 10 min at 4° C. The resulting supernatant was collected and stored at −20 °C for further experiments and referred to as the supercritical extract intestinal phase (SIP) or methanolic extract intestinal phase (MIP).
Flavonoid bioaccessibility was calculated according to Equation (1).
where the flavonoid content in the crude extract and intestinal phase is expressed in µg/g extract.

3.5. In Vitro Biological Activity of Oregano

3.5.1. Cellular Antioxidant Activity (CAA)
First, cell viability in Caco-2 was determined by the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) as described by Pacheco-Ordaz et al. [58]. Tested concentrations were 100, 200, 400, and 800 µg/mL. Absorbance was measured at 490 nm using a 96-well Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Cell viability was calculated using Equation (2).
where AbsT is the absorbance of the treatment, AbsB is the absorbance of the blank, and Absc is the absorbance of the control.
The cellular antioxidant activity was evaluated in Caco-2 cells following the method reported by Gutiérrez-Grijalva et al. [38]. Caco-2 cells were purchased from the American Type Culture Collection (ATCC® HTB-37™, Manassas, VA, USA). They were grown in Dulbecco’s Modified Eagle Medium (DMEM) (ATCC) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were seeded at 5 × 104 cell/well in a 96-well plate and allowed to adhere for 24 h. Then, cells were treated with 100 µL of samples at 200 µg/mL, containing 60 µM DCFH-DA, and incubated for 20 min at 37 °C. Afterward, treatment solutions were removed, and cells were washed twice with PBS. Finally, 100 µL of AAPH (500 µM) was added to each well, except for the blank and negative control wells. In a Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA), fluorescence emitted at 538 nm upon excitation at 485 nm was measured every 2 min for 90 min at 37 °C. The CAA values were calculated according to Equation (3).
where ∫SA is the integrated area under the sample fluorescence versus time curve, and ∫CA is the integrated area from the control curve.

3.5.2. Nitric Oxide Production in RAW 264.7 Macrophages
RAW 264.7 cell line (ATCC® TIB-71™-71) was purchased from the American Type Culture Collection. The cell viability of RAW 264.7 mouse macrophages was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay [59] using an extract concentration of 50 μg/mL (based on preliminary studies). Absorbance was measured at 490 nm using a 96-well Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Cell viability was calculated using Equation (2).
NO production was assessed to evaluate the anti-inflammatory potential of oregano extracts using the method of Antunes-Ricardo et al. [59]. In brief, RAW 264.7 mouse macrophages were cultured in DMEM solution supplemented with 5% fetal bovine serum (FBS), and 1% penicillin-streptomycin antibiotic (GIBCO, Carlsbad, CA, USA) and incubated in a 5% CO2 atmosphere at 37 °C. Cells were seeded at a density of 5 × 104 cells/well in a 96-well microplate and incubated for 24 h. Then, the intestinal phase at 50 µg/mL (based on preliminary studies) was added and incubated for 4 h. After that, half of the wells were stimulated with 1 μg/mL LPS from Escherichia coli O127B8 (Sigma-Aldrich, Burlington, MA, USA), while the other half was used as a control for each sample. After 18 h of incubation, the Griess Reagent System (Promega Corporation, Madison, WI, USA) was used to determine NO production by measuring nitrite levels in the culture medium. In brief, 100 µL of supernatant was transferred to a new plate, mixed with 10 µL of sulfanilamide, and incubated for 10 min. Following, 10 µL of N-1-naphthylethylenediamine dihydrochloride (NED) reagent was added and incubated for 10 min. Afterward, absorbance was measured at 550 nm on a Synergy HT plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). NO concentration was calculated using a sodium nitrite standard (0.78–50 μM) curve and expressed as NOx µM.

3.5.3. Glucose Uptake Assay in HepG2 Cells
The HepG2 human hepatocyte cell line was also obtained from the American Type Culture Collection (ATCC® HB-8065™). Cell viability of HepG2 cells was determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay using an extract concentration of 50 μg/mL (based on preliminary studies). Absorbance was measured at 490 nm using a 96-well Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Cell viability was calculated using Equation (2).
The glucose uptake analysis was carried out according to Huang et al. [53], with some modifications. Briefly, HepG2 cells were cultured in a 5% CO2 atmosphere at 37 °C in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded in a 96-well microplate at a cell density of 5 × 104 cells/well and incubated for 24 h. The medium was then replaced with DMEM containing 1% FBS, 25 mM D-glucose, and 4 mM L-glutamine (ATCC 30-2002, Manassas, VA, USA). After 24 h, half of the wells were treated with 50 nM insulin (Sigma-Aldrich, Burlington, MA, USA), and the remaining wells served as controls.
After 24 h of incubation, 50 μL of SIP or MIP at 50 μg/mL was added to the wells, with glibenclamide at 24.7 µg/mL or 50μM (Ultra Laboratorios, Guadalajara, Mexico) used as a positive control, and incubation continued for 4 h. Glucose uptake was measured using a commercial Glucose (GO) assay kit (Sigma-Aldrich, Burlington, MA, USA) according to the manufacturer’s instructions. The glucose concentration was calculated from a glucose curve and expressed in mM. Glucose uptake was calculated by subtracting the glucose concentration in wells with treatments from that in the blank wells (no cells).

3.6. Flavonoid Content by Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)
The flavonoid content in the extracts and their respective intestinal phases was quantified using the methodology by Picos-Salas et al. [19]. The analysis was performed with a UPLC class H system (Waters Corporation, USA) coupled with a quadrupole mass (G2-XS QT). The flavonoids were separated at 40 °C using a UPLC BEH C18 column (1.7 μm × 2.1 mm × 100 mm) with following gradient elution, water with formic acid 0.1% (A) and acetonitrile (B) as follows: 0 min, 95% (A); 5 min, 70% (A); 9 min, 30% (A); 14 min, 0% (A); 14.5 min, 0% (A); 15 min, 95% (A); and 16 min, 95% (A); at a flow rate of 0.3 mL/min. Electrospray (ESI) was employed for compound ionization in negative mode, utilizing multiple reaction monitoring for detection. The following conditions were used for the mass analysis: capillary voltage of 1.5 kV, sampling cone of 30 V, desolvation gas of 800 L/h, and a temperature of 500 °C, with a collision ramp of 0–30 V. Flavonoids were identified and quantified by comparing their retention times and peak areas to those of the corresponding standards (naringenin, phloridzin, cirsimaritin, apigenin, quercetin, and luteolin). The results were expressed as µg/g of extract. The abundance of each compound relative to the sum of the flavonoids tested (%) in the samples was calculated.

3.7. Prediction of Microspecies Distribution and Ionization State
To accurately assess the bioaccessibility and permeation potential of LG flavonoids, the ionization state (microspecies distribution) was predicted across the biologically relevant pH range of the gastrointestinal tract. The chemical structure of each flavonoid was analyzed using the Chemicalize platform [60]. Specifically, the resulting fractional distribution of neutral and charged species was calculated over a pH range spanning 1 to 7, simulating the conditions from the stomach to the lower intestine. This prediction is essential for interpreting the pH-dependent behavior of the compounds during the bioaccessibility and cell-based assays.

3.8. Statistical Analysis
All experiments were performed at least three times, and results were expressed as mean ± standard deviation. All data were analyzed using an analysis of variance (ANOVA) with two factors: supercritical and methanolic extract, each with two levels of crude extract and digested extract, unless stated. Mean comparisons were carried out by Tukey’s HSD test, unless noted, using the software Minitab 19 (Minitab LLC, State College, PA, USA), taking a level of p < 0.05 as significant.

4. Conclusions
This comparative study on the effects of flavonoid-rich extracts obtained from LG through supercritical CO2 extraction and conventional solvent extraction, followed by simulated gastrointestinal digestion, demonstrates that supercritical extraction offers significant advantages in terms of phytochemical profile and bioactivity. The supercritical CO2 extraction process yielded an extract with enhanced flavonoid content and bioactivity, particularly in anti-inflammatory potential, which may be related to its high naringenin concentration. Also, this technique mitigates the drawbacks of conventional extraction, such as low selectivity, and avoids undesirable reactions caused by light and oxygen during extraction. In addition, a synergistic effect could be affecting some bioactivities, including the CAA and glucose uptake. Compared to the MIP, the most relevant bioactivity of the SIP was the reduction of NO production in LPS-stimulated RAW 264.7 macrophages; however, the potential to increase glucose uptake in insulin-resistant HepG2 cells and to elicit antioxidant activity in Caco-2 cells should still be considered. Since the in vitro bioaccessibility results indicated that gastrointestinal digestion led to high rates of degradation of oregano flavonoids, there is a clear need for effective protection systems to prevent this degradation. Encapsulation strategies should be explored to enhance flavonoid stability and preserve their biological activity. Finally, future studies should focus on elucidating the molecular mechanisms involved and evaluating these extracts in in vivo models to validate their potential application as functional ingredients for the prevention and treatment of chronic degenerative diseases, such as diabetes and chronic inflammation.
A limitation of this study is the use of only six flavonoid standards for identification and quantification, even though they are among the most abundant flavonoids in LG. This approach may overlook the presence of other compounds that could contribute to the extract’s bioactivity.