Absolute Bioavailability and PK/PD of Quercetin in Normoglycemic and Alloxan-Induced Diabetic Rats

Abstract

This study aimed to determine the absolute bioavailability of quercetin and quantitatively evaluate its pharmacokinetic–pharmacodynamic (PK-PD) relationship regarding acute glucose-lowering effects in normoglycemic and alloxan-induced diabetic rats, addressing whether its in vivo efficacy is driven by the free aglycone or its biotransformed intermediates. Healthy and diabetic rats received single doses of quercetin either orally (75 mg/kg) or intravenously (38 mg/kg). Plasma concentrations of free quercetin were quantified using a validated HPLC-DAD method, and temporal PK-PD relationships between systemic exposure and the percentage variation of glycemia were mathematically evaluated employing Pearson correlation analysis. The absolute bioavailability of free quercetin was significantly impaired by the pathophysiological state, dropping from 59.7% in healthy rats to 40.9% in diabetic subjects. Despite this diminished systemic exposure, oral administration elicited significant hypoglycemic responses. Crucially, the Pearson correlation analysis revealed a pronounced temporal dissociation: the onset of glycemic reduction occurred independently of the maximal circulating concentration of free quercetin. Furthermore, intravenous delivery bypassed first-pass barriers and induced a markedly faster and deeper hypoglycemic effect (up to −47% in diabetic rats). Finally, the diminished bioavailability under diabetic conditions and the stark PK-PD temporal dissociation strongly suggest that quercetin’s acute antihyperglycemic effect is driven by rapid hepatic Phase II biotransformation, implicating conjugated metabolites (rather than the free aglycone) as the principal pharmacological effectors.

1. Introduction

Diabetes mellitus is a chronic metabolic disorder associated with persistent hyperglycemia and severe long-term complications, serving as a major global health challenge [1,2]. Current pharmacological treatments have been effective to a certain degree, but they may cause unwanted side effects or lose effectiveness over time, necessitating the investigation of new therapeutic approaches [3]. Natural compounds, and in particular dietary flavonoids, have been identified as potential candidates due to their multiple target effects on metabolism as well as their mechanisms to mitigate oxidative stress and systemic inflammation [4,5].

Among these, quercetin (3,3′,4′,5,7-pentahydroxyflavone) stands out as one of the most abundant and extensively studied flavonols [6]. Extensive in vitro and preclinical models have demonstrated its potent antidiabetic properties, including glycemic control improvement, insulin sensitivity enhancement, and pancreatic beta-cell preservation [7]. However, the clinical translation and in vivo efficacy of quercetin remain severely constrained by its unfavorable pharmacokinetic profile, a phenomenon historically described as the “flavonoid paradox” and “quercetin paradox” [8,9]. Characterized by high lipophilicity and poor aqueous solubility, the free aglycone undergoes rapid and extensive first-pass Phase II metabolism (primarily involving glucuronidation, sulfation, and methylation) in the gastrointestinal tract and liver upon oral administration [10,11]. This biotransformation results in exceptionally low systemic exposure to unconjugated (free) quercetin, raising fundamental questions about the actual chemical species responsible for its biological effects [12].

Although the disposition of quercetin has been extensively described, there remains a dearth of comprehensive pharmacokinetic–pharmacodynamic (PK-PD) studies directly correlating the systemic exposure of the free aglycone with its acute glucose-lowering efficacy [13]. Moreover, the majority of available pharmacokinetic data are derived from healthy animal models. This is a major limitation, as serious metabolic disorders such as diabetes profoundly alter essential physiological parameters for drug disposition, including gastrointestinal transit time, hepatic perfusion, and the expression of metabolizing enzymes [14]. As a result, the absolute bioavailability, combined with the temporal dynamics of quercetin’s hypoglycemic effect under pathophysiological conditions, could be markedly different from that in the normoglycemic state, a less-investigated variable.

To address this knowledge gap, the study’s research question was whether the in vivo antidiabetic efficacy of quercetin is directly driven by systemic exposure to the free aglycone or mediated by its rapid Phase II biotransformation. Based on the physiological alterations inherent to metabolic disorders, we hypothesized that the diabetic state would significantly impair the absolute bioavailability of free quercetin compared to healthy subjects. Furthermore, we postulated a temporal dissociation between the peak plasma concentration of the free aglycone and the maximal hypoglycemic response, which would strongly implicate a metabolite-driven pharmacological mechanism [15]. To test this hypothesis, the present study aimed to determine the absolute bioavailability of quercetin following oral and intravenous administration in both normoglycemic and alloxan-induced diabetic rat models. Through temporal alignment of plasma concentrations of free quercetin with the percentage variation of glycemia, this work provides the first direct PK-PD correlation designed to quantitatively evaluate the influence of the pathophysiological state on the systemic disposition and acute efficacy of this widely consumed flavonol.

2. Materials and Methods

2.1. Chemicals and Reagents

Quercetin dihydrate (≥98% purity) and alloxan monohydrate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Analytical-grade methanol, acetonitrile, and orthophosphoric acid were purchased from J.T. Baker (Ecatepec, Edo. de México, Mexico). Ultrapure water was obtained using a Milli-Q purification system (Millipore, Bedford, MA, USA). All reagents and solvents were of analytical or HPLC grade.

2.2. Animals

Male Wistar rats weighing 200–250 g were used to evaluate the absolute bioavailability of quercetin and its hypoglycemic effects. Rats were obtained from the Bioterium of the Facultad de Química, Universidad Nacional Autónoma del México (UNAM) and Bioterium of the Universidad Juárez Autónoma de Tabasco (UJAT), Mexico. Animals were bred and maintained in an accredited institutional animal facility under controlled environmental conditions, including a 12 h light/12 h dark photoperiod, a temperature of 25 ± 3 °C, and a relative humidity of 45–65%. Rats were provided with Zeigler Rodent chow and potable water ad libitum throughout the study.

All animal procedures adhered strictly to the Mexican Federal Regulations for Animal Care and Use in Research, as stipulated by the Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA) under the Official Mexican Standard NOM-062-ZOO-1999, which governs the ethical handling, housing, and experimental use of laboratory animals and was approved by the Institutional Ethics Committee from “Universidad Juárez Autónoma de Tabasco” (UJAT-001-2017).

2.3. Drug Preparation and Dosing Regimens

For the determination of absolute bioavailability, quercetin dihydrate was administered to overnight-fasted (16 h) Wistar rats by oral (intragastric) and intravenous routes. Oral administration was performed by intragastric gavage (IG) at a dose of 75 mg/kg, with quercetin dissolved in 2 mL of physiological saline solution (0.9% NaCl). To ensure homogeneity, the mixture was subjected to sonication in a water bath for 15 min at 25 °C immediately before administration. Dosing was conducted using a Kendall DOVER ROB-NEL (Medtronic Co., Minneapolis, MN, USA) urinary catheter (8 Fr, 16″ length) to ensure accurate and homogeneous delivery.

Intravenous administration was carried out via the tail vein, previously dilated using a thermostatic warming chamber (37 °C). Quercetin was administered at a dose of 38 mg/kg, dissolved in 0.3 mL of distilled water/Polysorbate 80 (10:1, v/v), using a BD Plastipak insulin syringe (Cuautitlán Izcalli, Edo. de México, Mexico). The solution was prepared by vortexing followed by 10 min of sonication to achieve a clear, particle-free solution suitable for bolus injection. The selected oral and intravenous doses were designed to enable the calculation of absolute bioavailability and to account for route-dependent pharmacokinetic differences.

Identical dosing regimens were applied in normoglycemic rats and in rats with alloxan-induced diabetes, following the same formulation, administration procedures, and fasting conditions.

2.4. Induction of Experimental Diabetes

Diabetes mellitus was induced by a single intraperitoneal (i.p.) injection of alloxan monohydrate (150 mg/kg) dissolved in sterile saline, following a 12 h fasting period. After 72 h, blood glucose levels were measured from the tail vein using an Accu-Chek glucometer (Roche Diagnostics, Mannheim, Germany). Rats with fasting blood glucose ≥ 250 mg/dL were considered diabetic and included in the study.

2.5. Pharmacokinetic Study Design and Blood Sampling

For oral bioavailability assessment in normoglycemic rats, blood samples were collected at 0, 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 240, 360, 420, and 480 min post-administration. For intravenous administration, sampling times were 0, 5, 15, 20, 25, 30, 50, 70, 90, 120, 180, 220, 240, 260, and 320 min. In all experiments, three rats were used per sampling time, resulting in 45 animals per experimental condition. Blood samples were obtained by cardiac puncture and centrifuged at 9860× g for 15 min to obtain plasma. Plasma samples were subsequently analyzed for free quercetin using the validated HPLC–DAD method.

An equivalent experimental design was applied to alloxan-induced diabetic rats, with oral sampling times of 0, 10, 30, 60, 75, 90, 105, 120, 180, 220, 240, 300, 360, 420, and 480 min, and intravenous sampling times identical to those used in normoglycemic animals.

2.6. Quercetin Extraction from Plasma Using Solid-Phase Extraction

Plasma quercetin concentrations were determined following a solid-phase extraction (SPE) protocol optimized for rat plasma. Extraction was performed using C18 Bakerbond cartridges (60 Å, 200 mg; J.T. Baker, Phillipsburg, NJ, USA) connected to a multiport vacuum manifold with controlled pressure (SPE-12G; J.T. Baker, USA). The extraction procedure consisted of cartridge conditioning, sample loading, removal of matrix interference through washing, and final elution of the analyte. Cartridges were first conditioned under vacuum with 10 mL of phosphate buffer (70 μM, pH 2.4), adjusted to maintain an approximate drip rate of 30 drops per minute. Subsequently, 500 μL of Wistar rat plasma was applied to the conditioned cartridges. Matrix cleansing was achieved by sequential washing with 6 mL of distilled water, followed by 6 mL of phosphate buffer. Afterward, the vacuum was left open for 5 min, allowing the sorbent bed to dry completely.

Quercetin was then eluted using 5 mL of methanol acidified with acetic acid. The eluate was evaporated to dryness under a gentle stream of nitrogen gas, and the resulting residue was reconstituted in 1 mL of acetonitrile. The reconstituted samples were immediately subjected to chromatographic analysis to quantify free (unconjugated) quercetin using high-performance liquid chromatography equipped with a diode-array detector (HPLC–DAD).

2.7. Chromatographic Conditions for HPLC-DAD Analysis of Quercetin

Quantitative analysis of plasma quercetin was performed using a High-Performance Liquid Chromatography (HPLC) system (Agilent Technologies 1200 Series, Santa Clara, CA, USA) equipped with a quaternary pump, autosampler, thermostatted column compartment, and diode-array detector (DAD). Chromatographic separation was achieved on a Synergy C18 reversed-phase column (150 × 4.6 mm, 4 μm particle size) under the conditions optimized in the original methodology. The mobile phase consisted of acetonitrile and 0.6% acetic acid (90:10, v/v). The column temperature was maintained at 20 °C, and the injection volume was 20 μL. Isocratic elution was applied at a flow rate consistent with method performance, and detection was carried out at 370 nm, corresponding to the absorbance maximum of quercetin. The DAD allowed simultaneous monitoring of spectral purity to ensure analyte specificity. Calibration curves were constructed in blank rat plasma following identical extraction procedures, covering a concentration range of 0–7.5 μg/mL with calibration levels at 0.5, 1.0, 2.5, 5.0, and 7.5 μg/mL.

2.8. Bioanalytical Method Validation

The bioanalytical method for the quantification of quercetin in rat plasma was fully validated in accordance with the U.S. Food and Drug Administration (FDA) Guidance for Industry: Bioanalytical Method Validation. Validation parameters included selectivity, linearity, sensitivity, accuracy, precision, recovery, and stability to ensure the reliability and reproducibility of the analytical procedure.

2.8.1. Linearity

Calibration curves were constructed in blank rat plasma and processed using the same solid-phase extraction procedure applied to study samples. The calibration model was established over a concentration range of 0–7.5 μg/mL, with calibration levels at 0.5, 1.0, 2.5, 5.0, and 7.5 μg/mL. Linearity was evaluated by linear regression analysis of the analytical response versus nominal concentration.

2.8.2. Limit of Detection (LOD) and Limit of Quantification (LOQ)

The limit of detection (LOD) and the lower limit of quantification (LOQ) were defined according to the FDA guidelines for bioanalytical method validation. LOD and LOQ were established based on signal-to-noise criteria, using approximate ratios of 3:1 for LOD and 10:1 for LOQ. The LOQ was operationally defined as the lowest concentration on the calibration curve meeting predefined criteria for accuracy and precision, as specified by FDA regulatory standards.

2.8.3. Precision

Intra-day (within-run) and inter-day (between-run) accuracy and precision were performed using quality control samples prepared at low (0.5 μg/mL), medium (1.0 μg/mL), and high (7.5 μg/mL) concentrations within the calibration range. Accuracy was defined as the percentage of nominal concentration recovered, while precision was expressed as the coefficient of variation (%CV).

2.8.4. Extraction Recovery

Recovery was assessed by comparing the analytical response obtained from quality control samples subjected to the complete extraction procedure with that from post-extraction spiked samples at equivalent nominal concentrations. For this purpose, blank control rat plasma free of quercetin was used to prepare both extracted QC samples and post-extraction spiked samples, thereby ensuring the absence of endogenous analyte interference. Recovery assessments were performed at low (0.5 μg/mL), medium (1.0 μg/mL), and high (7.5 μg/mL) concentration levels within the calibration range, following standardized bioanalytical validation practices.

2.9. Pharmacokinetic Analysis

Plasma concentration–time data of free quercetin obtained after oral (intragastric) and intravenous administration were analyzed using a non-compartmental approach, based on standard pharmacokinetic principles. Individual concentration–time profiles were constructed for each experimental condition (oral and intravenous administration in normoglycemic and alloxan-induced diabetic rats).

The maximum plasma concentration (C_max) and the corresponding time to reach C_max (T_max) were determined directly from the observed data. The area under the plasma concentration–time curve from time zero to the last measurable concentration (AUC_0–t) was calculated using the linear trapezoidal method. When appropriate, the area was extrapolated to infinity (AUC_0–∞) using the terminal phase of elimination.

The elimination rate constant (λz) was estimated by linear regression of the terminal log-linear portion of the concentration–time curve, and the elimination half-life (t½) was calculated as ln(2)/λz.

Absolute oral bioavailability (F) of quercetin was calculated by comparing the dose-normalized AUC values obtained after oral and intravenous administration according to the following equation:

$$F \left(\%\right)=\left({\displaystyle \frac{{AUC}_{oral}\times {Dose}_{iv}}{{AUC}_{iv}\times {Dose}_{oral}}}\right)$$

where AUC_oral and AUC_iv represent the areas under the curve after oral and intravenous administration, respectively, while Dose_oral and Dose_oiv correspond to the respective administered doses.

2.10. Assessment of Hypoglycemic Effect

The hypoglycemic effect of quercetin was assessed in normoglycemic rats and in rats with alloxan-induced diabetes using the same experimental groups and dosing regimens applied for the pharmacokinetic and bioavailability studies. Quercetin was administered at 75 mg/kg by the oral (intragastric) route and at 38 mg/kg by the intravenous route, following a 16 h fasting period with free access to water.

Baseline blood glucose concentrations were measured immediately before dosing (time 0). Subsequently, blood samples were collected concomitantly with pharmacokinetic sampling via tail vein incision. Glucose concentrations were determined using a portable Accu-Chek glucometer, based on the glucose oxidase–peroxidase (GOD–POD) method.

Blood glucose measurements were performed at the same time points defined for the corresponding bioavailability studies, after both oral and intravenous administration, allowing for direct temporal alignment between the glycemic response and plasma quercetin concentrations. For each administration route, treated groups were compared with vehicle-treated control groups, which received 5 mL/kg of physiological saline solution (oral route) or 1 mL/kg of saline/Polysorbate 80 (10:1, v/v) (intravenous route).

The hypoglycemic response was expressed as percentage variation of glycemia (%VG) relative to baseline values, calculated as:

$$\%VG=\left[{\displaystyle \frac{{Glu}_x-{Glu}_0}{{Glu}_0}}\right]\times 100$$

where Glu₀ represents baseline blood glucose concentration (blood glucose concentration measured at time 0 immediately before the administration of quercetin or vehicle), and Glu_x corresponds to the glucose concentration at each evaluated time point. Temporal profiles of %VG were constructed using SigmaPlot version 11.0.

2.11. Pharmacokinetic–Pharmacodynamic (PK–PD) Correlation

The relationship between the absolute bioavailability of quercetin and its hypoglycemic effect was evaluated by correlating plasma concentrations of free quercetin with the corresponding percentage variation of glycemia (%VG). Correlation analyses were performed independently for each route of administration (oral and intravenous) and for each glycemic condition (normoglycemic and alloxan-induced diabetic rats).

For each experimental condition, plasma quercetin concentrations were plotted on the x-axis. In contrast, %VG values were plotted on the y-axis, enabling the assessment of the association between hypoglycemic response and key pharmacokinetic parameters, including maximum plasma concentration (C_max), time to reach C_max (T_max), and total systemic exposure expressed as area under the concentration–time curve (AUC).

The strength and direction of the association between pharmacokinetic and pharmacodynamic variables were quantified using Pearson’s correlation coefficient (r). Correlation strength was interpreted according to predefined criteria: |r| < 0.5, weak association; |r| = 0.52–0.8, moderate association; and |r| > 0.8, strong association. This approach enabled an objective evaluation of the degree of association between systemic quercetin exposure and its hypoglycemic effect across administration routes and metabolic states.

2.12. Statistical Analysis

All data are expressed as mean ± standard deviation (SD) unless otherwise indicated. Statistical analyses were performed using SigmaPlot version 11.0. Comparisons between treatment groups and their corresponding vehicle-treated controls were conducted using analysis of variance (ANOVA), followed by appropriate post hoc tests when applicable.

Correlation analyses for PK–PD evaluation were performed using Pearson’s correlation test, as described above. A p-value < 0.05 was considered statistically significant for all analyses.

3. Results

3.1. Bioanalytical Validation and Quality Control

Exhaustive quality control ensures that plasma quercetin measurements are accurate and reproducible, supporting reliable estimation of pharmacokinetic parameters and PK–PD analyses. Accordingly, analytical performance was assessed against FDA-recommended validation criteria, ensuring that concentration–time profiles reflect biological disposition rather than methodological variability.

The HPLC-DAD method demonstrated high selectivity for the quantification of free quercetin in rat plasma. Given that the study targeted a single analyte, selectivity was further validated by assessing the peak spectral purity using the DAD system. Comparison of the UV-Vis spectra at the upslope, apex, and downslope of the quercetin peak confirmed the absence of co-eluting interferences from the plasma matrix. Method accuracy was established with a mean extraction recovery of 92.3 ± 4.9%, while system precision showed a relative standard deviation (RSD) of 2.18% over three consecutive analytical days. These parameters ensure the reliability of the calculated AUC and Cmax values.

3.2. Linearity, LOD, and LOQ

The linearity of the chromatographic system was assessed using quercetin standards prepared in acetonitrile over the range of 0.5–7.5 μg/mL and monitored at 370 nm. Mean peak areas increased proportionally with concentration, and least-squares regression yielded y = 45.36x + 2.2865 with r² = 0.9998. The dispersion across calibration levels was low (SD = 1.306; RSD = 2.808%). Based on the linearity data, the LOD and LOQ were calculated from the calibration curve (regression equation). The LOD was 0.0122 μg/mL, and the LOQ was 0.325 μg/mL, indicating that under the established chromatographic conditions, quercetin concentrations below 0.0122 μg/mL were not detectable, whereas concentrations ≥ 0.325 μg/mL could be quantified with acceptable precision and accuracy, consistent with FDA-recommended bioanalytical validation criteria.

3.3. Precision and Extraction Recovery

System precision was assessed across the calibration levels (0.5–7.5 μg/mL) over three consecutive analytical days, with triplicate injections performed at each level. The overall mean response was 46.6 (peak area), with an SD of 1.020 and an RSD of 2.187%, supporting low day-to-day and run-to-run variability of the chromatographic response and meeting FDA-recommended system performance criteria for quantitative bioanalysis. Finally, the method accuracy, expressed as percent recovery (%R) at representative concentrations (0.5, 2.5, and 7.5 μg/mL), yielded an overall mean %R of 92.3% with an SD of 4.9 and an RSD of 5.3% across replicates. These results indicate consistent assay performance across the evaluated concentration levels and are within FDA-recommended acceptance criteria for bioanalytical methods.

3.4. Oral Bioavailability of Free Quercetin in Normoglycemic Rats

In normoglycemic rats receiving intragastric quercetin (75 mg/kg), free quercetin was first quantifiable in plasma at 10 min (Figure 1A). Concentrations remained low during the early sampling window and then increased progressively, forming a slow ascending phase that culminated in a delayed peak. The maximum concentration (C_max) reached 4.91 ± 0.31 μg/mL at 240.0 ± 19.2 min (T_max). After this peak, concentrations dropped rapidly, approaching near-baseline levels by 420 min and becoming non-quantifiable at 480 min (below the assay LOQ). Systemic exposure was therefore integrated over 10–420 min, yielding an AUC (10–420 min) of 1086.09 ± 33.73 μg·min/mL.

3.5. Intravenous Bioavailability of Free Quercetin in Normoglycemic Rats

A markedly different profile was observed after intravenous administration of quercetin (38 mg/kg), with high circulating free quercetin detected at the first post-dose sampling point (9.8 μg/mL at 5 min). Concentrations then declined steeply during the early time course (approximately 7.3 μg/mL by 15 min) and continued to fall thereafter, producing a monotonic decrease toward low/near-baseline values by the late sampling points (Figure 1B). Non-compartmental analysis of the intravenous concentration–time curve gave an AUC_0–t of 920.34 ± 24.56 μg·min/mL, an elimination half-life (t½) of 52.39 ± 0.09 min, and a plasma clearance (CL) of 1.71 ± 0.13 L·h⁻¹·kg⁻¹ and a volume of distribution (Vd) of 2.15 ± 0.15 L/kg.

3.6. Absolute Bioavailability in Normoglycemic Rats

With both routes characterized in the same model, absolute bioavailability of free quercetin was computed by dose-normalized comparison of exposure after intragastric and intravenous administration (Figure 1E). Using the measured oral AUC (1086.09 μg·min/mL; 75 mg/kg) and intravenous AUC (920.34 μg·min/mL; 38 mg/kg), the calculated absolute bioavailability (F) of free quercetin was 0.597 (59.7%).

3.7. Oral Hypoglycemic Assay in Normoglycemic Rats

The oral glycemic profile showed modest fluctuations around baseline during the early post-dose phase, followed by a discrete hypoglycemic diminution relative to vehicle controls (physiological saline, 5 mL/kg). The reduction intensified between 75 and 120 min, reaching its deepest values at 105 and 120 min, where quercetin-treated rats achieved approximately −20% Variation of Glycemia (%VG, Figure 1C); these time points were significantly lower than vehicle (p < 0.01). Thereafter, %VG trended back toward baseline, with the treated and vehicle curves converging by the later time points.

3.8. Intravenous Hypoglycemic Assay in Normoglycemic Rats

After intravenous dosing, the %VG trajectory displayed a clear biphasic pattern: an early transient positive excursion (maximal at ~5 min) followed by a sustained decline into negative values. The hypoglycemic response became significant versus vehicle at 90 min and remained significant through 220 min (p < 0.01). The nadir occurred at 180 min (approximately −26% VG), after which %VG recovered progressively toward baseline, approaching control values by 260–320 min (Figure 1D). Vehicle-treated animals, in contrast, remained near baseline with relatively small-amplitude oscillations and no prolonged negative phase.

3.9. Oral Bioavailability of Quercetin in Alloxan-Induced Diabetic Rats

Intragastric administration of quercetin (75 mg/kg) generated a measurable plasma concentration–time profile of free quercetin. The analyte was detectable at early sampling times and increased progressively from 10 min onward, with concentrations rising through 60–105 min and reaching a maximum at 120 min. The observed C_max was 3.94 ± 0.99 μg/mL, with a T_max of 120 min (Figure 2A).

After the peak, plasma concentrations declined progressively during the late sampling phase. Free quercetin remained quantifiable up to 420 min, whereas at later time points, concentrations fell below the assay quantification range. Based on the concentration–time profile, oral systemic exposure was AUC = 742.88 ± 23.87 μg·min/mL.

Comparison with the corresponding oral profile in normoglycemic animals showed a clear temporal shift and reduced exposure. Specifically, the peak occurred earlier (T_max 120 min vs. 240 min) and was followed by lower concentrations during the post-peak phase (Figure 1A and Figure 2A). Consistent with this pattern, oral systemic exposure was lower than that measured in normoglycemic rats (742.88 ± 23.87 vs. 1086.09 ± 33.73 μg·min/mL), and the difference was statistically significant (Student’s t-test, p < 0.01).

3.10. Intravenous Bioavailability of Free Quercetin in Alloxan-Induced Diabetic Rats

Intravenous administration of quercetin (38 mg/kg) produced a rapidly declining plasma concentration–time profile. The highest concentration was observed at the first post-dose sampling point, with a C_max of 9.11 μg/mL at 5 min (Figure 2B), followed by a steep decrease over time. The decline was pronounced during the early post-administration phase, and free quercetin approached baseline values thereafter; concentrations were reported to fall below the method detection limit (LOD = 0.012 μg/mL) by approximately 90 min post-dose. Non-compartmental analysis yielded an elimination half-life (t½) of 28.19 ± 0.13 min, plasma clearance (CL) of 4.08 ± 0.22 L·h⁻¹·kg⁻¹, a volume of distribution (Vd) of 2.75 ± 0.18 L/kg, and systemic exposure (AUC) of 697.13 ± 12.34 μg·min/mL.

Relative to the intravenous profile in normoglycemic animals, concentrations were comparable during the initial phase (0–25 min), including the peak region (approximately 9.8 μg/mL in normoglycemic rats vs. 9.1 μg/mL here). Beyond this interval, however, the decline became more pronounced, and plasma concentrations were significantly lower at later time points (Student’s t-test, p < 0.01) (Figure 1B and Figure 2B).

This pattern was reflected in the exposure and disposition parameters. Intravenous AUC was significantly reduced compared with normoglycemic rats (697.13 ± 12.34 vs. 920.34 ± 24.56 μg·min/mL, p < 0.01), together with a shorter half-life and higher clearance (t½: 28.19 ± 0.13 vs. 52.39 ± 0.09 min; CL: 4.08 ± 0.22 vs. 1.71 ± 0.13 L·h⁻¹·kg⁻¹).

3.11. Absolute Bioavailability in Alloxan-Induced Diabetic Rats

Absolute bioavailability of free quercetin was estimated by dose-normalized comparison of the oral and intravenous exposure profiles (Figure 2E). Using the measured AUC values for oral administration (742.88 μg·min/mL, 75 mg/kg) and intravenous administration (697.13 μg·min/mL, 38 mg/kg), the calculated absolute bioavailability (F) was 0.409 (40.9%).

Under the experimental conditions used, this indicates that approximately 40% of the administered oral dose reached the systemic circulation as free quercetin when normalized to the intravenous reference. By comparison, the corresponding value in normoglycemic rats was F = 0.597 (59.7%), representing an 18.8 percentage-point decrease (approximately 31.5% relative reduction) in the diabetic model. This reduction was consistent with the lower systemic exposure observed in both the oral and intravenous profiles.

3.12. Oral Antidiabetic Assay in Alloxan-Induced Diabetic Rats

Quercetin administered intragastrically (75 mg/kg) produced a reduction in the percentage variation of glycemia (%VG) relative to the vehicle group (physiological saline, 5 mL/kg) when evaluated at the same time points used for the oral bioavailability study (Figure 2C). The temporal profile showed a relatively early onset of glycemic reduction, with statistically significant differences emerging at 60 min and persisting through 105 min post-administration (p < 0.01 vs. vehicle). Across this interval, the %VG curve of the quercetin-treated group displayed a more pronounced downward trajectory than the control group, with the strongest effect observed within the 60–105 min window. In contrast, vehicle-treated animals showed fluctuations around baseline values without a sustained reduction in comparable magnitude (Figure 2C). At later time points, the response in the quercetin-treated group progressively attenuated.

Comparison of the oral hypoglycemic response between normoglycemic and diabetic animals (Figure 1C and Figure 2C) showed a more pronounced effect in the diabetic model in terms of onset, duration, and magnitude of glycemic reduction. Specifically, diabetic rats exhibited a deeper decrease in %VG and a broader effect window than normoglycemic rats under the same quercetin dose (75 mg/kg). In addition, statistically significant differences were observed at more time points in the diabetic group, whereas in normoglycemic animals, the significant response was temporally more restricted.

Taken together, these results show that oral quercetin elicits an acute hypoglycemic effect in diabetic rats, with a more pronounced response than that observed in normoglycemic animals under the same experimental conditions.

3.13. Intravenous Antidiabetic Assay in Alloxan-Induced Diabetic Rats

Intravenous quercetin (38 mg/kg) produced a marked reduction in the percentage variation of glycemia (%VG) relative to the vehicle group (physiological saline, 5 mL/kg) when assessed at the same sampling times used for the intravenous bioavailability study (Figure 2D). Statistically significant reductions in %VG were observed from 30 min through 180 min post-administration (p < 0.01 vs. vehicle), indicating a rapid onset and sustained hypoglycemic response within this time window.

The %VG profile showed a pronounced downward trajectory after the early post-dose phase, with deeper reductions than those observed with oral administration. The maximal decrease reached approximately −47% VG after intravenous dosing, compared with approximately −40% VG after oral administration. In addition to the greater magnitude of the response, the duration of the significant effect was longer after intravenous administration (approximately 130 min) than after oral dosing (approximately 45 min) under the same experimental framework.

Comparison of the intravenous glycemic response between normoglycemic and diabetic animals (Figure 1D and Figure 2D) showed that the onset of glycemic reduction occurred earlier in the diabetic group, whereas the overall duration of the pharmacological effect was similar in both models (approximately 130 min). The principal difference between groups was observed in response magnitude, with the diabetic animals exhibiting a deeper %VG reduction across the significant time window. Taken together, these data indicate that intravenous quercetin elicits a rapid and sustained acute hypoglycemic response, with greater magnitude and longer duration than oral administration, and with a more pronounced effect in diabetic than in normoglycemic animals under the same dose-normalized experimental comparisons.

3.14. Pearson Correlation Between Plasma-Free Quercetin and Glycemic Response

Having characterized route-dependent exposure profiles and glycemic responses, we next quantified concentration–effect coupling by correlating plasma-free quercetin concentrations with the percentage of glycemic variation (%VG) using Pearson’s correlation analysis. Consistent with the time-course profiles, the maximal oral glycemic reductions occurred before the time of peak plasma exposure (T_max) and did not align with maximal free-quercetin concentrations. To account for the biphasic oral pharmacokinetic profile (ascending absorption and descending elimination), correlations were examined separately for each segment. During the absorption phase (alpha; Figure 3A), which encompasses the rising portion of the oral concentration–time curve (concentrations spanning ~0–4.144 μg/mL), the scatter points were widely dispersed around the regression line. Consequently, the relationship between concentration and %VG was weak and non-significant (r = −0.289, p > 0.05, y = −2.775x − 10.821). Notably, large negative %VG values occurred at intermediate concentrations rather than clustering at the upper end of the concentration range, mathematically mirroring the earlier observation that the onset of significant hypoglycemia did not coincide with peak systemic exposure. In contrast, the elimination phase (beta; Figure 3B), representing the descending portion of the oral profile, showed a more coherent trend. The regression line had a positive slope (r = 0.607, p < 0.05, y = 2.706x − 10.671), indicating a moderate and statistically significant association within this segment. However, the data points remained clustered in a narrow %VG range (approximately −6 to +4%), suggesting that the concentration–effect alignment during the beta phase occurred primarily during the post-peak period, when glycemic effects were less pronounced than those observed earlier in the time-course. For intravenous administration, the scatter plot (Figure 3C) showed a clear monotonic pattern across the analyzed concentration range (from ~9.109 down to ~0.516 μg/mL). The fitted regression displayed a steep negative slope (r = −0.871, p < 0.05, y = −5.267x + 14.746), indicating a strong and statistically significant inverse relationship between plasma free-quercetin concentration and %VG. More generally, more negative %VG value sizes clustered into the low end of the concentration range, consistent with the time-course results where maximum glycemic reductions appeared at later timepoints, long after plasma free quercetin had already dropped sharply from its first peak.

4. Discussion

The present study provides the first direct pharmacokinetic–pharmacodynamic (PK-PD) correlation of free quercetin in both normoglycemic and alloxan-induced diabetic models, yielding a fundamental pharmacological insight: the maximal acute hypoglycemic effect does not temporally align with the peak systemic exposure of the free aglycone. While our complete bioanalytical validation confirmed that quercetin is rapidly absorbed and reaches measurable plasma concentrations (yielding an absolute bioavailability of 59.7% in healthy rats and 40.9% in diabetic subjects), the robust inverse mathematical correlations (Pearson analysis) demonstrated a pronounced temporal dissociation. Specifically, the onset of significant glycemic reduction consistently occurred before or long after the maximal circulating concentrations of free quercetin, suggesting that the parent molecule itself is not the immediate or sole effector of the observed antidiabetic response (Figure 4). Furthermore, our calculated absolute bioavailability of 59.7% in normoglycemic rats is notably higher than the extremely low values (often <10%) cited in earlier literature [9,11].

The low systemic bioavailability of the quercetin aglycone is primarily attributed to its extremely poor aqueous solubility, lipophilic nature, and instability in gastrointestinal fluids [16,17,18]. Moreover, it is well established that a major obstacle to quercetin’s bioavailability is its rapid and extensive Phase II metabolism [19]. Specifically, the aglycone is subjected to immediate glucuronidation, sulfation, and methylation within the intestinal epithelial cells (enterocytes) before even reaching the portal circulation [20]. Therefore, the marked discrepancy in our higher calculated bioavailability likely stems from two crucial methodological factors. First, the optimized dosing formulation in our protocol effectively mitigated the typical solubility barriers in the intestinal lumen, a critical factor considering that absorption is highly dependent on the chemical form and vehicle [16,21]. Second, by implementing a highly rigorous early-phase blood-sampling schedule, we successfully captured the true absorption kinetics before the aggressive presystemic clearance in the enterocytes could completely deplete the parent compound [20]. Complementary to these factors, the robustness of our results was further ensured by the strategic optimization of the HPLC-DAD method. The use of an acidified mobile phase (0.6% acetic acid) was critical to stabilize the polyphenolic structure and ensure reproducible retention times, while the C18 solid-phase extraction (SPE) protocol was specifically optimized to eliminate matrix effects from the altered plasma composition of diabetic subjects. This analytical design achieved a sensitive limit of detection (0.0122 μg/mL), which was essential for the precise quantification of the terminal elimination phase. This approach provides a more accurate reflection of the intact aglycone reaching the systemic circulation.

Instead, these findings support a metabolite-mediated pharmacological mechanism. Quercetin is inherently susceptible to rapid and extensive Phase II biotransformation, driven primarily by UDP-glucuronosyltransferases (UGTs) in both the intestinal epithelium and the liver immediately following absorption [22,23]. Our in silico predictive models of xenobiotic biotransformation (MetaPrint2D), indicating a high probability of metabolic attack at the 3, 4′, and 7 hydroxyl positions, are entirely consistent with experimental in vitro and in vivo profiling. Studies demonstrate that hepatic and intestinal UGTs regioselectively conjugate these exact highly reactive sites, yielding massive pools of circulating phase II metabolites, predominantly quercetin-3-glucuronide (Q3G) and isorhamnetin-3-glucuronide [23,24,25].

The pharmacological relevance of this biotransformation is supported by contemporary literature. Historically, Phase II conjugates were dismissed as inactive excretion products; however, fundamental cellular studies have proven that specific glucuronides, such as Q3G, are actively transported into hepatic and muscle cells, where they are further processed, retaining intrinsic biological efficacy [26]. Specifically, contemporary molecular profiling and ex vivo models confirm that circulating Q3G directly stimulates the AMP-activated protein kinase (AMPK) pathway and modulates crucial metabolic signaling, subsequently enhancing cellular glucose uptake [10,25,27]. Consequently, the rapid enzymatic conversion of free quercetin into these bioactive conjugated derivatives fully explains the early onset of hypoglycemia observed before the aglycone T_max during oral administration, as well as the sustained hypoglycemic effect during the elimination phase when free quercetin levels have completely plummeted.

This metabolite-driven hypothesis is further corroborated by the striking pharmacodynamic differences observed between the administration routes. Despite receiving approximately half the dose (38 mg/kg) compared to the oral group (75 mg/kg), rats administered intravenous quercetin exhibited a significantly faster, deeper, and more prolonged hypoglycemic response. By bypassing the physical and enzymatic barriers of the gastrointestinal tract, most notably the extensive presystemic glucuronidation within enterocytes and intestinal efflux mechanisms [19,20], intravenous delivery introduces the entire dose of the free aglycone directly into the systemic circulation. This immediate bioavailability likely overwhelms initial distribution phases, presenting the hepatic Phase II metabolic machinery with a massive substrate load. This physiological response aligns with established pharmacokinetic models, where an intravenous bolus results in an extremely rapid systemic clearance of the free aglycone and an immediate, proportional surge in circulating conjugates, as the hepatic tissue rapidly processes the sudden influx of the parent compound [22,23,28]. Consequently, a rapid and voluminous pool of active conjugated metabolites is generated almost instantaneously, driving the acute and sustained drop in blood glucose levels (reaching up to −47% VG in diabetic subjects) that clearly outperformed the oral route [25].

Finally, this study highlights the critical influence of the pathophysiological state on the systemic disposition of flavonoids, demonstrating a significant impairment of quercetin’s absolute bioavailability in the diabetic model. The systemic availability of the free aglycone dropped substantially from 59.7% in normoglycemic rats to 40.9% in alloxan-induced diabetic subjects. This pronounced decrease is likely multifactorial, driven by the profound physiological and biochemical disruptions inherent to the diabetic state [29]. Experimental diabetes is known to induce gastrointestinal dysmotility, which can alter intestinal transit times and limit the optimal absorption window for highly lipophilic compounds [14]. More critically, the diabetic state significantly alters the expression and activity of hepatic drug-metabolizing enzymes [30]. The literature demonstrates that chronic hyperglycemia and its associated metabolic stress directly upregulate specific UDP-glucuronosyltransferase isoforms (most notably UGT1A1 and related families) in the hepatic tissue of diabetic rat models [31]. The impact of these physiological alterations on dietary polyphenols has been increasingly documented; for instance, Chen et al. (2017) [32] explicitly demonstrated that the diabetic state significantly alters the pharmacokinetic profile and Phase II metabolism of polyphenols, driving differential accumulation of conjugated metabolites compared to healthy models. This upregulation translates into a significantly increased capacity for rapid glucuronidation. Consequently, an exacerbated hepatic metabolic machinery intensifies the first-pass effect, clearing free quercetin from the bloodstream more rapidly. Paradoxically, this accelerated biotransformation not only explains the lower systemic exposure (AUC) of the free aglycone but also perfectly aligns with the earlier onset and greater magnitude of the hypoglycemic response observed in diabetic rats, as a faster clearance of the parent compound translates to a more rapid accumulation of the active conjugated metabolites (see proposed model in Figure 4).

While the present findings provide compelling in vivo and mathematical evidence for a metabolite-mediated antidiabetic effect, this study is not without limitations. The bioanalytical methodology employed (HPLC-DAD) was specifically validated for the precise and robust quantification of the free quercetin aglycone, which prevented the direct structural characterization and simultaneous tracking of its circulating Phase II conjugates. Although predictive in silico models strongly support rapid glucuronidation and sulfation at specific hydroxyl sites, future investigations employing advanced mass spectrometry techniques (LC-MS/MS) are warranted. Such studies would allow for the direct pharmacokinetic quantification of specific circulating metabolites (such as quercetin-3-glucuronide) and their individual evaluation in isolated pharmacodynamic models to definitively confirm their proportional contribution to the overall glucose-lowering response.

5. Conclusions

In conclusion, this study demonstrates that the absolute bioavailability of free quercetin is significantly impaired by the diabetic state, decreasing from 59.7% in normoglycemic rats to 40.9% in alloxan-induced diabetic models. Despite this reduced systemic exposure to the parent compound, pronounced hypoglycemic responses were observed across both administration routes. Crucially, the direct PK-PD evaluation revealed a clear mathematical and temporal dissociation between the peak plasma concentrations of the free aglycone and the maximal glucose-lowering effect. These quantitative findings indicate that the acute antihyperglycemic activity is not temporally driven by the circulating free quercetin itself. Instead, this distinctive pharmacokinetic behavior strongly suggests that the in vivo pharmacological effect is mediated by its rapid and extensive phase II biotransformation. Ultimately, this work highlights the critical impact of pathophysiological conditions on flavonoid disposition and establishes that conjugated metabolites, rather than the parent aglycone, are likely the primary biological effectors, warranting their targeted quantification in future mechanistic studies.

Despite these findings, a primary limitation of our study remains the lack of direct quantification for specific Phase II metabolites; therefore, future investigations employing advanced LC-MS/MS techniques are warranted to definitively characterize the circulating levels of conjugates such as quercetin-3-glucuronide and confirm their individual contribution to the observed antihyperglycemic effect. Furthermore, while the alloxan-induced model provides a robust controlled environment to study the influence of hyperglycemia on Phase II metabolism, future studies in Type 2 diabetes models are necessary to fully assess the clinical translation of these findings to the most prevalent form of the disease.