Quercetin and Derivatives Ameliorate Metabolic Disturbances by Regulating Gut Metabolite Profiles in Mice with Circadian Rhythm Disruption and High-Fat Diet

Abstract

Background: Amidst evolving modern lifestyles characterized by widespread circadian rhythm disturbances and high-fat dietary habits, the incidence of metabolic disorders continues to escalate. In recent years, plant-derived bioactive compounds have attracted considerable interest as therapeutic candidates, with quercetin and its derivatives demonstrating promising potential for the regulation of metabolism. Methods: This study employed a dual-induction model of metabolic dysregulation, elicited through both circadian rhythm disruption and a high-fat diet, to systematically evaluate the regulatory effects of quercetin and its derivatives on mice through dual stimulation by circadian rhythm disruption and a high-fat diet. Results and Conclusions: Non-targeted fecal metabolomics analysis indicates that quercetin and its derivatives significantly alter the intestinal metabolite profile in mice, alleviating metabolic abnormalities induced by circadian rhythm disruption and high-fat diet. These findings provide a theoretical basis for the future development of quercetin-based functional food products.

1. Introduction

Circadian rhythm (CR) refers to the intrinsic oscillation of biological processes that occur in approximately 24 h cycles. As a fundamental physiological feature of living organisms, they profoundly influence a wide range of human physiological functions, including cognitive performance, emotional regulation, and work efficiency. These rhythms are closely synchronized with environmental cues, particularly light–dark cycles. Disruption of the circadian rhythm leads to dysregulation of core clock gene expression, triggering various physiological and pathological changes [1]. For example, frequent shift work, trans-meridian travel, or irregular eating behaviors can cause misalignment between endogenous biological clocks and environmental signals, resulting in circadian rhythm disorders [2]. Persistent circadian disruption impairs glucose tolerance and insulin sensitivity and increases the risk of chronic metabolic diseases such as diabetes and hypertension. Currently, diabetes represents a critical global public health issue, with projections estimating that 1.3 billion individuals will be affected worldwide by 2050 [3], drawing the sustained attention of international health organizations.

Quercetin (Q), a representative flavonol compound with the molecular formula C₁₅H₁₀O₇, is widely distributed in vegetables and fruits, such as onions (68.5 mg/100 g), broccoli (13.6 mg/100 g), and cranberries (25 mg/100 g). It can be extracted using various methods, including solvent extraction, acid hydrolysis, and enzymatic transformation, with the enzymatic approach offering superior practicality [4]. Isoquercitrin (IQ), a major glycosylated derivative of quercetin, contains a sugar moiety linked via a β-glycosidic bond at the 3-hydroxyl position. Compared to its hydrophobic parent compound, isoquercitrin exhibits greater bioavailability in vivo and is more readily absorbed in the intestines [5]. Quercetin is commonly employed as a natural antioxidant in the food industry. When incorporated into lipid-rich products, such as dairy creams and processed meats, it inhibits lipid peroxidation and extends their shelf life [6]. In beverages such as fruit juices and yogurt, it enhances antioxidant capacity and promotes the bioavailability of coexisting nutritional components [7]. Collectively, quercetin and its derivatives exhibit a diverse range of biological activities. Elucidating their regulatory effects on nutritional health holds considerable scientific significance and has potential applications.

The natural flavonoid quercetin exhibits significant metabolic regulatory potential, but its mechanism of action in metabolic dysregulation induced by combined circadian rhythm disruption and high-fat diet remains incompletely elucidated. This study combines non-targeted metabolomics technology to investigate the mechanisms by which quercetin and its derivatives improve abnormal glucose and lipid metabolism in mice subjected to a combination of circadian rhythm disruption and a high-fat diet (Figure 1). Quercetin and its derivatives significantly improve the metabolite profile of mice, enhance the enrichment of signaling factors in pathways related to glucose and lipid metabolism, promote the secretion of specific receptor metabolites, and demonstrate potential for improving metabolic disorders involving glucose and lipids. These findings establish a theoretical foundation for future research on the therapeutic use of quercetin and its derivatives in managing metabolic disorders associated with circadian misalignment (e.g., shift work and sleep deprivation) and support the development and market advancement of quercetin-based functional foods.

2. Materials and Methods

2.1. Materials

2.1.1. Laboratory Animals

Specific pathogen-free (SPF) male C57BL/6 mice (28–29 days old) were obtained from the Guangdong Medical Laboratory Animal Center (production license number: SYXK [Guangdong] 2022-0002). The animals were housed in the SPF facility of Guangdong Pharmaceutical University (use license number: SYXK [Guangdong] 2022-0125) under controlled ambient conditions (21–25 °C). A 7-day acclimatization period was imposed under a 12 h light/12 h dark cycle (lights on at ZT0 = 08:00; off at ZT12 = 20:00), with ad libitum access to feed and water.

2.1.2. Primary Reagents and Instruments

Quercetin was sourced from Shanghai Macklin, Shanghai, China; isoquercetin from Shanghai Yuanye, Shanghai, China; and enzyme-modified isoquercetin (EMIQ) from Sichuan Shangrui Pharmaceutical, Chengdu, China. A high-fat diet was provided by Jiangsu Xietong Medicine Co., Ltd., Nanjing, China. Other key reagents and instruments included a BCA Protein Quantification Kit (Beyotime, Shanghai, China), polyvinylidene difluoride (PVDF) membrane (0.22 μm, Millipore, Burlington, MA, USA), high-speed refrigerated centrifuge (Sorvall ST 8R, Thermo Fisher, Waltham, MA, USA), automatic rapid sample grinder (JXFSTPRP-24, Shanghai Jingxin, Shanghai, China), UHPLC-Q Exactive HF-X mass spectrometer (Thermo Fisher), and ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA).

2.2. Methods

2.2.1. Animal Grouping and Intervention

Ninety mice were randomly allocated to ten groups (n = 9 per group): normal circadian rhythm control (Control, Con), circadian rhythm disruption (CRD), normal circadian rhythm with high-fat diet (Control+High-Fat Diet, Con+HFD), circadian rhythm disruption with high-fat diet (CRD+HFD), normal circadian rhythm with high-fat diet and quercetin (Con+HFD+Q), circadian rhythm disruption with high-fat diet and quercetin (CRD+HFD+Q), normal circadian rhythm with high-fat diet and isoquercetin (Con+HFD+IQ), circadian rhythm disruption with high-fat diet and isoquercetin (CRD+HFD+IQ), normal circadian rhythm with high-fat diet and enzyme-modified isoquercetin (Con+HFD+EMIQ), and circadian rhythm disruption with high-fat diet and enzyme-modified isoquercetin (CRD+HFD+EMIQ). The interventions were initiated after a 10-day acclimation period. Quercetin and its derivatives (Q, IQ, and EMIQ) were administered via oral gavage at a dose of 10⁻³ g/kg every other day at specified time points. The control groups (Con, CRD, Con+HFD, and CRD+HFD) received an equivalent volume of water via gavage.

2.2.2. Establishment of a Mouse Model of Circadian Rhythm Disorder and High-Fat Diet

A circadian rhythm disruption model was established using alternating light–dark cycles. Mice in the control group were maintained under a standard 12 h light/12 h dark cycle (12:12 LD), with lights on at 08:00 and off at 20:00. Mice in the disrupted rhythm groups were subjected to a repeating 4-day cycle consisting of two days under the standard 12:12 LD cycle followed by two days under a reversed 12:12 dark:light (DL) cycle. This simulated a shift-work-like pattern over a continuous 21-week period. Light exposure periods were defined using chronotime (ZT), where ZT0 indicated the beginning of the light phase (light phase: ZT0–ZT12; dark phase: ZT12–ZT24). Concurrently, mice in the high-fat diet groups were provided with a high-fat diet, whereas the control groups received standard chow. Throughout the experimental period, weekly assessments were conducted to monitor the behavioral state, locomotor activity, feed intake, and body weight. All data were systematically recorded during the feeding period.

2.2.3. Fecal Sample Collection

In the 21st week of the experiment, fresh fecal samples (approximately 50 mg per mouse) were collected from each group, placed in sterile EP tubes, immediately flash-frozen in dry ice, and subsequently stored at −80 °C for non-targeted metabolomics analysis.

2.2.4. Sample Preparation

A 15.0 mg portion of each sample was weighed in a 2 mL centrifuge tube. A 6 mm stainless steel grinding bead and 400 μL of extraction solution (methanol:water = 4:1, v/v), containing four internal standards (e.g., L-2-chlorophenylalanine at 0.02 mg/mL), were added. Samples were homogenized using a cryogenic tissue grinder for 6 min (−10 °C, 50 Hz), followed by cold ultrasonic extraction for 30 min (5 °C, 40 kHz). The samples were then incubated at −20 °C for 30 min and centrifuged at 13,000× g for 15 min at 4 °C. The supernatants were transferred into injection vials with insert tubes for subsequent instrumental analysis. Additionally, 20 μL of supernatant from each sample was pooled to generate a quality control (QC) sample.

2.2.5. UHPLC-Q Exactive HF-X Operating Conditions

Chromatographic Conditions [8]: An ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm i.d., 1.8 µm; Waters, Milford, CT, USA) was used. Mobile phase A consisted of 95% water and 5% acetonitrile containing 0.1% formic acid, and mobile phase B consisted of 47.5% acetonitrile, 47.5% isopropanol, and 5% water, also containing 0.1% formic acid. The injection volume was 3 μL, and the column temperature was maintained at 40 °C.

Mass Spectrometry Conditions [8]: Samples were analyzed using electrospray ionization (ESI) in both positive and negative ion modes. The spray voltages were set at 3400 V and −3000 V, respectively. The sheath gas flow rate was 60 arb, the auxiliary gas flow rate was 20 arb, the oven temperature was 350 °C, the capillary temperature was 320 °C, and the scan range was 70–1050 m/z.

2.3. Statistical Analysis

All 10 groups were initially assigned 9 mice each (n = 9). To ensure data stability, outliers (maximum and minimum values) were further removed prior to statistical analysis.

The raw data collected were imported into the metabolomics processing software Progenesis QI v. 2.0 (Waters Corporation, Milford, CT, USA) for data processing. They were then matched against public metabolite databases HMDB (http://www.hmdb.ca/) and Metlin (https://metlin.scripps.edu/), as well as an in-house library, to obtain metabolite information. Subsequently, intra-group sample reproducibility and inter-group sample variability were calculated to assess data quality. Finally, metabolic pathway annotation was performed using the KEGG database (https://www.kegg.jp/kegg/pathway.html, accessed on 16 January 2026) to identify pathways involving differentially expressed metabolites.

Differential metabolites were screened based on OPLS-DA output, applying the following thresholds: Variable Importance in Projection (VIP) ≥ 1, Fold Change (FC) ≥ 2 or ≤0.5, and p < 0.05. Metabolic pathway annotation was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

The data were organized and subjected to significance analysis using Excel 2021 and JMP 13. Experimental replicates were conducted with n ≥ 5, and the data are expressed as the mean ± standard deviation. Western blot band intensities were quantified using the ImageJ v. 1.41 software. Graphs and figures were generated using GraphPad Prism v. 9.5.1.

3. Results and Discussion

3.1. Effects of Quercetin and Its Derivatives on Body Weight in Mice

Long-term irregular lifestyles and high-sugar, high-fat diets readily disrupt physiological metabolism. At week 20 (Figure 2), weighing results revealed that under normal circadian rhythms, mice in the Con+HFD group exhibited significantly higher body weights than the Con group. Both quercetin and its derivative treatment groups significantly reduced body weight (p < 0.05), indicating that 20 weeks of intervention effectively suppressed HFD-induced weight gain and demonstrated clear regulatory improvements in mouse blood glucose levels.

However, under circadian rhythm disruption, the CRD+HFD group exhibited further significant weight gain compared to the Con+HFD group, confirming a synergistic weight-gain effect between high-fat diet and rhythm disruption. At this stage, only the CRD+HFD+EMIQ treatment group showed significantly lower body weight than the CRD+HFD group (p < 0.05), while quercetin and isoquercetin treatments failed to produce significant regulatory effects. This discrepancy is hypothesized to stem from EMIQ’s higher bioavailability, enabling its stable efficacy even under the pathological state of circadian disruption. The ineffectiveness of Q and IQ is presumed to be closely related to circadian rhythm disruption altering gut microbiota composition and regulating the intestinal metabolite profile, thereby impairing their absorption and functional expression. This aligns with the findings reported by Guo et al. [9].

3.2. Multivariate Statistical Analysis of Intergroup Differences in Gut Metabolites Among Mouse Groups

To investigate the differential effects of quercetin and its derivatives on gut metabolites in mice subjected to circadian rhythm disruption and a high-fat diet, principal component analysis (PCA) was applied to identify the primary sources of variation. By reducing multidimensional metabolic data into key principal components, PCA enabled the assessment of overall metabolic pattern shifts among the groups. This method facilitated the extraction of major metabolic features while minimizing noise, thereby enhancing analytical reliability and enabling the visual interpretation of group-specific differences [10].

In this study, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed to screen different metabolites, with variable importance in projection (VIP) > 1 and p < 0.05 set as the screening criteria for marker metabolites. As shown in Figure 3 and Figure 4, the cumulative contribution rates of the first and second principal components (PC1 + PC2) were 54.77% and 59.19%, respectively, indicating that these components jointly accounted for over half of the total variance. PCA plots revealed distinct separation of intestinal metabolic profiles between the control and intervention groups, indicating statistically significant metabolic differences (p < 0.05). The control group exhibited a tightly clustered intra-group distribution and low variance, suggesting a relatively stable metabolic state. In contrast, the HFD group displayed a broader dispersion and greater variability, indicating that the high-fat diet substantially altered the gut metabolic landscape. Notably, as shown in Figure 3, following intervention with quercetin and its derivatives, the sample clusters for the Con+HFD+Q, Con+HFD+IQ, and Con+HFD+EMIQ groups tended to shift toward the normal control group. A similar trend was observed in the CRD+HFD+EMIQ group (Figure 4), suggesting that quercetin and its derivatives partially restored the metabolic profiles disrupted by circadian rhythm disturbance and high-fat dietary intake.

A regression model linking metabolite expression levels with sample groupings was further constructed using partial least squares discriminant analysis (PLS-DA). The model demonstrated that all data points in Figure 5 fell within the 95% confidence interval, and distinct separation trends were observed between the quercetin-treated groups and the HFD group in both positive and negative ionization modes. The quercetin and derivative-treated groups exhibited greater divergence from the HFD group, supporting the modulatory effects of these compounds on glucose metabolism dysregulated by high-fat diet exposure. However, as depicted in Figure 5c,d, partial overlap was observed between the circadian-rhythm-disrupted quercetin-treated group and the HFD group in the positive ion mode, indicating less pronounced metabolite category differentiation. In contrast, in the negative ion mode, the CRD+HFD+EMIQ group demonstrated clear separation from the other groups (p < 0.05), implying significant differences in metabolic phenotypes across the experimental conditions.

To further validate the robustness of the PLS-DA model, a permutation test was conducted. The model performance was evaluated using cumulative explanatory power indices, with values approaching 1 indicating strong model stability, high adaptability, and predictive reliability [11]. As shown in Figure 6a,b, the cumulative explanatory powers R²X(cum) and R²Y(cum) in the cation mode were 0.480 and 0.405, respectively. In the anion mode, these values reached 0.798 and 0.633, respectively. For the circadian rhythm-disrupted group, R²X(cum) and R²Y(cum) in cationic mode were 0.518 and 0.407, while in anionic mode, they were 0.620 and 0.624, respectively (Figure 6c,d). A 200-iteration permutation test within the 95% confidence interval revealed a declining trend in the R² and Q² values with increasing permutation frequency, whereas the regression line demonstrated an upward trajectory. This outcome confirmed the model’s high stability and repeatability and indicated the absence of overfitting. R² and Q² are widely accepted indicators of model validation via permutation testing [12]. The interception of the Q² regression line with the Y-axis being less than 0.05 further confirmed the reliability of the model, making it suitable for subsequent metabolomics investigations.

3.3. Effects of Quercetin and Its Derivatives on Intestinal Metabolites in Mice

3.3.1. Effects of Quercetin and Its Derivatives on Metabolites in Mice with Normal Circadian Rhythms and Those on a High-Fat Diet

Using p < 0.05 and VIP_pred_OPLS-DA > 1 as screening criteria, Venn diagrams and volcano plots were employed to compare differential metabolites between quercetin and its derivatives and the control group and to identify shared and unique metabolites among groups. The results are shown in Figure 7. The number of metabolites shared between the control and Con+HFD, Con+HFD+Q, Con+HFD+IQ, and Con+HFD+EMIQ groups were 3364, 3452, 3360, and 3443, respectively. The Con+HFD group exhibited 751 significantly upregulated and 2613 significantly downregulated metabolites compared to the control group, indicating that a high-fat diet markedly suppressed intestinal metabolite expression in mice. In contrast, the quercetin and derivative-treated groups (Con+HFD+Q, Con+HFD+IQ, and Con+HFD+EMIQ) displayed 821, 893, and 939 upregulated metabolites, respectively, and 2631, 2467, and 2504 significantly downregulated metabolites, respectively, compared to the control group (Figure 7a). This overall pattern of differential regulation further suggests that appropriate supplementation with quercetin and its derivatives effectively enhances intestinal metabolite expression, mitigates metabolic disturbances induced by a high-fat diet, and exerts a regulatory effect on counteracting the downregulation of diet-associated metabolites.

Subsequent Venn diagram analysis enabled a comparative evaluation of the shared and unique metabolites between the experimental groups. Among the 4083 differentially expressed metabolites identified, 2786 were common to all four comparison groups, with the Con+HFD+EMIQ group sharing the highest number of metabolites (3443) with the control group (Figure 7b). Compared with the Con+HFD versus Con comparison, the Con+HFD+Q versus Con, Con+HFD+IQ versus Con, and Con+HFD+EMIQ versus Con comparisons contained 330, 382, and 470 unique differentially expressed metabolites, respectively. These findings indicate that quercetin and its derivatives substantially influence the gut metabolic profile in mice, thereby modulating the expression of microbe-derived metabolites. The presence of unique metabolites suggests that these compounds ameliorate high-fat diet-induced metabolic disorders through distinct and specific regulatory mechanisms.

3.3.2. Effects of Quercetin and Its Derivatives on Gut Metabolism in Circadian-Rhythm-Disrupted and High-Fat Diet-Fed Mice

This study investigated the regulatory effects of quercetin and its derivatives on gut metabolic products in mice subjected to circadian rhythm disruption and a high-fat diet. The volcano plot (Figure 8a) illustrates that the effects of oral administration of quercetin and its derivatives on gut metabolites were rapidly visualized compared to those in the CRD group. The CRD+HFD group exhibited 3369 differentially expressed gut metabolites, of which 741 were upregulated and 2628 were downregulated.

In contrast, the CRD+HFD+Q, CRD+HFD+IQ, and CRD+HFD+EMIQ groups showed 891, 807, and 822 upregulated metabolites, respectively, and 2491, 2584, and 2619 downregulated. When these findings were compared with those in Figure 7b, it was evident that the groups receiving quercetin and its derivatives under normal circadian conditions demonstrated a higher number of upregulated and lower number of downregulated metabolites. This trend indicates that circadian rhythm disruption may alter the abundance and composition of the mouse gut microbiota (e.g., phylum Firmicutes), thereby affecting the metabolite expression levels [13].

Pairwise comparisons using Venn diagram analysis (Figure 8b) further revealed that among the 4000 differentially expressed metabolites, 2807 were shared across all four comparison groups. Specifically, the CRD+HFD vs. CRD group shared 3080 differential metabolites with the CRD+HFD+Q vs. CRD group, 3113 with the CRD+HFD+IQ vs. CRD group, and 3061 with the CRD+HFD+EMIQ vs. CRD group. The numbers of unique differentially expressed metabolites in these comparisons were 302, 278, and 380, respectively. In contrast to the results shown in Figure 7b, the combined effects of circadian rhythm disruption and a high-fat diet led to a marked reduction in the number of unique metabolites regulated by quercetin and its derivatives. This suppression suggests that the regulatory capacity of these compounds on gut metabolites is diminished by circadian misalignment. These results underscore the essential role of circadian rhythm homeostasis in supporting the metabolic regulatory functions of quercetin and its derivatives. In summary, visual and statistical analyses of gut metabolites across groups revealed characteristic metabolic changes induced by quercetin and its derivatives under conditions of circadian disruption.

3.4. Analysis of Inter Group VIP Values for Differentially Metabolized Compounds in Mouse Intestines by Quercetin and Its Derivatives

3.4.1. Analysis of VIP Values for Intergroup Differences in Gut Metabolites of Quercetin and Its Derivatives in Normal Circadian Rhythm and High-Fat Diet Mice

The VIP value is a key statistical metric used to identify differential metabolites, reflecting the contribution of each metabolite to intergroup variation within multivariate models such as PLS-DA. Higher VIP values indicate a greater influence on group separation, making them critical for identifying potential biomarkers. In this study, orthogonal partial least squares discriminant analysis (OPLS-DA) was used as a supervised method. Through sevenfold cross-validation, the expression patterns and differential changes in metabolites across paired independent samples were assessed and predicted. The metabolites with the greatest impact were screened based on the VIP analysis of the first principal component. Cluster heatmaps and VIP bar charts were subsequently employed to visually compare expression patterns of differential metabolites across groups, providing deeper insight into metabolite–group associations. Furthermore, the integration of VIP values from multivariate analysis and p-values from univariate statistics allowed for a comprehensive visualization of metabolite importance and expression trends.

Analysis of the effects of quercetin and its derivatives on differential gut metabolites revealed that, relative to the control group, both the number and expression levels of intestinal metabolites were markedly reduced in the Con+HFD group. This finding indicates that a high-fat diet significantly suppresses intestinal metabolism. As shown in Figure 9a, gingerglycolipid B recorded the highest VIP score (2.77) and was identified as the most representative biomarker in the comparison between the Con and the Con+HFD groups. Gingerglycolipid B is a glycolipid molecule composed of carbohydrate and lipid moieties. Research indicates that its structural analog curcumin-glycolipid A enhances glucose uptake and mitigates damage caused by abnormal glucose metabolism by inducing IRS-1 and AMPK phosphorylation and upregulating PPARγ expression [14]. The present findings suggest that a high-fat diet suppresses the expression of gingerglycolipid B, thereby impairing glucose metabolism and contributing to metabolic disorders such as hyperglycemia.

Following intervention with quercetin and its derivatives, the expression levels of most intestinal metabolites were significantly elevated compared to those in the Con+HFD group. Among these, felotaxel, clobutinol, and 3α,17α-dihydroxy-5β-androstane displayed markedly higher VIP scores and emerged as the principal signature metabolites with strong discriminatory power (Figure 9b–d).

Previous studies have indicated that the primary metabolic pathways for felodipine and clonidine within the gut microbiota involve demethylation, whereas 3α,17α-dihydroxy-5β-androstane is produced via glucuronide metabolism. These pathways represent the key metabolic routes of flavonoid transformation. The accumulation of demethylated products may exert regulatory effects on the dysregulation of glucose metabolism. Flavonoids, including quercetin and its derivatives, exhibit antihyperglycemic activity by inhibiting α-glucosidase [15,16].

Moreover, methylation of the hydroxyl groups on the B-ring of quercetin, specifically at positions 3 and 4, yields methoxylated derivatives, such as 3-O-methylquercetin and 4-O-methylquercetin, which also exhibit hypoglycemic effects [17]. These metabolic transformations suggest that the gut microbiota modulates the biological activity of quercetin and its derivatives through structural modifications. These changes provide mechanistic insights into the ability of these compounds to regulate intestinal metabolism and improve glucose homeostasis.

3.4.2. Analysis of Intergroup VIP Values for Gut Metabolites Differentially Expressed in Mice with Circadian Rhythm Disorders and High-Fat Diets Following Administration of Quercetin and Its Derivatives

Disruption of circadian rhythms, such as through sleep deprivation or irregular feeding schedules, has been shown to disturb microbial rhythmicity, potentially resulting in dysbiosis and impairment of microbial metabolic function [1].

As illustrated in Figure 10, annohexocin displayed a notably high VIP score (2.6796) in the VIP bubble plot and was significantly downregulated in the Con+HFD group, indicating that its regulatory mechanism may be associated with exposure to a high-fat diet. Annohexocin is an acetylpyrazine compound isolated from Annona plants. According to Yiseul Son et al. [18], papaya extract modulates insulin signaling pathways by enhancing IRS-1 phosphorylation and promoting the membrane translocation of glucose transporter GLUT2. Moreover, papaya extract alleviated hyperglycemia-induced hepatic injury by upregulating AMPK phosphorylation and modulating the p-mTOR/mTOR ratio, thereby improving lipid metabolic disturbances in the livers of diabetic mice [19].

In addition, bullatanocin levels were significantly elevated in the CRD+HFD group compared with those in the CRD group. This observation is consistent with the findings of Hongling Du et al. [19], who identified bullatonocin in the serum of thrombotic rats, although its expression did not correlate directly with thrombosis inhibition by Rehmannia. These findings suggest that bullatanocin may not directly modulate thrombosis-related mechanisms but could influence glucose metabolism by altering the gut microbiota composition, including taxa such as Prevotella and Lactobacillus. Together, these results indicate that circadian rhythm disruption combined with high-fat diet intake modifies gut microbial communities and their metabolic outputs, thereby influencing the biosynthesis of metabolites involved in glucose regulation.

Further analysis was performed to evaluate the effects of quercetin and its derivatives on gut metabolite expression in mice subjected to both circadian rhythm disruption and a high-fat diet. The intervention groups demonstrated significant amelioration of gut metabolic profiles. As shown in Figure 10b–d, the CRD+HFD+Q and CRD+HFD+EMIQ groups exhibited significantly increased expression of 2-(4-hydroxy-3-sulfooxyphenyl) acetic acid and caffeic acid 3-glucoside compared to the CRD+HFD group. These findings are consistent with those reported by Parkar et al. [20]. Both compounds are phenolic acid derivatives that are likely generated through sulfation and microbial transformation pathways in the gastrointestinal tract. These metabolites possess antioxidant and anti-inflammatory properties and may exert regulatory effects on glycolipid metabolism by modulating host–microbiota interactions [21,22].

Selma et al. [23] also reported that bacterial genera, such as Clostridium and Eubacterium, can convert phenolic compounds into sulfate ester forms, thereby influencing host immune responses and metabolic pathways. Furthermore, the intestinal metabolite josamycin was significantly elevated in the CRD+HFD+IQ group compared to the CRD+HFD group. Research indicates that the biosynthesis of josamycin is associated with fatty acid metabolism and secondary metabolite synthesis pathways within the gut microbiota, leading to the generation of multiple hydroxylated and demethylated compounds. These modified metabolites are implicated in the regulation of glucose metabolism [14]. Collectively, these findings suggest that quercetin and its derivatives promote beneficial shifts in gut microbial composition and activity, thereby enhancing the biosynthesis of structurally diverse hydroxylated and demethylated metabolites associated with glycemic regulation.

3.5. Correlation Heatmap Analysis of Quercetin and Its Derivatives with Differential Metabolites in Mice Intestines

3.5.1. Correlation Heatmap Analysis of Quercetin and Its Derivatives with Differential Gut Metabolites in Mice with Normal Circadian Rhythms and Those on a High-Fat Diet

To visually illustrate the concentration trends of gut metabolites influenced by high-fat diet and the modulatory effects of quercetin and its derivatives, the top 30 most abundant metabolites identified in the study were selected for hierarchical cluster analysis. As shown in Figure 11, the metabolic profiles of the Con and HFD groups were distinctly separated, indicating significant differences in gut metabolite composition between mice fed with normal and high-fat diets. Following intervention with quercetin and its derivatives, the intestinal metabolite levels exhibited pronounced fluctuations and progressively converged to those of the Con group.

Compared to the Con group, the HFD group showed markedly elevated levels of various metabolites, including amino acids, indole compounds, and lipids. Among these, L-tyrosine, a key aromatic amino acid, plays a central role in protein synthesis, neurotransmitter biosynthesis, and hormone production. Previous studies have indicated that certain Bacteroidetes species significantly enhance L-tyrosine production, and its abnormal elevation is closely associated with impaired glucose metabolism [24]. Excessive tyrosine reacts with free radicals to form 3-nitrotyrosine, which damages pancreatic β-cells and contributes to insulin resistance. Moreover, elevated tyrosine levels can impair glucose homeostasis by enhancing gluconeogenic activity and inhibiting glucose clearance [25]. Aberrant tyrosine metabolism also promotes microbial utilization of tryptophan via the indolepyruvate pathway, thereby increasing indole compound production, as observed in the HFD group.

Following intervention with quercetin and its derivatives, the levels of L-tyrosine and indoline were significantly downregulated in the Con+ HFD + IQ and Con + HFD + EMIQ groups. Concurrently, these treatments markedly upregulated several beneficial metabolites, including bile acid compounds, diketones, and purine derivatives. Among these, the expression of the primary bile acid chenodeoxycholic acid (CDCA) was notably increased (Figure 11). Bile acids are cholesterol-derived metabolites synthesized in the liver. In addition to their established roles in lipid digestion and absorption, they are increasingly recognized as modulators of glucose metabolism. CDCA has been shown to enhance glucose uptake and utilization in peripheral tissues by activating FXR-mediated insulin signaling pathways, thereby ameliorating insulin resistance [26]. Furthermore, bile acids inhibit the activation of the NLRP3 inflammasome through the TGR5-cAMP-PKA signaling axis, contributing to the prevention of high-fat diet-induced glucose dysregulation [27].

Prolonged consumption of high-fat diets promotes the progression of non-alcoholic fatty liver disease (NAFLD). Studies have shown that quercetin significantly increases the abundance of Clostridium species in the gut, which may contribute to its anti-inflammatory and glucose regulatory effects [28]. Enhanced Clostridium abundance promotes bile acid secretion, which in turn suppresses hepatic lipid accumulation and mitigates the progression of insulin resistance [29].

3.5.2. Heatmap Analysis of Correlation Between Quercetin and Its Derivatives and Gut Metabolites in Circadian-Rhythm-Normal and High-Fat Diet-Fed Mice

In assessing the regulatory effects of quercetin and its derivatives on gut metabolites in mice subjected to circadian rhythm disruption and high-fat diet, it was observed that the combined impact of these stressors significantly increased the production of amino acids, indole compounds, lipids, and fatty acid amides within the gut microbiota (Figure 12). Notably, the CRD+HFD group exhibited further elevated linoleamide levels, a fatty acid amide, compared to the Con+HFD group. This finding indicates that circadian rhythm disruption exacerbates lipid metabolism dysregulation, resulting in fatty acid amide accumulation. These results are consistent with those reported by Amini M et al. [30], who demonstrated that circadian rhythm disruption induced by sleep deprivation in mice inhibits fatty acid amide hydrolase (FAAH) activity, leading to the abnormal accumulation of fatty acid amides. FAAH, a membrane-bound mammalian enzyme responsible for the synthesis and degradation of fatty acid amides, may exhibit diminished activity in the CRD+HFD group, which contributes to increased linoleamide levels. Prolonged high-fat diet intake has also been shown to elevate hepatic oxidative stress, promote the oxidative accumulation of fatty acids, and induce hepatic injury, further amplifying linoleamide deposition and aggravating disturbances in lipid metabolism [31].

Following treatment with quercetin and its derivatives, linoleamide production was substantially inhibited, accompanied by significant downregulation of amino acids, indole compounds, and lipids. Specifically, the expression of L-tyrosine, dihydroindole, and sphingosine was suppressed, mitigating glucose metabolism abnormalities and insulin resistance induced by high-fat diet exposure. These findings are consistent with the trends shown in Figure 11. Additionally, both quercetin and isoquercetin markedly enhanced the expression of tetrapyrrole compounds, including 3-hydroxy-12-oxocholan-24-oic acid and stercobilin. Among these, 3-hydroxy-12-oxocholan-24-oic acid, a bile acid derivative, plays a pivotal role in regulating lipid metabolism, maintaining cholesterol homeostasis, and supporting glucose regulation by modulating bile acid balance [32]. Overall, these findings demonstrate that quercetin and its derivatives not only suppress the overproduction of amino acids and lipids by the gut microbiota but also promote the synthesis of beneficial metabolites involved in glucose and lipid regulation, particularly bile acid derivatives.

3.6. KEGG Pathway Enrichment Analysis of Gut Differentially Expressed Metabolites in Mice Treated with Quercetin and Its Derivatives

3.6.1. KEGG Pathway Enrichment Analysis of Gut Differentially Expressed Metabolites in Mice with Normal Circadian Rhythms and Those Induced by High-Fat Diets Treated with Quercetin and Its Derivatives

To elucidate the mechanisms through which gut metabolites mediate the ameliorative effects of quercetin and its derivatives on impaired glucose metabolism, enrichment analyses of key signaling and metabolic pathways were conducted. KEGG pathways were used as the analytical framework, with reference species or their close relatives selected as the database for mapping differential metabolites. In total, 188 metabolic pathways were identified in the enrichment analysis of gut metabolites from mice under normal circadian rhythm and high-fat diet conditions, with varying degrees of enrichment. Among these, 26 pathways were significantly enriched (p < 0.05). As illustrated in Figure 12, seven pathways demonstrated both low p-values and high pathway impact (p < 0.001). The pathways, from highest to lowest enrichment, were primary bile acid biosynthesis, tryptophan metabolism, bile secretion, tyrosine metabolism, linoleic acid metabolism, caffeine metabolism, steroid hormone biosynthesis, and neuroactive ligand–receptor interactions. Notably, the differentially regulated primary bile acid biosynthesis and bile secretion pathways are closely associated with bile acid metabolism, corroborating the findings of Hu Jinrui, who reported similar pathway enrichment when studying the effects of mung-bean-derived antioxidant peptides on gut metabolites in mice fed a high-fat diet [33]. Among these, the primary bile acid biosynthesis pathway displayed the highest enrichment score (value = 7.018), indicating the substantial involvement of this pathway in the observed metabolic changes.

Abnormal bile acid metabolism induces insulin resistance and chronic inflammation, contributing to hyperglycemia and the development of diabetes. In the liver, cholesterol is enzymatically converted into primary bile acids, which are subsequently transformed by gut microbiota. These bile acids modulate the balance of the bile acid pool and regulate the FXR and G protein-coupled receptor 5 signaling pathways, thereby influencing glucose and lipid metabolism and insulin sensitivity [27,34]. Within this context, Bacteroides species act as key microbial agents in the transformation of primary bile acids into secondary bile acids, such as deoxycholic acid and cholic acid. This microbial conversion regulates bile acid pool homeostasis, supports microbial community structure, and promotes host metabolic health [35]. These findings suggest that quercetin and its derivatives enhance the secretion and metabolic transformation of bile acids by modulating the abundance of beneficial gut bacteria in mice fed a high-fat diet, thereby improving secondary bile acid synthesis and hepatobiliary circulation.

Additionally, prolonged intake of high-fat, high-sugar diets has been associated with neurotransmitter imbalances and cognitive decline, which increase the risk of neurological disorders such as memory impairment [36]. Metabolite enrichment analysis further revealed the significant involvement of the neuroactive ligand–receptor interaction pathway in the modulation of gut metabolites by quercetin and its derivatives. This pathway included 16 enriched metabolites, such as 5-hydroxytryptamine (5-HT), tryptophan, and N-oleoyldopamine, which are critical for regulating the central and peripheral nervous systems.

The enrichment of the neuroactive ligand–receptor interaction pathway and the detection of differential metabolites (e.g., 5-HT, tryptophan) suggest that quercetin and its derivatives may modulate the synthesis or metabolism of neurotransmitters. This aligns with previous studies reporting that quercetin could potentially enhance 5-HT, norepinephrine, and dopamine metabolism, as well as mitigate cognitive decline via SIRT1/NF-κB signaling [37]. The enzymes tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH), which catalyze the biosynthesis of serotonin and dopamine, respectively, function synergistically to maintain neurological and psychological homeostasis. Furthermore, Khadeeja Khan et al. [38] demonstrated that quercetin exhibits antidepressant properties by modulating neurotransmitter levels, cAMP signaling, and neuroactive ligand–receptor pathways [39]. Feng et al. [40] reported that quercetin and its derivative, quercetin-3-glucoside, are metabolized by Enterococcus faecium and Ruminococcus longum into short-chain fatty acids, including 3,4-dihydroxyphenylacetic acid, chloro-glucarol, butyrate, and acetate [41]. These metabolites stimulate intestinal chromaffin cells and upregulate TPH expression and enzymatic activity, thereby enhancing the conversion of tryptophan into 5-HT. The resulting serotonin can then cross the blood–brain barrier and influence central nervous system function. In this study, the tryptophan metabolic pathway was significantly enriched (p < 0.001), indicating that quercetin and its derivatives modulate the composition and activity of the gut microbiota in mice fed a high-fat diet. This regulation facilitates the production of neurotransmitter-like metabolites and contributes to maintaining the integrity of the intestinal barrier and homeostasis of the central nervous system.

3.6.2. KEGG Pathway Enrichment Analysis of Gut Metabolites Differentially Expressed in Mice with Circadian Rhythm Disruption and High-Fat Diet Induced by Quercetin and Its Derivatives

This study investigated the effects of quercetin and its derivatives on gut metabolites in mice subjected to the combined effects of circadian rhythm disruption and a high-fat diet. KEGG pathway enrichment analysis identified 215 metabolic pathways with varying degrees of enrichment, of which 37 pathways were statistically significantly enriched (p < 0.05). These included tryptophan metabolism, tyrosine metabolism, primary bile acid biosynthesis, and bile secretion, along with ten additional pathways of high significance. Notably, compared to the circadian rhythm control group (Figure 13), alanine, aspartate, and glutamate metabolism; protein digestion and absorption; and serotonergic synapse pathways were significantly enriched under the combined influence of circadian rhythm disruption and high-fat diet (p < 0.001). These findings suggest that circadian rhythm disruption modulates the regulatory efficacy of quercetin and its derivatives on gut microbiota diversity in mice fed a high-fat diet, consequently altering the activation of specific metabolic signaling pathways.

As illustrated in Figure 14, pathways related to alanine, aspartate, and glutamate metabolism, along with protein digestion and absorption, fall under the broader category of amino acid metabolism. Circadian rhythm disruption disturbs amino acid homeostasis, particularly affecting the metabolism of branched-chain amino acids (BCAAs). Such disruptions result in abnormal tissue distribution of amino acids, interfere with protein synthesis, and may contribute to the pathogenesis of metabolic disorders. Existing research has demonstrated that circadian misalignment caused by sleep disturbances disrupts the diurnal rhythmicity of plasma BCAA concentrations, including leucine, isoleucine, and valine [42]. Elevated BCAA levels have been reported to impair insulin signaling, diminish insulin sensitivity in peripheral tissues, and contribute to disturbances in glucose metabolism [43].

In addition, hyperglycemic mice subjected to circadian rhythm disruption exhibited dysbiosis of the gut microbiota, particularly in microbial taxa involved in amino acid metabolism. Altered composition and functional shifts have been observed in microbial genera, such as Bifidobacterium, Clostridium, and Bacteroides, all of which play key roles in modulating amino acid metabolic pathways [42].

These findings suggest that quercetin and its derivatives may influence host amino acid metabolism by modulating the composition and functional activity of gut microbiota. Supporting evidence from similar studies has shown that citrus-derived polymethoxyflavones regulate microbial BCAA metabolism and alleviate high-fat diet-induced metabolic syndromes [44]. Moreover, serotonergic synaptic pathway function is significantly influenced by circadian rhythm disruption. Previous studies have demonstrated that prolonged circadian misalignment impairs serotonin synthesis and release, reduces synaptic receptor sensitivity, induces cognitive deficits, and disrupts the gut microbiota composition. A high-fat diet further exacerbated these disruptions, diminishing the regulatory efficacy of quercetin and its derivatives on gut metabolic activity in mice with circadian rhythm disruption. Consequently, the impact of the intervention may be delayed or reduced under such combined stress conditions [45].

3.7. Section Limitation

Although non-targeted metabolomics holds significant research value for studying intestinal metabolites in mice, its inherent limitations—overreliance on database matching and algorithmic analysis to reflect metabolite changes—make it difficult to thoroughly validate metabolite functions. Mechanistic inferences based on pathway enrichment analysis remain insufficiently validated: the direct regulatory effects of specific gut microbiota and the functional relevance of key targets have yet to be verified using pharmacological tools. These limitations point the way forward for future research to enhance the application potential of quercetin and its derivatives in the intervention of metabolic diseases.

4. Conclusions

This study established murine models of circadian rhythm disruption and high-fat diet exposure to investigate the ameliorative effects of quercetin and its derivatives on aberrant glucose and lipid metabolism. Non-targeted fecal metabolomics revealed that the highest number of shared differential metabolites was observed between the control and Con+HFD groups. Compared with the control group, the HFD group exhibited a substantial reduction in metabolite expression, whereas the greatest increase in upregulated metabolites was observed in the groups treated with quercetin and its derivatives. Venn diagram analysis identified 2786 differentially expressed metabolites common across all comparison groups, with the greatest overlap (3443 metabolites) between the Con+HFD+EMIQ and control groups. Under circadian rhythm disruption, both the HFD and quercetin-treated groups exhibited similar trends, indicating that quercetin and its derivatives exert a modulatory effect on the gut microbiota, attenuating the metabolic suppression induced by a high-fat diet.

VIP value analysis revealed that gingerol-8-glucoside B was significantly downregulated in the HFD group compared to that in the control group, whereas felotaxel, clobutinol, and 3α,17α-dihydroxy-5β-androstane were among the most significantly upregulated metabolites in the Con+HFD+Q, Con+HFD+IQ, and Con+HFD+EMIQ groups, respectively. Additionally, compared with the CRD+HFD group, levels of 2-(4-hydroxy-3-sulfoxyphenyl)acetic acid and caffeic acid 3-glucoside were markedly elevated in the CRD+HFD+Q and CRD+HFD+EMIQ. Correlation heatmap analysis further confirmed that the high-fat diet promoted L-tyrosine and linoleamide accumulation, whereas quercetin and its derivatives suppressed the expression of both compounds and simultaneously elevated intestinal levels of chenodeoxycholic acid and stercosterol. KEGG pathway enrichment analysis demonstrated that quercetin and its derivatives primarily modulate amino acid and lipid metabolism by promoting bile acid secretion through the bile acid biosynthesis pathway and regulating neurotransmitter synthesis, such as 5-HT, via the neuroactive ligand–receptor interaction pathway. These findings suggest that quercetin and its derivatives enhance bile acid metabolite secretion by influencing gut microbial composition and related signaling pathways, thereby improving glucose metabolism.

Collectively, these results demonstrate the regulatory potential of quercetin and its derivatives in mitigating metabolic abnormalities by modulating intestinal metabolite levels in mice. These findings provide mechanistic insights into the interactions between quercetin, gut microbial metabolites, and glucose metabolism, highlighting the potential application of these compounds in the management of metabolic diseases. Future pharmacological investigations should explore the specific effects of quercetin and its derivatives on the targeted gut microbiota and their subsequent effects on host metabolic regulation. Further research is warranted to elucidate the molecular mechanisms underlying these effects, particularly the interactions between these compounds and circadian clock genes, metabolic enzymes, specific receptors, and intracellular signaling cascades. A more comprehensive understanding of these pathways may facilitate the development of circadian-gene-targeted therapeutic strategies, thereby offering novel theoretical frameworks and practical approaches for the personalized treatment of metabolic disorders associated with circadian rhythm disruption.