A Preliminary Study on the Effects of Fanfengling Dietary Supplement on Gut Antioxidant Capacity and Metabolomic Profile in Apis mellifera

TL;DR

This study shows that the dietary supplement Fanfengling improves gut health and metabolism in honeybees during a stressful spring period.

Contribution

The study demonstrates that Fanfengling enhances antioxidant capacity and energy metabolism in honeybees, offering a natural tool for apiculture.

Findings

Fanfengling boosts antioxidant defenses and energy metabolism in honeybees.

Metabolomic analysis revealed significant changes in compounds like hypoxanthine and quercetin 3-O-glucoside.

Dosing intervals affected physiological markers like ALP and T-AOC differently.

Abstract

This preliminary study investigated the effects of the dietary supplement Fanfengling on honeybee gut health during the critical spring brood-rearing period. We found that Fanfengling enhances antioxidant capacity and optimizes energy metabolism in honeybees, thereby improving their resilience to environmental stressors. These findings provide scientific support for the application of Fanfengling as a natural management tool to promote colony health in apiculture.

Honeybees experience significant physiological stress during spring brood-rearing due to fluctuating temperatures and forage scarcity, leading to oxidative damage and metabolic disruption. This study evaluated the effects of the dietary supplement Fanfengling on gut antioxidant capacity and metabolism in Apis mellifera during this critical period. Using physiological assays and LC-QTOF metabolomics, we measured key markers including SOD, SDH, ALP, T-AOC, and MDA. Metabolomic analysis detected 22,377 peaks and annotated 2799 metabolites, with significant alterations in hypoxanthine, delphinidin, and quercetin 3-O-glucoside. Enriched KEGG pathways included glutathione metabolism, pyrimidine metabolism, and ether lipid metabolism. Physiologically, Fanfengling enhanced antioxidant and energy metabolic status: Group B (dosed every 21 days) showed peak ALP and SDH/SOD activities, while Group A (dosed every other day) achieved the highest T-AOC. These results indicate that Fanfengling mitigates spring-induced oxidative stress by boosting antioxidant defenses and optimizing energy metabolism, supporting its potential use in apicultural health management.

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1. Introduction

Honeybees are critical pollinators underpinning global agricultural productivity and ecosystem stability [1,2]. However, global honeybee populations have experienced a sharp decline due to the combined effects of habitat fragmentation, pesticide exposure, pathogen pressure, and climate change [3,4]. Their gut microbiota constitutes a relatively simple yet specialized consortium dominated by core bacterial genera such as Gilliamella, Snodgrassella, Lactobacillus, and Bifidobacterium. These symbionts have co-evolved with their hosts, forming stable mutualistic relationships essential for nutrition, immune modulation, and pathogen defense [5]. These environmental challenges disrupt gut microbiota homeostasis, inducing dysbiosis that impairs nutrient metabolism, immune competence, and pathogen defense, thereby compromising bee health [6]. A dysbiotic microbiome impairs nutrient metabolism, weakens immune competence, reduces detoxification capacity, and heightens pathogen susceptibility, ultimately compromising both individual survival and colony fitness.

The spring brood-rearing period represents a critical period for colony expansion and population renewal in honeybees. During this time, fluctuating ambient temperatures coupled with forage scarcity (insufficient nectar and pollen) impose dual metabolic challenges: energetic stress and elevated oxidative burden. Oxidative stress triggers lipid peroxidation, protein carbonylation, and DNA damage, ultimately impairing bee longevity and reproductive fitness [7]. Consequently, maintaining gut homeostasis and metabolic integrity is essential for bees to withstand these environmental pressures. This underscores the strategic importance of developing targeted feed supplements as a viable intervention to mitigate physiological stress and enhance colony robustness in modern apiculture.

Fanfengling, a dietary supplement for honeybees, demonstrates potential in promoting colony reproduction and enhancing immune competence in commercial apiculture. Its constituent bioactive compounds include flavonoids, polysaccharides, and alkaloids [8], which have been shown in other animal models to possess antioxidant, anti-inflammatory, and gut microbiota-modulating properties [9]. However, systematic investigation of Fanfengling’s specific regulatory mechanisms on intestinal metabolite composition and antioxidant systems in honeybees, particularly during spring brood rearing, remains limited.

This study aims to mechanistically investigate how Fanfengling supplementation impacts gut antioxidant capacity and metabolic profiles in Apis mellifera during spring brood-rearing. By quantifying key physiological and metabolic markers, identifying differential metabolites and pathways, and elucidating regulatory mechanisms, we seek to understand Fanfengling’s role in mitigating oxidative stress and managing energy homeostasis. The results will provide a scientific basis for the rational application of Fanfengling in apicultural health management.

2. Materials and Methods

2.1. Experimental Colony Establishment

In early spring, healthy colonies were selected for the experimental groups based on standardized queen age, colony strength, and management history. Two colonies served as controls and four received Fanfengling supplementation. We selected two frames of bees from each colony for sampling.

Bee Source Statement: The honeybee colonies used in this study were maintained at the Apicultural Experimental Station of Shanxi Agricultural University. All colonies were managed using standard research-oriented beekeeping practices, with no commercial purchase involved.

Due to logistical constraints and the standardized management of colonies at the Apicultural Experimental Station, we selected two colonies per group as biological replicates. To support parallel analyses, gut tissues from worker bees in each colony were pooled separately for metabolomic analysis and biochemical assays, ensuring sufficient material for both platforms. For metabolomic analysis, each pooled sample was processed with three technical replicates to ensure measurement reliability. For biochemical assays, each pooled homogenate was analyzed with 15 technical replicates to enhance statistical robustness and reproducibility. This approach provided adequate tissue quantity for each analytical platform while maintaining within-colony homogeneity and reducing inter-colony variation. The pooling strategy follows established practices in honeybee metabolomics, where limited colony numbers are compensated by robust within-group sampling to detect consistent metabolic and physiological signatures [10].

2.2. Feeding Method

Upon completion of the feeding regimen on 20 March, 30 adult worker bees were collected from each colony. The experimental colonies were allocated to three groups: Group A (n = 2) received Fanfengling every other day from 25 February to 20 March; Group B (n = 2) received a single dose on 27 February; and the control group (n = 2) received no supplementation. Fanfengling was administered at 4 g per kilogram of 1:1 (w/v) sucrose syrup, delivering 2 g per frame and 4 g per colony per feeding. Control colonies received sucrose syrup only at equivalent volumes. Based on typical syrup consumption of 50–100 mL per frame per day in spring, we estimated that each bee ingested approximately 0.05–0.1 mg of Fanfengling per day in Group A, and 1–2 mg in the single-dose Group B. No adverse effects (e.g., mortality, reduced foraging) were observed during the trial.

Fanfengling is a commercially available honeybee dietary supplement produced in China. According to the manufacturer’s product specification sheet (Shanxi Bee Products Research Institute, Jinzhong, China), its primary bioactive constituents include flavonoids (e.g., quercetin, kaempferol glycosides), polysaccharides, and alkaloids derived from botanical extracts. Although the precise formulation is proprietary, these compound classes have independently been associated with antioxidant, anti-inflammatory, and immunomodulatory effects in insects and other animal models [11]. The presence of anthocyanins and flavonol glycosides in the supplement is consistent with the metabolomic detection of delphinidin, cyanidin-3-O-glucoside, and quercetin 3-O-glucoside in treated bee guts, suggesting that these compounds may be directly absorbed or metabolically transferred from the supplement. We therefore hypothesize that flavonoids—particularly quercetin glycosides and anthocyanins—are major contributors to the observed enhancement of antioxidant capacity and energy metabolism.

2.3. Sample Collection and Preparation

Upon feeding completion, 30 adult worker bees were collected from each colony and immediately frozen in liquid nitrogen. Samples were stored at −80 °C until analysis. For biochemical assays, whole gut tissues were dissected from frozen bees and homogenized in appropriate buffers. For metabolomic analysis, 50 mg of pooled gut tissue from each colony was extracted using 1 mL of ice-cold methanol:water (4:1, v/v) containing 0.1% formic acid. The mixture was homogenized with a bead mill (30 Hz, 2 min) and sonicated at 4 °C (40 kHz, 30 min), followed by centrifugation at 12,000× g for 10 min at 4 °C. The supernatant was filtered through a 0.22 μm PTFE membrane and transferred to LC-MS vials for analysis.

2.4. LC-MS Metabolomic Analysis

Metabolomic profiling was conducted using a Waters Xevo G2-XS QTOF mass spectrometer (Waters Corporation, Milford, MA, USA) coupled to an ACQUITY UPLC system and controlled by MassLynx V4.2 software. Data were acquired in MSe continuum mode, enabling simultaneous acquisition of low-energy (precursor ion) and elevated-energy (fragment ion) spectra within a single analytical run. Raw data were preprocessed using Progenesis QI software (version 3.0), including peak alignment, normalization to total ion intensity, and retention time correction. Quality control (QC) samples were analyzed every six injections to monitor system stability, with coefficient of variation < 20% required for metabolite inclusion. Metabolites were annotated by matching accurate mass and fragmentation patterns against KEGG, HMDB, and LipidMaps databases (mass tolerance ≤ 5 ppm). Differential metabolites were identified using orthogonal partial least squares discriminant analysis (OPLS-DA) with Variable Importance in Projection (VIP) > 1.0, fold change (FC) > 1.5 or < 0.67, and Student’s t-test p < 0.05. Model validation was performed using 200 permutation tests (p < 0.01). Significantly altered metabolic pathways were identified via KEGG enrichment analysis using a hypergeometric test (p < 0.05, FDR < 0.1).

Metabolites were identified based on accurate mass (mass error ≤ 5 ppm) and MS/MS fragmentation matching against public databases (KEGG, HMDB, LipidMaps). For key metabolites of interest (e.g., delphinidin, quercetin 3-O-glucoside, hypoxanthine), where commercial standards were unavailable, MS/MS spectra were manually verified against reference spectra from published literature to bolster identification confidence.

2.5. Biochemical Assays

Commercially available assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) were used to quantify superoxide dismutase (SOD; Cat. No. A001-3-2), succinate dehydrogenase (SDH; Cat. No. A022-1-1), alkaline phosphatase (ALP; Cat. No. A059-2-2), malondialdehyde (MDA; Cat. No. A003-1-2), and total antioxidant capacity (T-AOC; Cat. No. A015-1-2). All procedures were performed following the manufacturer’s instructions. Gut tissues dissected from six worker bees per colony were pooled and homogenized (1:10 w/v) in ice-cold 0.9% saline containing 0.1% Triton X-100. Homogenates were centrifuged at 4 °C for 10 min (8000× g for SOD, SDH, ALP, and MDA; 10,000× g for T-AOC). Supernatants were collected, and protein concentrations were determined via bicinchoninic acid (BCA) assay for normalization. All measurements were performed in six technical replicates per colony.

2.6. Statistical Analysis

Data were analyzed using SPSS 23.0. Normality and homogeneity of variance were confirmed using Shapiro–Wilk and Levene’s tests, respectively. Group means were compared by one-way ANOVA followed by the post hoc least significant difference (LSD) test. Results are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05. For metabolomic data, multivariate analysis and pathway enrichment were performed using MetaboAnalyst 5.0.

3. Results

3.1. PCA Analysis Reveals Distinct Gut Metabolite Profiles Induced by Fanfengling

To evaluate the overall impact of Fanfengling administration (Group A, every-other-day dosing) on honeybee gut metabolomes, principal component analysis was performed. Metabolomic profiling identified 14,597 peaks (1547 annotated metabolites) in positive ion mode and 7780 peaks (1252 metabolites) in the negative ion mode. PCA score plots revealed robust separation between Group A and Control Group C, with tight intra-group clustering (Figure 1). The first two principal components (PC1) explained 62.12% of total variance in positive ion mode (PC1: 43.72%, PC2: 18.40%) (Figure 1A) and 61.89% in negative ion mode (PC1: 45.84%, PC2: 16.05%) (Figure 1B). These results demonstrate that Fanfengling significantly altered intestinal metabolite composition. Model quality parameters (R^2^X = 0.85, Q^2^ = 0.76) indicated good fit and predictive capability. Permutation testing (200 permutations, p < 0.05) confirmed model validity, supporting subsequent OPLS-DA analysis [12].

3.2. OPLS-DA Identifies Fanfengling-Associated Metabolic Alterations

To identify Fanfengling-specific metabolite perturbations, we performed orthogonal partial least squares discriminant analysis (OPLS-DA) to maximize separation between treatment and control groups [13]. OPLS-DA score plots revealed clear discrimination in both ion modes (Figure 2A,B). Model quality parameters indicated robust goodness-of-fit (R^2^Y = 0.994 for positive mode and R^2^Y = 0.996 for negative mode) and predictive ability (Q^2^ > 0.7 for both modes), confirming reliable capture of treatment-induced metabolic variation [14]. Permutation testing (200 permutations) validated model significance, with all permuted models yielding significantly lower Q^2^ values (p < 0.01) and intercepts below zero (Figure 2C,D), thereby excluding overfitting. These results establish that the OPLS-DA models reliably reflect Fanfengling-specific metabolic effects, supporting subsequent differential metabolite screening based on Variable Importance in Projection (VIP) values [15].

3.3. Differential Metabolite Analysis

Fanfengling supplementation significantly altered the gut metabolome, yielding 151 differentially expressed metabolites in positive ion mode (88 upregulated, 63 downregulated) and 163 in negative ion mode (86 upregulated, 77 downregulated) (Figure 3).

Across both ion modes, a total of 1547 and 1252 metabolites were identified in the treatment groups, respectively (Table 1). Differential metabolites were screened using volcano plot analysis based on VIP > 1.0, |log_2_FC| > 0.585, and Student’s t-test p < 0.05 [16]. Among the most significantly altered metabolites (Table 2), several were linked to antioxidant stress and energy metabolism. In positive ion mode, upregulated anthocyanins (delphinidin, cyanidin-3-O-glucoside) are potent antioxidants that scavenge reactive oxygen species [17]. Positiveion mode identified 4 upregulated and 11 downregulated metabolites between the two groups, while negative ion mode showed 5 upregulated and 10 downregulated. Functional annotation linked to key processes including oxidative stress response, energy metabolism, and inflammatory regulation. For example, delphinidin and cyanin-3-O-glucoside—anthocyanins detected in positive ion mode—are well-established as potent antioxidant and anti-inflammatory agents. In negative ion mode, quercetin 3-O-glucoside, a flavonoid glycoside, known for its strong antioxidant capacity, was significantly increased. Conversely, hypoxanthine, a purine metabolite whose accumulation indicates oxidative stress, was markedly downregulated, suggesting enhanced energy homeostasis. Additionally, dUDP (pyrimidine metabolism) and beta-paxitriol (a triterpenoid) were altered, potentially reflecting modified nucleotide synthesis and cellular protection. These metabolomic changes provide mechanistic insight into Fanfengling’s antioxidant and metabolic benefits [18].

The complementary use of positive and negative ion modes broadened metabolite coverage: positive mode preferentially detected alkaloids and anthocyanins, whereas the negative mode excelled in identifying flavonoids and organic acids. This dual-mode strategy enhanced annotation confidence and facilitated more robust biological interpretation.

3.4. KEGG Pathway Enrichment Analysis

KEGG pathway enrichment analysis of differentially expressed metabolites found five significantly enriched pathways (p < 0.05): pyrimidine metabolism, ether lipid metabolism, glutathione metabolism, benzoic acid degradation, and tryptophan metabolism (Figure 4). Among these, pyrimidine metabolism exhibited the most pronounced statistical significance, indicating its central role in Fanfengling-induced metabolic modulation.

Pathway impact was evaluated using pathway topology analysis in MetaboAnalyst 5.0, which combines enrichment significance with the topological importance of metabolites within pathways. Glutathione metabolism and pyrimidine metabolism exhibited both high enrichment significance (−log_10_(p) > 3) and high pathway impact scores (>0.5), underscoring their pivotal role in the metabolic rewiring induced by Fanfengling.

3.5. Biochemical Indicators

Single-dose Fanfengling administration (Group B) significantly elevated intestinal ALP, SDH, and SOD activities compared to Group A and controls (p < 0.05; Figure 5A,B,E). MDA content showed the inverse pattern: Control > Group A > Group B (p < 0.05; Figure 5C). Conversely, Group A exhibited the highest total antioxidant capacity and Complex IV activity among all treatments (p < 0.05; Figure 5D).

4. Discussion

Metabolomics, a technology that comprehensively analyzes all metabolites within an organism and their regulatory patterns, facilitates the quantitative assessment of metabolite profiles and the exploration of their relationships with physiological and pathological alterations. This approach is widely applied to elucidate the molecular mechanisms underlying animal growth and development, reproduction, environmental adaptability, and stress resistance [19,20]. Ref. [21] utilized non-targeted metabolomics coupled with liquid chromatography-mass spectrometry (LC-MS) to demonstrate that glucose metabolism and fat digestion/absorption significantly influence metabolic pathways during the pre-pupal stage of the Apis mellifera. Subsequently, in the pupal stage, metabolites were found to be involved in regulating chitin and lipopolysaccharide precursor formation, as well as the biosynthesis of phenylalanine, tyrosine, and tryptophan.

This study systematically investigated the effects of supplementing Apis mellifera with Fanfengling during the critical spring brood-rearing period on intestinal antioxidant capacity and associated metabolic profiles. We integrated non-targeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) with physiological and biochemical indicators. Results demonstrated that BeeBoost supplementation significantly altered gut metabolite composition and enhanced antioxidant function in bees. A total of 151 and 163 differentially expressed metabolites were identified in positive and negative ion modes, respectively. Annotation of 15 significantly altered metabolites revealed key compounds closely linked to antioxidant defense and energy metabolism, including anthocyanins (delphinidin and cyanidin-3-O-glucoside), flavonoids (quercetin-3-O-glucoside), purine metabolites (hypoxanthine), dUDP, and β-guaiacol. Although the exact compositional profile of Fanfengling is not fully disclosed, its declared botanical-derived flavonoids and polysaccharides align with the metabolite signatures detected in treated bees. Quercetin-3-O-glucoside and delphinidin, both significantly upregulated in Group A, are known constituents of several medicinal plants commonly used in pollinator supplements [22,23]. Their accumulation in gut tissue likely reflects direct dietary intake and possibly partial metabolic conversion. These observations are consistent with earlier studies showing that dietary flavonoids are absorbed and exert systemic antioxidant effects in honeybees [24]. Bee pollen contains flavanols (e.g., quercetin, kaempferol) and anthocyanins, which contribute to its antioxidant potential and help mitigate oxidative damage from foraging flights and environmental stress [25]. Anthocyanin content and diversity in bee pollen are highly dependent on plant sources [26]. Anthocyanins exert potent antioxidant effects across diverse organisms [27]. In bees, flavonoids exhibit radical-scavenging, anti-inflammatory, antibacterial, antiviral, and anti-allergic activities, particularly during pharyngeal gland development, thereby supporting colony resilience during food shortages [28,29]. Quercetin and its derivatives (e.g., quercetin-3-O-glucoside) enhance antioxidant capacity in bees, improve resistance to imidacloprid, and modulate immune responses [30]. Hypoxanthine plays a dual role in bee physiology: it serves as an energy substrate for gut symbionts such as Snodgrassella alvi and Ralstonia, fueling the tricarboxylic acid (TCA) cycle to maintain microbial homeostasis; conversely, its levels fluctuate in association with queen bee ovarian development. During ovarian activation, hypoxanthine concentration decreases markedly due to upregulated purine nucleotide synthesis, potentially exerting negative feedback on oocyte maturation [31,32]. Similarly, dGDP (deoxyguanosine diphosphate) levels increase during ovarian activation, indicative of the purine metabolic reprogramming. The sesquiterpene β-guaiacol, widely distributed in plants, displays potent antibacterial, anti-inflammatory, anticancer, and neuroprotective properties. Propolis, produced by honeybees from collected plant resins mixed with wax and other secretions, is rich in diverse terpenoids [33]. The differential expression of these metabolites suggests that Fanfengling supplementation promotes flavonoid accumulation while attenuating markers of oxidative damage. It may also modulate nucleotide synthesis and cellular protection mechanisms. Collectively, these alterations indicate metabolic reprogramming that bolsters redox homeostasis and energy production—essential for sustaining colony vitality during high metabolic demand periods [34]. Overall, Fanfengling feeding profoundly reshapes the bee metabolome, inducing systemic shifts in metabolite abundances. This activates pathways involved in antioxidant defense and energy provision, thereby reinforcing redox balance and optimizing energy metabolism. Such changes provide a molecular basis for the observed improvements in physiological and biochemical indicators, establishing a metabolic framework for colony health maintenance during brood-rearing phases.

KEGG enrichment analysis further elucidated the systemic metabolic effects of Fanfengling supplementation, revealing significant enrichment in several key pathways, including glutathione metabolism, pyrimidine metabolism, ether lipid metabolism, benzoic acid degradation, and tryptophan metabolism. Among these, glutathione metabolism was especially prominent as a key part of the cell’s antioxidant defense system. The upregulation of this pathway aligns with the observed increases in SOD activity and T-AOC, as glutathione is essential for the regeneration of antioxidant enzymes and the direct neutralization of reactive oxygen species [35,36]. The enrichment of pyrimidine metabolism suggests enhanced nucleotide biosynthesis, which may support rapid cell proliferation and DNA repair mechanisms under stress conditions [37], consistent with the increased metabolic activity required during brood rearing. Furthermore, this enrichment may arise from both host nucleotide synthesis and microbial contributions, as bee gut symbionts such as Snodgrassella alvi and Gilliamella are known to engage in purine and pyrimidine cycling, thereby potentially modulating host energy homeostasis and stress resilience [38]. Future metatranscriptomic or gnotobiotic studies could further elucidate these microbiota-mediated mechanisms in greater detail.

The metabolomic findings were strongly corroborated by the physiological and biochemical assays. Specifically, Fanfengling supplementation led to a significant increase in SOD activity and total antioxidant capacity (T-AOC), along with a marked reduction in MDA content—a key indicator of lipid peroxidation. These results clearly demonstrate that Fanfengling enhances honeybees’ intrinsic antioxidant defense system, thereby mitigating oxidative damage [39]. Moreover, the significant elevation in alkaline phosphatase (ALP) and succinate dehydrogenase (SDH) activities in the treated groups indicates improved nutrient absorption and energy metabolism. ALP is involved in the hydrolysis of phosphate esters, facilitating the absorption of lipids, glucose, and other nutrients [40], while SDH is a key enzyme in the tricarboxylic acid (TCA) cycle, reflecting enhanced mitochondrial respiration and ATP production [41]. These adaptations are particularly crucial during spring brood rearing, when energy demands are high.

Notably, the metabolic and physiological responses to Fanfengling varied between the two feeding regimens. Group B (single dose) exhibited the highest levels of ALP, SDH, and SOD activities, along with the lowest MDA content, suggesting that a single, well-timed intervention may optimally stimulate antioxidant and energy metabolic pathways without causing metabolic overload [42]. This observation aligns with hormesis principles, wherein mild stressors promote resilience. In contrast, frequent dosing (Group A) may maintain baseline antioxidant capacity without eliciting further amplified peak responses, as indicated by its highest T-AOC levels. This dose-dependent metabolic plasticity aligns with previous studies on intermittent feeding models, which have shown that intermittent feeding enhances metabolic efficiency and stress resistance [43]. These findings underscore the need for precise calibration of Fanfengling administration to maximize its benefits while avoiding potential adverse effects.

Although our study revealed significant metabolic and physiological responses to Fanfengling supplementation, the limited number of colonies per group may constrain extrapolation to varied apicultural contexts. Future investigations should incorporate more colonies and environmental replicates to enhance statistical power and generalizability. Nonetheless, our pooling strategy for samples and application of rigorous multivariate analyses (PCA, OPLS-DA) were designed to minimize colony-level variability and emphasize robust treatment effects.

Taken together, these findings support the hypothesis that Fanfengling’s bioactivity is primarily driven by its flavonoid fraction, although synergistic contributions from polysaccharides and alkaloids cannot be excluded. Future studies should combine controlled dose–response feeding experiments with targeted quantification of specific compounds (e.g., via LC-MS/MS) to identify the precise active ingredient and establish causal mechanisms.

Overall, our study provides multi-level evidence that Fanfengling enhances honeybee health by modulating key metabolic pathways and strengthening antioxidant defenses. The upregulation of glutathione and pyrimidine metabolism, coupled with the accumulation of flavonoid antioxidants and the reduction in oxidative stress markers, illustrates a coherent mechanism through which Fanfengling mitigates the physiological challenges associated with spring brood rearing. These effects are further reflected in the improved nutrient absorption and energy metabolism, as evidenced by the elevated ALP and SDH activities [44]. By integrating metabolomic and physiological data, we have elucidated a potential pathway-metabolite-function axis through which Fanfengling exerts its beneficial effects. This not only advances our understanding of its mode of action but also provides a scientific basis for its rational application in apicultural practice. Although this study emphasized host metabolomic and biochemical responses, concurrent gut microbiota profiling (e.g., via 16S rRNA sequencing) would help determine whether Fanfengling’s effects are mediated by microbial community shifts. We advocate for such integrative analyses in future studies to comprehensively unravel the supplement’s mechanisms of action.

5. Conclusions

This study demonstrates that Fanfengling activates the antioxidant defense system in Italian honeybees by regulating key metabolic pathways (glutathione and pyrimidine metabolism) and altering specific metabolites such as hypoxanthine. This metabolic reprogramming directly enhanced physiological antioxidant capacity, as evidenced by elevated total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity, alongside reduced malondialdehyde (MDA) levels. These findings indicate that Fanfengling enhances bee stress resistance through an integrated pathway-metabolite-function mechanism, providing a natural strategy for spring colony health management. Future research should identify specific bioactive components and validate field efficacy to advance sustainable beekeeping practices. In particular, targeted LC-MS/MS quantification of key flavonoids and glutathione pathway intermediates will help confirm their absolute concentrations and reinforce the mechanistic insights proposed here.