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5 March 2026

15 Pages

Modulation of Glycemic Control by Vaccinium myrtillus Leaf Extract: Impact of Inulin Co-Administration

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Institute for Medicinal Plants Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia
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Institute of Medical and Clinical Biochemistry, Faculty of Medicine, University of Belgrade, Pasterova 2, 11000 Belgrade, Serbia
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Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
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Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Cartagena 130014, Colombia
Foods2026, 15(5), 903;https://doi.org/10.3390/foods15050903
This article belongs to the Special Issue Innovative Applications of Polyphenols, Flavonoids, and Other Bioactives from Plant Foods
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Abstract

Vaccinium myrtillus L. (bilberry) leaves have traditionally been used to manage hyperglycaemia in folk medicine. The combination of plant polyphenols with dietary fibres such as inulin may offer enhanced metabolic benefits; however, their combined effects remain insufficiently characterized. This study aimed to evaluate the antihyperglycaemic effects of bilberry leaf extract (VME) and its inulin-enriched formulation (VMEI), and to investigate their potential as functional food components for managing type 2 diabetes. VME and VMEI were assessed using in vitro enzyme inhibition and an in vivo Caenorhabditis elegans model to evaluate toxicity. High-performance liquid chromatography (HPLC) was used to profile major compounds. A three-month dietary intervention was conducted in mice fed a high-fat, high-sucrose (HFHS) diet to examine the glucose-lowering effects of the extracts. HPLC analysis identified chlorogenic acid as the major constituent, followed by quercetin derivatives. VMEI exhibited stronger α-glucosidase inhibition than VME, indicating synergistic activity. Rutin and hyperoside showed the highest inhibitory activity against α-amylase and α-glucosidase, respectively. In C. elegans, VME displayed moderate toxicity at higher doses, and both extracts reduced locomotion. In the mouse model, both VME and VMEI significantly reduced blood glucose levels in HFHS-fed mice, with effects comparable to healthy control. VMEI showed more pronounced improvements, still not statistically significant. Overall, the combination of bilberry leaf polyphenols with inulin demonstrated enhanced in vitro enzyme inhibition, while in vivo findings indicate potential antihyperglycaemic effects that warrant further investigation. These results support the continued exploration of VMEI as a functional food candidate for metabolic health management.

1. Introduction

Diabetes represents a major global public health concern, with the number of affected individuals increasing substantially each year [1]. According to the World Health Organization (WHO), the global diabetic population grew from 200 million in 1990 to 830 million in 2022, and it is projected to reach 1.3 billion by 2050. The rise in diabetes prevalence has been particularly rapid in low- and middle-income countries compared to high-income nations. Notably, over 95% of diabetes cases are classified as type 2 [2]. Lifestyle factors such as obesity and physical inactivity are among the primary contributors to the growing prevalence of the disease [3]. Diabetes can be managed by various approaches and its combination including antidiabetic medications, healthy diet, physical activity, and herbal remedies [4]. Vaccinium myrtillus L. (Ericaceae), also known as bilberry or European blueberry, is characterized by its high nutritional and medicinal properties. For this reason, it is one of the most important berries in Europe, both economically and for the food and pharmaceutical industries [5]. The fruits are used fresh and dried in the diet, but are also processed into various foods such as juices, jams and more. Both the fruit and the leaves are used in traditional medicine in various countries [6]. While there are official monographs for bilberry fruit—for example from the European Medicines Agency (EMA), the European Pharmacopoeia and the European Scientific Cooperative on Phytotherapy (ESCOP)—there is currently no official monograph for bilberry leaves [7,8,9]. Traditionally, bilberry leaves have been used primarily for the treatment of diabetes [6], but also as an astringent, diuretic and against inflammation, especially in the oral cavity [10]. Despite its wide traditional use in diabetes, there is limited and conflicting scientific data on the antidiabetic effect of bilberry leaves [11,12]. Several studies have investigated the antidiabetic potential of bilberry leaf extracts. Cignarella et al. reported a 26% reduction in plasma glucose levels and a 39% reduction in triglycerides in streptozotocin-diabetic rats treated with a dried hydroalcoholic extract [12]. Rau et al. found that bilberry extract inhibited α-amylase and activated PPAR-γ, suggesting antidiabetic properties [1]. A multi-herb formulation containing bilberry also lowered blood glucose and fructosamine levels in alloxan-induced diabetic mice. Sidorova et al. demonstrated that bilberry leaf extract lowered blood glucose levels and glycated hemoglobin levels and improved insulin sensitivity in both obese Zucker diabetic rats and streptozotocin-induced diabetic Wistar rats, along with a reduction in body weight in the ZDF model [13]. In addition to medicinal herbs, studies have also shown that a high-fibre diet can lead to improvements in diabetes [14]. Inulin, which is found in the tuber of Jerusalem artichoke, is a soluble, indigestible fibre [15]. Due to its indigestibility, it cannot cause an increase in blood glucose levels in humans after consumption, but can prevent a sharp increase in blood glucose levels and thus protect the islet cells [16]. Shao et al. showed a reduction in body weight, fasting blood glucose levels, total and HDL cholesterol and average daily food consumption in hyperglycaemic mice treated with inulin [17]. In addition, human clinical studies have shown a reduction in metabolic syndrome biomarkers following inulin supplementation [18], and inulin consumption has also been associated with an improvement in glucose metabolism and a lower risk of gestational diabetes mellitus [19]. In 2018, inulin was approved by the Food and Drug Administration (FDA) as an ingredient that can improve the nutritional value of various food products [20].
The aim of this study was to evaluate the antidiabetic potential of bilberry (Vaccinium myrtillus L.) leaf extract by in vitro and in vivo assays targeting hyperglycaemia and to investigate the potential synergistic effects of inulin enrichment on its antihyperglycaemic efficacy.

2. Materials and Methods

2.1. Plant Material and Extracts Preparation

Dried leaves of bilberry (Vaccinium myrtillus L., Ericaceae) were purchased from the Institute for Medicinal Plants Research “Dr. Josif Pančić” (Belgrade, Serbia; batch: 01540120). The leaves were ground in a laboratory mill and then subjected to percolation extraction for 24 h with a 50:50 ethanol/water mixture and a solid to solvent ratio of 1:2. After extraction, the ethanol was evaporated under vacuum at 50 °C using the Buchi rotavapor R-114 rotary evaporator. The dry matter of the resulting liquid extract was 23.4%, measured with the HB43S moisture analyser, Mettler Toledo(Greifensee, Switzerland).
The extract was divided into 2 parts, and inulin was added to one part at a concentration of 20% based on the dry mass. Before the lyophilization process, the prepared solution was heated to 30 °C and mixed with a magnetic stirrer until completely homogenized.
Bilberry extracts, without (VME) and with inulin (VMEI), were frozen at −80 °C for 24 h and then freeze-dried using the Beta 1–8 freeze dryer (Martin Christ, GmbH, Osteroide am Harz, Germany) at a temperature of −60 °C and a pressure of 0.011 mbar for 24 h and, to remove the capillary water residues, at −60 °C and a pressure of 0.0012 mbar for a further hour. After lyophilization process, the extracts were crushed into powders.

2.2. HPLC Analysis

Chromatographic analysis was carried out using an Agilent 1260 RR HPLC system (Agilent, Waldbronn, Germany) fitted with a diode-array detector operating between 190 and 550 nm. Separation was achieved on a Zorbax SB-C18 reversed-phase column (150 × 4.6 mm, 5 μm particle size; Agilent). The mobile phases consisted of (A) 1% (v/v) orthophosphoric acid in water and (B) acetonitrile. A gradient elution programme was applied as follows: 0–2.6 min, 90–85% A; 2.6–8 min, 85% A; 8–10.8 min, 85–80% A; 10.8–18 min, 80% A; 18–23 min, 80–70% A; 23–25 min, 70–50% A; 25–27 min, 50–30% A; 27–29 min, 30–10% A; 29–31 min, 10–0% A; and 31–34 min, 0% A. The flow rate was maintained at 0.8 mL/min, the column temperature was set to 40 °C, and the injection volume was 8 μL. Detection was performed at 260, 280, 320, and 360 nm. Compounds were identified by matching their retention times and UV spectra with those of corresponding reference standards. Quantitative determination was based on external calibration curves, and the results were expressed as micrograms per gram of dry weight (μg/g DW).

2.3. In Vitro Anti-Hyperglycaemic Assay

Bilberry extracts (with and without inulin) were dissolved in 80% ethanol to reach stock concentration of 1000 µg/mL. The initial concentrations of the individual analytical standard compounds (purchased from ChemFaces (Wuhan, Hubei, China)) were prepared in methanol as follows: chlorogenic acid (1200 µg/mL), isoquercitrin (1020 µg/mL), hyperoside (700 µg/mL), quercetin (175 µg/mL), quercitrin (1000 µg/mL) and rutin (1200 µg/mL). These stock solutions were subsequently used to prepare the working dilutions for both the α-glucosidase and α-amylase inhibition assays described below.

2.3.1. Alpha-Glucosidase Inhibition Assay

For the evaluation of α-glucosidase inhibition activity, the previously described protocol by Wan et al. was employed [21]. The reaction mixture contained 120 μL of the sample (S) and 20 μL α-glucosidase solution prepared by potassium phosphate buffer (0.1 M, pH 6.8) to reach a concentration of 0.5 U/mL. After pre-incubation (5 min at 37 °C), 20 μL of 5 mM pNPG employed as substrate was added and continuously incubated for 20 min at 37 °C. To terminate the reaction, 80 μL of 0.2 M sodium carbonate dissolved in potassium phosphate buffer was added. The absorbances were measured at 405 nm using the Multiskan Sky Thermo Scientific microplate reader (Vantaa, Finland). Acarbose was used as the positive control. The control (C) contained a buffer instead of the samples. To avoid the influence of sample colour, the absorbances of the test samples were corrected by the absorbance of the extract colour control (CC), containing the sample mixed with buffer. The measurements were performed in triplicate and the results are expressed as mean ± standard error.
The percentage of inhibition of α-glucosidase activity was calculated as follows:
Inhibition (%) = [C − (S − CC)/C] × 100.

2.3.2. Alpha-Amylase Inhibition Assay

This assay, which evaluates the ability of bilberry extract and individual compounds to inhibit α-amylase, was performed according to the protocol described by Zengin et al. [22]. In brief, the reaction mixture contained 25 μL of sample (S) and 50 μL of α-amylase enzyme solution dissolved in sodium phosphate buffer (0.1 M, pH 6.8 with 6 mM sodium chloride) to reach a concentration of 0.5 mg/mL. After pre-incubation (37 °C, 10 min). 50 μL of starch solution (0.1%) was added and incubation continued (10 min, 37 °C). The reaction was terminated by adding 25 μL 1M hydrochloric acid. To visualize the reaction, 100 μL of Lugol’s solution was added to the reaction mixture. The absorbance values were measured at 630 nm using a Multiskan Sky Thermo Scientific microplate reader (Vantaa, Finland). Acarbose obtained from Sigma-Aldrich (St. Louis, MO, USA) was used as a positive control. The enzyme control (EC) contained buffer instead of the sample, while the substrate control (SC) contained buffer instead of the enzyme. To avoid the influence of sample colour, the absorbances of the test samples were corrected by the absorbance of the sample colour control (CC) containing the sample mixed with buffer. Measurements were performed in triplicate and results are expressed as mean ± standard error. The percentage of inhibition of α-amylase activity was calculated as follows:
Inhibition (%) = [(S − CC) − EC)/SC] × 100.

2.4. Biological Effects of VME and VMEI Extracts in Caenorhabditis elegans

2.4.1. Preparation of Test Solutions

Stock solutions of VME and VMEI were prepared in dimethyl sulfoxide (DMSO) and subsequently diluted in K-medium (32 mM KCl and 51 mM NaCl in Milli-Q water, Merck KGaA, Darmstadt, Germany) to obtain final concentrations of 31.25, 62.5, 125, 250, 500, and 1000 μg/mL, each containing 0.5% DMSO. The negative control consisted of K-medium with 0.5% DMSO.

2.4.2. Strains and Handling Conditions

A preliminary assessment of the biological activity and toxicological protective profile of Vaccinium myrtillus leaf extract (VME), and in combination with inulin (VMEI) was conducted using Caenorhabditis elegans. The wild type Caenorhabditis elegans N2 strain (Caenorhabditis Genetics Center-CGC, University of Minnesota, Minneapolis, MN 55455, USA) was used for lethality, locomotion and toxicological protection assays. Worms were maintained at 20 °C under standard conditions on nematode growth medium plates (NGM), with Escherichia coli OP50 as food source [23,24]. Synchronized worm populations were obtained lysing gravid adults with a bleaching solution (NaOH 0.5 M, NaClO 1.05% in Milli-Q water), adapting the protocol described by Duran-Izquierdo et al. [25]. For the assays described below, four experiments were conducted, each including three technical replicates per treatment.

2.4.3. Lethality Assay

Nematodes at the L4 larval stage were exposed to VME and VMEI treatments, as well as to the negative control, for 24 h at 20 °C in 96-well plates, with 12 ± 2 worms per well. After exposure, the nematodes were examined under a stereo microscope and classified as alive if they exhibited spontaneous movement or responded to gentle mechanical stimulation, and as dead if no such response was observed [26].

2.4.4. Locomotion Assay

L4-stage nematodes were exposed to the extracts and the negative control for 24 h at 20 °C. For each treatment, 15 worms were individually evaluated, allowing each worm to move freely for 60 s prior to scoring in order to minimize startle-induced behavioural responses. The number of body bends over a 20-s interval was scored for each nematode, counting one bend each time the posterior bulb of the pharynx reached a maximum flexion in the direction opposite to the previous bend [27].

2.4.5. Toxicity Protection Assay

Synchronized L1-stage nematodes were initially exposed for 24 h to the extracts at the No Observed Adverse Effect Level (NOAEL) concentrations previously identified in the lethality assay (VME: 250 μg/mL; VMEI: 125 μg/mL), along with their respective negative controls. Following this pre-exposure, the worms were treated with increasing concentrations of CdCl2 (50, 100, 250, 500, 1000, 2000, and 4000 µM) and incubated for an additional 24 h. At the end of the exposure, the number of live and dead nematodes was counted [28].

2.5. In Vivo Anti-Hyperglycemic Assay

2.5.1. Animals

Male C57BL/6J mice (8 weeks old; 18–22 g) were used for this study. They were group-housed (four per cage) under standard conditions, with a 12-h light/dark cycle and a controlled temperature of 22 ± 3 °C. All procedures were in accordance with the ethical guidelines laid down in the Serbian legislation on animal research and the European Directive 2010/63/EU on the protection of animals used for scientific purposes. Authorisation for the study was obtained from the Ministry of Agriculture, Forestry and Water Management–Veterinary Directorate (authorisation number 003573855/2024/1).

2.5.2. Diets

At the beginning of the study, all healthy mice were randomly divided into four experimental groups (n = 8 per group). The first group was administered a high-fat diet with 20% lard and a 20% sucrose solution as drinking water (HFSD). The second and third groups received the same HFSD supplemented with either Vaccinium myrtillus leaf extract (HFSD + VME) or Vaccinium myrtillus leaf extract in combination with inulin (HFSD + VMEI), each at a dose of 150 mg/kg body weight. The fourth group served as a control and received a standard rodent pellet diet (SPRD) and water for the duration of the 90-day trial. Animals in the other groups received their respective treatments instead of water. All animals had unrestricted access to food throughout the study. The animals’ health, behaviour and activity were monitored daily, while food and water intake (including sucrose solution for the HFSD groups) and body weight were recorded weekly. Fasting blood glucose levels were determined twice—once after two months and again at the end of the three-month trial. Fasting blood glucose levels were determined using a glucometer (Prizma) with fresh tail vein blood (~20 μL per sample). At the end of the study, the animals were anesthetized with intravenous thiopental and humanely euthanised for pathological examination.

2.6. Data Analysis

Data are presented as mean ± standard error. The Shapiro–Wilk and Bartlett tests were used to assess normality and homogeneity of variances, respectively. Since the data did not follow a normal distribution, group differences were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test with Bonferroni adjustment. Toxicity protection responses to each CdCl2 concentration were compared between extract-treated groups and their respective controls using the Mann–Whitney U test. All statistical analyses were performed using GraphPad Prism version 8.0.1, and differences were considered statistically significant at p < 0.05.

3. Results

3.1. HPLC Analysis

Although the polyphenolic composition of bilberry fruits has been widely investigated, considerably less information is available regarding the quantitative profile of phenolic compounds in bilberry leaves, particularly in populations originating from Serbia. In this study, chlorogenic acid and selected quercetin derivatives in leaf extracts were quantified using high-performance liquid chromatography (HPLC), and the resulting values are presented in Table 1. The corresponding chromatograms are provided in the Supplementary Materials (Figure S1). Previous investigations [29] have likewise indicated that these compounds represent dominant phenolic constituents in bilberry leaves. Similarly, Tian et al. [30] identified caffeoylquinic acids as the principal phenolics in Finnish samples, with chlorogenic acid being the most abundant compound. Our findings are consistent with these reports, as chlorogenic acid was the major phenolic compound detected in both analyzed extracts.
Table 1. The amount of individual phenolics in bilberry (VME) and inulin-enriched bilberry extract (VMEI).
A slightly higher content of individual polyphenols was found in the inulin-free extracts. This finding can be attributed to the dilution effect due to the addition of inulin (20% inulin, based on the dry residue of the extract).

3.2. In Vitro Anti-Hyperglycaemic Activity

Given the long-standing use of bilberry leaves in traditional medicine for the management of diabetes, numerous phytochemical investigations, as well as in vivo and clinical studies, have been undertaken to assess their biological effects and elucidate potential mechanisms of action [11,31].
The data presented in Table 2 showed that the IC50 value of VMEI (501.59 µg/mL) was lower than that of VME in the α-glucosidase inhibition assay, suggesting a potentially enhanced inhibitory effect of the combined formulation. Both extracts showed weaker activity than the positive control acarbose. Bljajić et al. found that the hydroethanolic extract obtained from bilberry leaves had a strong inhibitory effect on the α-glucosidase activity, with the IC50 being statistically equal to that of acarbose (0.29 mg/mL and 0.50 mg/mL, respectively) [32]. The results obtained in the present study are in agreement with the findings of Bljajić et al. [32]. As shown in Figure 1a, the tested extracts inhibited the activity of this enzyme at all applied concentrations and in a dose-dependent manner. Similar values were obtained for all individual constituents in the range of concentrations tested (Figure 2). Among the individual constituents, the most active α-glucosidase inhibitor was hyperoside (IC50 value = 75.51 µg/mL), followed by rutin, quercetin, isoquercitrin, and chlorogenic acid, while quercitrin was the least active constituent (Table 2). Interestingly, all individual components showed a stronger inhibition of α-glucosidase than acarbose. Similar results were obtained by Lin et al., who also found that hyperoside exhibited stronger antioxidant and anti-α-glucosidase activity than other compounds from Crataegus pinnatifida [33]. Despite lower IC50 values compared to acarbose in vitro, the physiological relevance of these findings remains uncertain due to potential differences in bioavailability, metabolism, and achievable concentrations in vivo.
Table 2. In vitro inhibition of α-glucosidase and α-amylase by bilberry extracts (VME and VMEI), their individual components and acarbose, expressed as IC50 values (µg/mL).
Figure 1. Enzyme inhibiting activity of (a) α-glucosidase and (b) α-amylase by bilberry extracts (VME and VMEI) at different concentrations.
Figure 2. Enzyme inhibiting activity of α-glucosidase and α-amylase by individual components at different concentrations.
In the α-amylase inhibition assay, the two extracts showed enzyme inhibitory effects, but only at the highest applied concentrations (250, 500 and 1000 µg/mL) and in a dose-dependent manner (Figure 1b), and the inhibition did not reach 50% within the tested range. Consequently, IC50 values could not be determined, which limits the quantitative assessment of inhibitory potency and comparison with standard inhibitors. Future studies should include a broader concentration range to enable more precise evaluation. Cvetkova et al. reported on the anti-amylase activity of ethanol extracts and freeze-dried samples of the aerial parts and leaves of bilberry collected from two localities and found that the aerial parts showed stronger activity than the leaves [34]. Freeze-dried samples were more potent α-amylase inhibitors than ethanolic extracts, with IC50 values of 15.70 and 19.89 mg/mL for two localities, which is outside of the concentration range tested in the current study. Similar to α-glucosidase, the values obtained in the α-amylase inhibition assay clearly showed that VMEI has a stronger inhibitory effect compared to VME (Figure 1a,b). This is not surprising considering the results of Cardullo et al. [35]. These researchers found that both low-chain chicory inulin and long-chain topinambur inulin led to α-amylase inhibition with IC50 values of 0.45 and 0.50 mg/mL, respectively. Based on the results obtained, Cardullo et al. [35], concluded that the enrichment of spaghetti with inulin, which has an inhibitory effect on α-amylase, determines the hypoglycaemic properties of the pasta and thus lowers the corresponding IC50 value. The results presented in Table 2 also showed that the positive control acarbose and the individual components were active in a broader concentration range (Table 2). In this assay, the strongest inhibition against α-amylase was observed for rutin (IC50 value = 34.99 µg/mL), while other components showed significantly weaker inhibition. Among them, quercetin and isoquercitrin showed the IC50 values lower than positive control acarbose. However, their in vitro potency does not necessarily reflect in vivo efficacy. Within the range of concentrations tested, the individual components showed dose-dependent inhibition of α-amylase (Figure 2). In the study of Hendra et al. [36], rutin showed an inhibitory activity with IC50 value of 177.2 µg/mL, which was about five times higher than in our study, while the inhibition of α-glucosidase was stronger (IC50 value of 47.2 µg/mL compared to 92.04 µg/mL in our study).

3.3. Biological Effects of VME and VMEI Extracts in Caenorhabditis elegans

The results of the lethality and locomotion assays in C. elegans are shown in Figure 3 and Figure 4. The highest lethality percentages were observed in worms exposed to 500 μg/mL and 1000 μg/mL VME, reaching 6.7% and 20.2%, respectively. For VMEI, lethality occurred at a lower concentration and then progressively increased: 2.5% at 250 μg/mL, 5.3% at 500 μg/mL and 7.4% at 1000 μg/mL. These lethal effects at ≥250 μg/mL for VMEI and ≥500 μg/mL for VME suggest that combining the extract with inulin could modify its toxicity profile. This observation is partially consistent with previous reports indicating that combinations of plant extracts with polysaccharides can modify the bioavailability and biological activity of antioxidant compounds such as polyphenols [37,38,39], potentially altering their physiological effects. Therefore, findings in C. elegans are interpreted as a preliminary biological and toxicological profile, and not as direct evidence of glycemic control. Regarding locomotion, both VME and VMEI slightly reduced movement at 1000 μg/mL, showing a 21.4% decrease in body bends for VME and a 14% decrease for VMEI compared with their respective controls, although these differences were not statistically significant (Figure 4). Locomotion in C. elegans is a well-characterized physiological response regulated by the balance between excitatory cholinergic and inhibitory GABAergic neurotransmission to ensure coordinated muscle activity [40]. The minor inhibition observed at higher extract concentrations suggests potential interference with the nematode’s neuromuscular or enteric nervous system [41]; however, this result should be interpreted with caution, and since no significant differences were detected, it does not support conclusions regarding alterations in neurotransmission. Consistently, no clear differences attributable to inulin were observed for this endpoint, and these findings should therefore be regarded as model-specific biological observations rather than evidence of therapeutic efficacy.
Figure 3. Lethality in C. elegans exposed to VME (A), and VMEI (B). All tested concentrations and lethality values (%) are shown. * Significant differences (p < 0.05) compared to control (0 µg/mL).
Figure 4. Locomotion in C. elegans exposed to VME (A), and VMEI (B). No significant mean differences were found (p < 0.05) compared to control (0 µg/mL).
It is also important to consider that certain phytochemical compounds can modulate biological activity in C. elegans depending on their structure and concentration, and responses are not always linear or predictable, sometimes exhibiting hormetic effects characterized by beneficial outcomes at low doses and toxicity at higher ones [42]. For example, González-Paramás et al. evaluated an anthocyanin-rich bilberry fruit extract (Vaccinium myrtillus L.) at 5 and 10 μg/mL, finding that while 5 μg/mL provided protective effects against oxidative stress, the higher concentration of 10 μg/mL resulted in increased reactive oxygen species accumulation and higher mortality rates, indicating a hormetic response [43]. In contrast, Wang et al. reported that supplementing C. elegans with blueberry extract (Vaccinium spp.) at 50, 200, and 500 μg/mL improved motility and reduced lipofuscin accumulation in a dose-dependent manner without signs of hormesis [44]. These contrasting outcomes highlight that the biological activity of such phytochemical extracts may vary depending on factors such as the species or varieties within the same genus, the extraction methods, formulation, the specific biological endpoints assessed, and the additional molecular pathways modulated by the phytochemical compounds. Finally, neither VME nor VMEI provided protection against CdCl2-induced toxicity in C. elegans (Figure S2), indicating the absence of cytoprotective effects under the evaluated conditions. Accordingly, the present data do not support definitive mechanistic inferences regarding activation of stress response pathways. Cellular defence against cadmium requires coordinated detoxification systems, including phase II enzymes, metallothioneins, and ABC transporters, within regulatory networks involving transcription factors such as SKN-1 and DAF-16 through PMK-1 signalling [45]; however, these pathways were not directly evaluated in this study. Given that natural compounds can variably modulate these stress response pathways [46], the involvement of these routes should be regarded as hypothetical in the present work. Further targeted studies are therefore required to elucidate the protective potential of V. myrtillus extracts under cadmium stress and to identify the specific bioactive components responsible for these effects.

3.4. Effect of Vaccinium myrtillus Leaf Extracts Alone and in Combination with Inulin on Glucose Metabolism in High-Fat and Sucrose Diet (HFSD) in Mice

Previous in vivo studies have confirmed the antidiabetic activity of extracts from V. myrtillus leaves [13,40,47]. At the same time, bilberry leaves have shown that they have the potential to prevent various diabetic complications such as liver damage and nephropathy, and also play a role in the regulation of lipid metabolism [40]. In addition, studies in animal models have shown that inulin can help lower blood glucose levels and improve the condition of diabetics. Inulin-type fructans serve as low-calorie alternatives to sugar or fat and are classified as non-viscous, soluble and fermentable dietary fibres [48]. Miao et al. reported that dietary supplementation with inulin alleviated metabolic disorders in mice with gestational diabetes mellitus by modulating the RENT/AKT/IRS/GLUT4 signalling pathway [26]. Ghavidel et al. demonstrated that inulin can enhance the effect of resveratrol in Wistar rats with streptozotocin-induced diabetes by promoting a healthier intestinal microbiome and increasing the stability of resveratrol [49].
In our study, hyperglycaemia was induced in mice by administering a high-fat diet in combination with a 20% sucrose solution. As shown in Figure 5, this combination of lipotoxic and glucotoxic stress resulted in a significant increase in blood glucose levels by approximately 12.42% compared to the control group receiving a standard diet. The effects of tested VML extracts (with or without inulin) in the high-fat, high-sucrose diet (HFSD) on glycemic control were studied at two time points: 60 and 90 days. The results were compared with those of mice fed only the HFSD and those of mice fed a standard diet. Throughout the experiment, daily inspections revealed no noticeable differences between the animals in terms of appearance, behaviour or growth rate. Both the extract and the combination of extract and inulin showed a positive effect in lowering blood glucose levels at the second evaluation time point. At the first evaluation time point (after 60 days), blood glucose levels were lower compared to the HFSD group; however, the differences were not statistically significant.
Figure 5. The effect of VME and VMEI on a high-fat and -sucrose diet (HFSD) on the glycemic status of mice was followed after (A) 60 and (B) 90 days and compared with the effect of the HFSD alone (high-fat-diet treatment) and with the standard pellet diet (control). Different letters above the columns indicate statistically significant differences between groups (p < 0.05).
After three months of treatment, the administration of bilberry extracts alone or in combination with inulin led to a significant reduction in blood glucose levels, making them comparable to those of the control group. This indicates a possible complete recovery from the hyperglycaemic state. It is also known that a number of herbal medicines have a gradual effect, sometimes taking weeks or even months to become noticeable. The group receiving bilberry leaf extract in combination with inulin showed the most significant improvement after 90 days, with blood glucose levels comparable to those of the control group. However, no statistically significant difference was found between this group and the group receiving the extract without inulin. Sidorova et al. demonstrated that bilberry leaf extract normalized glycosylated hemoglobin levels in 50% of diabetic animals and significantly reduced blood glucose compared to untreated diabetic controls [47].

4. Conclusions

This study confirms the therapeutic potential of bilberry leaf extract, especially in combination with inulin, in the control of glucose metabolism and the inhibition of important carbohydrate-digesting enzymes. The observed inhibition of α-amylase and α-glucosidase by the extract and its dominant polyphenols, as well as the gradual but significant reduction in blood glucose levels in mice subjected to a high-fat and high-sucrose diet, emphasizes its promising effect in the treatment of early-stage diabetes. The most pronounced effects were observed in the group treated with the inulin-enriched bilberry leaf extract, indicating an enhanced effect on glycemic control compared to the extract alone. These results support the further development of functional formulations containing bilberry leaf extract and inulin as natural active ingredients for the prevention and supportive treatment of type 2 diabetes. A limitation of the present study is the absence of glucose tolerance testing, insulin level measurements, and the normalization of metabolic parameters to body weight. Inclusion of these assessments would provide a more comprehensive evaluation of the metabolic effects of bilberry leaf extract and its combination with inulin. Future studies are warranted to incorporate these analyses to better elucidate the mechanisms underlying the observed glycemic changes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15050903/s1, Figure S1. Chromatogram of VME recorded at 320 nm: 1—chlorogenic acid, 2—rutin, 3—hyperoside, 4—isoquercitrin, 5—quercitrin, 6—quercetin; Figure S2. Lethality in C. elegans exposed to the mixtures: VME-CdCl2 (A) and VMEI-CdCl2 (B). * Significant mean differences (p < 0.05) when compared to the control (0.5% DMSO).

Author Contributions

Conceptualization, J.Ž. and K.Š.; methodology, J.Ž., S.R., S.P., A.A.A., J.R.-O. and J.O.V.; software, J.R.-O., J.O.V. and A.A.A.; validation, J.Ž., S.R., S.P., A.A.A., J.R.-O. and J.O.V.; formal analysis, J.Ž., S.R., S.P., A.A.A., J.R.-O. and J.O.V.; investigation, J.Ž., S.R., S.P., A.A.A., J.R.-O. and J.O.V.; resources, J.Ž. and K.Š.; data curation, J.Ž., S.R., S.P., A.A.A., J.R.-O. and J.O.V.; writing—original draft preparation, J.Ž., A.A.A., J.R.-O. and J.O.V.; writing—review and editing, S.R., S.P., K.Š. and J.O.V.; visualization, J.R.-O. and J.O.V.; supervision, J.Ž., K.Š. and J.O.V.; project administration, J.Ž., S.R. and J.O.V.; funding acquisition, J.Ž., K.Š., S.R. and J.O.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

This animal study protocol was approved by the Ethics Committee of the Ministry of Agriculture, Forestry and Water Management–Veterinary Directorate (Approval No. 003573855/2024/1) on 30 December 2024.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials, further inquiries can be directed to the corresponding author.

Acknowledgments

The authors acknowledge their gratitude to the Ministry of Science, Technological Development and Innovation of the Republic of Serbia, contract number 451-03-136/2025-03/200003.

Conflicts of Interest

The authors declare no conflicts of interest.

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