4-Ortho-Mono-Fructosyl Phlorizin Reduces Sodium–Glucose Cotransporter 2 and Improves Metabolic Dysfunction in a High-Fat Diet/Streptozotocin-Induced Diabetic Mouse Model

1. Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from impaired insulin secretion, insulin resistance, or a combination of both mechanisms [1]. The disease is also associated with the progressive dysfunction of pancreatic β-cells, which contributes to the deterioration of glycemic control over time [1,2]. DM comprises several clinical categories, including type 1 diabetes, type 2 diabetes (T2DM), gestational diabetes, and less common forms such as monogenic diabetes and secondary diabetes. Among these, T2DM is the most prevalent, accounting for approximately 90% of all diabetes cases worldwide [1]. Over the past decade, the global burden of T2DM has increased dramatically, making it a major public health problem due to its strong association with long-term complications affecting the cardiovascular system, kidneys, liver, and overall metabolic homeostasis [3,4]. Currently, an estimated 589 million adults aged 20–79 years live with diabetes mellitus, representing approximately 11.1% of the global adult population, and this figure is projected to rise to nearly 852.5 million by 2050 [5].

The treatment for T2DM involves lifestyle changes, oral medications, and insulin therapy. Medications help to maintain blood sugar levels under control, but they can cause weight gain, risks of low blood sugar, and insufficient protection against cardiovascular, liver, and kidney complications, among others. This has led to the development of antidiabetic molecules that can regulate blood sugar levels while providing additional metabolic and cardiovascular benefits [6]. Sodium-glucose cotransporters (SGLTs), particularly SGLT1 and SGLT2, play a critical role in glucose homeostasis by mediating glucose reabsorption in the intestine and kidneys, respectively [7,8]. SGLT2, expressed in the proximal renal tubules, mediates ~90% of glucose reabsorption from tubular fluid; its inhibition blocks renal glucose reabsorption, increases urinary glucose excretion, and lowers blood glucose levels. Pharmacologically inhibiting these transporters has proven effective in improving glycemic control, leading to the development of selective SGLT2 inhibitors (gliflozins), which are now widely used in the clinical management of T2DM [8]. Recent studies have shown that SGLT-2 inhibitors exert beneficial effects on renal and cardiovascular function and display hepatoprotective properties, including reductions in hepatic steatosis and improvements in the lipid profile [9].

One of the first compounds identified as an SGLT inhibitor was phlorizin, a naturally occurring dihydrochalcone described in the 19th century as a dual SGLT1/SGLT2 inhibitor with marked antidiabetic activity [10,11]. However, despite its efficacy, its clinical application has been limited by its low water solubility, low oral bioavailability, and rapid metabolic degradation [12].

To overcome these pharmacokinetic limitations, 4-ortho-mono-fructosyl phlorizin (4PHF); a novel fructosylated derivative of phlorizin, was obtained via enzymatic biocatalysis. This structural modification was previously designed to improve phlorizin solubility and bioavailability [13,14], with the aim of retaining its known SGLT2 inhibitory activity [12].

In the context of T2DM, comprehensive evaluation of biochemical and histopathological parameters is essential to characterize the therapeutic potential of new antidiabetic agents [15]. Historically, T2DM treatment has primarily focused on lowering blood glucose levels. However, more recent therapeutic approaches have shifted toward addressing the underlying pathophysiological disturbances associated with the disease, particularly by reducing cardiorenal risk and promoting organ protection [9]. Furthermore, the liver and kidneys are key target organs in the pathophysiology of T2DM [16]. The liver plays a central role in glucose and lipid metabolism and is highly susceptible to metabolic dysregulation, while the kidneys are crucial in glucose reabsorption and are a primary target of diabetic complications such as nephropathy [2,17].

Therefore, in this study the effects of 4PHF were assessed in a murine model, emphasizing its potential benefits on metabolic abnormalities and the histological protection of the liver and kidneys. Furthermore, 4PHF modulation of SGLT2 renal tissue expression was accomplished to obtain mechanistic information of glucose reabsorption inhibition. Consequently, the present study aimed to evaluate the antidiabetic efficacy of 4PHF in an experimental diabetic mouse model induced by a high-fat diet (HFD) and streptozotocin (STZ) by assessing its effects on glycemia, urinary glucose excretion, lipid profile, hepatic and renal histopathology, and renal SGLT2 expression.

2. Materials and Methods

All reagents were obtained from Sigma-Aldrich-Merck (Darmstadt, Germany), unless otherwise specified.

2.1. Enzymatic Synthesis and Purification of 4-Ortho-Mono-Fructosyl Phlorizin (4PHF)

2.1.1. Enzymatic Synthesis and Purification

Biocatalysis was carried out following previously reported methodologies [13,18]. In brief, the reaction mixture contained fructosyltransferase from Paraburkholderia phymatum (KesL) (0.5 U/mL), kindly provided by the Institute of Biotechnology (IBT, Cuernavaca, Morelos, Mexico), sucrose (500 mM) as the fructosyl donor, and 99% pure phlorizin (25 mM) in acetate buffer (5 mM, pH 5.8) as the acceptor substrate. The final reaction volume was 500 mL. The reaction was incubated at 37 °C for 24 h and subsequently terminated by heating in a water bath, at 90 °C for 10 min.

2.1.2. HPLC-UV Analysis

After the reaction was stopped, phlorizin bioconversion was determined from the relative decrease in substrate concentration, quantified by high-performance liquid chromatography with ultraviolet detection (HPLC–UV), according to the following equation: Bioconversion (%) = [(C₀ − C_f)/C₀] × 100, where C₀ and C_f represent the initial and final phlorizin concentrations, respectively [14]. A solution of the lyophilized sample was prepared in water at a final concentration of 10 mg/L and analyzed using an Agilent Technologies (Santa Clara, CA, USA) Infinity 1260 HPLC system equipped with a Phenomenex (Torrance, CA, USA) Luna C18 column (5 μm, 100 Å, 150 mm × 4.6 mm) and an Agilent G4212 60007 UV detector (6 MPa). Chromatographic separation was achieved using a binary gradient of 0.05% formic acid in water (solvent A) and acetonitrile with 0.05% formic acid (solvent B) at a flow rate of 0.8 mL/min and a column temperature of 40 °C, gradient elution conditions: 90% A-10% B (min 0), 50% A-50% B (1-20 min), 5% A-95% B (20-25 min), 90% A-10% B (25-30 min). Total injection time was 30 min [14]. Analyses were performed in triplicate. Residual phlorizin was detected at 280 nm and quantified using an external calibration curve (0.05–1.0 mM; R² ≥ 0.99) prepared with a phlorizin standard.

2.1.3. UPLC-ESI-QTOF-MS/MS Analysis

The final reaction mixture was analyzed by ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC–ESI–QTOF–MS/MS) and subsequently purified by flash chromatography using a Flash Manager C-615 system (BÜCHI, Switzerland).

Chromatographic analysis of the reaction products was performed using a Waters Acquity UPLC H-Class system (Waters, Milford, MA, USA) equipped with a degasser, quaternary pump, autosampler, and column heater. Separation was achieved at 40 °C using Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) from Waters, with an injection volume of 10 μL. The mobile phase consisted of (A) water containing 0.3% (v/v) formic acid and (B) acetonitrile containing 0.3% (v/v) formic acid, which were delivered at a flow rate of 0.3 mL/min. The gradient program was as follows: 0–1 min, 95% A; 1–11 min, linear decrease from 95% to 30% A; 11–12 min, return to 95% A; and 12–15 min, re-equilibration at 95% A. Mass spectrometric analysis was conducted using a Acquity Xevo G2-XS QToF mass spectrometer from Waters, equipped with an electrospray ionization (ESI) source operating in negative ion mode. Spectra were acquired over a mass range of m/z 50–1000. The operating parameters were set as previously reported: capillary voltage 2.5 kV, cone voltage 40 V, source temperature 100 °C, desolvation temperature 250 °C, and collision energy 6 eV [19].

2.1.4. Flash Chromatography

The products obtained from the enzymatic reaction with the highest conversion were purified by flash chromatography using a Flash Manager C-615 system (BÜCHI, Switzerland), equipped with a dual A/B pumping system and coupled to a C-660 fraction collector (BÜCHI, Switzerland). The separation was performed on a FlashPure EcoFlex C18 column (20 g capacity). Before loading the sample, the column was equilibrated with 50 mL of Milli-Q ultrapure water at a flow rate of 3 mL/min for 5 min, followed by conditioning with 50 mL of 100 % acetonitrile under the same flow rate and time condition, then sugars were eluted with water during 15 min at 3 mL/min, followed by phenolic fructosides elution with a gradient from 0 to 20 % of acetonitrile (30 min at 3 mL/min), subsequently a gradient from 20 to 30 % of acetonitrile was applied (30 min at 3 mL/min).

The purification process was monitored by thin-layer chromatography (TLC). Chromatographic spots corresponding to UV-absorbing compounds, including 4PHF, were visualized under UV illumination at 280 nm. Carbohydrate-containing compounds were subsequently detected by derivatization with an orcinol-based reagent under acidic conditions, followed by heating for color development [19].

2.2. In Vivo Antidiabetic Activity of 4PHF

2.2.1. Ethical Approval and Animals

All experimental procedures were conducted in accordance with the guidelines of the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) (No. 2024-0C2). Animal handling followed the recommendations of the Official Mexican Standard NOM-062-ZOO-1999 [20].

Twenty-seven male CD1 mice (5 weeks old, 28 ± 4.76 g) were used in this study and obtained from the Bioterio Morelos (Morelos, Mexico). The animals were housed under controlled environmental conditions (12:12 h light–dark cycle, 22 °C, constant humidity) with free access to food and water.

2.2.2. HFD/STZ-Induced Experimental Diabetes Model

To induce DM a slightly modified HFD/STZ model was used [21]. Twenty-one mice were fed ad libitum with a HFD (4.6 kcal/g and 45% kcal from fat) acquired from Dyets, #102238 (Bethlehem, PA, USA) for 28 consecutive days. On day 29, a single intraperitoneal injection of STZ at a dose of 150 mg/kg body weight was performed to induce diabetes. STZ solution was freshly prepared in 0.1 M citrate buffer (pH 4.8) and administered following a 4 h fasting period. The overall mortality rate during diabetes induction was 15%. To reduce the risk of fatal hypoglycemia following STZ administration mice received 10% sucrose solution instead of drinking water for 24 h after STZ injection. Moreover, mortality rates above 20% have been proposed as indicative of excessive experimental severity, requiring implementation of additional animal welfare measures [22].

Hyperglycemia was confirmed 72 h after STZ administration by determining blood glucose levels from tail vein blood using a portable glucometer One Touch, Lifescan (Malvern, PA, USA). Animals with fasting blood glucose levels ≥150 mg/dL were considered diabetic [22]. Fasting blood glucose was subsequently monitored weekly for 28 days under 4 h fasting conditions.

The six remaining animals were included in the negative control group that received a standard diet (3.4 kcal/g, 14.1% energy from fat) that was obtained from Safe (Augy, France).

2.2.3. Experimental Design and Treatments

Diabetic mice were randomly assigned to four experimental groups (n = 6 per group). Mice in each experimental group were housed in polycarbonate cages of appropriate dimensions [20]: the negative control group comprising healthy mice (NC), the diabetic control group (DC) that were administered distilled water orally; the positive control group, comprising of diabetic control mice treated orally with dapagliflozin (10 mg/kg) (DAPA) (AstraZeneca®); and a group including diabetic mice treated orally with 4PHF (130 mg/kg). The dose was selected based on a previous study in which a medium dose of phlorizin (60 mg/kg) was administered to rats [23]. The equivalent mouse dose was calculated using the standard body surface area-based conversion factors [24]. Treatments were administered once daily by oral gavage for 28 consecutive days, as previously reported [23]. Body weight was recorded weekly throughout the experimental period. Food and water consumption were determined over a 24-h period by calculating the difference between the amounts provided and the respective amounts remaining.

2.2.4. Assessment of Urinary Glucose Excretion, Blood Sampling and Biochemical Analyses

On day 26 of treatment, mice were individually placed in metabolic cages and spontaneously eliminated urine was collected over a 6-hour period to assess glucose levels in the urine using the RANDOX glucose kit (Crumlin, UK) [25]. On day 29, following completion of the treatment period, fasting serum glucose was evaluated with a portable glucometer as described previously. Then, the animals were euthanized by cervical dislocation, and whole blood samples were collected and centrifuged at 1,500 ×g for 10 min at room temperature to obtain serum for biochemical analyses. Serum samples were frozen at -20°C and processed within 1 week. Serum levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) were quantified using commercial enzymatic kits from RANDOX, according to the manufacturer's instructions. All measurements were performed in duplicate [21,26]. Alterations in biochemical parameters have been previously described in HFD/STZ- induced diabetes model (26).

2.2.5. Histological Analysis of the Liver and Kidneys

Liver and kidney tissues were fixed in 4% paraformaldehyde, dehydrated through graded ethanol solutions (70%, 80%, 96%, and 100%), and embedded in paraffin blocks at 56–60 °C. Tissue sections (4 μm thick) were obtained using a microtome. Five liver sections per animal were stained with hematoxylin and eosin (H&E), whereas five kidney sections per animal were stained with periodic acid–Schiff (PAS) and counterstained with hematoxylin. Measurements obtained from the five sections were averaged to generate a single value per animal for each tissue. Histological evaluation was performed using a BestScope B2048T optical microscope (Beijing, China) equipped with an EP200 digital camera at magnifications of 400×.

Liver histopathology was evaluated using a semi-quantitative scoring system [27], where scores were assigned as follows: 0 (normal), 1 (mild alterations), 2 (moderate alterations), and 3 (severe alterations). The parameters assessed included hepatocellular degeneration, inflammatory infiltration, cytoplasmic vacuolization, vascular congestion, and sinusoidal alterations, and five H&E-stained sections per animal were analyzed.

Renal histopathology was assessed in PAS-stained sections using a semi-quantitative scoring system adapted from Fioretto and Mauer [28], scoring tubular atrophy, tubular dilation, PAS-positive tubular staining, glomerular damage, and inflammatory infiltration using the same 0–3 scale, with five renal sections analyzed per animal.

2.2.6. Immunohistochemical Detection of SGLT2 in Renal Cortex

Immunohistochemical analysis of SGLT2 expression was performed on five renal sections per animal following a previously described protocol [29], with minor modifications. Sections were deparaffinized, rehydrated through graded ethanol and xylol solutions (100, 90, 80, 70%), and incubated with a blocking solution containing 0.1 M PBS, 5% bovine serum albumin, and Triton X-100 0.5% for 2 h at 4 °C. They were then incubated overnight at 4 °C with a rabbit anti-SGLT2 primary antibody (ab37296) diluted at a 1:500 ratio obtained from ABCAM (Waltham, MA, USA).

After washing, the sections were incubated with a biotinylated goat anti-rabbit IgG (BA-100) from Vector Laboratories (Newark, CA, United States) for 2 h at room temperature, followed by incubation with the avidin–biotin–peroxidase complex (ABC, Vector Laboratories) for 1 h. Immunoreactivity was visualized using a 3,3′-diaminobenzidine (DAB) solution (0.05%) in the presence of hydrogen peroxide.

SGLT2 expression was quantified as the percentage of SGLT2-positive tubular area per field, following modified methods described by Wang et al. (2017) [30]. Five renal cortical fields per animal were analyzed using ImageJ software 2.17.0-0 (Bethesda, MD, USA). Following the evaluation of immunoreactivity, tissue sections were counterstained with PAS to improve visualization.

2.3. Statistical Analysis

All results are presented as mean ± standard error of the mean (SEM). Data were analyzed using one-way ANOVA for comparisons among independent groups and repeated-measures ANOVA for longitudinal data, using GraphPad Prism software (version 8.0.1). Post hoc comparisons were performed using Dunnett’s multiple -comparison test, p < 0.05 was considered statistically significant, and the DC group considered as the reference.

3. Results

3.1. Biocatalytic Synthesis, Purification, and Characterization of Fructosylated Phlorizin Derivatives

HPLC–UV analysis was used to determine phlorizin bioconversion at the end of the reaction and to assess the chromatographic purity of both the individual 4PHF-containing fractions and the final pooled preparation after lyophilization. The enzymatic reaction resulted in a bioconversion of 81.98 % as determined by HPLC-UV from the relative decrease in substrate concentration between the beginning and the end of the reaction.

UPLC–ESI–QTOF–MS/MS analysis of the final reaction mixture in negative-ion mode revealed multiple chromatographic peaks tentatively assigned to residual phlorizin and fructosylated phlorizin derivatives (Figure S1 and Table S1). As reported in Table S1, the peak at 8.83 min exhibited an ion at m/z 481.197, tentatively assigned to the formate adduct of phlorizin, [C₂₁H₂₄O₁₀ + HCOO]⁻. A peak at 7.94 min exhibited an ion at m/z 643.222, tentatively assigned to the formate adduct of monofructosylated phlorizin, [C₂₇H₃₄O₁₅ + HCOO]⁻. Additional peaks at 7.92 and 7.41 min, with ions at m/z 643.225 and 643.212, respectively, were tentatively associated with monofructosylated species. Their possible isomeric relationships require further structural characterization. Peaks at 7.51 and 6.82 min exhibited ions at m/z 805.249 and 805.253, respectively, provisionally assigned to the formate adducts of difructosylated phlorizin, [C₃₃H₄₄O₂₀ + HCOO]⁻. These preliminary assignments suggest the formation of mono- and difructosylated products during the enzymatic reaction.

The reaction mixture was subsequently purified by flash chromatography, and fractions 5–25 were collected. TLC analysis revealed UV-absorbing spots and positive staining with an orcinol-based reagent, supporting the presence of carbohydrate-containing compounds in these fractions (Figure S2). Fractions selected on the basis of their TLC profiles were further analyzed by HPLC–UV to assess their composition and chromatographic purity.

Fractions 5–25 were pooled, concentrated by rotary evaporation, and lyophilized to obtain the preparation administered to the animals. HPLC–UV analysis of the reconstituted lyophilized preparation at 280 nm revealed a predominant peak assigned to 4PHF at 12.7 min, a peak corresponding to residual phlorizin at 15.8 min, and additional peaks assigned to other fructosylated derivatives of phlorizin (Figure 1), the chemical structures of phlorizin and 4PHF are also included. The phlorizin standard exhibited a single sharp peak at approximately 15.8 min (Figure S3), whereas the analyzed fractions displayed a predominant peak at approximately 12.7 min, assigned to 4PHF. The structure of 4PHF was previously elucidated by nuclear magnetic resonance spectroscopy (18), and the corresponding nuclear magnetic resonance spectra are presented in Figure S4 and Figure S5. The relative peak areas were 94.50% for 4PHF, 2.11% for residual phlorizin, and 3.39% for the other identified fructosylated derivatives (Table 1). These percentages represent relative chromatographic peak areas rather than mass fractions. Thus, the preparation administered to the animals consisted predominantly of 4PHF, with residual phlorizin and minor amounts of other fructosylated derivatives.

3.2. Effects of 4PHF on Body Weight, Food Intake, and Water Consumption

Once diabetes was confirmed in male CD-1 mice showing fasting hyperglycemia (≥150 mg/dL), the treatments were initiated. The body weight of the NC group increased 20.58 % by study completion, whereas the DC group exhibited a reduction in mean body weight (−7.33 %) by study completion (Figure 2a). In contrast, the 4PHF-treated group showed a moderate decrease in body weight (−3.67 %), whereas mice in the DAPA group maintained relatively stable body weight, with a slight increase of 0.40 %. However, these differences were not statistically significant among the groups.

Water intake varied among the experimental groups (Figure 2b). The DC group had a daily average water intake of 31.32 ± 5.41 mL, which was apparently higher than that of the NC group (15.33 ± 0.52 mL); however, this difference did not reach statistical significance. Similarly, water intake in the 4PHF-treated group (18.58 ± 1.15 mL) and the DAPA-treated group (17.63 ± 1.98 mL) did not differ significantly from that of the DC group.

Average food intake was significantly higher in the DC group than in the NC group (76.31 ± 4.30 kcal/day vs. 52.32 ± 3.89 kcal/day, respectively; p < 0.01; Figure 2c). In contrast, food intake in the 4PHF- and DAPA-treated groups (71.02 ± 5.01 and 70.57 ± 4.30 kcal/day, respectively), did not differ significantly from that of the DC group.

3.3. Effects of 4PHF on Fasting Blood Glucose Levels

Weekly monitoring of fasting blood glucose levels revealed sustained hyperglycemia in the DC group throughout the experimental period, reaching approximately 460 mg/dL on day 28, corresponding to a modest reduction of 19.11% from baseline values. In contrast, the group treated with 4PHF exhibited a progressive reduction in blood glucose levels, reaching values ​​of approximately 315–325 mg/dL at the end of the study, which represents a 36.44% decrease (p < 0.05 vs. DC). Treatment with dapagliflozin resulted in a more pronounced hypoglycemic effect, with glucose levels of approximately 220–230 mg/dL by day 28, corresponding to a 56.68% reduction (p < 0.01 vs. DC) (Table 2).

3.4. Effects of 4PHF on Urinary Glucose Excretion

Urinary glucose levels differed significantly among the experimental groups (Figure 4). As expected, the NC group showed minimal glucose excretion, whereas the DC group exhibited a significant increase (p < 0.001), reaching approximately 70 mg/dL. In contrast, treatment with 4PHF resulted in a significant increase in urinary glucose excretion, reaching values ​​of approximately 190 mg/dL (p < 0.001 vs. DC). A more pronounced renal glucose excretion was observed in the dapagliflozin-treated group, which showed urinary glucose levels of approximately 270 mg/dL (p < 0.001 vs. DC), which is significantly higher than those observed in the 4PHF-treated group (p < 0.001).

3.5. Effects of 4PHF on Lipid Profile

Serum lipid profile analysis revealed significant differences between the diabetic DC group and the treated groups (Figure 5). Specifically, the DC group showed a significant increase in total cholesterol levels when compared to the NC group (205.16 ± 3.86 mg/dL vs. 73.27 ± 1.36 mg/dL; p < 0.001), respectively, while treatment with 4PHF significantly reduced them to 93.28 ± 4.84 mg/dL compared to DC, interestingly cholesterol levels in 4PHF-treated group and NC group were similar. The DAPA group showed a similar response (p < 0.001 vs. DC).

In addition, serum triglyceride concentrations were also significantly elevated (p < 0.01) in DC mice at 191.5 ± 14.18 mg/dL when compared to the NC group (105.17 ±10.23). 4PHF treatment significantly reduced triglyceride concentrations compared to DC (91.83 ± 8.70 vs. 191.5 ± 14.18; p < 0.001). Similarly, the DAPA group exhibited lower triglyceride levels (p < 0.001 vs. DC).

Regarding HDL, the DC group exhibited a significant decrease (p < 0.05) vs. NC group, 36.93 ± 2.97 mg/dL and 41.36 ± 0.57 mg/dL, respectively. The HDL levels were significantly increased in the 4PHF-treated group (53.39 ± 3.58 mg/dL) compared to the DC group (36.93 ± 2.97 mg/dL) (p < 0.001). Likewise, the DAPA group also showed higher HDL levels (p < 0.001 vs. DC). Furthermore, the LDL levels of the DC group were higher than NC (64.44 ± 2.61 mg/dL and 44.03 mg/dL ± 2.46, respectively; p < 0.01). The 4PHF group significantly reduced LDL levels (42.76 ± 2.80 mg/dL) relative to the DC animals (64.44 ± 2.61 mg/dL) (p < 0.001), and this effect was also observed in the DAPA group (p < 0.001 vs. DC).

3.6. Liver Weight and Hepatic Histopathological Evaluation

Liver weight did not differ significantly among the groups (Figure 6a). However, the liver weights of the 4PHF and DAPA-treated groups were numerically closer to those of the NC group than those of the DC group.

Histological examination of liver sections stained with hematoxylin and eosin (H&E) revealed significant pathological alterations in the DC group in contrast to NC group, including extensive vacuolization, vascular congestion, and hepatic architecture disruption (Figure 7). In contrast, both DAPA- and 4PHF-treated groups exhibited preserved hepatic morphology, with reduced structural alterations and more organized hepatic parenchyma relative to the DC group. Semi-quantitative analysis demonstrated significant reductions in vacuolization, vascular congestion, hepatocellular degeneration, inflammatory infiltration, and sinusoidal alterations in the 4PHF group when compared to the DC group (p < 0.05 to p < 0.001). The DAPA group also showed significant improvements (p < 0.01 to p < 0.001) when compared to DC animals. Notably, 4PHF showed effects comparable to DAPA across all evaluated parameters.

3.7. Renal Weight and Histopathological Evaluation

The weight of the left and right kidneys did not differ significantly among the groups as previously shown in Figure 6b and Figure 6c. However, the left kidney weight of the 4PHF group was numerically closer to those of the NC group than those of the DC group.

PAS-stained renal sections from the DC group revealed significant differences compared with the NC group (p < 0.01 to p < 0.001) with respect to tubular and glomerular alterations, including tubular dilation, accumulation of PAS-positive material, glomerular damage, inflammatory infiltration, and tubular atrophy (Figure 8). Treatment with 4PHF significantly attenuated these pathological features, with semi-quantitative scoring indicating significant reductions in tubular dilation (p < 0.01), PAS-positive staining (p < 0.001), glomerular damage (p < 0.01), inflammation/infiltration (p < 0.001), and tubular atrophy (p < 0.001) compared to DC animals. The magnitude of renal protection observed in the 4PHF-treated group was comparable to that of the DAPA group.

3.8. Effects of 4PHF on Renal SGLT2 Expression

Immunohistochemical analysis revealed significantly higher and intense SGLT2 immunoreactivity in the renal cortex of DC mice, with widespread staining in proximal tubules (Figure 9) relative to the NC group. In contrast, the group exhibited a marked reduction in SGLT2 staining intensity, and quantitative analysis demonstrated a significant decrease in SGLT2-positive area in 4PHF-treated animals compared to the NC group (p < 0.001). The 4PHF treatment reduced SGLT2 expression to approximately 7%, while treatment with DAPA reduced it further to about 5%.

The number of SGLT2-positive tubules per field in the 4PHF-treated group showed an intermediate reduction that did not reach statistical significance, whereas DAPA treatment reduced them when compared to NC mice (p < 0.01).

4. Discussion

The present study employed an integrated strategy combining enzymatic biocatalysis for the generation of fructosylated phlorizin derivatives with subsequent biological evaluation in an experimental diabetic mouse model induced by HFD and STZ. The biocatalytic results demonstrate that the KesL enzyme efficiently catalyzed fructosyl transfer reactions, generating a mixture of mono-, di-, and higher-order fructosylated derivatives as shown previously [18]. The formation of multiple fructosylated species is consistent with the transfructosylation activity reported for fructosyltransferases [13,31] and reflects the capacity of KesL to utilize phlorizin as an acceptor substrate. Although heterogeneous, the resulting product mixture was effectively enriched by flash chromatography, thereby allowing subsequent biological evaluation. The chromatographic complexity observed for the fructosylated products compared with the precursor compound further supports successful enzymatic modification [31]. The presence of a predominant derivative suggests preferential fructosylation at specific hydroxyl positions, consistent with previous reports describing regioselective enzymatic fructosylation of polyphenolic compounds [32]. The predominant fructosylated fraction, designated as 4PHF, represents a structurally modified derivative of phlorizin with potentially improved biological properties.

Fructosylation can modify the physicochemical properties of phenolic compounds relative to their native forms. In the present study, the enzymatic reaction achieved a phlorizin bioconversion of 81.98 %, reflecting substrate consumption rather than the specific yield of 4PHF. Following purification, the resulting preparation exhibited a chromatographic purity of 94.5% for 4PHF, compared with approximately 74.5% 4PHF in the enriched fraction reported in our previous work [18]. The higher purity achieved in this study was attributed to optimization of the purification and enrichment procedure. Nevertheless, further optimization of the reaction conditions is needed to minimize the formation of secondary products.

Comparable enzymatic transfructosylation efficiencies have been reported in the literature; for instance, levansucrase-catalyzed reactions using different bacterial systems achieved maximum yields of 58% with Vibrio natriegens LS2, 36% with Novosphingobium aromaticivorans LS3, and 18% with Pseudomonas graminis LS4 [33]. Other modifications such as galactosylation of several phenolic compounds, including phlorizin via permeabilized cells from Pantoea anthophila have been reported to reach conversion yields of up to 41.8% [19]. These studies highlight the versatility of enzymatic glycosylation strategies for modifying phenolic compound structures, and other studies have demonstrated that the versatility of enzymatic fructosylation of phenolic acceptors is highly dependent on the catalytic system employed and the reaction conditions [31].

4PHF has been reported to exhibit activity against key therapeutic targets implicated in T2DM pathogenesis, including dipeptidyl peptidase-4 and the insulin receptor [34]. More recently, our research group demonstrated that 4PHF inhibits α-amylase in vitro assays, while in silico analyses suggested a slightly stronger predicted binding affinity toward α-glucosidase than toward α-amylase [18]. The organoprotective effects observed in the present animal study may therefore be associated with the biological activities previously reported in vitro and in silico. It is possible to affirm that 4PHF has a dual effect by inhibiting enzymes responsible for carbohydrate digestion, and the prevention of renal glucose reabsorption. Although the precise mechanisms remain to be elucidated, our findings collectively support the potential of modified phlorizin derivatives such as 4PHF to exhibit improved biological properties. It has been reported that some chalcone derivatives show better biological activities [35], particularly antidiabetic activity, as determined by a method quantifying glucose consumption by adipocytes [36].

The present study demonstrates that the fructosylated phlorizin derivative 4PHF exerts a multifaceted pharmacological profile in an experimental diabetic mouse model induced by HFD and STZ. Although its antihyperglycemic activity was moderate compared with dapagliflozin, treatment consistently improved fasting blood glucose while producing marked increases in urinary glucose excretion, significant improvements in lipid metabolism, attenuation of hepatic and renal injury, and reduced renal SGLT2 expression. These findings highlight the functional relevance of the structural modification achieved through fructosylation and suggest that the biological activity of 4PHF extends beyond glucose lowering alone.

The partial preservation of body weight observed in 4PHF-treated animals is consistent with improved metabolic stability under chronic hyperglycemic conditions. In uncontrolled T2DM, persistent hyperglycemia promotes proteolysis and lipolysis, leading to progressive weight loss [37]. The ability of 4PHF to attenuate this effect without altering food intake suggests an improvement in systemic metabolic efficiency rather than appetite suppression, and similar neutral effects on food intake have been reported following phlorizin administration in diabetic rodent models [38].

Dyslipidemia is a central metabolic abnormality in DM and contributes significantly to cardiovascular risk and the progression of diabetic complications [39]. In the present study, 4PHF significantly reduced serum total cholesterol, triglycerides, and LDL concentrations while increasing HDL levels, indicating an overall improvement in metabolic homeostasis. These changes are particularly noteworthy because the lipid profile achieved after 4PHF treatment approached that reported for healthy male ICR (CD-1) mice, especially for total cholesterol, while triglyceride concentrations were markedly reduced and HDL levels were partially restored [40]. Although direct comparisons between independent studies should be interpreted cautiously due to differences in experimental protocols, these findings suggest that 4PHF effectively counteracts diabetes-associated dyslipidemia and promotes the recovery of a near-physiological lipid profile. Similar lipid-lowering effects have previously been reported for phlorizin and related flavonoids in diabetic models [23]. Improvements in lipid homeostasis have been associated with reduced glucotoxicity and enhanced insulin sensitivity, which contribute to normalization of hepatic lipid metabolism [41,42]. Furthermore, Zhang et al. demonstrated that phlorizin administration in HFD/STZ-induced diabetic rats significantly reduced fasting blood glucose, total cholesterol, triglycerides, and LDL-C while increasing HDL-C, accompanied by attenuation of hepatic lipid accumulation and restoration of pancreatic morphology, emphasizing that simultaneous correction of hyperglycemia and dyslipidemia is essential for limiting diabetes progression [23].

Interestingly, the favorable biochemical profile observed after 4PHF treatment was accompanied by a significant increase in urinary glucose excretion. Under diabetic conditions, chronic hyperglycemia induces adaptive SGLT2 overexpression in proximal tubular cells, thereby enhancing renal glucose reabsorption and perpetuating metabolic imbalance [12,43]. The ability of 4PHF to promote urinary glucose excretion suggests that the inhibitory activity of its phlorizin precursor is preserved [11], although with lower potency than the selective SGLT2 inhibitor dapagliflozin. Additionally, metabolic improvements associated with SGLT2 inhibition have been reported by Tahara et al., who demonstrated that a high dose of ipragliflozin not only improved hyperglycemia but also significantly reduced serum triglyceride levels from 194 ± 5.9 mg/dL in untreated HFD/STZ-induced diabetic mice to 128 ± 11 mg/dL in the treated group. In the present study, serum triglyceride levels in diabetic mice were comparable (191.5 ± 5.8 mg/dL); however, treatment with 4PHF resulted in an even greater reduction, reaching 91.8 ± 3.5 mg/dL, which is lower than the levels reported for ipragliflozin in the aforementioned study. A similar trend was observed for total plasma cholesterol. Tahara et al. reported that untreated diabetic mice exhibited total plasma cholesterol levels of 146 ± 8 mg/dL, which decreased to 117 ± 5 mg/dL following treatment with a high dose of ipragliflozin. Likewise, in the present study, untreated diabetic mice exhibited elevated total cholesterol levels (205.2 ± 3.8 mg/dL), which were markedly reduced to 93.28 ± 2.0 mg/dL following 4PHF treatment [26]. Our findings are consistent with those reported by Tahara et al. (2013), supporting the notion that SGLT2 inhibition by phlorizin derivatives contributes to the improvement of diabetes- and obesity-associated metabolic abnormalities.

Importantly, the renal histopathological improvements observed following 4PHF treatment appeared proportionally greater than the reduction in fasting blood glucose, suggesting that its renoprotective activity may not be exclusively explained by glycemic control. The combined administration of HFD and STZ has previously been reported to induce pronounced structural alterations in the kidneys [44]. Tubular dilation, PAS-positive material accumulation, inflammatory cell infiltration, and tubular atrophy are hallmarks of early diabetic nephropathy [45]. The significant attenuation of these lesions indicates that 4PHF confers substantial renal protection. Previous studies have also demonstrated that phlorizin attenuates renal injury and oxidative stress in experimental diabetes [46]. However, because oxidative stress biomarkers and inflammatory cytokines were not evaluated in the present study, the contribution of these mechanisms remains speculative and should be investigated in future studies.

Hepatic protection represents another important finding of this work. T2DM-associated liver injury is driven by glucotoxicity, lipid accumulation, oxidative stress, and inflammatory activation [47]. The significant reduction in hepatocellular degeneration, vacuolization, vascular congestion, and inflammatory cell infiltration observed after 4PHF administration suggests improved hepatic metabolic homeostasis. Together with the favorable changes in serum lipid profile, these observations are consistent with previous reports describing hepatoprotective effects of phlorizin and flavonoid-derived compounds [23], as well as with the improvements in hepatic steatosis reported for both phlorizin and SGLT2 inhibitors in experimental diabetes.

Immunohistochemical analysis further demonstrated that 4PHF significantly reduced renal SGLT2 overexpression, a pathological adaptation commonly observed in diabetic complications [30]. While dapagliflozin acts as a potent and selective SGLT2 inhibitor, 4PHF appeared to moderately reduce SGLT2 expression without complete suppression. This regulatory profile may partially explain the moderate antihyperglycemic effect associated with substantial metabolic and tissue protection observed in the present study.

Because several molecular targets have already been identified for phlorizin and related dihydrochalcones [48], additional studies evaluating oxidative stress and inflammatory mediators, and other signaling pathways will be required to fully elucidate the mechanism of action of 4PHF. On the other hand, the hypoglycemic activity of SGLT2 inhibitors is largely independent of pancreatic insulin secretion [42]. A previous study evaluated the effects of phlorizin on insulin-stimulated glucose uptake in MKR mice and reported no improvement [49]. In the present study, circulating insulin concentrations were not measured. However, the assessment of serum insulin levels together with insulin resistance would provide valuable insight into the metabolic effects of phlorizin derivative 4PHF in the treatment of experimental diabetes.

A previous study reported that male mice are more frequently used than female mice in experimental diabetes models [50]. Consistent with this trend, the present study included only male mice, highlighting the need for future studies to incorporate female mice to address sex-related differences in diabetes and treatment response.

5. Conclusions

The biocatalytic synthesis and purification of 4PHF was successfully achieved, and its biological activity was demonstrated in an experimental diabetic mouse model induced by HFD and STZ. Treatment with 4PHF significantly improved dyslipidemia, increased urinary glucose excretion, and diabetes-associated liver and kidney damage when compared to diabetic control group. Furthermore, 4PHF emerges as a promising therapeutic candidate for diabetes mellitus by reducing renal SGLT2 expression, improving metabolic dysfunction, and attenuating diabetes-associated hepatic and renal injury. These findings provide a solid experimental basis for future studies aimed at elucidating the underlying molecular mechanisms of 4PHF and evaluating its potential to prevent diabetes-related complications.