Synergistic inhibition of α‐glucosidase and α‐amylase by phenolic compounds isolated from olive oil by‐products

Abstract

BACKGROUND

The inhibition of carbohydrate‐digesting enzymes, α‐glucosidase and α‐amylase, is a key strategy in managing postprandial hyperglycemia in type 2 diabetes. Natural phenolic compounds isolated from olive mill by‐product (alperujo), such as hydroxytyrosol (HT) and trans‐p‐coumaroyl secologanoside (comselogoside, COM), have shown potential in this regard. However, the individual effect of the 3,4‐dihydroxyphenylglycol (DHPG), another potent phenolic antioxidant naturally found in olive fruit with the same ortho‐diphenolic structure as HT, but with an additional hydroxyl group in the β position, and their combination with HT and COM on these enzymes remain underexplored.

RESULTS

The present study evaluated the inhibitory effects of HT, DHPG and COM on α‐glucosidase and α‐amylase. DHPG exhibited the strongest inhibition of α‐glucosidase (IC₅₀ = 288.2 μm), comparable to the anti‐diabetic drug acarbose (IC₅₀ = 281.9 μm), whereas HT and COM showed moderate activity. Binary combinations of HT and DHPG (1:1) displayed synergistic inhibition of both enzymes, significantly enhancing their efficacy. Conversely, DHPG and COM combinations resulted in antagonistic effects on α‐amylase inhibition, although they showed a significant inhibitory effect on α‐glucosidase (IC₅₀ = 100.7 μm).

CONCLUSION

These findings highlight the potential of phenolic compounds derived from olive oil by‐products as natural alternatives for postprandial glucose control. The synergistic interactions between specific compounds suggest an enhanced therapeutic effect that could be used for diabetes management. Further studies are needed to elucidate the mechanisms underlying these interactions and assess their in vivo efficacy. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and pancreatic beta‐cell dysfunction, leading to persistent hyperglycemia. Its global prevalence is rising, largely driven by obesity and sedentary lifestyles, and it is associated with severe complications such as cardiovascular diseases, neuropathies and nephropathies. ¹ , ² Current management strategies primarily involve lifestyle modifications and hypoglycemic agents, including α‐glucosidase and α‐amylase inhibitors (e.g. acarbose), which delay carbohydrate digestion and reduce postprandial glycemic peaks. ³

However, the prolonged use of these drugs can cause adverse effects such as gastrointestinal discomfort, highlighting the need for safer and more effective natural alternatives. In this context, phenolic compounds derived from olive oil by‐products, such as hydroxytyrosol (HT) and oleuropein have gained prominence because of their antioxidant and inhibitory effects on carbohydrates digestive enzymes. ⁴ More recently, other phenolic compound isolated from olive mill waste, the trans‐p‐coumaroyl secologanoside (comselogoside), have shown potential inhibition of α‐glucosidase and α‐amylase. ⁵ However, because the synergistic action of various compounds often enhances efficacy in extracts compared to isolated compounds, the potential effects of hydroxytyrosol (HT) and comselogoside (COM), along with 3,4‐dihydroxyphenylglycol (DHPG), comprising another abundant phenolic compound in olives, remain largely unexplored.

The present study aims to evaluate the inhibitory activity of HT, DHPG and COM, both individually and in combination, on α‐glucosidase and α‐amylase to determine potential synergistic effects. Although HT and COM have been previously studied by other researchers for their inhibitors effect on these enzymes, DHPG has not yet been explored in this context. A synergistic approach could enhance therapeutic efficacy, reduce the required doses and minimize potential adverse effects, providing a promising natural alternative for T2DM management.

MATERIALS AND METHODS

Chemicals

α‐glucosidase from Saccharomyces cerevisiae, porcine pancreatic α‐amylase, 4‐nitrophenyl β‐d‐glucopyranoside, starch from potato, 3,5‐dinitrosalicylic acid, potassium sodium tartrate and acarbose were purchased from Sigma‐Aldrich Química S.A. (Madrid, Spain). Alperujo (olive mill solid by‐product resulting from the biphasic extraction of olive oil) was provided by an experimental oil mill in the Instituto de la Grasa (CSIC) (Seville, Spain).

Preparation of olive ethanol extract from alperujo

Alperujo (500 g) was extracted with 1 L of 50% ethanol twice at 80 °C for 60 min. After filtration using filter paper, the ethanolic extract was concentrated and eliminate the ethanol under vacuum by rotary evaporation to a final volume of 200 mL. The resulting extract was defatted using 200 mL of n‐hexane twice to remove all possible oil traces. The trace of n‐hexane present in the aqueous fraction was eliminated by evaporation under vacuum. Samples were stored at 4 °C until analysis.

Isolation HT, DHPG and COM from olive extracts

HT and DHPG were isolated and purified using a patent chromatographic system process described by Fernández‐Bolaños et al. ⁶ to obtain HT (7.73 mg mL⁻¹) and DHPG (13.57 mg mL⁻¹) with a purity of 95% and 90%, respectively, in water.

To isolation of COM the defatted ethanolic extract was fractionated using Amberlite XAD‐16 (25 × 3 cm) (Sigma‐Aldrich, St Louis, MO, USA) chromatography followed by a separation on a Sephadex LH‐20 (18 × 2 cm) (Cytiva, Marlborough, MA, USA) chromatography to give a fraction of COM 8.93 mg mL⁻¹ with a purity of 95% in MeOH 50%. ⁷ Purified compounds were stored at 4 °C until analysis.

In vitro α‐glucosidase inhibitory activity

The α‐glucosidase inhibitory activity was assessed using a spectrophotometry method, adapted from the procedure reported by Kim et al. ⁸ with minor modifications. The assay as performed in triplicate, where 20 μL of the sample (HT, DHPG, COM and mixtures of these compounds) at varying concentrations was added to a 96‐well microplate. Following this, 120 μL of 100 mm phosphate buffer (pH 6.8) and 60 μL of 5 mm 4‐nitrophenyl‐α‐d‐glucopyranoside (pNPG) in phosphate buffer were added to each well (A ₁). A positive control (A ₀) was prepared for each assay, consisting of 120 μL of phosphate buffer, 20 μL of 11.5% DMSO and 60 μL of pNPG. The mixtures were incubated at 37 °C for 5 min. Then, 30 μL of α‐glucosidase enzyme from S. cerevisiae (G5003; Sigma‐Aldrich; 0.1 U mL⁻¹ in phosphate buffer) was added. For each sample, a reaction blank (A ₂) was prepared without the enzyme. The absorbance was measured at 400 nm at the start of the reaction (t = 0) and again after 30 min of incubation at 37 °C (t = 30). The absorbance values for A ₀, A ₁ and A ₂ were calculated by subtracting the absorbance at t = 0 from that at t = 30. The percentage inhibition of the enzyme activity was calculated using:

$$\text{Inhibitory percentage}\left(\%\right)=\left(1-\frac{A_1-A_2}{A_0}\right)\times 100$$

The concentrations of HT, DHPG, COM, their mixtures (at 1:1 and 10:1 ratios) and acarbose (as the positive control) that produced 50% inhibition of enzyme activity (IC₅₀) were determined by plotting the inhibition percentage against inhibitory concentration (mg mL⁻¹). The IC₅₀ values were obtained through logarithmic regression analysis, based on the average of three independent experiments.

In vitro α‐amylase inhibitory activity

The inhibitory activity of porcine pancreatic α‐amylase was evaluated using a spectrophotometry method adapted from the procedure reported by Adisakwattana et al. ⁹ with slight modifications. The assay was performed in triplicate, where 25 μL of the sample (HT, DHPG, COM and mixtures of these compounds) at varying concentrations and 25 μL of the enzyme solution (1 mg mL⁻¹ of α‐amylase from Bacilus subtilis 165 BAU g⁻¹; MP Biomedical, Santa Ana, CA, USA) were added to wells of a 96‐well microplate (A ₃). A negative control (A ₁) was prepared for each assay, consisting of 25 μL of phosphate buffer and 25 μL of the enzyme to represent 100% activity. Blanks without the enzyme were prepared for both the samples (A ₄) and the negative control (A ₂). The mixtures were incubated at room temperature (25 °C) for 10 min. Following this, 25 μL of 0.5% (w/v) starch solution in 1 m phosphate buffer (pH 6.9) was added, and the plate was incubated again at 25 °C for 10 min. ¹⁰ , ¹¹ After the incubation, 50 μL of dinitrosalicylic acid (DNS) reagent were added, and the plate was heated in a water bath at 100 °C for 5 min. Once the plate was cooled, the absorbance was measured at 540 nm using a Multiskan FC spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The percentage of α‐amylase inhibition was calculated using:

$$I\left(\%\right)=1–\frac{\left(A_1-A_2\right)}{\left(A_3-A_4\right)}$$

The concentration of HT, DHPG, COM and acarbose required to inhibit the activity of the enzyme by 50% (IC₅₀) was calculated by lineal regression analysis.

Interaction and statistical analysis

The phenolic compounds (HT, DHPG and COM) were tested in binary combinations at various concentrations in both the α‐glucosidase and α‐amylase inhibition assays. The interaction between the phenolic compounds was evaluated by comparing the experimental inhibitory activity with the theoretical (calculated) values, using the following equation adapted from Skroza, et al. ¹² :

$$\text{Difference}\left(\%\right)=\left(\frac{\text{Combination}\mathrm{ab}\times 100}{\text{Individual}\mathrm{a}+\text{Individual}\mathrm{b}}\right)-100$$

where ‘combination ab’ is the experimentally measured inhibitory activity of the phenolic mixture, and ‘Individual a’ and ‘Individual b’ are the inhibition values of each compound tested individually.

If the experimentally measured value of the mixture exceeds the theoretical value [Difference (%) > 1], this suggests a synergistic interaction between the phenolic compounds. If the Difference (%) < 1, this indicates an antagonistic effect. A Difference (%) close to 0 suggests no interaction between the compounds.

The combination index (CI) was determined based on the median‐effect principle, as developed by Chou and Talalay ¹³ using CompuSyn (https://www.combosyn.com). The CI equation is:

$$\mathrm{CI}=\frac{\mathrm{Da}}{\mathrm{Dam}}+\frac{\mathrm{Db}}{\mathrm{Dbm}}$$

where Da and Db represent the doses of inhibitors ‘a’ and ‘b’ in the combination, whereas Dam and Dbm are the doses of each inhibitor individually required to achieve the same level of inhibition (IC₅₀). The fractional inhibition of each compound in the combination was calculated as:

$$\mathit{fa}=\frac{{\mathit{IC}}_{50\mathrm{a}}}{{\mathit{IC}}_{50\mathrm{a}}+{\mathit{IC}}_{50\mathrm{b}}}\mathit{fb}=\frac{{\mathit{IC}}_{50\mathrm{b}}}{{\mathit{IC}}_{50\mathrm{a}}+{\mathit{IC}}_{50\mathrm{b}}}$$

and the doses of inhibitors ‘a’ and ‘b’ in the combinations were calculated as:

$$\mathit{Da}=\mathit{fa}•{\mathit{IC}}_{50\mathrm{a}}\mathit{Db}=\mathit{fb}•{\mathit{IC}}_{50\mathrm{b}}$$

And, finally, CI was determined using:

$$\mathrm{CI}=\frac{\mathit{Da}}{{\mathit{IC}}_{50\mathrm{a}}}+\frac{\mathit{Db}}{{\mathit{IC}}_{50\mathrm{b}}}$$

Based on the CI value, the interaction was classified as synergistic (CI < 1), additive (CI = 1), or antagonistic (CI > 1).

The dose reduction index (DRI) indicates how much the doses of each compound in the combination can be reduced at the same time as maintaining efficacy. ¹⁴ The DRI was calculated using the following formula:

$$\text{DRIa}=\frac{\mathit{IC}50a}{\mathrm{Dab}}\text{DRIb}=\frac{\mathit{IC}50b}{\mathrm{Dab}}$$

where IC50a and IC50b are the IC₅₀ values of compounds ‘a’ and ‘b’ individually, and Dab represents the doses of compounds ‘a’ and ‘b’ in the combinations.

A DRI value greater than 1 (DRI > 1) indicates a favourable dose reduction with retained efficacy, suggesting synergism. A DRI of 1 indicates that the dose in the combination is the same as when administered individually (additive effect), whereas a DRI less than 1 (DRI < 1) suggests that higher doses are required to achieve the same effect, indicating antagonism.

All experiment were conducted in triplicate and the data are expressed as the mean ± standard deviation (SD).

RESULTS

In vitro inhibition of α‐glucosidase and α‐amylase

To evaluate the in vitro anti‐diabetic activity of the most abundant phenolic molecules in olive fruits (HT, DHPG and COM), the inhibitory effects on α‐glucosidase and α‐amylase were assessed and compared with acarbose, a synthetic drug commonly used in the management of type‐2 diabetes, which served as the positive control for both enzymes. As shown in Fig. 1, all three phenolic compounds, along with acarbose, inhibited both enzymes in a concentration‐dependent manner within the tested ranges: 10–1300 μg mL⁻¹ for α‐glucosidase and 2–140 μg mL⁻¹ for α‐amylase. The regression equations and the R ² values for α‐glucosidase (logarithmic) and α‐amylase (linear) are also provided.

Figure 1.

Concentration‐dependent inhibition of α‐glucosidase (a) and α‐amylase (b) by hydroxytyrosol (HT), 3,4‐dihydroxyphenylglycol (DHPG) and comselogoside (COM) isolated from olive mill by‐product (alperujo), and acarbose (positive control). The logarithmic and lineal equations, along with their respective R ² values, are shown for α‐glucosidase and α‐amylase inhibition, respectively. Error bar represent the SD of the mean from three independent experiments, each performed in triplicate.

Previous studies have demonstrated that HT is an effective inhibitor of α‐glucosidase from S. cerevisiae and a mild inhibitor of porcine pancreatic α‐amylase. ⁴ , ¹⁵ By contrast, COM has shown only weak to moderate activity against α‐glucosidase. ⁵ However, to the best of our knowledge, this is the first study to investigate the inhibitory effects of COM on α‐amylase and of DHPG on both α‐glucosidase and α‐amylase.

The results were expressed as IC₅₀ values (Table 1), representing the inhibitory potency of the tested compounds.

Table 1.

Inhibition activities (IC₅₀ values in μg mL⁻¹ and μm) of α‐glucosidase and α – amylase by HT, DHPG and COM isolated from alperujo, compared with acarbose (positive control)

	Inhibitory α‐glucosidase		Inhibitory α‐amylase
	IC50 (μg mL−1)	μm	IC50 (μg mL−1)	μm
Hydroxytyrosol (HT)	192 ± 5	1244 ± 32	171.7 ± 39.5	1115.0 ± 53.4
3,4‐Dihydroxyphenylglycol (DHPG)	48.9 ± 1.8	286 ± 11	70.13 ± 2.60	412.5 ± 15.2
Comselogoside (COM)	655 ± 36	1221 ± 67	338.2 ± 34.4	631.0 ± 64.1
Acarbose	186 ± 40	288 ± 62	3.77 ± 8.70	5.84 ± 1.08

p‐NPG and starch were used as substrates for the α‐glucosidase and αamylase assays, respectively.

Note: IC₅₀, concentration causing 50% inhibition. Results are expressed as the mean ± SD in triplicate.

In this study, the α‐glucosidase inhibitory activities of HT and COM were found to be weak to moderate, with IC₅₀ values approximately four times higher than that of acarbose. Only DHPG exhibited inhibitory activity comparable to acarbose. It is important to note that IC₅₀ values for pure compounds are expressed in micromolar (μM) to allow for accurate comparisons of molecular potency, especially when the compounds differ significantly in molecular weight.

For α‐amylase inhibition, the IC₅₀ values of all three compounds exceeded 400 μm, whereas acarbose showed a much lower value of 5.8 μm. The data suggest that, although HT inhibits α‐glucosidase and α‐amylase similarly, DHPG has a stronger effect on α‐glucosidase inhibition than on α‐amylase and COM inhibits α‐amylase more effectively than α‐glucosidase. These findings do not match the results published by Hadrich et al. ⁴ and Dekdouk et al., ¹⁵ who reported that HT is a potent inhibitor of α‐glucosidase, with inhibition levels similar to acarbose in the study by Hadrich et al. ⁴ and lower in the study by Dekdouk et al. ¹⁵ . However, our results on HT are consistent with those of Mwakalukwa et al., ¹⁶ who reported an IC₅₀ > 1000 μg mL⁻¹ for α‐glucosidase and IC₅₀ > 500 μg mL⁻¹ for α‐amylase, with acarbose showing values similar to those found in our study.

Interaction analysis of α‐glucosidase and α‐amylase inhibition of binary mixtures

The interactions between HT and DHPG in equimolar (1:1) and 10:1 mixtures, as well as HT and COM in 10:1 and 1:1 ratios, and DHPG and COM (1:1), were evaluated. A 10:1 ratio was initially selected to reflect the natural proportion of these phenolic compounds in olives, where the amount of HT is typically 10 times higher than that of DHFG and COM.

To draw any conclusion about potential interaction within the binary mixture of phenolic compounds, the CI and DRI were determined for α‐glucosidase and α‐amylase inhibition using CompuSyn, ¹¹ a system widely used for the evaluation of drug combination. The CI quantitatively assesses the combined effect to determine whether the mixture provides more or less inhibition than the individual components. DRI indicates how much the doses of each compound in the combination can be reduced without compromising effectiveness.

For α‐glucosidase, all the tested combination, HT:DHPG (1:1), HT:COM (10:1 and 1:1) and DHPG:COM (1:1), significantly enhanced inhibition compared to the individual compounds, based on the median‐effect equation with their IC₅₀ (Table 2).

Table 2.

Inhibition activities (IC₅₀ values in μg mL⁻¹ and μm) of α‐glucosidase and αamylase by the mixture of HT, DHPG and COM on α‐glucosidase and α‐amylase

	IC50 (μg mL−1)	μm	CI	DRI	Effect
α‐glucosidase
HT and DHPG (10:1)	137 ± 2	882 ± 13	1.15	1.6:3.6	Additive
HT and DHPG (1:1)	44.6 ± 3.8	278 ± 25	0.36	8.9:2.1	Synergism
HT and COM (10:1)	77.7 ± 17.7	474 ± 109	0.38	2.9:28.4	Synergism
HT and COM (1:1)	96.1 ± 8.6	401 ± 38	0.32	6.2:6.1	Synergism
DHPG and COM (1:1)	26.30 ± 1.04	101 ± 4	0.13	5.7:24.3	Synergism
α‐amylase
HT and DHPG (10:1)	85.5 ± 9.8	550 ± 63	0.72	2.2:8.2	Synergism
HT and DHPG (1:1)	20.4 ± 4.3	126 ± 26	0.16	17.7:6.5	Synergism
DHPG and COM (1:1)	293 ± 43	1131 ± 16	2.17	0.7:1.1	Antagonism

Note: p‐NPG and starch were used as substrates for the α‐glucosidase and α‐amylase assays, respectively.

The other CI values were below 0.4, demonstrating a clear synergistic effect. Notably, the DHPG:COM (1:1) combination had a CI of 0.13, suggesting a strongly synergistic interaction between DHPG and COM. In this case, based the median inhibition of α‐glucosidase with an IC₅₀ of 100.7 μm, showed that this combination was a potent inhibitor, with a lower IC₅₀ than acarbose (281 μm). Additionally, the HT and DHPG combination, with an IC₅₀ of 278.5 μm, close to that of acarbose, exhibited strong synergism at lower concentrations (up to 20 μg mL⁻¹), whereas higher concentrations suggested clear antagonism. This interaction between phenolic compounds was finally demonstrated by comparing the experimental inhibitory activity of the combination of HT and DHPG with the theoretical (calculated) values, which were calculated using the contribution of individual inhibitory effects of each compound (Fig. 2). Higher experimental values of the combination compared to theoretical ones suggest a potentially synergistic effect.

Figure 2.

Comparison of experimental and theoretical inhibition of α‐glucosidase and interaction effects in mixtures of 1:1 ratios of HT and DHPG at different concentrations. A positive difference (%) > 0 suggests a potential synergistic effect, a negative difference (%) < 0 indicates an antagonistic effect, and a difference (%) ≈ 0 represents an additive effect (no interaction).

Additionally, the DRI for the mixtures was evaluated. For instance, in the HT and DHPG (1:1) combination, an 8.9‐fold lower dose of HT and 2.1‐fold lower dose of DHPG were required to achieve the same inhibition of α‐glucosidase as the individual compounds (Table 2).

For the α‐amylase activity, the HT:DHPG (1:1) combination enhanced inhibition compared with the individual components, with a CI value of 0.16, indicating a strong synergistic effect. However, the DHPG:COM combination, which had shown significant synergism for α‐glucosidase, exhibited a CI of 2.17 for α‐amylase and no enhancement of inhibition, indicating antagonistic activity. Similarly, in the same synergistic combination, a 17.7‐fold reduction in the dose of HT and 6.5‐fold reduction in the dose of DHPG were needed to achieve the same inhibitory effect on α‐amylase compared to the doses of individual substances (Table 2).

DISCUSSION

The results indicate that DHPG exhibits the highest inhibitory capacity on α‐glucosidase, with an IC₅₀ comparable to that of acarbose, suggesting strong hypoglycemic potential. By contrast, HT and COM exhibited more moderate activity, consistent with previous studies ⁵ , ¹⁶ emphasizing their antioxidant properties over their inhibitory effects on digestive enzymes.

For α‐amylase, the IC₅₀ values were significantly higher compared to acarbose, indicating lower inhibitory efficacy. Although this reduced inhibition may be advantageous in minimizing gastrointestinal side effects, such as flatulence and diarrhea resulting from abnormal bacterial fermentation in the colon, it is important to ensure that inhibition is not too weak to exert a meaningful therapeutic effect. ¹⁷ , ¹⁸ Effective management of postprandial hyperglycemia requires a balanced inhibition that reduces carbohydrate digestion without causing undesirable adverse effects. Therefore, selecting α‐glucosidase inhibitors with minimal but adequate impact on α‐amylase activity is preferable. Given these considerations, DHPG emerges as a promising candidate, potentially offering a safer and effective alternative to synthetic inhibitors.

Binary combination analysis revealed a pronounced synergistic effect between HT and DHPG in a 1:1 ratio, significantly enhancing α‐glucosidase and α‐amylase inhibition at the same time as reducing their IC₅₀ values. This suggests a favorable interaction, particularly at lower concentrations, which may improve therapeutic efficacy at the same time as minimizing the required dosage. Likewise, the combinations of DHPG with COM in a 1:1 ratio showed synergistic inhibition of α‐glucosidase, as evidenced by its CI value of 0.13. This result indicates a powerful synergy, with the mixture achieving a lower IC₅₀ than acarbose. However, this last combination DHPG:COM (1:1) showed an antagonistic effect on α‐amylase inhibition (CI = 2.17), indicating that certain interactions may reduce inhibitory efficacy. This highlights the importance of thoroughly evaluating compound combinations before proposing therapeutic formulations.

From a mechanistic perspective, the superior α‐glucosidase inhibitory activity of DHPG compared to HT suggests that the additional OH‐ group at the β position of the aliphatic chain of DHPG enhances its interactions with the enzyme's active site. In the case of COM, its secoiridoid and trans‐p‐coumaroyl moieties may play a role in enzyme interaction through hydrophobic and hydrogen bonding interactions. ¹⁹ , ²⁰ However, its inhibitory efficacy appears weaker relatived to DHPG, highlighting structural differences that influence enzyme binding affinity and inhibition efficacy.

Beyond their enzymatic effects, HT and DHPG have demonstrated synergistic activity in other biological processes, including inhibition of platelet aggregation and lipid peroxidation, ²¹ as well as reducing oxidative stress and cardiovascular biomarkers. ²² Given the strong link between type‐2 diabetes and cardiovascular complications, this synergy could contribute not only to glucose regulation but also to cardiovascular protection, further supporting their therapeutic potential.

CONCLUSIONS

The results support the potential of olive oil by‐products as a source of bioactive compounds with significant anti‐diabetic properties. Although individual phenolic compounds such as HT, DHPG and COM show moderate inhibitory effects on α‐glucosidase and α‐amylase, with DHPG being the most active of the three, their combinations, particularly HT and DHPG (1:1), display strong synergistic effects that enhance enzyme inhibition. This suggests that a multi‐compound approach could offer more effective management of postprandial hyperglycemia than single compounds alone. Furthermore, the ability to reduce the dosage of each compound in synergistic combinations could minimize side effects and enhance the therapeutic value of these natural products. It is worth noting, however, that the α‐glucosidase used in this study is of yeast origin and may differ significantly from the human intestinal enzyme in terms of structure and activity. Therefore, although the findings provide valuable preliminary insights, they should be interpreted with caution regarding human applications. Future studies should focus on elucidating the precise molecular interactions underlying these synergies, validating the results with human‐recombinant enzymes, and testing their efficacy in vivo models to fully realize their potential as natural alternatives to synthetic anti‐diabetic drugs.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.