Chiang Mai Journal of Science

Hydrophobic Binding and Inhibition Mechanism of 2,3-Dihydroxychalcones against α-Glucosidase

1. INTRODUCTION

     Type 2 diabetes (T2D) characterized by high glucose levels has affected millions of people worldwide due to its serious complications such as cardiovascular, kidney and blindness diseases [1, 2]. Reducing glucose levels in blood through blocking carbohydrate digestion has been a goal to treat T2D [3]. α-Glucosidase is involved in the hydrolysis of disaccharides and oligosaccharides into α-D-glucose [4, 5]. Thus, α-glucosidase inhibition is one of the effective approaches to control hyperglycemia [6]. Currently, the anti-diabetic drugs such as acarbose, voglibose and miglitol have been used as α-glucosidase inhibitors [7-9]. However, these drugs available commercially are associated with side effects such as meteorism, diarrhea and flatulence [10]. Consequently, much effort has been undertaken to screen new α-glucosidase inhibitors with safer properties.
     Chalcones (1,3-diphenyl-2-propene-1-one analogues) are widely isolated in natural sources such as plants and flowers [11]. Chemically, the structure of chalcones is composed of two aromatic rings (A and B rings) connected to each other by an α,β-unsaturated carbonyl linker. Interestingly, these characteristics exhibited potent biological activities [12, 13]. In the recent past, chalcones have been reported as promising anti-diabetic candidates [6, 14-16]. The previous results revealed that the degree of the hydroxylation and/or the presence of electron-donating groups in two phenyl rings mainly contributed to the anti-diabetic activities [15, 17]. It was found that the introduction of hydroxy groups at C-2,4 or C-3,4-positions in the ring B improved anti-diabetic activities [17-19]. Keeping in that respect and in continuation of our previous work on chalcones, we selected chalcones bearing hydroxy groups at C-2,3 positions, named 2,3-dihydroxychalcones (Figure 1) to explore the effects of these groups on the α-glucosidase inhibition.
     Numerous analogues of chalcone found in plants contain hydroxy/methoxyl groups or occur in a form of glycosides. In contrast, chalcone templates bearing fluorine (F) are rare in nature. Interestingly, fluorinated organic compounds have exhibited promising biological activities, and thus fluorine-containing molecules have been synthesized to evaluate the effects on biological properties [20-22]. In that respect, we are focusing on chalcones F-substituted in the ring A (Figure 1) to examine the effects of fluoro groups on the α-glucosidase inhibitory activity. It is worth noting that the binding contacts and inhibition mechanism of 2,3-dihydroxychalcones against α-glucosidase were also investigated by using a combination of fluorescence and absorption spectral analysis.

2. MATERIALS AND METHODS

2.1 Materials
     α-glucosidase generated by Saccharomyces cerevisiae Type I, lyophilized powder, ≥10 units/mg protein, para-nitrophenyl-α-D-glucopyranoside (pNPG, ≥ 99%) and 8-anilino-1-naphthalenesulfonic acid (ANS, ≥ 97%) were purchased from Sigma-Aldrich (St, Louis, USA). Acarbose (95%) and dimethyl sulfoxide (DMSO, ≥99.9%) were obtained from Thermo Fisher Scientific (USA). \( \mathrm{K_2HPO_4 \cdot 3H_2O} \) (≥ 99%) and \( \mathrm{KH_2PO_4} \) (≥ 99.5%) and \( \mathrm{Na_2CO_3} \) (≥ 99.8%) were purchased from Xilong (China). Four 2,3-dihydroxychalcones named Non-F, 3′-F, 4′-F and 3′,4′-diF (Figure 1) were previously synthesized by our group [23].

2.2 In vitro α-Glucosidase Assay
     The α-glucosidase inhibition assay was performed using reported procedures [24, 25]. Mixture with a volume of \(180\ \mu\mathrm{L}\) including \(20\ \mu\mathrm{L}\) α-glucosidase (0.3 U/mL), final concentrations in wells of acarbose (6, 24, 96 and 384 μM) or 2,3-dihydroxychalcones (Non-F: 20, 40, 60 and 80 μM; 3′-F: 6, 9, 12 and 15 μM; 4′-F: 25, 50, 75 and 100 μM; 3′,4′-diF: 5, 10, 15 and 20 μM) and phosphate buffer solution (50 mM, pH 6.9, DMSO 2%, v/v) was added to each well of the 96-well plate and incubated at \(37^\circ\mathrm{C}\) for 30 min. The enzyme reaction was initiated by adding \(20\ \mu\mathrm{L}\) pNPG solution (1.0 mM). The reaction was carried out at \(37^\circ\mathrm{C}\) for 30 min. Finally, \(20\ \mu\mathrm{L}\) \( \mathrm{Na_2CO_3} \) solution (125 mM) was added to stop the enzyme reaction. The absorbance was determined at 405 nm in the Elisa microplate reader (JP Selecta, 2100C, Spain). The α-glucosidase inhibitory activity of each inhibitor was calculated by percent inhibition (I%) = \( \left( \frac{\mathrm{OD}_{\mathrm{control}} - \mathrm{OD}_{\mathrm{inhibitor\ treated}}}{\mathrm{OD}_{\mathrm{control}}} \right) \times 100\% \), where OD is the optical density at 405 nm. Each concentration of inhibitors was tested in three separate wells and in triplicate (\(n = 3 \times 3\)). For each experiment, the mean OD value obtained from the three wells was used to calculate the inhibition percentage. A dose–response curve (I% versus concentration) was then plotted, and the \( \mathrm{IC_{50}} \) (the half maximal inhibitory concentration) value was determined by regression analysis. Finally, the mean \( \mathrm{IC_{50}} \) value from the three independent experiments was calculated, and the standard deviation (SD) of \( \mathrm{IC_{50}} \) was determined using the STDEV function in Excel.

2.3 Intrinsic Fluorescence Measurements of α-Glucosidase
     A series of solutions (2 mL) containing α-glucosidase (2.7 U/mL) and inhibitors with various concentrations was stirred for 2 min at \(37^\circ\mathrm{C}\). The concentration ranges of inhibitors (Non-F: 20–80 μM; 3′-F: 6–13 μM; 4′-F: 30–75 μM; 3′,4′-diF: 8–20 μM) were chosen on the basis of the \( \mathrm{IC_{50}} \) values. The solutions were then transferred to a 1.0 cm quartz cuvette. At an excitation wavelength of 295 nm, the intrinsic fluorescence spectra of α-glucosidase were recorded using a Horiba spectrofluorometer (FluoroMax-4, Horiba, Japan) in the range 300–450 nm. Both slit widths for excitation and emission were 2.0 nm. The enzyme fluorescence was deducted from the fluorescence background of the phosphate buffer. Due to the inner filter effect [26], the fluorescence intensity of α-glucosidase at each inhibitor concentration was corrected for the absorption of the inhibitor at 295 nm (eq. 1).

\( F_{\mathrm{corr}} = 10^{\varepsilon [I]/2} \, F_{\mathrm{mean}} \)   (1)

Where, \( F_{\mathrm{corr}} \) and \( F_{\mathrm{mean}} \) refer to the corrected and mean fluorescence intensities. The \( F_{\mathrm{mean}} \) was an average fluorescence intensity of three scans at each inhibitor concentration. The \( \varepsilon \) value denotes the molar extinction coefficient of the inhibitor at 295 nm and \( [I] \) is the inhibitor concentration.

2.4 Hydrophobic Experiments of α-Glucosidase using ANS
     Each 2 mL solution containing α-glucosidase (2.7 U/mL), ANS (12.5 μM) and inhibitor at a given concentration (Non-F: 0, 20.0, 40.0 μM; 3′-F: 0, 4.0, 8.0 μM; 4′-F: 0, 8.0, 24.0 μM; 3′,4′-diF: 0, 4.1, 8.2 μM) was transferred to a 1.0 cm quartz cuvette after stirring for 2 min at \(37^\circ\mathrm{C}\) to equilibrate. The mixed solutions were excited at 375 nm and their fluorescence spectra were detected over a wavelength range of 400–600 nm. Slit widths for both excitation and emission were 2.0 nm. The fluorescence spectra of α-glucosidase, ANS and inhibitors alone were also scanned in the same experimental conditions. The fluorescence intensity was an average intensity of three scans after subtracting the buffer fluorescence.

2.5 Determination of Inhibition Mechanism against α-Glucosidase
     The same procedure for the in vitro α-glucosidase assay was carried out to determine inhibition types of inhibitors against α-glucosidase. The OD value of a mixture in each well with α-glucosidase (0.6 U/mL) and pNPG at increasing concentrations (5.0, 6.7, 10.0, 20.0 mM) was read at 405 nm in the absence and presence of inhibitors (Non-F: 0–30 μM; 3′-F: 0–9 μM; 4′-F: 0–6 μM; 3′,4′-diF: 0–40 μM, acarbose: 0–200 μM). The inhibition mechanism and the Michaelis–Menten constant (\(K_m\)) of 2,3-dihydroxychalcones were determined using the Lineweaver–Burk plot of equation (2) [27, 28].

\( \frac{1}{V} = \frac{K_m}{V_{\max}} \frac{1}{[S]} + \frac{1}{V_{\max}} \)   (2)

Here, \( V \) is the enzyme reaction rate depending on substrate concentration. \( V_{\max} \) and \( [S] \) are the maximum reaction rate and the substrate concentration, respectively. \( K_m \) is the Michaelis–Menten constant.

3. RESULTS AND DISCUSSION

3.1 Inhibitory Activities of 2,3-Dihydroxychalcones against α-Glucosidase
     In present work, a panel of four 2,3-dihydroxychalconoids was tested, concerning their ability to inhibit α- glucosidase activity using acarbose as a positive control. For comparison, concentration-response experiments were carried out to obtain the half-maximal inhibitory concentration (IC₅₀, μM) values for each compound. As illustrated in Figure 2, 2,3-dihydroxychalcones displayed a more inhibitory effect on α-glucosidase activity than that of acarbose (IC₅₀ = 69.58+2.04 μM). Among the 2,3-dihydroxychalcones evaluated, compound 3',4'-diF exhibited the highest inhibition against α-glucosidase with an IC₅₀ value of 5.34+0.11 μM, in turn, a close IC₅₀ value of 3'-F (IC₅₀ = 6.84+0.33 μM), which improved by 13-fold and 10-fold over the standard acarbose in this assay. In general, the activity was increased when the ring A of 2,3-dihydroxychalcone (Non-F, IC₅₀ = 60.68+0.85 μM) was fluorinated, indicating that the fluorination in the A ring made a positive contribution to activity. Compound 3'-F possessing a meta-fluoro group in ring A was found to be active with an IC50 value of 6.840.33 μM, while compound 4'-F (IC₅₀ = 48.94+0.19 μM) with a fluoro group at para-position exhibited a decreased inhibition against α-glucosidase. Interestingly, in comparison with 3′-F and 4′-F, compound 3′,4′-diF (\( \mathrm{IC_{50}} = 5.34 \pm 0.11 \ \mu M \)) being fluorinated at both meta and para-positions showed a close activity to 3′-F, revealing the crucial role of 3′-F group in ring A on the α-glucosidase inhibition. Furthermore, the fluoro-substitution at both 3′ and 4′ positions in the ring A may contribute to the increased activity of 3′,4′-diF through synergistic effect.
     The position of hydroxy groups in chalcones are key factors for their α-glucosidase inhibition. Considering the effects of hydroxy groups in the ring B of chalcones on the inhibitory activity, Cai et al. reported that the synergistic contribution of 2,4-dihydroxyl or 3,4-dihydroxy groups led to an increased inhibition compared to a positive control, 1-deoxynojirimycin [17, 18]. Herein, in order to explore the effects of fluoro groups in ring A on α-glucosidase inhibition, new fluorinated chalcones, which possess absolute 2,3-dihydroxy substituents in ring B were selected. In comparison with acarbose, we suggested that the α-glucosidase inhibition of active 2,3-dihydroxychalcones can be due to the synergistic effects of fluoro and 2,3-dihydroxy groups in two rings on α-glucosidase.
     Most of the previous literature reported the effects of hydroxy and/or methoxy substituents in chalcones on the inhibition against α-glucosidase [18, 19]. Meanwhile, fluorinated organic compounds possess promising bioactivities [20–22], however, it is not predictable the role of fluorine substituents in enhancing the bioactivity [21]. The structure–activity relationship (SAR) analysis has been considered as a straightforward approach to elucidate the effects of fluoro groups on bioactivity. Herein, we demonstrated for the first time that the fluorination in ring A boosted up the α-glucosidase inhibition and the 3′-F motif mainly contributed to the activity.
     The in vitro enzyme assay provided preliminary insights into inhibitory activity, however, it does not account for factors such as metabolic stability and cellular uptake, which may limit its relevance to in vivo efficacy [29]. In our present results, the promising in vitro α-glucosidase inhibitory activity observed in fluorinated chalcones suggests their potential as effective antidiabetic candidates for further in vivo evaluation. The effects imparted by fluorination on enhanced lipophilicity, metabolic stability and hydrogen bonding formation that affect molecule absorption and distribution were described [20]. The fluorinated inhibitors may exhibit improved pharmacokinetic behavior compared with their non-fluorinated counterparts. Future studies therefore focus on assessing their in vivo antidiabetic efficacy and toxicity profiles to confirm the biological relevance of the fluorine substitution.

3.2 Contacts between 2,3-Dihydroxychalcones and α-Glucosidase
     A variety of molecular interactions can be detected via the fluorescence intensity reduction of a fluorophore, i.e., fluorescence quenching. This process has been widely applied in biochemical systems. The contacts of inhibitors and enzyme can be detected by fluorescence change of enzyme without and with inhibitors [30]. The intrinsic fluorescence of enzyme originates from amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). In our experiments, we selected Trp in α-glucosidase as a fluorophore to explore the contacts of 2,3-dihydroxychalcones with the α-glucosidase. As shown in Figure 3A-D, when excited at 295 nm, the fluorescence of chalcones alone was negligible in the emission range of 300-450 nm, i.e., the chalcones were insensitive to the excitation at 295 nm. In contrast, the fluorescence intensity of α-glucosidase was observed with a maximum peak near 345 nm. In addition, the enzyme fluorescence was quenched with increasing the concentration of 2,3-dihydroxychalcones, revealing the interaction of chalcones and α-glucosidase, which confirmed the inhibition of inhibitors on α-glucosidase.

3.3 Hydrophobic Binding between 2,3-Dihydroxychalcones and α-Glucosidase
     Hydrophobic contact between inhibitor and enzyme can be proved by adding an extrinsic fluorescent probe, 8-anilino-1-naphthalenesulfonic acid (ANS) to enzyme solution. The ANS probe binds with high affinity to the hydrophobic surface of the enzyme to form a fluorescent complex, [enzyme/ANS] [31, 32]. The fluorescence quenching measurements on the [enzyme/ANS] complex in the presence of inhibitors can reveal the accessibility of the inhibitor to enzyme via hydrophobic competition [33-34]. As depicted in Figure 4A-D, the addition of ANS to α-glucosidase solution resulted in the fluorescence signal in the range from 400 to 600 nm after excitation at 375 nm, indicating that the non-covalent [α-glucosidase/ANS] complex was significantly formed between ANS probe and enzyme. In addition, when excited at 375 nm, the emission of ANS alone can be distinguished from the fluorescence of the complex, while there were barely emission signals of chalcones and α-glucosidase under the same excitation condition.
     When treated with 2,3-dihydroxychalcones, the fluorescence intensity of [α-glucosidase/ANS] complex was reduced in an inhibitor concentration dependent manner (Figure 4A-D). The observation suggested that there is a competition between chalcone inhibitors and ANS on the surface hydrophobicity of α-glucosidase. This resulted in a decrease in the amount of complex by adding inhibitors. In other words, chalcones bind to a hydrophobic domain and thus, reduce the hydrophobic surface of α-glucosidase. These results were also observed when α-glucosidase was treated with other inhibitors such as xanthohumol [33], genistein [34], butyl-isobutyl-phthalate [35]. Such inhibitor induced hydrophobic decrease supports the crucial role of the hydrophobic surface at the active site of α-glucosidase.

3.4 Inhibition Mechanisms of 2,3-Dihydroxychalcones against α-Glucosidase
     At the final step in this research, the inhibition kinetics of α-glucosidase by Non-F, 3'-F, 4'-F and 3',4'-diF was studied by absorbance measurements at 405 nm. The inhibition mechanism of each chalcone was determined by plotting the reciprocal of the rate (1/v) of product formation vs the reciprocal of the substrate concentration (1/[pNPG]) in the presence of various concentrations of inhibitors, thus obtaining the Lineweaver-Burk plots (Figure 5) [27, 28]. As shown in Figure 5A, C and D, the lines of Lineweaver-Burk plots obtained for Non-F, 4'-F and 3',4'-diF intersected the y-axis in the first quadrant, indicating that these chalcones act as competitive inhibitors against α-glucosidase, i.e., the inhibitors bind to the same catalytic site as the substrate (pNPG). In addition, as observed in Figure 5E, the positive control acarbose presented a type of competitive inhibition, in line with reported results [36-38]. The Lineweaver-Burk lines for 3'-F (Figure 5B) gave a series of lines converging on the same point on the x axis in the second quandrant, revealing that a non-competitive mode existed in α-glucosidase with respect to 3'-F, i.e., a. binding site different from the one of substrate. In eq (2), the Michaelis–Menten constant Km, a kinetic parameter is equal to substratrate concentration required to reach half of the vmax value. This parameter was used as an indication of the binding affinity (strength) of enzyme to its substrate [28, 39]. For competitive types (Figure 5A, C and D), an increase in the concentration of inhibitors (Non-F, 4'-F and 3',4'-diF) resulted in an increase in Km value (-1/Km = -1/[pNPG], i.e., a lower affinity for the substrate. Considering a non-competitive inhibitor 3'-F, the binding of 3'-F and pNPG is completely independent. Therefore, the reaction rate reduced with increasing the inhibitor concentration, while Km remained constant. As shown in Figure 5B, the calculated value of Km was 2.3×10^-4 M (-1/Km = -1/[pNPG] = -4.27+0.11 mM^-1) in our experimental condition.

4. CONCLUSIONS

     In conclusion, we selected 2,3-dihydroxychalcones containing fluorine in ring A as inhibitors against α-glucosidase. Chalcones were potent inhibitors of α-glucosidase, showing IC₅₀ values lower than the pharmaceutical agent acarbose (IC₅₀ = 69.58+2.04 µM). The introduction of a fluoro group at the 3'-position in ring A (3'-F, IC₅₀ = 6.84+0.33 µM) greatly contributed to the α-glucosidase inhibitory activity. In addition, the fluorine substitution at both meta- and para-positions (3',4'-diF, IC₅₀ = 5.34+0.11 µM) boosted up the inhibition through a synergy. The fluorescence quenching experiments indicated chalcones directly bind to enzyme and reduce the hydrophobicity of enzyme. Moreover, binding types elucidated the specific inhibition mechanisms of chalcones against α-glucosidase. The results of the Lineweaver-Burk plots pointed out that Non-F, 4'-F and 3',4'-diF function as competitive inhibitors, whereas 3'-F inhibits the α-glucosidase activity in a non-competitive manner. Based on reported results for the first time, these chalcones can serve as promising inhibitors for further in vivo anti-diabetes assay.

ACKNOWLEDGEMENTS

     This work belongs to the project grant No: T2025-93 funded by Ho Chi Minh City University of Technology and Engineering, Vietnam.

AUTHOR CONTRIBUTIONS

     Khoi Dinh Dang: Methodology, Writing - Reviewing and Editing. Phuong Ho: Formal analysis, Writing - Original draft preparation. Bich Van Thi Pham: Formal analysis, Writing - Reviewing and Editing. Hao Minh Hoang: Conceptualization, Data curation, Supervision, Methodology, Software, Writing - Original draft preparation, Writing - Reviewing and Editing.

CONFLICT OF INTEREST STATEMENT

     The authors declare that they hold no competing interests.

REFERENCES

[1] Nathan D.M., Long-term complications of diabetes mellitus. The New England Journal of Medicine, 1993; 328(23): 1676–1685. DOI 10.1056/NEJM199306103282306.

[2] Duckworth W., Abraira C., Moritz T., Reda D., Emanuele N., Reavene P.D., et al., Glucose control and vascular complications in veterans with type 2 diabetes. The New England Journal of Medicine, 2009; 360(2): 129–139. DOI 10.1056/NEJMoa0808431.

[3] Chadha G.S. and Morris M.E., An extended minimal physiologically based pharmacokinetic model: Evaluation of type II diabetes mellitus and diabetic nephropathy on human IgG pharmacokinetics in rats. The AAPS Journal, 2015; 17(6): 1464–1474. DOI 10.1208/s12248-015-9810-0.

[4] Poongunran J., Perera H.K.I., Jayasinghe L., Fernando I.T., Sivakanesan R., Araya H., et al., Bioassay-guided fractionation and identification of α-amylase inhibitors from Syzygium cumini leaves. Pharmacognosy Research, 2017; 55(1): 206–211. DOI 10.1080/13880209.2016.1257031.

[5] Kumar V., Prakash O., Kumar S., and Narwal S., α-glucosidase inhibitors from plants: A natural approach to treat diabetes. Pharmacognosy Reviews, 2011; 5(9): 19–29. DOI 10.4103/0973-7847.79096.

[6] Mukhtar A., Shah S., Kanwal, Khan K.M., Khan S.U., Zaibes S., et al., Synthesis of chalcones as potential α-glucosidase inhibitors, in-vitro and in-silico studies. ChemistrySelect, 2021; 6(37): 9933–9940. DOI 10.1002/slct.202102434.

[7] Kumar Singla R., Singh R. and Kumar Dubey A., Important aspects of post-prandial antidiabetic drug, acarbose. Current Topics in Medicinal Chemistry, 2016; 16(23): 2625–2633. DOI 10.2174/1568026616666160414123500.

[8] Scott L.J. and Spencer C.M., Miglitol: A review of its therapeutic potential in type 2 diabetes mellitus. Drugs, 2000; 59(3): 521–549. DOI 10.2165/00003495-200059030-00012.

[9] Kaku K., Efficacy of voglibose in type 2 diabetes. Expert Opinion on Pharmacotherapy, 2014; 15(8): 1181–1190. DOI 10.1517/14656566.2014.918956.

[10] Phung O.J., Effect of noninsulin antidiabetic drugs added to metformin therapy on glycemic control, weight gain, and hypoglycemia in type 2 diabetes. JAMA, 2010; 303(14): 1410. DOI 10.1001/jama.2010.405.

[11] Ardianah B., Chalcones bearing N,O and S-heterocycles: Recent notes on their biological significances. Journal of Applied Pharmaceutical Science, 2019; 9(8): 117–129. DOI 10.7324/JAPS.2019.90816.

[12] Wang G., Piva De Silva G., Wiebe N.E., Fehr G.M. and Dauis R.L., Development of a metal-free amine oxidation method utilizing DEAD chemistry. RSC Advances, 2017; 7(7): 48848–48852. DOI 10.1039/C7RA09165F.

[13] Sahu N.K., Bahadara S.S., Choudhary J. and Kohli D.V., Exploring pharmacological significance of chalcone scaffold: A review. Current Medicinal Chemistry, 2012; 19(2): 209–225. DOI 10.2174/092986712803414132.

[14] Pham T.B.V., Le T.H., Nguyen T.B.T., Vo T.N., Le M.T., Ho P., et al., Solvent-free, microwave-assisted, solid-catalyzed synthesis and α-glucosidase inhibition of chalcones. Vietnam Journal of Chemistry, 2023; 61(3): 325–332. DOI 10.1002/vjch.202200151.

[15] Mahapatra D.K., Asati V. and Bharti S.K., Chalcones and their therapeutic targets for the management of diabetes: Structural and pharmacological perspectives. European Journal of Medicinal Chemistry, 2015; 92: 839–865. DOI 10.1016/j.ejmech.2015.01.051.

[16] Yoon G., Lee W., Kim S.N. and Cheon S.H., Inhibitory effect of chalcones and their derivatives from Glycyrrhiza inflata on protein tyrosine phosphatase 1B. Bioorganic & Medicinal Chemistry Letters, 2009; 19(17): 5155–5157. DOI 10.1016/j.bmcl.2009.07.054.

[17] Jung S.H., Park S.Y., Kim-Pak Y., Lee H.K., Park K.S., Shin K.H., et al., Synthesis and PARP-gamma ligand-binding activity of the new series of 2′-hydroxychalcone and thiazolidinedione derivatives. Chemical and Pharmaceutical Bulletin, 2006; 54(3): 368–371. DOI 10.1248/cpb.54.368.

[18] Cai C.Y., Rao L., Rao Y., Guo J.X., Xiao Z.Z., Cao J.Y., et al., Analogues of xanthones—chalcones and bis-chalcones as α-glucosidase inhibitors and anti-diabetes candidates. European Journal of Medicinal Chemistry, 2017; 130: 51–59. DOI 10.1016/j.ejmech.2017.02.007.

[19] Ramman A., Bhaskar B.V., Venkateswarlu N., Gu W. and Zyranov G.V., Design, synthesis, docking and biological evaluation of chalcones as promising antidiabetic agents. Bioorganic Chemistry, 2020; 95: 103527. DOI 10.1016/j.bioorg.2019.103527.

[20] Smart B.E., Fluorine substituent effects (on bioactivity). Journal of Fluorine Chemistry, 2001; 109(1): 3–11. DOI 10.1016/S0022-1139(01)00375-X.

[21] Isanbor C. and O’Hagan D., Fluorine in medicinal chemistry: A review of anti-cancer agents. Journal of Fluorine Chemistry, 2006; 127(3): 303–319. DOI 10.1016/j.jfluchem.2006.01.011.

[22] Shah F.H., Wanjare B.D. and Kim S.J., Synthesis, characterization and in silico biological and anticancer activity of 3-(2-fluorophenyl)-N-(4-fluorophenyl)-7H-[1,2,4] triazolo[3,4-b][1,3,4] thiadiazin-6-amine. Chiang Mai Journal of Science, 2022; 49(2): 377–386. DOI 10.12982/CMJS.2022.038.

[23] Ho P., Nguyen T.L., Nguyen T.N.A., Ha A.Q.T. and Hoang H.M., Fluorine containing chalcones as anticancer agents: Solvent-free synthesis and cytotoxicity against HepG2 cancer cells. Science & Technology Development Journal, 2024; 27(4): 3623–3632. DOI 10.32508/stdj.v27i4.4350.

[24] Tsujii E., Murai M., Shiragami N. and Takatsuki A., Necristine is a potent inhibitor of α-glucosidases, demonstrating activities similarly at enzyme and cellular levels. Biochemical and Biophysical Research Communications, 1996; 220(2): 459–466. DOI 10.1006/bbrc.1996.0427.

[25] Saddique F.A., Ahmad M., Ashfaq U.A., Mansha A. and Khan S.G., Alpha-glucosidase inhibition and molecular docking studies of 4-hydroxy-N′-benzylidene-1-phenylethylidene-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides. Chiang Mai Journal of Science, 2021; 48(2): 460–469.

[26] Phillips S.R., Wilson L.J. and Borkman R.F., Acrylamide and iodide fluorescence quenching as a structural probe of tryptophan microenvironment in bovine lens crystallins. Current Eye Research, 1986; 5(8): 611–620. DOI 10.3109/02713688609015126.

[27] Lineweaver H. and Burk D., The determination of enzyme dissociation constants. Journal of the American Chemical Society, 1934; 56(3): 658–666. DOI 10.1021/ja01318a036.

[28] Kaewnarak K. and Rakriyatham N., Inhibitory effects of phenolic compounds in Ocimum sanctum extract on the α-glucosidase activity and the formation of advanced glycation end-products. Chiang Mai Journal of Science, 2017; 44(1): 203–214.

[29] Karunarathna S.C., Patabendige N.M., Lu W., Dai D. and Hapuarachchi K.K., Exploring the health benefits of Ganoderma mechamisms of action and anti-diabetic activities. Chiang Mai Journal of Science, 2025; 52(6): e2025080. DOI 10.12982/CMJS.2025.080.

[30] Lakowicz J.R., Principles of Fluorescence Spectroscopy, 3^rd Edn., Springer, 2006.

[31] Cardullo N., Muccilli V., Pulvirenti L., Cornu A., Pouvségu L., Deffieux D., et al., C-glucosidic ellagitannins and galloylated glucoses as potential functional food ingredients with anti-diabetic properties: A study of α-glucosidase and α-amylase inhibition. Food Chemistry, 2020; 313: 126099. DOI 10.1016/j.foodchem.2019.126099.

[32] Feroz S.R., Mohamad S.B., Bakri Z.S.D., Malek S.N.A. and Tayyab S., Probing the interaction of a therapeutic flavonoid, pinocembrin with human serum albumin: Multiple spectroscopic and molecular modeling investigations. PLoS One, 2013; 8(10): e76067. DOI 10.1371/journal.pone.0076067.

[33] Liu M., Yin H., Liu G., Dong J., Qian Z. and Miao J., Xanthohumol, a prenylated chalcone from beer hops, acts as an α-glucosidase inhibitor in vitro. Journal of Agricultural and Food Chemistry, 2014; 62(24): 5548–5554. DOI 10.1021/jf500426z.

[34] Nga V.T. and Hao H.M., Inhibition kinetics and mechanism of genistein against α-glucosidase. Vietnam Journal of Chemistry, 2024; 62(4): 493–499. DOI 10.1002/vjch.202200173.

[35] Liu M., Zhang W., Qiu L. and Lin X., Synthesis of butyl-isobutyl-phthalate and its interaction with α-glucosidase in vitro. Journal of Biochemistry, 2011; 149(1): 27–33. DOI 10.1093/jb/mvq110.

[36] Proença C., Freitas M., Ribeiro D., Oliveira E.F.T., Sousa J.L.C., Tomé S.M., et al., α-glucosidase inhibition by flavonoids: an in vitro and in silico structure–activity relationship study. Journal of Enzyme Inhibition and Medicinal Chemistry, 2017; 32(1): 1216–1228. DOI 10.1080/14756366.2017.1368503.

[37] Son H.U., Yoon E.K., Yoo C.Y., Park C.H., Bae M.A., Kim T.H., et al., Effects of synergistic inhibition on α-glucosidase by phytoalexins in soybeans. Biomolecules, 2019; 9(12): 828–841. DOI 10.3390/biom9120828.

[38] Calder P.C. and Geddes R., Acarbose is a competitive inhibitor of mammalian lysosomal acid α-D-glucosidases. Carbohydrate Research, 1989; 191(1): 71–78. DOI 10.1016/0008-6215(89)85047-5.

[39] Rogers A. and Gibson Y., Enzyme Kinetics: Theory and Practice; in Schneider J., ed., Plant Metabolic Networks. Springer New York, 2009: 71–103. DOI 10.1007/978-0-387-78745-9_4.

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