Use of biphenyl compounds in inhibiting α-glucosidase activity and application in lowering blood sugar

Abstract

The present invention provides the use of biphenyl compounds in inhibiting the activity of α-glucosidase and their application in reducing blood glucose, belonging to the technical field of biomedicine. The present invention provides the application of biphenyl compounds as α-glucosidase inhibitors and their application in reducing blood glucose levels. The biphenyl compounds of the present invention can significantly inhibit the activity of α-glucosidase, wherein the IC50 value of THB is 11.52 μM, the dissociation constant is 3.91×10-5 M, and the inhibitory effect of THB on α-glucosidase is concentration-dependent, with the highest inhibition rate reaching 96.50%. The present invention also evaluates the safety and in vivo blood glucose-lowering activity of the biphenyl compounds, which can effectively reduce the postprandial blood glucose level in vivo and have the potential to be used as drugs for the treatment of diabetes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of biphenyl compounds in inhibiting α-glucosidase activity and their application in reducing blood sugar. Background Art

[0002] Biphenyl compounds are formed by connecting two phenyl groups through a single bond and have applications in many fields. For example, in industry, they are often used as high-temperature heat transfer media to transfer heat in hot oil circulation systems; they are raw materials for the engineering plastic polysulfone and are also used to manufacture fuels, engineering plastics, and high-energy fuels, etc.; they can also be used as solvents, especially in fields such as coatings, inks, and adhesives, and can effectively dissolve many organic substances to improve the processing performance of products. Biphenyl is also an important intermediate for synthesizing some organic pesticides such as the rodenticide diphacinone and bromadiolone, etc. At the same time, in the medical field, biphenyl and its derivatives are used in drug synthesis. For example, in the synthesis of certain anti-tumor drugs, biphenyl is an important intermediate, but the research on biphenyl itself in medicine, especially in reducing blood sugar, is extremely rare.

[0003] 3,4,5-Trihydroxybiphenyl (THB) is a biphenyl compound with a hydroxyl group connected to the 3, 4, and 5 positions of one of the benzene rings respectively, and its molecular formula is C 12 H 10 O3. THB has multiple hydroxyl groups, and these hydroxyl groups can provide hydrogen atoms to combine with free radicals in the body, thereby playing an antioxidant role, reducing the damage of free radicals to cells, and contributing to the prevention and treatment of diseases related to oxidative stress, such as cardiovascular diseases, neurodegenerative diseases, etc. THB also has anti-inflammatory activity. It can play an anti-inflammatory role by inhibiting the activation of inflammation-related signaling pathways and reducing the release of inflammatory mediators, and has potential application value in the treatment of inflammatory diseases such as arthritis and enteritis. Through research, it is found that THB also has a certain inhibitory effect on certain bacteria and fungi, which may contribute to the development of new antibacterial drugs for the treatment of bacterial and fungal infectious diseases. At the same time, THB is also an important intermediate for synthesizing drugs with various specific structures and properties. It can be seen that THB has a good application and research foundation, but there is less research on other aspects of THB. Summary of the Invention

[0004] The present invention provides the use of biphenyl compounds in inhibiting α-glucosidase activity and their application in reducing blood sugar. The biphenyl compounds have strong inhibitory activity against α-glucosidase and can significantly reduce the postprandial blood sugar level.

[0005] The present invention provides the application of a biphenyl compound or a pharmaceutically acceptable salt of the biphenyl compound in the preparation of an α-glucosidase activity inhibitor. The biphenyl compound has the structure shown in Formula I:

[0006] , formula I;

[0007] In the structure shown in formula I, R1 includes any one of -H, -OH, and -OMe; R2 includes any one of -H, -OH, and -OMe; R3 includes any one of -H, -OH, and -OMe; R4 includes -H or -OMe.

[0008] In a preferred embodiment of the present invention, the concentration of the biphenyl compound in the α-glucosidase activity inhibitor is not less than 1.57 μM.

[0009] The present invention also provides an α-glucosidase activity inhibitor, the active ingredient of which includes the biphenyl compound shown in formula I or a pharmaceutically acceptable salt of the biphenyl compound.

[0010] The present invention also provides the use of the biphenyl compound shown in formula I or a pharmaceutically acceptable salt of the biphenyl compound in the preparation of a hypoglycemic drug.

[0011] The present invention also provides the use of the biphenyl compound shown in formula I or a pharmaceutically acceptable salt of the biphenyl compound in the preparation of a drug for preventing and / or treating diabetes.

[0012] In a preferred embodiment of the present invention, the diabetes includes diabetes targeting α-glucosidase.

[0013] The present invention also provides a drug for preventing and / or treating diabetes, the active ingredient of which includes the biphenyl compound shown in formula I or a pharmaceutically acceptable salt of the biphenyl compound, and also includes a pharmaceutically acceptable carrier.

[0014] In a preferred embodiment of the present invention, the biphenyl compound includes 3,4,5-trihydroxybiphenyl.

[0015] Beneficial effects: The present invention provides the use of the biphenyl compound shown in formula I as an α-glucosidase inhibitor and in reducing blood glucose levels. The biphenyl compound of the present invention can bind to α-glucosidase. For example, THB has a medium-strength binding affinity with α-glucosidase, and its dissociation constant (KD value) is 3.91×10 -5 M, and the binding shows concentration dependence. The biphenyl compound of the present invention can significantly inhibit α-glucosidase activity. For example, the IC 50 value of THB is 11.52 μM, and the inhibitory effect of THB on α-glucosidase has concentration dependence, and the highest inhibition rate reaches 96.50%. The present invention also evaluated the in vivo hypoglycemic activity of THB. THB can effectively reduce the postprandial blood glucose level in vivo and has the potential to be used as a diabetes treatment drug. Description of the Drawings

[0016] Figure 1 Statistical chart of the inhibitory activity of THB against α-glucosidase (n = 3);

[0017] Figure 2 Result graph of the inhibitory kinetics of THB against α-glucosidase. In the figure, A: Curve of the relationship between the enzymatic reaction rate (ν) and the concentration of [α-glucosidase]; B: Real-time kinetic process of the inhibition of α-glucosidase activity; C: Lineweaver-Burk double-reciprocal plot of THB inhibiting α-glucosidase; D: Second-order curve of THB inhibiting α-glucosidase;

[0018] Figure 3 Result graph of the inhibitory effect of the combination of THB and acarbose on α-glucosidase. The numbers marked above the combination line are the combination index (CI) values calculated using CompuSyn software;

[0019] Figure 4 Result graph of the SPR analysis of the binding affinity between α-glucosidase and THB;

[0020] Figure 5 Circular dichroism spectrum. In the figure, A: Circular dichroism spectrum of α-glucosidase; B: Circular dichroism spectrum of the complex of α-glucosidase and THB;

[0021] Figure 6 Result graph of the molecular docking analysis simulation of THB and α-glucosidase. In the figure, A: Structure of the THB-α-glucosidase complex; B: Binding site of THB on the surface of α-glucosidase; C: Two-dimensional interaction mode of THB binding to α-glucosidase; D: THB molecules in the binding pocket;

[0022] Figure 7 Result graph of the molecular dynamics simulation of the complex of α-glucosidase and THB. In the figure, A: RMSD of α-glucosidase and its THB complex; B: RMSF of α-glucosidase and THB; C: Formation of hydrogen bonds between α-glucosidase and THB;

[0023] Figure 8 Result graph of the toxicity verification of THB. In the figure, A: Survival rate of MDA-MB-231 cells after exposure to THB (0 - 500 μM) for 24 h; B: Survival rate of HepG2 cells after exposure to THB (0 - 500 μM) for 24 h;

[0024] Figure 9It is the change curve of postprandial blood glucose (PBG) in mice after sucrose loading; the data are expressed as mean ± standard deviation, n = 6; compared with the control group, *p < 0.05, ***p < 0.001, ****p < 0.0001; Con: control group; Acar: acarbose group;

[0025] Figure 10 is the incremental area under the blood glucose curve (AUC 0~120min ) after sucrose administration; the data are expressed as mean ± standard deviation, n = 6; compared with the control group, *p < 0.05, ***p < 0.001, ****p < 0.0001; Con: control group; Acar: acarbose group. Specific embodiments

[0026] The present invention provides the use of a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound in the preparation of an α-glucosidase activity inhibitor, and the biphenyl compound has the structure shown in Formula I:

[0027] , Formula I;

[0028] In the structure shown in Formula I, R1 includes any one of -H, -OH and -OMe; R2 includes any one of -H, -OH and -OMe; R3 includes any one of -H, -OH and -OMe; R4 includes -H or -OMe.

[0029] In one embodiment of the present invention, the inhibitory activity of the biphenyl compound on α-glucosidase was verified. In particular, the inhibitory effect of 3,4,5-trihydroxybiphenyl (THB) on α-glucosidase is concentration-dependent. When the concentration increases from 1.57 μM to 50 μM, the inhibitory effect gradually enhances, and the highest inhibition rate reaches 96.50%. After measurement, the IC 50 value is 11.52 μM, confirming the strong inhibitory activity of the THB on α-glucosidase.

[0030] The structural formula of the THB of the present invention is shown in Formula II:

[0031] , Formula II.

[0032] The pharmaceutically acceptable salt of the biphenyl compound of the present invention can be a pharmaceutically acceptable sodium salt, potassium salt or calcium salt formed by combining with NaOH, KOH or Ca(OH)2.

[0033] The present invention also provides an α-glucosidase activity inhibitor, and the active ingredient includes the biphenyl compound shown in Formula I or a pharmaceutically acceptable salt of the biphenyl compound.

[0034] In one embodiment of the present invention, it is verified that the THB has both in vitro inhibitory activity and in vivo inhibitory activity against α-glucosidase. Therefore, the THB can be prepared into a corresponding inhibitor capable of inhibiting the activity of α-glucosidase. The present invention does not have any special limitations on other excipient components of the inhibitor, as long as the inhibitory activity of THB against α-glucosidase can be demonstrated after binding with the THB.

[0035] The present invention also provides the use of the biphenyl compound shown in Formula I or a pharmaceutically acceptable salt of the biphenyl compound in the preparation of a hypoglycemic drug.

[0036] In one embodiment of the present invention, through an oral experiment on mice, the postprandial hypoglycemic effects of THB and acarbose were compared. The results showed that at 60 min after administration, the postprandial blood glucose levels of the high-dose THB group (dosage 8.3 mg / kg) and the acarbose group were both significantly lower than those of the control group; the area under the blood glucose curve (AUC) of the high-dose THB group and the acarbose group was also significantly lower than that of the control group. This indicates that the THB can effectively reduce the postprandial blood glucose level in vivo and can be applied to the preparation of hypoglycemic drugs.

[0037] The present invention does not have any special limitations on the dosage form and excipients of the drug, and it can be prepared using conventional pharmaceutical dosage forms in the art, such as tablets, powders, capsules, injections, or oral liquids, etc.

[0038] The present invention also provides the use of the biphenyl compound shown in Formula I or a pharmaceutically acceptable salt of the biphenyl compound in the preparation of a drug for preventing and / or treating diabetes.

[0039] In a preferred embodiment of the present invention, the diabetes includes diabetes targeting α-glucosidase.

[0040] The present invention also provides a drug for preventing and / or treating diabetes, the active ingredient of which includes the biphenyl compound shown in Formula I or a pharmaceutically acceptable salt of the biphenyl compound, and also includes a pharmaceutically acceptable carrier.

[0041] In order to further illustrate the present invention, the uses of the biphenyl compound provided by the present invention in inhibiting the activity of α-glucosidase and in hypoglycemia are described in detail below in combination with examples, but they should not be construed as limiting the protection scope of the present invention.

[0042] In the embodiments of the present invention, the α-glucosidase (purity ≥ 90.0%) and acarbose (purity ≥ 98%) used were purchased from MedChemExpress, USA, and p-nitrophenyl-α-D-glucopyranoside (p-NPG) (purity ≥ 98%) was purchased from Yuanye Bio-Technology Co., Ltd., Shanghai. THB was isolated and extracted from the leaves of Sorbus pohuashanensis and was disclosed in the article (Song C, Wang X, Yang J, Kuang Y, Wang Y, Yang S, Qin J, Guo L. Antifungal Biphenyl Derivatives from Sorbus pohuashanensis Leaves Infected by Alternaria tenuissi and Their Effect against Crop Pathogens. Chem Biodivers. 2021 May;18(5):e2100079. doi: 10.1002 / cbdv.202100079. Epub 2021 Apr 6. PMID: 33821531.).

[0043] Example 1 Determination of the inhibition rate of THB

[0044] After mixing 2 μL of the biphenyl compound solution (final concentration 100 μM) with phosphate buffer solution (PBS, pH 6.8), 20 μL of α-glucosidase solution was added and the reaction was carried out in a 96-well microplate for 15 minutes. Subsequently, 20 μL of 10 mmol / L p-nitrophenyl-α-D-glucopyranoside (pNPG) substrate solution was added and incubation was continued for 30 minutes. Finally, 80 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction, and the absorbance value was measured at a wavelength of 405 nm using a microplate reader.

[0045] The results are shown in Table 1. At a concentration of 100 μM, HC-2 had the strongest inhibitory effect on α-glucosidase, with an inhibition rate of 98.90%, showing significant α-glucosidase inhibitory activity.

[0046] Table 1 Inhibition rates of HC series compounds on α-glucosidase at 100 μM

[0047]

[0048] After mixing the THB series concentration solution (final concentration 0.157 - 50 μM, 2 μL) with phosphate buffer (PBS, pH 6.8), add α-glucosidase solution (20 μL) and react in a 96-well microplate for 15 min. Subsequently, add 10 mmol / L pNPG substrate solution (20 μL) and continue to incubate for 30 min. Finally, add 0.2 mol / L Na2CO3 solution (80 μL) to terminate the reaction, and measure the absorbance value at a wavelength of 405 nm using a microplate reader.

[0049] The results are as Figure 1 shown. THB exhibited significant α-glucosidase inhibitory activity, and the inhibitory effect of THB on α-glucosidase was concentration-dependent. When the concentration increased from 1.57 μM to 50 μM, the inhibitory effect gradually enhanced, and the highest inhibition rate reached 96.50%. Its IC 50 value was 11.52 μM, confirming the potent inhibitory activity of THB on α-glucosidase.

[0050] Example 2 Inhibition Kinetics of THB on α-Glucosidase

[0051] Refer to the protocol of Example 1. In the enzyme kinetics study, the absorbance of the mixed system of different concentrations of THB and α-glucosidase was measured, and a curve was plotted with the enzyme concentration on the abscissa to evaluate the reversibility of THB inhibition. In the substrate kinetics experiment, the absorbance of the mixed system of different THB concentrations and different substrate concentrations was recorded. The Lineweaver-Burk double reciprocal plot method was used to determine the inhibition type and calculate the inhibition constant (Ki). The Ki value of non-competitive inhibition was calculated by the following formula:

[0052] (1);

[0053] (2);

[0054] (3);

[0055] (4);

[0056] In the formula: v represents the initial reaction rate, [I] represents the inhibitor concentration, and [S] represents the substrate concentration.

[0057] The parameter α represents the ratio of the non-competitive inhibition constant to the competitive inhibition constant, and in pure non-competitive inhibition, α = 1.

[0058] The concentration of p-nitrophenyl glucoside (pNPG) was fixed at 10 mM, and the concentrations of THB were 0, 3.13, 6.25, 12.15, 25.00, 50.00 μM (corresponding to curves a→f in sequence). The relationship curves of the residual enzyme activity (v) and the enzyme concentration ([E]) at different inhibitor concentrations are shown as Figure 2 in A. All concentration curves are straight lines starting from the origin, and the slope decreases with the increase of the inhibitor concentration, indicating that the inhibition of THB is reversible. As a control, for irreversible inhibition, the curves corresponding to higher inhibitor concentrations should be parallel to the control group (same slope) and intersect at the same point on the abscissa ([E]).

[0059] The inhibition mechanism of THB on α-glucosidase was elucidated by constructing the Lineweaver-Burk double-reciprocal curve ( Figure 2 in C). The intersection points of all fitted straight lines are located on the x-axis. With the increase of the THB concentration, the slope of the curve gradually increases, while the Km value remains relatively stable (0.69 ± 0.02 mM, Table 2), and at the same time, Vmax decreases from 2.99 to 2.60 μM·min -1 . It indicates that THB acts as a non-competitive inhibitor of α-glucosidase. Further, a secondary curve was obtained by plotting the slope against [THB], and this linear relationship indicates that THB binds to a single inhibitory site on the enzyme molecule. In addition, the calculated K i value is 26.26 ± 4.95 μM, indicating a strong binding affinity between THB and α-glucosidase.

[0060] Table 2 The kinetic parameters of α-glucosidase change in a concentration-dependent manner with the change of inhibitor concentration

[0061]

[0062] Example 3 Study on the real-time inactivation kinetics of THB on α-glucosidase

[0063] A mixed system containing α-glucosidase (10 μg·mL -1 ), PBS buffer, and THB solutions at different concentrations (1.57 - 9.38 μM) was prepared in a 96-well plate. The mixed system was incubated at 37 °C for 0, 15, 30, 60, 120, and 180 min respectively. After the pre-incubation, 20 μL of 10 mM P-NPG solution was added to initiate the reaction. After reacting for 30 min, the absorbance at 405 nm was immediately measured. The kinetic curve was plotted with the steady-state rate of substrate hydrolysis (Vt / V0) as the ordinate and the corresponding pre-incubation time as the abscissa.

[0064] In the concentration range of 1.57 - 9.38 μM, no significant change in enzyme activity was observed during the co-incubation of THB with α-glucosidase ( Figure 2 in B). The enzyme inactivation induced by THB rapidly reached an equilibrium state, indicating that THB could rapidly bind to the enzyme and cause the loss of its catalytic activity, and this process did not show detectable kinetic characteristics.

[0065] Example 4 Synergistic inhibitory effect of THB and acarbose on α-glucosidase

[0066] The combination index (CI) method was used to evaluate the interaction between THB and acarbose in inhibiting α-glucosidase. The CI value was calculated by CompuSyn software, and its calculation formula is as follows:

[0067] (5);

[0068] In this equation: (Dx)1 and (Dx)2 represent the concentrations required for THB and acarbose to produce specific inhibitory effects alone, respectively; (D)1 and (D)2 represent the corresponding concentrations required when the two drugs are used in combination to achieve the same inhibitory effect. According to the CI value, the nature of drug interaction is determined: CI > 1.1 indicates antagonism, 0.9 ≤ CI ≤ 1.1 suggests additive effect, and CI < 0.9 characterizes synergistic inhibitory effect.

[0069] The Chou-Talalay method was used to evaluate the synergistic inhibitory effect of THB and acarbose on α-glucosidase. The results are as Figure 3 shown. All tested combinations showed significantly enhanced inhibitory activity compared with single drug use (treatment with THB or acarbose alone). Quantitative analysis showed that the CI values were between 0.32 and 1.05, confirming the synergistic or additive effect of these two compounds in inhibiting α-glucosidase. From the perspective of the mechanism of action, this effect stems from their different binding modes on the enzyme molecule: acarbose, as a classical competitive inhibitor, occupies the active center of α-glucosidase, while THB exerts non-competitive inhibition by binding to the allosteric site. This complementary binding mechanism enables the two inhibitors to interact with the enzyme simultaneously, thus achieving synergistic inhibition of catalytic activity.

[0070] Example 5 SPR experiment

[0071] SPR analysis was performed using a Biacore 8K surface plasmon resonance instrument. First, the surface of the CM5 sensor chip was activated with an NHS / EDC mixture, and then α-glucosidase was immobilized using an Amine Coupling Kit (GE Healthcare Life Sciences, catalog number BR100050). The affinity assay was strictly performed according to the manufacturer's specifications, and a reference channel without coupled protein was set up to subtract non-specific binding. Analytes were injected at a constant flow rate of 30 μL / min with a concentration gradient of 6.25 - 100 μM, and the response value changes during the binding-dissociation process were monitored in real time. Finally, the SPR curve was fitted based on the 1:1 Langmuir binding model using Biacore Insight Evaluation software to calculate the binding constant and kinetic parameters.

[0072] The results are as Figure 4 shown. SPR analysis indicated that the binding of THB to α-glucosidase was concentration-dependent, and its dissociation constant (KD value) was 3.91×10 -5 M, suggesting a medium-strength binding affinity between the two.

[0073] Example 6 Circular dichroism experiment

[0074] The circular dichroism (CD) spectrum was measured using a Jasco J-810 circular dichroism spectrometer. To detect secondary structure changes, the measurement was carried out in the far-ultraviolet range (190 - 260 nm). The instrument parameters were set as follows: optical path 1 nm, scanning rate 50 nm / min. The experiment used a 3 mM fixed-concentration THB solution, and the α-glucosidase concentration was maintained at 0.05 μg / μL. The CD data were analyzed using CDNN software to quantitatively analyze the changes in the secondary structure components of α-glucosidase.

[0075] The CD spectral analysis of α-glucosidase showed characteristic negative peaks at 210 nm and 216 nm (Figure 5), indicating α-helical and β-sheet secondary structures, respectively. After quantitative analysis of the CD spectrum using CDNN software, significant structural changes were found after binding to THB (Table 3). The secondary structure composition of native α-glucosidase was 14.70% α-helical, 7.80% antiparallel β-sheet, 3.00% parallel β-sheet, 27.30% β-turn, and 46.80% random coil.

[0076] After binding to THB, obvious structural reorganization was observed: the α-helical content decreased from 14.70% to 6.30%, and the β-turn content decreased from 27.30% to 17.30%. At the same time, the content of ordered structural elements increased significantly. The content of antiparallel β-sheet increased from 7.80% to 19.50%, the content of parallel β-sheet increased from 3.00% to 3.10%, and the content of random coil increased from 46.80% to 50.10%. These structural transformations indicate that the ligand induced the compression of the overall conformation of the enzyme, which may lead to steric hindrance at the active site. The observed structural reorganization may hinder the accessibility of the substrate and the correct orientation of the catalytic pocket, thereby reducing the catalytic efficiency of the enzyme. It is demonstrated that the binding of THB induces significant structural reorganization.

[0077] Table 3 Results of circular dichroism spectroscopy

[0078]

[0079] Example 7 Molecular docking

[0080] Molecular docking simulations were carried out using AutoDock 4.2 (https: / / autodock.scripps.edu / ). The crystal structure of Saccharomyces cerevisiae α-glucosidase (PDB ID: 3A4A, resolution 1.60 Å, wild type) was selected for the experiment, and this structure was obtained from the Protein Data Bank (https: / / www.rcsb.org / ). Before docking, the protein structure was preprocessed, including: removing water molecules, adding hydrogen atoms, assigning atomic charges, and removing the co-crystallized ligand maltose. The three-dimensional structure of THB was constructed by ChemDraw3D and optimized by energy minimization using Open Babel.

[0081] In the docking simulation, α-glucosidase was used as a rigid receptor, while the THB ligand remained completely flexible. The Site Finder tool in MOE (Molecular Operating Environment) software was used to predict the active site, and the potential binding conformations were explored by the Lamarckian genetic algorithm (LGA) built into AutoDock. The optimal binding configuration was screened according to the lowest docking score of the enzyme-ligand complex, and the key interaction characteristics, including hydrogen bonds, hydrophobic interactions, and other important binding site interactions, were further analyzed using BIOVIA Discovery Studio 2021.

[0082] First, the reliability of the docking protocol was verified by redocking the co-crystallized ligand (maltose), and the obtained root mean square deviation (RMSD) was 0.9821 Å, significantly lower than the acceptable threshold of 2.0 Å, confirming that this computational method can accurately reproduce the experimentally observed binding conformation. Subsequently, the docking analysis of THB with Saccharomyces cerevisiae α-glucosidase (PDB ID: 3A4A) showed that THB had excellent binding affinity, with a docking score of -5.4340, comparable to that of the reference compound maltose (-5.9706). As Figure 6 shown in A-B, THB formed a rich interaction network within the enzyme active site, including: hydrogen bonding, π-cation interaction, π-anion interaction, π-alkyl interaction, and van der Waals forces. In addition, hydrogen bonds were formed between THB and four key residues, ASP-69, ASP-215, GLU-277, and TYR-158 ( Figure 6 shown in C-D), and these interactions are likely to play an important regulatory role by inducing conformational changes in the enzyme.

[0083] Example 8 Molecular Dynamics Simulation

[0084] The most favorable docking pose of the THB-α-glucosidase complex determined by molecular docking in Example 7 was selected as the starting conformation for MD analysis. All simulations were performed using GROMACS V2020 and the AMBER03 force field, and the ligand topology was generated by the Sobtop software. A simulation box was constructed centered on the protein-ligand complex, and the minimum distance from the edge of the simulation box was ensured to be 10 Å. The system was filled with TIP3P water molecules and 16 Na + ions were added for neutralization. Energy minimization was performed using the steepest descent algorithm until equilibrium was reached. Subsequently, the system underwent a two-step equilibration process: first, a 1 ns canonical ensemble (NVT) simulation, and then a 1 ns isothermal-isobaric (NPT) simulation. Both steps were carried out at 310.15 K and 1.0 ATM. Finally, a 200 ns molecular dynamics simulation was performed using the equilibrated system, and the obtained trajectory was analyzed in detail.

[0085] Considering the inherent flexibility of the receptor and ligand, performing MD simulations on the docking complex can improve and verify the accuracy of the molecular docking results. For this purpose, the present invention performed MD simulations on the optimized docking complex within a time range of 200 ns. The stability of the protein-ligand complex was evaluated by monitoring the RMSD (root mean square difference) value of the Cα atoms relative to the initial conformation of the protein. As Figure 7As shown in A, during the MD generation stage (0 - 50 ns), the RMSD value fluctuates between 0.0 and 2.0 nm. After 50 ns, the RMSD value tends to be stable, with only minor fluctuations within the range of 2.0 to 3.0 nm.

[0086] As Figure 7 As shown in B, the X - axis represents the residue number, and the Y - axis corresponds to the RMSF (root - mean - square fluctuation) value. It is worth noting that the amino acid residues in the ranges of 120 - 160, 450 - 470, and 550 - 580 show relatively high RMSF values, indicating greater flexibility. These regions may be related to the loop structures in the protein, and due to the absence of small - molecule binding, these regions usually exhibit higher mobility. In contrast, the RMSF values of the remaining regions are all below 30 nm, indicating that the conformational changes occurring during the MD simulation are minimal. This observation emphasizes the overall stability of the THB protein complex because most of the protein structure remains rigid and well - defined throughout the simulation.

[0087] The average number of hydrogen bonds formed between THB and α - glucosidase during the 200 - ns simulation was analyzed ( Figure 7 in C). THB and α - glucosidase formed up to 6 hydrogen bonds, and 4 of these hydrogen bonds remained constant within the time range of 0 - 175 ns. These findings are consistent with the molecular docking results, highlighting the crucial role of hydrogen bonds as the main determinant of the binding between THB and α - glucosidase.

[0088] Example 9 MM / PBSA Binding Free Energy Calculation

[0089] The binding free energy of the THB-α-glucosidase complex was calculated using the molecular mechanics / Poisson-Boltzmann surface area (MM-PBSA) method. According to the calculation method established by Lolok et al. (Lolok, N., Sumiwi, S. A., Muhtadi, A., et al. (2021). Molecular docking and molecular dynamics studies of bioactive compounds contained in noni fruit (Morinda citrifolia L.) against human pancreatic α-amylase. Journal of Biomolecular Structure and Dynamics. 40(15), 7091-8.), the Poisson-Boltzmann equation was used for the calculation of the polar solvation energy, and the grid spacing was set to 0.5 Å; the solvent environment was selected as the aqueous solution system, and the dielectric constant was set to 80 accordingly; the non-polar solvation energy was estimated by calculating the solvent accessible surface area, and the probe radius was set to 1.4 Å. The binding free energy of the complex (ΔGbind) was obtained by subtracting the sum of the free energy of the unbound receptor (ΔGrec) and the free energy of the free ligand (ΔGlig) from the free energy of the complex (ΔGcomplex).

[0090] As shown in Table 4, the van der Waals force, electrostatic interaction, and non-polar solvation energy of the THB-α-glucosidase complex were all negative values, indicating that these interactions were favorable for binding; while the polar solvation energy was positive, suggesting that the polar solvation effect might partially weaken the binding of THB to α-glucosidase. It is worth noting that the calculated binding free energy was -15.38 ± 1.96 kcal / mol, confirming that THB could bind to α-glucosidase spontaneously and efficiently. This value was significantly lower than that of another natural biphenyl compound, honokiol (-4.9 kcal / mol), which was probably attributed to the smaller molecular size of THB. These calculation results were highly consistent with the previous molecular docking and molecular dynamics simulation results, further verifying the stability and high affinity of the THB-α-glucosidase complex.

[0091] Table 4 Molecular interactions of THB-α-glucosidase

[0092]

[0093] Example 10 Prediction of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties

[0094] Analysis was performed using the Swiss ADME online platform (http: / / www.swissadme.ch / ), and the prediction results were evaluated according to Lipinski's five rules. These rules stipulate that the molecular weight of the compound should be ≤500 Da; the logarithm of the lipophilicity-hydrophilicity partition coefficient (log P) < 5; the number of hydrogen bond donors (HBD) ≤ 5; and the number of hydrogen bond acceptors (HBA) < 10. In addition, the admetSAR tool (http: / / lmmd.ecust.edu.cn / admetsar2) was used to predict the toxicity characteristics of THB, so as to comprehensively evaluate its pharmacological potential.

[0095] HepG2 and MDA-MB-231 cells were seeded into 96-well plates at a density of 8×10 3 cells per well. After the cells adhered overnight, they were treated with different concentrations (0, 31.25, 62.5, 125, 250, and 500 μM) of THB for 24 h. After treatment, the CCK-8 kit was used to detect cell viability. Finally, the absorbance value of each well was measured at a wavelength of 450 nm using a microplate reader, and quantitative analysis was performed.

[0096] The ADMET properties of the compound THB were evaluated using Swiss ADME and ADMET SAR tools. The detailed results are shown in Tables 5 and 6. The analysis results indicate that THB conforms to Lipinski's "5" rule and has a high gastrointestinal (GI) absorption rate, indicating its good drug-like properties. In addition, THB did not exhibit AMES toxicity (mutagenicity), nor any carcinogenic, hepatotoxic, nephrotoxic, respiratory, reproductive, or hemolytic toxicity, highlighting its non-toxic characteristics. The cytotoxicity test further confirmed the low toxicity of THB. After 24 hours of exposure, its IC 50 values for MDA-MB-231 and HepG2 cells exceeded 250 μM ( Figure 8 ). In terms of drug interactions, THB was identified as a non-inhibitor of CYP2D6 and CYP3A4. This means that THB will not interfere with the metabolism of CYP2D6 and CYP3A4 substrates. In summary, the ADMET characteristics of THB highlight its potential as an α-glucosidase inhibitor for the treatment of diabetes, and its good pharmacokinetics and safety also support this.

[0097] Table 5 Evaluation of the drug-like properties of compound THB according to Lipinski's "5" rule

[0098]

[0099] Table 6 Prediction of the pharmacokinetics and biosafety of compound THB

[0100]

[0101] Example 11 Oral Sucrose Tolerance Test in Mice

[0102] Male ICR mice at 4 weeks of age and weighing 20 - 25 g were selected for the experiment. All SPF - level animals were housed in a temperature - controlled environment with a 12 - hour light / dark cycle, and had free access to standard pellet feed and drinking water. A 1 - week adaptive feeding was carried out before the experiment.

[0103] After the adaptive feeding, the mice were randomly divided into 4 groups (6 mice in each group). After a 12 - hour fast, they were intragastrically administered with:

[0104] ① Normal saline (blank control group);

[0105] ② Acarbose (15 mg / kg, positive control group);

[0106] ③ Low - dose THB group (2.76 mg / kg);

[0107] ④ High - dose THB group (8.3 mg / kg).

[0108] 15 minutes after administration, a sucrose load (3 g / kg body weight) was intragastrically administered. Tail vein blood was collected at 30, 60, and 120 min after the sucrose load, and a Yuetai brand blood glucose meter was used to measure the blood glucose value. The area under the blood glucose curve (AUC) within 120 min was calculated by the trapezoidal method to evaluate the overall blood glucose response.

[0109] The results are as Figure 9 shown. After the sucrose load, the blood glucose level of the mice first increased, reached the peak at 30 min, and then gradually decreased. At 60 min after administration, the post - prandial blood glucose levels in the high - dose THB group and the acarbose group were significantly lower than those in the control group. In addition, the area under the blood glucose curve (AUC) in the high - dose THB group and the acarbose group was also significantly lower than that in the control group ( Figure 10 ). It indicates that THB can effectively reduce the post - prandial blood glucose level in vivo.

[0110] Although the above - mentioned embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of 3,4,5-trihydroxybiphenyl or a pharmaceutically acceptable salt thereof in the preparation of a hypoglycemic drug.

2. Use of 2,3,4,5 - trihydroxybiphenyl or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating diabetes, characterized in that, The diabetes mellitus includes diabetes mellitus targeting α-glucosidase.

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