Application of biphenyl compound in inhibiting activity of alpha-glucosidase and application of biphenyl compound in reducing blood sugar

Abstract

The invention provides application of a biphenyl compound in inhibiting activity of alpha-glucosidase and application of the biphenyl compound in reducing blood sugar, and belongs to the technical field of biological medicine. The invention provides an application of a biphenyl compound as an alpha-glucosidase inhibitor and an application of the biphenyl compound in reducing the blood sugar level. The biphenyl compound can significantly inhibit the activity of alpha-glucosidase, the IC50 value of THB is 11.52 [mu] M, the dissociation constant is 3.91 * 10 <-5 > M, the inhibition effect of THB on alpha-glucosidase has concentration dependence, and the highest inhibition rate reaches 96.50%. The safety and in-vivo hypoglycemic activity of the biphenyl compound are evaluated, the postprandial blood sugar level can be effectively reduced in vivo, and the biphenyl compound has the potential of serving as a diabetes treatment medicine and the value of preparing auxiliary hypoglycemic food.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and specifically relates to the use of biphenyl compounds in inhibiting alpha-glucosidase activity and in reducing blood sugar. Background Art

[0002] Biphenyl compounds are composed of two phenyl groups connected by a single bond. They are used 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 the raw materials for engineering plastics polysulfones and are also used to make fuels, engineering plastics and high-energy fuels. They can also be used as solvents, especially in the fields of coatings, inks and adhesives. They can effectively dissolve many organic substances and improve the processing performance of products. Biphenyl is also an important intermediate in the synthesis of some organic pesticides such as rodenticides such as brodifacoum and brodifacoum. At the same time, in the field of medicine, biphenyl and its derivatives are used in drug synthesis. For example, in the synthesis of some anti-tumor drugs, biphenyl is an important intermediate, but there is very little research on biphenyl itself in medicine, especially in lowering blood sugar.

[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. Its molecular formula is C 12 H 10 O 3 . THB has multiple hydroxyl groups, which can provide hydrogen atoms and combine with free radicals in the body, thereby playing an antioxidant role, reducing the damage of free radicals to cells, and helping to prevent and treat diseases related to oxidative stress, such as cardiovascular diseases, neurodegenerative diseases, etc. THB also has anti-inflammatory activity. It can inhibit the activation of inflammation-related signaling pathways, reduce the release of inflammatory mediators, and exert anti-inflammatory effects. It has potential application value in the treatment of inflammatory diseases such as arthritis and enteritis. Studies have found that THB also has a certain inhibitory effect on certain bacteria and fungi, which may help to develop new antibacterial drugs for the treatment of bacterial and fungal infectious diseases. At the same time, THB is also an important intermediate for the synthesis of drugs with a variety of specific structures and properties. It can be seen that THB has a good application and research basis, but there is little research on THB in other aspects. Summary of the invention

[0004] The present invention provides the use of a biphenyl compound in inhibiting α-glucosidase activity and in reducing blood sugar. The biphenyl compound has a strong inhibitory activity on α-glucosidase and can significantly reduce the postprandial blood sugar level.

[0005] 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, wherein the biphenyl compound has a structure shown in Formula I: , Formula I; In the structure shown in Formula I, R 1 Including any one of -H, -OH and -OMe; R 2 Including any one of -H, -OH and -OMe; R 3 Including any one of -H, -OH and -OMe; R 4 Includes -H or -OMe.

[0006] 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.

[0007] The present invention also provides an α-glucosidase activity inhibitor, wherein the active ingredient comprises a biphenyl compound shown in Formula I or a pharmaceutically acceptable salt of the biphenyl compound.

[0008] The present invention also provides the use of the biphenyl compound or the pharmaceutically acceptable salt of the biphenyl compound having the structure shown in Formula I in the preparation of a drug for lowering blood sugar.

[0009] The present invention also provides the use of the biphenyl compound or the pharmaceutically acceptable salt of the biphenyl compound having the structure shown in Formula I in the preparation of food for assisting in lowering blood sugar.

[0010] The present invention also provides the use of a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound having a structure shown in Formula I in the preparation of a drug for preventing and / or treating diabetes.

[0011] In a preferred embodiment of the present invention, the diabetes mellitus includes diabetes mellitus targeting α-glucosidase.

[0012] The present invention also provides a food composition for assisting in lowering blood sugar, wherein the active ingredient in the food composition comprises a biphenyl compound having a structure shown in Formula I or a pharmaceutically acceptable salt of the biphenyl compound.

[0013] The present invention also provides a drug for preventing and or treating diabetes, wherein the active ingredient comprises a biphenyl compound or a pharmaceutically acceptable salt of the biphenyl compound having a structure as shown in Formula I, and also comprises 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 the use in lowering blood sugar levels. The biphenyl compound of the present invention can bind to α-glucosidase, such as THB, which has a moderate binding affinity with α-glucosidase, and its dissociation constant (KD value) is 3.91×10 -5 M, and the binding is concentration-dependent. The biphenyl compound of the present invention can significantly inhibit the activity of α-glucosidase, such as the IC of THB. 50 The value is 11.52 μM, 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 in vivo hypoglycemic activity of THB. THB can effectively reduce postprandial blood sugar levels in vivo, has the potential to be used as a diabetes treatment drug, and has the value of preparing auxiliary hypoglycemic foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the statistical graph of the inhibitory activity of THB on α-glucosidase (n=3); Figure 2 The results of the inhibition kinetics of THB on α-glucosidase are shown in Figure 1. A: the relationship curve between the enzyme reaction rate (ν) and the [α-glucosidase] concentration; B: the real-time kinetic process of α-glucosidase activity inhibition; C: Lineweaver-Burk double reciprocal plot of THB inhibiting α-glucosidase; D: the secondary curve of THB inhibiting α-glucosidase; Figure 3 This is the result diagram of the inhibition of α-glucosidase by the combination of THB and acarbose. The numbers marked above the combination line are the combined index (CI) values ​​calculated by CompuSyn software. Figure 4 This is the SPR analysis result of the binding affinity between α-glucosidase and THB; Figure 5 Circular dichroism spectra, A: circular dichroism spectrum of α-glucosidase; B: circular dichroism spectrum of α-glucosidase and THB complex; Figure 6 The simulation results of molecular docking analysis of THB and α-glucosidase are shown in Figure A: THB-α-glucosidase complex structure; B: THB binding site on the surface of α-glucosidase; C: two-dimensional interaction mode of THB binding to α-glucosidase; D: THB molecule in the binding pocket; Figure 7The molecular dynamics simulation results of the complex of α-glucosidase and THB are shown in 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; Figure 8 The toxicity verification results of THB are shown in Figure A: The survival rate of MDA-MB-231 cells after exposure to THB (0-500 μM) for 24 hours; B: The survival rate of HepG2 cells after exposure to THB (0-500 μM) for 24 hours; Fig. 9 The curve of postprandial blood glucose (PBG) changes in mice after sucrose loading; 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; Fig.10 The area under the blood glucose curve (AUC 0~120min ) Data are expressed as mean ± SD, n = 6; compared with the control group, *p < 0.05, ***p < 0.001, ****p < 0.0001; Con: control group; Acar: acarbose group. DETAILED DESCRIPTION

[0017] 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, wherein the biphenyl compound has a structure shown in Formula I: , Formula I; In the structure shown in Formula I, R 1 Including any one of -H, -OH and -OMe; R 2 Including any one of -H, -OH and -OMe; R 3 Including any one of -H, -OH and -OMe; R 4 Includes -H or -OMe.

[0018] In one embodiment of the present invention, the inhibitory activity of the biphenyl compound on α-glucosidase was verified, especially the inhibitory effect of 3,4,5-trihydroxybiphenyl (THB) on α-glucosidase was concentration-dependent. When the concentration increased from 1.57 μM to 50 μM, the inhibitory effect gradually increased, and the highest inhibition rate reached 96.50%. After determination, IC 50 The value was 11.52 μM, confirming the potent inhibitory activity of THB against α-glucosidase.

[0019] The structural formula of THB of the present invention is shown in Formula II: , Formula II.

[0020] The pharmaceutically acceptable salt of the biphenyl compound of the present invention can be prepared by mixing with NaOH, KOH or Ca(OH) 2 Pharmaceutically acceptable sodium salt, potassium salt or calcium salt formed by combination.

[0021] The present invention also provides an α-glucosidase activity inhibitor, wherein the active ingredient comprises a biphenyl compound shown in formula I or a pharmaceutically acceptable salt of the biphenyl compound.

[0022] In one embodiment of the present invention, it is verified that the THB has both in vitro and in vivo inhibitory activity against α-glucosidase, and therefore, the THB can be prepared into a corresponding inhibitor that can inhibit the activity of α-glucosidase. The present invention does not specifically limit other auxiliary ingredients of the inhibitor, as long as they can be combined with the THB to show the inhibitory activity of THB against α-glucosidase.

[0023] The present invention also provides the use of the biphenyl compound or the pharmaceutically acceptable salt of the biphenyl compound shown in formula I in the preparation of a drug for reducing blood sugar.

[0024] In one embodiment of the present invention, an oral experiment on mice was conducted to compare the postprandial hypoglycemic effects of THB and acarbose. The results showed that at 60 minutes after administration, the postprandial blood glucose levels of the high-dose THB group (dosage 8.3 mg / kg) and the acarbose group were significantly lower than those of the control group; the blood glucose area under the 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 postprandial blood glucose levels in vivo and can be used to prepare blood glucose-lowering drugs.

[0025] The present invention has no special limitation on the dosage form and auxiliary materials of the drug, and the drug can be prepared using conventional drug dosage forms in the art, such as tablets, powders, capsules, injections or oral solutions.

[0026] The present invention also provides the use of the biphenyl compound or the pharmaceutically acceptable salt of the biphenyl compound shown in formula I in the preparation of food for assisting in lowering blood sugar.

[0027] In one embodiment of the present invention, it is confirmed that THB has the effect of inhibiting α-glucosidase activity in vitro, and in another embodiment, it is confirmed that THB has the effect of lowering blood sugar in vivo. Therefore, the THB can be used as an effective ingredient of functional foods such as health foods to assist in lowering blood sugar.

[0028] 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.

[0029] In a preferred embodiment of the present invention, the diabetes mellitus includes diabetes mellitus targeting α-glucosidase.

[0030] The present invention also provides a food composition for assisting in lowering blood sugar, wherein the active ingredient in the food composition comprises a biphenyl compound as shown in formula I or a pharmaceutically acceptable salt of the biphenyl compound.

[0031] The present invention does not specifically limit the types and sources of the remaining raw materials of the food composition except the THB, and the food composition can be prepared using conventional edible ingredients in the art.

[0032] The present invention also provides a drug for preventing and or treating diabetes, wherein the active ingredient comprises a biphenyl compound or a pharmaceutically acceptable salt of the biphenyl compound as shown in Formula I, and also comprises a pharmaceutically acceptable carrier.

[0033] In order to further illustrate the present invention, the use of the biphenyl compound provided by the present invention in inhibiting α-glucosidase activity and in reducing blood sugar is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] The α-glucosidase (purity ≥ 90.0%) and acarbose (purity ≥ 98%) used in the examples of the present invention were purchased from MedChemExpress, USA, and p-nitrophenol-α-D-pyranoglucoside (p-NPG) (purity ≥ 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. 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. AntifungalBiphenyl Derivatives from Sorbus pohuashanensis Leaves Infected by Alternariatenuissi and Their Effect against Crop Pathogens. Chem Biodivers. 2021 May;18(5):e2100079. doi: 10.1002 / cbdv.202100079. Epub 2021 Apr 6. PMID: 33821531.).

[0035] Example 1 Determination of THB inhibition rate After mixing 2 μL of biphenyl compound solution (final concentration 100 μM) with phosphate buffer (PBS, pH 6.8), α-glucosidase solution (20 μL) was added to a 96-well microplate for 15 minutes. Subsequently, 10 mmol / L p-nitrophenol-α-D-pyranoglucoside (pNPG) substrate solution (20 μL) was added and incubated for another 30 minutes. Finally, 0.2 mol / L Na 2 CO 3 The reaction was terminated by adding 80 μL of the solution, and the absorbance was measured at 405 nm using an enzyme reader.

[0036] 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.

[0037] Table 1 Inhibition rate of HC series compounds on α-glucosidase at 100 μM

[0038] After mixing THB series concentrations (final concentration 0.157~50 μM) solution (2 μL) with phosphate buffer (PBS, pH 6.8), α-glucosidase solution (20 μL) was added to a 96-well microplate for 15 min. Then, 10 mmol / L pNPG substrate solution (20 μL) was added and incubated for 30 min. Finally, 0.2 mol / L Na 2 CO 3 The reaction was terminated by adding 80 μL of the solution, and the absorbance was measured at 405 nm using an enzyme reader.

[0039] The results are as follows Figure 1 As shown in the results, 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 increased, and the highest inhibition rate reached 96.50%. Its IC 50 The value was 11.52 μM, confirming the potent inhibitory activity of THB against α-glucosidase.

[0040] Example 2 Inhibition kinetics of THB on α-glucosidase Refer to the scheme of Example 1. In the enzyme kinetics study, the absorbance of the mixed system of THB with different concentrations and α-glucosidase was measured, and a curve was drawn with the enzyme concentration as the horizontal axis 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: (1); (2); (3); (4); Where: v represents the initial reaction rate, [I] represents the inhibitor concentration, and [S] represents the substrate concentration.

[0041] The parameter α represents the ratio of the noncompetitive inhibition constant to the competitive inhibition constant, and in pure noncompetitive inhibition α=1.

[0042] The concentration of p-nitrophenol glucoside (pNPG) was fixed at 10 mM, and the concentrations of THB were 0, 3.13, 6.25, 12.15, 25.00, and 50.00 μM (corresponding to curves a→f, respectively). The relationship between the residual activity (v) of the enzyme and the enzyme concentration ([E]) at different inhibitor concentrations is shown in the figure. Figure 2 As shown in Figure A, all concentration curves are straight lines starting from the origin, and the slope decreases as the inhibitor concentration increases, indicating that the inhibitory effect of THB is reversible. As a control, if it is irreversible inhibition, the curve corresponding to the higher inhibitor concentration should remain parallel to the control group (same slope) and intersect at the same point on the horizontal axis ([E]).

[0043] The inhibitory mechanism of THB on α-glucosidase was elucidated by constructing Lineweaver-Burk double reciprocal curve ( Figure 2 C). The intersection points of all fitted straight lines are located on the x-axis. As the THB concentration increases, the slope of the curve gradually increases, while the Km value remains relatively stable (0.69 ± 0.02 mM, Table 2), while Vmax decreases from 2.99 to 2.60 μM·min -1 This indicates that THB acts as a noncompetitive inhibitor of α-glucosidase. Further, the slope was plotted against [THB] to obtain a second-order curve, and this linear relationship indicates that THB binds to a single inhibitory site on the enzyme molecule. In addition, the calculated K i The value was 26.26±4.95 μM, indicating that there was a strong binding affinity between THB and α-glucosidase.

[0044] Table 2 Kinetic parameters of α-glucosidase showed concentration-dependent changes with inhibitor concentration

[0045] Example 3 Real-time inactivation kinetics of α-glucosidase by THB Prepare a 96-well plate containing α-glucosidase (10 μg mL -1 ), PBS buffer and different concentrations of THB solution (1.57~9.38 μM). The mixed system was incubated at 37℃ for 0, 15, 30, 60, 120 and 180 min. After the pre-incubation, 20 μL of 10 mM P-NPG solution was added to start the reaction. After the reaction lasted for 30 min, the absorbance at 405 nm was immediately measured. The kinetic curve was drawn with the steady-state rate of substrate hydrolysis (Vt / V0) as the ordinate and the corresponding pre-incubation time as the abscissa.

[0046] No significant changes in enzyme activity were observed during the co-incubation of THB with α-glucosidase in the concentration range of 1.57-9.38 μM ( Figure 2 (B). THB-induced enzyme inactivation quickly reached equilibrium, indicating that THB could rapidly bind to the enzyme and cause the loss of its catalytic activity without any detectable kinetic characteristics.

[0047] Example 4 Synergistic inhibitory effect of THB and acarbose on α-glucosidase The combination index (CI) method was used to evaluate the interaction between THB and acarbose in inhibiting α-glucosidase. The CI value was calculated using CompuSyn software, and the calculation formula is as follows: (5); In this equation: (Dx) 1 and (Dx) 2 Respectively represent the concentrations required for THB and acarbose to produce specific inhibitory effects when acting alone; (D) 1 and (D) 2 It represents the corresponding concentration required to achieve the same inhibitory effect when the two drugs are used together. The nature of drug interaction is determined based on the CI value: CI>1.1 indicates an antagonistic effect, 0.9≤CI≤1.1 indicates an additive effect, and CI<0.9 indicates a synergistic inhibitory effect.

[0048] The Chou-Talalay method was used to evaluate the synergistic inhibitory effect of THB and acarbose on α-glucosidase. Figure 3As shown, all tested combinations showed significantly enhanced inhibitory activity compared to single drug treatment (THB or acarbose alone). Quantitative analysis showed that the CI values ​​were between 0.32 and 1.05, confirming that the two compounds had a synergistic or additive effect in inhibiting α-glucosidase. From the perspective of mechanism of action, this effect stems from the differentiated binding modes of the two on the enzyme molecule: acarbose, as a classic competitive inhibitor, occupies the active center of α-glucosidase, while THB exerts a non-competitive inhibitory effect by binding to the allosteric site. This complementary binding mechanism enables the two inhibitors to interact with the enzyme simultaneously, thereby achieving synergistic inhibition of catalytic activity.

[0049] Example 5 SPR experiment SPR analysis was performed using a Biacore 8K surface plasmon resonance instrument. First, the surface of the CM5 sensor chip was activated with a mixture of NHS / EDC, and then α-glucosidase was immobilized using an amine coupling kit (Amine Coupling Kit, GE Healthcare Life Sciences, catalog number BR100050). The affinity determination strictly followed the manufacturer's operating specifications, and a reference channel for uncoupled protein was set to deduct non-specific binding. The analyte was injected with a concentration gradient of 6.25-100 μM and a constant flow rate of 30 μL / min, and the response value changes of the binding-dissociation process were monitored in real time. Finally, the Biacore Insight Evaluation software was used to fit the SPR curve based on the 1:1 Langmuir binding model to calculate the binding constant and kinetic parameters.

[0050] The results are as follows Figure 4 As shown in Figure 2, SPR analysis showed that the binding of THB to α-glucosidase was concentration-dependent, and its dissociation constant (KD value) was 3.91×10 -5 M, indicating that the two have a moderate binding affinity.

[0051] Example 6 Circular dichroism experiment The circular dichroism (CD) spectra were measured using a Jasco J-810 circular dichroism spectrometer. To detect changes in the secondary structure, the measurements were performed in the far-ultraviolet band (190-260 nm). The instrument parameters were set as follows: pathlength 1 nm, scanning rate 50 nm / min. A 3 mM fixed concentration THB solution was used in the experiment, and the α-glucosidase concentration was kept 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.

[0052] CD spectrum 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. Quantitative analysis of the CD spectrum using CDNN software revealed that the structure changed significantly after binding with THB (Table 3). The secondary structure of native α-glucosidase consists of 14.70% α-helical, 7.80% antiparallel β-sheet, 3.00% parallel β-sheet, 27.30% β-turn, and 46.80% random coil.

[0053] After binding with THB, a significant 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, with the content of antiparallel β-sheet increasing from 7.80% to 19.50%, the content of parallel β-sheet increasing from 3.00% to 3.10%, and the content of random coil increasing from 46.80% to 50.10%. These structural transitions indicate that the ligand induces a compression of the overall conformation of the enzyme, which may lead to steric hindrance of 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 a significant structural reorganization.

[0054] Table 3 Circular dichroism results

[0055] Example 7 Molecular docking AutoDock 4.2 (https: / / autodock.scripps.edu / ) was used for molecular docking simulation. The crystal structure of Saccharomyces cerevisiae α-glucosidase (PDB ID: 3A4A, resolution 1.60 Å, wild type) was used in the experiment, which was obtained from the Protein Data Bank (https: / / www.rcsb.org / ). The protein structure was preprocessed before docking, including: removing water molecules, adding hydrogen atoms, assigning atomic charges, and removing the co-crystallization ligand maltose. The three-dimensional structure of THB was constructed using ChemDraw3D, and Open Babel was used for energy minimization optimization.

[0056] In the docking simulation, α-glucosidase acts as a rigid receptor, while the THB ligand remains completely flexible. The Site Finder tool in the MOE (Molecular Operating Environment) software was used to predict the active site, and the potential binding conformations were explored using the Lamarckian genetic algorithm (LGA) built into AutoDock. The optimal binding configuration was screened based on the minimum docking score of the enzyme-ligand complex, and BIOVIA Discovery Studio 2021 was further used to analyze key interaction features, including hydrogen bonds, hydrophobic interactions, and other important binding site interactions.

[0057] First, the reliability of the docking scheme was verified by redocking the co-crystallized ligand (maltose), and the root mean square deviation (RMSD) obtained was 0.9821 Å, which was significantly lower than the acceptable threshold of 2.0 Å, confirming that the computational method can accurately reproduce the binding conformation observed in the experiment. Subsequent 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, which was comparable to the reference compound maltose (-5.9706). Figure 6 As shown in Figure AB, THB forms a rich interaction network in the enzyme active site, including hydrogen bonding, π-cation interaction, π-anion interaction, π-alkyl interaction, and van der Waals force. In addition, THB forms hydrogen bonds with four key residues, ASP-69, ASP-215, GLU-277, and TYR-158 ( Figure 6 These interactions are likely to play an important regulatory role by inducing conformational changes in the enzyme.

[0058] Example 8 Molecular dynamics simulation 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 AMBER03 force field, and the ligand topology was generated by Sobtop software. A simulation box was constructed with the protein-ligand complex as the center, and the minimum distance to the edge of the simulation box was ensured to be 10 Å. The system was filled with TIP3P water molecules, and 16 Na +The ions were neutralized. 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, followed by a 1 ns isothermal and isobaric (NPT) simulation. Both steps were performed at 310.15 K and 1.0 ATM. Finally, a 200 ns molecular dynamics simulation was performed using the equilibrated system, and the trajectories obtained from the simulation were analyzed in detail.

[0059] Considering the inherent flexibility of receptors and ligands, MD simulations of docked complexes can improve and verify the accuracy of molecular docking results. To this end, the present invention performed MD simulations on the optimized docked complexes within a time range of 200 ns. The stability of the protein-ligand complex was evaluated by monitoring the RMSD (root mean square deviation) value of the Cα atom relative to the initial conformation of the protein. Figure 7 As shown in Figure 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 slight fluctuations in the range of 2.0 to 3.0 nm.

[0060] like Figure 7 As shown in Figure 2B, the X-axis represents the residue number and the Y-axis corresponds to the RMSF (root mean square fluctuation) value. It is noteworthy that the amino acid residues in the range of 120~160, 450~470, and 550~580 show higher RMSF values, indicating greater flexibility. These regions may be related to the loop structures in the protein, which usually show higher fluidity due to the absence of small molecule binding. In contrast, the RMSF values ​​of the remaining regions were all below 30 nm, indicating that minimal conformational changes occurred during the MD simulation. This observation emphasizes the overall stability of the THB protein complex, as most of the protein structure remained rigid and well-defined throughout the simulation.

[0061] The average number of hydrogen bonds formed between THB and α-glucosidase during the 200 ns simulation was analyzed ( Figure 7 (C), THB α-glucosidase formed up to 6 hydrogen bonds, 4 of which remained constant over the time range of 0-175 ns. These findings are consistent with the molecular docking results, highlighting the key role of hydrogen bonds as the main determinant of THB binding to α-glucosidase.

[0062] Example 9 Calculation of MM / PBSA binding free energy The molecular mechanics / Poisson-Boltzmann surface area (MM-PBSA) method was used to calculate the binding free energy of the THB-α-glucosidase complex. According to the calculation method established by Lolok et al. (Lolok, N., Sumiwi, SA, 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 polar solvation energy was calculated using the Poisson-Boltzmann equation, and the grid spacing was set to 0.5Å; the solvent environment selected the aqueous solution system, and the corresponding dielectric constant was set to 80; the non-polar solvation energy was estimated by calculating the solvent accessible surface area, and the probe radius was set to 1.4Å. The complex binding free energy (ΔGbind) was obtained by deducting the sum of the unbound receptor free energy (ΔGrec) and the free ligand free energy (ΔGlig) from the complex free energy (ΔGcomplex).

[0063] As shown in Table 4, the van der Waals force, electrostatic interaction and nonpolar solvation energy of the THB-α-glucosidase complex are all negative, indicating that these interactions are conducive to binding; while the polar solvation energy is positive, suggesting that the polar solvation effect may partially weaken the binding of THB to α-glucosidase. It is worth noting that the calculated binding free energy is -15.38±1.96 kcal / mol, confirming that THB can spontaneously and efficiently bind to α-glucosidase. This value is significantly lower than that of another natural biphenyl compound and magnolol (-4.9 kcal / mol), which is likely due to the smaller molecular size of THB. These calculation results are highly consistent with previous molecular docking and kinetic simulation results, further verifying the stability and high affinity of the THB-α-glucosidase complex.

[0064] Table 4 THB-α-glucosidase molecular interactions

[0065] Example 10 ADMET (absorption, distribution, metabolism, excretion and toxicity) property prediction The Swiss ADME online platform (http: / / www.swissadme.ch / ) was used for analysis, and the prediction results were evaluated according to the Lipinski five rules. The rule stipulates that the molecular weight of the compound should be ≤500Da; the logarithm of the lipid-water partition coefficient (log P) should be <5; the number of hydrogen bond donors (HBD) should be ≤5; and the number of hydrogen bond acceptors (HBA) should be <10. In addition, the admetSAR tool (http: / / lmmd.ecust.edu.cn / admetsar2) was used to predict the toxicity characteristics of THB, thereby comprehensively evaluating its pharmacological potential.

[0066] HepG2 and MDA-MB-231 cells were cultured at 8 × 10 3 The cells were seeded in a 96-well plate at a density of 100 cells / mL. After the cells adhered overnight, they were treated with different concentrations of THB (0, 31.25, 62.5, 125, 250 and 500 μM) for 24 hours. After treatment, the cell viability was detected using a CCK-8 kit, and the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader for quantitative analysis.

[0067] The Swiss ADME and ADMET SAR tools were used to evaluate the ADMET properties of compound THB, and the detailed results are shown in Tables 5 and 6. The analysis results showed that THB complies with Lipinski's "5" rule and has a high gastrointestinal (GI) absorption rate, indicating that it has good drug-like properties. In addition, THB did not show AMES toxicity (mutagenicity) nor any carcinogenicity, hepatotoxicity, nephrotoxicity, respiratory toxicity, reproductive toxicity, or hemolytic toxicity, emphasizing its non-toxic characteristics. The low toxicity of THB was further confirmed by cytotoxicity tests, and its IC 20 for MDA-MB-231 and HepG2 cells was 0.54 after 24 hours of exposure. 50 Values ​​exceeding 250 μM ( Figure 8 ). In terms of drug interactions, THB was determined to be a non-inhibitor of CYP2D6 and CYP3A4. This means that THB does not interfere with the metabolism of CYP2D6 and CYP3A4 substrates. In conclusion, the ADMET characteristics of THB highlight its potential as an α-glucosidase inhibitor for the treatment of diabetes, which is also supported by its good pharmacokinetics and safety profile.

[0068] Table 5 Evaluation of the drug-like properties of compound THB according to Lipinski's "5" rule

[0069] Table 6 Predicted pharmacokinetics and biosafety of compound THB

[0070] Example 11 Oral sucrose tolerance test in mice Male ICR mice aged 4 weeks and weighing 20-25 g were used in the experiment. All SPF animals were housed in a temperature-controlled environment with a 12-hour light / dark cycle and had free access to standard pelleted feed and drinking water. A one-week adaptive feeding was performed before the experiment.

[0071] After the adaptive feeding, the mice were randomly divided into 4 groups (6 mice in each group) and given oral administration after fasting for 12 hours: ①Normal saline (blank control group); ②Acarbose (15 mg / kg, positive control group); ③THB low-dose group (2.76 mg / kg); ④THB high-dose group (8.3 mg / kg).

[0072] Fifteen minutes after administration, sucrose loading (3 g / kg body weight) was administered by gavage. Blood was collected from the tail vein at 30, 60, and 120 minutes after sucrose loading, and blood glucose levels were measured using a Yuyue brand blood glucose meter. The area under the blood glucose curve (AUC) within 120 minutes was calculated by the trapezoidal method to evaluate the overall blood glucose response.

[0073] The results are as follows Fig. 9 As shown in the figure, after sucrose loading, the blood glucose level of mice first increased, reached a peak at 30 minutes, and then gradually decreased. At 60 minutes after administration, the postprandial blood glucose levels of the high-dose THB group and the acarbose group were significantly lower than those of the control group. In addition, the blood glucose area under the curve (AUC) of the high-dose THB group and the acarbose group was also significantly lower than that of the control group ( Fig.10 ). This indicates that THB can effectively reduce postprandial blood sugar levels in the body.

[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound in the preparation of an α-glucosidase activity inhibitor, characterized in that: The biphenyl compound has a structure shown in Formula I: , Formula I; 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.

2. The application according to claim 1, characterized in that: The concentration of the biphenyl compound in the α-glucosidase activity inhibitor is not less than 1.57 μM.

3. An α-glucosidase activity inhibitor, characterized in that The active ingredient includes a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound, wherein the biphenyl compound has a structure shown in Formula I: , Formula I; 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.

4. Use of a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound in the preparation of a drug for reducing blood sugar, characterized in that: The biphenyl compound has a structure shown in Formula I: , Formula I; 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.

5. Use of a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound in the preparation of a food for assisting in lowering blood sugar, characterized in that: The biphenyl compound has a structure shown in Formula I: , Formula I; 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.

6. Use of a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound in the preparation of a drug for preventing and / or treating diabetes, characterized in that: The biphenyl compound has a structure shown in Formula I: , Formula I; 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.

7. The use according to claim 6, characterized in that: The diabetes mellitus includes diabetes mellitus targeting α-glucosidase.

8. A food composition for assisting in lowering blood sugar, characterized in that: The active ingredient in the food composition includes a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound, and the biphenyl compound has a structure shown in Formula I: , Formula I; 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.

9. A drug for preventing and / or treating diabetes, characterized in that: The active ingredient includes a biphenyl compound or a pharmaceutically acceptable salt of a biphenyl compound, and also includes a pharmaceutically acceptable carrier; The biphenyl compound has a structure shown in Formula I: , Formula I; 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.

10. The drug according to claim 9, characterized in that: The biphenyl compound includes 3,4,5-trihydroxybiphenyl.

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