Methylphenol compounds, methods of making and using the same

Abstract

The application discloses methyl phenolic compounds, a preparation method and application thereof. Six new methyl phenolic compounds are separated from the skin of eleocharis tuberosa, and the compounds all show good inhibitory activity on alpha-glucosidase, and are stronger than the clinical first-line drug acarbose. The inhibitory activity of compound 3 is the best, enzyme kinetics research shows that the compound 3 plays a role as a reversible mixed inhibitor, ADME predicts that the compound 3 has good drug properties, including high gastrointestinal absorption and no CYP450 enzyme inhibition. The cytotoxicity experiment shows that the six compounds have no obvious growth inhibition on human embryonic kidney cells HEK293 at a concentration of 100 muM, and have good safety. The compounds can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, and are used for preparing a health care product for reducing blood sugar or a drug for treating diabetes. The preparation method is simple, easy to operate, suitable for industrial production, and is helpful to the high-value reuse of the skin of eleocharis tuberosa.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and in particular to methylphenolic compounds, their preparation methods, and applications. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia, primarily caused by insulin secretion defects or impaired biological action. With changing global lifestyles and an aging population, the incidence of diabetes is rising annually, becoming a major public health problem seriously threatening human health. Long-term hyperglycemia can lead to complications in multiple organs, including the cardiovascular system, kidneys, and retina, placing a heavy burden on patients' families and society.

[0003] In diabetes treatment strategies, controlling postprandial blood glucose levels is crucial. Alpha-glucosidase is a key enzyme on the brush border of the chorionic villi of the small intestine, responsible for catalyzing the breakdown of oligosaccharides and polysaccharides into monosaccharides (such as glucose), thereby promoting the absorption of carbohydrates in the intestine. Therefore, alpha-glucosidase inhibitors, by competitively inhibiting the activity of this enzyme, delay the digestion and absorption of carbohydrates, effectively reducing postprandial blood glucose peaks in diabetic patients, and are currently a widely used class of oral hypoglycemic drugs in clinical practice.

[0004] Currently, the main α-glucosidase inhibitors used in first-line clinical practice include acarbose, voglibose, and miglitol. Although these synthetic drugs have achieved significant efficacy in controlling blood sugar, they still have many limitations in practical application: Limited activity and high dosage: Existing drugs have relatively weak inhibitory activity against α-glucosidase, and often require high dosages to achieve the desired therapeutic effect.

[0005] Significant side effects: Due to the use of high doses and drug residues at non-target sites, undigested carbohydrates are fermented by bacteria in the colon, often leading to severe gastrointestinal adverse reactions such as bloating, abdominal pain, and diarrhea, which affects patient compliance.

[0006] Drug resistance issue: Long-term use of synthetic drugs with a single mechanism of action may lead to reduced efficacy or drug resistance in some patients.

[0007] In light of the aforementioned issues, the search for novel α-glucosidase inhibitors with novel structures, stronger activity, and higher safety profiles from natural products has become an important direction in current drug development. Naturally derived compounds typically possess better biocompatibility and multi-target regulatory potential.

[0008] Water chestnut ( Eleocharis dulcis(Burm. f.) Trin. ex Hensch. is a perennial aquatic plant widely cultivated in Asia. Its enlarged underground corm, called water chestnut (also known as horse chestnut, water caltrop, black yam, etc.), can be eaten raw as a fruit, used as a vegetable, or used to extract starch. The corm also has medicinal uses, stimulating appetite, detoxifying, aiding digestion, and promoting gastrointestinal health, thus possessing both edible and medicinal value. Modern chemical and pharmacological studies show that it is rich in polyphenols, polysaccharides, and other active ingredients, exhibiting antioxidant, antibacterial, anti-inflammatory, and anticancer biological activities. Water chestnut peel is a byproduct of water chestnut processing and is often discarded as waste, causing environmental pollution and a significant waste of water chestnut peel resources. Research on the chemical composition and biological activity of water chestnut peel can improve its utilization rate and economic value, which is of great significance to the development of the water chestnut industry. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention extracts a class of methylphenolic compounds with novel chemical structures from water chestnut peel. These compounds exhibit stronger inhibitory activity against α-glucosidase than the existing clinical drug acarbose, and can be used to prevent or treat hyperglycemia or diabetes.

[0010] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: This invention provides a class of methylphenol compounds, wherein the compounds are selected from one or more of compounds 1, 2, 3, 4a, 4b and 5, and have the structural formulas shown in formulas 1-3, 4a, 4b and 5: .

[0011] Preferably, the compound is compound 3, with the following structural formula. Compound 3 exhibits the strongest inhibitory activity against α-glucosidase.

[0012] .

[0013] The present invention also provides the use of the methylphenolic compounds in the preparation of α-glucosidase inhibitors.

[0014] The present invention also provides the use of the methylphenolic compounds in the preparation of hypoglycemic drugs or foods.

[0015] The present invention also provides the use of the methylphenolic compounds in the preparation of drugs for the prevention or treatment of diabetes.

[0016] As a further description of the above scheme: the application uses the above compound or its pharmaceutically acceptable salts, esters, or stereoisomers as active ingredients; the content of the active ingredient is 0.1-99.0% (w / w).

[0017] As a further description of the above scheme: the compound is used in combination with a pharmaceutically acceptable carrier, adjuvant or excipient to prepare an injection, injection, powder, tablet, oral liquid, capsule or granule.

[0018] The present invention also provides a method for preparing the methylphenolic compound, the method comprising the following steps: Step (1) Take the dried and crushed water chestnut peel, extract it with an organic solvent, and recover the solvent to obtain the total extract; Step (2) The total extract was dissolved in a small amount of water and separated by D101 macroporous resin. It was eluted sequentially with methanol or ethanol and water at a volume ratio of 0:100-95:5. The eluted fraction with a volume ratio of ethanol-water or methanol-water of 65-75:35-25 was concentrated under reduced pressure and then loaded onto a normal-phase silica gel column. It was eluted with dichloromethane / methanol at a volume ratio of 1:0-0:1. Under the detection of thin-layer chromatography, 10 fractions Fr. 1~Fr. 10 were obtained. Step (3) The Fr. 8 fraction was separated by silica gel column chromatography with dichloromethane / methanol as eluent at a ratio of 2:1 to 0:1. The fractions were combined by thin-layer chromatography to obtain nine fractions Fr. 8-1 to Fr. 8-9. The Fr. 8-6 fraction was separated by Sephadex LH-20 gel chromatography column with dichloromethane / methanol at a ratio of 1:1. The fractions were then purified by semi-preparative HPLC to obtain compounds 1-3.

[0019] As a further description of the above scheme: Fr. 2 fraction was separated by gel chromatography, eluted with dichloromethane / methanol at a volume ratio of 1:1, and the fractions were combined by thin-layer chromatography to obtain five fractions Fr. 2-1 to Fr. 2-5; Fr. 2-4 fraction was purified by semi-preparative HPLC, eluted with acetonitrile / water mobile phase to obtain compounds 4 and 5; among them, compound 4 was separated by chiral HPLC column, eluted with n-hexane / isopropanol at a ratio of 76:24 to obtain a pair of enantiomers 4a and 4b.

[0020] As a further description of the above scheme: the organic solvent in step (1) is selected from one or more of the following: ethyl acetate, acetone, n-butanol, ethanol, methanol, aqueous n-butanol, aqueous acetone, aqueous ethanol and aqueous methanol; the mass-volume ratio of the water chestnut peel to the organic solvent is 1:1-15.

[0021] This invention isolated and identified six novel methylphenolic compounds from water chestnut peel, which exhibited good inhibitory activity against α-glucosidase. Compound 3 showed a significant inhibitory concentration (IC50) against α-glucosidase. 50The concentration of compound 3 was 10.30 ± 0.27 μM, significantly superior to the clinical drug acarbose. Enzyme kinetic studies showed that compound 3 functioned as a reversible mixed inhibitor, and ADME prediction indicated good drug-like properties, including high gastrointestinal absorption and no CYP450 enzyme inhibition. Cytotoxicity experiments showed that compound 3 had no significant growth inhibitory effect on human embryonic kidney cells HEK293 at a concentration of 100 μM, demonstrating good safety and promising potential for the development of hypoglycemic drugs.

[0022] Compared with the prior art, the present invention has the following beneficial effects: Compounds 1-5 provided by this invention, especially the novel compound 3, exhibit extremely strong α-glucosidase inhibitory activity. Experimental data show that the compound's IC50... 50 All were significantly lower than the positive control drug acarbose. This means that the dosage of the compounds of this invention can be significantly reduced to achieve the same hypoglycemic effect, thereby potentially reducing drug side effects.

[0023] This invention, through enzyme kinetic studies, confirms that the core compound 3 exhibits reversible mixed-type inhibition of α-glucosidase with a defined inhibition constant. This well-defined mechanism indicates that the compound can effectively interfere with enzyme-substrate binding and catalytic processes, laying a solid theoretical foundation for subsequent drug molecule structure optimization and formulation development.

[0024] This invention also successfully achieved the chiral resolution of compound 4, obtaining enantiomers 4a and 4b with single configurations, and found that both exhibited excellent activity. This not only reveals the potential influence of the chiral center on biological activity, but also provides a material basis for the development of high-purity, high-specificity chiral drugs.

[0025] The compounds of this invention are derived from natural products and, compared to fully synthetic drugs, generally have better biocompatibility and potentially lower toxicity, which aligns with the current trend in the development of natural drugs and has promising clinical application prospects and market value. Attached Figure Description

[0026] Figure 1 The process flow diagrams for the preparation of compounds 1-5 are shown. Figure 2 For compound 1 1 H NMR spectrum; Figure 3 For compound 1 13 C NMR spectrum; Figure 4 The HR-ESI-MS spectrum of compound 1 is shown below. Figure 5 For compound 2 1 H NMR spectrum; Figure 6 For compound 2 13 C NMR spectrum; Figure 7 Here is the HR-ESI-MS spectrum of compound 2; Figure 8 For compound 3 1 H NMR spectrum; Figure 9 For compound 3 13 C NMR spectrum; Figure 10 Here is the HR-ESI-MS spectrum of compound 3; Figure 11 For compound 4 1 H NMR spectrum; Figure 12 For compound 4 13 C NMR spectrum; Figure 13 Here is the HR-ESI-MS spectrum of compound 4; Figure 14 The ECD spectra of compounds 4a / 4b are shown. Figure 15 For compound 5 1 H NMR spectrum; Figure 16 For compound 5 13 C NMR spectrum; Figure 17 Here is the HR-ESI-MS spectrum of compound 5; Figure 18 Experiments were conducted to determine the reversibility of α-glucosidase inhibition by compound 3, the inhibition model, and the determination of Ki and Ki′. In the figures, a is the v-[E] fitting curve for compound 3; b is the 1 / v-1 / [pNPG] fitting curve for compound 3; c is the 1 / v-[I] fitting curve for compound 3; d is the [pNPG] / v-[I] fitting curve for compound 3; e is the slope-[I] fitting curve for compound 3 obtained from a Lineweaver-Burk plot; f is the Y-intercept-[I] fitting curve for compound 3 obtained from a Lineweaver-Burk plot. All data are the mean ± standard deviation of three independent experiments. Figure 19 The three-dimensional and two-dimensional visualizations show the molecular docking of compound 3 with α-glucosidase protein. In the three-dimensional diagram, the compound molecule is shown in blue, the interacting amino acid residues are shown in yellow, hydrogen bonds are shown in purple, π-π stacking interactions are shown in green, and cation-π interactions are shown in red. Figure 20The results are shown in the molecular dynamics simulations of compound 3 and α-glucosidase protein. Specifically, a) is the root mean square fluctuation (RMSF) curve: a lower RMSF value indicates less fluctuation in the amino acid residues during binding; b) is the hydrogen bond fluctuation curve: more hydrogen bonds indicate stronger interactions and higher structural stability; c) is the radius of gyration (Rg) curve: a smaller Rg value indicates a more compact conformation and no significant expansion during the simulation; d) is the root mean square deviation (RMSD) curve: a lower RMSD value indicates less structural change and higher stability of the complex; e) is the solvent accessible surface area (SASA) curve: a lower and more stable SASA value indicates tighter conformational folding and a more stable state during binding; f) is the free energy distribution during the complex binding process: each trough corresponds to an equilibrium state, and the deeper the trough, the lower the free energy. Figure 21 ADME prediction results for compound 3: Bioavailability radar map obtained from the SwissADME online tool; Figure 22 Results of toxicity assays of compounds 1-5 on HEK293 cells; Note: compound concentration was 100 μM; data are expressed as mean ± standard deviation, n=3. Detailed Implementation

[0027] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these solutions are provided to make the understanding of the disclosure of the present invention more thorough and complete.

[0028] Unless otherwise stated, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and does not limit the scope of the invention in any way. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] Example 1: Preparation method of compounds 1-5 Preparation process flow of compounds 1-5: Based on experimental results, the preparation process flow of compounds 1-5 is summarized as follows: Figure 1 As shown, the specific operation is as follows.

[0030] Take dried water chestnut peel, crush it, and extract it three times with 3 times the amount (m / v) of 80% ethanol. Combine the extracts and recover the solvent under reduced pressure to obtain the total extract.

[0031] The total extract was dissolved in a small amount of water and separated by D101 macroporous resin, eluting sequentially with 0%, 30%, 70%, and 95% ethanol-water as mobile phases. The 70% ethanol-water fraction was concentrated under reduced pressure and then loaded onto a normal-phase silica gel column, eluted with a gradient of dichloromethane / methanol (volume ratios of 1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, 4–6 column volumes for each gradient). The fractions were combined to obtain 10 fractions Fr. 1–Fr. 10 under thin-layer chromatography (developing solvent: petroleum ether / ethyl acetate).

[0032] Fraction Fr. 8 was separated by silica gel column chromatography using dichloromethane / methanol as eluent (2:1, 1.5:1, 1:1, 1:2, 1:5, 0:1, 3-5 column volumes for each gradient), and the fractions were combined by thin-layer chromatography to obtain nine fractions Fr. 8-1 to Fr. 8-9. Fraction Fr. 8-6 was separated by Sephadex LH-20 gel chromatography column (dichloromethane / methanol 1:1), and then purified by semi-preparative HPLC using acetonitrile / water at a volume ratio of 30:70 to obtain compounds 1-3.

[0033] Fraction Fr. 2 was separated by gel permeation chromatography (dichloromethane / methanol 1:1), and the fractions were combined by thin-layer chromatography to obtain five fractions Fr. 2-1 to Fr. 2-5. Fraction Fr. 2-4 was purified by semi-preparative HPLC, eluting with acetonitrile / water at a volume ratio of 38:64 to obtain compounds 4 and 5. Compound 4 was separated by chiral HPLC (CHIRALPAK AD-H column (4.6 mm × 250 mm, 5 µm) with n-hexane / isopropanol (76:24) as the mobile phase to obtain a pair of enantiomers 4a and 4b.

[0034] Example 2: Structural characterization of compounds 1-5 Physical constants and spectral data of compound 1: pale yellow amorphous powder; - 8.00 ( c 0.12,MeOH); IR (KBr) ν max 3436cm -1 2959cm -1 2926cm -1 2857cm -1 1635cm -1 1384cm -1 1200cm -1 1073cm -1 and 1041 cm -1 ; 1 H NMR and 13C1NMR data are shown in Table 1; HR-ESI-MS m / z 497.1646 [M +CF3COO] - (Calculated value: C) 21 H 27 F3O 10 (497.1640). Compound 1 1 The H NMR spectrum is shown in [reference]. Figure 2 Compound 1 13 The C NMR spectrum is shown below. Figure 3 The HR-ESI-MS spectrum of compound 1 is shown below. Figure 4 .

[0035] Physical constants and spectral data of compound 2: pale yellow amorphous powder; -44.55 ( c 0.11,MeOH); IR (KBr) ν max 3436cm -1 2970cm -1 2877cm -1 1636cm -1 1610cm -1 1384cm -1 1166cm -1 and 1073 cm -1 ; 1 H NMR and 13 C1NMR data are shown in Table 1; HR-ESI-MS m / z 387.1226 [M + Cl] - (Calculated value: C) 18 H 24 O7Cl, 387.1216). Compound 2 1 The H NMR spectrum is shown in [reference]. Figure 5 Compound 2 13 The C NMR spectrum is shown below. Figure 6 The HR-ESI-MS spectrum of compound 2 is shown below. Figure 7 .

[0036] Physical constants and spectral data of compound 3: pale yellow amorphous powder; -44.25 ( c 0.08,MeOH); IR (KBr) ν max 3438cm -1 2959cm -1 2855cm -1 1635cm -1 1384cm -11086cm -1 and 1037cm -1 ; 1 H NMR and 13 C1NMR data are shown in Table 1; HR-ESI-MS m / z 481.1687 [M + CF3COO] - (Calculated value: C) 21 H 27 F3O9, 481.1691). Compound 3 1 The H NMR spectrum is shown in [reference]. Figure 8 Compound 3 13 The C NMR spectrum is shown below. Figure 9 The HR-ESI-MS spectrum of compound 3 is shown in [reference needed]. Figure 10 .

[0037] Physical constants and spectral data of compound 4: yellow amorphous powder; IR (KBr) ν max 3435cm -1 2959cm -1 2856cm -1 1638cm -1 1603cm -1 1465cm -1 1304cm -1 1162cm -1 and 1111 cm -1 ; 1 H NMR and 13 C10 NMR data are shown in Table 2; HR-ESI-MS m / z 315.0876 [M - H] - (Calculated value: C) 17 H 15 O6, 315.0874). Compound 4 1 The H NMR spectrum is shown in [reference]. Figure 11 Compound 4 13 The C NMR spectrum is shown below. Figure 12 The HR-ESI-MS spectrum of compound 4 is shown in [reference needed]. Figure 13 Compounds 4a and 4b were obtained after resolution. To further determine their absolute configuration, the ECD spectra of compounds 4a / 4b were calculated using time-dependent density functional theory (TDDFT), as shown below. Figure 14 Compound 4a after resolution: a yellow amorphous powder; +22.67 (c 0.06, MeOH). Compound 4b: yellow amorphous powder; -19.71 ( c0.07,MeOH).

[0038] Physical constants and spectral data of compound 5: pale yellow needle-like crystals; -0.46 ( c 0.13, MeOH); IR (KBr) ν max 3431cm -1 2957cm -1 2875cm -1 and 1650 cm -1 ; 1 H NMR and 13 C10 NMR data are shown in Table 2; HR-ESI-MS m / z 193.0495 [M + H] + (Calculated value: C) 10 H9O 4， 193.0495); Compound 5 1 The H NMR spectrum is shown in [reference]. Figure 15 Compound 5 13 The C NMR spectrum is shown below. Figure 16 The HR-ESI-MS spectrum of compound 5 is shown below. Figure 17 .

[0039] Table 1. Compounds 1-3 1 H and 13 C NMR data

[0040] Note: Data were determined using deuterated methanol as the solvent. 1 H NMR 600 MHz, 13 C NMR 150 MHz).

[0041] Table 2. Compounds 4 and 5 1 H and 13 C NMR data

[0042] Note: Data were determined using deuterated DMSO as the solvent. 1 H NMR 600 MHz, 13 C NMR 150 MHz).

[0043] The final chemical structural formulas of compounds 1-5 are as follows: .

[0044] Example 3: α-glucosidase inhibitory activity of compounds 1-5 Alpha-glucosidase belongs to the class of oligosaccharide hydrolases. Its inhibitors reduce carbohydrate degradation and delay carbohydrate digestion and absorption by competitively inhibiting the action of glycosidases on the villi membrane of the small intestinal epithelium. This effectively lowers the peak postprandial blood glucose concentration in diabetic patients, thus achieving the goal of blood glucose control. Alpha-glucosidase inhibitors (such as acarbose) have become a widely used class of oral hypoglycemic drugs in clinical practice. Alpha-glucosidase inhibitor activity screening can be performed by detecting the in vitro enzymatic reaction between the enzyme and its substrate 4-Nitrophenyl α-D-glucopyranoside (PNPG, a maltose analogue). After the substrate is added to the enzyme reaction, it is catalyzed by the enzyme to decompose into p-nitrophenol (PNP) and glucose. PNP is a colored substance with maximum absorption at 405 nm, which can be measured using an enzyme-linked immunosorbent assay (ELISA) reader. The inhibitory activity of the sample can be calculated based on the OD value.

[0045] 2) Experimental methods α-glucosidase and PNPG were diluted to 0.25 U / mL and 2.5 mM, respectively, using PBS. Simultaneously, the test sample was diluted to an appropriate concentration using PBS. 60 μL of PBS, 10 μL of test sample, 10 μL of α-glucosidase, and 20 μL of PNPG were sequentially added to a 96-well plate, with six replicates per sample. The plate was incubated at 37°C for 50 min, and 40 μL of Na₂CO₃ (0.1 M) was added to quench the reaction. The absorbance of the reaction mixture was recorded at 405 nm, and the α-glucosidase inhibitory activity (%) relative to the blank was calculated.

[0046] Inhibition rate % = [ 1 - (As-Ab) / (Ac-Ab) ] × 100%; As -- Sample absorbance; Ac -- Blank absorbance; Ab -- Background absorbance.

[0047] Acarbose was used as a positive control in the experiment.

[0048] 3) Experimental Results Table 3. α-glucosidase inhibitory activities of compounds 1-5 and acarbose.

[0049] As shown in Table 3, compounds 1-5 all exhibited good α-glucosidase inhibitory activity, which was stronger than that of the first-line clinical drug acarbose. Among them, the new compound 3 showed the strongest inhibitory activity against α-glucosidase.

[0050] As shown in Table 3, compounds 1-5 all exhibited good α-glucosidase inhibitory activity, which was stronger than that of the first-line clinical drug acarbose. Among them, compound 3 showed the strongest inhibitory activity against α-glucosidase.

[0051] 4) Enzyme kinetic results To elucidate the mechanism of action of compound 3, this study investigated its kinetics of inhibiting α-glucosidase. The reversibility of inhibition and the inhibition model are as follows: Figure 18 As shown in a–f. Under the condition of fixed substrate concentration and varying enzyme concentration, the initial reaction rate plots of different concentrations of compound 3 (5 μM, 10 μM, 20 μM, and 40 μM) present a set of straight lines passing through the origin with gradually decreasing slopes. Figure 18 a). This indicates that compound 3 is a reversible inhibitor of α-glucosidase. In experiments with fixed enzyme concentrations and varying substrate concentrations, Lineweaver–Burk plots of different concentrations of compound 3 (5 μM, 10 μM, 20 μM, and 40 μM) show a set of intersecting straight lines in the third quadrant (a). Figure 18 (b) This indicates that compound 3 inhibits α-glucosidase activity via a mixed-mode approach, binding to both the free enzyme and the enzyme-substrate complex. By plotting the slope of the line against the inhibitor concentration in a Lineweaver–Burk plot, the dissociation constant (Ki) of compound 3 binding to the free enzyme was determined to be 15.08 μM. By plotting the intercept of the line against the inhibitor concentration in a Lineweaver–Burk plot, the dissociation constant Ki' of compound 3 binding to the enzyme-substrate complex was found to be 4.34 μM, indicating a stronger affinity for the enzyme-substrate complex.

[0052] 5) Molecular docking results of compound 3 The glucose unit of compound 3 forms stable hydrogen bonds with key residues at the α-glucosidase active site, including Ser157, Tyr158, and Arg315 (bond lengths of 1.9 Å, 2.0 Å, and 1.9 Å, respectively). These hydrogen bonds are crucial for stabilizing the enzyme-ligand complex, indicating that the glucose unit is an essential structure for the inhibition of α-glucosidase activity by compound 3. Furthermore, compound 3 forms carbon-hydrogen interactions with Asp307 and Phe314, and hydrophobic interactions with Val216, Phe178, Phe159, and Phe303, further enhancing the conformational stability of the complex. Simultaneously, compound 3 exhibits van der Waals interactions with numerous surrounding residues (such as Glu277 and Leu313). Figure 19Meanwhile, the calculated MM-GBSA binding energy is as low as −29.97 kcal / mol, reflecting the strong binding affinity between compound 3 and α-glucosidase. In summary, these multimodal interactions collectively constitute a robust binding network, maintaining the stability of the complex and supporting its potent inhibitory activity.

[0053] 6) Molecular dynamics simulation results Molecular dynamics simulations showed that the α-glucosidase-compound 3 complex exhibited high thermodynamic and structural stability during the 100 ns simulation. Root mean square fluctuation (RMSF) analysis indicated that the protein core region was highly rigid (RMSF < 0.3 nm for most residues). Figure 20 a) Only the surface ring region and the N-terminal region exhibit localized flexibility, which ensures the structural integrity of the active sites. Hydrogen bond analysis shows an average of approximately 3.2 hydrogen bonds (a). Figure 20 (b) Two to four hydrogen bonds remained stable for most of the simulation time, indicating that hydrogen bonding is the key driving force for maintaining binding stability. Correspondingly, the radius of gyration (Rg) increased from approximately 2.41 nm to approximately 2.47 nm within the first 10 ns, subsequently stabilizing between 2.46 and 2.48 nm. Figure 20 c); The root mean square deviation (RMSD) increased from approximately 0.75 nm to approximately 0.80 nm within the first 10 ns, and then stabilized in the range of 0.78–0.82 nm. Figure 20 d); Solvent-accessible surface area (SASA) increased from approximately 225 nm in the first 10 ns. 2 Increased to approximately 250nm 2 It then stabilized at 245–260 nm. 2 between( Figure 20 e). All these parameters exhibit a rapid equilibrium process and low volatility, confirming that the complex maintains a tight and stable folded conformation. Free energy landscape plots constructed using RMSD (0.70–0.82 nm) and Rg (2.40–2.50 nm) as reaction coordinates ( Figure 20 f) shows a funnel-shaped topology and a distinct global energy minimum (0.30 kJ / mol), which confirms that the complex adopts a single thermodynamically dominant conformation and demonstrates the high thermodynamic stability of the α-glucosidase and compound 3 complex.

[0054] Example 4 ADME prediction of compound 3 Based on comprehensive drug absorption, distribution, metabolism, and excretion (ADME) property predictions and drug-likeness analysis, compound 3 exhibits good potential for development as an orally active lead compound. (In the biopharmaceutics radar chart...) Figure 21 In the diagram, the red distorted hexagon representing compound 3 lies entirely within the pink shaded region representing the optimal property range. This compound possesses balanced physicochemical properties: moderate lipophilicity (consensus oil-water partition coefficient Log P). o / w=1.43), and relatively high topological polar surface area (TPSA=108.61 Å). 2 Furthermore, its predicted good water solubility lays a solid foundation for its bioavailability. Key pharmacokinetic predictions show that the compound has high gastrointestinal absorption efficiency and does not inhibit any major cytochrome P450 enzymes, suggesting high oral bioavailability and a low risk of drug-drug interactions. Regarding drug-likeness, compound 3 fully complies with mainstream evaluation criteria such as Lipinski's Rule of Five, Veber's Rule, and Egan's Rule, with no rule violations; its bioavailability radar chart shows that all key parameters are within the ideal range.

[0055] Example 5 Cytotoxicity assay of compounds 1-5 To evaluate the biosafety of these compounds, this study further used the CCK-8 assay to detect their cytotoxicity against human embryonic kidney 293 (HEK293) cells. The results are as follows: Figure 22 As shown, the key active compounds screened in this study did not exhibit significant cytotoxicity to normal mammalian cells, laying a safety foundation for their subsequent activity studies and potential applications.

[0056] In summary, the methylphenolic compounds provided by this invention can safely and effectively inhibit α-glucosidase; thereby enabling the preparation of hypoglycemic drugs or foods; or the preparation of drugs for the prevention or treatment of diabetes.

[0057] The present invention also provides pharmaceutical compositions or hypoglycemic food compositions containing said compound 3. The compound 3 is formulated into different dosage forms with pharmaceutically acceptable salts and pharmaceutically acceptable carriers, adjuvants, additives, or excipients.

[0058] Example 6 Preparation of Injection Solution Take compound 3, add water for injection as usual, filter, fill and sterilize to prepare an injection solution.

[0059] Example 7 Preparation of Injection Take compound 3, dissolve or suspend it in sterile water for injection, stir well, filter with a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature, and then aseptically seal to obtain the injection.

[0060] Example 8 Powder Preparation Compound 3 was taken and added to the excipient in a weight ratio of 9:1 to prepare a powder.

[0061] Example 9 Tablet Preparation Take compound 3 and add it to the excipient in a weight ratio of 1:5 to 1:10, then granulate and compress the mixture into tablets.

[0062] Example 10 Preparation of oral liquid Compound 3 was prepared into an oral liquid using conventional oral liquid preparation methods.

[0063] Example 11 Preparation of capsules, granules, or powders Take compound 3 and add it to the excipient in a weight ratio of 3:1 to 5:1 to prepare capsules, granules or powders.

[0064] Example 12 Food Preparation Compound 3 is prepared by combining it with conventional food additives.

[0065] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention extends to all other methods and applications having the same function.

Claims

1. A methylphenol compound, characterized in that, The compound is selected from one or more of compound 1, compound 2, compound 3, compound 4a, compound 4b and compound 5, and has the structural formulas shown in formulas 1-3, 4a, 4b and 5: 。 2. The methylphenolic compound according to claim 1, characterized in that, The compound is compound 3, with the following structural formula: 。 3. The use of the methylphenolic compound of claim 1 in the preparation of α-glucosidase inhibitors.

4. The use of the methylphenolic compound according to claim 1 in the preparation of hypoglycemic drugs or food.

5. The use of the methylphenolic compound according to claim 1 in the preparation of drugs for the prevention or treatment of diabetes.

6. The application according to any one of claims 3-5, characterized in that, The application uses the compound of claim 1 or its pharmaceutically acceptable salt, ester, or stereoisomer as the active ingredient; the content of the active ingredient is 0.1-99.0%.

7. The application according to any one of claims 3-5, characterized in that, The compound is used in combination with a pharmaceutically acceptable carrier, adjuvant, or excipient to formulate an injection, injection, powder, tablet, oral liquid, capsule, or granule.

8. The method for preparing the methylphenolic compound according to claim 1, characterized in that, The method is operated as follows: Step (1) Take the dried and crushed water chestnut peel, extract it with an organic solvent, and recover the solvent to obtain the total extract; Step (2) The total extract was dissolved in water and separated by D101 macroporous resin. It was eluted sequentially with methanol or ethanol and water at a volume ratio of 0:100-95:

5. The eluted fraction with a volume ratio of ethanol-water or methanol-water of 65-75:35-25 was concentrated under reduced pressure and loaded onto a normal-phase silica gel column. It was eluted with dichloromethane / methanol at a volume ratio of 1:0-0:

1. Under the detection of thin-layer chromatography, 10 fractions Fr. 1~Fr. 10 were obtained. Step (3) The Fr. 8 fraction was separated by silica gel column chromatography with dichloromethane / methanol as eluent at a ratio of 2:1 to 0:

1. The fractions were combined by thin-layer chromatography to obtain nine fractions Fr. 8-1 to Fr. 8-9. The Fr. 8-6 fraction was separated by Sephadex LH-20 gel chromatography column with dichloromethane / methanol at a ratio of 1:

1. The fractions were then purified by semi-preparative HPLC to obtain compounds 1-3.

9. The method for preparing methylphenolic compounds according to claim 8, characterized in that, Fr. 2 fractions were separated by gel permeation column chromatography, eluted with dichloromethane / methanol at a volume ratio of 1:1, and analyzed by thin-layer chromatography to obtain five fractions Fr. 2-1 to Fr. 2-5; Fr. 2-4 fractions were purified by semi-preparative HPLC, eluted with acetonitrile / water mobile phase, to obtain compounds 4 and 5; Compound 4 was separated by elution with a chiral HPLC column using hexane / isopropanol at a ratio of 76:24 to obtain enantiomers 4a and 4b.

10. The method for preparing methylphenolic compounds according to claim 8, characterized in that, The organic solvent in step (1) is selected from one or more of the following: ethyl acetate, acetone, n-butanol, ethanol, methanol, aqueous n-butanol, aqueous acetone, aqueous ethanol and aqueous methanol; the mass-volume ratio of water chestnut peel to organic solvent is 1:1-15.

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