Hypoglycemic active compound in ginger as well as screening method and application of hypoglycemic active compound
Through the virtual screening method of quantum chemistry and molecular docking, compounds with lowering blood sugar in ginger were screened out, solving the problems of low screening efficiency and insufficient accuracy in the prior art, and achieving efficient screening of compounds and significant lowering blood sugar.
Patent Information
- Application Number
- CN202510261029.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-07-22
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to efficiently screen out spicy compounds with lowering blood sugar activity in ginger, and the single molecular docking method is insufficient, resulting in failure of activity verification.
The virtual screening method used for quantum chemistry and molecular docking was used, and preliminary screening and molecular docking were combined with quantum chemistry methods for verification. Compounds with lowering blood sugar activity were screened out, and their activity was verified through C2C12 cell experiments.
The efficiency and hit rate of compound screening were improved. The screened 6-gingerol, 6-gingerenol, 8-gingerenol, 10-gingerenol and gingerone phenol had higher blood sugar-lowering activities than insulin, which had a significant effect of promoting glucose absorption and had a wide range of application prospects.
Smart Images
Figure CN120356557A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of pharmaceutical chemistry, and particularly relates to a hypoglycemic active compound in ginger, a screening method thereof, and applications thereof. Background Art
[0002] Diabetes is a chronic disease, and various complications caused by it have become the main causes of disability and premature death of patients. Therefore, the research on drugs in the field of diabetes is particularly important. In the treatment of diabetes, compared with synthetic drugs, the application of natural hypoglycemic substances in plants has the advantages of being easily acceptable, being able to be taken for a long time, and having small toxic and side effects.
[0003] Ginger (Zingiber officinale Roscoe) is an aromatic plant that can be used both as medicine and food, and has extremely high medicinal value. As shown in Table 1, the pungent compounds in ginger are mainly classified into gingerol, shogaol, paradol, zingerone according to the functional group structure. Gingerol is the component that contributes the most to the pungent substances in ginger. The structure of gingerol is unstable and will change during drying or cooking, generating shogaol with strong reactivity. Shogaol contains α,β-unsaturated ketone, which is the structural basis for its strong biological activity. Under the action of microorganisms, the unsaturated bond in shogaol will be reduced and further transformed into paradol. The molecular structure of zingerone is relatively small and does not exist in fresh ginger, but fresh ginger can produce it through the retro-aldol reaction of gingerol after drying or roasting. At present, studies have shown that the molecular activity of zingerone is relatively low among the pungent compounds in ginger.
[0004] Table 1 Types of pungent compounds in ginger
[0005] The TRPV1 channel is a homotetramer composed of four identical subunits, each subunit including a transmembrane region, an intracellular N-terminus, and an intracellular C-terminus. The intracellular N-terminus of the TRPV1 channel consists of an ankyrin repeat domain (ARD), a linker domain, and a pre-S1 helix. Among them, the ARD has six ankyrin (ANK) repeat sequences (residues 101 - 364). ATP binding to the ARD sensitizes the channel, generating a larger current under the action of specific activators such as capsaicin. The C-terminus of the TRPV1 channel contains a TRP domain and a β-sheet region. The TRP domain is close to the pore and consists of 30 residues, playing a key role in subunit tetramerization and channel function. The transmembrane region of the TRPV1 channel includes six transmembrane helices S1 - S6, among which the S1 - S4 transmembrane helices and the S4 - S5 linker are conserved domains. The S1 - S4 of the TRPV1 channel is called the voltage sensor-like domain (VSLD). S5, the pore helix, and the S6 helix form the central pore of the channel. The loop region between S5 and the pore helix covers the top of the pore, called the turret. The vanilloid binding pocket is located below the transmembrane region, composed of S3, S4, the S4 - S5 linker, and S5 and S6 of adjacent subunits, which is the binding site for vanilloid compounds such as capsaicin, RTX, piperine, and gingerol. In the ligand-unbound state (apo), it usually binds to phosphatidylinositol, see Figure 1-2 .
[0006] Mutational studies have found that the important residues for the binding of the TRPV1 protein receptor to capsaicin are: Y511 and S512 on S3, M547 and T550 on S4. Some researchers have determined the "head-down, tail-up" orientation of capsaicin in the vanilloid binding pocket, that is, the vanillyl head of capsaicin points downward to the S4 - S5 linker, and the fatty acid chain tail points upward to S4. The binding of capsaicin depends on hydrogen bonds and van der Waals interactions. The tail of capsaicin mainly provides van der Waals interactions, while the hydroxyl group of the vanillyl head and the carbonyl oxygen of the amide group in the neck form hydrogen bonds with E570 and T550 respectively. However, the capsaicin complex structure of other researchers does not show the interaction between T550 and the neck. They believe that the role of T550 is the transmission of conformational waves. The binding of capsaicin causes the movement of the S4 - S5 linker, which in turn promotes the movement of S6 and the opening of the lower gate of the channel. Among other vanilloid compounds, piperine does not form stable hydrogen bonds with T550 and E570, but directly interacts with T670 on S6 to control the opening of the channel, see Figure 3 .
[0007] In the process of drug research and development, molecular docking is often used to study intermolecular interactions and is a theoretical simulation method for predicting receptor-ligand binding modes and affinities. Molecular docking usually calculates the interactions between ligands and receptors using empirical force fields or scoring functions, and the corresponding parameters come from the default of the force field, which has the advantage of fast calculation speed and can quickly screen a small molecule database of millions of levels. However, a single molecular docking method often shows insufficient accuracy, resulting in failed activity verification. And the final result after theoretical screening based on quantum chemistry may be a co-amorphous state or a physical mixture, and a single screening method cannot meet the screening of actual active compounds. Therefore, the screening method combining quantum chemistry and molecular docking is more practical in the actual drug screening process, reducing the screening times, narrowing the screening range, and increasing the screening efficiency. In addition, there has been less research on the hypoglycemic activity of pungent compounds in ginger in the past.
[0008] Based on this, it is very meaningful to develop a method based on quantum chemistry and molecular docking for compound screening to more efficiently screen out pungent compounds with hypoglycemic activity. Summary of the Invention
[0009] In view of the above problems, the present invention provides a hypoglycemic active compound in ginger, its screening method and application, virtually screens the pungent compounds in ginger by quantum chemistry and molecular docking methods to obtain active compounds, and verifies the hypoglycemic activity of the active compounds through in vitro experiments.
[0010] In order to achieve the above object, the technical solutions adopted by the present invention are as follows: An object of the present invention is to provide a screening method for hypoglycemic active compounds in ginger, specifically including the following steps: S1: Obtain candidate compounds through a virtual screening method; S2: Evaluate the hypoglycemic activity of the candidate compounds to obtain target active compounds.
[0011] Further, in the step S1, the virtual screening method is a virtual screening method combining quantum chemistry method and molecular docking method.
[0012] Furthermore, the quantum chemistry method is as follows: The molecular geometry of the pungent compounds in ginger is preliminarily geometrically optimized using the Molecular Mechanics (MM2) method to obtain the initial conformation. The initial conformation is imported into the Gaussianview software, and the B3LYP6-311G(d) basis set is selected. Using the Density Functional Theory (DFT) method, it is submitted to Gaussian09 for opt+freq calculation to obtain the lowest energy conformation of the molecule and its structural energy parameters.
[0013] Furthermore, the molecular docking method is as follows: The molecular structure of the compound is downloaded and constructed through PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ). After optimization in the Pymol software, it is stored in the PDB format. Then, the file is opened with the Autodock Tools software to adjust the charge number and stored in the PDBQT format. The TRPV1 protein structure is downloaded from the PDB database (http: / / www.rcsb.org / ). After preprocessing such as water removal, hydrogen addition, and charge calculation, the receptor structure for docking is obtained and stored in the PDBQT format. The molecular docking software AutoDock Tools is used to dock TRPV1 with the pungent compounds in ginger to obtain the receptor-ligand conformation in the docking state, and the hydrogen bond length and docking activation energy are calculated.
[0014] Further, the specific process of obtaining the candidate compounds through virtual screening in step S1 includes: (1) The compounds are preliminarily screened by the quantum chemistry method. The reaction activity is mainly evaluated from three aspects: the net charge distribution and atomic charge difference, the frontier molecular orbital energy level difference △E, and the bond dissociation enthalpy. The atomic charge differences, frontier orbital energy differences, and bond dissociation enthalpies of the pungent compounds in ginger are sorted respectively, and priority limits are set for the atomic charge differences, frontier orbital energy differences, and bond dissociation enthalpies. According to the principles of quantum chemistry, the priority of bond dissociation enthalpy is higher than that of atomic charge difference, which is higher than that of frontier orbital energy difference. According to the evaluation results, the first preliminary screening database is obtained. (2) The compounds are synchronously screened by the molecular docking method. The optimization method uses the genetic algorithm, and the evaluation function uses the semi-empirical free energy evaluation function. Scoring and ranking are performed according to the docking score, and candidate compounds are obtained by evaluating the interaction mode between the docking molecule and the receptor protein, thus obtaining the second preliminary screening database. (3) The candidate compounds are obtained by integrating the results of (1) and (2).
[0015] Further, in step S2, the hypoglycemic activity evaluation method is the C2C12 cell glucose uptake experimental method.
[0016] Another object of the present invention is to provide a hypoglycemic active compound screened by the screening method of the hypoglycemic active compound in the above-mentioned ginger. The active compound is a compound having the structure shown below or a pharmaceutically acceptable salt thereof: .
[0017] Another object of the present invention is to provide an application of the hypoglycemic active compound in the above-mentioned ginger in the preparation of a hypoglycemic drug. The hypoglycemic activities of the active compounds 6-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, paradol, and zingerone are all higher than that of insulin.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: In the present invention, virtual screening is carried out by quantum chemistry and molecular docking methods to obtain target compounds, and the hypoglycemic activities of the target compounds are verified. The specific advantages are as follows: (1) By using the virtual screening method based on the combination of quantum chemistry and molecular docking, time and cost are saved, and compared with a single screening method, the screening efficiency and the hit rate of compounds are improved; (2) The in vitro C2C12 cell glucose uptake experiment confirms that 6-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, and paradol can significantly promote the glucose uptake of insulin-resistant myotubes, zingerone has no obvious promoting effect on the glucose uptake of insulin-resistant myotubes, and the glucose uptake of these compounds is higher than that of insulin. In addition, the order of the promoting effect on glucose uptake ability is: 6-gingerol > 6-shogaol > 8-shogaol > 10-shogaol > paradol > zingerone > insulin. Therefore, this type of compound has great application prospects in the prevention or treatment of hypoglycemic diseases. Description of the Drawings
[0019] Figure 1 is a schematic diagram of the functional domain of the TRPV1 channel; Figure 2 is a three-dimensional structural diagram of the functional domain of the TRPV1 channel; Figure 3 is a schematic diagram of the binding of capsaicin to the TRPV1 channel; Figure 4 is the optimized molecular conformation of the pungent compound in the ginger of the present invention; Figure 5 is the dipole moment and atomic charge distribution of the pungent compound in the ginger of the present invention; Figure 6 is a schematic diagram of the LUMO orbital of the pungent compound in the ginger of the present invention; Figure 7 is a schematic diagram of the HOMO orbital of the pungent compound in the ginger of the present invention; Figure 8It is a schematic diagram of the binding of the pungent compound in ginger of the present invention to the TRPV1 channel; Figure 9 It is the toxicity result of DMSO of the present invention; Figure 10 It is the CCK8 cytotoxicity result of the pungent compound in ginger of the present invention; Figure 11 It is (A) the effect of different pungent compounds in ginger on the glucose uptake ability of cells in the resting state; (B) the effect of different pungent compounds in ginger on the glucose uptake ability of cells under insulin resistance; Note: Figure 9 In, compared with the control group: ****, p < 0.0001; Figure 10 In, compared with the DMSO group: *, p < 0.1; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; Figure 11 In, compared with the positive control group: ***, p < 0.001; ****, p < 0.0001; compared with the model group: ##, p < 0.01; , p < 0.001; #, p < 0.0001. Detailed implementation manners
[0020] In order to make the purpose and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0021] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.
[0022] Example 1 Computational simulation analysis of pungent compounds in ginger using quantum chemistry methods S1, Download the structures of pungent compounds in ginger from the Pubchem website, construct the initial geometric models of 9 pungent compounds in ginger using Chemdraw software, and perform preliminary geometric optimization of the molecular geometric configurations using the molecular mechanics method (Molecular Mechanics.MM2); S2, Input the optimized configuration results into Gaussian09 and use the density functional theory method (Density Functional Theory, DFT), and perform full geometric optimization using the basis set (DFT) B3LYP / 6-311G(d). The optimization results are until no imaginary frequencies appear, that is, the dominant conformations of each molecule are obtained, as shown in Figure 4.
[0023] S3, after obtaining the dominant conformation, the Gaussian09 density functional DFT method and the B3LYP / 6-311G (d) basis set are used to perform corresponding quantum chemical calculations on the molecule, and the atomic charge distribution, molecular energy and dipole moment are calculated. According to molecular structure theory, the greater the charge difference between atoms, the easier it is for electrons to transition, the easier it is for atomic bonds to dissociate, and chemical reactions to occur. Conversely, chemical reactions are not likely to occur. In quantum chemistry, sites with larger charge differences are generally considered to be molecular active sites. Figure 5 As can be seen from Table 2, the atomic charge difference is ranked as 6-shogaol > 8-shogaol > 6-shogaol > zingerone, and the atomic charge difference of the phenolic hydroxyl bond of other substances is not obvious.
[0024] Table 2 The atomic charge differences at the phenolic hydroxyl groups of spicy ginger compounds
[0025] S4, infer the reaction activity center and the size of the reaction activity based on the frontier molecular orbital and orbital energy difference. When a molecule undergoes a chemical reaction, it is only related to those molecular orbitals with active "valence electrons", namely the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). These orbits are called molecular frontier orbitals. Quantum chemistry molecular orbital theory believes that the distribution of the highest occupied orbital HOMO and the lowest unoccupied orbital LUMO can intuitively indicate the main site of action of antioxidant free scavenging activity. When substituents are substituted at different positions, due to the different electron donating and electron withdrawing abilities of different substituents, the π electron cloud distribution becomes uneven, and the corresponding HOMO orbital distribution and LUMO orbital distribution change accordingly. In the HOMO diagram, the electron cloud density will increase for groups with strong electron donating ability; in the LUMO diagram, the electron cloud density will increase for groups with strong electron withdrawing ability.
[0026] Ginger is a spicy compound with a polyphenol structure, so according to the first reaction principle, it is easy to undergo nucleophilic reaction, so the active center is located at the HOMO orbital. Figure 6 and Figure 7 It can be seen that the HOMO orbitals of spicy ginger compounds are mainly concentrated around the phenolic hydroxyl bond, indicating that this may be the reaction active center. It can be seen from the figure that when Gingerol is converted to Shogaol, the HOMO electron cloud density increases, the electron donating ability is enhanced, and the reaction activity increases. When Gingerol is converted to Paradol and Zingerone structures, the HOMO electron cloud density decreases, the electron donating ability decreases, and the reaction activity decreases.
[0027] According to molecular orbital theory, the frontier molecular orbital energy level difference △E (△E=EHOMO-ELUMO) is an important theoretical parameter to characterize the activity of a molecule. Among them, EHOMO is the highest occupied orbital energy, which characterizes the ability of a molecule to push electrons. The larger the EHOMO value, the stronger its ability to push electrons; ELUMO is the lowest unoccupied orbital energy, which characterizes the ability of a molecule to absorb electrons. The smaller the ELUMO value, the stronger its ability to absorb electrons. The smaller the frontier molecular orbital energy level difference △E value, the easier it is for electrons in the molecule to transition, and the stronger the reaction activity; conversely, the weaker the reaction activity. It can be obtained from Table 3 that the order of frontier orbital energy level difference △E is: 8-shogaol <10-shogaol <6-shogaol <12-shogaol <6-shogaol <10-shogaol <8-shogaol <gingerol < gingerol < zingerone, that is, the order of reaction activity is speculated to be: 8-shogaol > 10-shogaol > 6-shogaol > 12-shogaol > 6-shogaol > 10-shogaol > 8-shogaol > gingerol > zingerone.
[0028] Table 3 Frontier orbital energies and energy ranges of spicy compounds in ginger
[0029] S5, infer the reaction activity center based on the bond dissociation enthalpy. Bond dissociation enthalpy is one of the most commonly used parameters to characterize reaction activity. It refers to the energy that needs to be absorbed during the breaking process of a chemical bond or the energy released during the synthesis of a chemical bond. It can directly reflect the stability of the chemical bond and thus the difficulty of the chemical reaction. The smaller the bond dissociation enthalpy, the easier it is for the chemical bond to break or form, and the easier it is for the reaction to occur. As can be seen from Table 4, based on the bond dissociation enthalpy, the reaction activity is inferred to be 6-gingerol>8-shogaol>10-shogaol>shogaol>12-shogaol>10-shogaol>6-shogaol>gingerone>8-shogaol.
[0030] Table 4 Bond dissociation enthalpy of spicy compounds in ginger
[0031] Example 2 Visualized analysis of the docking of spicy compounds in ginger with TRPV1 protein receptor molecules S1, Molecular structures of gingerols, shogaols, zingerones, and zingeronephenols were downloaded and constructed through PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ). After optimization in Pymol software, they were stored in PDB format, and then opened with Autodock Tools software to adjust the charge number and stored in PDBQT format.
[0032] S2. The TRPV1 protein structure was downloaded from the PDB database (http: / / www.rcsb.org / ). After preprocessing such as water removal, hydrogen addition, and charge calculation, the receptor structure for docking was obtained and stored in the PDBQT format.
[0033] S3. The molecular docking software AutoDock Tools was used to dock TRPV1 with ginger pungent compounds, obtaining the receptor-ligand conformation in the docking state, and calculating the hydrogen bond length and docking activation energy.
[0034] S4. The binding form of ginger pungent compounds to the TRPV1 protein receptor was visually displayed ( Figure 8 ). The binding mode of ginger pungent compounds is similar to that of capsaicin. 6G, 10G, 12G, P, and Z all adopt the "head-down, tail-up" binding mode. The head forms hydrogen bonds with the receptor protein, and the tail fatty chain interacts with surrounding amino acids through van der Waals forces to play a stabilizing role. 6G, 10G, P, and Z are respectively connected to the N-terminus of ARG-559 to form hydrogen bonds. In addition, the binding modes of different compounds to TRPV1 are slightly different. The vanillyl head and neck of 10G also form hydrogen bonds with the N-terminus of GLU-572 and TYR-513 respectively, which are all key sites that can activate the TRPV1 channel; the binding site of 12G has been rarely reported. In this experiment, it forms hydrogen bonds with the N-termini of ARG-493 and ASN-439; the Z molecule has a small structure and a short fatty chain, so the tail binds to the O-terminus of the ASN-553 end closer to the head to form a hydrogen bond. The binding modes of 6S, 8S, 8G, and 10S are relatively similar. The tails of 6S and 8G form hydrogen bonds with the O-terminus of THR-552, the head of 8S forms a hydrogen bond with the O-terminus of GLU-572, the head of 10S forms a hydrogen bond with the O-terminus of GLU-515, and the neck forms a hydrogen bond with the O-terminus of TYR-557. Different binding modes may affect the binding stability of the receptor and ligand. Based on the hydrogen bond length and binding mode, it is preliminarily judged that 6G, 10S, P, and Z bind more stably to the TRPV1 protein.
[0035] Table 5 Amino acid types and H-bond lengths of different ginger pungent substances binding to TRPV1
[0036] S5. Evaluate the binding and interaction between ginger pungent compounds and TRPV1 protein receptor according to the binding free energy. The binding free energy in the docking results is an indicator to measure the binding stability of molecules. A lower binding free energy value usually indicates a more stable binding between molecules. As can be seen from Table 6, the order of binding energy is 6-gingerol = paradol < 10-gingerol = zingerone < 12-gingerol < 6-shogaol < 8-shogaol < 10-shogaol = 8-gingerol. Therefore, according to the molecular docking experiment, the order of binding stability between ginger pungent compounds and TRPV1 receptor is: 6-gingerol = paradol > 10-gingerol = zingerone > 12-gingerol > 6-shogaol > 8-shogaol > 10-shogaol = 8-gingerol.
[0037] Table 6 Docking binding energy between pungent compounds in ginger and TRPV1
[0038] Example 3 S1. Using quantum chemical calculations, optimize the molecules with MM2 force field and perform DFT functional optimization calculations with B3LYP6-311G(d) basis set, and screen the target compounds by combining the net charge distribution, atomic charge difference, frontier orbital energy gap, and bond dissociation enthalpy. The screening method is to sort the atomic charge difference, frontier orbital energy gap, and bond dissociation enthalpy of pungent compounds in ginger respectively. The sorting method uses the pythonsorted function, and priority limits are set for the atomic charge difference, frontier orbital energy gap, and bond dissociation enthalpy. According to the principles of quantum chemistry, the priority of bond dissociation enthalpy is higher than that of atomic charge difference, which is higher than that of frontier orbital energy gap. According to the screening principle of the present invention, five compounds screened by the present invention are 6-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, and paradol for subsequent experimental verification.
[0039] S2. Using molecular docking, perform molecular docking of different ginger pungent compounds with TRPV1 using Pymol and Autodock. The optimization method uses the genetic algorithm, and the evaluation function uses the semi-empirical free energy evaluation function. According to the docking scores, 6-gingerol, 10-gingerol, 12-gingerol, paradol, and zingerone are screened for subsequent verification.
[0040] S3. In summary, combining the results of the two computational simulations, the ginger pungent compounds finally determined for mechanism verification are 6-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, paradol, and zingerone.
[0041] Example 4 Verification of hypoglycemic activity of pungent compounds in ginger S1, DMSO toxicity experiment. To study the toxic effects of the solvent DMSO, the CCK8 method was used to determine the viability of C2C12 cells at different volume fractions of DMSO. The C2C12 cells were cultured to 80% confluence, the cell concentration was calculated by hemocytometry, and diluted to 3×10 5 / mL with complete medium. 100 μL of the diluted cell suspension and DMSO solutions with volume fractions of 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, and 6% were added to 96-well plates, cultured for 24 h, the liquid was aspirated, washed with PBS, medium containing 10% CCK8 was added, cultured for 1 h, and the absorbance was measured at 450 nm to calculate cell viability. The cell viability calculation formula: (absorbance of experimental group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) * 100%.
[0042] Experimental results: As Figure 9 shown, DMSO at different volume fractions has different degrees of influence on cell viability. DMSO can be considered to have no effect on cell viability when the volume fraction is below 1%. In subsequent cell drug administration experiments, the volume fraction of DMSO applied is below 0.1%, so the solvent toxicity effect can be excluded.
[0043] S2, CCK8 experiments on different pungent compounds in ginger. To study the toxic effects of 6G, 6S, 8S, 10S, P, and Z and select an appropriate drug administration range to avoid cell death. The cells were cultured to 80% confluence, the cell concentration was calculated by hemocytometry, and diluted to 3×10 5 / mL with complete medium. The drugs were dissolved in DMSO as drug stock solutions, and the drug stock solutions were diluted with complete medium as drug working solutions. 100 μL of the diluted cell suspension and drug working solutions with different final concentrations were added to 96-well plates, cultured for 24 h, the liquid was aspirated, washed with PBS, medium containing 10% CCK8 was added, cultured for 1 h, and the absorbance was measured at 450 nm to calculate cell viability. The cell viability calculation formula: (absorbance of experimental group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) × 100%.
[0044] Experimental results: As Figure 10As shown, when the final concentration of 6G is below 10 μg / mL, the drug has little effect on the cells. Therefore, a concentration of 10 μg / mL was selected for subsequent experiments. The drug 6S has a strong cytotoxic effect on C2C12 cells. When the final concentration is below 1.5 μg / mL, the drug has little effect on the cells. A concentration of 1.5 μg / mL was selected for subsequent experiments. The drug 8S has a strong cytotoxic effect on C2C12 cells. When the final concentration is below 3 μg / mL, the drug has little effect on the cells. A concentration of 3 μg / mL was selected for subsequent experiments. The cytotoxic effect of the drug 10S on C2C12 cells is weaker than that of 6S and 8S, but stronger than that of 6G. When the final concentration is below 3 μg / mL, the drug has little effect on the cells. A concentration of 3 μg / mL was selected for subsequent experiments. P also has a strong cytotoxic effect on C2C12 cells. The cells are more sensitive to changes in drug concentration. When the final drug concentration is below 3 μg / mL, it has little effect on the cells. A concentration group of 3 μg / mL was selected for subsequent experiments. The cytotoxic effect of drug Z on C2C12 cells is weaker than that of P. When the final drug concentration is below 5 μg / mL, it has little effect on the cells. A concentration of 5 μg / mL was selected for subsequent experiments.
[0045] S3. The ability of C2C12 cells to uptake glucose was determined by the 2-NBDG method. After culturing C2C12 cells to 80% density, they were induced to differentiate for 8 days in a medium containing 2% horse serum. After differentiating into myotubes, the cells were starved for 6 h in a 0.2% BSA medium. The cells were divided into a negative control group (CON group), a positive control group (INS group), a model group (MOD group), and drug groups (6G group, 6S group, 8S group, 10S group, P group, Z group). The model group and drug groups were added with a medium containing 0.5 mM palmitic acid (PA) to induce the establishment of an insulin resistance cell model, and the other groups were treated with a control medium without palmitic acid. After culturing for 24 h, the cells were washed with PBS and starved for 1 h in a sugar-free BSA medium. 30 min before the end of starvation, the positive control group, model group, and drug groups were added with an insulin working solution to make the final concentration of insulin 100 nM. After the drug treatment, 2-NBDG was dissolved in a 0.2% BSA sugar-free DMEM medium at a final concentration of 80 μM. The medium containing 2-NBDG was added to the wells to incubate the cells, and the incubation was carried out in a 37 °C cell culture incubator for 30 min. After the incubation, the wells were taken out, the medium was aspirated, and the cells were washed 3 times with sterile PBS. Then, a multifunctional microplate reader was used to measure the fluorescence intensity of each well under the conditions of an excitation wavelength of 485 and an emission wavelength of 520 nm. Another Blank group was set up, and the treatment method was that there were no cells in the wells, no drugs were added, and an equal amount of PBS was added before detection. The relative glucose uptake of each group of cells was calculated.
[0046] Relative glucose uptake = (FISample - FlBlank) / (FIcontrol - FlBlank) Experimental results: As Figure 11 shown, through the relative glucose uptake, it was found that under the normal resting state of cells, the compounds all extremely significantly increased the glucose uptake of C2C12 myotubes, and the compound [6]-gingerol had the best effect among the six compounds, and the increasing effect was similar to that of insulin. Compared with normal C2C12 myotubes, the glucose uptake ability of C2C12 myotubes in the insulin resistance model was significantly reduced under insulin stimulation, which indicated that we successfully established an insulin resistance model. The compounds [6]-gingerol, [6]-shogaol, [8]-shogaol,
[10] -shogaol could extremely significantly promote the glucose uptake of insulin-resistant myotubes, [6]-paradol could significantly promote it and reached statistical significance, while [6]-gingerone had no obvious promoting effect. The order of the promoting effect on glucose uptake ability was: [6]-gingerol > [6]-shogaol > [8]-shogaol >
[10] -shogaol > [6]-paradol > [6]-gingerone > insulin.
[0047] In summary, in this example, compound screening was carried out based on the methods of quantum chemistry and molecular docking, and the hypoglycemic activity was evaluated by the glucose uptake ability of C2C12 cells. The results showed that the hypoglycemic activities of the compounds [6]-gingerol, [6]-shogaol, [8]-shogaol,
[10] -shogaol, [6]-paradol, [6]-gingerone were all higher than that of insulin, and they had great application prospects in the prevention or treatment of hypoglycemic diseases.
[0048] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A screening method for hypoglycemic active compounds in ginger, characterized in that: Specifically, it includes the following steps: S1: Obtain candidate compounds through virtual screening methods; S2: Evaluate the hypoglycemic activity of the candidate compounds to obtain target active compounds.
2. The screening method of hypoglycemic active compounds in ginger according to claim 1, wherein: In step S1, the virtual screening method is a virtual screening method combining quantum chemistry method and molecular docking method.
3. The screening method of hypoglycemic active compounds in ginger according to claim 2, wherein: The quantum chemistry method is as follows: Use the molecular mechanics method to perform preliminary geometric optimization on the molecular geometric configuration of ginger pungent compounds to obtain the initial conformation; Import the initial conformation into the Gaussianview software, select the B3LYP6-311G(d) basis set and use the density functional theory method, and submit it to Gaussian09 for opt+freq calculation to obtain the lowest energy conformation of the molecule and its structural energy parameters.
4. The screening method of hypoglycemic active compounds in ginger according to claim 2, wherein: The molecular docking method is as follows: Download and construct the molecular structure of the compound through PubChem; Optimize and store it in PDB format in the Pymol software, and then open the file with the Autodock Tools software to adjust the charge number and store it in PDBQT format; The TRPV1 protein structure is downloaded from the PDB database, and after pre-treatment such as water removal, hydrogen addition, and charge calculation, the dockable receptor structure is obtained and stored in PDBQT format; Use the molecular docking software AutoDock Tools to dock TRPV1 with ginger pungent compounds to obtain the receptor-ligand conformation in the docking state, and calculate the hydrogen bond length and docking activation energy.
5. A screening method for hypoglycemic active compounds in ginger according to claim 1, characterized in that: In step S1, obtaining candidate compounds through virtual screening methods specifically includes: Preliminarily screen the compounds through the quantum chemistry method, and mainly evaluate the reaction activity from three aspects: the net charge distribution and atomic charge difference, the frontier molecular orbital energy level difference △E, and the bond dissociation enthalpy: Sort the atomic charge difference, frontier orbital energy level difference, and bond dissociation enthalpy of the pungent compounds in ginger respectively, and limit the priority of the atomic charge difference, frontier orbital energy level difference, and bond dissociation enthalpy. According to the quantum chemistry principle, the priority of bond dissociation enthalpy is higher than that of atomic charge difference which is higher than that of frontier orbital energy level difference, and obtain the first preliminary screening database according to the evaluation results; Synchronously screen the compounds through the molecular docking method. The optimization method uses the genetic algorithm, and the evaluation function uses the semi-empirical free energy evaluation function. Sort according to the docking score, and obtain candidate compounds by evaluating the interaction mode between the docking molecule and the receptor protein, and obtain the second preliminary screening database; Integrate the results of (1) and (2) to obtain candidate compounds.
6. The screening method of hypoglycemic active compounds in ginger according to claim 1, characterized in that: In step S2, the hypoglycemic activity evaluation method is the C2C12 cell glucose uptake experimental method.
7. A hypoglycemic active compound obtained by the screening method of a hypoglycemic active compound in ginger as described in any one of claims 1-6, characterized in that: The active compound is a compound with the following structure or its pharmaceutically acceptable salt: 。 8. Use of a hypoglycemic active compound in ginger for preparing a hypoglycemic drug according to claim 7, characterized in that: The hypoglycemic activities of the active compounds 6-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, paradol, and zingerone are all higher than that of insulin.