KR20240011490A - Pharmaceutical composition for preventing or treating diabetes by DPP4 hyperexpression comprising Glycyrrhiza uralensis derived Licochalcone A as an active ingredient - Google Patents

Abstract

The present invention relates to licorice ( Glycyrrhiza uralensis )-derived licochalcone A as an active ingredient in the pharmaceutical composition for preventing or treating diabetes overexpressing DPP4 (dipeptidyl peptidase 4). It was confirmed that licochalcone A isolated and extracted from licorice exhibits a DPP4 inhibitory effect. As a result, it can be usefully used as a composition for preventing, treating, or improving DPP4 overexpression diabetes.

Description

Pharmaceutical composition for preventing or treating diabetes by DPP4 hyperexpression comprising Glycyrrhiza uralensis derived Licochalcone A as an active ingredient}

The present invention is licorice ( Glycyrrhiza It relates to a pharmaceutical composition for preventing or treating diabetes overexpressing DPP4 (dipeptidyl peptidase 4), which contains licochalcone A derived from uralensis as an active ingredient.

Diabetes is a chronic metabolic disease with a rapidly increasing impact on people worldwide. Diabetes affected 463 million people worldwide in 2019, and is expected to increase to 578 million in 2030 and 700 million in 2045. Additionally, diabetes is estimated to account for approximately 10% of deaths worldwide. Among them, type 2 diabetes mellitus (T2DM) is the most common, accounting for approximately 90% of patients. Diabetes is characterized by insufficient pancreatic insulin production and/or insulin resistance in peripheral tissues, and chronic hyperglycemia associated with diabetes is associated with long-term damage to vital organs such as the eyes, kidneys, nerves, and heart.

“Incretin” refers to a group of hormones that include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which, upon ingestion, stimulate the production of insulin by the pancreas. It acts on cells and is produced in the intestines. DPP4 (dipeptidyl peptidase 4) reduces circulating active GLP-1 and GIP within 1 minute, and DPP4 inhibitors are drugs that promote their action by inhibiting DPP4 and enhancing active GLP-1 and GIP levels. Therefore, DPP4 inhibitors have emerged as a new therapy for the treatment of type 2 diabetes. However, current DPP4 inhibitors have several side effects that limit their practical application. Therefore, there is an emerging therapeutic need for new DPP4 inhibitors derived from natural resources such as herbs and plants.

Licorice ( Glycyrrhiza) uralensis ) is well known as a medicinal plant belonging to the leguminosae family. To date, various biologically active ingredients have been identified from licorice, and the main components include triterpene saponin and flavonoid. Various pharmacological effects of licorice and its active ingredients have been reported, but the anti-diabetic effect of licorice and its active ingredients through DPP4 inhibition has not yet been reported.

1. Republic of Korea Patent No. 10-1060942 (published on August 24, 2011)

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes overexpressing dipeptidyl peptidase 4 (DPP4), containing licochalcone A as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or improving diabetes overexpressing DPP4 (dipeptidyl peptidase 4) containing licochalcone A as an active ingredient.

To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating diabetes overexpressing DPP4 (dipeptidyl peptidase 4) containing licochalcone A as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving diabetes overexpressing DPP4 (dipeptidyl peptidase 4) containing licochalcone A as an active ingredient.

According to the present invention, licorice ( Glycyrrhiza uralensis ), it was confirmed that licochalcone A (licochalcone A), isolated and extracted, exhibits an inhibitory effect on DPP4 (dipeptidyl peptidase 4), so that it can be usefully used as a composition for preventing, treating or improving DPP4 overexpression diabetes.

Figure 1 shows the results of analyzing the interaction between licochalcone A (LicA) (a) and siragliptin (b) and dipeptidyl peptidase 4 (DPP4) amino acid residues.
Figure 2 shows the results of analyzing the global energy of the LicA+DPP4 and sitagliptin+DPP4 complexes for GLP1/GIP (Glucagon like peptide 1/Glucose dependent insulinotropic polypeptide).
Figure 3 shows the results of molecular dynamics simulation of the LicA+DPP4 and sitagliptin+DPP4 complexes. (A) shows the RMSD of the enzyme, (B) the RMSD of the ligand, (C) the radius of gyration, and (D) the RMSF framework of the enzyme.
Figure 4 shows the results of analyzing the relative inhibition rate of DPP4 according to the concentration of LicA.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating diabetes overexpressing dipeptidyl peptidase 4 (DPP4), comprising licochalcone A as an active ingredient.

The lycochalcone A is licorice ( Glycyrrhiza uralensis ) and can inhibit DPP4 activity.

In addition, the lycochalcone A may interact with one or more DPP4 residues selected from the group consisting of Arg123, His124, Glu203, Glu204, Ile205, Phe206, Gly207, Phe355, Arg356, Tyr548, Tyr663, Tyr667, Arg670 and Asn711. However, it is not limited to this.

Additionally, the licochalcone A can activate GLP1 (Glucagon like peptide 1) or GIP (Glucose dependent insulinotropic polypeptide).

The pharmaceutical composition of the present invention is prepared in unit dose form or in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person skilled in the art. It can be manufactured by internalizing it.

The pharmaceutically acceptable carriers are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Includes, but is not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

In the present invention, the content of additives included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

The pharmaceutical compositions include injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, paste agents, and cataplasmase agents. It may be formulated in the form of one or more external skin preparations selected from the group consisting of, but is not limited to this.

The pharmaceutical composition of the present invention may additionally contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, alginate, polyvinyl pyrrolidone. It includes, but is not limited to, binders such as talc, calcium stearate, lubricants such as hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, and surfactants such as polysorbate, cetyl alcohol, glycerol, etc. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method. For oral administration, it can be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it can be formulated as an injection, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.

The dosage of the pharmaceutical composition of the present invention is determined by the patient's condition, weight, age, gender, health, dietary constitution specificity, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and The range may vary depending on the drug form and can be appropriately selected by a person skilled in the art. For example, it may range from about 0.1 to 10,000 mg/kg, but is not limited and may be administered once to several times a day.

The pharmaceutical composition may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically applied) depending on the desired method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition, and those skilled in the art will know that it is effective for the desired treatment. Dosage can be easily determined and prescribed. The pharmaceutical composition of the present invention may be administered once a day, or may be administered in several divided doses.

In addition, the present invention provides a health functional food composition for preventing or improving diabetes overexpressing DPP4 (dipeptidyl peptidase 4) containing licochalcone A as an active ingredient.

The present invention can be generally used with commonly used foods.

The food composition of the present invention can be used as a health functional food. The term “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Health Functional Food Act, and “functionality” refers to food that is related to the structure and function of the human body. It means ingestion for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects.

The health functional food composition may contain common food additives, and its suitability as a “food additive” is determined in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety, unless otherwise specified. The decision is made based on the specifications and standards for the item.

Items listed in the “Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as subchromic pigment, licorice extract, crystalline cellulose, high-liquid pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in the form of capsules, hard capsules can be manufactured by mixing and filling the composition according to the present invention with additives such as excipients in a regular hard capsule, and soft capsules can be manufactured by mixing and filling the composition according to the present invention. It can be manufactured by mixing with additives such as excipients and filling it with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

Definitions of terms such as excipients, binders, disintegrants, lubricants, coagulants, flavoring agents, etc. are described in literature known in the art and include those with the same or similar functions. There is no particular limitation on the type of food, and it includes all health functional foods in the conventional sense.

In the present invention, the term “prevention” refers to all actions that inhibit or delay DPP4 overexpression diabetic disease by administering the composition according to the present invention.

In the present invention, the term “treatment” refers to any action that improves or beneficially changes the symptoms of DPP4 overexpression diabetic disease by administering the composition according to the present invention.

In the present invention, the term “improvement” refers to any action that improves the bad condition of DPP4 overexpression diabetic disease by administering the composition according to the present invention.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[ Experiment example 1] Protein and ligand preparation

The crystal structure of dipeptidyl peptidase-4 (hereinafter referred to as DPP4, PDB ID: 4FFW) was obtained through the protein databank, and the three-dimensional structure of licochalcone A (hereinafter referred to as LicA) (CID: 5318998) was searched in the PubChem database.

[ Experiment example 2] Molecular docking Docking )

Ligands were docked into DPP4 using AutoDock 4.2. To minimize the energy utilization of the ligand, the MMFF94 force field was used, and docking calculations were performed on the target protein (DPP-4). An affinity map measuring 40 × 40 × 40° was created using the grid tool (auto grid tool) with the goal of controlling the grid of the DPP4 catalytic site. The X, Y, and Z values were kept at 34.36, 48.98, and 40.19, respectively. The initial position, orientation, and twist of the ligand were determined randomly. Each docking experiment consisted of 100 individual runs, and each run was programmed to terminate after a maximum of 2,500,000 energy evaluations. The final figures (values) were derived using Discovery Studio 2021.

[ Experiment example 3] Protein-protein interactions ( PPIs )

Protein-protein docking simulations were performed using the web version of PatchDock (https://bioinfo3d.cs.tau.ac.il/PatchDock/) and FireDock (http://bioinfo3d.cs.tau.ac.il/ It was ranked separately with FireDock/). PatchDock generated 100 predictions for each interaction and submitted them to FireDock to select the 10 best solutions based on global energy.

[ Experiment example 4] Molecular dynamics simulation

Through studies of the internal motion of proteins, many biological functions and dynamic mechanisms have been identified. Molecular dynamics (hereinafter referred to as MD) simulations of the LicA+DPP4 and sitagliptin+DPP4 complexes were performed using GROMACS 2019.6 at 300K, and the GROMOS 9643a1 force-field was used. The topology and force field parameters of the compounds (LicA and sitagliptin) were generated using the PRODRG server, and the charge of the topology file was manually modified. Place the LicA+DPP4 and sitagliptin+DPP4 complexes in a cubic box with an initial dimension of 8 nm filled with water molecules, and use the gmx editconf module to generate boundary conditions and a gmx solvent module for dissolution. The edge of the box was set to be located 1 nm away from the molecule. To dissolve the protein, the Simple Point Charge (spc216) water model was used. Using the particle mesh Ewald method, the interaction between the compound and DPP4 was confirmed through energy-grps in the molecular dynamics parameter (mdp) file, and the MD system was minimized using the steepest descent (1500 steps). Afterwards, the temperature was raised (0 to 300 K) over a 100-ps equilibrium period under periodic boundary conditions at a constant volume. The equilibration process was completed through two stages, NVT ensemble and NPT ensemble, and the final production stage (100 ns) was performed at 300 K. MD trajectories were analyzed using the GROMACS analysis module, and a graphical presentation of the 3D model was created using Discovery Studio 2021 and PyMOL.

[ Experiment example 5] DPP4 Enzyme activity assay ( in vitro )

To confirm the DPP4 inhibitory activity of LicA, a DPP4 inhibitor screening kit (Sigma Aldrich, St. Louis, MO, USA) was used. Cleavage of the fluorescent product (μex = 360nm, μem = 460nm), which was proportional to the enzyme activity present in the well, was measured using a microwell plate reader. Fluorescence emission was measured in kinetic mode over time in a 96-well plate with a black well bottom for 30 minutes, and the following equation 1 was used to calculate the relative inhibition ratio. ΔF/ΔT represents the change in fluorescence during a given time interval, and the graph was prepared using Google Sheets.

[Equation 1]

[ Example One] LicA and DPP4's Interaction analysis

According to Experimental Examples 3 and 4, the interaction between LicA and DPP4 was confirmed, and as shown in Figure 1, it was confirmed that LicA strongly binds to DPP4. Specifically, LicA interacted with the residues of DPP4: Arg123, His124, Glu203, Glu204, Ile205, Phe206, Gly207, Phe355, Arg356, Tyr548, Tyr663, Tyr667, Arg670, and Asn711, of which Glu203 was LicA. and formed a hydrogen bond, and the binding energy of LicA and DPP4 was -6.16 kcal/mol.

On the other hand, the control sitagliptin interacted with the residues of DPP4: Arg123, Glu203, Glu204, Ile205, Phe206, Gly207, Phe355, Arg356, Ser631, Tyr632, Tyr663, Tyr667, and Asn711. The binding energy of DPP4 was found to be -6.70 kcal/mol.

In conclusion, it was confirmed that Arg123, Glu203, Glu204, Ile205, Phe206, Gly207, Phe355, Arg356, Tyr663, Tyr667, and Asn711 are DPP4 residues that commonly interact with LicA and sitagliptin.

[ Example 2] Protein-protein interactions ( PPIs ) analyze

According to Experimental Example 3, as a result of applying PPIs to the LicA + DPP4 and sitagliptin + DPP4 complexes through GLP1/GIP (Glucagon like peptide 1/Glucose dependent insulinotropic polypeptide), as shown in Figure 2, the two complexes It was confirmed that GLP1/GIP was activated. Specifically, DPP4 had a global energy of -84.60 kcal/mol when bound to GLP1, but in the presence of LicA and sitagliptin, the global energy decreased to -74.53 and -55.14 kcal/mol, respectively. In addition, DPP4 had a global energy of -62.42 kcal/mol when bound to GIP, but in the presence of LicA and sitagliptin, the global energy decreased to -23.17 and -45.30 kcal/mol, respectively.

[ Example 3] LicA + DPP4 and Sitagliptin + DPP4 Trajectory analysis of the complex

In general, RMSD provides an inference about the extent of deviation of an atomic group (protein, ligand, or ligand-protein complex) with respect to the respective initial reference structure, as MD results show that DRMSD values reflect the backbone of the DPP4 enzyme.

Therefore, the stability profiles of LicA+DPP4 and sitagliptin+DPP4 complexes were monitored using the GROMACS command gmx_rmsd to estimate their respective RMSD values throughout the simulation run, as shown in Figure 3, LicA+ The average RMSD values of DPP4 and sitagliptin+DPP4 complexes were 0.251nm and 0.335nm, respectively, confirming that the LicA+DPP4 complex exhibits a more stable trajectory than the sitagliptin+DPP4 complex.

In addition, in order to investigate the ligand dynamics within the catalytic pocket of DPP4, the trajectories of the two complexes were visually checked, and as shown in Figure 3, both compounds (LicA and sitagliptin) showed similar interaction patterns. It was confirmed that stable binding appears in the catalytic pocket of DPP4. Additionally, the Rg plot showed little difference between the DPP4 enzyme and the compounds, indicating that the DPPT enzyme is stable with these compounds.

Additionally, as a result of identifying specific residues interacting with the compound using an RMSF plot, a higher fluctuation region was seen at residues 239 to 247, as shown in Figure 3, and the LicA+DPP4 complex There was significant variation in the region 239~247, and it was confirmed that the highest variation occurred in the residue Ser-243. This means that the DPP4 enzyme was stabilized by binding to the compounds (LicA and sitagliptin).

[ Example 4] LicA DPP4 Inhibitory activity assay

According to Experimental Example 5, the DPP4 inhibitory activity of LicA was confirmed, and as shown in FIG. 4, it was confirmed that LicA inhibited DPP4 activity in a concentration-dependent manner (IC ₅₀ = 347.93 μM).

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

A pharmaceutical composition for preventing or treating diabetes overexpressing DPP4 (dipeptidyl peptidase 4), comprising licochalcone A as an active ingredient. The method of claim 1, wherein the lycochalcone A is licorice ( Glycyrrhiza uralensis ) A pharmaceutical composition characterized in that it is isolated or extracted from The pharmaceutical composition according to claim 1, wherein the licochalcone A inhibits DPP4 activity. The method of claim 1, wherein the lycochalcone A is one or more DPP4 residues selected from the group consisting of Arg123, His124, Glu203, Glu204, Ile205, Phe206, Gly207, Phe355, Arg356, Tyr548, Tyr663, Tyr667, Arg670 and Asn711. Pharmaceutical compositions characterized in that they interact. The pharmaceutical composition according to claim 1, wherein the licochalcone A activates GLP1 (Glucagon like peptide 1) or GIP (Glucose dependent insulinotropic polypeptide). A health functional food composition for preventing or improving diabetes overexpressing DPP4 (dipeptidyl peptidase 4) containing licochalcone A as an active ingredient.