TW201244728A - Use of glycyrrhizic acid in preparing drug compound for type II diabetes and medical composition for treating type II diabetes - Google Patents

Abstract

A use of glycyrrhizic acid in preparing drug compound for type II diabetes that using the glycyrrhizic acid as the active ingredient for treating type II diabetes to make the cell expressing the glucose transporter 4, and strengthening the transport the glucose from extracellular domain to intracellular domain; and a medical composition for treating type II diabetes comprises glycyrrhizic acid and acceptable carriers in medicine.

Description

201244728 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to the use of a glycyrrhetinic acid, in particular to a licorice-human fee for the preparation of a medicament for the treatment of a second-type diabetes drug. [Prior Art] Licorice, alias sweet grass, firecracker, sweet root, and open grass, is the root and rhizome of the leguminous herbaceous licorice (also Fish.). Traditionally, Chinese herbal medicine has widely used licorice as a medicinal material for regulating cold and heat medicines, and its sweet taste can overwhelm the tingling and bitterness of other medicinal materials. Therefore, Chinese medicine practitioners often add licorice to the medicine list to improve the taste of other medicinal materials. The main component of licorice is the triterpenoid Glycyrrhizic acid, also known as Giycyrrhizin, which is the sweet source of licorice. Glycyrrhiza Ssl is produced by β-D-Gravial Hydrase (p_D_giucuronidase) and produces Glucuronic acid and a molecule of 18β-glycyrrhetic acid. This glycyrrhetinic acid Glycyrrhizic acid is an important pharmacologically active ingredient in licorice, and glycyrrhizic acid accounts for about 5~11% of the main components of licorice. 'Glycyrrhetinic acid accounts for 3~7〇/〇. Please refer to the chemical structure of glycyrrhetinic acid shown in Figure 1. Glycyrrhetinic acid, also known as G1ycyrrhetinic acid, is a white crystalline powder that is odorless and tasteless. The molecular formula is (^ over 46〇4, molecular weight is 47). 〇64, not in the water and good lipophilicity. In recent years, studies have found that glycyrrhizic acid must be decomposed into glycyrrhetinic acid before being absorbed by the small intestine, and more than 97% of glycyrrhetinic acid obtained by hydrolysis of glycyrrhizic acid is丨叩_Glycyrrhetinic acid, the rest is 18α_glycyrrhetic acid, of which 18 licorice has a very good pharmacological activity, and 201244728 is better than glycyrrhizic acid and 18 α-glycyrrhetinic acid. Natural medicine for the treatment of viral hepatitis and HIV virus' and glycyrrhetinic acid is also active for other physiological effects: 1. Humoral regulation: glycyrrhetinic acid has a deoxysonic cortex-like effect The amount of sodium and sodium is reduced, the discharge is increased, the normal ratio of blood in the middle and the discharge is maintained, and the excretion of water is regulated, and the amount of intracellular fluid and external fluid is maintained. 2. Immunomodulation · · Glycyrrhetinic acid It inhibits the activation and proliferation of tau lymphocytes and giant scorpion cells, and strengthens the effect of cortisone to exert anti-free properties. In addition, glycyrrhetinic acid and its derivatives can inhibit lipoxygenase and bad lactase and inhibit inflammation. The formation of sexual mediators produces anti-allergic effects; 3. Antispasmodic effect. Glycyrrhetinic acid inhibits the activity of cytochrome ρ isozymes, and reduces the toxicity of toxic heat and carcinogens. Phase II metabolic enzymes (Phasenenzyme) Activity, increase = :=Γ! The combination of metabolic enzymes 'accelerates the discharge of toxic drugs and carcinogens in Zhao, reduces its damage to the liver; plays a dual role: the glycyrrhetinic acid on some chemical substances caused by the moon film (4) And guinea pig stress gastric bleeding have inhibitory effect. 5. Tumor inhibition: no matter the acidity of the small 岐 st disaggregated water, licorice ^ ^ mu, the side spoon has an inhibitory effect, and, with the evening high or 3-曱 斗 二 · · dimethyl dimethyl a dimethyl = = = = ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( = = = = = = = = = = = = = = = = = = = Type of diabetes drug-based 'Xizhi drop, (four) with machine ;=要= 201244728 Abdominal (four) cells affinity for diabetes drugs, thereby increasing the sensitivity of p cells to (d) sugar, promoting " fine running to secrete sputum to allow cells to absorb glucose, or (2) improving peripheral tissue for 姨 island The sensitivity of the hormone increases the effect of the cells on the absorption of glucose. And the second known hypoglycemic drugs are mostly synthetic drugs. The synthetic drugs usually have many side effects and cause a great burden on the patient's liver. The present invention is directed to a use of glycyrrhetinic acid for the preparation of a pharmaceutical compound for treating type 1 diabetes, wherein glycyrrhetinic acid is capable of guiding cells to express glucose transporters. The performance of 4 proteins (Gluc〇se (10) 4, GLUT4 for short) enhances the transfer of glucose from the extracellular to the intracellular cells to lower blood sugar. * The second object of the present invention is to provide a use of glycyrrhetinic acid for the preparation of a compound for treating a second type of diabetes, wherein the glycyrrhetinic acid, the cell surface glucose transporter 4 protein exhibits (four) treatment or prevention of blood sugar south. The Department of Medicine provides a treatment for type 2 diabetes. In the eighth, glycyrrhetinic acid acts as a pharmaceutically active ingredient in combination with the insulin receptor of the organism, which enhances the action of cells to move glucose from the cell to the cell. The purpose of this is to provide a kind of treatment for type 2 diabetes: Le, · and compound ' Among them, glycyrrhetinic acid is a natural compound, which can reduce the burden on the liver of *. ~ 201244728 In order to achieve the above object, the technical content of the present invention includes: private-species licorice (4) to prepare and cure (4) type 2 diabetes drug compounding „ licorice- auxoic acid as treatment (4) type 2 diabetes drug combination The sputum component 'actuates the glucose transporter 4 protein, which enhances the cell's transfer of sputum from the extracellular to the intracellular. The pharmaceutical composition for treating type 2 diabetes contains hypoallic acid and is pharmaceutically acceptable. The carrier of the glycyrrhetinic acid is a concentration of 10 to 2 〇 0 micromolar. The pharmaceutical composition is a dosage form of a tablet, a powder, a granule, a pill, a capsule or a liquid. The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings in which: FIG. The use of the second type of diabetes music material s material 'specially the glycyrrhetinic acid system as the treatment of the second type of diabetes drug compound active age, (four) with the money body combined with the guide cells for pancreas The related signal transmission pathway, and the cells express the glucose transporter 4 eggs (GLUT4), and spread the 4 eggs to the cell membrane, enhance the cells to transfer glucose from the extracellular to the intracellular and 0_ =::==== 201244728 Hepatocarcinoma cell lines were co-cultured, and the following tests were performed separately: (A) cell viability test, (B) glucose uptake rate test, (c) glucose transporter 4 protein expression test (D) Insulin receptor binding assay, (8) Muscle X body matrix protein activation assay, and (F) Glycyrrhetinic acid signal transduction pathway assay, etc. ' Record the cell physiology changes of the liver cancer cell line. This example is selected Human hepatoma cell line (HepG2 cells) was purchased and purchased from Taiwan Hsinchu Corporation Legal People's Food Industry Development Research Institute, and its registration number is CCRC 60025. This example is the liver cancer cell. The strain was cultured in DMEM medium (Dulbecco, s modified Engle's medium) at a temperature of 37 ° C under 5% carbon dioxide gas until the cancer cells proliferated to the cultured barn. After eight minutes, a buffer solution is taken, and the proliferation cultured cancer cells are washed from the wall of the culture vessel into the buffer to facilitate cell counting of the liver cancer cell line, and subsequent glucose absorption rate, glucose transporter. 4 protein expression assay, insulin receptor binding assay, insulin receptor matrix protein activation assay and glycyrrhetinic acid message transmission pathway assay. More specifically, this example is the proliferation of cancer cells in 10% The DMEM medium of fetal bovine serum albumin, after the cancer cells grow to 70% of the culture container, repeatedly rinse the oyster culture with a commercial trypsin-EDTA buffer to wash the adherent cancer cells. Subsequent experiments were performed in chymotrypsin-EDTA buffer. (Α) Cell viability assay to confirm that the glycyrrhetinic acid of the present invention does not have a cytotoxic effect on cells. In this example, the cell viability is determined by the cell viability staining method (MTT assay) 201244728, and the mitochondria of living cells are utilized. The succinate dehydrogenase (dehydrogenase) contained in the metabolites is metabolized in the yellow MTT [ 3-(4,5-cimethylthiazol-2_yl)-2,5-diphenyltetrazolium bromide (MTT) for a certain reaction time. The tetrazolium in MTT is converted into a blue product formazan accumulated in the cells, and then added with DMSO to dissolve formazan to measure the content of formazan in the cells. Formazan has an absorption value at a wavelength of 570 nm, when the number of living cells is increased (the cell The more vigorous the mitochondrial respiration, the higher the succinate dehydrogenase activity, the higher the amount of MTT metabolized to formazan, the higher the absorption at 570 nm, so the absorbance at 570 nm can be used to estimate cell viability. To confirm that the glycyrrhetinic acid of the present invention does not have cytotoxicity. Please refer to Fig. 2 and Table 1. In this example, 9 groups of cancer cells (each group containing 1×10 /5 cells/ml of HepG2 cells), respectively, with 〇, 10, 20, 30, 40, 50 , 60, 80 and 1 〇〇 micromolar concentration (μΜ) of glycyrrhetinic acid co-cultured at 37 ° C, 5% carbon dioxide gas 'the 9 groups of cancer cells in the order of no added glycyrrhetinic acid control Group AA to group A8 and group A1 to A8 were cultured for 1 hour, and cell viability test was performed to confirm the survival rate of each group of HepG2 cells. Difference 1 Table: Glucose Absorption Rate of Group A0 to A8 (〇/〇) Group A0 A1 A2 A3 A4 A5 A6 A7 A8 Survival Rate (%) 100 126 120 118 116 126 127 117 103 Measured in Group A0 The absorbance of the HepG2 cells in the A〇 group was 100%, and the survival rate of HepG2 cells in the other groups was higher than II5%. Except the A8 group, the remaining groups were significantly different from the A group. 201244728

Poor, ((4) this). Therefore, the cell viability staining method of the present invention shows that the glycyrrhetinic acid of the present invention does not cause cytotoxicity, and the cell activity of the HePG2 cells of the experimental group having a concentration of less than 1〇〇μΜ is more than 115% higher, even having the effect of promoting cell growth. Therefore, it was confirmed that the glycyrrhetinic acid of the present invention does not cause a toxic effect on cancer cells. The X (B) glucose absorption rate test is also the glycyrrhetinic acid of the present invention, which has the function of promoting the transfer of glucose from the extracellular to the intracellular side of the cells. In this embodiment, a grape-like surface-2-NBDG [2- (N_(7_nitrobenz_2)amino 2 deoxygiucose] test the effect of cells absorbing glucose. The glucose analog can scatter light with a wavelength of 528 nm under a light source with a wavelength of 485 nm. The cells absorb the glucose analog in the cell at a wavelength of The 485 nm light source excites the glucose analog and measures the absorbance at a wavelength of 528 nm to estimate the efficiency of glucose absorption by each group of cells. 凊 Referring to Figures 3 and 2, this example is a group of 6 cancer cells (each The group contained lxl〇5 cells/ml of HepG2 cells, respectively, with 〇, 1〇, 20, 30, 40 and 50 micromolar concentrations (μΜ) of glycyrrhetinic acid at a temperature of 37 C, 5% dioxide gas Under the condition of co-cultivation, the 6 groups of cancer cells were sequentially selected from the control group No. 1 and the 5 groups in which the glycyrrhetinic acid was not added, and the glucose analog was added to each group of cancer cells after 1 hour of culture. In the same condition After incubation for 30 minutes, the cells were washed with PBS buffer solution to remove the glucose analog and the remaining medium which were not absorbed by the cells and excited by the light source at a wavelength of 485 nm, and the absorbance at a wavelength of 528 nm was measured. 201244728 ～ Glucose absorption rate (0/〇) B2 B3 B4 273 307 B5 373 Absorption rate 丨, "w early--and after adding the glucose analog, not (4) 3G minutes of incubation, the direct operating wavelength is like (10) absorbance as Benchmark (set to 1 G ()% glucose analog absorption rate), the BG group of the control group and the glucose analog absorption rate of the mth to B5 groups of the experimental group have a positive side tendency with the concentration of the licorice reading, confirming the present invention Glycyrrhetinic acid has the effect of promoting the absorption of glucose by cells. (C) The expression of glucose transporter 4 protein is tested to confirm that the present day licorice has the function of promoting the expression of glucose transporter 4 eggs, so that the cells (tetra) glucose are extracellularly From the town to the cell, this embodiment is a glucose transporter of the cells by Western blotting method (Westem g) _ the glycyrrhetinic acid or insulin (UK) nM of the present invention Now the amount of 'confirmed that the increase from the reservoir licorice read grape sugar transporter 4 (iv) proteins promote cell performance grape sugar transfer from extracellular to intracellular. In March, as shown in Fig. 4 and Table 3, in the present example, 6 groups of cancer cells (each group containing 1 x 10 cells/ml of HepG2 cells) and 30 μL of glycyrrhetinic acid at a temperature of 37 were used. (:, 5% dioxygenated WEB conditions were co-cultured for 10, 20, 30, 60 and 120 hours. The 6 groups of cancer cells were sequentially selected as the control group CG group and the experimental μ group α to C5. The protein was subjected to Western blotting method, and the GLlm protein content of each group was quantified as the optimal action time of the milk of the present milk. In addition, there were 6 groups of insulin (l〇〇nM) control groups, respectively. The same conditions were cultured in the c〇~C5 group. The 6 groups of insulin control groups were the ic〇 group, the 201224,448 group, the iC2 group, the iC3 group, the iC4 group, and the iC5 group, respectively. The glycyrrhetinic acid in this test does have the same effect as insulin, and both have the effect of improving the performance of GLUT4 protein. More specifically, this embodiment is a cancer cell culture solution of the above C〇~C5 group and the iCO~iC5 group. After removing the culture solution, the cancer cells are washed with a pBS buffer solution, and then the PBS buffer solution is removed, and then diluted in a protein lysis buffer at a certain ratio (preferably 1:10) for cell disruption treatment. This embodiment is oscillated for 30 minutes at temperature. 'After centrifugation at 100 rpm for 10 minutes to obtain protein in the cells; and then analyzed by a commercial protein quantitative kit (Bi〇_Rad protein assay reagent; Bio-Rad lab, USA) by each group (CO~C5) And iCO~iC5 group) The total amount of protein extracted by the cells, taking the same amount of protein, using Western anti-GLUT4 antibody (santa Cruz Biotechnology, hic, USA) for Western blot analysis. Table 3: C0~C5 Group GUJT4 performance multiplier array C0 Cl C2 C3 C4 C5 multiple 1 6.8 4.4 3.1 2.8 1 group iCO iCl iC2 iC3 iC4 iC5 multiple 1 5.2 4.1 3.2 3.4 2.8 Please refer to the Western blotting protein shown in Figure 4 a The staining result (the experimental control protein of the present example was β-actin), and the GLUT4 signal of the control group (the group c〇) was used as the reference, and the GLUT4 expression amount of the experimental group (the ci~C5 group) was compared with the licorice time. The action time of acid has a positive correlation trend, especially within the 0th to the minute, even if the cell exhibits GLUT4, the GLUT4 signal of the group of 12-15 201244728 is 6.8 times of the control group (C0 group.) signal. 'And in the C2~C4 group Although the GLUT4 signal is lower than that of the C1 group, it is higher than the GLUT4 signal of the control group. Please also refer to the Western blotting protein staining result shown in Figure 4b (the experimental control protein of this example is β-actin). Based on the GLUT4 signal of the control group (iCO group), the GLUT4 expression level of the experimental group (iCl~iC5 group) has a positive correlation with the action time of insulin, especially within the first 10 minutes. The GLUT4 signal of the i-th group was 5.2 times that of the control group (iCO group), and the GLUT4 signal of the group C2 to C4 was higher than the GLUT4 signal of the control group, confirming the present invention. The glycyrrhetinic acid does have the effect of promoting the expression of the glucose transporter 4 protein in the cell, thereby promoting the cell to transfer glucose from the extracellular to the intracellular. (D) Insulin receptor binding assay to confirm that the glycyrrhetinic acid of the present invention achieves the effect of promoting the absorption of glucose by the cells by binding to the insulin receptor, and the present embodiment is based on the first and second insulin resistance receptors. The antibody is labeled with insulin, and the fluorescent cursor calibration of the antibody is detected by a first-class cytometer (Becton Dickinson; FACScan, California, USA), and the result is that the glycyrrhetinic acid competes with the grade 1 antibody for the insulin receptor binding site. The binding rate of the grade 1 antibody to the insulin receptor' further inferred the binding rate of glycyrrhetinic acid to the insulin receptor. This example tests the binding rate of insulin receptors for different doses of glycyrrhetinic acid (D1) and (D2) different durations of action. (D1) For the different glycyrrhetinic acid action time and insulin receptor binding rate, please refer to the 5th and 6th and 4th tables. In this example, 7 groups of cancer cells are used (each group contains lxl〇5 cens/ml). HepG2 cells), and

S-13—201244728 3(^]^ glycyrrhetinic acid or 1()()11]^Tengdaosu co-cultivate 〇, 0.5, i or 3 hours at a temperature of 37. 〇, 5% carbon dioxide gas, please Refer to Table 3 for the group of 7 cancer cells and the test conditions, and the 7 groups of cancer cells are in the control group D1-0 group (without adding glycyrrhetinic acid or insulin) and the experimental group D1-1 In the group D-1, the cell culture medium of each group was subjected to flow cytometry analysis, and the glycyrrhetinic acid or the binding ratio of insulin to insulin receptor of the present invention was confirmed. Table: D1-0 to D1-6 The test group has a combined rate of chromium ruthenium and sputum" and D1-0 D1-1 D1-2 D1-3 D1-4 D1-5 D1-6 as Ig·min (min) 0 0.5 1 3 1 3 3 glycyrrhetinic acid ( 3〇μΜ) -- + + + _ — + Tengdaosu (100ηΜ) ———— + + + Light quantity (%) 53.5 81.8 88.9 98.0 83.7 98.5 97.6 Fluorescent drop (%) —— 28.3 35.4 44.5 30.2 45.0 44.1 Anti-sense binding rate (%) 100 37.6 22.4 2.35 34.1 1.18 3.53 In more detail, in this example, the cells of the above groups were washed with a PBS buffer solution, and then centrifuged at 100 rpm for 5 minutes. The cells were washed with a cell buffer (0.5 g BSA in 100 ml of PBS buffer) and dissolved back into the cell buffer, and shaken for 10 minutes before adding an anti-insulin receptor class 1 antibody (Anti-InsR). Antibody; BD, USA) was reacted at 4 ° C for 1 hour, and then reacted with a grade 2 antibody (anti-mouse IgG-FITC; BD, USA) at a temperature of 4 ° C for 0.5 hours, then washed with PBS buffer solution. Thereafter, the cells are re-dissolved into 1 ml of PBS, and the antibody fluorescence amount test can be performed by the flow cytometer, and the amount of fluorescence represents the amount of pancreatic acid (4) which is not combined with glycyrrhetinic acid or pancreatic A-protein in 201244728, The extent to which the amount of fluorescence measured by the cytometer is shifted to the left represents the degree of binding of the antibody to the insulin receptor, that is, the amount of binding of the licorice or the A receptor to the insulin receptor is increased. ...the flow cytometry fluorescence amount diagram shown in Figures 5 and 6, based on the peak position of the fluorescence amount of the control group (the third group), the reference line (the dotted line shown in the figure) is set, and the comparison is recorded. Group (D1_〇 group) and experimental group (D1-1 to D1-6 group) g: on the base Percentage of camp light on the left side of the guideline. When the amount of fluorescence of each experimental group is shifted to the left of the baseline, the amount of fluorescence decreases, and based on the amount of fluorescence of the control group (5) 5%, it is determined as licorice reading or insulin. The amount of binding to the insulin receptor, the amount of fluorescence of each experimental group divided by the amount of fluorescence of the control group, the antibody binding rate of each experimental group was obtained (the higher the anti-coincidence rate, the licorice or the insulin and insulin The lower the binding rate of the receptors, the f group is tested by different glycosic acid, and the binding rate of glycyrrhetinic acid to the receptor is related to the glycyrrhetinic acid (4). A positive correlation trend, and the D1-4 and D1-5 groups were tested with insulin for different durations of action', indicating that the binding rate of insulin to the receptor of the island of insulin also has a positive phase-potential with the concentration of insulin, and The result of D1 is that it can bind to the insulin receptor regardless of glycyrrhetinic acid or insulin. (D2) Different glycyrrhetinic acid doses and insulin receptor binding rates are clear. Referring to Figures 7 and 8 and Table 5, in this example, 7 groups of cancer cells are used (each group contains 匕1〇5 (^113). /1111]9;卬(}2 cells), co-cultured with 30, 100, 200 μg glycyrrhetinic acid or 5〇, 1〇〇, fine insulin at 37 C, 5% carbon dioxide gas for 3 hours , —15 — 201244728 Please refer to the 5th episode of the 7 groups of cancer cells and test conditions, and let the Hai 7 group of cancer cells in the same group as the control group D2-0 (without adding glycyrrhetinic acid or ventral island) And the group of D2-1 to D2-6 of the group of mice. After the cell culture solution of each group was subjected to flow cytometry analysis, it was confirmed that the different concentrations of glycyrrhetic acid or insulin and insulin receptor of the present invention were combined. Table 5. Table D, D2_0~D2-6 test conditions and antibody combination group D2-0 D2-1 D2-2 D2-3 D2-4 D2-5 D2-6 Dosage - 30μΜ ΙΟΟμΜ 200μΜ 50ηΜ ΙΟΟηΜ 200ηΜ Antibody Fluorescence (%) 60.3 73.9 93.8 92.5 89.3 89.7 93.5 Fluorescent Decrease (〇/〇) —— 13.6 33.5 32.2 29.0 29.4 33.2 Antibody binding rate (%) _ 100 62.9 8.6 13.4 21,4 20,0 8.9 In more detail, in this example, the cells of the above groups are washed with a PBS buffer solution, and the same as described in (5). The flow cytometry cell front step = step of antibody fluorescence amount test, and the amount of fluorescence represents the number of insulin receptors that are not bound to glycyrrhizin or insulin, and the fluorescence 畺 is measured by flow cytometry to the left. The degree of shift represents the extent to which the binding of the antibody to the insulin receptor is decreased, that is, the amount of binding of glycyrrhetinic acid or insulin to the insulin receptor is increased. The monthly flow cell shown in Figures 7 and 8 is referred to. Schematic diagram of the amount of fluorescence of the instrument, based on the wave of the fluorescence of the control group (D2), set a soil line (the dotted line of the picture) to record the control group (D2_〇 group) and experiment The group (the first brain ~ 〇 2_6 group) falls on the left side of the baseline, the amount of light I scores, the amount of each of the real frequency, the leftward shift to the county, the amount of light, and the amount of light in the camp, and the amount of light in the control group. (10) Correction as a benchmark, 201244728 combination of grass money or insulin and insulin receptor The amount: each: the body divided by the amount of camp light of the control group to obtain the islets of each experimental group (antibody binding (four) Gaoyu licorice reading or the lower the binding rate of Tengdaosu with I, and -), wherein, 02 The -1~D2-3 group is different from the test 'showing the glycyrrhetinic acid and insulin receptor!) 2 mouth 4^ has a positive correlation trend with the concentration of glycyrrhetinic acid, while the first (four)/group is Insulin, which is not tested, is tested to show islet trends. The binding rate of the body of the body also has a positive correlation with the concentration of insulin. * The licorice of the present invention has the ability to bind to the insulin receptor by the different action time and dose of the present example, and thus has the function of activating insulin downstream signaling. (Ε) Insulin Receptor Matrix Protein Activation Assay The message transmission triggered by insulin is carried out via several highly regulated ear-external networks, such as the ΡΙ3Κ message delivery pathway and the ΜΑΡΚ message transmission pathway. More specifically, when insulin binds to the insulin receptor, it inactivates the insulin receptor substrate proteins (irs proteins), which are related kinases (Kinase) that cause downstream signaling pathways: acidification. In order to confirm that the glycyrrhetinic acid of the present invention has the function of inducing insulin-related insulin signaling pathway, this embodiment detects the glycyrrhetinic acid-induced insulin receptor matrix protein (IRS-1 protein) by the western blotting method. The phenomenon of activation, that is, the insulin receptor matrix protein is acidified, confirming that the glycyrrhetinic acid system has a function of inducing downstream signaling of the insulin receptor. In this example, the insulin receptor matrix protein activation was tested for different action times of (1) glycyrrhetinic acid and (E2) 17 - 201244728 different doses. (E1) Different glycyrrhetinic acid action time and insulin receptor-based activation, please refer to Figures 9a and 9b and Tables 6 and 7, shown in this example, 12, and cancer cells (each group contains 1χ105) Cells/ml of HepG2 cells, 6 of which were co-cultured with Μ, 10, 20, 30, 60 and 120 J at 30 °C and 5 °/Q carbon dioxide gas respectively. Refer to the 6th table for the group of 6 groups of cancer cells and the test conditions, and the 5th 6th group of cancer cells were sequentially in the control group E1〇 group (without adding licorice) and the only group E1-1 ~E1-5 group, extracting its total protein for Western blotting method' to confirm the activation effect of the different action time of the glycyrrhetinic acid of the present invention on IRS-1. In addition, please refer to the % map and the 7th table, which are the other 6 groups of cancer cells in the present embodiment (the iE1_〇~iE1_5 group, wherein the iE1〇 group is a control group containing no insulin) with insulin (1〇〇). nM) The activation test of jRSd was carried out as a control group to confirm that the IRS_1 activation effect of the glycyrrhetinic acid of the present invention was effective. More specifically, the present embodiment is a cancer cell culture solution of the above E1_〇~E1_5 group and the iEl-Ο~iEl_5 group, and after removing the culture solution, washing the cancer cells with a pBS buffer solution, and then removing the cancer cells. After PBS buffer solution, diluted in a liter ratio (preferably 1 · 1 〇) in a gold lysis buffer for cell rupture treatment. This example is incubated at a temperature of 4 ° C for 30 minutes, and then at 1000 rpm. Centrifuge at a speed of 1 minute to obtain intracellular protein; then analyze by a group of commercial protein quantitative reagents (Bio-Rad protein assay reagent; Bio-Rad lab, USA)

The total protein extracted from the cells of the El-0~El-5 group and the iEl-〇~iEl-5 group was taken as an amount of 18, 201244728, taking the same amount of protein, using - commercial anti-multiple commercial anti-IRS-1 antibody (Lab Visi USA) Method analysis. Know the western ink point action time (min) 0 10 20 m bismuth 1 glycyrrhetinic acid (3〇μΜ) E1-0 E1-1 E1-2 El-3 insulin (ΙΟΟηΜ) iEl-Ο iEl-Ι iEl-2 iEl-3

-, 丨 '丨, 刀如. π 曰 曰 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色 染色The acid 7 time has a positive correlation trend 'specially within the first to the first minute to produce, the activation of the IRS-1' and continued until the 12th minute, the activation of the Chinese fruit (Group E1-5), In addition, the nickname of the unphosphorylated insulin receptor matrix (IRS-1) has a negative correlation with the action time, especially in the E1-5 group. Please refer to the Western blotting protein staining result shown in Figure 9b (the experimental control protein of this example is β-actin), and the phosphorylated insulin receptor matrix protein of the iEl-Ο to iEl-5 group (p _ ; The [RS _丨) signal has a positive correlation with the insulin action time, especially in the first to the first minute, the IRS-1' can be activated immediately and the effect of activating the IRS-1 is continued until the 120th minute. In addition, the signal of the unphosphorylated insulin receptor matrix protein IRS has a negative correlation with the action time, especially the performance of the iEEl-5 group; the insulin of the iEl group activates the IRS- The signal of 1 also has a positive correlation trend. 201244728 There is a promotion of the above-mentioned singularity. The effect of the licorice money on the date of the hair and the insulin 〆 = = = = = = = = = = = = = = = = = = = = , 'tongue (four) (f) glycyrrhetinic acid (four) amount and insulin receptor matrix protein / ancient relationship έ makeup ^ ~ 1G map and 帛 7 table, this example will be 6 groups of cancer two groups The cells containing the coffee (four) ^ are widely separated from the = 0, 10, 2 (), 3 (), 4 () and 5 _ glycyrrhetinic acid at temperature. - The cells were co-cultured for 2 hours under conditions of emulsified carbon gas. Please refer to the group 7 and the test conditions of the 6 groups of cancer cells, and the 6 groups of cancer cells were sequentially selected as a control group E2_〇 group and experiment. The group of the group, the extract...the heart protein was subjected to the Western blotting method, and the activation effect of the different doses of the glycyrrhetinic acid of the present invention on the IRS-1 was confirmed. More details δ 'This example is to take the above groups of cells after washing the cells with a PBS buffer solution, and the Western blotting process described in (E1) to carry out the activation test of IRS-1 to confirm the different effects of the present invention. The dose of glycyrrhetinic acid has the effect of activating the IRS-1. Table 6: Test strip of group E? Group Ε 2-0 Ε 2-1 Ε 2-2 Ε 2-3 Ε 2-4 Ε 2-5 dose of glycyrrhetinic acid (μΜ) 0 10 20 30 40 50 Please refer to paragraph 10. The Western blotting method protein staining result shown in the figure (the experimental control protein of the present embodiment is β-actin), and the phytochemical island polypeptide receptor matrix protein (ρ-IRS-1) signal of the Ε2·〇 to e2_5 group The system has a positive correlation with the dosage of glycyrrhetinic acid, especially in the E2-3 and E2-4 groups. Its activation is 201244728. The effect of IRS-1 is most significant. In addition, the unphosphorylated insulin receptor matrix protein (IRS) The signal has a negative correlation with the action time, and it is confirmed that the glycyrrhetinic acid of the present invention does have the effect of promoting the activation of the I r S -1 by the cells, thereby inducing the effect of the cells entering the insulin receptor downstream. (F) Glycyrrhetinic acid message transmission method The insulin-induced message transmission includes the PI3K message transmission pathway, which mainly controls glucose uptake, glycogen synthesis, etc. to regulate the amount of glucose in the blood; and MAPK The message transmission pathway promotes cell growth and activates the kinase of the PI3K signaling pathway to synthesize proteins. In order to confirm that the glycyrrhetinic acid of the present invention does have an insulin-inducing pathway for inducing cells, this embodiment measures the Akt kinase of the glycyrrhetinic acid and PI3K signaling pathway of the present invention and the MAPK message transmission by Western blotting method. Activation of p38, jNK, and erk kinases (ie, phosphorylation of each kinase) promotes cell expression of GLUT4. 4 Refer to Fig. 10 and Table 7'. In this example, array cancer cells (HepG2 cells containing lxlO5 cells/ml in each group) were firstly used in each test group (FI) Akt kinase, (F2) ERK. Inhibitors of kinase, (F3) p38 kinase and (F4) JNK kinase are added to the cancer cell culture medium, and after adding the glycyrrhetinic acid of the present invention at different times, the kinases of various signaling pathways are confirmed by Western blotting method. activation. More detailed s 'this example is taken as shown in the seventh table of each group of cancer cell culture medium, with the (E1) test treatment step to obtain a total group of cancer cells of the total protein '5 Hai F1 group to - Anti-p-Akt kinase multi-strain antibody and primary antibody 201244728 ===body (10)F2 anti-卩 激酶 kinase multi-strain against two strains of dikinase multi-strain; the F3 group-anti-"38 夕 几 and - ί ̄ ̄ 38 kinase multi-strain antibody; the first F4 group is the primary antibody _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The target kinases were analyzed by Western blotting. ~~7^, ρ order group information, delivery route test conditions\( min ) Inhibitor Akt ERK P38 kinase 0 F1-0 10 20 F1-2 30 60 120 F2-0 F2-1 F3-0 F3-1 F3-2

Fl-3 Fl-4 F1-5 F2-3 F2-4 F2-5 F3-3 F3-4 F3-5 Please refer to the Western blotting protein staining results shown in Figure 11, the F1 and F2 kinases There is a phenomenon of acidification, especially the phosphorylation effect of the F1_2 and F1-3 groups is the most obvious, and the acidification effect of the F2_3 and F2 4 groups is the most obvious, which shows that the glycyrrhetinic acid of the present invention passes through 2〇~6 GLUT4 was induced via the PI3K/Akt kinase signaling pathway and the MAPK/ ERK kinase signaling pathway during the minute of action, as the kinases of the F3 and F4 groups were not phosphorylated, indicating that the p38 and JNK kinases are induced GLUT4 showed no performance; this test confirmed that the glycyrrhetinic acid of the present invention has an effect of inducing a PI3K/Akt kinase message transduction pathway and a MAPK/ERK kinase message transmission pathway. Please refer to the 12th, 13th and 9th tables again. This embodiment will be 6

S—22 — 201244728 Group of cancer cells were first co-cultured with Akt, erk, _, qing, and kinases for 1 hour, followed by glycyrrhetinic acid (30 μΜ) ^37 °C, 5% Under the conditions of carbon dioxide gas, 12 small-inch 乂 、, 屋 (7) test procedures were carried out, and commercial anti-GLUT4 antibody was used for Western blotting analysis. Test conditions Group Glycyrrhizae ^30μΜ:) Stimulant F5-0 F5-1 F5-2 F5-3 F5-4 F5-5

Akt

ERK p38

JNK GLUT4 performance (multiple) per ^ refer to Figure 12 shown in Figure 12, the real touch (4) (10) ώ), from the (four) map of the performance of the bar graph, the F5_〇 group is not added glycyrrhetinic acid or inhibition ^ In the control group, the GLUT4 showed a multiple of ι times, the (5) group did not add a side inhibition (4) added licorice Wei positive control tilt, its gluT4ί 4 Μ times, the F5 rhyme F5_5 time added service and κ _ inhibitor, # GLUT4 The amount of performance = significant: difference, and the - her group added: P38 stimulating_formulation, there is no significant difference with the woven group, the certificate (4) corresponds to the above test about each _ acidification, thereby γ licorice The hypoacid has a signal transduction pathway that induces a ρΐ3κ-exciting relationship, and the effect of GLUT4 in the hyperactive cell. The glycyrrhetinic acid can be prepared as a pharmaceutical active ingredient (active theory) to treat a second type of urinary pharmaceutical composition, 3.04 1.35 1.27

S—23 201244728 can be administered to various biological individuals individually or in combination with at least one commercial or stagnation component in a variety of ways, the carrier being capable of being an excipient or a diluent. The agent can be selected as a mosquito, and the agent can be called but not limited to steaming water or glycerin. The pharmaceutical composition is administered orally to various organisms on a regular basis. Furthermore, the pharmaceutical composition may be in the form of a rotatory agent, a powder, a medicinal granule, a pill, a capsule or a liquid, wherein the pharmaceutical composition contains 1 G to a finely lyced glycyrrhetinic acid, and the pharmaceutical composition is helpful. Cell (4) Glucose is transferred from extracellular to immortal and achieves the efficacy of treating type 2 diabetes. - The present invention is a use of glycyrrhetinic acid for the preparation of a compound for treating a second type of diabetes, wherein the glycyrrhetinic acid can induce the expression of the glucose transporter 4 protein and enhance the transfer of the sugar from the extracellular to the intracellular 'To achieve the effect of lowering blood sugar. The present invention is a use of glycyrrhetinic acid for the preparation of a medicament for the treatment of a second type of diabetes drug, wherein the glycyrrhetinic acid enables the cell to exhibit the expression of the grape = sugar transporter 4 protein for the treatment or prevention of hyperglycemia. The present invention relates to a pharmaceutical composition for treating type 2 diabetes, wherein glycyrrhetinic acid is a pharmaceutically active ingredient that binds to an islet receptor of an organism, and enhances the action of cells to transfer glucose from the cell to the cell. The present invention is a pharmaceutical group for treating type 2 diabetes, and the glycyrrhetinic acid system is a natural drug compound, which has the function of transferring glucose from the extracellular to the intracellular, and reducing the liver collapse of the patient.

The present invention has been disclosed in the above-described preferred embodiments without departing from the spirit and scope of the present invention. And the modification is still the hair of the present invention: 'The scope of protection of the invention is subject to the definition of the patent scope of the application in May.

[The diagram briefly illustrates the chemical structure of the glycyrrhetinic acid of J invention. Dijon··No = a bar graph of the dose of the grass f acid to the cell survival rate. Bar chart. Face _ (four) read __ like absorption rate egg === action time on glucose transporter 4 J remuneration between glucose transporter 4 egg flow I:::: coffee time and phosphatine slender: Figure 'Figure: different licorice A bar graph of the flow cytometry and the insulin receptor binding rate between the time of subacid action and the insulin receptor binding rate. (5) The first graph of the binding rate of licorice reading dose to insulin receptor: different acid acid side such as (four) acidified insulin receptor

25 S 201244728 Protein staining of matrix proteins. Figure 9b: Protein staining of different insulin action times and phosphorylated insulin receptor matrix proteins. Figure 10: Protein staining of different glycyrrhetinic acid doses and phosphorylated insulin receptor matrix proteins. Figure 11: Kinase protein staining of different signaling pathways. Figure 12: Different signal-transmission pathways of kinase inhibitors and GLUT4 protein staining. Figure 13: Bar graph of protein expression in different signaling pathways of kinase inhibitors and GLUT4. [Main component symbol description] [None] —26 —

Claims (1)

201244728 VII. Application for Patent Park: Buzi acid is used to prepare medicinal herbs for the treatment of type 2 diabetes drug compound 2, in order to treat the second hybrid drug synthesis, so that the cells express glucose transporter 4 protein, enhance," The cell cells transfer glucose from the extracellular to the intracellular. - A composition for the treatment of type 2 diabetes (IV) and a pharmaceutically acceptable carrier. Each has a Ganzhuo 酉夂4: Shen:: The use of the glycyrrhetinic acid according to the above 2, wherein the application of the glycyrrhetinic acid is 1〇~2〇0 micromolar concentration (μΜ). The invention relates to the treatment of type 2 diabetes, and the Q substance 'where' the pharmaceutical composition is a preparation, a powder, a granule, a granule, a capsule or a liquid. S-27.