Medicinal substance for reducing generation of methylglyoxal and application thereof

Abstract

The invention discloses an application of glycyrrhizic acid in preparation of a medicine for reducing generation of methylglyoxal under a high glucose condition, the harm of endogenous methylglyoxal is reduced, and the high glucose condition is 18-25 mmol / L or fasting blood glucose is 10 mmol / L or above. The glycyrrhizic acid directly acts on the target glucose-6-phosphate isomerase to improve the enzyme activity of the glucose-6-phosphate isomerase under a high glucose condition, so that glucose metabolism is regulated and controlled, and the generation of methylglyoxal and D-lactic acid is reduced; the pharmaceutical composition is used for preventing or treating diabetes related complications, the related complications comprise diabetic nephropathy, diabetic eye diseases or diabetic atherosclerosis, and a new technical scheme is provided for preventing or treating the diabetes related complications.

Description

Technical Field

[0001] The invention relates to a medicinal substance for reducing the generation of methylglyoxal and an application thereof, in particular to glycyrrhizic acid and its salts for improving the enzyme activity of glucose-6-phosphate isomerase under high sugar conditions to reduce the generation of methylglyoxal and D-lactic acid, belonging to the field of medical technology. Background Art

[0002] Methylglyoxal (MG), also known as methylglyoxal and 2-oxopropanal, has a CAS number of 78-98-8. Methylglyoxal can be divided into exogenous and endogenous sources. Exogenous methylglyoxal can be ingested through diet, accounting for about 1%. Endogenous methylglyoxal mainly comes from triose phosphates, namely 3-phosphoglyceraldehyde and dihydroxyacetone phosphate, in the process of glycolysis. It is dephosphorylated by non-enzymatic processes or degraded by the enediol intermediates at the active site of triose phosphate isomerase, accounting for about 90% of the total methylglyoxal; other minor sources include degradation of glycated proteins, lipid peroxidation, catabolism of threonine, and oxidation of acetone in ketone body catabolism during diabetic ketoacidosis.

[0003] Under normal physiological conditions, the content of methylglyoxal in the body is maintained at a low level, and about 0.1% to 0.4% of glucose will be converted into methylglyoxal. Under pathological conditions such as diabetes, gluconeogenesis, and glycerol neogenesis, the content of methylglyoxal will increase. Diabetes will reduce the activity of glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis disorders and accumulation of triose phosphate. At the same time, the elimination of triose phosphate is slowed down due to the decrease in the activity of glyoxal metabolizing enzyme Ⅰ (Glyoxalase Ⅰ, GLO1), which together lead to an increase in the content of methylglyoxal, aggravating glycation and leading to the occurrence and development of complications.

[0004] Methylglyoxal is a highly active α-carbonyl aldehyde that can irreversibly modify protein residues and nucleic acids to form advanced glycation end products (AGEs) and DNA adducts. The increase of methylglyoxal and advanced glycation end products is a key factor in the development of the disease. Reducing the generation of methylglyoxal and accelerating its metabolism can reduce its toxicity.

[0005] Aminoguanidine is a strong scavenger of methylglyoxal, the dicarbonyl of which can react with guanidine. Aminoguanidine can effectively reduce the production of advanced glycation end products and protect diabetic nephropathy, retinopathy and neuropathy in diabetic animal models. However, in two large clinical trials, aminoguanidine failed to reduce the doubling time of serum creatinine in type 1 diabetes (T1D), resulting in experimental failure. In clinical trials of type 2 diabetes (T2D), the trial was terminated due to adverse reactions such as gastrointestinal and liver function abnormalities.

[0006] Pyridoxamine is an analog of vitamin B6 that has the function of scavenging methylglyoxal and can effectively reduce the carbonylation induced by methylglyoxal, but its effect is weaker than that of aminoguanidine. At the same time, pyridoxamine can increase the activity of GLO1. In a phase II clinical trial, pyridoxamine not only inhibited the formation of advanced glycation end products, but also significantly improved the renal function of patients with T2D.

[0007] Metformin has a certain effect in reducing the production of methylglyoxal. Metformin inhibits hepatic gluconeogenesis and increases cellular glucose uptake. Improving glucose metabolism and controlling hyperglycemia may be the first step to reduce the accumulation of methylglyoxal. The structure of metformin is similar to the methylglyoxal scavenger aminoguanidine, and it can also capture methylglyoxal. Studies have shown that metformin can reduce systemic plasma methylglyoxal levels and increase the levels of metformin-MG imidazolinone compounds in urine samples.

[0008] In terms of increasing GLO1 activity and accelerating methylglyoxal metabolism, the combination of trans-resveratrol and citrus peel can increase GLO1 activity in peripheral blood mononuclear cells by 22%, while reducing plasma methylglyoxal by 37%. Polyphenols can also increase the expression of GLO1. In a randomized, double-blind, placebo-controlled, crossover trial, quercetin was found to reduce plasma methylglyoxal concentrations in healthy (pre-) hypertensive men and women. However, this effect cannot be explained by increased GLO1 expression, so quercetin's reduction of methylglyoxal may be its clearance effect. Summary of the invention

[0009] The purpose of the present invention is to obtain a medicinal substance with high safety and the ability to reduce the generation of endogenous methylglyoxal under high sugar conditions, so as to provide a new technical solution and option for the prevention or treatment of diabetes-related complications. The Chinese medicine licorice is listed as a top-grade product in the "Shennong Bencao Jing", has the function of "harmony of all medicines", and is widely used in clinical practice; glycyrrhizic acid is the core active ingredient of licorice, and has been developed into a variety of medicines as an active ingredient, which are mostly used in clinical practice for liver protection, anti-inflammatory, etc. At present, there are no reports on the use of glycyrrhizic acid to reduce the generation of endogenous methylglyoxal under high sugar conditions. The glycyrrhizic acid provided by the present invention can be used to reduce the generation of endogenous methylglyoxal under high sugar conditions, and can prevent and treat diabetes-related complications, such as diabetic nephropathy, diabetic eye disease, and diabetic atherosclerosis, or alleviate the clinical symptoms of the above diseases.

[0010] The technical solution adopted to achieve the technical purpose of the present invention is:

[0011] Application of glycyrrhizic acid in the preparation of drugs for reducing the generation of methylglyoxal under high sugar conditions. Endogenous methylglyoxal mainly comes from triose phosphates, namely 3-phosphoglyceraldehyde and dihydroxyacetone phosphate, in the glycolysis process, which is dephosphorylated by non-enzymatic processes or degraded by enediol intermediates at the active site of triose phosphate isomerase, accounting for about 90% of the total amount of methylglyoxal. Glycyrrhizic acid can reduce the generation of endogenous methylglyoxal in the glycolysis pathway under high sugar conditions.

[0012] Preferably, the high sugar condition is a fasting blood sugar level of 10 mmol / L or more. SD male rats were modeled with streptozotocin (35 mg / Kg of streptozotocin was injected intraperitoneally for two consecutive days), and the fasting blood sugar of the tail vein was measured with a Roche blood glucose meter after fasting for 12 hours for about two weeks after modeling. Rats with a fasting blood sugar value greater than 10.0 mmol / L were selected as diabetic model rats, and the fasting blood sugar could reach 33 mmol / L.

[0013] Preferably, the high sugar condition is 18-25 mmol / L. In the in vitro experiment, HepG2 cells were cultured using RPMI-1640 basal medium, and the glucose concentration of RPMI-1640 basal medium was about 11 mmol / L. The blank group was cultured using RPMI-1640 basal medium, and the model group and the glycyrrhizic acid administration group needed to add glucose injection to the culture medium to increase the glucose concentration to 7 mmol / L or 14 mmol / L.

[0014] Furthermore, the glycyrrhizic acid can also reduce the production of D-lactic acid under high sugar conditions. The enzymes that affect the production and elimination of triose phosphate in the glycolysis pathway are crucial to the content of endogenous methylglyoxal. Glucose is phosphorylated to 6-phosphate glucose (Glucose-6-phosphate, G6P) by hexokinase 2, G6P is isomerized to 6-phosphate fructose by glucose-6-phosphate isomerase (GPI), and then converted to 1,6-diphosphate fructose by phosphofructokinase L, and the product is then catalyzed by fructose diphosphate aldolase A to produce triose phosphate. Triose phosphate includes 3-phosphoglyceraldehyde and dihydroxyacetone phosphate, of which 3-phosphoglyceraldehyde is converted to 1,3-diphosphate glyceride by glyceraldehyde-3-phosphate dehydrogenase. The above-mentioned enzymes such as hexokinase 2, glucose-6-phosphate isomerase, phosphofructokinase L, and fructose diphosphate aldolase A jointly regulate the production of endogenous methylglyoxal. At the same time, methylglyoxal in the body is mainly eliminated through the glyoxalase system. First, methylglyoxal reacts with reduced glutathione to form hemithioacetal through a non-enzymatic reaction; secondly, it is catalyzed by glyoxalase I to generate SD-lactylglutathione; finally, SD-lactylglutathione is hydrolyzed by glyoxalase II to non-toxic D-lactic acid (D-Lacticacid, D-LA).

[0015] Furthermore, the glycyrrhizic acid increases the enzymatic activity of glucose-6-phosphate isomerase under high sugar conditions and reduces the generation of methylglyoxal and D-lactic acid. Surface plasmon resonance experiments show that glycyrrhizic acid solutions with concentrations of 25 to 0.78 μmol / L have binding responses with GPI protein, and the equilibrium constant KD value of glycyrrhizic acid and GPI protein is 16.46 μM, and the standard deviation SE (KD) of the KD value is 2.6×10 -6 , indicating that glycyrrhizic acid has a good combination with GPI protein and can mediate GPI protein. After glycyrrhizic acid mediates GPI protein, it increases the GPI enzyme activity of HepG2 cells under high sugar conditions, thereby regulating the glucose metabolic pathway, reducing the production of methylglyoxal and D-lactic acid, and thus reducing the harm caused by methylglyoxal under high sugar conditions.

[0016] Furthermore, the drug is a drug for preventing and treating complications related to diabetes, and the related complications include one or more of diabetic nephropathy, diabetic eye disease or diabetic atherosclerosis. Under pathological conditions such as diabetes, gluconeogenesis, and glycerol neogenesis, the content of methylglyoxal will increase. Diabetes will reduce the activity of glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, leading to glycolysis disorders and accumulation of triose phosphate. At the same time, the reduction in the activity of glyoxal metabolic enzyme I leads to a slow elimination of triose phosphate, which together leads to an increase in the content of methylglyoxal, while aggravating glycation and leading to the occurrence and development of diabetic complications. Methylglyoxal can also cause intracellular protein glycation, causing pathological changes in specific proteins, including hemoglobin, crystallin, mitochondrial proteins and histones. Methylglyoxal and advanced glycation end products can glycosylate and modify type IV collagen and laminin rich in structures such as renal blood vessels, glomerular basement membranes and mesangium, forming intramolecular / intermolecular cross-links, destroying their spatial scaffold structure, causing thickening of the glomerular capillary basement membrane and increased mesangial permeability. Methylglyoxal can also reduce its hypoglycemic effect by changing the structure of insulin and inhibiting its signaling pathway through glycosylation reactions.

[0017] Further, the drug contains 25 to 150 mg of glycyrrhizic acid or glycyrrhizic acid salt, and the glycyrrhizic acid salt is calculated as glycyrrhizic acid. The content of glycyrrhizic acid or glycyrrhizic acid salt in the drug is the specification of the drug, that is, the content of glycyrrhizic acid or glycyrrhizic acid salt contained in a single unit preparation. In actual application, multiple unit preparations can be used according to the effective amount for treatment.

[0018] Furthermore, the glycyrrhizic acid salt is monopotassium glycyrrhizinate, diammonium glycyrrhizinate, glycyrrhizin zinc, monoammonium glycyrrhizinate or magnesium isoglycyrrhizinate. Glycyrrhizic acid contains a carboxyl group in its structure and is acidic to a certain extent. According to the requirements of the dosage form, it can form a salt with a suitable base.

[0019] Furthermore, the medicine also contains a pharmaceutically acceptable carrier or excipient.

[0020] Further, the dosage form of the medicine is tablets, hard capsules, granules, pills or injections. Those skilled in the art can prepare the above-mentioned dosage form by combining the glycyrrhizic acid or glycyrrhizic acid salt with a pharmaceutically acceptable carrier or adjuvant according to the actual needs in a conventional manner. The above-mentioned pharmaceutically acceptable carrier or adjuvant includes: disintegrant, lubricant, diluent, wetting agent, adhesive, colorant, coating material, antioxidant, stabilizer, flavoring agent, preservative, osmotic pressure regulator, pH regulator, etc., such as diluent, one or more combinations of starch, compressible starch, dextrin, microcrystalline cellulose, lactose. Those skilled in the art can recognize that some pharmaceutically acceptable excipients can provide more than one function and provide optional functions, depending on how much of the excipient is present in the preparation and which other excipients are present in the preparation.

[0021] The skilled person has the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable pharmaceutical excipients for the appropriate amount of the present invention. In addition, there are a large number of resources available to those skilled in the art that describe pharmaceutically acceptable excipients and are used to select suitable pharmaceutically acceptable pharmaceutical excipients (i.e., excipients). Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Hand book of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

[0022] Glycyrrhizic acid belongs to triterpenoid compounds, and its molecular formula is C 42 H 62 O 16 , molecular weight 821, is one of the main active ingredients of Chinese medicine liquorice. Glycyrrhizic acid can be obtained by extraction and purification from Chinese medicine liquorice, or it can be purchased. Extraction and purification are both conventional methods in the art, such as immersion, percolation, decoction, reflux or ultrasound; purification methods such as silica gel column chromatography, gel column chromatography or reverse phase column chromatography.

[0023] Definitions and explanations of terms

[0024] The term "effective amount" or "therapeutically effective amount" refers to an amount of the compound of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases as defined below). The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the subject's weight and age, the severity of the disease condition, or the mode of administration, which can be easily determined by a person of ordinary skill in the art. The specific therapeutically effective amount varies depending on the following factors: the specific compound selected, the dosage regimen relied on, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.

[0025] The term "specification" refers to the weight (or potency) or content (%) or filling amount of the main drug contained in each vial, tablet or other unit preparation.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the standard meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0027] Unless otherwise specified, the present invention adopts conventional methods of molecular pharmacology, biochemistry or pharmacological detection, and each step and condition can refer to conventional operation steps and conditions in the art.

[0028] The beneficial effects of the present invention are:

[0029] (1) Glycyrrhizic acid can reduce the production of endogenous methylglyoxal and D-lactic acid under high sugar conditions, reduce the harm of methylglyoxal, and can be used to prevent or treat diabetes-related complications such as diabetic nephropathy, diabetic eye disease or diabetic atherosclerosis.

[0030] (2) Glycyrrhizic acid can directly act on the target GPI and has good in vitro binding, with an equilibrium constant of KD = 16.46 μM. Glycyrrhizic acid can increase the enzyme activity of GPI under high glucose conditions, thereby regulating the glucose metabolic pathway, thereby improving the utilization of glucose by HepG2 cells under high glucose conditions, reducing the production of methylglyoxal and D-lactic acid, and thus reducing the harm caused by methylglyoxal under high glucose conditions.

[0031] (3) Glycyrrhizic acid can reduce the levels of D-lactic acid, uric acid, creatinine and blood urea nitrogen in the serum of diabetic rats. Renal H&E staining also showed that glycyrrhizic acid can improve the renal function of diabetic rats and alleviate diabetic nephropathy.

[0032] (4) Glycyrrhizic acid has good safety. When the concentration of glycyrrhizic acid reaches 1 mmol / L, it shows certain cytotoxicity to HepG2 cells (48 h). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a graph showing the effect of glycyrrhizic acid on the survival rate of HepG2 cells (48h).

[0034] Figure 2 This is the SPR docking fitting curve diagram of glycyrrhizic acid and GPI protein.

[0035] Figure 3 This is the result graph of GPI enzyme activity at 9 minutes.

[0036] Figure 4 This is a statistical chart of the test results of serum uric acid (UA) content in diabetic rats.

[0037] Figure 5 This is a statistical chart of the test results of serum creatinine (Cr) content in diabetic rats.

[0038] Figure 6This is a statistical chart of the test results of serum urea nitrogen (BUN) content in diabetic rats.

[0039] Figure 7 H&E staining of the kidney of diabetic rats. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand and implement the present invention, the present invention is further described below in conjunction with specific examples, but the present invention is not limited to the scope of the examples. The experimental methods used in the examples are conventional methods unless otherwise specified; the experimental animals, materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0041] Example 1: Investigation of safe concentration of glycyrrhizic acid

[0042] 1 cell plating

[0043] Sterilize the instruments and clean bench with ultraviolet for 30 minutes, transfer the cultured cells to the clean bench, aspirate the culture medium in the culture dish, and then wash 1-2 times with PBS, 2.0-3.0 mL each time. Add about 1.5 mL of trypsin to the cell culture dish, disperse it evenly, and then put it into the incubator for digestion. During this period, observe whether the cells are deformed or detached from the wall under a microscope. After the cells are detached from the wall, add 3.0 mL of complete culture medium to terminate the digestion and blow and wash with a pipette, then transfer the cells to a 5 mL centrifuge tube, centrifuge at 1300-1500 rpm for 5 minutes, and discard the supernatant. Resuspend the cells with an appropriate amount of RPMI-1640 complete culture medium. Take an appropriate amount of cell resuspension and dilute it with complete culture medium and aspirate 10 μL to count the cells using a hemocytometer, and adjust the cell count to 6×10 4 ~8×10 4 cells / mL, and add 100 μL to each well of the 96-well plate. Add 100 μL of PBS to each well outside the well where the cells were added, and then place the 96-well plate in an incubator at 37°C and 5% CO 2 nourish.

[0044] 2. Drug administration

[0045] The drug can be administered after the cells attach to the wall. Glycyrrhizic acid is prepared into a 4mmol / L stock solution with complete culture medium, and an appropriate amount of the stock solution is diluted with complete culture medium in equal proportions to form 2, 1, 0.5, and 0.25mmol / L glycyrrhizic acid solutions. The safe drug concentration range of glycyrrhizic acid is 0.25-4mmol / L. Before administration, the residual culture medium in each well is aspirated, and 100μL of complete culture medium is directly added to the blank control well, and 100μL of drug-containing culture medium with different drug concentrations is added to the glycyrrhizic acid-treated well. At the same time, 100μL of complete culture medium is used as the zero control well.

[0046] 3. Survival rate determination

[0047] After 48 hours of drug administration, the residual medium or drug-containing medium in each well was aspirated, and 100 μL of complete medium was added to each well, followed by 10 μL of cell counting reagent (Cell Counting Kit-8, CCK-8), and incubated at 37°C for 2 hours. The absorbance value (optical density, OD) at 450 nm was measured with an ELISA reader to calculate the cell survival rate.

[0048] Cell survival rate = (OD value of drug-treated well - OD value of zero-adjusted control well) / (OD value of blank control well - OD value of zero-adjusted control well) × 100%

[0049] The experimental results of HepG2 cell survival rate (48h) showed that the cells showed certain toxicity only when the concentration of glycyrrhizic acid reached 1mmol / L, and glycyrrhizic acid had good safety.

[0050] Example 2: Glucose consumption measurement (addition of 14 mmol / L glucose)

[0051] 1. Drawing of standard curve

[0052] According to the operation method of the glucose colorimetric test kit (GOD-POD method), a series of 0-30 mmol / L glucose standard solutions were prepared. The amount of each standard solution was 3 μL, and then 300 μL of enzyme working solution was added. The wells were incubated at 37°C for 15 min, and the OD value of each well was measured at 505 nm using an enzyme reader. Two replicate wells were prepared for each concentration, and the standard curve was drawn with the absolute OD value of purified water as 0. The glucose colorimetric test kit (UX05X8B47759) was purchased from Wuhan Elerite Biotechnology Co., Ltd.

[0053] 2 Determination of test solution

[0054] An appropriate amount of cells (about 800,000) were plated in a 6-well plate, and blank group, model group, and glycyrrhizic acid group were set up respectively. After the cells adhered to the wall, the culture medium was completely aspirated, and the culture medium was replaced with RPMI-1640 basal medium, with a total volume of 1 mL per well. The concentrations of added glucose and drugs in the blank group, model group, and glycyrrhizic acid group are shown in Table 1. The glucose concentration in the initial culture medium and the culture medium after each group of cells was cultured for 12 hours was determined by referring to the glucose colorimetric test kit, and then the glucose consumption of HepG2 cells was investigated.

[0055] Table 1 Glucose and glycyrrhizic acid concentrations in the culture medium of each group

[0056]

[0057] The fitting standard curve of the absolute absorbance value Y of the glucose standard solution and the concentration X of the standard solution is Y=0.0434X-0.0087 (r=0.9992), which indicates that the absolute absorbance OD value of glucose in the range of 0-30mmol / L has a good linear relationship with its concentration.

[0058] The 12h glucose consumption of the blank group was 3.52±0.04mmol / L, and the glucose consumption of the model group was 3.547±0.51mmol / L. There was no statistical difference between the model group and the blank group, indicating that the utilization of glucose by HepG2 cells under the induction of 14mmol / L glucose was not significantly changed. The glucose consumption of the glycyrrhizic acid group (administration group) increased to 5.663±0.94mmol / L, which was significantly higher than that of the model group, and there was a statistical difference between the two groups (P=0.0127), indicating that glycyrrhizic acid improved the utilization of glucose by HepG2 cells under high glucose conditions.

[0059] Example 3: L-lactic acid detection

[0060] Referring to the grouping of "Example 2 Determination of Glucose Consumption", the content of L-lactic acid in the culture medium after 12 hours of HepG2 cell culture was determined according to the operation method of the L-lactic acid (LA) colorimetric test kit, and then the production of L-lactic acid in HepG2 cells was investigated. The L-lactic acid colorimetric test kit (UX10RNZ48004) was purchased from Wuhan Elaruite Biotechnology Co., Ltd.

[0061] The fitting standard curve of the absolute absorbance value Y of the L-lactic acid standard solution and the concentration X of the standard solution is Y=0.1941X+0.0544 (r=0.9947), and the concentration range of the L-lactic acid standard solution is 0-7 mmol / L.

[0062] The L-lactic acid content in the cell culture medium of the blank group and the model group was 5.30±0.26 and 5.39±0.1mmol / L, respectively, and there was no statistical difference between the model group and the blank group. The L-lactic acid content in the glycyrrhizic acid group increased to 6.86±0.07mmol / L, and the L-lactic acid production was significantly higher than that in the model group, and there was a statistical difference between the two groups (P<0.0001). This indicates that glycyrrhizic acid increased the production of L-lactic acid in HepG2 cells and enhanced the glycolysis of HepG2 cells. This is consistent with the trend of glycyrrhizic acid increasing glucose consumption, indicating that glycyrrhizic acid improved the glycolysis of HepG2 cells.

[0063] Example 4: In vitro binding experiment of glycyrrhizic acid and GPI protein

[0064] Surface Plasmon Resonance (SPR) is a biosensor analysis and detection technology based on biosensor chips, which can be used to analyze the binding mode between drugs and proteins. SPR technology has the advantages of real-time and no need to label proteins. This study used CM5 chip amino-coupled protein, where channel 1 was the reference channel and channel 4 was the detection channel to verify the in vitro binding and binding strength of glycyrrhizic acid and GPI. The specific steps are:

[0065] (1) Preparation of GPI protein working solution: Take 2 μL of 1.0 mg / mL GPI protein solution and dilute it with 10 mmol / L sodium acetate (pH 4.5) to a final concentration of 20 μg / mL to obtain 100 μL of GPI protein working solution.

[0066] (2) Preparation of glycyrrhizic acid working solution: accurately weigh 9.5 mg of glycyrrhizic acid and add DMSO to prepare a 10 mmol / L glycyrrhizic acid mother solution; take 10 μL of the glycyrrhizic acid mother solution, dilute it to 100 μmol / L with 1×PBST buffer, centrifuge at 12000 rpm for 5 min, take the supernatant, and then dilute it with 1×PBST buffer (containing 1% DMSO) in sequence to obtain a series of glycyrrhizic acid working solutions with concentrations of 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μmol / L.

[0067] (3) GPI protein coupling: The CM5 chip was first activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) solution for 7 min, and then the GPI protein working solution was flowed through the detection channel at 10 μL / min for 10 min to couple the GPI protein to the chip. After GPI protein coupling, the detection channel and reference channel were blocked with ethanolamine for 7 min.

[0068] (4) Working solution test: Place different concentrations of glycyrrhizic acid working solutions into the sample rack in turn and set the positions of each sample. The injection time is 180 seconds, the waiting time is 60 seconds, and the test is run.

[0069] (5) Data analysis: The steady-state equilibrium analysis model was used to fit the binding curve and obtain the equilibrium constant KD.

[0070] The fitting curve of glycyrrhizic acid solution and GPI protein showed that the equilibrium constant KD value of glycyrrhizic acid and GPI protein was 16.46 μM, and the standard deviation SE (KD) of KD value was 2.6×10 -6 This indicates that glycyrrhizic acid has a good binding with GPI protein, indicating that glycyrrhizic acid can mediate GPI protein.

[0071] Example 5: GPI enzyme activity detection

[0072] Referring to the modeling and administration methods in "Example 2 Determination of Glucose Consumption", the cells were cultured in a 6-well plate for 12 hours and then digested. The cells were extracted with the extract provided by the kit, 2 wells of cells were added with 250 μL of the extract, and then ultrasonically disrupted (200 W, 5 min, ultrasonic for 2 s and stop for 5 s). After centrifugation at 12000 rpm for 10 min, the supernatant was taken for GPI enzyme activity determination. The GPI enzyme activity in the HepG2 cell extract was determined by referring to the GPI kit operation method, and the size of the GPI enzyme activity of each group of samples was investigated. The phosphoglucose isomerase (PGI) / phosphoglucose isomerase (PHI) kit (20221206001) was purchased from Shanghai Jianglai Biotechnology Co., Ltd.

[0073] The GPI enzyme activity of each group of cell fluid at 9 minutes was measured. The GPI enzyme activity of HepG2 cell fluid in the blank group was 94.35±2.43nmol / min / mg prot, and the GPI enzyme activity of the model group was 82.43±6.63nmol / min / mg prot, which was lower than that of the blank group, and there was a significant difference between the blank group and the blank group (P=0.0461), that is, 14mmol / L glucose reduced the GPI enzyme activity of HepG2 cells, resulting in a decrease in the efficiency of catalyzing glucose. The GPI enzyme activity of the glycyrrhizic acid group was 95.63±3.90nmol / min / mg prot, which was higher than that of the model group, and there was a statistical difference between the model group and the model group (P=0.0308). It shows that glycyrrhizic acid can increase the GPI enzyme activity of HepG2 cells under high sugar conditions, thereby regulating the glucose metabolic pathway, thereby improving the utilization of glucose by HepG2 cells under high sugar conditions, reducing the production of methylglyoxal and D-lactic acid, and thus improving the harm caused by methylglyoxal under high sugar conditions.

[0074] Example 6: Investigation of glucose concentration

[0075] HepG2 cells were cultured in a 10 cm diameter culture dish. After the cells adhered to the wall or grew to an appropriate number (about 6 million), the residual RPMI-1640 complete medium in the culture dish was aspirated, and a blank group, model group 1, and model group 2 were set up.

[0076] Blank group: only RPMI-1640 basal medium was given, and the theoretical value of glucose concentration was 11.1mmol / L;

[0077] Model group 1: 5% glucose injection was added to RPMI-1640 basal culture medium to a concentration of 7 mmol / L (the volume of 5% glucose injection accounted for approximately 2.5% of the total volume).

[0078] Model group 2: 5% glucose injection was added to RPMI-1640 basal culture medium to a concentration of 14 mmol / L.

[0079] The final volume of each dish was 4 mL. After modeling or drug administration, the dish was heated to 5% CO 2 , cultured in a 37°C constant temperature incubator for 8 h. After the culture was completed, the culture medium supernatant was centrifuged at 12000 rpm for 10 min to obtain the test solution.

[0080] After 8h of cell culture, the content of D-lactic acid in the culture medium of model group 1 and model group 2 increased significantly after the addition of 7mmol / L (model group 1) and 14mmol / L (model group 2) glucose. The amount of D-lactic acid produced in the culture medium of blank group, model group 1 and model group 2 was 0.0386±0.0062, 0.3266±0.066 and 0.4483±0.044mmol / L, respectively. There were significant differences between model group 1 and model group 2 compared with the blank group, with P values ​​of 0.0006 and less than 0.0001, respectively. At the same time, there was also a significant difference between model group 1 and model group 2 (P=0.0410), indicating that the higher the glucose concentration, the higher the D-lactic acid content in the culture medium, and it showed a certain dose dependence. Both 7mmol / L and 14mmol / L glucose can be used for the induction of HepG2 cells, that is, the high glucose condition is 18-25mmol / L.

[0081] Example 7: Investigation of Glycyrrhizic Acid Dosage Concentration

[0082] Referring to "Example 6 Investigation of Glucose Concentration", a blank group, a model group and a glycyrrhizic acid group were set up respectively. The concentration of added glucose in the model group was 7mmol / L and 14mmol / L, and the concentration of glycyrrhizic acid ranged from 62.5 to 500μmol / L. The dosage concentration of glycyrrhizic acid was screened by detecting the amount of D-lactic acid generated by glycyrrhizic acid at different concentrations under glucose induction. The D-lactic acid (LA) colorimetric test kit (GY04PHZV1809) was purchased from Wuhan Elerite Biotechnology Co., Ltd.

[0083] Drawing of standard curve:

[0084] (1) Preparation of standard solution: 10 mmol / L D-lactic acid standard solution (reagent 5) was diluted with purified water to prepare a series of standard solutions with concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.35, and 0.4 mmol / L.

[0085] (2) Preparation of enzyme working solution: Mix the buffer solution (reagent 1) and the enzyme stock solution (reagent 2) in a volume ratio of 100:1 and use it immediately.

[0086] (3) Take 100 μL of standard solution of different concentrations and add them to 96-well plates, with 2 replicates for each concentration. Add 50 μL of enzyme working solution and 10 μL of color developer (reagent 3) to each well, and incubate at 37°C for 10 min. After the incubation, add 90 μL of stop solution (reagent 4), shake for 5 s, and measure the OD value of each well at 530 nm with an ELISA reader.

[0087] The standard curve was drawn with the absolute OD value of purified water as 0. The fitted standard curve of the absolute absorbance value Y of the standard solution and the concentration X of the standard solution was Y=0.833X+0.0047 (r=0.9958). This indicates that the absolute absorbance OD value of D-lactic acid in the range of 0-0.4 mmol / L has a good linear relationship with its concentration.

[0088] Determination of OD value of each group of solutions

[0089] Take 100 μL of each sample to be tested and add it to a 96-well plate. Then add 50 μL of enzyme working solution and 10 μL of color developer to each well, and then incubate at 37°C for 10 min. After the incubation, add 90 μL of stop solution and shake for 5 seconds. Measure the OD value of each group of solutions at 530 nm with an ELISA instrument, take the absolute OD value of purified water as 0, and calculate the content of D-lactic acid.

[0090] HepG2 cells were cultured for 8 hours under the condition of 7mmol / L glucose. Glycyrrhizic acid in the range of 62.5-500μmol / L could reduce the production of D-lactic acid (n=3). The production of D-lactic acid in the blank group and model group was 0.0310±0.0057 and 0.3243±0.0640mmol / L, respectively. The production of D-lactic acid in the glycyrrhizic acid groups with concentrations of 62.5, 125, 250, and 500μmol / L were 0.0478±0.0090, 0.0421±0.0124, 0.0371±0.0145, and 0.0328±0.0097mmol / L, respectively. Glycyrrhizic acid groups with different concentrations could reduce the production of D-lactic acid, and there were significant differences compared with the model group (P<0.0001).

[0091] HepG2 cells were cultured for 8 hours under the condition of adding 14mmol / L glucose, and glycyrrhizic acid in the range of 62.5-500μmol / L could also reduce the production of D-lactic acid (n=1). The production of D-lactic acid in the blank group and model group was 0.0713mmol / L and 0.6476mmol / L, respectively, and the production of D-lactic acid in the glycyrrhizic acid groups with the concentrations of 62.5, 125, 250 and 500μmol / L were 0.3713, 0.3703, 0.0792, 0.1052mmol / L, respectively.

[0092] Example 8: Determination of glucose consumption (addition of 7 mmol / L glucose)

[0093] A blank group, a model group, and a glycyrrhizic acid group were set up, and the glucose concentration in the initial culture medium and the culture medium of each group after the cells were cultured for 8 hours were measured to investigate the 8-hour glucose consumption of HepG2 cells. The added glucose concentration and glycyrrhizic acid concentration of the blank group, the model group, and the glycyrrhizic acid group are shown in Table 2.

[0094] Table 2 Glucose and glycyrrhizic acid concentrations in the culture medium of each group

[0095]

[0096] After 8 hours of culture, the 8-hour glucose consumption of the blank group was 5.59±0.10mmol / L, and the glucose consumption of the model group was 5.32±0.58mmol / L. There was no statistical difference between the model group and the blank group, indicating that the utilization of glucose by HepG2 cells under high glucose conditions did not change significantly. The glucose consumption of the glycyrrhizic acid group increased to 6.91±0.34mmol / L, which was significantly higher than that of the model group, and there was a statistical difference with the model group (P=0.0059), indicating that glycyrrhizic acid improved the utilization of glucose by HepG2 cells under high glucose conditions.

[0097] Example 9: Determination of AGEs content

[0098] Referring to the grouping of "Determination of Glucose Consumption in Example 8", the content of AGEs in HepG2 cell fluid was determined according to the operation method of the human advanced glycation end products (AGEs) enzyme-linked immunosorbent assay kit, and the content of AGEs was converted into the content of AGEs corresponding to 1 mmol / L glucose consumed. The AGEs ELISA kit (121622006103431216) was purchased from Shanghai Jianglai Biotechnology Co., Ltd.

[0099] The content of AGEs generated per 1mmol / L glucose consumed in the cell fluid of the blank group, model group and glycyrrhizic acid group was 21.95±3.10, 38.10±3.56 and 29.02±0.79ng / mL, respectively. There was a statistical difference between the model group and the blank group (P=0.0009), and there was also a statistical difference between the model group and the glycyrrhizic acid group (P=0.0162). This shows that the generation of AGEs is increased under high sugar conditions, and the glycyrrhizic acid can also reduce the generation of AGEs under high sugar conditions.

[0100] Example 10: Animal Experiment

[0101] 1. Experimental Animals

[0102] Fifty-six SPF-grade male SD rats were purchased from the Experimental Animal Center of China Three Gorges University (license number: SCXK(E)2022-0012). They weighed 140-170 g and were housed in the SPF-grade environment of the Experimental Animal Center of Hubei University of Chinese Medicine, with 4 rats in each cage. After 10 days of adaptive feeding, experimental research was carried out. The ethical number for animal experiments was: HUCMS202209005.

[0103] 2. Experimental methods

[0104] 2.1 Establishment and grouping of diabetic animal models

[0105] 2.1.1 Establishment of diabetic animal models

[0106] SD male rats were fed with basal diet and fasted for 12 h before modeling. Streptozotocin (STZ) was prepared with citric acid buffer solution with pH 4.4-4.6, and the concentration of STZ was 14 mg / mL. SD rats were intraperitoneally injected with STZ at a dose of 35 mg / Kg for two consecutive days for modeling. About two weeks after modeling, after fasting for 12 h, the fasting blood glucose of the tail vein was measured with a Roche blood glucose meter. Rats with fasting blood glucose values greater than 10.0 mmol / L were selected as diabetic model rats, and 40 diabetic model rats were obtained.

[0107] 2.1.2 Grouping of diabetic animal models

[0108] Except for 6 rats in the normal control group, the 40 diabetic model rats were randomly divided into 5 groups: the model control group, the low-dose glycyrrhizic acid group (GL-L), the medium-dose glycyrrhizic acid group (GL-M), the high-dose glycyrrhizic acid group (GL-H), and the metformin group (MET), with 8 rats in each group. The drug was administered once a day for a period of 4 weeks.

[0109] (1) The normal control group (Normal contol, NC) was intragastrically administered with 0.5% sodium carboxymethylcellulose aqueous solution at a volume of 6 mL per kilogram of rats.

[0110] (2) The model control group (Diabetic control, DC) was intragastrically administered with 0.5% sodium carboxymethylcellulose aqueous solution at a volume of 6 mL per kilogram of rats.

[0111] (3) The metformin group (Metformin, MET) was intragastrically administered at a dose of 150 mg / kg / d, and the intragastric volume was 6.4 mL per kilogram of rats. The dosing dose of the positive drug metformin group was combined with the clinical use dose of Glucophage and referred to relevant literature.

[0112] (4) The high, medium and low dose groups (GL-H, GL-M and GL-L) of glycyrrhizic acid were administered orally at doses of 96, 48 and 24 mg / kg / d, respectively. The oral administration volume of low, medium and high concentrations was 6 mL per kg of mouse. The dosage of the medium dose group was based on the clinical dosage of glycyrrhizic acid diammonium capsules and referenced to relevant literature.

[0113] 2.2 Sampling and organ index

[0114] After 4 weeks of administration, the rats were fasted for 12 hours, weighed and tested for fasting blood sugar. Then, they were anesthetized with 2% sodium pentobarbital (40 mg / kg) and blood was collected from the abdominal aorta. The blood was allowed to stand at 4°C for 2 hours and then centrifuged at 3000 rpm for 10 minutes. Serum was collected for the determination of biochemical indicators. The kidneys were taken and weighed, and a portion was fixed with 4% paraformaldehyde for H&E staining.

[0115] 2.3 Detection of fasting blood glucose (FBG) in diabetic rats

[0116] After 28 days of administration, the FBG in the model group was 28.67±1.62mmol / L, which was significantly higher than that in the blank group (5.0±0.52mmol / L), and there was a significant difference between the two groups (P<0.0001); the FBG in the low-dose glycyrrhizic acid group, the medium-dose glycyrrhizic acid group, the high-dose glycyrrhizic acid group, and the metformin group were 24.60±1.18, 13.83±1.26, 15.30±3.86, and 16.83±6.40mmol / L, respectively. The medium-dose glycyrrhizic acid group, the high-dose glycyrrhizic acid group, and the metformin group could effectively reduce the FBG of diabetic rats, and there were significant differences with the model group, with P values ​​of 0.0011, 0.0027, and 0.0071, respectively. This indicates that medium and high doses of glycyrrhizic acid can reduce the FBG of diabetic rats.

[0117] 2.4 Determination of D-lactic acid

[0118] The content of D-lactic acid in rat serum was determined by referring to the D-lactic acid colorimetric test kit.

[0119] After 28 days of administration, the D-lactic acid content in the model group was 0.2568±0.0182mmol / L, which was significantly higher than that in the blank group (0.1816±0.0443mmol / L), and there was a significant difference between the two groups (P=0.0421). The D-lactic acid content in the low-dose glycyrrhizic acid group, the medium-dose glycyrrhizic acid group, the high-dose glycyrrhizic acid group, and the metformin group was 0.1919±0.0379, 0.1979±0.0556, 0.1349±0.0526, and 0.0823±0.0296mmol / L, respectively. Glycyrrhizic acid and metformin both had the effect of reducing D-lactic acid to varying degrees, and there was a significant difference between the high-dose glycyrrhizic acid group and the metformin group and the model group (P=0.0003).

[0120] 2.5 Renal function biochemical index detection

[0121] The contents of uric acid, creatinine and blood urea nitrogen in rat serum were determined by referring to the uric acid, creatinine and blood urea nitrogen test kit methods. Uric acid (UA) test kit (20221124) and creatinine (Cr) determination kit (20221128) were purchased from Nanjing Jiancheng Bioengineering Institute, and urea colorimetric test kit (DP05P4DH5756) was purchased from Wuhan Elerite Biotechnology Co., Ltd.

[0122] The serum uric acid levels of diabetic rats in the blank group, model group, low-dose glycyrrhizic acid group, medium-dose glycyrrhizic acid group, high-dose glycyrrhizic acid group and metformin group were 11.32±1.73, 25.70±5.56, 19.48±5.29, 15.84±3.59, 17.41±4.51 and 15.80±2.37μmol / L, respectively; the creatinine levels were 26.67±7.00, The contents of uric acid, creatinine and urea nitrogen were 48.37±11.17, 38.92±8.10, 27.33±6.63, 28.44±8.15 and 28.31±11.78μmol / L, respectively; the contents of urea nitrogen were 6.55±0.75, 14.23±0.67, 12.97±1.27, 10.67±2.51, 11.78±2.01 and 9.70±1.12mmol / L, respectively. The results of uric acid, creatinine and urea nitrogen test showed that the uric acid, creatinine and urea nitrogen in the model group were significantly increased compared with the blank group and there was a statistical difference, indicating that the renal function of rats was damaged to a certain extent under high glucose conditions. Compared with the model group, the high-dose and medium-dose glycyrrhizic acid groups and the positive group could significantly reduce the levels of uric acid and creatinine, and the medium-dose glycyrrhizic acid group and the positive group could significantly reduce the content of urea nitrogen, indicating that medium-dose and high-dose glycyrrhizic acid have a certain protective effect on the renal function of diabetic rats.

[0123] 2.6H&E dyeing

[0124] Paraffin embedding and sectioning: The tissues were dehydrated with 70-100% ethanol and xylene in different concentrations until transparent, then waxed, embedded, and trimmed. They were then cut into slices about 5 μm thick using a microtome. After spreading, the slices were fixed on glass slides for subsequent experiments. Three samples were randomly selected from each group for kidney testing.

[0125] The renal tissues of diabetic rats were stained with H&E and observed. The normal control group (NC) had clear glomerular structures, obvious cystic cavity spacing, and normal renal tubules; the rats in the model group (DC) had increased numbers of cells in the glomeruli, thickened glomerular basement membranes, swollen glomeruli, and narrowed cystic cavities. The glycyrrhizic acid group and the metformin group had clearer glomerular contours, fewer cells in the glomeruli, and more obvious cystic cavity spacing. This indicates that glycyrrhizic acid can protect the renal function of diabetic rats.

[0126] The experimental data of the present invention were statistically analyzed and plotted using GraphPad Prism 8.0 and Ai software. The experimental data were expressed as mean ± SD, and then One-way ANOVA was used for one-way analysis of variance, followed by Tukey's test for pairwise comparisons, with P < 0.05 indicating statistically significant differences. Compared with the blank group, *: P < 0.05, **: P < 0.01, ****: P < 0.0001; compared with the model group, #: P < 0.05, ##: P < 0.01, ###: P < 0.001.

Claims

1. Application of glycyrrhizic acid in the preparation of drugs for reducing the production of methylglyoxal under high sugar conditions.

2. The use according to claim 1, characterized in that: The high sugar condition is a fasting blood sugar level of 10 mmol / L or more.

3. The use according to claim 1, characterized in that: The high sugar condition is 18-25 mmol / L.

4. The use according to claim 1, characterized in that: The glycyrrhizic acid can also reduce the generation of D-lactic acid under high sugar conditions.

5. The use according to claim 1 or 4, characterized in that: The glycyrrhizic acid improves the enzyme activity of glucose-6-phosphate isomerase under high sugar conditions and reduces the generation of methylglyoxal and D-lactic acid.

6. The use according to claim 1, characterized in that: The medicine is a medicine for preventing and treating diabetes-related complications, and the related complications include diabetic nephropathy, diabetic eye disease or diabetic atherosclerosis.

7. The use according to claim 1, characterized in that: The medicine contains glycyrrhizic acid or glycyrrhizic acid salt with a specification of 25 to 150 mg, and the glycyrrhizic acid salt is calculated as glycyrrhizic acid.

8. The use according to claim 7, characterized in that: The glycyrrhizic acid salt is monopotassium glycyrrhizinate, diammonium glycyrrhizinate, glycyrrhizin zinc, monoammonium glycyrrhizinate or magnesium isoglycyrrhizinate.

9. The use according to claim 1, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier or excipient.

10. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, hard capsule, granule, pill or injection.

Images