Medicinal composition derived from liquorice and application thereof

Abstract

The invention discloses an application of a medicinal composition in preparation of a medicine for reducing generation of methylglyoxal under a high glucose condition, the medicinal composition comprises neoisoliquiritin, comastol, glycyrrhizic acid and glycyrrhetinic acid monoglucuronide, and the high glucose condition is 18-25 mmol / L. The invention further discloses a preparation method of the medicinal composition. The medicinal composition disclosed by the invention can be used for reducing the generation of endogenous methylglyoxal and D-lactic acid; 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 present invention relates to a medicinal composition derived from liquorice and an application thereof, in particular to the preparation of the medicinal composition of liquorice and its use in reducing the generation of endogenous methylglyoxal and D-lactic acid under high sugar conditions, and the application of the medicinal composition of liquorice in preparing drugs for diabetic nephropathy, diabetic eye disease or diabetic atherosclerosis and other diabetes-related complications, 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 pharmaceutical composition 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 medicinal liquorice is listed as a top grade in the Shennong's Herbal Classic, has the function of "harmony among various medicines", and is widely used in clinical practice. At present, there is no report on the use of the Chinese medicinal liquorice to reduce the generation of endogenous methylglyoxal under high sugar conditions. The medicinal composition of liquorice provided by the present invention contains compounds such as glycyrrhizic acid, which can be used to reduce the generation of endogenous methylglyoxal and D-lactic acid under high sugar conditions, and can prevent or 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] The invention discloses a pharmaceutical composition for use in the preparation of a drug for reducing the generation of methylglyoxal under high sugar conditions, wherein the pharmaceutical composition comprises neoisoliquiritigenin, comaistol, glycyrrhizic acid and monoglucuronic acid glycyrrhetinic acid. Endogenous methylglyoxal mainly originates from triose phosphates, i.e., 3-phosphoglyceraldehyde and dihydroxyacetone phosphate, in the glycolysis process, which are 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 pharmaceutical composition further comprises liquiritigenin, isoliquiritigenin and formononetin.

[0013] Furthermore, 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 pharmaceutical composition 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 content of glycyrrhizic acid in the drug is 25-150 mg. The content of glycyrrhizic acid in the drug is the specification of the drug, that is, the content of glycyrrhizic acid contained in a single unit preparation. In actual application, multiple unit preparations can be used according to the effective dose for treatment.

[0015] Furthermore, the pharmaceutical composition 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-Lactic acid, D-LA).

[0016] Furthermore, the drug is a drug for preventing or 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 reduces 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 reduced activity of glyoxal metabolic enzyme I leads to a slow elimination of triose phosphate, which together lead 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 glycation of intracellular proteins, 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, which are rich in renal blood vessels, glomerular basement membranes and mesangial structures, to form intramolecular / intermolecular cross-links, destroying their spatial scaffolding 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] Furthermore, the medicine comprises a therapeutically effective amount of a pharmaceutical composition. In actual application, multiple unit preparations can be used according to the therapeutically effective amount.

[0018] Furthermore, the medicine also contains a pharmaceutically acceptable carrier or excipient.

[0019] Further, the dosage form of the drug is tablets, hard capsules, granules or pills. Those skilled in the art can prepare the above dosage form by combining the pharmaceutical composition of the formulation specification with a pharmaceutically acceptable carrier or excipient according to the conventional method according to actual needs. The above-mentioned pharmaceutically acceptable carrier or excipient includes: disintegrant, lubricant, diluent, wetting agent, adhesive, colorant, coating material, antioxidant, stabilizer, flavoring agent, preservative, 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, which depends on how much of the excipient is present in the preparation and which other excipients are present in the preparation.

[0020] 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 Hand book of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

[0021] Glycyrrhizic acid, liquiritin, isoliquiritin, formononetin, neoisoliquiritin, comaistol and monoglucuronic acid glycyrrhetinic acid can be obtained by extraction and purification from the Chinese medicinal liquorice, or can be purchased. Extraction and purification are 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. The pharmaceutical composition can be obtained by separation and purification of the liquorice extract, or can be prepared by using the compounds contained in the pharmaceutical composition.

[0022] Definitions and explanations of terms

[0023] The term "effective amount" or "therapeutically effective amount" refers to the amount of the pharmaceutical composition 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 the disease condition to be 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 will vary 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.

[0024] The term "specification" refers to the weight (or potency) or content (%) or loading of the main drug in each vial, tablet or other unit preparation. In the present invention, the main drug is a pharmaceutical composition.

[0025] 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.

[0026] 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.

[0027] The beneficial effects of the present invention are:

[0028] (1) The pharmaceutical composition can reduce the production of D-lactic acid under high sugar conditions and reduce the harm of methylglyoxal.

[0029] (2) The glycyrrhizic acid in the pharmaceutical composition 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] (3) The glycyrrhizic acid in the pharmaceutical composition can reduce the levels of D-lactic acid, uric acid, creatinine and blood urea nitrogen in the serum of diabetic rats; kidney H&E staining also shows that glycyrrhizic acid has the effect of improving the renal function of diabetic rats and alleviating diabetic nephropathy.

[0031] (4) The glycyrrhizic acid in the pharmaceutical composition can increase the reduced glutathione content and total superoxide dismutase activity in the serum of diabetic rats, thereby improving the oxidative stress of diabetes and alleviating diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the result of screening the active parts of licorice.

[0033] Figure 2 This is the result diagram of segmented screening of licorice active flow.

[0034] Figure 3 This is the result of screening the active ingredients of licorice.

[0035] Figure 4 It is the total ion current diagram of the pharmaceutical composition in negative ion mode.

[0036] Figure 5 It is the total ion current of the pharmaceutical composition in the positive ion mode.

[0037] Figure 6 This is a statistical chart of the test results of serum uric acid (UA) content in diabetic rats.

[0038] Figure 7 This is a statistical chart of the test results of serum creatinine (Cr) content in diabetic rats.

[0039] Figure 8 This is a statistical chart of the test results of serum urea nitrogen (BUN) content in diabetic rats.

[0040] Fig. 9 H&E staining of the kidney of diabetic rats.

[0041] Figure symbols: 1. Liquoricein; 2. Isoliquiritigenin; 3. Formononetin; 4. Neoisoliquiritigenin; 5. Comestol; 6. Glycyrrhizic acid; 7. Monoglucuronic acid glycyrrhetinic acid. DETAILED DESCRIPTION

[0042] 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.

[0043] Example 1: Preparation of a pharmaceutical composition

[0044] 200g of licorice coarse powder was extracted with 8 times and 6 times of 85% (V / V) ethanol under reflux for 1h, and the extracts were combined to obtain about 2.5L of extract. After the extract was recovered under reduced pressure, about 200mL of concentrated solution was obtained. The concentrated solution was extracted with petroleum ether 4 times, ethyl acetate 7 times, and water-saturated n-butanol 5 times, and the volume of petroleum ether, ethyl acetate, water-saturated n-butanol and concentrated solution was about 1:1 each time. The extracts and water were recovered under reduced pressure, and the extracts were dried under vacuum to obtain 1.301g of licorice petroleum ether extract, 3.765g of ethyl acetate extract, 8.852g of n-butanol extract, and 37.501g of water extract.

[0045] 0.999g of the extract of the n-butanol part was dissolved in methanol and then mixed with silica gel (200-300 mesh). The amount of silica gel used for mixing was about 2 times the weight of the n-butanol part. After mixing, the sample was placed on a silica gel column. The amount of silica gel used for separation was about 20 times the weight of the n-butanol part. The eluent was a dichloromethane-methanol system. It was eluted with dichloromethane-methanol (12:1), dichloromethane-methanol (10:1), and dichloromethane-methanol (8:1) in sequence. Finally, methanol was used to press the column. The amount of dichloromethane-methanol (12:1), dichloromethane-methanol (10:1), and dichloromethane-methanol (8:1) was 90mL (starting from the exit of the lowest color band), 150mL, and 120mL, respectively. A fraction was collected every 7-9mL. A total of 46 fractions (No.1-46) were collected, and the 47th fraction (No.47) was a methanol column fraction. Based on the thin layer chromatography of each fraction, similar fractions were combined to obtain fraction A (No. 1-13) 19 mg, fraction B (No. 14-29) 63.7 mg, fraction C (No. 30-45) 228 mg, and fraction D (No. 47) 482.6 mg. Fraction B is the pharmaceutical composition of the present invention.

[0046] Example 2: Screening of active parts of licorice

[0047] 1 Grouping and drug administration

[0048] HepG2 cells were cultured in a culture dish with a diameter of 10 cm. 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, a model group, and a drug-treated group were set up.

[0049] Blank group: only RPMI-1640 basal medium was given, and the theoretical value of glucose concentration was 11.1mmol / L;

[0050] Model group: 5% glucose injection was added to RPMI-1640 basal medium to a volume of 7 mmol / L (the volume of 5% glucose injection accounted for approximately 2.5% of the total volume).

[0051] Drug administration group: After adding 5% glucose injection to RPMI-1640 basal medium to a volume of 7 mmol / L, an appropriate volume of drug solution (accounting for 0.1% of the final volume) was added.

[0052] The experimental results of the effects of petroleum ether, ethyl acetate, n-butanol, water and total extract of licorice on the survival rate of HepG2 cells (24h) showed that the petroleum ether fraction showed obvious cytotoxicity to HepG2 cells at a concentration of 200μg / mL; the ethyl acetate fraction showed obvious cytotoxicity at concentrations of 100μg / mL and 200μg / mL. The IC values ​​of the petroleum ether fraction and the ethyl acetate fraction were calculated.50 The concentrations of n-butanol fraction, water fraction and total extract of licorice showed no obvious cytotoxicity to HepG2 cells in the concentration range of 25-200 μg / mL. According to the cell survival rate results of each fraction on HepG2 cells, in order to ensure that there are enough cells when screening the active fractions and the cell amounts of each fraction are roughly consistent, the drug concentrations of petroleum ether fraction and ethyl acetate fraction were set to 50 μg / mL, and the cell survival rate was about 90%; the drug concentrations of n-butanol fraction, water fraction and total extract were set to 200 μg / mL, and the cell survival rate was above 90%. All contain 0.1% cell-grade DMSO.

[0053] The final volume of each dish was 4 mL. After modeling or drug administration, the cells were cultured in a 5% CO2, 37°C constant temperature incubator for 8 h. After the culture was completed, the culture supernatant was centrifuged at 12000 rpm for 10 min to obtain the test solution.

[0054] 2. Determination of OD value of test solution

[0055] 2.1D-Lactic Acid Standard Curve Drawing

[0056] (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.

[0057] (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.

[0058] (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.

[0059] The standard curve was drawn with the absolute OD value of purified water as 0.

[0060] The fitting standard curve of the absolute absorbance value Y of the D-lactic acid standard solution and the concentration X of the standard solution is 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. The D-lactic acid (LA) colorimetric test kit (GY04PHZV1809) was purchased from Wuhan Elerite Biotechnology Co., Ltd.

[0061] 2.2 Determination and calculation of test solution

[0062] A total of 7 groups of test solutions were prepared, including blank group (Control), model group (Model), petroleum ether extract, ethyl acetate extract, n-butanol extract, water extract, and total extract group, with 3 samples in each group. 100 μL of each of the blank group, model group, and drug group were added to a 96-well plate, and 50 μL of enzyme working solution and 10 μL of color developer were added to each well, and then incubated at 37°C for 10 minutes. After the incubation, 90 μL of stop solution was added and shaken for 5 seconds. The OD value of each group of solutions at 530 nm was measured by an enzyme reader, and the absolute OD value of purified water was taken as 0 and the content of D-lactic acid was calculated.

[0063] The content of D-lactic acid in the culture medium of the blank group was 0.0412±0.0085mmol / L, and the content of D-lactic acid in the culture medium of the model group was significantly increased, reaching 0.2798±0.0256mmol / L, and there was a significant difference compared with the blank group (P<0.0001), indicating that 7mmol / L glucose modeling agent can increase the content of D-lactic acid in the culture medium of HepG2 cells. Compared with the model group, the D-lactic acid content in the cell culture medium of the total extract group was 0.2138±0.0124mmol / L, which was reduced but there was no significant difference (P=0.1597); the D-lactic acid content in the petroleum ether part group and the ethyl acetate part group remained basically unchanged, which were 0.3329±0.0444 and 0.2631±0.0692mmol / L, respectively, and there was no significant difference compared with the model group; the D-lactic acid content in the n-butanol part group and the water part group were 0.1356±0.0069 and 0.1802±0.0135mmol / L, respectively, which were reduced to varying degrees and there were significant differences, indicating that the n-butanol part and the water part can reduce the production of D-lactic acid in HepG2 cells induced by 7mmol / L glucose.

[0064] Example 3: Screening of Licorice Active Flow Segments

[0065] Referring to "Grouping and Dosing in Example 2", the dosing group was flow fractions A, B, C, and D in the n-butanol part, the final drug concentration was 100 μg / mL, and the basal culture medium contained 0.1% cell-grade DMSO. After dosing, each group was cultured in a 5% CO2, 37°C constant temperature incubator for 8 hours, and the culture supernatant was centrifuged at 12000 rpm for 10 minutes to obtain the test solution. A total of 6 groups of test solutions were prepared, including blank group, model group, flow fraction A group (Fraction A), flow fraction B group (Fraction B), flow fraction C group (Fraction C), and flow fraction D group (Fraction D), with 3 samples in each group.

[0066] The determination of D-lactic acid was the same as in "Determination and calculation of test solution in Example 2", and the content of D-lactic acid in each group of test solution was calculated. The content of D-lactic acid in the cell culture medium of the blank group was 0.0365±0.0063mmol / L, and the content of D-lactic acid in the culture medium of the model group was significantly increased compared with the blank group, and the content was 0.3303±0.0066mmol / L, and there was a significant difference with the blank group (P<0.0001). Compared with the model group, among the four flow segment administration groups, only the flow segment B group had a reduced content of D-lactic acid, which was 0.1834±0.0358mmol / L, and there was a significant difference with the model group (P=0.0012); the content of D-lactic acid in the culture fluid of flow segment A group, flow segment C group and flow segment D group was 0.4876±0.0321, 0.3787±0.0405, 0.3386±0.0458mmol / L, respectively, and none of them had the activity of reducing the production of D-lactic acid. It can be seen that flow segment B can reduce the production of D-lactic acid in HepG2 cells induced by 7mmol / L glucose, so the n-butanol site flow segment B is an active flow segment, that is, the pharmaceutical composition of the present invention.

[0067] Example 4: Analysis of ingredients of licorice medicinal composition

[0068] 1 Preparation of test solution

[0069] Preparation of the test solution of the medicinal composition: accurately weigh 1.3 mg of the medicinal composition (active flow segment B), dissolve it in 6.5 mL of chromatographic methanol to prepare a 200 μg / mL solution, centrifuge at 12000 rpm for 10 min, and take the supernatant for liquid-mass analysis.

[0070] Preparation of reference substance mixed solution: accurately weigh 1.2 mg of reference substances liquiritin, 1.5 mg of licorice chalcone B, 1.1 mg of licorice chalcone, 1.3 mg of isoliquiritigenin, 1.2 mg of glycyrrhizic acid, 1.4 mg of licorice chalcone A and 1.3 mg of glabridin, and dissolve them in 1.2, 1.5, 1.1, 1.3, 1.2, 1.4 and 1.3 mL of chromatographic methanol, respectively, to prepare 1 mg / mL reference substance mother solutions; take 20 μL of each reference substance mother solution, mix them evenly, and then add 800 μL of chromatographic methanol to dilute to obtain a mixed standard mother solution; take 200 μL of the mixed standard mother solution, add 1.2 mL of chromatographic methanol to dilute to obtain a 3 μL / mL mixed reference substance solution, centrifuge at 12000 rpm for 10 min, and take the supernatant for liquid-mass analysis.

[0071] 2LC-MS / MS conditions

[0072] 2.1 Chromatographic conditions

[0073] Waters C 18 (100mm×2.1mm,1.7μm) chromatographic column, 0.1% formic acid water (A)-acetonitrile (B) as mobile phase, gradient elution. Gradient elution program is 0-8min, 19%-19% B; 8-35min, 19%-50% B; 35-36min, 50%-100% B; 36-40min, 100%-100% B. Column temperature 30℃; DAD detector, detection wavelength 254nm; flow rate 0.3mL / min; injection volume 2μL.

[0074] 2.2 Mass spectrometry conditions

[0075] ESI source ion source. Positive and negative ion mode acquisition, MS E The mode collected primary mass spectrometry ions and secondary mass spectrometry ions. The capillary voltage was 2500 V, the injection cone voltage was 40 V, the ESI ion source temperature was 500 °C, the nebulizer gas flow rate was 500 L / h, the scanning interval was 0.1 s, the mass-to-charge ratio range was m / z 50-900, and leucine enkephalin was used as a real-time molecular weight calibration.

[0076] 2.3 Test results

[0077] MS EThe primary mass spectrometry quasi-molecular ion peaks and secondary mass spectrometry fragment ion peaks of the pharmaceutical composition and mixed reference solution in positive and negative ion modes were collected. The quasi-molecular ion peaks and the fragmentation rules were compared in combination with the fragmentation rules and the chemical components contained in licorice to determine the potential compounds in the pharmaceutical composition. Through the analysis of liquid-mass, 7 compounds were identified from the pharmaceutical composition, numbered 1-7 as liquiritin, isoliquiritin, formononetin, neoisoliquiritin, comestol, glycyrrhizic acid and monoglucuronic acid glycyrrhetinic acid, among which the compounds liquiritin and glycyrrhizic acid had the same retention time, quasi-molecular ion peaks and fragment ion peaks as the reference substances liquiritin and glycyrrhizic acid under the same conditions, further confirming that the pharmaceutical composition contained liquiritin and glycyrrhizic acid. Information on the 7 compounds is shown in Table 1.

[0078] Table 1 Compounds identified from pharmaceutical compositions by LC-MS

[0079]

[0080] References:

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[0082] [2] Ma Haijuan, Gao Jian, Zhang Yali, et al. Based on HPLC-MS n Rapid identification of licorice components and mass spectrometry fragmentation pathways[J]. Chinese Journal of Traditional Chinese Medicine (formerly Acta Pharmacopoeia) 2018, 33(3): 1120-1123.

[0083] [3] Zhang Wei, Jiang Shu, Qian Dawei, et al. Study on the interaction between formononetin and human intestinal bacteria[J]. Acta Pharmaceutica Sinica, 2014, 49(8): 1162-1168.

[0084] [4] Seong SH, Kim BR, Cho ML, et al. Phytoestrogen Coumestrol SelectivelyInhibits Monoamine Oxidase-A and AmyloidβSelf-Aggregation[J]. Nutrients, 2022, 14(18):1-11.

[0085] [5] Dai Yan. Study on separation and purification of monoglucuronic acid glycyrrhetinic acid[D]. Nanjing University of Technology, 2006.

[0086] [6]Guangguo Tan, Zhenyu Zhu, Hai Zhang, et al. Analysis of phenolic and triter penoid compounds in licorice and rat plasma by high-performance liquid chro matography diode-array detection, time-of-flight mass spectrometry and quadrup ole ion trap mass spectrometry[J]. Rapid Commun. Mass Spectrom, 2010, 24: 209-218.

[0087] Example 5: Screening of chemical components

[0088] Referring to "Grouping and Dosing in Example 2", the dosing concentrations of glycyrrhizic acid, liquiritin, formononetin and isoliquiritin were all 125 μmol / L, and the basal culture medium contained 0.2% cell-grade DMSO. After dosing, each group was cultured in a 5% CO2, 37°C constant temperature incubator for 8 hours, and the culture supernatant was centrifuged at 12000 rpm for 10 minutes to obtain the test solution. A total of 6 groups of test solutions were prepared, including the blank group, model group, liquiritin group, isoliquiritin group, formononetin group and glycyrrhizic acid group, with 3 samples in each group.

[0089] The determination of D-lactic acid was the same as in "Determination and calculation of test solution in Example 2", and the content of D-lactic acid in each group of test solution was calculated. The content of D-lactic acid in the cell culture fluid of the blank group and the model group was 0.1257±0.0142 and 0.2281±0.0257mmol / L, respectively. The content of D-lactic acid in the model group was significantly increased, and there was a significant difference with the blank group (P=0.0159). Compared with the model group, among the four compound administration groups, only the glycyrrhizic acid group had a lower content of D-lactic acid, which was 0.1115±0.0195mmol / L, and there was a significant difference with the model group (P=0.0062); the content of D-lactic acid in the liquiritin group, formononetin group and isoliquiritigenin group was 0.2377±0.0618, 0.2534±0.0219 and 0.2828±0.0155mmol / L, respectively, which was not significantly different from the model group. In addition, there was no significant difference in the content of D-lactic acid in the cell culture medium between the liquiritin group and the isoliquiritigenin group (P=0.5116). This shows that glycyrrhizic acid in the pharmaceutical composition has the effect of reducing the production of D-lactic acid in HepG2 cells under high sugar conditions.

[0090] Example 6: Glucose consumption measurement (addition of 14 mmol / L glucose)

[0091] 1. Drawing of standard curve

[0092] 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, incubated at 37 ° C for 15 min, and the OD value of each well was measured at 505 nm using an enzyme reader. There were 2 replicate wells 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.

[0093] 2 Determination of test solution

[0094] An appropriate amount of cells (about 800,000) were plated in a 6-well plate, and a blank group, a model group, and a glycyrrhizic acid group were set up respectively. After the cells adhered to the wall, the culture medium was aspirated and the culture medium was replaced with RPMI-1640 basal culture medium, with a total volume of 1 mL per well. The concentration of added glucose and drug concentrations of the blank group, model group, and glycyrrhizic acid group are shown in Table 2. The survival rate of HepG2 cells with glycyrrhizic acid was investigated in the drug concentration range of 0.25-4mmol / L (48h). The experimental results showed that the cells showed certain toxicity only when the concentration of glycyrrhizic acid reached 1mmol / L, and the median lethal concentration was 1.47mmol / L.

[0095] Table 2 Glucose and glycyrrhizic acid concentrations in the culture medium of each group

[0096]

[0097] The glucose concentration in the initial culture medium and in the culture medium of each group of cells after further culture for 12 hours was determined by referring to the glucose colorimetric test kit, and then the glucose consumption of HepG2 cells was investigated.

[0098] 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.

[0099] 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.

[0100] Example 7: L-lactic acid detection

[0101] Referring to the grouping of "Example 6 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.

[0102] 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.

[0103] 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.

[0104] Example 8: Investigation of Glycyrrhizic Acid Dosage Concentration

[0105] Referring to "Grouping and Administration of Example 2", 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 administration concentration of glycyrrhizic acid was screened by detecting the reduction of D-lactic acid production by glycyrrhizic acid at different concentrations under glucose induction.

[0106] 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).

[0107] 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.

[0108] Example 9: Determination of glucose consumption (addition of 7 mmol / L glucose)

[0109] 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 3.

[0110] Table 3 Glucose and glycyrrhizic acid concentrations in the culture medium of each group

[0111]

[0112]

[0113] 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.

[0114] Example 10: Determination of the Content of AGEs

[0115] Refer to the grouping in "Example 9: Determination of Glucose Consumption". According to the operation method of the human advanced glycation end products (AGEs) enzyme-linked immunosorbent assay kit, determine the content of AGEs in the HepG2 cell lysate, and convert the content of AGEs into the content of AGEs corresponding to the consumption of 1 mmol / L glucose. The AGEs ELISA kit (121622006103431216) was purchased from Shanghai Jianglai Biotechnology Co., Ltd.

[0116] The contents of AGEs generated per 1 mmol / L glucose consumed in the cell lysates of the blank group, model group, and glycyrrhizic acid group were 21.95 ± 3.10, 38.10 ± 3.56, and 29.02 ± 0.79 ng / 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 indicates that the generation of AGEs was increased under high-glucose conditions, and glycyrrhizic acid can reduce the AGEs generation induced by high glucose.

[0117] Example 11: Animal Experiment

[0118] 1. Experimental Animals

[0119] 56 SPF-grade male SD rats were purchased from the Experimental Animal Center of China Three Gorges University (license number: SCXK(E)2022-0012). Their body weights were 140 - 170 g, and they were fed 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, the experimental study was carried out. The ethical number for the animal experiment was: HUCMS202209005.

[0120] 2. Experimental Methods

[0121] 2.1 Establishment and Grouping of Diabetic Animal Models

[0122] 2.1.1 Establishment of Diabetic Animal Models

[0123] Male SD rats were fed with basal diet and fasted for 12 h before modeling. Streptozotocin (STZ) was prepared with a citric acid buffer solution at 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 in 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.

[0124] 2.1.2 Grouping of Diabetic Animal Models

[0125] In addition to 6 normal control groups, 40 diabetic model mice were randomly divided into 5 groups: model control group, glycyrrhizic acid low-dose group (GL-L), glycyrrhizic acid medium-dose group (GL-M), glycyrrhizic acid high-dose group (GL-H) and metformin group (MET), with 8 mice in each group. The drugs were administered once a day for 4 weeks.

[0126] (1) The normal control group (NC) was intragastrically administered with 6 mL / kg of 0.5% sodium carboxymethylcellulose aqueous solution.

[0127] (2) The model control group (Diabetic control, DC) was intragastrically administered with 6 mL / kg of 0.5% sodium carboxymethylcellulose aqueous solution.

[0128] (3) Metformin (MET) group was gavaged at a dose of 150 mg / kg / d, with a gavage volume of 6.4 mL per kg of mouse. The dosage of the positive drug metformin group was based on the clinical dosage of Glucophage and referenced to relevant literature.

[0129] (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.

[0130] 2.2 Sampling and organ index

[0131] 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.

[0132] 2.3 Determination of reduced glutathione (GSH) content in diabetic rats

[0133] The content of reduced glutathione in rat serum was determined by referring to the operation method of the reduced glutathione colorimetric test kit. The reduced glutathione colorimetric test kit (NW03XP649789) was purchased from Wuhan Elaruite Biotechnology Co., Ltd.

[0134] After 28 days of administration, the serum GSH content of 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 was 321.9±20.3, 160.7±5.7, 248.7±6.9, 177.6±2.8, 171.0±9.7 and 290.5±70.2 μmol / L, respectively. The GSH content in the model group decreased compared with the blank group, and there was a statistical difference (P=0.0003), indicating that the oxidative stress produced by diabetes can reduce the GSH content in the serum of rats. The low-dose glycyrrhizic acid group and the metformin group can increase the GSH content in the serum of diabetic rats, and there is a significant difference with the model group, with P values ​​of 0.0371 and 0.0023, respectively. It shows that the low-dose glycyrrhizic acid group increases the GSH content in the serum of diabetic rats and can improve the oxidative stress of diabetes.

[0135] 2.4 Determination of total superoxide dismutase (T-SOD) activity in diabetic rats

[0136] The activity of total superoxide dismutase in rat serum was determined by referring to the operation method of the total superoxide dismutase colorimetric test kit. The total superoxide dismutase colorimetric test kit (UX14LRX21757) was purchased from Wuhan Elaruite Biotechnology Co., Ltd.

[0137] After 28 days of administration, the activities of serum T-SOD 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 322.9±31.9, 285.5±24.0, 372.1±66.9, 326.9±17.1, 300.7±30.1 and 383.2±55.5U / mL, respectively. The activity of T-SOD in the model group was slightly decreased compared with the blank group, but there was no statistical difference (P>0.05). The low-dose glycyrrhizic acid group and metformin group could increase the activity of T-SOD, and there were significant differences with the model group, with P values ​​of 0.0320 and 0.0121, respectively. The low-dose glycyrrhizic acid group could increase the activity of T-SOD in the serum of diabetic rats and improve diabetic oxidative stress.

[0138] 2.5 Determination of D-lactic acid

[0139] The content of D-lactic acid in rat serum was determined by referring to the D-lactic acid colorimetric test kit.

[0140] 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).

[0141] 2.6 Renal function biochemical index detection

[0142] 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.

[0143] 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.

[0144] 2.7 H&E staining

[0145] 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.

[0146] 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.

[0147] 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. Use of a pharmaceutical composition in the preparation of a drug for reducing the generation of methylglyoxal under high sugar conditions, the pharmaceutical composition comprising neoisoliquiritigenin, comaistol, glycyrrhizic acid and monoglucuronic acid glycyrrhetinic acid.

2. The use according to claim 1, characterized in that: The pharmaceutical composition also contains liquiritigenin, isoliquiritigenin and formononetin.

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 content of glycyrrhizic acid in the medicine is 25-150 mg.

5. The use according to claim 1 or 2, characterized in that: The pharmaceutical composition can also reduce the generation of D-lactic acid under high sugar conditions.

6. The use according to claim 1, characterized in that: The medicine is a medicine for preventing or 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 comprises a therapeutically effective amount of a pharmaceutical composition.

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

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

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