Application of 4-methylesculetin, a potential active ingredient of xiasangju, in the preparation of drugs for the prevention and treatment of diabetes

Abstract

The present invention discloses the application of 4-methyl esculetin, a potential active ingredient of xiasangju, in the preparation of a drug for preventing and treating diabetes, and belongs to the field of medicine. The present invention provides the application of 4-methyl esculetin in the preparation of a drug for preventing and treating diabetes, which can promote the consumption of glucose by cells, reduce the levels of ROS and MDA in liver tissue, and increase the levels of SOD and GSH-PX in liver tissue. The present invention is based on xiasangju, studies its drug components, clarifies the active ingredients of the drug and the dosage, improves the safety of drug administration, and also plays an important role in preventing and treating diabetic complications.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of 4-methylesculetin, a potential active ingredient of xiasangju, in the preparation of drugs for preventing and treating diabetes. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar levels due to relative insulin deficiency or insulin resistance. Diabetic patients have disorders in fat and protein metabolism, and clinically they may present with polyuria, polydipsia, polyphagia, and weight loss. Long-term hyperglycemia is prone to acute complications such as ketoacidosis or chronic complications such as vascular and neurological complications. Among them, type II diabetes, also known as non-insulin-dependent diabetes, has normal or increased insulin secretion, but the liver, adipose tissue, and skeletal muscle have reduced insulin sensitivity, i.e., insulin resistance. Most patients are adults. The main pathogenesis of type II diabetes is as follows: (1) Most cases of type II diabetes are caused by defects in the receptors on the cell membrane of insulin target cells, which reduce the sensitivity of insulin binding to the receptors, making it impossible for the body to fully absorb and utilize extracellular glucose, leading to increased blood sugar levels. (2) Insulin receptor substrate defects. Insulin receptor substrates are adapter proteins in the cytoplasm that mainly connect to various effector molecules such as insulin receptors to mediate the cell's response to insulin. (3) Insulin signal transduction defects. In the process of insulin secretion and signal transmission, insulin signal transmission is a series of processes involving multiple signal proteins. The development of drugs to treat diabetes is a continuous research and development direction in this field.

[0003] The current treatment of diabetes is mainly to control blood sugar and reduce complications. The main hypoglycemic drugs are oral Western medicine hypoglycemic drugs and injection of insulin analogs, but because they need to be used continuously and have obvious side effects, the use of traditional Chinese medicine has become an important choice for diabetes treatment in recent years.

[0004] For example, Chinese patent CN117815340A discloses a Chinese medicine composition for lowering blood sugar and its preparation method, wherein the Chinese medicine composition comprises a main ingredient and a side ingredient, and the main ingredient and the side ingredient are made of the following Chinese medicinal materials in parts by weight: the main ingredient comprises: 30 parts of astragalus, 35 parts of lycium bark, 25 parts of wolfberry, 25 parts of ginseng, 30 parts of schisandra chinensis, 20 parts of coptis root, 15 parts of mulberry leaf, 15 parts of platycodon, 25 parts of polygonatum, and 10 parts of liquorice; the side ingredients comprise: 20-40 parts of kudzu root, 15-35 parts of hawthorn, 15-20 parts of tuckahoe, 15-35 parts of atractylodes, 15 -35 parts, polygonatum 0-40 parts, catharanthus roseus 0-30 parts, yam 10-30 parts, wild chrysanthemum 20-25 parts, selfheal 10-30 parts, rehmannia glutinosa 20-40 parts, corn silk 30-55 parts, salvia miltiorrhiza 25-35 parts, honeysuckle 10-15 parts, anemarrhena asphodeloides 0-45 parts, trichosanthes root 0-45 parts, ophiopogon japonicus 0-45 parts. The blood sugar lowering traditional Chinese medicine composition has a good auxiliary blood sugar lowering effect, does not contain chemical synthetic drugs, and does not add hormones. However, the existing traditional Chinese medicine composition has many types of ingredients, and the effective ingredients are unclear, and the specific addition amount is also unclear.

[0005] Xiasangju is made from three medicinal materials: Prunella vulgaris, mulberry leaves, and wild chrysanthemum. It has the effects of clearing the liver and improving eyesight, dispersing wind and heat, removing dampness and arthritis, and relieving sores. Modern research has found that the main chemical components of Prunella vulgaris include triterpenes, steroids, flavonoids, and phenylpropanoids, which have a wide range of pharmacological effects such as lowering blood sugar, lowering blood pressure, and anti-tumor. Mulberry leaves are rich in chemical components, mainly including flavonoids, alkaloids, phenols, sterols, stilbenes, polysaccharides, and volatile oils, which have biological activities such as lowering blood sugar, anti-inflammatory, antibacterial, antiviral, anti-aging, and anti-cancer. Wild chrysanthemum contains chemical components such as volatile oils, flavonoids, polysaccharides, organic acids, amino acids, and trace elements, and has physiological activities such as antibacterial, anti-inflammatory, analgesic, anti-tumor activity, and antioxidant activity. Previous studies have found that Xiasangju can effectively improve various indicators of diabetic mice, but the existing technology has not yet clarified its active ingredients. In order to further expand the use of Xiasangju, it is necessary to further study the effective ingredients of Xiasangju. Summary of the invention

[0006] Based on the deficiencies in the prior art, the present invention studies the drug components of xiasangju, finds that 4-methylesculetin has a good effect in lowering blood sugar, and provides an application of 4-methylesculetin in the preparation of drugs for preventing and treating diabetes.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] Application of 4-methylesculetin in the preparation of drugs for preventing and treating diabetes.

[0009] The diabetes is type II diabetes.

[0010] The medicine is a medicine for improving abnormal glucose and lipid metabolism.

[0011] The drug is a drug that increases cellular glucose consumption.

[0012] The drug is a drug that increases the level of liver oxidative stress.

[0013] The medicine is a medicine for reducing the levels of ROS and MDA in liver tissue.

[0014] The medicine is a medicine for increasing the levels of SOD and GSH-PX in liver tissue.

[0015] The medicine comprises 4-methylesculetin and pharmaceutically acceptable excipients.

[0016] "Pharmaceutically acceptable excipients" refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated for safety and are included in drug preparations in addition to active ingredients. The same pharmaceutical excipient can be used in drug preparations for different routes of administration and has different effects and uses. The pharmaceutical excipients added to the drugs provided by the present invention can play the role of excipient, carrier or stability improvement. In addition, they also have important functions such as solubilization, solubilization or sustained release.

[0017] Typical but non-limiting excipients include: one or more of solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, and diluents.

[0018] Fillers include starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; flavoring agents include sweeteners and various flavors; preservatives include parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include PEG6000, PEG4000, insect wax, etc.

[0019] The pharmaceutical dosage forms of the present invention include tablets, capsules, solutions, granules, pills and powders.

[0020] The dosage of 4-methylesculetin in the drug is 6.3-17.5 μM.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] A new use of 4-methylesculetin has been found through research, which can promote the consumption of glucose by cells, reduce the levels of ROS and MDA in liver tissue, and increase the levels of SOD and GSH-PX in liver tissue. Therefore, 4-methylesculetin can be used to make hypoglycemic drugs; the present invention is based on xiasangju, studies its drug components, clarifies the active ingredients of the drug and the dosage, improves the safety of drug administration, and also plays an important role in preventing and treating diabetic complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effects of different concentrations of insulin and different action time on the proliferation activity of HepG2 cells;

[0024] in: Figure 1 A in the middle is the proliferation activity of HepG2 cells at 24 h; Figure 1 Middle B is the proliferation activity of HepG2 cells at 48h; Figure 1 Middle C is the proliferation activity of HepG2 cells at 72 h;

[0025] Figure 2 The effects of different concentrations of insulin and different action time on glucose consumption of HepG2 cells;

[0026] in: Figure 2 A in the middle is the glucose consumption of HepG2 cells at 24 h; Figure 2 Middle B is the glucose consumption of HepG2 cells at 48 h; Figure 2 Middle C is the glucose consumption of HepG2 cells at 72 h;

[0027] Figure 3 Glucose consumption at different time points after unloading of the IR-HepG2 cell model;

[0028] Figure 4 The effect of 4-methylesculetin on the viability of IR-HepG2 cells;

[0029] in: Figure 4 A in the middle is the cell viability of HepG2 cells at 24 h in the first CCK-8 experiment; Figure 4 Middle B is the cell viability of HepG2 cells at 48 h in the first CCK-8 experiment; Figure 4 Middle C is the cell viability of HepG2 cells at 72 h in the first CCK-8 experiment; Figure 4 D in the middle is the cell viability of HepG2 cells at 24 h in the second CCK-8 experiment; Figure 4 Middle E is the cell viability of HepG2 cells at 48 h in the second CCK-8 experiment; Figure 4 Middle F is the cell viability of HepG2 cells at 72 h in the second CCK-8 experiment;

[0030] Note: Compared with the control group, P*<0.05, P**<0.01.

[0031] Figure 5 is the effect of 4-methylesculetin on glucose consumption and glycogen content in IR-HepG2 cells;

[0032] in: Figure 5 A in the middle is glucose consumption; Figure 5 B is the glycogen content;

[0033] Note: Compared with the Control group, P*<0.05, P**<0.01; compared with the Model group, P#<0.05, P##<0.01.

[0034] Figure 6 The effect of 4-methylesculetin on improving oxidative stress in IR-HepG2 cells;

[0035] in: Figure 6 A in the middle is the MDA content; Figure 6 B is the SOD content; Figure 6 C in the middle is the GSH-PX content; Figure 6 Where D is FI content;

[0036] Note: Compared with the Control group, P*<0.05, P**<0.01; compared with the Model group, P#<0.05, P##<0.01. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with specific embodiments, which are intended to explain the present invention rather than to limit it.

[0038] Xiasangju granules produced by Guangzhou Baiyunshan Xingqun (Pharmaceutical) Co., Ltd. contain 4-methylesculetin involved in the present invention.

[0039] 1. Purpose of the experiment

[0040] HepG2 cells were used as test subjects and 4-methylesculetin as the test drug. The effects of 4-methylesculetin on the activity, glucose consumption, glycogen content and oxidative stress indexes of the HepG2 cell IR model induced by high glucose and high insulin were observed, and the effects of 4-methylesculetin on improving glucose consumption and glycogen synthesis, alleviating oxidative stress damage and increasing the ability to resist oxidative stress in the HepG2 cell IR model were evaluated.

[0041] 2. Experimental Materials

[0042] 2.1 Experimental cells

[0043] Human hepatocellular carcinoma (HepG2) cells were purchased from Wuhan Pronocell Life Science Co., Ltd., catalog number: CL-0103.

[0044] 2.2 Main experimental reagents

[0045] Table 1 Main reagents and consumables

[0046]

[0047] 2.3 Experimental instruments

[0048] Table 2 Main instruments

[0049]

[0050] 3. Experimental methods:

[0051] 3.1 HepG2 cell culture

[0052] (1) Cell recovery

[0053] Take out the cryovial containing HepG2 cell line from the -80℃ refrigerator, quickly put it into a 37℃ water bath, and shake the cryovial quickly to thaw it quickly. After the cryopreservation solution is completely dissolved, wipe the wall of the cryopreservation tube with an alcohol cotton ball, transfer the thawed cell solution to a 15mL centrifuge tube containing 3mL complete culture medium in the clean bench, blow it evenly, and centrifuge it at 1000 rpm for 5min at room temperature. After centrifugation, discard the supernatant, add 1mL of complete culture medium, blow it evenly, transfer it to a T25 cell culture flask containing 4mL complete culture medium, shake it evenly with the 8-shaped method, and place it in a 37℃ incubator containing 5% CO2 for culture. After 24 hours, observe the cell adhesion growth.

[0054] (2) Cell passaging

[0055] Observe the cells under a microscope. When the cell growth density reaches more than 80%, the cells can be subcultured. Take the culture flask out of the incubator, discard the old culture medium, add 2 mL of PBS buffer, gently shake the culture flask to wash away cell debris or dead cells, and wash twice. Discard the PBS buffer, add 1 mL of 0.25% trypsin, and digest in the incubator for about 2 min. Observe under a microscope. When the cytoplasm shrinks and the cell connection is loose, add 2 mL of complete culture medium to terminate the digestion. Use a pipette to gently blow and mix the cells. Transfer the cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 rpm at room temperature for 5 min. After centrifugation, discard the supernatant, add a certain volume of complete culture medium to resuspend the cells, gently blow and mix, subculture at a ratio of 1:3 or 1:4, continue to culture, and take cells in the logarithmic growth phase for the next experiment.

[0056] (3) Cell cryopreservation

[0057] Observe the cells under a microscope. When the cell confluence reaches more than 80%, they can be frozen. Take the culture flask out of the incubator, discard the old culture medium, add 2 mL PBS buffer, shake the culture flask gently, wash away cell debris or dead cells, and wash twice. Discard the PBS buffer, add 1 mL 0.25% trypsin, and place it in the incubator for digestion for about 2 min. Observe under a microscope. When the cells are completely detached from the flask wall and the cells are loosely connected, add 2 mL complete culture medium to terminate the digestion. Use a pipette to gently blow and mix the cells. Transfer the cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 rpm at room temperature for 5 min. After centrifugation, discard the supernatant, add 4°C pre-cooled serum-free cell freezing solution to resuspend the cells, gently blow and mix the cells with a pipette, and dispense into 2 mL sterile cryopreservation tubes, 1 mL per tube, seal the cap with a sealing film, and mark the cell name, freezing date, experimenter and other information on the cryopreservation tube, and store it directly in a -80°C refrigerator or liquid nitrogen.

[0058] 3.2 Establishment of HepG2 cell insulin resistance (IR) model in vitro

[0059] (1) Preparation of main reagents

[0060] a. Complete culture medium: MEM (containing NEAA) basal culture medium + 10% FBS + 1% P / S

[0061] b. Preparation of high-glucose MEM-EBSS complete medium:

[0062] Preparation of 25 mmol / L glucose solution: Weigh 3.5 g of glucose (analytical grade) and dissolve it in 15 mL of MEM complete culture medium. After complete dissolution, filter through a 0.22 μm microporous filter membrane to obtain the solution.

[0063] c. Preparation of insulin solution:

[0064] The molecular weight of insulin is 5808, and 1 IU (international unit) of insulin injection is equivalent to 0.035 mg of anhydrous human insulin. Take 415 μL of 40 IU / mL insulin injection and add high-glucose MEM-EBSS complete medium (25 mmol / L) to make 1 mL 1×10 -4 mol / L insulin solution. Then the gradient dilution was made to a final concentration of 1×10 -5 , 1×10 -6 , 1×10 -7 , 1×10 - 8 mol / L insulin solution. Prepare it before use.

[0065] (2) Optimal concentration and duration of action for inducing IR in HepG2 cells

[0066] a. Effects of different insulin concentrations and different action times on cell proliferation activity

[0067] HepG2 cells in the logarithmic growth phase were taken and 1×10 4 Cells / well were inoculated in a 96-well culture plate. They were divided into a control group and an insulin group, with 6 replicates in each group. After incubation in a 37°C, 5% CO2 saturated humidity incubator for 24 h, the culture medium was aspirated and replaced with serum-free culture medium for starvation treatment for 12 h, and then the culture medium was aspirated and washed three times with 100 μL PBS buffer solution. Normal culture medium was added to the normal control group, and freshly prepared culture medium containing 25 mmol / L glucose and 1×10 insulin were added to the model group. -4 , 1×10 -5 , 1×10 -6 , 1×10 -7 , 1×10 -8 mol / L culture medium, incubated in a 37℃, 5% CO2 saturated humidity incubator for 24 h, 48 h and 72 h respectively. The culture medium was discarded, and 10 μL CCK-8 was added to each well according to the instructions of the CCK-8 cell proliferation-toxicity detection kit. The cells were cultured in a 37℃, 5% CO2 incubator for 2 h, and the absorbance value at 450 nm of each well was measured by a microplate reader. The growth of cells was detected for 24 h, 48 h and 72 h, respectively, to investigate the effects of different concentrations of insulin and different action times on cell proliferation activity.

[0068] b. Effects of different insulin concentrations and durations on cellular glucose consumption

[0069] The cell culture treatment method is the same as (1). After incubation for 24 h, the supernatant was aspirated. The glucose determination kit (glucose oxidase method) was used to add samples according to the detection steps in the kit manual. The OD value of each well was measured on a microplate reader at 505 nm. The glucose content in the cell supernatant of each well was calculated according to the formula [glucose concentration (mmol / L) = (OD sample tube-OD blank tube) / (OD standard tube-OD blank tube) × 5.55 mmol / L]; the glucose consumption was calculated, glucose consumption = 25 mmol / L-cell supernatant glucose content. The insulin action concentration and action time with the minimum cell glucose consumption and no obvious effect on cell proliferation were selected as the optimal insulin concentration and optimal action time for model replication.

[0070] c. Stable duration of HepG2 cell IR model

[0071] The model was established according to the optimal insulin concentration and optimal insulin action time that were screened out, and then the control group and insulin group cells were placed in normal culture medium containing 25 mmol / L glucose but no insulin for a further 24 h, 48 h and 72 h. The glucose content in the cell supernatant was determined using a glucose determination kit (glucose oxidase method), and the glucose consumption of each group of cells was calculated to determine the stability of the IR state of the cell model.

[0072] 3.3 Determination of cell viability using CCK-8 method to determine the concentration range of 4-methylesculetin

[0073] HepG2 cells in the logarithmic growth phase and in good growth condition were taken, digested with trypsin, and complete culture medium was added to adjust the cell concentration to 6.5×10 4 Cells / mL were inoculated in a 96-well plate at a volume of 100 μL per well. 100 μL of medium without cells was used as a blank group. 100 μL of PBS was added to the surrounding wells of the 96-well plate to reduce the edge effect. The plate was incubated at 37°C and 5% CO2 for 24 h. After the cells adhered to the wall, the original medium was discarded. After washing with PBS twice, 100 μL of different concentrations of drug solution was added to each well of the drug group. Six parallel wells were set for each concentration. 100 μL of medium containing 0.1% DMSO was added to the control group. After 24, 48, and 72 h of culture, 10 μL of CCK-8 solution was added to each well and incubated in an incubator for 0.5 h. The operation was protected from light. The optical density (OD) of each well at a wavelength of 450 nm was detected using an ELISA reader. The cell survival rate (%) was calculated according to formula (5).

[0074] Cell viability (%) = [OD （实验） -OD （空白） ] / [OD（对照） -OD （空白） ]×100 (5)

[0075] Note: OD （实验） : OD value of the wells containing cells, culture medium, drugs, and CCK-8; OD （对照） : OD value of the well containing cells, culture medium, CCK-8, and no drug; OD （空白） : OD values ​​of wells without cells or drugs and with culture medium and CCK-8.

[0076] 3.4 Pharmacological effects of 4-methylesculetin on the anti-T2DM active ingredients on IR-HepG2 cells

[0077] (1) Grouping and dosing

[0078] Normal control group (Control group): normal HepG2 cell suspension inoculated in complete culture medium; model group (Model group): high glucose and high insulin (IR-HepG2) culture medium; positive drug group (Met group): IR-HepG2 + metformin; drug-treated group: IR-HepG2 + 4-methylesculetin high, medium, and low doses. HepG2 cells in the logarithmic growth phase were obtained, digested with trypsin, and made into single cell suspensions. The cells were counted and the cell density was adjusted to 1×10 5 Cells / well, final volume 100μL, inoculated on culture plates of different specifications according to the test indicators, and cultured. After incubation in a 37℃, 5% CO2 incubator for 24 h, the culture medium was aspirated and replaced with serum-free culture medium for starvation treatment for 12 h, then the culture medium was aspirated and washed 1-2 times with PBS buffer solution. Complete culture medium was added to the control group, and freshly prepared culture medium containing 25 mmol / L glucose, 1×10 -6 mol / L insulin culture medium, the final volume was 200μL, and then placed in a 37℃, 5% CO2 incubator for 24 hours to establish an in vitro HepG2 cell IR model. After 24 hours of induction, the original culture medium was discarded, washed 1-2 times with PBS buffer solution, and metformin and different concentrations of +4-methylesculetin solution were added according to the above grouping, and then placed in a 37℃, 5% CO2 incubator for drug treatment for 24 hours.

[0079] (2) Biochemical index detection

[0080] The contents of glucose, glycogen, SOD, GSH-PX and MDA in each group of cells were detected according to the instructions of the biochemical kit.

[0081] (3) Reactive oxygen species (ROS)

[0082] a. Cell collection: aspirate the culture medium, digest the cells with trypsin, add complete culture medium to stop digestion, collect into centrifuge tubes, centrifuge at 1000 rpm for 5 min, discard the supernatant, and keep the cell pellet. Add PBS, wash twice, centrifuge at 1000 rpm for 5 min, aspirate the supernatant, and keep the cell pellet for determination.

[0083] b. Resuspend cells: Dilute the probe DCFH-DA to a concentration of 10 µM with PBS, and resuspend the cell pellet with the diluted probe to prepare a cell suspension.

[0084] c. Negative control tube: one tube contains cells without probe but only PBS.

[0085] d. Incubate the cells at 37°C for 30 min; invert and mix every 3-5 minutes to ensure full contact between the probe and the cells.

[0086] e. Collect the single cell suspension after incubation (probe labeling), centrifuge at 1000 rpm for 5 min, aspirate the supernatant, wash twice with PBS, and collect the cell pellet by centrifugation for fluorescence detection.

[0087] f. Resuspend the collected cell pellet in PBS and use it for detection.

[0088] g. The wavelength was set to the optimal emission wavelength of 525 nm and measured by flow cytometry.

[0089] 4. Experimental results

[0090] 4.1 Establishment of HepG2 cell insulin resistance (IR) model in vitro

[0091] (1) Safety study of insulin action concentration and duration

[0092] Effects of different insulin concentrations and different action times on the proliferation activity of HepG2 cells. Figure 1 Compared with the control group, 10 -8 -10 -5 mol / L insulin was applied to HepG2 cells for 24 h, 48 h, 10 -8 -10 -6 There was no statistical difference in the proliferation activity of HepG2 cells after 72 h of insulin treatment; however, 10 -4 mol / L insulin was applied to HepG2 cells for 24h, 48h and 72h, 10 -5 When HepG2 cells were treated with 100 mol / L insulin for 72 h, the proliferation activity of HepG2 cells was significantly decreased (P<0.05 or P<0.01).

[0093] Table 3 Effects of different insulin concentrations and different action times on the proliferation activity of HepG2 cells ( ±s, n=6)

[0094]

[0095] Note: Compared with the control group, P * <0.05, P ** <0.01.

[0096] (2) Effects of different insulin concentrations and action times on glucose consumption in HepG2 cells

[0097] From Table 4 and Figure 2 Compared with the control group, 10 -6 , 10 -5 and 10 -4 When 10 mol / L insulin was applied to HepG2 cells for 24h and 48h, their glucose consumption was significantly reduced (P<0.01), but there was no statistical difference in glucose consumption when the cells were applied for 72h. -8 and 10 -7 There was no statistical difference in glucose consumption when the cells were treated with insulin of 10 mol / L for 24 h, 48 h and 72 h. Combining the results in Table 3 and Table 4, the optimal concentration of insulin prepared by the IR model of HepG2 was determined to be 10 -6 mol / L, the action time is 24 h.

[0098] Table 4 Effects of different concentrations of insulin and different action time on glucose consumption of HepG2 cells ( ±s, n=6)

[0099]

[0100] Note: Compared with the control group, P*<0.05, P**<0.01.

[0101] (3) Stability of insulin resistance in HepG2 cell model

[0102] 10 -6 After 24 h of action of 1.5 mol / L insulin, the glucose consumption of IR-HepG2 cells in normal culture medium at different time points. Figure 3 It can be seen that compared with the control group, the glucose consumption of cells in the 24 h and 48 h insulin groups was significantly reduced (P<0.01), and there was no statistical difference in glucose consumption between the two groups at 72 h. Therefore, the IR-HepG2 cell model established by this method can last for 48 h.

[0103] Table 5 Glucose consumption at different time points after unloading of IR-HepG2 cell model ( ±s, n=6)

[0104]

[0105] Note: Compared with the control group, P * <0.05, P ** <0.01.

[0106] 4.2 Determination of 4-methylesculetin dosage

[0107] In order to determine the safe dose range of 4-methylesculetin, the survival rate of HepG2 cells was measured by CCK-8 assay. Figure 4 As shown in Figures AC, compared with the Control group, there was no statistical difference in the viability of HepG2 cells in the 4-methylesculetin (6.25μM, 12.5μM) groups at 24 h, 48 h, and 72 h, no statistical difference in the 25μM group at 24 h and 48 h, no statistical difference in the 50μM and 100μM groups at 24 h, and the cell viability of the 200μM and 400μM groups at 24 h, 48 h, and 72 h was significantly decreased (P<0.01), indicating that the survival rate of HepG2 cells in the 4-methylesculetin (6.25μM, 12.5μM, 25μM, 50μM, 100μM, 200μM, 400μM) groups gradually decreased with the increase of drug concentration and the extension of action time.

[0108] To further determine the highest safe dose of 4-methylesculetin, the lowest toxic dose of 25 μM was selected as the maximum dose, and the second CCK-8 experiment was performed with a 0.7-fold decreasing dose, as shown in Table 7 and Figure 4 As shown in DF, compared with the Control group, there was no statistical difference in the viability of HepG2 cells in the 4-methylesculetin (6.0025μM, 8.575μM, 12.25μM, 17.5μM) groups at 24 h, 48 h, and 72 h, and there was no statistical difference in the viability of HepG2 cells in the 25μM group at 24 h and 48 h, and the viability of HepG2 cells decreased significantly at 72 h (P<0.05).

[0109] Based on the above results, subsequent experiments were conducted with 17.5 μM 4-methylesculetin solution as the high dose (4-QYT-H), 10.5 μM 4-methylesculetin solution as the medium dose (4-QYT-M), and 6.3 μM 4-methylesculetin solution as the low dose (4-QYT-L).

[0110] Table 6 Effects of 4-methylesculetin on the viability of IR-HepG2 cells ( ±s, n=6)

[0111]

[0112] Note: Compared with the control group, P * <0.05, P ** <0.01.

[0113] Table 7 Effects of 4-methylesculetin on the viability of IR-HepG2 cells ( ±s, n=6)

[0114]

[0115] Note: Compared with the control group, P * <0.05, P ** <0.01.

[0116] 4.3 Effects of 4-methylesculetin on glucose consumption and glycogen content in IR-HepG2 cells

[0117] As shown in Table 8 and Figure 5 As shown in the results, compared with the Control group, the glucose consumption in the Model group was significantly decreased (P<0.01), and compared with the Model group, the glucose consumption in the 4-QYT-H group, 4-QYT-M group, 4-QYT-L group, and Met group was significantly increased (P<0.01). Compared with the Control group, the glycogen content in the Model group was significantly decreased (P<0.01). Compared with the Model group, the glycogen content in the 4-QYT-H group, 4-QYT-M group, 4-QYT-L group, and Met group was significantly increased (P<0.01).

[0118] Table 8 Effects of 4-methylesculetin on glucose production and glycogen content in IR-HepG2 cells ( ±s, n=6)

[0119]

[0120] Note: Compared with the Control group, P * <0.05, P ** <0.01; compared with the Model group, P # <0.05, P ## <0.01.

[0121] 4.4 Effect of 4-methylesculetin on reducing ROS generation and improving oxidative stress in IR-HepG2 cells

[0122] As shown in Table 9 and Figure 6As shown in the figure, compared with the Control group, the contents of ROS and MDA in the Model group were significantly increased (P<0.01), and the contents of SOD and GSH-PX were significantly decreased (P<0.01). Compared with the Model group, the contents of ROS and MDA in the 4-QYT-H group, 4-QYT-M group, 4-QYT-L group and Met group were significantly decreased (P<0.01), and the contents of SOD and GSH-PX were significantly increased (P<0.05 or P<0.01).

[0123] Table 9 Effect of 4-methylesculetin on improving oxidative stress in IR-HepG2 cells ( ±s, n=6)

[0124]

[0125] Note: Compared with the Control group, P * <0.05, P ** <0.01; compared with the Model group, P # <0.05, P ## <0.01.

[0126] According to the above test results, 4-methylesculetin can significantly increase the glucose consumption of IR-HepG cells and increase the glycogen synthesis; significantly improve the antioxidant stress capacity of IR-HepG cells, reduce the levels of ROS and MDA, and increase the levels of SOD and GSH-PX. The high-dose 4-methylesculetin group has better effects, indicating that 4-methylesculetin can be used to make hypoglycemic drugs.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of 4-methylesculetin, a potential active ingredient of xiasangju, in the preparation of drugs for the prevention and treatment of type 2 diabetes.

2. The use according to claim 1, characterized in that: The medicine is a medicine for improving abnormal glucose and lipid metabolism.

3. The use according to claim 1, characterized in that: The drug is a drug that increases cellular glucose consumption.

4. The use according to claim 1, characterized in that: The drug is a drug that increases the level of liver oxidative stress.

5. The use according to claim 4, characterized in that: The medicine is a medicine for reducing the levels of ROS and MDA in liver tissue.

6. The use according to claim 4, characterized in that: The medicine is a medicine for increasing the levels of SOD and GSH-PX in liver tissue.

7. The use according to claim 1, characterized in that: The medicine comprises 4-methylesculetin and pharmaceutically acceptable excipients.

8. The use according to claim 1, characterized in that: The dosage form of the medicine is tablets, granules, pills or capsules.

9. The use according to claim 1, characterized in that: The dosage of 4-methylesculetin in the drug is 6.3-17.5 μM.

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