Dihydroisocoumarin compound as well as preparation method and application thereof

Abstract

The invention relates to a preparation method and application of a dihydroisocoumarin compound (3R, 4R)-3-(beta-D-glucopyranoloxymethyl)-3, 4-dihydro-5, 6, 7-trihydroxy-4-(3 '-methoxy-4'-hydroxyphenyl)-1H-[2]-benzopyran-1-ketone in dogwood, which can be used for effectively solving the medication problem of diabetes mellitus, and adopts the technical scheme that the dosage of the dihydroisocoumarin compound (3R, 4R)-3-(beta-D-glucopyranoloxymethyl)-3, 4-dihydro-5, 6, 7-trihydroxy-4-(3 '-methoxy-4'-hydroxyphenyl)-1H-[2]- The chemical structure of the dihydroisocoumarin compound is as follows: # imgabs0 #, the dihydroisocoumarin compound disclosed by the invention can have a certain improvement effect on insulin-induced HepG2 cell injury, and can be used as a medicine for treating diabetes mellitus, and the medicine can be a monomer or a derivative thereof, and can be used for preparing a medicine for treating diabetes mellitus. The invention is an innovation of the dihydroisocoumarin compound in dogwood and the application of the dihydroisocoumarin compound.

Description

1. Technical Field

[0001] The invention relates to the field of medicine, in particular to a dihydroisocoumarin compound extracted from Cornus officinalis and a preparation method and application thereof. 2. Background Technology

[0002] Diabetes is a chronic metabolic disease characterized by elevated blood sugar levels due to insulin deficiency (type 1 diabetes) or insulin resistance (type 2 diabetes). According to the International Diabetes Federation (IDF), approximately 537 million people aged 20-79 years worldwide will have diabetes in 2021, and this number is expected to rise to 783 million by 2045. Diabetes has a significant impact on human health and may lead to multiple complications involving multiple organ systems. These complications include cardiovascular disease, nephropathy, retinopathy, neuropathy, and diabetic foot ulcers, which pose a serious threat to quality of life and life expectancy. Current drug interventions for diabetes management mainly include insulin therapy and oral hypoglycemic drugs such as metformin, sulfonylureas, and thiazolidinediones. However, these treatments have certain limitations. Insulin therapy may require injections and has poor compliance, while oral medications may cause gastrointestinal adverse reactions and are associated with the risk of hypoglycemia and weight gain. Therefore, there is an urgent need to develop innovative anti-diabetic drugs to improve treatment outcomes and enhance patient well-being.

[0003] Cornus officinalis fruit is a traditional Chinese medicine widely used to treat a variety of diseases, including dizziness, tinnitus, weakness of waist and knees, impotence, spermatorrhea, enuresis, frequent urination, and internal heat and thirst. Chemical studies have shown that Cornus officinalis fruit contains iridoid glycosides, tannins, flavonoids, small amounts of volatile oils, organic acids and triterpenoids. Pharmacological studies have shown that it has multiple therapeutic effects such as hypoglycemic, antioxidant, anti-inflammatory, anti-tumor, neuroprotective and hepatoprotective. Many pharmacological studies focus on the anti-diabetic effects of Cornus officinalis. For example, the study by Han and He showed that oral administration of Cornus officinalis extract (300 mg / kg every 2 days, 400 mg / kg per day) and isolated loganin, morroniside and ursolic acid (200 mg / kg per day) significantly reduced the fasting blood glucose level of diabetic rats and alleviated the symptoms of diabetes such as polydipsia, polyphagia, polyuria and weight loss (Y. Han, H. W. Jung, Y. K. Park, Selective therapeutic effect of Cornus officinalis fruits on the damage of different organs in STZ-induced diabetic rats, Am J Chin Med 42 (2014) 1169-82; K. He, S. Song, Z. Zou, M. Feng, D. Wang, Y. Wang, X. Li, X. Ye, The hypoglycemic and synergistic effect of loganin, morroniside, and ursolic acidisolated from the fruits of Cornus officinalis, Phytother Res 30 (2016) 283-91.). The study by Peng et al. showed that some secoiridoid compounds in Cornus officinalis fruits may have potential α-glucosidase inhibitory activity (ZCPeng, J.He, XGPan, J.Zhang, YMWang, XSYe, CYXia, WWLian, Y.Yan, XLHe, WKZhang, JKXu, Secoiridoid dimers and their biogenic precursors from the fruits of Cornus officinalis with potential therapeutic effects on type 2 diabetes, Bioorg Chem 117 (2021) 105399.).The anti-DM effects of Cornus officinalis extracts and effective parts are clear, but there is no research on the anti-DM activity of dihydroisocoumarins in Cornus officinalis. III. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a dihydroisocoumarin compound extracted from Cornus officinalis and its preparation method and application, which can effectively solve the medication problem of type 2 diabetes.

[0005] The technical solution provided by the present invention is that the dihydroisocoumarin compound extracted from Cornus officinalis, (3R, 4R)-3-(β-D-pyranose glucose oxymethyl)-3,4-dihydro-5,6,7-trihydroxy-4-(3'-methoxy-4'-hydroxyphenyl)-1H-[2]-benzopyran-1-one (Compound 1), has the chemical structural formula:

[0006]

[0007] Its preparation method is:

[0008] 1) 10-100 kg of Cornus officinalis pulp is extracted with 2-10 times amount of deionized water at 80-100° C. by reflux extraction for 1-3 times, and the extract is concentrated under reduced pressure to form an extract. The extract is dissolved in water and passed through a D-101 macroporous adsorption resin column, and eluted with ultrapure water and 95% ethanol respectively.

[0009] 2) The 95% ethanol eluate was concentrated under reduced pressure at low temperature to form an extract, dissolved in water, and extracted with petroleum ether, ethyl acetate and n-butanol for 3-10 times in sequence. The n-butanol extraction part was concentrated under pressure to form an extract, dissolved in water, passed through a Diaion HP-20 macroporous adsorption resin column, and eluted with ethanol-water (0:100, 20:80, 30:70, 40:60, 95:5; v / v) gradient to obtain 5 parts (BF1-BF5).

[0010] 3) The BF2 fraction obtained in step 2) was passed through an MCI column and eluted with methanol-water (0:100, 20:80, 40:60, 60:40, 100:0; v / v) to obtain 5 fractions (BF2a-BF2e). The BF2a fraction was passed through a Sephadex LH-20 column and gradient eluted with methanol-water (20:80, 50:50, 70:30, 100:0; v / v) to obtain 5 fractions (BF2a1-BF2a5).

[0011] 4) BF2a4 obtained in step 3) was passed through a Sephadex LH-2 column and eluted with methanol-water (4:6-8:2; v / v) to obtain 9 fractions (BF2a4a-BF2a4i). BF2a4c was passed through a silica gel column and eluted with dichloromethane-methanol (12:1-5:1, v / v) to obtain 5 fractions (BF2a4c1-BF2a4c5).

[0012] 5) The BF2a4c3 fraction obtained in step 4) was passed through a silica gel column and eluted with dichloromethane-methanol (10:1, v / v) to obtain 6 fractions (BF2a4c3a-BF2a4c3f). BF2a4c3d was passed through a silica gel column and eluted with dichloromethane-methanol (10:1-0:1; v / v) to obtain 5 fractions (BF2a4c3d1-BF2a4c3d5).

[0013] 6) BF2a4c3d4 was purified by semi-preparative HPLC (9% methanol, 3 mL / min), the chromatographic column was COSMOSIL C18-MS-II column (5 μm, 10×250 mm), the mobile phase was 19% MeCN, and the flow rate was 3 mL / min. Compound 1 (t R =39.7min).

[0014] The invention relates to the use of the dihydroisocoumarin compounds in the preparation of drugs for treating type 2 diabetes.

[0015] The present invention also provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the dihydroisocoumarin compound according to claim 1 or a pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite or mixture thereof.

[0016] The dihydroisocoumarin compound (3R, 4R)-3-(β-D-pyranose glucose oxymethyl)-3,4-dihydro-5,6,7-trihydroxy-4-(3'-methoxy-4'-hydroxyphenyl)-1H-[2]-benzopyran-1-one of the present invention is obtained by using cornus officinalis pulp through solvent extraction, macroporous adsorption resin chromatography, silica gel column chromatography, gel column chromatography, MCI column chromatography, Sephadex LH-20 gel column chromatography, preparative liquid phase separation and other steps. The preparation method is simple and easy, and the product has high purity. The results of pharmacological activity studies show that the compound can significantly increase the glucose consumption of insulin-induced HepG2 cell insulin resistance model and significantly promote the glucose uptake of HepG2 cell insulin resistance model, indicating that the compound has a potential effect in treating diabetes and is of great significance in the development of anti-diabetic drugs. IV. Description of the drawings

[0017] Figure 1 The dihydroisocoumarin compound 1 of the present invention 1 H-NMR spectrum.

[0018] Figure 2 The dihydroisocoumarin compound 1 of the present invention 13 C-NMR spectrum.

[0019] Figure 3 It is the DEPT-135 spectrum of dihydroisocoumarin compound 1 of the present invention.

[0020] Figure 4 The dihydroisocoumarin compound 1 of the present invention 1 H- 1 H COSY spectrum.

[0021] Figure 5 It is the HSQC spectrum of the dihydroisocoumarin compound 1 of the present invention.

[0022] Figure 6 It is the HMBC spectrum of the dihydroisocoumarin compound 1 of the present invention.

[0023] Figure 7 It is the NOESY spectrum of the dihydroisocoumarin compound 1 of the present invention.

[0024] Figure 8 It is the UV spectrum of dihydroisocoumarin compound 1 of the present invention.

[0025] Fig. 9 It is the IR spectrum of the dihydroisocoumarin compound 1 of the present invention.

[0026] Fig.10 It is the HRESIMS spectrum of the dihydroisocoumarin compound 1 of the present invention.

[0027] Fig.11 The CD spectrum of dihydroisocoumarin compound 1 of the present invention.

[0028] Fig.12 This is a bar graph showing the effect of the dihydroisocoumarin compound 1 of the present invention on the viability of HepG2 cells.

[0029] Fig.13 It is a line graph of glucose consumption of HepG2 cells by dihydroisocoumarin compound 1 of the present invention.

[0030] Fig.14 This is a flow cytometric imaging diagram of glucose uptake by HepG2 cells by the dihydroisocoumarin compound 1 of the present invention.

[0031] Fig.15This is a fluorescence image of the dihydroisocoumarin compound 1 of the present invention on the expression of the insulin receptor INSR in HepG2 cells.

[0032] Fig.16 The structural diagram of a dihydroisocoumarin compound 1 of the present invention. V. Specific implementation methods

[0033] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0034] Embodiment 1:

[0035] In a specific implementation of the present invention, a method for preparing dihydroisocoumarin comprises the following steps:

[0036] 1) 10 kg of Cornus officinalis pulp was extracted twice with 3 times amount of deionized water at 90°C under reflux, and the extract was concentrated under reduced pressure to form an extract. The extract was dissolved in water and passed through a D-101 macroporous adsorption resin column, and eluted with ultrapure water and 95% ethanol respectively.

[0037] 2) The 95% ethanol eluate was concentrated under reduced pressure at low temperature to form an extract, dissolved in water, and extracted six times with petroleum ether, ethyl acetate and n-butanol in sequence. The n-butanol extraction fraction was concentrated under pressure to form an extract, dissolved in water, passed through a Diaion HP-20 macroporous adsorption resin column, and eluted with ethanol-water (0:100, 20:80, 30:70, 40:60, 95:5; v / v) gradient to obtain five fractions (BF1-BF5).

[0038] 3) The BF2 fraction obtained in step 2) was passed through an MCI column and eluted with methanol-water (0:100, 20:80, 40:60, 60:40, 100:0; v / v) to obtain 5 fractions (BF2a-BF2e). The BF2a fraction was passed through a Sephadex LH-20 column and gradient eluted with methanol-water (20:80, 50:50, 70:30, 100:0; v / v) to obtain 5 fractions (BF2a1-BF2a5).

[0039] 4) BF2a4 obtained in step 3) was passed through a Sephadex LH-2 column and eluted with methanol-water (4:6-8:2; v / v) to obtain 9 fractions (BF2a4a-BF2a4i). BF2a4c was passed through a silica gel column and eluted with dichloromethane-methanol (12:1-5:1, v / v) to obtain 5 fractions (BF2a4c1-BF2a4c5).

[0040] 5) The BF2a4c3 fraction obtained in step 4) was passed through a silica gel column and eluted with dichloromethane-methanol (10:1, v / v) to obtain 6 fractions (BF2a4c3a-BF2a4c3f). BF2a4c3d was passed through a silica gel column and eluted with dichloromethane-methanol (10:1-0:1; v / v) to obtain 5 fractions (BF2a4c3d1-BF2a4c3d5).

[0041] 6) BF2a4c3d4 was purified by semi-preparative HPLC (9% methanol, 3 mL / min), the chromatographic column was COSMOSIL C18-MS-II column (5 μm, 10×250 mm), the mobile phase was 19% MeCN, and the flow rate was 3 mL / min. Compound 1 (t R =39.7min).

[0042] Embodiment 2:

[0043] In a specific implementation of the present invention, a method for preparing dihydroisocoumarin comprises the following steps:

[0044] 1) 10 kg of Cornus officinalis pulp was extracted three times with 3 times amount of deionized water at 90° C. The extract was concentrated under reduced pressure to form an extract, which was dissolved in water and passed through a D-101 macroporous adsorption resin column and eluted with ultrapure water and 95% ethanol, respectively.

[0045] 2) The 95% ethanol eluate was concentrated under reduced pressure at low temperature to form an extract, dissolved in water, and extracted four times with petroleum ether, ethyl acetate and n-butanol in sequence. The n-butanol extraction fraction was concentrated under pressure to form an extract, dissolved in water, passed through a Diaion HP-20 macroporous adsorption resin column, and eluted with ethanol-water (0:100, 20:80, 30:70, 40:60, 95:5; v / v) gradient to obtain five fractions (BF1-BF5).

[0046] 3) The BF2 fraction obtained in step 2) was passed through an MCI column and eluted with methanol-water (0:100, 20:80, 40:60, 60:40, 100:0; v / v) to obtain 5 fractions (BF2a-BF2e). The BF2a fraction was passed through a Sephadex LH-20 column and gradient eluted with methanol-water (20:80, 50:50, 70:30, 100:0; v / v) to obtain 5 fractions (BF2a1-BF2a5).

[0047] 4) BF2a4 obtained in step 3) was passed through a Sephadex LH-2 column and eluted with methanol-water (4:6-8:2; v / v) to obtain 9 fractions (BF2a4a-BF2a4i). BF2a4c was passed through a silica gel column and eluted with dichloromethane-methanol (12:1-5:1, v / v) to obtain 5 fractions (BF2a4c1-BF2a4c5).

[0048] 5) The BF2a4c3 fraction obtained in step 4) was passed through a silica gel column and eluted with dichloromethane-methanol (10:1, v / v) to obtain 6 fractions (BF2a4c3a-BF2a4c3f). BF2a4c3d was passed through a silica gel column and eluted with dichloromethane-methanol (10:1-0:1; v / v) to obtain 5 fractions (BF2a4c3d1-BF2a4c3d5).

[0049] 6) BF2a4c3d4 was purified by semi-preparative HPLC (9% methanol, 3 mL / min), the chromatographic column was COSMOSIL C18-MS-II column (5 μm, 10×250 mm), the mobile phase was 19% MeCN, and the flow rate was 3 mL / min. Compound 1 (t R =39.7min).

[0050] Embodiment 3:

[0051] In a specific implementation of the present invention, a method for preparing dihydroisocoumarin comprises the following steps:

[0052] 1) 10 kg of Cornus officinalis pulp was extracted three times with 1 times amount of deionized water at 90° C. The extract was concentrated under reduced pressure to form an extract, which was dissolved in water and passed through a D-101 macroporous adsorption resin column and eluted with ultrapure water and 95% ethanol, respectively.

[0053] 2) The 95% ethanol eluate was concentrated under reduced pressure at low temperature to form an extract, dissolved in water, and extracted 8 times with petroleum ether, ethyl acetate and n-butanol in sequence. The n-butanol extraction fraction was concentrated under pressure to form an extract, dissolved in water, passed through a Diaion HP-20 macroporous adsorption resin column, and eluted with ethanol-water (0:100, 20:80, 30:70, 40:60, 95:5; v / v) gradient to obtain 5 fractions (BF1-BF5).

[0054] 3) The BF2 fraction obtained in step 2) was passed through an MCI column and eluted with methanol-water (0:100, 20:80, 40:60, 60:40, 100:0; v / v) to obtain 5 fractions (BF2a-BF2e). The BF2a fraction was passed through a Sephadex LH-20 column and gradient eluted with methanol-water (20:80, 50:50, 70:30, 100:0; v / v) to obtain 5 fractions (BF2a1-BF2a5).

[0055] 4) BF2a4 obtained in step 3) was passed through a Sephadex LH-2 column and eluted with methanol-water (4:6-8:2; v / v) to obtain 9 fractions (BF2a4a-BF2a4i). BF2a4c was passed through a silica gel column and eluted with dichloromethane-methanol (12:1-5:1, v / v) to obtain 5 fractions (BF2a4c1-BF2a4c5).

[0056] 5) The BF2a4c3 fraction obtained in step 4) was passed through a silica gel column and eluted with dichloromethane-methanol (10:1, v / v) to obtain 6 fractions (BF2a4c3a-BF2a4c3f). BF2a4c3d was passed through a silica gel column and eluted with dichloromethane-methanol (10:1-0:1; v / v) to obtain 5 fractions (BF2a4c3d1-BF2a4c3d5).

[0057] 6) BF2a4c3d4 was purified by semi-preparative HPLC (9% methanol, 3 mL / min), the chromatographic column was COSMOSIL C18-MS-II column (5 μm, 10×250 mm), the mobile phase was 19% MeCN, and the flow rate was 3 mL / min. Compound 1 (t R =39.7min).

[0058] The pharmacological activity results of the dihydroisocoumarin compounds prepared by the present invention show that they can significantly increase the glucose consumption of the insulin-induced HepG2 cell insulin resistance model, and can be used as a drug for treating type 2 diabetes. The relevant experimental data are as follows:

[0059] 1. Structural identification of the compounds of the present invention:

[0060] Compound 1 is an amorphous powder (MeOH). Its molecular formula was determined by HRESIMS to be C 23 H 26 O 13 (m / z533.1286,[M+Na] +). Its infrared spectrum showed the presence of hydroxyl (3457 cm-1) and carbonyl (1682 cm-1). Its NMR data were very similar to those of (3S,4S)3-(β-D-glucopyranosyloxymethyl)-3,4-dihydro-5,6,7-trihydro-xy-4-(4′-hydroxy-3′-methoxyphenyl)-1H-[2]-benzopyran-1-one [AM Abdulmagid, VN Laurence, G. Moroy, C. Moretti, C. Lavaud, Dihydroisocoumarin glucosides from stem bark of Caryocar glabrum, Phytochemistry 68 (2007) 2439-2443.], indicating that compound 1 may have the same skeleton as the reported compound, which was verified by 2D NMR spectroscopy. 1 H NMR, 13 CNMR, 1 H- 1 Its planar chemical structure was confirmed by H COSY, HSQC and HMBC spectra. Its attached sugar group was confirmed to be D-glucose by chiral HPLC analysis. 3 J H-1″,H-2″ (7.7Hz) confirmed that its glucoside group was in β-configuration. Its NOESY spectrum showed correlations between H-3a / H-4, H-1” / H-4, H-3 / H-2’, and H-3 / H-6’, indicating that H-3 and the 3-methoxy-4-hydroxyphenyl group were located on the same side of the lactone ring (α orientation), while H-4 and the β-D-pyranose glucopyranose oxymethyl group were located on the other side (β orientation). The absolute configuration of the compound was determined by comparing the experimental ECD spectrum with the calculated ECD spectrum. Therefore, the structure of compound 2 was determined to be (3R,4R)-3-(β-D-pyranose glucopyranose oxymethyl)-3,4-dihydro-5,6,7-trihydroxy-4-(3'-methoxy-4'-hydroxyphenyl)-1H-[2]-benzopyran-1-one.

[0061] Compound 1 1 H-NMR and 13 The C-NMR spectrum data are shown in Table 1.

[0062] Table 1. Compound 1 1 H NMR (500 MHz, CD 3 OD) and 13 C NMR spectral data

[0063]

[0064]

[0065] *no=The signal was not observed

[0066] 2. Anti-diabetic effects of dihydroisocoumarin compounds of the present invention

[0067] 1) CCK-8 assay to determine the effect of compound 1 on the cell viability of HepG2 cells

[0068] HepG2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were seeded in 96-well plates at a density of 1×10 5 / well. Preparation of insulin resistance (IR): HepG2 cells were starved in serum-free DMEM for 24 hours. After 24 hours of culture, the culture medium was changed to serum-free DMEM containing 20 μM insulin and incubated for another 24 hours. Then, DMEM culture medium, metformin culture medium (10 μM) or culture medium containing monomeric compounds (10.0 μM) were added respectively. After incubation for 24 hours, the culture medium was replaced, and 100 μL of new serum-free culture medium was added. Then, 10 μL of CCK-8 reagent was added, and the cells were incubated at 37°C for 1 hour. The optical density (OD) value of each well was measured at 450 nm using a microplate spectrophotometer. Results are shown in Fig.12 Compound 1 had no significant effect on the cell viability of HepG2 cells at doses of 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, and 20.0 μM.

[0069] 2) Determination of relative glucose consumption

[0070] HepG2 cells (1×105 cells / well) cultured in 96-well plates were treated with insulin and compound 1, and the culture medium was collected and the glucose concentration was determined using a glucose assay kit. The glucose content of the original DMEM culture medium minus the glucose content of the experimental group culture medium was used to obtain the glucose consumption (GC). The relative glucose consumption (RGC) was calculated as: RGC = GC / OD.

[0071] Results Fig.13 Compound 1 can significantly increase the relative glucose consumption of HepG2 insulin resistance model in a dose-dependent manner, with the best effect being at a dose of 20.0 μM.

[0072] 3) 2-NBDG probe method to detect glucose uptake ability

[0073] HepG2 cells (1×10 5 / mL, 1mL / well), treated with insulin and compound 1; 10.0μM) and heated for 24h as described above. Then gently pour out the culture medium, add 1.0mL DMEM culture medium to each well, add glucose uptake fluorescent probe (2-NBDG, 20.0μM), and incubate in a cell culture incubator for 1h. Collect the cells and transfer them to the flow tube, wash them twice with PBS, and add 500μL DRAQ5 (1:500) nuclear dye to resuspend the cells. Detect the cell fluorescence intensity with Flow Sight.

[0074] Results Fig.14 The fluorescence of the cells in the compound 1 group was significantly enhanced, indicating that compound 1 can significantly improve the glucose uptake ability of the HepG2 insulin resistance model.

[0075] 4) Cell immunofluorescence detection of insulin receptor INSR expression

[0076] The cells were 1×10 5 The cells were seeded in the Cell Carrier at a density of 100 μL / well. TM -96 plates, compound 1 was administered at a dose of 10.0 μM, and the method was the same as 1). After 24 h of incubation, the cells were fixed with 4% paraformaldehyde (room temperature, 20 minutes). They were then blocked with 10% goat serum (room temperature, 90 minutes), followed by incubation with INSR (1:500) and Tubulin (1:1000) at 4°C overnight, and goat anti-rabbit IgG H&L (Alexa Fluor) was added. 488) and goat anti-mouse IgG H&L (Alexa 594) for secondary antibody pre-adsorption (1:1000), incubated at 37°C for 1 h, and then DAPI was used to re-stain the cell nucleus. The fluorescence intensity was measured using a high-content cell imaging system, with excitation wavelengths of 488 nm and 597 nm, respectively. The relative expression of the protein was normalized by Harmony 4.8. The results are shown in Fig.15 The fluorescence of the cells in the compound 1 group was significantly enhanced, indicating that compound 1 can significantly increase the number of insulin receptors (INSR) on the cell membrane of the HepG2 insulin resistance model and promote the transport and utilization of glucose.

[0077] The invention adopts the pulp of Cornus officinalis, a plant of the genus Cornus of the family Cornaceae, and obtains a novel dihydroisocoumarin compound through solvent extraction, macroporous adsorption resin column chromatography, MCI gel, Sephadex LH-20 column chromatography, silica gel column chromatography, preparative high performance liquid separation and the like. The pharmacological activity results show that the compound can significantly increase the glucose consumption of an insulin-induced HepG2 cell insulin resistance model, increase the number of insulin receptors (INSR) in the cell membrane of the HepG2 insulin resistance model, increase the glucose uptake by the cells, and increase the glucose consumption. The compound can be used as a drug for treating type 2 diabetes, is an innovation in dihydroisocoumarin and its use, and has good application and promotion value.

Claims

1. A dihydroisocoumarin compound extracted from Cornus officinalis, characterized in that: The novel compound (3R, 4R)-3-(β-D-glucopyranose oxymethyl)-3,4-dihydro-5,6,7-trihydroxy-4-(3'-methoxy-4'-hydroxyphenyl)-1H-[2]-benzopyran-1-one (1) has the chemical structural formula:

2. The method for preparing a dihydroisocoumarin compound extracted from Cornus officinalis according to claim 1, characterized in that: The following steps are involved: 1) 10-100 kg of Cornus officinalis pulp is extracted with 2-10 times amount of deionized water at 80-100° C. by reflux extraction for 1-3 times, and the extract is concentrated under reduced pressure to form an extract. The extract is dissolved in water and passed through a D-101 macroporous adsorption resin column, and eluted with ultrapure water and 95% ethanol respectively. 2) The 95% ethanol eluate was concentrated under reduced pressure at low temperature to form an extract, dissolved in water, and extracted with petroleum ether, ethyl acetate and n-butanol for 3-10 times in sequence. The n-butanol extraction part was concentrated under pressure to form an extract, dissolved in water, passed through a Diaion HP-20 macroporous adsorption resin column, and eluted with ethanol-water (0:100, 20:80, 30:70, 40:60, 95:5; v / v) gradient to obtain 5 parts (BF1-BF5). 3) The BF2 fraction obtained in step 2) was passed through an MCI column and eluted with methanol-water (0:100, 20:80, 40:60, 60:40, 100:0; v / v) to obtain 5 fractions (BF2a-BF2e). The BF2a fraction was passed through a Sephadex LH-20 column and gradient eluted with methanol-water (20:80, 50:50, 70:30, 100:0; v / v) to obtain 5 fractions (BF2a1-BF2a5). 4) BF2a4 obtained in step 3) was passed through a Sephadex LH-2 column and eluted with methanol-water (4:6-8:2; v / v) to obtain 9 fractions (BF2a4a-BF2a4i). BF2a4c was passed through a silica gel column and eluted with dichloromethane-methanol (12:1-5:1, v / v) to obtain 5 fractions (BF2a4c1-BF2a4c5). 5) The BF2a4c3 fraction obtained in step 4) was passed through a silica gel column and eluted with dichloromethane-methanol (10:1, v / v) to obtain 6 fractions (BF2a4c3a-BF2a4c3f). BF2a4c3d was passed through a silica gel column and eluted with dichloromethane-methanol (10:1-0:1; v / v) to obtain 5 fractions (BF2a4c3d1-BF2a4c3d5). 6) BF2a4c3d4 was purified by semi-preparative HPLC (9% methanol, 3 mL / min), the chromatographic column was COSMOSIL C18-MS-II column (5 μm, 10×250 mm), the mobile phase was 19% MeCN, and the flow rate was 3 mL / min. Compound 1 (t R =39.7min).

3. Use of the coumarin compound according to claim 1 in the preparation of a drug for treating type 2 diabetes.

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