A preparation method and application of diphenyl ether compounds

Abstract

The invention discloses a preparation method and application of a diphenyl ether compound, wherein the diphenyl ether compound includes 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol, which can be efficiently prepared from marine microorganisms. Experiments have shown that 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol can inhibit the accumulation of triglycerides and the activity of α-glucosidase during the differentiation of 3T3-L1 preadipocytes. The compound can be used to prepare anti-obesity, lipid-lowering and glucose-lowering medicines and health products, functional foods, and food additives.

Description

Technical Field

[0001] The present invention relates to the technical field of marine biology, and in particular to a method for preparing a diphenyl ether compound derived from marine Aspergillus and an application thereof in preparing a lipid-lowering and blood sugar-lowering drug. Background Art

[0002] With the rapid development of the global economy, the incidence of obesity is increasing day by day. It is estimated that by 2050, the global obesity prevalence rate will increase to 60% among adult men, 50% among adult women, and 25% among children under 16 years old. Obesity will lead to a series of complications, such as diabetes, cardiovascular disease, myocardial infarction, hypertension, hyperlipidemia, etc. Obesity is one of the most important factors causing diabetes.

[0003] Obesity is the result of a disruption in the balance between energy intake and consumption. Excessive excess energy is stored in adipocytes in the form of triglycerides, causing the adipocytes to gradually grow larger. When the adipocyte volume grows to a certain extent, new pre-adipocytes will continue to proliferate in preparation for the storage of triglycerides, ultimately leading to the continuous enlargement of adipose tissue and weight gain. At present, inhibiting the proliferation of adipocytes, the proliferation and differentiation of pre-adipocytes is an important idea for anti-obesity. Fat accumulation is a key step in the development of obesity. Controlling this step will effectively control the progression of obesity, and the core process of obesity pathophysiology is excessive fat deposition. 3T3-L1 pre-adipocytes induce fat accumulation in vitro and differentiate into mature adipocytes. They are currently recognized cell lines for studying the fat deposition process in the country and are widely used in the study of drug lipid-lowering and anti-obesity activity mechanisms.

[0004] Diabetes is a common endocrine metabolic disease characterized by glucose metabolism disorder caused by insufficient insulin secretion or peripheral tissue insensitivity to insulin. Polysaccharides ingested by the human body are broken down by various enzymes and then enter the blood through the small intestine for cell metabolism. Therefore, inhibiting various enzymes in the process of sugar decomposition is one of the treatment ideas for diabetes. α-Glucosidase is a type of glycosidase that is mainly distributed on the brush edge of the chorionic villi of the small intestine and can degrade the α-1,4 glycosidic bonds of a series of oligosaccharides such as sucrose, maltose, and lactose. α-Glucosidase inhibitors can competitively inhibit glucosidase, thereby inhibiting the decomposition of polysaccharides and sucrose, inhibiting the absorption of carbohydrates in the small intestine, and slowing down the increase in postprandial blood sugar. Patients with type 2 diabetes often have postprandial hyperglycemia, which is prone to macrovascular and microvascular complications. Strict control of postprandial hyperglycemia can significantly reduce the occurrence and development of patients' vascular complications (coronary heart disease, atherosclerosis, diabetic nephropathy, etc.). Studies have shown that α-glucosidase inhibitors can improve postprandial blood glucose and glycosylated hemoglobin levels in diabetic animal models, improve glucose and lipid metabolism, and delay or prevent the occurrence and development of kidney, retinal, neuropathy, and ischemic myocardial infarction. Therefore, α-glucosidase inhibitors can not only reduce fasting and postprandial blood glucose, but also reduce the risk of macrovascular and microvascular lesions in patients, and reduce the morbidity and mortality of diabetic complications. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing diphenyl ether compounds in view of the technical defects existing in the prior art.

[0006] Another object of the present invention is to provide the use of marine fungus-derived phenyl ether compounds in reducing blood lipids and blood sugar.

[0007] The technical solution adopted to achieve the purpose of the present invention is:

[0008] A method for preparing a diphenyl ether compound, wherein the diphenyl ether compounds are

[0009] 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol, whose structural formula is shown in formula (I):

[0010]

[0011] The diphenyl ether compound is isolated from the fermentation culture of the strain Aspergillus versicolor.

[0012] In the above technical scheme, the preparation method comprises the following steps:

[0013] Step 1, preparing a rice fermentation product of the strain Aspergillus versicolor, and soaking it in ethyl acetate to obtain an extract of the fermentation product of the strain;

[0014] Step 2, using dichloromethane-methanol as the mobile phase, separating the extract by silica gel column chromatography, combining the fractions according to TLC analysis, and obtaining four components Fr.1 to Fr.4;

[0015] Component Fr.2 was purified by silica gel column chromatography, and dichloromethane-ethyl acetate was used as the mobile phase for gradient elution to obtain 8 components Fr.2.1 to Fr.2.8; component Fr.2.1 was purified by ODS column, and methanol-water was used for gradient elution to obtain 6 components Fr.2.1.1 to Fr.2.1.6; component Fr.2.1.3 was purified by silica gel column chromatography, and petroleum ether-ethyl acetate was used for isocratic elution to obtain compound

[0016] 3,7-dihydroxy-1,9-dimethyldibenzofuran;

[0017] The component Fr.3 was separated by silica gel column chromatography and gradient elution with dichloromethane-ethyl acetate to obtain three components Fr.3.1 to FR.3.3; Fr.3.2 was separated by ODS column and gradient elution with methanol-water to obtain four components (Fr.3.2.1 to FR.3.2.4); FR.3.2.1 was gradient eluted with petroleum ether-ethyl acetate to obtain the compound aspermutarubrol.

[0018] In the above technical solution, the rice fermentation product of the strain Aspergillus versicolor in step 1 of the preparation method is obtained by the following method:

[0019] The strain Aspergillus versicolor is inoculated on a PDA plate for culture, then the agar containing bacteria is cut off from the PDA plate and inoculated on MB seed culture solution for culture to obtain seed solution, and the seed solution is inoculated on a rice culture medium for culture to obtain a rice culture medium fermentation product;

[0020] The extract of the fermentation product of the strain in step 1 is obtained by the following method: crushing the fermentation product of the rice culture medium into pieces, soaking in ethyl acetate, recovering the ethyl acetate, and obtaining the extract of the fermentation product of the strain.

[0021] In the above technical scheme, the preparation method is to inoculate the strain Aspergillus versicolor on a PDA plate, place it in a constant temperature incubator at 25-30°C and culture it for 3-7 days; cut the bacteria-containing agar from the plate and inoculate it in MB seed culture solution, culture it at 25-30°C and 180-240rpm for 4 days to obtain seed solution; inoculate the seed solution into a rice culture medium, place it at 25-30°C, and culture it statically for 25-35 days.

[0022] In the above technical solution, the volume ratio of dichloromethane to methanol in the mobile phase in step 2 of the preparation method is 100:0 to 0:100.

[0023] In the above technical scheme, the volume ratio of dichloromethane-ethyl acetate in the mobile phase for eluting component Fr.2 in step 2 of the preparation method is 3:1-1:1.

[0024] In the above technical scheme, the volume ratio of methanol-water in the mobile phase for eluting component Fr.2.1 in step 2 of the preparation method is 40:60 to 100:0.

[0025] In the above technical scheme, in step 2 of the preparation method, the volume ratio of dichloromethane to ethyl acetate in the mobile phase for eluting component Fr.3 is 3:1 to 1:1, and when separating Fr.3.2 by ODS column, the volume ratio of methanol to water is 30:70 to 100:0.

[0026] Another aspect of the present invention is the use of diphenyl ether compounds in the preparation of lipid-lowering, blood sugar-lowering and / or anti-obesity drugs, health products, functional foods and / or food additives, characterized in that the diphenyl ether compounds are 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol, respectively, and their structural formulas are shown in formula (I):

[0027]

[0028] Another aspect of the present invention is a lipid-lowering, glucose-lowering and / or anti-obesity drug, comprising an active ingredient and a pharmaceutically acceptable excipient or carrier, wherein the active ingredient is any one of the diphenyl ether compounds 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol;

[0029] 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol, whose structural formula is shown in formula (I):

[0030]

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention discloses a method for preparing a compound of formula (1) from marine fungus Aspergillus versicolor, which can simply, effectively and quickly obtain a large amount of diphenyl ether compounds 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol.

[0033] 2. The present invention discloses that diphenyl ether compounds 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol can inhibit the accumulation of triglycerides and inhibit the activity of glucosidase during the differentiation of 3T3-L1 preadipocytes. The compounds can be used to prepare anti-obesity, lipid-lowering and glucose-lowering medicines and health products, functional foods, and food additives.

[0034] The Genbank accession number of the fungus Aspergillus versicolor GXIMD02004 of the invention is OP999340, and the strain is deposited in Guangxi University of Chinese Medicine with the deposit number GXIMD02004. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Compound 3,7-dihydroxy-1,9-dimethyldibenzofuran H NMR spectrum

[0036] Figure 2 Compound 3,7-dihydroxy-1,9-dimethyldibenzofuran C NMR spectrum

[0037] Figure 3 H NMR spectrum of compound aspermutarubrol

[0038] Figure 4 C NMR spectrum of compound aspermutarubrol

[0039] Figure 5 Compounds inhibit proliferation of 3T3-L1 preadipocytes

[0040] Figure 6 Dose-effect curve of compounds inhibiting 3T3-L1 preadipocyte differentiation

[0041] Figure 7Dose-effect curve of the compound 3,7-dihydroxy-1,9-dimethyldibenzofuran inhibiting α-glucosidase

[0042] Figure 8 Dose-effect curve of the compound aspermutarubrol inhibiting α-glucosidase DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] Embodiment 1 The preparation method of diphenyl ether compound comprises the following steps:

[0045] (1) Seed preparation and large-scale fermentation: The strain Aspergillus versicolor GXIMD02004 was taken out of a refrigerator at 4°C, inoculated on a PDA plate, and placed in a constant temperature incubator at 25°C for 5 days. The agar containing bacteria was cut from the plate and inoculated into a conical flask containing MB seed culture solution, and cultured at 28°C and 180rpm for 4 days to obtain seed solution. The seed solution was inoculated into rice culture medium and cultured at 25°C for 30 days. The fermentation product of the rice culture medium was crushed into pieces, immersed in ethyl acetate 5 times, each time for 4 days, and the ethyl acetate was recovered to obtain an extract of the fermentation product of the strain.

[0046] (2) Separation of diphenyl ether compounds: The extract was separated and extracted by silica gel column chromatography, with dichloromethane-methanol (100:0-0:100, V / V) as the mobile phase. The fractions were combined according to TLC analysis to obtain 4 components (Fr.1-Fr.4). Fr.2 was purified by silica gel column chromatography, with dichloromethane-ethyl acetate (3:1-1:1, V / V) as the mobile phase for gradient elution, to obtain 8 components (Fr.2.1-Fr.2.8). Fr.2.1 was purified by ODS column, with methanol-water (40:60-100:0, V / V) as the gradient elution, to obtain 6 components (Fr.2.1.1-Fr.2.1.6). Fr.2.1.3 was purified by silica gel column chromatography, with petroleum ether-ethyl acetate (5:1, V / V) as the isocratic elution, to obtain the compound 3,7-dihydroxy-1,9-dimethyldibenzofuran. Fr.3 was separated by silica gel column chromatography, and three components (Fr.3.1 to FR.3.3) were obtained by gradient elution with dichloromethane-ethyl acetate (3:1 to 1:1, V / V). Fr.3.2 was separated by ODS column, and four components (Fr.3.2.1 to FR.3.2.4) were obtained by gradient elution with methanol-water (30:70 to 100:0, V / V). FR.3.2.1 was obtained by gradient elution with petroleum ether-ethyl acetate (5:1, V / V) to obtain the compound aspermutarubrol.

[0047] The structural formulas of 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol are as shown in formula (1):

[0048]

[0049] Example 2 Experiment on the inhibition of proliferation of preadipocytes 3T3-L1 by compounds 1 and 2

[0050] Logarithmic phase preadipocytes 3T3-L1 cells were inoculated into 96-well culture plates at a density of about 3000 cells / well, cultured in high-glucose DMEM medium containing 10% calf serum, and cultured in a CO2 incubator at 37°C for 24 hours. After the cells adhered, DMEM complete culture medium containing different concentrations of compounds was replaced and cultured for a long time, so that the final concentrations of 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol were 50, 100, 200, 300, 400, and 500 μg / mL, respectively. The control group was a DMEM complete culture medium without compounds, and the blank group was a complete culture medium with an equal volume. Each group was set up with 3 parallel replicates. After 48 hours, the culture medium was discarded, 100 μL of the prepared MTT reagent was added, and the cells were placed in an incubator for incubation for 4 hours. The absorbance value (OD value) of each group was measured at a wavelength of 450 nm, and the survival rate was calculated. The results are as follows. Figure 5 As shown, 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol had no significant inhibitory effect on the proliferation of preadipocytes 3T3-L1 at concentrations below 200 μg / mL, and had a weaker inhibitory effect on the proliferation of preadipocytes 3T3-L1 at concentrations of 300-500 μg / mL.

[0051] Example 3 Experiment on the inhibitory activity of compounds 1 and 2 on 3T3-L1 preadipocyte differentiation

[0052] The test compound was prepared into a 2mg / mL stock solution with DMSO, and diluted into working solutions of different concentrations during the test; 0.1% DMSO was used as a blank control, and berberine was used as a positive control. Adipocytes 3T3-L1 in the logarithmic growth phase were inoculated in a 96-well plate. After the cells were completely fused, a certain concentration of the test compound was added to the induction differentiation medium I (DMEM medium supplemented with 10% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, 10μg / mL insulin, 1μM dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine and 10ng / mL biotin) and cultured for 48h, and no compound was added to the blank control. Then, a certain concentration of the test compound was added to induce differentiation medium II (DMEM complete medium containing 10μg / mL insulin) and cultured for 72h. The culture medium was removed, the cells were washed twice with PBS, and fixed with 10% formalin for 30min. The fixative was discarded, the cells were washed twice with PBS, and stained with Oil Red O for 15 min. The morphology and lipid droplets of adipocytes were observed under an inverted microscope, and the stained cells were treated with isopropanol and the OD value was measured at 490 nm.

[0053] The compounds 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol could inhibit the differentiation of 3T3-L1 cells to varying degrees at concentrations of 12.5-200 μg / mL and had a certain dose-effect relationship, with IC50 values ​​of 45.84 μg / mL and 42.59 μg / mL, respectively.

[0054] Example 4 Experiment on the inhibition of α-glucosidase activity by compounds 1 and 2

[0055] The compound was dissolved in 10% DMSO to prepare a 1mg / mL stock solution. The positive drug acarbose was dissolved in PBS buffer to prepare a 11mg / mL stock solution. The experiment was performed in a 96-well plate, and 20μL of α-glucosidase (0.2U / mL), 4-nitrobenzene-α-D-pyranoglucoside (2.5mM), and the test compound were added to each well. PBS buffer was used to replace α-glucosidase in the control wells, and PBS buffer was used to replace the test compound in the blank wells. Three replicate wells were set. Incubate at 37°C for 15min, add 80μL Na2CO3 (0.2M) to each well to terminate the reaction, and measure the OD value at 405nm.

[0056] Compounds 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol can inhibit α-glucosidase activity to varying degrees at concentrations of 10-333μg / mL and have a certain dose-effect relationship, with IC50 values ​​of 172.5μg / mL and 35.8μg / mL, respectively. The IC50 value of the positive drug acarbose is 12.1μg / mL. Compared with the positive drugs, compounds 3,7-dihydroxy-1,9-dimethyldibenzofuran and aspermutarubrol have a certain inhibitory effect on α-glucosidase activity.

[0057] 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. Use of diphenyl ether compounds in the preparation of lipid-lowering, blood sugar-lowering and / or anti-obesity drugs, characterized in that: The diphenyl ether compounds are 3,7-dihydroxy-1,9-dimethyldibenzofuran or aspermutarubrol, and their structural formulas are shown in formula (I):

2. The use according to claim 1, characterized in that The preparation method of 3,7-dihydroxy-1,9-dimethyldibenzofuran or aspermutarubrol comprises the following steps: Step 1, preparing a rice fermentation product of the strain Aspergillus versicolor, and soaking it in ethyl acetate to obtain an extract of the fermentation product of the strain; Step 2, using dichloromethane-methanol as the mobile phase, and using silica gel column chromatography to separate the extract to obtain four components Fr.1 to Fr.4; Component Fr.2 was purified by silica gel column chromatography, and dichloromethane-ethyl acetate was used as the mobile phase for gradient elution to obtain 8 components Fr.2.1 to Fr.2.8; component Fr.2.1 was purified by ODS column, and methanol-water was used for gradient elution to obtain 6 components Fr.2.1.1 to Fr.2.1.6; component Fr.2.1.3 was purified by silica gel column chromatography, and petroleum ether-ethyl acetate was used for isocratic elution to obtain compound 3,7-dihydroxy-1,9-dimethyldibenzofuran; The component Fr.3 was separated by silica gel column chromatography, and three components Fr.3.1 to FR.3.3 were obtained by gradient elution with dichloromethane-ethyl acetate. Fr.3.2 was separated by ODS column, and four components Fr.3.2.1 to FR.3.2.4 were obtained by gradient elution with methanol-water. FR.3.2.1 was eluted with petroleum ether-ethyl acetate to obtain the compound aspermutarubrol.

3. The use according to claim 2, characterized in that The rice fermentation product of the strain Aspergillus versicolor in step 1 is obtained by the following method: The strain Aspergillus versicolor is inoculated on a PDA plate for culture, then the agar containing bacteria is cut off from the PDA plate and inoculated on MB seed culture solution for culture to obtain seed solution, and the seed solution is inoculated on a rice culture medium for culture to obtain a rice culture medium fermentation product; The extract of the fermentation product of the strain in step 1 is obtained by the following method: crushing the fermentation product of the rice culture medium into pieces, soaking in ethyl acetate, recovering the ethyl acetate, and obtaining the extract of the fermentation product of the strain.

4. The use according to claim 3, characterized in that The strain Aspergillus versicolor is inoculated on a PDA plate, and placed in a constant temperature incubator at 25-30°C for 3-7 days; the bacteria-containing agar is cut from the plate and inoculated into MB seed culture solution, and the solution is cultured at 25-30°C and 180-240rpm for 4 days to obtain seed solution; the seed solution is inoculated into a rice culture medium, and the solution is placed at 25-30°C for static culture for 25-35 days.

5. The use according to claim 2, characterized in that The volume ratio of dichloromethane to methanol of the mobile phase in step 2 is 100:0 to 0:

100.

6. The use according to claim 2, characterized in that In step 2, the volume ratio of dichloromethane-ethyl acetate in the mobile phase for eluting component Fr.2 is 3:1-1:

1.

7. The use according to claim 2, characterized in that In step 2, the volume ratio of methanol to water in the mobile phase for eluting component Fr.2.1 is 40:60 to 100:

0.

8. The use according to claim 2, characterized in that In step 2, the volume ratio of dichloromethane to ethyl acetate in the mobile phase for eluting component Fr.3 is 3:1 to 1:1, and when separating Fr.3.2 by ODS column, the volume ratio of methanol to water is 30:70 to 100:0.

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