Hydroxyl-exposed urolithin A-8-O galactoside as well as synthesis method and application thereof

Abstract

The invention discloses hydroxyl-exposed urolithin A-8-O galactoside as well as a synthesis method and application thereof, and belongs to the technical field of organic synthesis. The synthesis method of urolithin A-8-O galactoside comprises the following steps: reacting 3-hydroxy-6-bromo-benzaldehyde with benzyl bromide, oxidizing the product by hydrogen peroxide, and coupling the oxidized product with resorcinol to obtain hydroxyl-exposed urolithin A; galactose is subjected to acetic anhydride substitution and hydrogen bromide substitution in sequence to obtain brominated galactose pentaacetate; and carrying out an oxygen glycosylation reaction on the urolithin A with the exposed hydroxyl and bromo-galactose pentaacetate, and removing a hydroxyl protecting group from a product to obtain the urolithin A-8-O galactoside with the exposed hydroxyl. The preparation method has the beneficial effects that the problem that the urolithin A is insoluble in water is solved, and the product has good blood sugar and blood fat reducing capacity; the process route is reasonable in design, convenient to operate, low in cost of used basic raw materials and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to a compound and a synthesis method and application thereof, in particular to urolithin A-8-O galactoside and a synthesis method thereof and application thereof in the preparation of hypoglycemic and hypolipidemic drugs, belonging to the technical field of organic synthesis. Background Art

[0002] Urolithin A (UA) is a 3,8-dihydroxy-dibenzo-6-one compound obtained by the body's ingestion of ellagitannin compounds and the removal of hydroxyl groups through metabolism by different types of microorganisms in the intestine. The structure of Urolithin A is shown below:

[0003]

[0004] A large number of studies have shown that urolithin A has good biological activity, such as anti-obesity, anti-cancer, anti-inflammatory, hypoglycemic, and hypolipidemic effects. As a natural product, urolithin A has low biological toxicity. Studies have shown that adding 5% urolithin A to food will not cause harm to tissues and organs. Urolithin A has passed the US food safety certification, which means that urolithin A can be legally used as a food additive in the United States.

[0005] Although urolithin A has good biological activity, its content in some organisms is extremely low and it is insoluble in water (because it contains a large conjugated structure), which greatly limits its application in medicine. Summary of the invention

[0006] To address the deficiencies of the prior art, the present invention aims to modify urolithin A, provide a water-soluble urolithin A compound, and a method for synthesizing the urolithin A compound, and also provide a new use of the urolithin A compound.

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

[0008] A method for synthesizing urolithin A-8-O-galactoside with exposed hydroxyl groups comprises the following steps:

[0009] (1) 3-Hydroxy-6-bromo-benzaldehyde undergoes a substitution reaction with benzyl bromide to obtain 2-bromo-5-benzylbenzaldehyde;

[0010] (2) hydrogen peroxide oxidizes 2-bromo-5-benzylbenzaldehyde to obtain 2-bromo-5-benzylbenzoic acid;

[0011] (3) 2-bromo-5-benzylbenzoic acid is coupled with resorcinol to obtain urolithin A with exposed hydroxyl groups;

[0012] (4) Acetic anhydride replaces the hydroxyl group of galactose to obtain galactose pentaacetic acid;

[0013] (5) replacing the anomeric group of galactose pentaacetate with hydrogen bromide to obtain bromogalactose pentaacetate;

[0014] (6) the exposed hydroxyl group of urolithin A undergoes an oxygen glycosylation reaction with bromogalactose pentaacetate to obtain urolithin A-8-O-galactoside without exposed hydroxyl group;

[0015] (7) The urolithin A-8-O-galactoside without exposed hydroxyl group is deprotected to obtain the urolithin A-8-O-galactoside with exposed hydroxyl group.

[0016] Preferably, in step (1), 3-hydroxy-6-bromo-benzaldehyde and benzyl bromide undergo a substitution reaction to obtain 2-bromo-5-benzylbenzaldehyde, which specifically comprises: dissolving 3-hydroxy-6-bromo-benzaldehyde and potassium carbonate in N,N-dimethylformamide, adding benzyl bromide, 3-hydroxy-6-bromo-benzaldehyde, potassium carbonate (1.5 equivalents) and benzyl bromide in a molar ratio of 1:1.5:1.05, reacting at 80°C for 4h, cooling to room temperature after the reaction is completed, and sequentially adding water for suction filtration, dissolving the filter cake with ethyl acetate, suction filtration, and concentrating and drying the filtrate to obtain 2-bromo-5-benzylbenzaldehyde.

[0017] Preferably, in step (2), the method for oxidizing 2-bromo-5-benzylbenzaldehyde with hydrogen peroxide to obtain 2-bromo-5-benzylbenzoic acid is specifically as follows: dissolving 2-bromo-5-benzylbenzaldehyde in acetonitrile, adding sodium dihydrogen phosphate and hydrogen peroxide, stirring at 25° C. for 2 hours, adding sodium chlorite, the molar ratio of 2-bromo-5-benzylbenzaldehyde, hydrogen peroxide and sodium chlorite is 1:2:1.4, reacting at 25° C. for 8 hours, and after the reaction is completed, acidifying the reaction solution with concentrated hydrochloric acid, filtering with suction, and drying the filter cake to obtain 2-bromo-5-benzylbenzoic acid.

[0018] Preferably, in step (3), the method for coupling 2-bromo-5-benzylbenzoic acid with resorcinol to obtain urolithin A with exposed hydroxyl groups is as follows: 2-bromo-5-benzylbenzoic acid, resorcinol and sodium hydroxide are dissolved in water, heated to reflux for 1 hour, copper sulfate is added, the molar ratio of 2-bromo-5-benzylbenzoic acid, resorcinol, sodium hydroxide and copper sulfate is 1:3:4:3, heated to reflux for 0.5 hour, and after the reaction is completed, it is cooled to room temperature and filtered, the filter residue is dissolved in ethyl acetate, hydrochloric acid is added and stirred, the stratification is allowed to stand, the organic phase is collected, and vacuum concentrated and dried to obtain urolithin A with exposed hydroxyl groups.

[0019] Preferably, in step (4), the method for replacing the hydroxyl group of galactose with acetic anhydride to obtain galactose pentaacetic acid is specifically as follows: dissolving galactose in acetic anhydride, the molar ratio of galactose to acetic anhydride being 1:10, adding H2SO4 dropwise, reacting at 25°C for 2h, and after the reaction is completed, adding dichloromethane and a large amount of saturated sodium bicarbonate aqueous solution in an equal volume to the acetic anhydride to the reaction solution, stirring continuously until no more bubbles are generated, collecting the organic phase, and sequentially drying with anhydrous sodium sulfate, concentrating, recrystallizing, filtering, and drying the crystals to obtain galactose pentaacetic acid.

[0020] Preferably, in step (5), the method for replacing the anomeric group of galactose pentaacetic acid with hydrogen bromide to obtain brominated galactose pentaacetate is specifically as follows: dissolving galactose pentaacetic acid in dichloromethane, adding hydrogen bromide, the molar ratio of galactose pentaacetic acid to hydrogen bromide is 1:10, reacting at 25°C for 2h, adding cold saturated sodium bicarbonate aqueous solution after the reaction is completed, collecting the organic phase, and sequentially drying over anhydrous sodium sulfate, concentrating, recrystallizing, filtering, and drying the crystals to obtain brominated galactose pentaacetate.

[0021] Preferably, in step (6), the method for reacting naked hydroxyl urolithin A with brominated galactose pentaacetate to obtain non-naked hydroxyl urolithin A-8-O-galactoside is as follows: dissolving tetrabutylammonium bromide in a mixed solvent of chloroform and water, adding naked hydroxyl urolithin A and potassium carbonate, dissolving brominated galactose pentaacetate in dichloromethane, adding the dissolved brominated galactose pentaacetate to the dissolved naked hydroxyl urolithin A, In the method, the molar ratio of tetrabutylammonium bromide, urolithin A with exposed hydroxyl group, potassium carbonate and bromogalactose pentaacetate is 1:1:3:1, and the reaction is carried out at 40°C for 8 hours. After the reaction is completed, saturated sodium bicarbonate aqueous solution and dichloromethane are added to the reaction solution for extraction, and the organic phase is collected, and dried over anhydrous sodium sulfate, dry-loaded on silica gel, the column portion is collected, recrystallized, filtered, and the crystals are dried to obtain urolithin A-8-O-galactoside with unexposed hydroxyl group.

[0022] Preferably, in step (7), the method for removing the hydroxyl protecting group of urolithin A-8-O galactoside without exposed hydroxyl group to obtain urolithin A-8-O galactoside with exposed hydroxyl group is specifically as follows: dissolving urolithin A-8-O galactoside without exposed hydroxyl group in a mixed solvent of dichloromethane and methanol, adding a carbon-supported palladium metal catalyst, introducing hydrogen, reacting at 8 times atmospheric pressure and 40° C. for 40 h to remove the benzyl group, cooling the reaction solution to room temperature and filtering, adding potassium carbonate to the filtrate, the molar ratio of urolithin A-8-O galactoside without exposed hydroxyl group, carbon-supported palladium metal catalyst and potassium carbonate is 1:0.2:1, reacting at 25° C. for 2 h to remove the acetyl group, and after the reaction is completed, filtering, dissolving the crude product, separating by column, collecting the eluate, and drying are performed in sequence to obtain urolithin A-8-O galactoside with exposed hydroxyl group.

[0023] A urolithin A-8-O-galactoside with exposed hydroxyl group is synthesized by any of the above methods.

[0024] The use of the above-mentioned naked hydroxyl group urolithin A-8-O galactoside in the preparation of hypoglycemic and / or hypolipidemic drugs.

[0025] The present invention is beneficial in that:

[0026] (1) The present invention obtains urolithin A-8-O-galactoside with exposed hydroxyl groups by glycosylation modification of urolithin A, thereby solving the problem that urolithin A is insoluble in water and promoting the research on urolithin A compounds in the development of low-side effect drugs;

[0027] (2) The present invention studies the pharmacological activity of naked hydroxyl urolithin A-8-O galactoside and finds that naked hydroxyl urolithin A-8-O galactoside has good hypoglycemic and hypolipidemic abilities, can be used to prepare hypoglycemic and hypolipidemic drugs, and has positive significance for the prevention and treatment of diabetes;

[0028] (3) The method for synthesizing the naked hydroxyl group-containing urolithin A-8-O-galactoside provided by the present invention has a reasonable process route design, convenient operation, and uses low-cost basic raw materials, which is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the synthetic route of urolithin A-8-O-galactoside with exposed hydroxyl group;

[0030] Figure 2 This is the H-NMR spectrum of urolithin A-8-O-galactoside with exposed hydroxyl groups;

[0031] Figure 3 This is the C NMR spectrum of urolithin A-8-O-galactoside with exposed hydroxyl group;

[0032] Figure 4 This is the result of HepG2 cell survival test;

[0033] Figure 5 This is a graph showing the results of the test for promoting glucose absorption in HepG2 cells. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0035] 1. Modification of Urolithin A

[0036] In order to improve the water solubility of urolithin A, the present invention performs glycosylation modification on urolithin A, and the final compound obtained is urolithin A-8-O-galactoside with exposed hydroxyl groups, and its structure is shown below:

[0037]

[0038] The synthetic route of naked hydroxyl group urolithin A-8-O-galactoside is as follows Figure 1 As shown, the synthesis method is as follows:

[0039] Step 1: Substitution reaction of 3-hydroxy-6-bromo-benzaldehyde with benzyl bromide

[0040] 20mmol 3-hydroxy-6-bromo-benzaldehyde (Compound 1) and 30mmol potassium carbonate (1.5 equivalents) were dissolved in 25mL N, N-dimethylformamide (DMF), and then 21mmol benzyl bromide (1.05 equivalents) was added, and the reaction was carried out at 80°C for 4h. After the reaction was completed, it was cooled to room temperature, 50mL of water was added and filtered, the filter cake was dissolved with ethyl acetate, filtered, and the filtrate was concentrated and dried to obtain 2-bromo-5-benzylbenzaldehyde (Compound 2) as a light yellow solid with a yield of 4.4g (15mmol) and a molar yield of 75.0%.

[0041] Step 2: Hydrogen peroxide oxidizes compound 2

[0042] 13.6 mmol of compound 2 was dissolved in 92 mL of acetonitrile, and then 12 mmol of sodium dihydrogen phosphate and 27.2 mmol of hydrogen peroxide (2.0 equivalents) were added, and the mixture was stirred at 25°C for 2 h. Then 19 mmol of sodium chlorite (1.4 equivalents) was added, and the reaction was continued at 25°C for 8 h. After the reaction was completed, the reaction solution was acidified with 4 mL of concentrated hydrochloric acid, and the filter cake was dried after suction filtration to obtain 2-bromo-5-benzylbenzoic acid (compound 3) as a white powder with a yield of 3.2 g (10.4 mmol) and a molar yield of 76.5%.

[0043] Step 3: Compound 3 is coupled with resorcinol

[0044] 9.8mmol of compound 3, 29.4mmol of resorcinol (3.0 equivalents) and 39.2mmol of sodium hydroxide (4.0 equivalents) were dissolved in 30mL of water, heated to reflux for 1h, and then 29.4mmol of copper sulfate (3.0 equivalents) was added, and the heating and reflux were continued for 0.5h. After the reaction was completed, it was cooled to room temperature, filtered, and the filter residue was dissolved with ethyl acetate. 300mL of 1M hydrochloric acid was added and stirred for 2h. After standing and stratification, the organic phase was collected, concentrated and dried in vacuo to obtain urolithin A (compound 4) with exposed hydroxyl groups as a light brown solid powder with a yield of 2.9g (8.7mmol) and a molar yield of 88.7%.

[0045] Step 4: Replace the hydroxyl group of galactose (Compound 5) with acetic anhydride

[0046] 56 mmol of compound 5 was dissolved in 560 mmol of acetic anhydride (10.0 equivalents), and then 6 drops of H2SO4 were added dropwise, and the reaction was carried out at 25°C for 2 h. After the reaction was completed, dichloromethane (DCM) and a large amount of saturated sodium bicarbonate aqueous solution in an equal volume to the acetic anhydride were added to the reaction solution, and the mixture was stirred continuously until no more bubbles were generated. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was recrystallized using a DCM / ethyl acetate system (the volume ratio of DCM to ethyl acetate was 1:15), and the crystals were filtered and dried to obtain galactose pentaacetic acid (compound 6), a white fluffy solid with a yield of 20.3 g (52 mmol) and a molar yield of 92.9%.

[0047] Step 5: Substitution of the anomeric group of compound 6 with hydrogen bromide

[0048] 52 mmol of compound 6 was dissolved in 256 mL of dichloromethane (DCM), and then 520 mmol of hydrogen bromide (10.0 equivalents, dissolved in acetic acid, concentration of 6.5 M) was added, and the reaction was carried out at 25°C for 2 h. After the reaction was completed, a cold saturated aqueous sodium bicarbonate solution was added, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was then recrystallized using a DCM / petroleum ether (PE) system (the volume ratio of DCM and PE was 1:15). The crystals were filtered and dried to obtain brominated galactose pentaacetate (compound 7), a white fluffy solid, with a yield of 24.2 g (44 mmol) and a molar yield of 84.6%.

[0049] Step 6: Compound 4 and compound 7 undergo glycosylation reaction

[0050] 10 mmol of tetrabutylammonium bromide (TBAB, 1.0 equivalent) was dissolved in 40 mL of a mixed solvent of chloroform and water (the volume ratio of chloroform to water was 1:1), and then 10 mmol of compound 4 and 30 mmol of potassium carbonate (3.0 equivalent) were added. 10 mmol of compound 7 (1.0 equivalent) was dissolved in 100 mL of dichloromethane, and the dissolved compound 7 was added to the dissolved compound 4, and the reaction was carried out at 40° C. for 8 h. After the reaction was completed, a saturated aqueous sodium bicarbonate solution and DCM were added to the reaction solution for extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, and added with silica gel for dry loading, and separated on a 300-400 mesh silica gel column (V DCM :V EA=100:1~30:1), collect the part passing through the column, use DCM and PE to recrystallize (the volume ratio of DCM and PE is 1:15), filter the crystals and dry them to obtain urolithin A-8-O galactoside (compound 8) without exposed hydroxyl groups as a white flocculent solid with a yield of 1.3 g (2.7 mmol) and a molar yield of 27.2%.

[0051] Step 7: Removal of the hydroxyl protecting group from compound 8

[0052] 2.7 mmol of compound 8 was dissolved in 25 mL of a mixed solvent of dichloromethane and methanol (the volume ratio of dichloromethane to methanol was 1:4), and then 0.54 mmol of carbon-supported palladium metal catalyst (0.2 equivalent) was added, and hydrogen was introduced. The benzyl group was removed at 8 times the atmospheric pressure and 40°C for 40 h. The reaction solution was cooled to room temperature and filtered. 2.7 mmol of potassium carbonate (1.0 equivalent) was added to the obtained filtrate, and the acetyl group was removed at 25°C for 2 h. After the reaction was completed, a small amount of methanol was used as an eluent for suction filtration to obtain a crude product. The crude product was dissolved in a mixed solvent of dimethyl sulfoxide (DMSO) and water (the volume ratio of DMSO to water was 1:4), and then separated by column separation using a dextran gel LH20 filler and methanol as an eluent. The eluate was collected and dried to obtain urolithin A-8-O galactoside (compound 9) with exposed hydroxyl groups, with a yield of 0.8 g (2.0 mmol) and a molar yield of 73.3%.

[0053] The H NMR spectrum of compound 9 is shown in Figure 2 , NMR carbon spectrum see Figure 3 .Depend on Figure 2 and Figure 3 It can be determined that compound 9 is urolithin A-8-O-galactoside with naked hydroxyl group.

[0054] It was calculated that the total yield of urolithin A-8-O-galactoside with naked hydroxyl group was 8.0%.

[0055] 2. Detection of water solubility of urolithin A-8-O-galactoside with exposed hydroxyl groups

[0056] Different amounts of naked hydroxyl-exposed urolithin A-8-O-galactoside (Compound 9) were added to 1 mL of water to observe the dissolution of naked hydroxyl-exposed urolithin A-8-O-galactoside (Compound 9).

[0057] It was observed that when the concentration of urolithin A-8-O-galactoside (Compound 9) with exposed hydroxyl groups was 5 mg / mL or less, there was no obvious precipitation in the aqueous solution.

[0058] Conclusion: Urolithin A-8-O-galactoside (compound 9) with exposed hydroxyl group has good water solubility.

[0059] 3. Study on the pharmacological activity of naked hydroxyl group urolithin A-8-O-galactoside 1. ABTS + Clearing ability

[0060] ABTS + The scavenging capacity was determined by:

[0061] (1) Determine the absorbance of the sample group: Take 50 μL of urolithin A solution of appropriate concentration, urolithin A-8-O-galactoside solution with exposed hydroxyl group, and quercetin (positive control) solution, and mix them with 150 μL of ABTS· + The solution was mixed evenly on a 96-well ELISA plate, reacted at 25°C in the dark for 6 min, and the absorbance of the reaction solution at 734 nm was measured. s ;

[0062] (2) Determine the absorbance value of the control group: without adding ABTS + The solution was used as the control group. The urolithin A solution, the urolithin A-8-O-galactoside solution with exposed hydroxyl groups, and the quercetin solution were kept at 25°C in the dark for 6 min. The absorbance value A of each solution at 734 nm was measured. j ;

[0063] (3) Determine the absorbance value of the control group: Use the sample solvent instead of the sample solution as the control group, take 50 μL of the sample solvent and mix it with 150 μL of 7 mM ABTS· + The solution was mixed evenly on a 96-well ELISA plate and kept in dark at 25°C for 6 min. The absorbance value A of each solution at 734 nm was measured. c ;

[0064] (4) Determine the absorbance of the blank group: replace ABTS with the sample solvent. + The blank group was prepared by taking 50 μL of urolithin A solution, urolithin A-8-O-galactoside solution with exposed hydroxyl groups, and quercetin solution, respectively, and mixing them with 150 μL of sample solvent. The mixture was incubated at 25 °C in dark for 6 min, and the absorbance of each solution at 734 nm was measured. b ;

[0065] (5) Calculate the ABTS of each sample according to the following formula: + Clearance:

[0066]

[0067] (6) According to the ABTS concentration of each sample + The clearance rate was determined by ABTS· + Scavenging ability: ABTS of each sample + IC 50 Value (ABTS+ The concentration of the sample when the clearance rate reaches 50%) is expressed.

[0068] IC 50 The smaller the value, the greater the ABTS + The stronger the removal ability, the lower the IC 50 The larger the value, the higher the ABTS + The weaker the cleaning ability.

[0069] 2. α-Glucosidase Inhibition Ability

[0070] The α-glucosidase inhibition ability was determined as follows:

[0071] (1) Determination of the absorbance of the sample group: 50 μL of urolithin A solution of appropriate concentration, urolithin A-8-O-galactoside solution with exposed hydroxyl group, and acarbose (positive control) solution were mixed with 50 μL of 1 μM α-glucosidase solution on a 96-well ELISA plate, reacted at 25°C for 10 min, and then 50 μL of 0.5 mM 4-nitrophenyl-α-D-pyranoglucoside solution was added, reacted at 25°C for 30 min, and the absorbance of the reaction solution at 405 nm was measured. s ;

[0072] (2) Determination of the absorbance of the control group: Take the control group without adding α-glucosidase solution, take 50 μL of urolithin A solution, naked hydroxyl urolithin A-8-O-galactoside solution and acarbose solution, let it stand at 25°C for 10 min, add 50 μL of 0.5 mM 4-nitrophenyl-α-D-pyranoglucoside solution, react at 25°C for 30 min, and measure the absorbance of the reaction solution at 405 nm. j ;

[0073] (3) Determination of the absorbance of the control group: The sample solvent was used instead of the sample solution as the control group. 50 μL of the sample solvent was mixed with 50 μL of 1 μM α-glucosidase solution on a 96-well ELISA plate, and the mixture was allowed to stand at 25°C for 10 min. 50 μL of 0.5 mM 4-nitrophenyl-α-D-pyranoglucoside solution was added and the mixture was reacted at 25°C for 30 min. The absorbance of the reaction solution at 405 nm was determined. c ;

[0074] (4) Determination of the absorbance of the blank group: The sample solvent was used instead of the α-glucosidase solution as the blank group. 50 μL of urolithin A solution, urolithin A-8-O-galactoside solution with exposed hydroxyl groups, and acarbose solution were mixed with 50 μL of the sample solvent, respectively. The mixture was allowed to stand at 25°C for 10 min. 50 μL of 0.5 mM 4-nitrophenyl-α-D-pyranoglucoside solution was added and reacted at 25°C for 30 min. The absorbance of the reaction solution at 405 nm was determined. b ;

[0075] (5) Calculate the α-glucosidase inhibition rate of each sample according to the following formula:

[0076]

[0077] (6) The α-glucosidase inhibition ability of each sample was determined according to the α-glucosidase inhibition rate of each sample at different concentrations: the α-glucosidase inhibition ability of each sample was expressed as IC 50 The value (the concentration of the sample when the α-glucosidase inhibition rate reaches 50%) is shown.

[0078] IC 50 The smaller the value, the stronger the α-glucosidase inhibition ability; conversely, the IC 50 The larger the value, the weaker the α-glucosidase inhibition ability.

[0079] 3. Pancreatic lipase inhibition ability

[0080] The pancreatic lipase inhibitory ability was determined as follows:

[0081] (1) Determination of absorbance of sample group: 50 μL of urolithin A solution of appropriate concentration, urolithin A-8-O-galactoside solution with exposed hydroxyl group, and orlistat (positive control) solution were mixed with 50 μL of 0.5 g / L lipase solution on a 96-well ELISA plate, reacted at 25°C for 10 min, then 50 μL of 0.1 mM 4-methylumbelliferyl oleate solution was added, reacted at 25°C for 30 min, and then 100 μL of 0.1 M citrate buffer solution (pH 4.2) was added to terminate the reaction. The absorbance of the reaction solution at an excitation wavelength of 355 nm and an emission wavelength of 460 nm was measured using a fluorescence spectrometer. s ;

[0082] (2) Determination of the absorbance of the control group: Take the solution without lipase as the control group, take 50 μL of urolithin A solution, urolithin A-8-O-galactoside solution with exposed hydroxyl group and orlistat solution, let it stand at 25°C for 10 min, add 50 μL of 0.1 mM 4-methylumbelliferyl oleate solution, react at 25°C for 30 min, then add 100 μL of 0.1 M citrate buffer solution (pH 4.2) to terminate the reaction, and use a fluorescence spectrometer to measure the absorbance of the reaction solution at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. j ;

[0083] (3) Determination of the absorbance value of the control group: The sample solvent was used instead of the sample solution as the control group. 50 μL of the sample solvent was taken and mixed with 50 μL of 0.5 g / L lipase solution on a 96-well ELISA plate. The plates were allowed to stand at 25°C for 10 min. 50 μL of 0.1 mM 4-methylumbelliferyl oleate solution was added and reacted at 25°C for 30 min. 100 μL of 0.1 M citrate buffer solution (pH 4.2) was added to terminate the reaction. The absorbance value A of the reaction solution at an excitation wavelength of 355 nm and an emission wavelength of 460 nm was measured using a fluorescence spectrometer. c ;

[0084] (4) Determination of the absorbance of the blank group: Tris-HCl buffer was used instead of lipase solution as the blank group. 50 μL of urolithin A solution, naked hydroxyl urolithin A-8-O-galactoside solution and orlistat solution were taken and mixed with 50 μL Tris-HCl buffer on a 96-well ELISA plate, respectively. The plates were allowed to stand at 25°C for 10 min, 50 μL of 0.1 mM 4-methylumbelliferyl oleate solution was added, and the reaction was allowed to proceed at 25°C for 30 min. 100 μL of 0.1 M citrate buffer solution (pH 4.2) was added to terminate the reaction. The absorbance of the reaction solution at an excitation wavelength of 355 nm and an emission wavelength of 460 nm was measured using a fluorescence spectrometer. b ;

[0085] (5) Calculate the pancreatic lipase inhibition rate of each sample according to formula (2);

[0086] (6) Determine the pancreatic lipase inhibition ability of each sample according to the pancreatic lipase inhibition rate of each sample at different concentrations: The pancreatic lipase inhibition ability of each sample is expressed as IC 50 The value (the concentration of the sample when the pancreatic lipase inhibition rate reaches 50%) is shown.

[0087] IC 50 The smaller the value, the stronger the pancreatic lipase inhibition ability; conversely, the IC 50 The larger the value, the weaker the pancreatic lipase inhibition ability.

[0088] 4. Cytotoxicity

[0089] The cytotoxicity was determined by:

[0090] (1) Determination of the absorbance of the experimental group (addition of urolithin A or urolithin A-8-O-galactoside with exposed hydroxyl groups) and the positive control group (addition of metformin): HepG2 cells were cultured in complete medium at 37°C and 5% carbon dioxide until the logarithmic growth phase, and then the density of the cell fluid was adjusted to 2×10 5 Cells / mL were added to the ELISA plate at a volume of 100 μL / well and cultured for 24 h. Then, 10 μL of different concentrations of urolithin A solution (31 μM, 63 μM, 125 μM) or urolithin A-8-O-galactoside solution (31 μM, 63 μM, 125 μM) with exposed hydroxyl groups was added to each well of the experimental group, and 10 μL of different concentrations of metformin solution (62 μM, 125 μM, 250 μM) was added to each well of the positive control group. The plates were cultured for 24 h, and the absorbance of each reaction solution at 450 nm was measured. s ;

[0091] (2) Determination of the absorbance value of the control group: The solvent used to dissolve the samples (urolithin A, naked hydroxyl urolithin A-8-O-galactoside, metformin) was used instead of the sample solution as the control group. Specifically, HepG2 cells were placed in complete culture medium at 37°C and 5% carbon dioxide to the logarithmic growth phase, and then the density of the cell solution was adjusted to 2×10 5 cells / mL, the cell solution was taken on the ELISA plate at a volume of 100 μL / well and cultured for 24 hours. Then, 10 μL of the solvent used to dissolve the sample was added to each well and cultured for 24 hours. Finally, the absorbance value A of each reaction solution at 450 nm was measured. c ;

[0092] (3) Determination of the absorbance value of the blank group: The complete culture medium was used instead of the cell solution as the blank group. Specifically, 100 μL / well of the complete culture medium was placed on the ELISA plate and cultured for 24 h. Then, 10 μL of the solvent used to dissolve the sample was added to each well and cultured for 24 h. Finally, the absorbance value A of each reaction solution at 450 nm was measured. b ;

[0093] (4) Calculate the cell survival rate according to the following formula:

[0094] Cell survival rate (%) = ((A s -A b ) / (A c -A b ))×100% (3)

[0095] The higher the cell survival rate, the lower the cytotoxicity; conversely, the lower the cell survival rate, the greater the cytotoxicity.

[0096] 5. Promote the ability of HepG2 cells to absorb glucose

[0097] The method for determining the ability of HepG2 cells to absorb glucose is as follows:

[0098] (1) HepG2 cells were cultured in complete culture medium at 37°C and 5% carbon dioxide until the logarithmic growth phase, and then the cell density was adjusted to 2×10 5 cells / mL, and the cell solution was taken on the ELISA plate at a volume of 100 μL / well and divided into 4 groups: normal group, model group, experimental group and positive control group, and cultured for 12 hours;

[0099] (2) The supernatant of each group was removed, and then 100 μL of complete medium containing 0.5 μM insulin and no serum was added to the model group, experimental group, and positive control group, and 100 μL of complete medium containing no insulin and serum was added to the normal group. The culture was continued for 36 h, and the supernatant of each group was removed again. The HepG2 cells were repeatedly washed 2-3 times with phosphate buffer;

[0100] (3) The experimental group was added with 10 μL of different concentrations of urolithin A solution (31 μM, 63 μM, 125 μM) or naked hydroxyl urolithin A-8-O-galactoside solution (31 μM, 63 μM, 125 μM), the positive control group was added with 10 μL of metformin solution (200 μM), the normal group and the model group were added with 10 μL of the solvent used to dissolve the sample, and at the same time, 10 μL of the solvent used to dissolve the sample was added to the blank well of the ELISA plate as the blank group, and then each group was added with 100 μL of DMEM culture medium containing 1% (w / v) antibiotics and 10% (v / v) PBS, and culture was continued for 24 h. The supernatant of the blank group, normal group, model group, experimental group and positive control group was taken to determine the glucose content (mmol);

[0101] (4) Calculate the glucose consumption of the normal group, model group, experimental group and positive control group according to the following formula:

[0102] Glucose consumption (mmol) = glucose content of blank group - glucose content of normal group, model group, experimental group or positive control group

[0103] The greater the glucose consumption, the stronger the ability of HepG2 cells to absorb glucose; conversely, the smaller the glucose consumption, the weaker the ability of HepG2 cells to absorb glucose.

[0104] Urolithin A, naked hydroxyl group urolithin A-8-O-galactoside and ABTS· +The results of the determination of the scavenging ability, α-glucosidase inhibition ability and pancreatic lipase inhibition ability are shown in Table 1.

[0105] Table 1 ABTS + Results of scavenging ability, α-glucosidase and pancreatic lipase inhibition ability assays

[0106]

[0107] Note: IC 50 The values ​​with different superscript letters (a, b, c) indicate significant differences among the samples.

[0108] As shown in Table 1, the naked hydroxyl group of urolithin A-8-O-galactoside has better α-glucosidase inhibition ability than the positive control, and higher pancreatic lipase inhibition ability than urolithin A. The ABTS· + There was no significant difference in the clearance ability.

[0109] The results of cell viability and glucose consumption of urolithin A, naked hydroxyl urolithin A-8-O-galactoside and metformin (positive control) are shown in Figure 4 and Figure 5 .

[0110] Depend on Figure 4 It can be seen that after treatment with the same concentration of exposed hydroxyl group urolithin A-8-O galactoside or urolithin A, the cell survival rate corresponding to the former is higher than the cell survival rate corresponding to the latter, that is, at the same concentration, the cytotoxicity of exposed hydroxyl group urolithin A-8-O galactoside is lower than that of urolithin A.

[0111] Depend on Figure 5 It can be seen that after treatment with the same concentration of exposed hydroxyl group urolithin A-8-O-galactoside or urolithin A, the glucose consumption corresponding to the former is higher than that corresponding to the latter. That is to say, at the same concentration, the ability of exposed hydroxyl group urolithin A-8-O-galactoside to promote HepG2 cells to absorb glucose is higher than that of urolithin A to promote HepG2 cells to absorb glucose.

[0112] Conclusion: The urolithin A-8-O-galactoside with exposed hydroxyl group synthesized by the method provided by the present invention has good blood sugar and blood lipid lowering ability.

[0113] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for synthesizing urolithin A-8-O-galactoside with exposed hydroxyl groups, characterized in that: The following steps are involved: (1) 3-Hydroxy-6-bromo-benzaldehyde undergoes a substitution reaction with benzyl bromide to obtain 2-bromo-5-benzylbenzaldehyde; (2) hydrogen peroxide oxidizes 2-bromo-5-benzylbenzaldehyde to obtain 2-bromo-5-benzylbenzoic acid; (3) 2-bromo-5-benzylbenzoic acid is coupled with resorcinol to obtain urolithin A with exposed hydroxyl groups; (4) Acetic anhydride replaces the hydroxyl group of galactose to obtain galactose pentaacetic acid; (5) replacing the anomeric group of galactose pentaacetate with hydrogen bromide to obtain bromogalactose pentaacetate; (6) the exposed hydroxyl group of urolithin A undergoes an oxygen glycosylation reaction with bromogalactose pentaacetate to obtain urolithin A-8-O-galactoside without exposed hydroxyl group; (7) The urolithin A-8-O-galactoside without exposed hydroxyl group is deprotected to obtain the urolithin A-8-O-galactoside with exposed hydroxyl group.

2. The method according to claim 1, characterized in that In step (1), 3-hydroxy-6-bromo-benzaldehyde and benzyl bromide undergo a substitution reaction to obtain 2-bromo-5-benzylbenzaldehyde, specifically: 3-Hydroxy-6-bromo-benzaldehyde and potassium carbonate were dissolved in N, N-dimethylformamide, benzyl bromide was added, and the molar ratio of 3-hydroxy-6-bromo-benzaldehyde, potassium carbonate (1.5 equivalents) and benzyl bromide was 1:1.5:1.05, and the reaction was carried out at 80°C for 4 hours. After the reaction was completed, it was cooled to room temperature and filtered by adding water, the filter cake was dissolved in ethyl acetate, filtered by suction, and the filtrate was concentrated and dried to obtain 2-bromo-5-benzylbenzaldehyde.

3. The method according to claim 1, characterized in that In step (2), the method of oxidizing 2-bromo-5-benzylbenzaldehyde with hydrogen peroxide to obtain 2-bromo-5-benzylbenzoic acid is specifically as follows: Dissolve 2-bromo-5-benzylbenzaldehyde in acetonitrile, add sodium dihydrogen phosphate and hydrogen peroxide, stir at 25°C for 2h, add sodium chlorite, the molar ratio of 2-bromo-5-benzylbenzaldehyde, hydrogen peroxide and sodium chlorite is 1:2:1.4, react at 25°C for 8h, and after the reaction is completed, acidify the reaction solution with concentrated hydrochloric acid, filter with suction, and dry the filter cake to obtain 2-bromo-5-benzylbenzoic acid.

4. The method according to claim 1, characterized in that: In step (3), the method for coupling 2-bromo-5-benzylbenzoic acid with resorcinol to obtain urolithin A with exposed hydroxyl groups is specifically as follows: Dissolve 2-bromo-5-benzylbenzoic acid, resorcinol and sodium hydroxide in water, heat and reflux for 1 hour, add copper sulfate, the molar ratio of 2-bromo-5-benzylbenzoic acid, resorcinol, sodium hydroxide and copper sulfate is 1:3:4:3, heat and reflux for 0.5 hour, cool to room temperature after the reaction is completed, filter in sequence, dissolve the filter residue with ethyl acetate, add hydrochloric acid and stir, let stand to separate, collect the organic phase, concentrate and dry in vacuo to obtain urolithin A with exposed hydroxyl group.

5. The method according to claim 1, characterized in that In step (4), the method of replacing the hydroxyl group of galactose with acetic anhydride to obtain galactose pentaacetic acid is specifically as follows: Dissolve galactose in acetic anhydride in a molar ratio of galactose to acetic anhydride of 1:10, add H2SO4 dropwise, and react at 25°C for 2h. After the reaction is completed, add dichloromethane in an amount equal to that of acetic anhydride and a large amount of saturated sodium bicarbonate aqueous solution to the reaction solution, and stir continuously until no more bubbles are generated. Collect the organic phase, and sequentially dry it with anhydrous sodium sulfate, concentrate it, recrystallize it, filter it, and dry the crystals to obtain galactose pentaacetic acid.

6. The method according to claim 1, characterized in that In step (5), the method for replacing the anomeric group of galactose pentaacetic acid with hydrogen bromide to obtain brominated galactose pentaacetate is specifically as follows: Galactose pentaacetic acid was dissolved in dichloromethane, and hydrogen bromide was added, the molar ratio of galactose pentaacetic acid to hydrogen bromide was 1:10, and the reaction was carried out at 25°C for 2 hours. After the reaction was completed, a cold saturated sodium bicarbonate aqueous solution was added, and the organic phase was collected, and dried over anhydrous sodium sulfate, concentrated, recrystallized, filtered, and dried crystals were sequentially performed to obtain brominated galactose pentaacetate.

7. The method according to claim 1, characterized in that In step (6), the method of reacting the exposed hydroxyl group urolithin A with bromogalactose pentaacetate to obtain the unexposed hydroxyl group urolithin A-8-O-galactoside is as follows: Tetrabutylammonium bromide is dissolved in a mixed solvent of chloroform and water, and urolithin A with naked hydroxyl group and potassium carbonate are added. Bromogalactose pentaacetate is dissolved in dichloromethane, and the dissolved bromogalactose pentaacetate is added to the dissolved urolithin A with naked hydroxyl group, and the molar ratio of tetrabutylammonium bromide, urolithin A with naked hydroxyl group, potassium carbonate and bromogalactose pentaacetate is 1:1:3:

1. The reaction is carried out at 40°C for 8 hours. After the reaction is completed, saturated sodium bicarbonate aqueous solution and dichloromethane are added to the reaction solution for extraction, and the organic phase is collected, and dried over anhydrous sodium sulfate, silica gel dry loading, column-passing part is collected, recrystallized, filtered, and the crystals are dried to obtain urolithin A-8-O galactoside with unexposed hydroxyl group.

8. The method according to claim 1, characterized in that In step (7), the method for removing the hydroxyl protecting group of urolithin A-8-O-galactoside without exposed hydroxyl group to obtain urolithin A-8-O-galactoside with exposed hydroxyl group is specifically as follows: The urolithin A-8-O galactoside without exposed hydroxyl group is dissolved in a mixed solvent of dichloromethane and methanol, a carbon-supported palladium metal catalyst is added, hydrogen is introduced, and the benzyl group is removed by reacting at 8 times the atmospheric pressure and 40°C for 40 hours. The reaction solution is cooled to room temperature and filtered, and potassium carbonate is added to the filtrate. The molar ratio of the urolithin A-8-O galactoside without exposed hydroxyl group, the carbon-supported palladium metal catalyst and potassium carbonate is 1:0.2:

1. The acetyl group is removed by reacting at 25°C for 2 hours. After the reaction is completed, suction filtration, dissolution of the crude product, column separation, collection of the eluate, and drying are performed in sequence to obtain the urolithin A-8-O galactoside with exposed hydroxyl group.

9. A urolithin A-8-O-galactoside with exposed hydroxyl groups, characterized in that: The compound is synthesized by the method according to any one of claims 1 to 8.

10. Use of the urolithin A-8-O-galactoside with exposed hydroxyl groups according to claim 9 in the preparation of hypoglycemic and / or hypolipidemic drugs.

Images