Naked hydroxyl group-containing urolithin A-8-O-galactoside and its synthesis method and application

Abstract

The invention discloses naked hydroxyl urolithin A 8 O galactoside and its synthesis method and application, and belongs to the field of organic synthesis technology. The synthesis method of the urolithin A 8 O galactoside comprises the following steps: 3 hydroxy 6 bromo benzaldehyde reacts with benzyl bromide, and the product is oxidized by hydrogen peroxide and then coupled with resorcinol to obtain naked hydroxyl urolithin A; galactose is successively replaced by acetic anhydride and hydrogen bromide to obtain brominated galactose pentaacetate; naked hydroxyl urolithin A undergoes oxygen glycosidation reaction with brominated galactose pentaacetate, and the product removes the hydroxy protecting group to obtain naked hydroxyl urolithin A 8 O galactoside. The present invention is beneficial in that: the problem that urolithin A is insoluble in water is solved, and the product has good hypoglycemic and hypolipidemic abilities; the process route is rationally designed, easy to operate, and the basic raw materials used are low in cost 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 derived from ellagitannin compounds after ingestion by the body and subsequent metabolism by various microorganisms in the intestines. The structure of UA is shown below:

[0003]

[0004] Numerous studies have demonstrated that urolithin A possesses promising biological activities, including anti-obesity, anti-cancer, anti-inflammatory, and hypoglycemic and lipid-lowering properties. As a natural product, urolithin A exhibits low toxicity, with studies demonstrating that adding 5% urolithin A to food does not harm tissues or organs. Urolithin A has received US Food Safety Certification, meaning it is legally permitted 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, while also providing new uses for the urolithin A compound.

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

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

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

[0010] (2) hydrogen peroxide oxidizing 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) Urolithin A with exposed hydroxyl groups undergoes an O-glycosylation reaction with bromogalactose pentaacetate to obtain Urolithin A-8-O-galactoside with unexposed hydroxyl groups;

[0015] (7) The hydroxyl protecting group of the urolithin A-8-O galactoside without exposed hydroxyl group is removed 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 completion of the reaction, 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 2h, 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 8h, and after completion of the reaction, 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 naked hydroxyl urolithin A is specifically as follows: 2-bromo-5-benzylbenzoic acid, resorcinol and sodium hydroxide are dissolved in water, heated under 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 under reflux for 0.5 hour, and after the reaction is completed, cooled to room temperature and filtered in sequence, 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 naked hydroxyl urolithin A.

[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, and reacting at 25°C for 2h. After the reaction is completed, adding dichloromethane and a large amount of saturated sodium bicarbonate aqueous solution in an amount equal to that of 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 and 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 the naked hydroxyl group urolithin A with bromogalactose pentaacetate to obtain the naked hydroxyl group urolithin A-8-O galactoside is as follows: dissolving tetrabutylammonium bromide in a mixed solvent of chloroform and water, adding the naked hydroxyl group urolithin A and potassium carbonate, dissolving the bromogalactose pentaacetate in dichloromethane, adding the dissolved bromogalactose pentaacetate to the dissolved naked hydroxyl group urolithin A, In the method, the molar ratio of tetrabutylammonium bromide, naked hydroxyl urolithin A, 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 unexposed hydroxyl urolithin A-8-O galactoside.

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

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

[0024] The use of the aforementioned 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 of urolithin A being 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 group urolithin A-8-O galactoside and finds that naked hydroxyl group urolithin A-8-O galactoside has good hypoglycemic and hypolipidemic abilities and can be used to prepare hypoglycemic and hypolipidemic drugs, which 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 groups;

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

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

[0034] The present invention will be 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. 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] 20 mmol of 3-hydroxy-6-bromo-benzaldehyde (Compound 1) and 30 mmol of potassium carbonate (1.5 equivalents) were dissolved in 25 mL of N,N-dimethylformamide (DMF), and then 21 mmol of benzyl bromide (1.05 equivalents) was added. The reaction was carried out at 80°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, 50 mL of water was added, and the mixture was filtered. The filter cake was dissolved in 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 4.4 g (15 mmol) 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. The mixture was stirred at 25°C for 2 h, and then 19 mmol of sodium chlorite (1.4 equivalents) was added. 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, filtered, and the filter cake was dried 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] 9.8 mmol of compound 3, 29.4 mmol of resorcinol (3.0 equivalents) and 39.2 mmol of sodium hydroxide (4.0 equivalents) were dissolved in 30 mL of water and heated under reflux for 1 h. Then, 29.4 mmol of copper sulfate (3.0 equivalents) was added and the heating and reflux were continued for 0.5 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filter residue was dissolved with ethyl acetate and 300 mL of 1 M hydrochloric acid was added and stirred for 2 h. After standing and stratification, the organic phase was collected and concentrated under vacuum to obtain urolithin A (compound 4) with exposed hydroxyl groups as a light brown solid powder with a yield of 2.9 g (8.7 mmol) 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. The reaction was carried out at 25°C for 2 h. After the reaction was completed, an equal volume of dichloromethane (DCM) and a large amount of saturated sodium bicarbonate aqueous solution were added to the reaction solution. 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. The crude product was recrystallized using a DCM / ethyl acetate system (the volume ratio of DCM to ethyl acetate was 1:15). The crystals were filtered and dried to obtain galactose pentaacetic acid (compound 6) as 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. 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) as 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 glycosidation 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 equivalents) 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. The organic phase was collected, dried over anhydrous sodium sulfate, and dry-loaded on silica gel. The sample was separated on a 300-400 mesh silica gel column (V DCM :V EA=100:1~30:1), the column fraction was collected, and recrystallized using DCM and PE (the volume ratio of DCM and PE was 1:15). The crystals were filtered and dried 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). 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 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 filtrate, and the acetyl group was removed at 25°C for 2 h. After the reaction was completed, the crude product was filtered with a small amount of methanol as the eluent 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 Sephadex LH20 packing and methanol as the eluent. The eluate was collected and dried to obtain the naked hydroxyl group of urolithin A-8-O-galactoside (compound 9) in 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 exposed hydroxyl group.

[0054] The total yield of naked hydroxyl group-urolithin A-8-O-galactoside was calculated to be 8.0%.

[0055] 2. Detection of the 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, and the dissolution of naked hydroxyl-exposed urolithin A-8-O-galactoside (Compound 9) was observed.

[0057] It was observed that when the concentration of naked hydroxyl group urolithin A-8-O galactoside (Compound 9) was 5 mg / mL or below, no obvious precipitation occurred in the aqueous solution.

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

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

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

[0061] (1) Determine the absorbance of the sample group: take 50 μL of appropriate concentration of urolithin A solution, naked hydroxyl urolithin A-8-O galactoside solution and quercetin (positive control) solution, and mix them with 150 μL of 7 mM ABTS· + The solution was mixed evenly on a 96-well ELISA plate, reacted at 25°C in the dark for 6 minutes, 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 naked hydroxyl group urolithin A-8-O galactoside solution and the quercetin solution were kept in the dark at 25°C for 6 min. The absorbance of each solution at 734 nm was measured. j ;

[0063] (3) Determine the absorbance value of the control group: replace the sample solution with the sample solvent as the control group, take 50 μL of the sample solvent and mix it with 150 μL of ABTS with a concentration of 7 mM. + The solution was mixed evenly on a 96-well ELISA plate and kept in the dark at 25°C for 6 min. The absorbance 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, naked hydroxyl urolithin A-8-O galactoside solution and quercetin solution, respectively, and mixing them with 150 μL of sample solvent. The mixture was incubated at 25 °C in the 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 rate:

[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 cleaning capacity 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 clearance ability is; on the contrary, IC 50 The larger the value, the greater the ABTS + The weaker the cleaning ability.

[0069] 2. α-glucosidase inhibition ability

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

[0071] (1) Determination of the absorbance of the sample group: 50 μL of appropriate concentration of urolithin A solution, naked hydroxyl urolithin A-8-O galactoside solution and acarbose (positive control) solution were mixed with 50 μL of 1 μM α-glucosidase solution on a 96-well microtiter plate, and reacted at 25°C for 10 min. Then, 50 μL of 0.5 mM 4-nitrophenyl-α-D-pyranoside solution was added and reacted at 25°C for 30 min. 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 minutes, add 50 μL of 0.5 mM 4-nitrophenyl-α-D-pyranoglucoside solution, react at 25°C for 30 minutes, 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. The plates were 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 plates were 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, naked hydroxyl urolithin A-8-O galactoside solution, and acarbose solution were mixed with 50 μL of 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 measured. 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 based on 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 values ​​(the concentration of the sample when the α-glucosidase inhibition rate reaches 50%) are 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 the absorbance of the sample group: 50 μL of appropriate concentrations of urolithin A solution, naked hydroxyl urolithin A-8-O galactoside solution, and orlistat (positive control) solution were mixed with 50 μL of 0.5 g / L lipase solution on a 96-well ELISA plate, and reacted at 25°C for 10 min. Then, 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 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 control group without lipase solution, take 50 μL of urolithin A solution, naked hydroxyl urolithin A-8-O galactoside solution 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 measure the absorbance of the reaction solution at an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a fluorescence spectrometer. j ;

[0083] (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 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 the plates were 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 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 mixed with 50 μL Tris-HCl buffer on a 96-well ELISA plate, and 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 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) The pancreatic lipase inhibition ability of each sample was determined based on the pancreatic lipase inhibition rate of each sample at different concentrations: the pancreatic lipase inhibition ability of each sample was expressed as IC 50 The values ​​(the concentration of the sample when the pancreatic lipase inhibition rate reaches 50%) are 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 (added with urolithin A or urolithin A-8-O-galactoside with exposed hydroxyl groups) and the positive control group (added with metformin): HepG2 cells were cultured in complete 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 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 naked hydroxyl urolithin A-8-O galactoside solution (31 μM, 63 μM, 125 μM) 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 cells were cultured for 24 h. Finally, the absorbance of each reaction solution at 450 nm was measured. s ;

[0091] (2) Determination of the absorbance 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 cultured in complete medium at 37°C and 5% carbon dioxide until 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 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. c ;

[0092] (3) Determination of the absorbance 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 of each reaction solution at 450 nm was measured. b ;

[0093] (4) Calculate cell viability 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 placed on an ELISA plate at a volume of 100 μL / well and divided into four groups: normal group, model group, experimental group, and positive control group, and cultured for 12 h;

[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 no serum was added to the normal group. The cells were cultured for 36 h, and the supernatant of each group was removed again. The HepG2 cells were washed repeatedly with phosphate buffer for 2-3 times;

[0100] (3) The experimental group was added with 10 μL of urolithin A solution of different concentrations (31 μM, 63 μM, 125 μM) or urolithin A-8-O-galactoside solution with exposed hydroxyl groups (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. 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 a blank group. Then, each group was added with 100 μL of DMEM culture medium containing 1% (w / v) antibiotics and 10% (v / v) PBS. The 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 measure 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 of each positive control +The results of the determination of 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 tests

[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, and the ABTS· + There was no significant difference in clearance ability.

[0109] The results of cell viability and glucose consumption of urolithin A, naked hydroxyl group 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 naked 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 naked 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 naked 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 naked 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 naked hydroxyl group-exposed urolithin A-8-O-galactoside synthesized by the method provided by the present invention has good hypoglycemic and hypolipidemic abilities.

[0113] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A method for synthesizing naked hydroxyl group urolithin A-8-O galactoside, characterized in that: The following steps are involved: (1) 3-Hydroxy-6-bromo-benzaldehyde reacts with benzyl bromide to produce 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 pentaacetate; (5) Hydrogen bromide replaces the anomeric group of galactose pentaacetate to obtain brominated galactose pentaacetate; (6) The exposed hydroxyl group of urolithin A undergoes an O-glycosylation reaction with bromogalactose pentaacetate to obtain the unexposed hydroxyl group of urolithin A-8-O-galactoside; (7) removing the hydroxyl protecting group of the urolithin A-8-O-galactoside without exposed hydroxyl groups to obtain the urolithin A-8-O-galactoside with exposed hydroxyl groups; The structures of naked hydroxyl group urolithin A, brominated galactose pentaacetate, unnaked hydroxyl group urolithin A-8-O-galactoside, and naked hydroxyl group urolithin A-8-O-galactoside are shown below: ； ； ； 。 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. The specific method is: 3-Hydroxy-6-bromo-benzaldehyde and potassium carbonate were dissolved in N,N-dimethylformamide, and benzyl bromide was added. The molar ratio of 3-hydroxy-6-bromo-benzaldehyde, potassium carbonate and benzyl bromide was 1:1.5:1.

05. The mixture was reacted at 80°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature and filtered with water, the filter cake was dissolved in ethyl acetate, filtered, 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 for oxidizing 2-bromo-5-benzylbenzaldehyde to obtain 2-bromo-5-benzylbenzoic acid is specifically as follows: 2-bromo-5-benzylbenzaldehyde was dissolved in acetonitrile, sodium dihydrogen phosphate and hydrogen peroxide were added, and the mixture was stirred at 25°C for 2 hours. Sodium chlorite was added, and the molar ratio of 2-bromo-5-benzylbenzaldehyde, hydrogen peroxide and sodium chlorite was 1:2:1.

4. The mixture was reacted at 25°C for 8 hours. After the reaction was completed, the reaction solution was acidified with concentrated hydrochloric acid, filtered, and the filter cake was dried to obtain 2-bromo-5-benzylbenzoic acid.

4. The method according to claim 1, wherein 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 were dissolved in water, heated under reflux for 1 hour, copper sulfate was added, the molar ratio of 2-bromo-5-benzylbenzoic acid, resorcinol, sodium hydroxide and copper sulfate was 1:3:4:3, and the mixture was heated under reflux for 0.5 hour. After the reaction was completed, the mixture was cooled to room temperature and filtered, the residue was dissolved in ethyl acetate, hydrochloric acid was added and stirred, the mixture was allowed to stand and separate, the organic phase was collected, and vacuum concentrated and dried to obtain urolithin A with exposed hydroxyl groups.

5. The method according to claim 1, wherein In step (4), the method for replacing the hydroxyl group of galactose with acetic anhydride to obtain galactose pentaacetate is specifically as follows: Galactose is dissolved in acetic anhydride in a molar ratio of galactose to acetic anhydride of 1:10, H2SO4 is added dropwise, and the reaction is carried out at 25°C for 2 hours. After the reaction is completed, dichloromethane with an equal volume to the acetic anhydride and a large amount of saturated sodium bicarbonate aqueous solution are added to the reaction solution, and the mixture is stirred continuously until no more bubbles are generated. The organic phase is collected and dried over anhydrous sodium sulfate, concentrated, recrystallized, filtered, and the crystals are dried to obtain galactose pentaacetate.

6. The method according to claim 1, characterized in that In step (5), the method for replacing the anomeric group of galactose pentaacetate with hydrogen bromide to obtain brominated galactose pentaacetate is specifically as follows: Galactose pentaacetate was dissolved in dichloromethane, and hydrogen bromide was added, with the molar ratio of galactose pentaacetate to hydrogen bromide being 1:

10. The mixture was reacted at 25°C for 2 hours. After the reaction was completed, a cold saturated aqueous sodium bicarbonate solution was added, and the organic phase was collected and dried over anhydrous sodium sulfate, concentrated, recrystallized, filtered, and the crystals were dried 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 naked hydroxyl urolithin A and potassium carbonate are added. Bromogalactose pentaacetate is dissolved in dichloromethane, and the dissolved bromogalactose pentaacetate is added to the dissolved naked hydroxyl urolithin A. The molar ratio of tetrabutylammonium bromide, naked hydroxyl urolithin A, 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. The organic phase is collected, dried over anhydrous sodium sulfate, dry-loaded on silica gel, the column portion is collected, recrystallized, filtered, and the crystals are dried to obtain unexposed hydroxyl urolithin A-8-O galactoside.

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

1. The reaction was carried out at 25°C for 2 hours to remove the acetyl group. After the reaction was completed, the urolithin A-8-O galactoside with exposed hydroxyl groups was filtered, the crude product was dissolved, the product was separated by column, the eluate was collected and dried to obtain the urolithin A-8-O galactoside with exposed hydroxyl groups.

9. A urolithin A-8-O-galactoside with exposed hydroxyl groups, characterized in that: The structure is as follows: 。 10. Use of the naked hydroxyl group urolithin A-8-O-galactoside according to claim 9 in the preparation of hypoglycemic or hypolipidemic drugs.

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