An alpha-glucosidase inhibitor and its preparation method and application

Abstract

The application discloses a natural alpha-glucosidase inhibitor and a preparation method and application thereof, and the natural alpha-glucosidase inhibitor is 3,3'-dimethoxy ellagic acid extracted from a sapium sebiferum. The application is specifically implemented according to the following steps: preparing a sapium sebiferum extract; and separating and purifying the sapium sebiferum extract to obtain 3,3'-dimethoxy ellagic acid. The application obtains a novel natural alpha-glucosidase inhibitor 3,3'-dimethoxy ellagic acid, and the inhibition effect of the 3,3'-dimethoxy ellagic acid on alpha-glucosidase is obvious at a concentration of 4 mg / mL, so that the 3,3'-dimethoxy ellagic acid provides a lead compound for research and development of a novel natural anti-diabetic drug.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and drug preparation technology, and relates to a natural α-glucosidase inhibitor, as well as the preparation method and application of the above-mentioned inhibitor. Background Technology

[0002] Alpha-glucosidase is a digestive enzyme located on the brush border membrane of the small intestine. It catalyzes the final step in carbohydrate digestion, breaking down oligosaccharides into absorbable monosaccharides. Inhibiting the activity of this enzyme can delay carbohydrate absorption, thereby effectively reducing postprandial blood glucose levels. Therefore, alpha-glucosidase inhibitors have become one of the first-line drugs for the clinical treatment of type 2 diabetes, such as acarbose and voglibose. However, currently used alpha-glucosidase inhibitors often have side effects such as gastrointestinal discomfort, bloating, abdominal pain, and diarrhea. Some patients even experience adverse reactions such as abnormal liver function, limiting their long-term clinical application. Therefore, finding highly effective and low-toxicity alpha-glucosidase inhibitors from natural products has become a hot topic in drug research. *Triadica sebifera* (Linnaeus) Small is a woody plant belonging to the genus *Triadica* in the family Euphorbiaceae. The *Chinese Pharmacopoeia* records that it has diuretic, anti-edema, detoxifying, and insecticidal effects, and can be used to treat ascites due to liver cirrhosis, difficulty in urination and defecation, etc. However, the *National Compendium of Chinese Herbal Medicines* indicates that it has slight toxicity. As an important economic tree species, the white waxy coating on the seeds of the Chinese tallow tree can be used to make "skin oil," and the kernel oil is called "clear oil." As early as the *Qimin Yaoshu* and *Nongzheng Quanshu*, there were records of its cultivation, stating that "collecting seeds to extract oil is very beneficial to the people," highlighting its significant economic value before the industrial era. Based on this research background, this invention isolated a natural compound, 3,3'-dimethoxyellagic acid, with α-glucosidase inhibitory activity from the traditional Chinese medicine Chinese tallow tree. Its preparation method and biological activity were systematically studied, providing a scientific basis and lead compound for the development of novel natural anti-diabetic drugs. Summary of the Invention

[0003] The first objective of this invention is to provide a natural α-glucosidase inhibitor, which has strong α-glucosidase inhibitory activity.

[0004] A second objective of this invention is to provide a method for preparing the aforementioned inhibitor.

[0005] A third objective of this invention is to provide the use of the above-mentioned inhibitors in antidiabetic drugs.

[0006] The first technical solution adopted in this invention is a natural α-glucosidase inhibitor, wherein the inhibitor is 3,3'-dimethoxyellagic acid extracted from Sapium sebiferum.

[0007] The second technical solution adopted in this invention is a method for preparing a natural α-glucosidase inhibitor, which is specifically implemented according to the following steps: Step 1: Prepare the Chinese tallow tree extract; Step 2: The Sapium sebiferum extract obtained in Step 1 is separated and purified to obtain 3,3'-dimethoxyellagic acid.

[0008] The second technical solution adopted in this invention is further characterized by the following: The preparation process in step 1 is as follows: Step 1.1: After drying and pulverizing the whole herb of Chinese tallow tree in the shade, extract the total extract with 75% industrial ethanol; Step 1.2: Disperse the extract obtained in Step 1.1 thoroughly in water, and defatt it with 3 times its volume of petroleum ether to obtain the petroleum ether fraction; Step 1.3: Add 3 times the volume of ethyl acetate to the raffinate obtained in step 1.2 for extraction, and evaporate to dryness to obtain the ethyl acetate fraction.

[0009] Step 2 is as follows: Step 2.1: Dissolve the ethyl acetate extract obtained in Step 1.3, add 200-300 mesh silica gel and stir until well mixed. The mass ratio of ethyl acetate extract to silica gel is 1:2. After the solvent has evaporated and the sample is dry, grind the sample into powder using a mortar and pestle for later use. Step 2.2: Normal-phase silica gel column chromatography was performed with gradient elution using petroleum ether-ethyl acetate (10:1, 5:1, 3:1, 2:1, 1:1, v / v) followed by methanol (5%, 10%, 20%, 30%, v / v), with 6 L of solvent used for each gradient wash. Step 2.3: Dry the effluent from each concentration gradient in Step 2.2 using a rotary evaporator; Step 2.4: The results of each part in Step 2.3 were analyzed by TLC thin-layer plate spotting, UV lamp, and sulfuric acid ethanol color development. The separated components were combined and finally merged into 5 components, labeled as Fr.1: PE:EA = 10:1 and 5:1, 29.2 g; Fr.2: PE:EA = 3:1 and 2:1, 52.7 g; Fr.3: PE:EA = 1:1, 39.6 g; Fr.4: PE+EA:MeOH = 9:1, 42.9 g; Fr.5: PE+EA:MeOH = 8:2, 38.4 g. Step 2.5: Using silica gel as the stationary phase, Fr.3 was eluted with a gradient eluent and collected. After collection, the eluent was dried using a rotary evaporator. The collected components were then labeled and subjected to TLC. The TLC results were observed under UV light. The TLC was then developed with sulfuric acid ethanol or iodine reagent. Based on the ratio shift values ​​shown on the TLC plate, components with similar compounds were combined to obtain 14 sub-components: Fr.1-1, Fr.1-2, Fr.1-3, Fr.1-4, Fr.1-5, Fr.1-6, Fr.1-7, Fr.1-8, Fr.1-9, Fr.1-10, Fr.1-11, Fr.1-12, Fr.1-13, and Fr.1-14. Step 2.6: Fr.-10 was first separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient elution with a methanol-water system containing 0.1% formic acid as the eluent. The gradient elution was performed according to the following time program: 60:40 → 40:60 in 40 min, v / v, followed by 40:60 → 0:100 in 20 min, 4 mL / min. A fraction was collected at a retention time of 23.4 min. This fraction was further separated by reversed-phase amino column chromatography using a gradient elution with a methanol-water system containing 0.1% formic acid as the eluent. The gradient elution was performed according to the following time program: 70:30 → 55:45 in 40 min, v / v, followed by 55:45 → 0:100 in 20 min, 4 mL / min, yielding compound 1. Compound 1 was identified as 3,3'-dimethoxyellagic acid by NMR and mass spectrometry.

[0010] In step 2.2, the volume ratios of petroleum ether to ethyl acetate are 10:1, 5:1, 3:1, 2:1, and 1:1, respectively, and the volume ratios of methanol in the petroleum ether and ethyl acetate mixed solution are 9:1 and 8:2, respectively.

[0011] In step 2.3, the eluent is a mixed solution of PE-EtOAc and EtOAc-MeOH at different concentrations. The polarity of the mixed solution is increased by increasing the proportion of the more polar solvent by 20% for each gradient.

[0012] The third technical solution adopted in this invention is: the application of a natural α-glucosidase inhibitor in the preparation of antidiabetic drugs.

[0013] The third technical solution adopted in this invention is characterized in that the inhibitor exhibits significant α-glucosidase inhibitory activity in in vitro enzyme activity experiments.

[0014] The beneficial effects of this invention are: 3,3'-dimethoxyellagic acid, a natural compound with α-glucosidase inhibitory activity, was isolated from the traditional Chinese herbal medicine *Sapium sebiferum*, providing a new lead compound and scientific basis for the development of natural antidiabetic drugs.

[0015] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 This is the 1H NMR spectrum of 3,3'-dimethoxyellagic acid, a natural α-glucosidase inhibitor of the present invention. Figure 2 This is the carbon NMR spectrum of 3,3'-dimethoxyellagic acid, a natural α-glucosidase inhibitor of the present invention. Figure 3 This invention relates to the chemical structural formula of 3,3'-dimethoxyellagic acid, a natural α-glucosidase inhibitor. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0018] This embodiment provides a method for preparing a natural α-glucosidase inhibitor, which is implemented according to the following steps: Step 1: Plant Extraction Step 1.1: After drying and pulverizing the whole herb of Chinese tallow tree in the shade, extract the total extract with 75% industrial ethanol; Step 1.2: Disperse the extract thoroughly in water, and degrease with 3 times its volume of petroleum ether to obtain the petroleum ether fraction; Step 1.3: Add 3 times the volume of ethyl acetate to the extract phase from Step 1.2 for extraction, and evaporate to dryness to obtain the ethyl acetate fraction.

[0019] Step 2: Separation and purification Step 2.1: Add the ethyl acetate extract obtained in step 1.3 to solvents such as ethyl acetate, petroleum ether, and methanol to dissolve it. Mix it with dry silica gel (200-300 mesh) at a mass ratio of 1:1 and stir evenly so that the sample can be completely adsorbed by the silica gel. After the solvent has evaporated and the sample is dry, grind the sample into powder with a mortar and pestle for later use. Step 2.2: Select a suitable glass column (10 × 56 cm), first pack the dry silica gel into the glass column, then use a vacuum pump to remove air bubbles from the column, and load the sample with silica gel adsorbed onto the silica gel column using the dry loading method. Use a mixed solution of PE-EA and EA-MeOH with different polarities as the eluent to perform gradient elution on the silica gel column, with 6 L of solvent for each ratio. Step 2.3: Dry the effluent of each proportion using a rotary evaporator to obtain 7 components; Step 2.4: Based on the TLC thin-layer plate spotting, UV lamp, and sulfuric acid-ethanol color development, the separated components were combined and finally merged into 5 components, labeled as Fr.1 (PE:EA = 10:1 and 5:1, 29.2 g), Fr.2 (PE:EA = 3:1 and 2:1, 52.7 g), Fr.3 (PE:EA = 1:1, 39.6 g), Fr.4 (PE+EA:MeOH = 9:1, 42.9 g), and Fr.5 (PE+EA:MeOH = 8:2, 38.4 g). Step 2.5: Dissolve Fr.2 in various organic solvents such as methanol and ethyl acetate, then mix it with silica gel at a ratio of 2:1. Pack the column using a dry method, using silica gel as the stationary phase. After continuously pressurizing with a vacuum pump to remove air bubbles, load the sample using a dry method. Use mixed solutions of PE-EtOAc (100:0→0:100) and EtOAc-MeOH (100:0→0:100) at different concentrations as eluents. Increase the polarity of the mixed solution by 20% for each gradient using a more polar solvent (EtOAc or methanol). Prepare 4 L of each solution for gradient elution of the column. Collect the solutions washed down with eluents of different polarities, evaporate to dryness using a rotary evaporator, and collect them in 25 mL sample vials, clearly labeling each. Each component was spotted by TLC, and the results were observed under UV light. Then, sulfuric acid ethanol or iodine reagent was used for color development. Based on the ratio shift values ​​shown by the TLC plate, components with similar compounds were combined to obtain 14 sub-components (denoted as Fr.1-1 to Fr.1-16). Fr.1-1 (0.76 g), Fr.1-2 (0.25 g), Fr.1-3 (2.8 g), Fr.1-4 (0.98 g), Fr.1-5 (0.17 g), Fr.1-6 (0.19 g), Fr.1-7 (0.41 g), Fr.1-8 (2.66 g), Fr.1-9 (0.27g), Fr.1-10 (0.64 g), Fr.1-11 (2.72 g), Fr.1-12 (1.63 g), Fr.1-13 (1.18 g), Fr.1-14 (0.64 g); Step 2.6: Fr.1-10 (0.64 g) was first separated by gradient elution using reversed-phase high-performance liquid chromatography (RP-C18 column; 5 μm; 10 × 250 mm). A methanol-water system containing 0.1% formic acid was used as the eluent, and the gradient elution was performed according to the following time program: 60:40 → 40:60 in 40 min, v / v, followed by 40:60 → 0:100 in 20 min, 4 mL / min. A fraction was collected at a retention time of 23.4 min. This fraction was further separated by reversed-phase amino column chromatography (YMC-Pack NH2, S-5, 10 × 250 mm) using gradient elution: a methanol-water system containing 0.1% formic acid was used as the eluent, and the following time program was set: 70:30 → 55:45 in 40 min, v / v, followed by 55:45 → 0:100 in 20 min, elution at 4 mL / min, to obtain compound 1 (18 mg, tR = 18.09 min).

[0020] Step 3: Compound Structure Identification Step 3.1: Slightly yellow powder, and only 1 spot is visible on the TLC thin-layer plate; Step 3.2: Based on the 1H, 13C NMR (C2D6SO, 400 MHz) NMR spectrum data (see... Figure 1 and Figure 2 Based on mass spectrometry ([M−H]- m / z = 329.037) and comparison with literature, compound 1 was identified as 3,3'-dimethoxyellagic acid, with the chemical formula shown below. Figure 3 As shown.

[0021] Step 4: α-Glucosidase Inhibitor Activity Test Step 4.1: Accurately weigh 4.5606 g of KH₂PO₄ and dissolve it in deionized water, then dilute to 100 mL using a volumetric flask; weigh 7.6470 g of K₂HPO₄ and dissolve it in deionized water, then dilute to 100 mL using a volumetric flask. Mix the two solutions and dilute to 500 mL in a volumetric flask, adjusting the pH to 6.7. Step 4.2: Accurately weigh 1.00 mg of α-glucosidase (50 U / mg) powder, dissolve it in 50 mL of PBS, and obtain a 1 U / mL α-glucosidase solution. Step 4.3: Accurately weigh 15.06 mg of α-p-nitrophenol glucoside (PNPG) powder, dissolve it in 10 mL of PBS to obtain a 5 mM α-p-nitrophenol glucoside solution. Step 4.4: Weigh 0.54 g of Na2CO3 solid powder and dissolve it in 50 mL of distilled water to obtain a 0.2 M Na2CO3 solution; Step 4.5: Weigh 2.00 mg of 3,3'-dimethoxyellagic acid sample and quantify it to 4 mg / mL using DMSO; Step 4.6: Set up the test group: First, add 50 μL of PBS solution to a 96-well plate, then add 1 μL of 4 mg / mL sample and 20 μL of α-glucosidase solution, and incubate at 37°C for 15 min. After removing the 96-well plate, add 20 μL of PNPG to each well and incubate at 37°C for 20 min. After the reaction is complete, add 50 μL of sodium carbonate to stop the reaction, and then measure the absorbance (AS) of each well at 405 nm using a microplate reader.

[0022] Step 4.7: Set up a blank control group: Add 50 μL of PBS solution to each well of a 96-well plate, followed by 1 μL of 4 mg / mL sample and 20 μL of PBS. Incubate at 37°C for 15 min. After removing the plate, add 20 μL of PNPG to each well and incubate at 37°C for 20 min. After the reaction is complete, add 50 μL of sodium carbonate to stop the reaction and measure the absorbance (AS) of each well at 405 nm using a microplate reader.

[0023] Step 4.8: Setting up a negative control group: First, add 50 μL of PBS solution to each well of a 96-well plate, followed by 1 μL of sample solvent DMSO and 20 μL of α-glucosidase solution. Incubate at 37°C for 15 min. After removing the plate, add 20 μL of PNPG to each well and incubate at 37°C for 20 min. After the reaction is complete, add 50 μL of sodium carbonate to stop the reaction and measure the absorbance (AS) of each well at 405 nm using a microplate reader.

[0024] Table 1. Inhibitory activity of 3,3'-dimethoxyellagic acid against α-glucosidase Step 4.9: Calculate IC50. Table 1 shows the inhibition results of 3,3'-dimethoxyellagic acid on α-glucosidase. It can be seen that 3,3'-dimethoxyellagic acid has a strong inhibitory effect on α-glucosidase at 4 mg / mL. Through the above method, this invention provides a flavonoid compound with significant inhibitory activity against α-glucosidase, which can provide a potential lead compound for the development of novel natural antidiabetic drugs.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A natural α-glucosidase inhibitor, characterized in that, The inhibitor is 3,3'-dimethoxyellagic acid extracted from Chinese tallow tree.

2. A method for preparing a natural α-glucosidase inhibitor as described in claim 1, characterized in that, The specific steps are as follows: Step 1: Prepare the Chinese tallow tree extract; Step 2: The Sapium sebiferum extract obtained in Step 1 is separated and purified to obtain 3,3'-dimethoxyellagic acid.

3. The method for preparing a natural α-glucosidase inhibitor according to claim 2, characterized in that, The preparation process in step 1 is as follows: Step 1.1: After drying and pulverizing the Chinese tallow tree, extract the total extract with 75% industrial ethanol; Step 1.2: Disperse the extract obtained in Step 1.1 thoroughly in water, and defatt it with 3 times its volume of petroleum ether to obtain the petroleum ether fraction; Step 1.3: Add 3 times the volume of ethyl acetate to the raffinate obtained in step 1.2 for extraction, and evaporate to dryness to obtain the ethyl acetate fraction.

4. A method for preparing an α-glucosidase inhibitor according to claim 3, characterized in that, Step 2 specifically involves: Step 2.1: Dissolve the ethyl acetate extract obtained in Step 1.3, add 200-300 mesh silica gel and stir until well mixed. The mass ratio of ethyl acetate extract to silica gel is 1:

1. After the solvent has evaporated and the sample is dry, grind the sample into powder using a mortar and pestle for later use. Step 2.2: Different concentration gradients of petroleum ether-ethyl acetate and ethyl acetate-methanol were added sequentially to the chromatographic column for gradient elution, with 6 L of solvent added for each concentration. Step 2.3: Dry the effluent from each concentration gradient in Step 2.2 using a rotary evaporator; Step 2.4: The results of each part in Step 2.3 were analyzed by TLC thin-layer plate spotting, UV lamp, and sulfuric acid ethanol color development. The separated components were combined and finally merged into 5 components, labeled as Fr.1, Fr.2, Fr.3, Fr.4, and Fr.

5. Step 2.5: Using silica gel as the stationary phase, Fr.2 was eluted and collected using a gradient eluent. After collection, the eluent was dried using a rotary evaporator. The collected components were then labeled and subjected to TLC. The results were observed under UV light. The TLC was then developed using sulfuric acid ethanol or iodine reagent. Based on the ratio shift values ​​displayed on the TLC plate, the components with similar compounds were combined to obtain 14 sub-components, which were designated as Fr.1-1 to Fr.1-14. Step 2.6: Fr.1-10 was first separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient elution system with a methanol-water system containing 0.1% formic acid as the eluent. The gradient elution was performed according to the following time program: 60:40→40:60 in 40 min, v / v, followed by 40:60→0:100 in 20 min, 4 mL / min. A fraction was collected at a retention time of 23.4 min. This fraction was further separated by reversed-phase amino column chromatography using a gradient elution system with a methanol-water system containing 0.1% formic acid as the eluent. The gradient elution was performed according to the following time program: 70:30→55:45 in 40 min, v / v, followed by 55:45→0:100 in 20 min, 4 mL / min, yielding 3,3'-dimethoxyellagic acid.

5. The method for preparing a natural α-glucosidase inhibitor according to claim 4, characterized in that, In step 2.2, the volume ratios of petroleum ether to ethyl acetate are 10:1, 5:1, 3:1, 2:1, and 1:1, respectively, and the volume ratios of methanol in the mixed solution of petroleum ether and ethyl acetate are 9:1 and 8:2, respectively.

6. The method for preparing a natural α-glucosidase inhibitor according to claim 4, characterized in that, In step 2.3, the eluent is a mixed solution of PE-EtOAc and EtOAc-MeOH at different concentrations, and the polarity of the mixed solution is increased by increasing the proportion of the more polar solvent by 20% for each gradient.

7. The use of a natural α-glucosidase inhibitor according to any one of claims 1 to 6 in an antidiabetic drug.

8. The application according to claim 7, characterized in that, 3,3'-Dimethoxyellagic acid exhibits significant inhibitory effects on α-glucosidase at a concentration of 4 mg / mL.

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