Polygonatum sibiricum extract and application thereof in preparation of medicine for treating diabetes
By using cooked tannin n-butanol extract as an α-glucosidase inhibitor, the problem of poor inhibition of α-glucosidase in the prior art was solved, and a stronger α-glucosidase inhibition effect was achieved, effectively reducing postprandial blood sugar.
Patent Information
- Application Number
- CN202510405169.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art has the problem of poor inhibition of α-glucosidase activity in the treatment or prevention of diabetes, and it is difficult to effectively reduce postprandial blood sugar.
By using cooked polypoxen extract, especially cooked polypoxen n-butanol extract, as an α-glucosidase inhibitor, the activity of α-glucosidase is inhibited, thereby reducing the absorption of carbohydrates in the small intestine.
Cooked Polygonatum n-butanol extract has a stronger inhibitory effect of α-glucosidase, which significantly improves the effect of inhibiting α-glucosidase activity compared with other extracts such as aqueous extract, ethyl acetate extract and residual aqueous extract.
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Abstract
Description
Technical Field
[0001] The present invention relates to a polygonatum sibiricum extract and its application in the preparation of drugs for treating diabetes, and particularly relates to that the polygonatum sibiricum extract can inhibit the activity of α-glucosidase, belonging to the field of medical technology. Background Art
[0002] The pathogenesis of diabetes includes insufficient insulin secretion (insulin secretion defect) and reduced sensitivity of insulin target sites in vivo tissues and organs (insulin action defect), resulting in elevated blood sugar, and accompanied by diseases such as dyslipidemia. α-Glucosidase inhibitors can inhibit the absorption of carbohydrates in the small intestine and can reduce postprandial blood sugar. α-Glucosidase inhibitors are mainly used for patients with postprandial blood sugar elevation and diabetes with carbohydrates as the main food. The commercially available α-glucosidase inhibitors include acarbose (Glucobay) and voglibose (Basen). α-Glycosidase inhibitors can also be used in combination with hypoglycemic drugs with other mechanisms of action to enhance the hypoglycemic effect, such as insulin, biguanides, sulfonylureas, and thiazolidinediones (TZDs). Taking α-glucosidase inhibitors alone before meals can also effectively prevent pre-meal hypoglycemia.
[0003] Polygonatum sibiricum is the dried rhizome of Polygonatum sibiricum, Polygonatum kingianum and Polygonatum cyrtonema of the genus Polygonatum in the Liliaceae family, and has the effects of nourishing yin and moistening the lungs, tonifying the spleen and replenishing qi, and nourishing the kidney and filling essence. The chemical components of polygonatum sibiricum include polysaccharides, saponins, flavonoids, anthraquinone compounds, etc., among which polygonatum polysaccharides and saponins are important active components of polygonatum sibiricum. Summary of the Invention
[0004] The invention purpose of the present invention is to provide a processed polygonatum sibiricum extract and its application in the preparation of drugs for treating or preventing diabetes in view of the problems existing in the prior art in the treatment or prevention of diabetes.
[0005] The purpose of the present invention is achieved by the following technical solutions:
[0006] In the first aspect, the present invention provides a polygonatum sibiricum extract for the preparation of drugs for treating diabetes, and the polygonatum sibiricum extract is an aqueous extract of processed polygonatum sibiricum or a n-butanol extract of the aqueous extract of processed polygonatum sibiricum.
[0007] Further, the aqueous extract of processed polygonatum sibiricum is prepared by extracting processed polygonatum sibiricum powder with 8 - 12 times of water for 2 - 3 times and concentrating to 0.48 g / mL, calculated by the raw drug amount of processed polygonatum sibiricum.
[0008] Further, the n-butanol extract of processed polygonatum sibiricum is an extract obtained by extracting the aqueous extract of processed polygonatum sibiricum with n-butanol and then concentrating.
[0009] Further, the polysaccharide percentage content of the processed polygonatum sibiricum is 15% - 25%.
[0010] Second aspect, the present invention provides an application of polygonatum extract in the preparation of drugs for treating diabetes.
[0011] Furthermore, the polygonatum extract is an α-glucosidase inhibitor.
[0012] α-Glucosidase is a class of enzymes that can catalyze the hydrolysis of α-glucosyl from the non-reducing end of substrates containing α-glucosidic bonds, hydrolyze glucosidic bonds to generate glucose, including α-amylase located in the intestinal lumen, maltase, α-dextrinase, and sucrase on the brush border of the upper small intestine, etc., and is an essential class of enzymes in the biological sugar metabolism pathway. α-Glucosidase inhibitors can inhibit the absorption of carbohydrates in the small intestine and reduce postprandial blood glucose. α-Glucosidase inhibitors are mainly used for patients with diabetes whose blood glucose rises after meals and whose staple food is carbohydrates.
[0013] Term definitions and explanations
[0014] Unless otherwise specified, all technical terms and scientific terms used herein have the standard meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a certain term, the definition in this article shall prevail.
[0015] The beneficial effects of the present invention are as follows:
[0016] 1. The n-butanol extract of processed polygonatum in the present invention has a good effect of inhibiting α-glucosidase activity, and its effect of inhibiting α-glucosidase activity is better than that of the water extract of polygonatum, ethyl acetate extract, and the remaining water extract. The n-butanol extract of processed polygonatum is obtained by the methods of water extraction and n-butanol extraction of processed polygonatum.
[0017] 2. The water extract of processed polygonatum has an α-glucosidase inhibitory effect.
[0018] 3. The n-butanol extract of processed polygonatum has a stronger α-glucosidase inhibitory effect. Description of the drawings
[0019] Figure 1 It is a diagram of the extraction method of polygonatum extract.
[0020] Figure 2 It is a diagram of the test results of the α-glucosidase inhibitory rate of different extraction parts of processed polygonatum. 1. Ethyl acetate extract of processed polygonatum 2. N-butanol extract of processed polygonatum 3. Remaining water extract of processed polygonatum 4. Water extract of processed polygonatum 5. Acarbose positive drug
[0021] Figure 3 It is a diagram of the test results of the α-glucosidase inhibitory rate of different extraction parts of raw polygonatum. 1. Ethyl acetate extract of raw polygonatum 2. N-butanol extract of raw polygonatum 3. Remaining water extract of raw polygonatum 4. Water extract of raw polygonatum 5. Acarbose positive drug Detailed implementation manners
[0022] To enable those skilled in the art to better understand the present invention and be able to implement it, the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the scope of the described embodiments. Unless otherwise specified, the test methods used in the embodiments are all conventional methods; the experimental animals, materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.
[0023] Example 1: Extraction method of processed polygonatum sibiricum extract and in vitro α-glucosidase inhibitory effect experiment
[0024] Extraction method of processed polygonatum sibiricum extract and preparation of other solutions used in the experiment
[0025] Preparation of total water extract of processed polygonatum sibiricum: Take 10 g of processed polygonatum sibiricum powder in a 500 mL beaker, add 100 mL of distilled water, heat to boiling, boil for 15 min, keep warm for 5 min, repeat three times, filter while it is hot, make the obtained filtrate up to 250 mL, and place it in a 4 °C refrigerator for later use.
[0026] Sample solution: Take 60 mL of the total water extract of processed polygonatum sibiricum in a 250 mL separating funnel, add 60 mL of ethyl acetate, shake, place the separating funnel on an iron ring and let it stand still to separate layers. Drain the lower ethyl acetate layer from the lower end of the separating funnel, pour out the upper aqueous layer from the upper mouth of the separating funnel, save the lower ethyl acetate layer, pour the aqueous layer poured out from the upper mouth back into the separating funnel, add 60 mL of ethyl acetate again, and repeat the operation for a total of three extractions to obtain a total of 180 mL of ethyl acetate extract. Pour the 60 mL water layer from the previous step back into the separating funnel and extract with n-butanol three times, 60 mL each time. Transfer the remaining aqueous solution, ethyl acetate extract, and n-butanol extract after extraction to 250 mL round-bottom flasks for later use.
[0027] Concentrate the water extract of processed polygonatum sibiricum, ethyl acetate extract, n-butanol extract, and remaining aqueous solution of processed polygonatum sibiricum to dryness using a rotary evaporator. See the appendix for the solution preparation method. Figure 1 .
[0028] Transfer the ethyl acetate extract of processed polygonatum sibiricum, n-butanol extract of processed polygonatum sibiricum, remaining aqueous solution after extraction of processed polygonatum sibiricum, and water extract of processed polygonatum sibiricum placed in the round-bottom flask after evaporation to dryness to 5 mL each with 5 mL of PBS phosphate buffer solution at pH 7.0. A total of four sample solutions are obtained, namely: water extract of processed polygonatum sibiricum, ethyl acetate extract of processed polygonatum sibiricum, n-butanol extract of processed polygonatum sibiricum, and remaining water extract of processed polygonatum sibiricum. Calculated according to the raw drug amount of processed polygonatum sibiricum, the concentration of the solution is 0.48 g / mL.
[0029] The preparation method of the raw polygonatum sibiricum extract is the same as that of the processed polygonatum sibiricum, obtaining the water extract of raw polygonatum sibiricum, the ethyl acetate extract of raw polygonatum sibiricum, the n-butanol extract of raw polygonatum sibiricum, and the remaining water extract of raw polygonatum sibiricum. Calculated according to the amount of raw polygonatum sibiricum, the concentration of the solution obtained is 0.48 g / mL.
[0030] Preparation of α-glucosidase solution: It is prepared using PBS phosphate buffer at pH 7.0, and the concentration of the prepared α-glucosidase solution is 125 U / mL. In the experiment, the α-glucosidase solution is diluted 20 times with PBS phosphate buffered saline at pH 7.0, and the diluted α-glucosidase concentration is 6.25 U / mL. The prepared α-glucosidase solution is diluted 20 times for standby.
[0031] Acarbose positive drug: Take out acarbose from refrigeration at 4°C, take 0.02 g of acarbose powder, pour it into a 1.5 mL EP tube, and dissolve it with 1 mL of PBS phosphate buffered saline at pH 7.0 to obtain an acarbose solution with a concentration of 20 g / L.
[0032] p-Nitrophenyl-α-D-glucopyranoside: Take out the pNPG sample from freezing at -20°C, weigh 0.9 mg of pNPG, and dissolve it with 1 mL of PBS phosphate buffered saline at pH 7.0 to obtain a pNPG solution with a concentration of 0.9 g / L, which is prepared and used immediately.
[0033] Preparation of the terminating solution Na2CO3 solution: Take 2 CO 3 1.06 g in a 200 mL small beaker, dissolve it with 100 mL of distilled water, and prepare and use it immediately.
[0034] In vitro α-glucosidase inhibitory effect experiment
[0035] Sample addition: Respectively pipette 10 μL of the sample solution into a 96-well plate, add 20 μL of PBS phosphate buffer at pH 7.0, and then add 20 μL of the α-glucosidase solution to fully mix the sample and the enzyme. There are two replicates for each sample.
[0036] Control well: Add 10 μL of the sample solution, 60 μL of PBS phosphate buffer, and 150 μL of Na2CO3 solution to the 96-well plate.
[0037] Incubation: Place the 96-well plate in a water-jacketed constant temperature incubator at 37°C for 15 min, take it out and quickly place it in a laminar flow hood.
[0038] Substrate addition: Add 20 μL of the pNPG solution to the 96-well plate. Each time after adding, pipette and blow 2 - 3 times to fully mix the substrate and the enzyme. React at a constant temperature of 37°C in a water-jacketed constant temperature incubator for 10 min, take it out and quickly place it in a laminar flow hood.
[0039] Add the termination solution: Use a 1 mL pipette tip on a 100 - 1000 μL pipette to aspirate 150 μL of Na2CO3 solution and add it to each well. Let it stand at room temperature for 5 min to rapidly terminate the reaction.
[0040] Detect the absorbance: Use a multi - functional microplate reader to detect the absorbance at 405 nm.
[0041] The calculation and graphing of experimental data are performed using Graphpad 8.0.2 software. Formula: Inhibition rate = (A 空白 - A 样品 ) / A 空白 ×100% (A 空白 is the absorbance of the system without adding the sample, and A 样品 is the absorbance of the system with the added sample).
[0042] The experimental results are shown in Appendix Figure 2 and Appendix Figure 3 .
[0043] The experimental results show that the average inhibition rate of the n - butanol extract of processed Polygonatum sibiricum is 54.35%, the average inhibition rate of the water extract of processed Polygonatum sibiricum is 33.08%. The average inhibition rate of the ethyl acetate extract of processed Polygonatum sibiricum is 7.07%, and the average inhibition rate of the remaining water extract of processed Polygonatum sibiricum is 16.15%. The average inhibition rate of the n - butanol extract of raw Polygonatum sibiricum is 0.64%, the average inhibition rate of the ethyl acetate extract of raw Polygonatum sibiricum is 3.49%, the average inhibition rate of the remaining water extract of raw Polygonatum sibiricum is 3.48%, and the average inhibition rate of the water extract of raw Polygonatum sibiricum is 7.36%. The water extract of processed Polygonatum sibiricum has an α - glucosidase inhibitory effect, and the n - butanol extract of processed Polygonatum sibiricum has a stronger α - glucosidase inhibitory effect.
[0044] Example 2: Determination of the content of Polygonatum polysaccharide
[0045] Drawing of the glucose standard curve
[0046] Preparation of the glucose standard solution: Weigh 0.033 g of anhydrous glucose precisely, transfer it to a 100 mL volumetric flask, and make up the volume with distilled water. Take 7 10 mL graduated stoppered test tubes, transfer the liquid in the 100 mL volumetric flask to the graduated stoppered test tubes according to the volumes of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL, and mark the test tubes. Add 2 mL of distilled water to each graduated stoppered test tube.
[0047] Preparation of the anthrone - sulfuric acid solution: Weigh 0.2 g of anthrone precisely and dissolve it in 100 mL of concentrated sulfuric acid to prepare a 0.2% anthrone - sulfuric acid solution.
[0048] Anthrone-sulfuric acid color reaction: Place a stoppered graduated test tube in an ice-water bath. Slowly add anthrone-sulfuric acid solution drop by drop to the stoppered graduated test tube using a dropper until the 10 mL mark is reached. After cooling for 10 min, take it out and let it cool to room temperature. Transfer the stoppered graduated test tube to a boiling water bath and keep it boiling for 10 min. Take it out and, when the temperature drops slightly, transfer the stoppered graduated test tube to an ice-water bath and cool for 10 min, then take it out.
[0049] Absorbance measurement of glucose standard solution: After turning on the UV spectrophotometer and preheating for 30 min, scan the baseline with the blank group. Then, take 0.1 mL of the sample in the stoppered graduated test tube for spectral scanning. The wavelength at the maximum absorbance in the range of 500 nm - 700 nm is taken as the maximum absorption wavelength (measured as 577 nm). Then, measure the absorbance of each concentration. At the maximum absorption wavelength (577 nm), zero the instrument with the blank group. Set each sample to be detected three times and take the average value. Make a scatter plot of concentration-absorbance for the obtained samples, and the standard curve is: y = 0.0996x + 0.0029 (R 2 = 0.994)
[0050] Determination of polysaccharide content in samples
[0051] Treatment of samples: Take 0.25 g of the finely powdered sample dried to constant weight and place it in a round-bottom flask. Add 150 mL of 80% ethanol and reflux in a water bath for 1 h (82 °C). Filter while it is hot and wash three times with 10 mL of hot 80% ethanol each time. Transfer the residue and filter paper to the flask, add 150 mL of distilled water, reflux in a 92 °C water bath for 1 h, filter while it is hot, wash three times with 10 mL of hot water each time. Combine all the filtrates and washings, and make up the volume to 500 mL with distilled water (extract raw and processed Polygonatum sibiricum Redoute once each).
[0052] Dilution of sample solutions: Dilute the extracted processed Polygonatum sibiricum Redoute extract and raw Polygonatum sibiricum Redoute extract by 5 times and 10 times respectively.
[0053] Sample anthrone-sulfuric acid color reaction: Using a 2 mL pipette, transfer 2 mL from the 10-fold dilution of raw polygonatum rhizome to a 10 mL graduated stoppered test tube, and transfer 2 mL from the 5-fold dilution of raw polygonatum rhizome to a 10 mL graduated stoppered test tube; Using a 2 mL pipette, transfer 2 mL from the 10-fold dilution of processed polygonatum rhizome to a 10 mL graduated stoppered test tube, and transfer 2 mL from the 5-fold dilution of processed polygonatum rhizome to a 10 mL graduated stoppered test tube. Also, take a 10 mL graduated stoppered test tube and transfer 2 mL of distilled water with a 2 mL pipette as the blank control group, and label each graduated stoppered test tube. Place the graduated stoppered test tubes in an ice-water bath and slowly add the anthrone-sulfuric acid solution dropwise to the graduated stoppered test tubes until the 10 mL mark is reached. After cooling for 10 min, take them out and cool to room temperature. Transfer the graduated stoppered test tubes to a boiling water bath and keep boiling for 10 min. Take them out and wait for the temperature to drop slightly, then transfer the graduated stoppered test tubes to an ice-water bath to cool for 10 min and take them out.
[0054] Absorbance measurement: After turning on the ultraviolet spectrophotometer and preheating for 30 min, set the wavelength to 577 nm and zero with the blank control sample. Then, measure the absorbance values of each sample in ascending order of concentration and substitute them into the glucose standard curve. The polysaccharide content of raw polygonatum rhizome can be calculated to be 6.95% and that of processed polygonatum rhizome is 16.00%. Using the same method, measure the polysaccharide content in the water extract, ethyl acetate extract, n-butanol extract, and remaining water extract of processed polygonatum rhizome in Example 1. The results are as follows: the polysaccharide concentration of the n-butanol extract of processed polygonatum rhizome is 0.0000 mg / mL, the polysaccharide concentration of the ethyl acetate extract of processed polygonatum rhizome is 20.2008 mg / mL, the polysaccharide concentration of the remaining water extract of processed polygonatum rhizome is 62.0281 mg / mL, and the total polysaccharide concentration of the water extract of processed polygonatum rhizome is 42.0884 mg / mL.
Claims
1. A polygonatum extract, characterized in that The polygonatum sibiricum extract is the water extract of cooked polygonatum sibiricum or the n-butanol extract of the water extract of cooked polygonatum sibiricum.
2. The polygonatum sibiricum extract according to claim 1, characterized in that The cooked polygonatum water extract is prepared by extracting cooked polygonatum powder with 8-12 times water for 2-3 times, and concentrating it to prepare 0.48g / mL, calculated based on the raw drug amount of cooked polygonatum.
3. The polygonatum sibiricum extract according to claim 2, characterized in that The cooked polygonatum sibiricum n-butanol extract is an extract obtained by extracting the cooked polygonatum sibiricum water extract with n-butanol and then concentrating it.
4. The polygonatum sibiricum extract according to claim 1, characterized in that The polysaccharide content of the cooked polygonatum is 15%-25%.
5. Use of the polygonatum sibiricum extract according to claim 1 in preparing a drug for treating diabetes.
6. The use according to claim 5, characterized in that: The polygonatum sibiricum extract is an alpha-glucosidase inhibitor.