Application of 2-linolenyl-rac-glycerol in alpha-glucosidase inhibition or blood sugar reduction

Abstract

According to the specific embodiment of the invention, the application of the 2-linolenyl-rac-glycerol in preparation of the alpha-glucosidase inhibitor or the hypoglycemic product is provided, and the 2-linolenyl-rac-glycerol has an alpha-glucosidase inhibiting effect, can be effectively used as the alpha-glucosidase inhibitor and plays a role in the hypoglycemic effect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to application of 2-linolenoyl-rac-glycerol in alpha-glucosidase inhibition or blood sugar reduction. Background Art

[0002] α-glucosidase is an important glycoside hydrolase that is widely present in organisms and is responsible for hydrolyzing the non-reducing ends of polysaccharides to produce α-D-glucose. It mainly acts on the digestion and absorption of carbohydrates in the human body, especially in the intestines. α-glucosidase promotes the conversion of carbohydrates into glucose, thereby affecting postprandial blood sugar levels.

[0003] α-glucosidase inhibitors (such as acarbose and voglibose) inhibit the activity of α-glucosidase in the small intestinal mucosa, delaying the absorption of carbohydrates, thereby reducing postprandial blood sugar. These drugs are suitable for diabetic patients with improper blood sugar control, especially those with type 2 diabetes. Not only that, long-term use of α-glucosidase inhibitors can reduce glycated hemoglobin (HbA1c) levels and reduce the risk of diabetic complications. In addition, studies have also shown that α-glucosidase inhibitors have multiple functions such as reducing insulin resistance, protecting cardiovascular health, and improving intestinal health.

[0004] At present, α-glucosidase inhibitors such as acarbose, voglibose and miglitol are clinical drugs for diabetic patients, which are mainly obtained by artificial synthesis through microbial fermentation. These drugs have obvious side effects such as gastrointestinal bloating, diarrhea, abdominal discomfort, etc. Therefore, it is of great significance to further develop α-glucosidase inhibitors, especially the development of safe and effective natural α-glucosidase inhibitors from natural plant resources. Summary of the invention

[0005] The inventors of the present invention found in the process of studying the extract of the Herba Lycoris Radiatae that the Herba Lycoris Radiatae extract and the component 2-linolenoyl-rac-glycerol in the Herba Lycoris Radiatae extract have a significant inhibitory effect on α-glucosidase. Based on this, a specific embodiment of the present invention provides an application of 2-linolenoyl-rac-glycerol in α-glucosidase inhibition or blood sugar reduction. The specific scheme is as follows:

[0006] Application of 2-linolenoyl-rac-glycerol in preparing alpha-glucosidase inhibitors or blood sugar lowering products.

[0007] Optionally, the 2-linolenoyl-rac-glycerol is the only active ingredient that inhibits α-glucosidase or lowers blood sugar.

[0008] The 2-linolenoyl-rac-glycerol according to a specific embodiment of the present invention has an α-glucosidase inhibitory effect, is effective as an α-glucosidase inhibitor, and plays a role in hypoglycemic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Comparison results of α-glucosidase inhibition rates of samples in Example 1;

[0010] Figure 2 Comparison results of α-glucosidase inhibition rates of samples in Example 2. DETAILED DESCRIPTION

[0011] A specific embodiment of the present invention provides the use of 2-linolenoyl-rac-glycerol in the preparation of α-glucosidase inhibitors or blood sugar-lowering products.

[0012] In the application of the specific embodiment of the present invention, the structural formula of the 2-linolenoyl-rac-glycerol is C 21 H 38 O4, CAS number is 3443-82-1, the specific structure is as follows:

[0013] .

[0014] In the application of the specific embodiment of the present invention, in some specific embodiments, the hypoglycemic product is a health food, food or a drug for preventing and treating hyperglycemia or diabetes-related diseases with hypoglycemic effect. In some specific embodiments, the hypoglycemic product also includes excipients; the excipients include but are not limited to at least one of lubricants, surfactants, disintegrants, wetting agents, adhesives, fillers, excipients, absorption promoters, adsorption carriers and diluents; in some specific embodiments, the dosage form of the hypoglycemic product includes tablets, capsules, granules, liquid preparations, pills or powders, etc., and any common dosage form in the art is acceptable; in some specific embodiments, the active ingredient or main active ingredient in the hypoglycemic product is 2-linolenoyl-rac-glycerol, in some specific embodiments, the active ingredient in the hypoglycemic product may also include a combination with other active ingredients, and in some specific embodiments, the 2-linolenoyl-rac-glycerol is the only active ingredient for inhibiting α-glucosidase or hypoglycemic.

[0015] The application of the specific embodiments of the present invention, in some specific embodiments, the 2-linolenoyl-rac-glycerol has a significant inhibitory effect on α-D-glucosidase, and has an inhibition rate on α-D-glucosidase that is equivalent to or higher than that of acarbose at the same concentration. In some specific embodiments, the concentration of the 2-linolenoyl-rac-glycerol is 100 μg / ml, and the inhibition rate on α-D-glucosidase is above 90%.

[0016] In the application of the specific embodiments of the present invention, in some specific embodiments, the 2-linolenoyl-rac-glycerol is extracted from Thunbergia schrenkiana, and in some specific embodiments, the 2-linolenoyl-rac-glycerol is obtained by chemical synthesis or extraction from other natural plants.

[0017] The present invention is further described below by means of the best specific embodiments.

[0018] The inhibition rate was determined by:

[0019] 1. Solution preparation:

[0020] (1) Phosphate buffer (pH 6.8)

[0021] (2) 0.2M sodium carbonate solution.

[0022] (3) 0.2U / mL α-glucosidase solution (pH 6.9): Accurately weigh α-glucosidase and prepare a 10U / mL enzyme solution with phosphate buffer. When required for the experiment, use phosphate buffer to prepare an enzyme solution of the corresponding concentration.

[0023] (4) 2 mM nitrobenzene-α-D-glucoside solution (pNPG): Accurately weigh an appropriate amount of pNPG and prepare a 20 mM stock solution with DMSO. When required for the experiment, use phosphate buffer to prepare a solution of the corresponding concentration.

[0024] (5) Sample: Accurately weigh an appropriate amount of sample and prepare a sample stock solution of corresponding concentration using phosphate buffer as required for the experiment.

[0025] 2. Prepare a black 96-well plate, add 60 μL 0.2 U / ml α-glucosidase and 10 μL sample solution, and incubate at 37°C for 10 min.

[0026] 3. Add 80 μL of substrate 2 mM pNPG to start the reaction and incubate at 37°C for 20 min.

[0027] 4. Add 50 μL of 0.2 M Na2CO3 solution to terminate the reaction.

[0028] 5. Measure the absorbance (A) value at 405 nm.

[0029] 6. The absorbance of the sample group is As, the absorbance of the blank control using buffer instead of sample is Ab, and the absorbance of the negative control using phosphate buffer instead of α-glucosidase solution is Ac.

[0030] α-Glucosidase inhibition rate (%) = (Ab−(As−Ac)) / Ab × 100%.

[0031] Example 1

[0032] The flowers and buds of the Herba Lycoris Radiatae are extracted by cold soaking with 75% ethanol at room temperature, and then concentrated to dryness under reduced pressure to obtain a Herba Lycoris Radiatae crude extract; the Herba Lycoris Radiatae crude extract is applied to an MCI chromatography column (MCI GEL CHP 20P, 75-150um, Mitsubishi Chemical, Japan), and gradient eluted with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution and 100% methanol aqueous solution in sequence, with 3 column volumes for each gradient elution, to obtain Herba Lycoris Radiatae refined components, which are represented by RC1-RC6 respectively.

[0033] Specifically, RC1 is the fraction eluted with water after MCI chromatography separation;

[0034] RC2 is the fraction eluted with 20% methanol in water after MCI chromatography separation;

[0035] RC3 is the fraction eluted with 40% methanol in water after MCI chromatography separation;

[0036] RC4 is the fraction eluted with 60% methanol in water after MCI chromatography separation;

[0037] RC5 is the fraction eluted with 80% methanol in water after MCI chromatography separation;

[0038] RC6 is the fraction eluted with 100% methanol after MCI chromatography separation.

[0039] The inhibition rate of the crude extract of the Herba Lycopodii and the Herba Lycopodii refined extracts RC1-RC6 was measured, and acarbose was used as a control. The acarbose sample was prepared in the following manner: an appropriate amount of acarbose powder was accurately weighed and prepared into a 10 mg / mL stock solution with DMSO. When required for the experiment, a secondary mother solution of corresponding concentration was prepared with PBS buffer. The measurement results are shown in Table 1 and Figure 1 shown.

[0040] Table 1 Comparison of α-glucosidase inhibition rates of samples in Example 1

[0041]

[0042] As shown in Table 1 above and Figure 1 The results showed that the crude extract of Herba Lycopodii has excellent inhibitory effect on α-D-glucosidase. The inhibitory effect on α-D-glucosidase at a concentration of 100 μg / ml is basically equivalent to the inhibitory effect of acarbose at a concentration of 1000 μg / ml. In further refined extracts, RC1 did not show an inhibitory effect on α-D-glucosidase. Starting from RC2, the same concentration had the same inhibitory effect on α-D-glucosidase as acarbose, indicating that when the volume concentration of methanol is 20%, the active ingredients containing inhibitory effect on α-D-glucosidase can be effectively eluted. RC3-RC6 showed better α-D-glucosidase inhibition than the crude extract, indicating that through MCI chromatographic separation, the elution fraction with a methanol volume concentration of more than 40% can successfully further obtain a more excellent α-D-glucosidase inhibitory effect of the Herba Lycopodii. In particular, RC3, RC5 and RC6 showed particularly excellent α-D-glucosidase inhibition. At a concentration of 100 μg / ml, the α-D-glucosidase inhibition rate was more than 90%, and at a concentration of 10 μg / ml, the α-D-glucosidase inhibition rate was more than 30%, among which RC3 reached more than 50%.

[0043] Example 2

[0044] The RC2-RC6 of Example 1 were further purified, separated and analyzed:

[0045] The extract RC2 was loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9:1-8:2), petroleum ether-isopropanol (volume ratio 8:2-7:3) and ethyl acetate:methanol 9.8:0.2. The separated compound component (Compound) was represented by C4.

[0046] The extract RC4 was loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9.8:0.2-8:2), dichloromethane-isopropanol (volume ratio 9.8:0.2-9:1), and petroleum ether-isopropanol (volume ratio 8:2-6:4); the extract was loaded onto a Sephdex column and eluted with methanol, and the separated compound components were represented as C3.

[0047] The extract RC5 was loaded onto a silica gel column and eluted with dichloromethane-acetone (volume ratio 9:1-8:2), dichloromethane-methanol (volume ratio 9:1), and petroleum ether-acetone (volume ratio 8:2-7:3). The separated compound components were represented by C1

[0048] The extract RC6 was loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9.8:0.2-9:1), dichloromethane-acetone, petroleum ether-acetone (volume ratio 9:1-7:3), and petroleum ether-ethyl acetate (volume ratio 9:1-8:2). It was then loaded onto a Sephdex column and eluted with dichloromethane-methanol (volume ratio 1:1). After multiple separations, the compound components obtained were represented as C2, C5, and C6, respectively.

[0049] Specifically, compound C1: 1,2',3',4',6'-O-pentaacetyl-3-O-trans-p-coumaroylsucrose, CAS No: 1392307-46-8, i.e. Mumeose D, Chinese name: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumaroylsucrose, structural formula: C 31 H 38 O 18 , the specific structure is as follows:

[0050]

[0051] Compound C2: 2', 3', 4', 6'-tetra-O-acetyl-3-O-(E)-p-coumaroylsucrose, CAS No: 1603815-30-0, Chinese name: 2', 3', 4', 6'-tetra-O-acetyl-3-O-(E)-p-coumaroylsucrose, structural formula: C 25 H 32 O 15 , the specific structure is as follows:

[0052] .

[0053] Compound C3: mumeose K, CAS No: 2132384-01-9, structural formula: C 25 H 32 O 15 , the specific structure is as follows:

[0054] .

[0055] Compound C4: daucosterol linoleate, CAS No: 79380-30-6, Chinese name: daucosterol linoleate, 3-O-(6'-O-linoleyl-β-D-glucose)-β-sitosterol; structural formula: C 53 H 90 O7, the specific structure is as follows:

[0056] .

[0057] Compound C5: Glycerol 1-eicosanoate 3-(4E,7E,10E-hexadecatrienoate), CAS No: 1415934-71-2, Chinese name: Glycerol-1-eicosanoate-3-(4E,7E,10E-hexadecatrienoate); Structural formula: C 39 H 70 O5, the specific structure is as follows:

[0058] .

[0059] Compound C6: 1,3-dihydroxylpropyl-(9Z,12Z)-octadeca-9,12-dienate, CAS No: 3443-82-1; Chinese name: 2-linolenoyl-rac-glycerol; structural formula: C 21 H 38 O4, the specific structure is as follows:

[0060]

[0061] The inhibition rate of the crude extract of Herba Lycopodii and C1-C6 was measured, and acarbose was used as a control. The results are shown in Table 2 and Figure 2 shown.

[0062] Table 2 Comparison of α-glucosidase inhibition rates of samples in Example 2

[0063]

[0064] As shown in Table 2 above and Figure 2 The results show that C1-C5 did not show an inhibitory effect on α-D-glucosidase, and C6, i.e., 2-linolenoyl-rac-glycerol, had a better inhibitory effect on α-D-glucosidase at a concentration of 100 μg / ml than acarbose at a concentration of 1000 μg / ml.

[0065] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. Application of 2-linolenoyl-rac-glycerol in the preparation of α-glucosidase inhibitors or blood sugar lowering products.

2. The use according to claim 1, characterized in that: The 2-linolenoyl-rac-glycerol is the only active ingredient for inhibiting α-glucosidase or lowering blood sugar.

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