A chitosan oligosaccharide unsaturated fatty acid salt derivative, its preparation method and application

Abstract

This invention relates to the fields of functional foods and pharmaceuticals, specifically disclosing a chitosan oligosaccharide unsaturated fatty acid salt derivative, its preparation method, and its application. The chitosan oligosaccharide unsaturated fatty acid salt derivative is a compound represented by formula (1). The specific preparation steps are as follows: chitosan oligosaccharide and unsaturated fatty acid undergo an ionic reaction to prepare the chitosan oligosaccharide unsaturated fatty acid salt derivative represented by formula (1), wherein the molar ratio of chitosan oligosaccharide to unsaturated fatty acid is 1:1 to 1:3. This derivative uses chitosan oligosaccharide, which has excellent biocompatibility, as its backbone, and binds the unsaturated fatty acid groups to the amino cation at the 2-position of the chitosan oligosaccharide in an anionic-cationic combination, thereby preparing the chitosan oligosaccharide unsaturated fatty acid salt derivative. This derivative simultaneously possesses the characteristics of both chitosan oligosaccharide and unsaturated fatty acid compounds, and can significantly inhibit the activity of α-glucosidase, thereby playing an auxiliary role in lowering blood sugar.

Description

Technical Field

[0001] This invention relates to the fields of functional foods and pharmaceuticals, specifically to a chitosan oligosaccharide unsaturated fatty acid salt derivative, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a chronic disease caused by insufficient insulin secretion, leading to impaired carbohydrate metabolism. Currently, there is no cure. Official data shows that the number of people with diabetes worldwide has been increasing daily in recent years. The progression of the disease and the outbreak of related complications have caused great distress to many diabetic patients. Therefore, finding effective drugs based on therapeutic targets for diabetes is urgently needed. Alpha-glucosidase is a widely distributed enzyme in the human body. Its main function is to break down ingested glucose polymers to produce glucose, thereby increasing the glucose content in the body and causing blood sugar levels to rise. Therefore, inhibiting the activity of this enzyme can lower blood sugar.

[0003] Chitosan oligosaccharides are degradation products of chitosan. Generally, chitosan oligosaccharides with a degree of polymerization between 2 and 20 are collectively referred to as chitosan oligosaccharides. Currently, almost all commercially available chitosan oligosaccharides are prepared from the degradation of chitosan. They are abundant in source and possess various biological activities, making them a highly valuable bio-oligosaccharide for research. At present, biological agents and materials prepared from chitosan oligosaccharide raw materials cover multiple industries such as food, medicine, pesticides, and cosmetics. With the development of green, low-carbon, and healthy living, the trend of developing and utilizing chitosan oligosaccharides as raw materials will further become prominent in various fields.

[0004] Unsaturated fatty acids are a collective term for a class of fatty acids containing unsaturated bonds. They include a variety of acid compounds. Studies have shown that unsaturated fatty acids are a class of high-quality fatty acids that are beneficial to human health. For example, some fatty acids are essential components of biological membranes and have the activity of lowering blood lipids and blood sugar.

[0005] The amino groups of chitosan oligosaccharides and the carboxyl groups of unsaturated fatty acids can form chitosan oligosaccharide salts, and the biological activity of chitosan oligosaccharides is significantly enhanced after salt formation. Therefore, by selecting unsaturated fatty acids with hypoglycemic activity and combining them with chitosan oligosaccharides, it is expected to achieve a synergistic effect of hypoglycemic activity while ensuring the hypoglycemic activity of each. To this end, a chitosan oligosaccharide unsaturated fatty acid salt derivative, its preparation method and application are provided. Summary of the Invention

[0006] The purpose of this invention is to address the deficiencies of the prior art by providing a chitosan oligosaccharide unsaturated fatty acid salt derivative, its preparation method, and its application, thereby resolving the problems raised in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a chitosan oligosaccharide unsaturated fatty acid salt derivative, wherein the chitosan oligosaccharide unsaturated fatty acid salt derivative is a compound represented by formula (1):

[0008]

[0009] R in the formula is:

[0010]

[0011] The value of n for chitosan oligosaccharide ranges from 2 to 20.

[0012] A method for preparing a chitosan oligosaccharide unsaturated fatty acid salt derivative, the specific steps are as follows: chitosan oligosaccharide and unsaturated fatty acid undergo an ionic reaction to prepare the chitosan oligosaccharide unsaturated fatty acid salt derivative shown in formula (1), wherein the molar ratio of chitosan oligosaccharide to unsaturated fatty acid is 1:1 to 1:3.

[0013] As a preferred technical solution of the present invention, the degree of polymerization of the chitosan oligosaccharide raw material used is between 2 and 20, and its molecular weight is applicable in the range of 500-5000 Da.

[0014] As a preferred embodiment of the present invention, the raw materials for the unsaturated fatty acids are α-linolenic acid (ALA), γ-linolenic acid (GLA), conjugated linoleic acid (CLA), docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA).

[0015] Application of a chitosan oligosaccharide unsaturated fatty acid salt derivative in inhibiting α-glucosidase activity.

[0016] The application of a chitosan oligosaccharide unsaturated fatty acid salt derivative in the preparation of hypoglycemic products, wherein the products are functional foods, health foods, foods for special medical purposes, or new drugs.

[0017] A blood sugar lowering product, wherein the product contains a pharmacologically effective concentration of chitosan oligosaccharide unsaturated fatty acid salt derivative.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: the derivative uses chitosan oligosaccharide with excellent biocompatibility as the backbone, and the unsaturated fatty acid groups are combined with the amino cation at the 2-position of the chitosan oligosaccharide in the form of anionic and cationic combination, thereby preparing a chitosan oligosaccharide unsaturated fatty acid salt derivative. This derivative has the characteristics of both chitosan oligosaccharide and unsaturated fatty acid compounds, and can significantly inhibit the activity of α-glucosidase, thereby playing an auxiliary role in lowering blood sugar. Attached Figure Description

[0019] Figure 1A synthetic route diagram for chitosan oligosaccharide unsaturated fatty acid salts provided in an embodiment of the present invention;

[0020] Figure 2 The infrared spectrum of the chitosan oligosaccharide raw material used in this invention is shown below. Based on the peak positions and structural characteristics of chitosan oligosaccharides, the characteristic peaks in the spectrum can be correlated with the characteristic groups of chitosan oligosaccharides as follows: 3395 cm⁻¹ -1 The peak at 2924 cm⁻¹ represents the stretching vibration absorption of the -OH group in chitosan oligosaccharides. -1 The absorption peak for the stretching vibration of CH is at 1582 cm⁻¹. -1 The peak at 1415 cm⁻¹ is the characteristic absorption peak of the -NH₂ group on chitosan oligosaccharide. -1 The peak at 1084 cm⁻¹ represents the absorption peak of the bending vibration of CH₂ on chitosan oligosaccharides. -1 The peak at this point represents the stretching vibration absorption peak of CO on chitosan oligosaccharide.

[0021] Figure 3 The infrared spectrum of chitosan oligosaccharide α-linolenic acid provided in Example 1 of this invention;

[0022] Figure 4 The infrared spectrum of chitosan oligosaccharide γ-linolenic acid provided in Example 1 of this invention;

[0023] Figure 5 The infrared spectrum of chitosan oligosaccharide conjugated linoleate provided in Example 1 of this invention;

[0024] Figure 6 The infrared spectrum of chitosan oligosaccharide docosahexaenoic acid salt provided in Example 1 of this invention;

[0025] Figure 7 The infrared spectrum of chitosan oligosaccharide eicosapentaenoate provided in Example 1 of the present invention;

[0026] exist Figure 3-7 In the infrared spectra of the various chitosan oligosaccharide unsaturated fatty acid salt products, in addition to retaining the characteristic peaks of chitosan oligosaccharides, characteristic absorption peaks of unsaturated fatty acids also appeared, such as in the 670-1000 cm⁻¹ range. -1 The out-of-plane bending vibration absorption peak of the double CH bond appears within the range, at 1640 cm⁻¹. -1 The characteristic absorption peak of the newly emerging carbon-carbon double bond in the vicinity is in the 3000-3400 cm⁻¹ range. -1 The absorption peaks within the range become sharper and stronger, indicating that they are absorption peaks of CH bonds in unsaturated fatty acids, which proves the successful synthesis of chitosan oligosaccharide unsaturated fatty acid salt derivatives. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0028] Example 1: According to Figure 1 The process described above prepares the compound shown in formula (1).

[0029] Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; specific steps are as follows:

[0030] Measure 0.02 mol of α-linolenic acid and place it in a 50 mL round-bottom flask. Then add 0.02 mol of sodium hydroxide, followed by 10 mL of deionized water. Stir at room temperature for 2 h until dissolved. Then weigh 0.01 mol of chitosan oligosaccharide (mW 500-2000 Da) and add it to the above reaction. Stir at room temperature for 4 h. After the reaction is complete, precipitate the product with anhydrous ethanol. After washing repeatedly with anhydrous ethanol and filtering, the product is obtained. Freeze-dry at -40 °C to constant weight to obtain chitosan oligosaccharide α-linolenic acid salt.

[0031] Example 2: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0032] 0.01 mol of α-linolenic acid was measured and placed in a 50 mL round-bottom flask. Then, 0.01 mol of sodium hydroxide was added, followed by 10 mL of deionized water. The mixture was stirred at room temperature for 1 h until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 500-2000 Da) was weighed and added to the above reaction mixture. The mixture was stirred at room temperature for 3 h. After the reaction was completed, the product was precipitated with anhydrous ethanol. After washing with anhydrous ethanol several times and filtering, the product was obtained and freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide α-linolenic acid salt.

[0033] Example 3: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0034] 0.03 mol of γ-linolenic acid was measured and placed in a 50 mL round-bottom flask. Then, 0.03 mol of sodium hydroxide was added, followed by 15 mL of deionized water. The mixture was stirred at room temperature for 2 h until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 500-2000 Da) was weighed and added to the above reaction mixture. The mixture was stirred at room temperature for 6 h. After the reaction was completed, the product was precipitated with anhydrous ethanol. After washing with anhydrous ethanol several times and filtering, the product was obtained and freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide γ-linolenic acid salt.

[0035] Example 4: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0036] 0.01 mol of γ-linolenic acid was measured and placed in a 50 mL round-bottom flask. Then, 0.01 mol of sodium hydroxide was added, followed by 10 mL of deionized water. The mixture was stirred at room temperature for 1 h until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 500-2000 Da) was weighed and added to the above reaction mixture. The mixture was stirred at room temperature for 4 h. After the reaction was completed, the product was precipitated with anhydrous ethanol. After washing with anhydrous ethanol several times and filtering, the product was obtained and freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide γ-linolenic acid salt.

[0037] Example 5: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0038] 0.01 mol of conjugated linoleic acid was placed in a 50 mL round-bottom flask, followed by 0.01 mol of sodium hydroxide and then 10 mL of deionized water. The mixture was stirred at room temperature for 2 hours until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 500-1000 Da) was weighed and added to the reaction mixture. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the product was precipitated with anhydrous ethanol. After repeated washing with anhydrous ethanol and filtration, the product was obtained and freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide conjugated linoleate.

[0039] Example 6: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0040] 0.02 mol of conjugated linoleic acid was placed in a 50 mL round-bottom flask, followed by 0.02 mol of sodium hydroxide, and then 12 mL of deionized water. The mixture was stirred at room temperature for 2 h until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 1000-2000 Da) was weighed and added to the reaction mixture. The mixture was stirred at room temperature for 5 h. After the reaction was complete, the product was precipitated with anhydrous ethanol, followed by repeated washing with anhydrous ethanol and filtration to obtain the final product. The final product was lyophilized at -40 °C to constant weight to obtain chitosan oligosaccharide conjugated linoleate.

[0041] Example 7: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0042] 0.02 mol of docosahexaenoic acid was placed in a 50 mL round-bottom flask, followed by 0.02 mol of sodium hydroxide and then 15 mL of deionized water. The mixture was stirred at room temperature for 2 hours until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 500-1000 Da) was weighed and added to the reaction mixture. The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the product was precipitated with anhydrous ethanol. After repeated washing with anhydrous ethanol and filtration, the product was obtained and freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide docosahexaenoic acid salt.

[0043] Example 8: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0044] 0.01 mol of docosahexaenoic acid was placed in a 50 mL round-bottom flask, followed by 0.01 mol of sodium hydroxide and then 10 mL of deionized water. The mixture was stirred at room temperature for 2 hours until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 2000-3000 Da) was weighed and added to the reaction mixture. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the product was precipitated with anhydrous ethanol. After repeated washing with anhydrous ethanol and filtration, the product was obtained and freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide docosahexaenoic acid salt.

[0045] Example 9: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0046] 0.01 mol of eicosapentaenoic acid was placed in a 50 mL round-bottom flask, followed by 0.01 mol of sodium hydroxide and then 10 mL of deionized water. The mixture was stirred at room temperature for 2 h until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 1000-2000 Da) was weighed and added to the reaction mixture. The mixture was stirred at room temperature for 4 h. After the reaction was complete, the product was precipitated with anhydrous ethanol. The product was then washed repeatedly with anhydrous ethanol and filtered to obtain the final product. The final product was freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide eicosapentaenoic acid salt.

[0047] Example 10: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivatives; the specific steps are as follows:

[0048] 0.03 mol of eicosapentaenoic acid was placed in a 50 mL round-bottom flask, followed by 0.03 mol of sodium hydroxide and then 15 mL of deionized water. The mixture was stirred at room temperature for 2 h until dissolved. Then, 0.01 mol of chitosan oligosaccharide (mW 500-1500 Da) was weighed and added to the reaction mixture. The mixture was stirred at room temperature for 6 h. After the reaction was complete, the product was precipitated with anhydrous ethanol. After repeated washing with anhydrous ethanol and filtration, the product was obtained and freeze-dried at -40 °C to constant weight to obtain chitosan oligosaccharide eicosapentaenoic acid salt.

[0049] Application example: α-glucosidase is a widely distributed enzyme in living organisms. Its main function is to break down polysaccharides ingested by the body to obtain glucose, thereby increasing the glucose content in the body and raising blood glucose concentration. Therefore, inhibiting α-glucosidase activity can reduce the glucose content in the human body and lower blood glucose concentration.

[0050] Based on the above experimental principles, the experimental raw materials used in this invention and the prepared chitosan oligosaccharide unsaturated fatty acid salt derivatives were selected, and their inhibitory activity against α-glucosidase was determined through the following experimental process.

[0051] First, accurately weigh 80 mg of the sample to be tested and dissolve it in 8 mL of PBS buffer to prepare a sample solution with an initial concentration of 10 mg / mL. Then, dilute the sample solution stepwise with PBS buffer to obtain sample solutions with concentrations of 2.0, 4.0, 6.0, 8.0, and 10 mg / mL.

[0052] Prepare 1 U / mL α-glucosidase solution, pNPG solution, and 0.1 mol / L sodium carbonate solution using PBS buffer for later use.

[0053] Experimental group: The test solutions were added to 96-well plates at a concentration of 50 μL / well. Then, 50 μL of α-glucosidase solution was added to each well, and the plates were incubated at 37°C for 10 minutes. After the incubation period, 50 μL of pNPG solution was added to each well, and the plates were incubated at 37°C for 20 minutes. Finally, 50 μL of sodium carbonate solution was added, and the plates were mixed thoroughly. The absorbance of the solution was measured at 405 nm.

[0054] Negative control group: The α-glucosidase solution in the experimental group was replaced with an equal volume of PBS buffer, and the remaining reagents and procedures were kept the same as those in the experimental group.

[0055] Blank control group: The test samples in the experimental group were replaced with an equal volume of PBS buffer, and the other reagents and procedures were kept the same as those in the experimental group.

[0056] The absorbance of each group was obtained through the above experimental procedures. Then, the α-glucosidase inhibition rate of each test compound at the corresponding concentration can be obtained using the following calculation formula:

[0057]

[0058] Among them, A 样品 This refers to the absorbance measured for the corresponding sample solution in the experimental group, A. 对照 This refers to the absorbance obtained by measuring the solution without α-glucosidase in the negative control group, A. 空白 This refers to the absorbance measured in the blank control group.

[0059] Table 1: Inhibition rate (%) of each sample against α-glucosidase

[0060]

[0061] The data in Table 1 lead to the following conclusions: The single chitosan oligosaccharide raw material exhibits very low inhibitory activity against α-glucosidase. At low test concentrations, it shows almost no inhibitory activity, and even at the highest test concentration of 10 mg / mL, its inhibition rate is only 2.4347%. In contrast, the five unsaturated fatty acid salt derivatives prepared in this invention show significantly improved inhibitory activity against α-glucosidase, with the inhibition rate gradually increasing with concentration, exhibiting a concentration-dependent effect. Among them, chitosan oligosaccharide docosapentaenoic acid salt showed the strongest enzyme inhibition rate, exceeding 30%. Therefore, it can be concluded that the unsaturated fatty acid salt derivatives prepared in this invention possess certain hypoglycemic activity. As a novel product with a simple preparation process and green, natural, and edible raw materials, it has potential development and application value in the subsequent development of hypoglycemic drugs.

[0062] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A chitosan oligosaccharide unsaturated fatty acid salt derivative, characterized in that: The derivative is obtained by forming an ionic salt from chitosan oligosaccharide and unsaturated fatty acid through an ionic reaction; the degree of polymerization of the chitosan oligosaccharide is 2-20, and the molar ratio of chitosan oligosaccharide to unsaturated fatty acid is 1:1-1:3; the unsaturated fatty acid is docosahexaenoic acid.

2. The use of the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 1 in the preparation of drugs that inhibit α-glucosidase activity.

3. The application of the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 1 in the preparation of hypoglycemic products, characterized in that: The product is a functional food or a drug.

4. The application of the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 1 in the preparation of hypoglycemic products, characterized in that: The product in question is a health food product.

5. A hypoglycemic product, characterized in that: The chitosan oligosaccharide unsaturated fatty acid salt derivative of claim 1 contains an effective concentration.

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