Chitosan oligosaccharide unsaturated fatty acid salt derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of functional food, medicine and health care, and particularly discloses a chitosan oligosaccharide unsaturated fatty acid salt derivative and a preparation method and application thereof.The chitosan oligosaccharide unsaturated fatty acid salt derivative is a compound shown in the formula (1), and the preparation method comprises the specific preparation steps that chitosan oligosaccharide and unsaturated fatty acid are subjected to an ionic reaction, and the chitosan oligosaccharide unsaturated fatty acid salt derivative is obtained. The chitosan oligosaccharide unsaturated fatty acid salt derivative shown in the formula (1) is prepared, and the molar ratio of chitosan oligosaccharide to unsaturated fatty acid is 1: 1-1: 3. According to the derivative, chitosan oligosaccharide with excellent biocompatibility is used as a framework, and an unsaturated fatty acid group is combined to 2-position amino positive ions of the chitosan oligosaccharide in a zwitterion combination mode, so that the chitosan oligosaccharide unsaturated fatty acid salt derivative is prepared. The derivative has the characteristics of two compounds of chitosan oligosaccharide and unsaturated fatty acid at the same time, and can remarkably inhibit the activity of alpha-glucosidase, so that the effect of assisting in reducing blood sugar is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of functional foods and medical health care, and in particular to a chitosan oligosaccharide unsaturated fatty acid salt derivative and a preparation method and application thereof. Background Art

[0002] Diabetes is a chronic disease that causes metabolic disorders of carbohydrates in the human body due to insufficient insulin secretion. There is currently no cure. Official data show that the number of diabetes patients has increased day by day worldwide in recent years. The development of the disease and the outbreak of related complications have caused great distress to the majority of diabetic patients. Therefore, it is urgent to find specific drugs based on the treatment targets of diabetes. Among them, α-glucosidase is a biological enzyme widely present in the human body. Its main function is to decompose glucose polymers ingested into the human body to produce glucose, thereby increasing the glucose content in the human body and causing blood sugar to rise. Therefore, if the activity of this enzyme is inhibited, it can have the effect of lowering blood sugar.

[0003] Chitosan oligosaccharides are degradation products of chitosan. Usually, chitosan oligosaccharides with a degree of polymerization between 2-20 are collectively referred to as chitosan oligosaccharides. At present, the common chitosan oligosaccharides on the market are almost all prepared by degradation of chitosan. They are rich in sources and have multiple biological activities. They are a kind of biological oligosaccharides with great research value. At present, biological preparations and materials prepared from chitosan oligosaccharide raw materials have covered multiple industries such as food, medicine, pesticides, and cosmetics. With the development of green, low-carbon and healthy life, the trend of chitosan oligosaccharides being developed and utilized as raw materials will be further highlighted in various fields.

[0004] Unsaturated fatty acids are a general term for fatty acids containing unsaturated bonds, which 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] Chitosan oligosaccharide salt compounds can be formed by using the amino groups of chitosan oligosaccharides and the carboxyl groups of unsaturated fatty acids, and the biological activity of chitosan oligosaccharides will be significantly improved after being salted. Therefore, unsaturated fatty acids with hypoglycemic activity are selected and combined with chitosan oligosaccharides. On the premise of ensuring the hypoglycemic activity of both, it is expected to achieve a superimposed effect of hypoglycemic activity. For this purpose, a chitosan oligosaccharide unsaturated fatty acid salt derivative, a preparation method and application thereof are provided. Summary of the invention

[0006] The purpose of the present invention is to provide a chitosan oligosaccharide unsaturated fatty acid salt derivative and a preparation method and application thereof in view of the defects of the prior art, so as to solve the problems raised by the above background technology.

[0007] To achieve the above object, 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] Where R is:

[0010]

[0011] Among them, the value range of chitosan oligosaccharide n is 2-20.

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

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

[0014] As a preferred technical solution of the present invention, the raw material of the unsaturated fatty acid is α-linolenic acid (ALA), γ-linolenic acid (GLA), conjugated linoleic acid (CLA), docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA).

[0015] The invention discloses an application of a chitosan oligosaccharide unsaturated fatty acid salt derivative in inhibiting the activity of alpha-glucosidase.

[0016] The invention discloses an application of a chitosan oligosaccharide unsaturated fatty acid salt derivative in the preparation of a product for reducing blood sugar. The product is a functional food, a health food, a food for special medical purposes or a new drug.

[0017] A blood sugar lowering product contains chitosan oligosaccharide unsaturated fatty acid salt derivatives in pharmacologically effective concentrations.

[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 skeleton, and combines the unsaturated fatty acid group to the amino cation at the 2-position of chitosan oligosaccharide in the form of anion-cation combination, thereby preparing a chitosan oligosaccharide unsaturated fatty acid salt derivative, and the derivative has the characteristics of both chitosan oligosaccharide and unsaturated fatty acid compounds, can significantly inhibit the activity of α-glucosidase, and thus play an auxiliary effect in lowering blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2 The infrared spectrum of the chitosan oligosaccharide raw material used in the present invention, according to the peak position on the spectrum and the structural characteristics of chitosan oligosaccharide, the characteristic peaks in the figure can be corresponded to the characteristic groups of chitosan oligosaccharide as follows: 3395cm -1 The stretching vibration absorption peak of chitosan oligosaccharide -OH group is 2924cm -1 The stretching vibration absorption peak of CH is 1582 cm -1 -NH 2 The characteristic absorption peak of the group is 1415cm -1 CH 2 Bending vibration absorption peak, 1084cm -1 The position is the stretching vibration absorption peak of CO on chitosan oligosaccharide;

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

[0022] Figure 4 The infrared spectrum of chitosan oligosaccharide γ-linolenate provided in Example 1 of the present invention;

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

[0024] Figure 6 The infrared spectrum of chitosan oligosaccharide docosahexaenoate provided in Example 1 of the present 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 addition to the characteristic peaks of chitosan oligosaccharide, the infrared spectra of the unsaturated fatty acid salt products also showed characteristic absorption peaks of unsaturated fatty acids, such as 670-1000cm -1 The out-of-plane bending vibration absorption peak of the double bond CH bond appears in the range of 1640 cm -1 The characteristic absorption peak of the newly appeared carbon-carbon double bond is at 3000-3400cm -1 The absorption peak in the range becomes sharper and the intensity increases, indicating the absorption peak of the CH bond in the unsaturated fatty acid, which proves the successful synthesis of the unsaturated fatty acid salt derivative of chitosan oligosaccharide. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention are described in detail below in conjunction with 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 making a clearer and more definite definition of the protection scope of the present invention.

[0028] Example 1: According to Figure 1 The process prepares the compound represented by formula (1).

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

[0030] 0.02 mol of α-linolenic acid was measured and placed in a 50 mL round-bottom flask, and then 0.02 mol of sodium hydroxide was added, followed by 10 mL of deionized water, and stirred at room temperature for 2 hours until dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 500-2000Da) was weighed and added to the above reaction, and stirred at room temperature for 4 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C to obtain chitosan oligosaccharide α-linolenic acid salt.

[0031] Embodiment 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, and then 0.01 mol of sodium hydroxide was added, followed by 10 mL of deionized water, and stirred at room temperature for 1 hour until it was dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 500-2000Da) was weighed and added to the above reaction, and stirred at room temperature for 3 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C to obtain chitosan oligosaccharide α-linolenic acid salt.

[0033] Embodiment 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, and then 0.03 mol of sodium hydroxide was added, followed by 15 mL of deionized water, and stirred at room temperature for 2 hours until dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 500-2000Da) was weighed and added to the above reaction, and stirred at room temperature for 6 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C to obtain chitosan oligosaccharide γ-linolenic acid salt.

[0035] Embodiment 4: Preparation method of chitosan oligosaccharide unsaturated fatty acid salt derivative; 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, and then 0.01 mol of sodium hydroxide was added, followed by 10 mL of deionized water, and stirred at room temperature for 1 hour until it dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 500-2000Da) was weighed and added to the above reaction, and stirred at room temperature for 4 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C 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 measured and placed in a 50 mL round-bottom flask, and then 0.01 mol of sodium hydroxide was added, followed by 10 mL of deionized water, and stirred at room temperature for 2 hours until it was dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 500-1000Da) was weighed and added to the above reaction, and stirred at room temperature for 4 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C 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 measured and placed in a 50 mL round-bottom flask, and then 0.02 mol of sodium hydroxide was added, followed by 12 mL of deionized water, and stirred at room temperature for 2 hours until it was dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 1000-2000Da) was weighed and added to the above reaction, and stirred at room temperature for 5 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C 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 measured and placed in a 50 mL round-bottom flask, and then 0.02 mol of sodium hydroxide was added, followed by 15 mL of deionized water, and stirred at room temperature for 2 hours until dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 500-1000Da) was weighed and added to the above reaction, and stirred at room temperature for 5 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C 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 measured and placed in a 50 mL round-bottom flask, and then 0.01 mol of sodium hydroxide was added, followed by 10 mL of deionized water, and stirred at room temperature for 2 hours until dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 2000-3000Da) was weighed and added to the above reaction, and stirred at room temperature for 4 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C 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 measured and placed in a 50 mL round-bottom flask, and then 0.01 mol of sodium hydroxide was added, followed by 10 mL of deionized water, and stirred at room temperature for 2 hours until dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 1000-2000Da) was weighed and added to the above reaction, and stirred at room temperature for 4 hours. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40°C to obtain chitosan oligosaccharide eicosapentaenoate.

[0047] Embodiment 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 measured and placed in a 50 mL round-bottom flask, and then 0.03 mol of sodium hydroxide was added, followed by 15 mL of deionized water, and stirred at room temperature for 2 h until dissolved, and then 0.01 mol of chitosan oligosaccharide (mW 500-1500Da) was weighed and added to the above reaction, and stirred at room temperature for 6 h. After the reaction was completed, the product was precipitated with anhydrous ethanol, and then washed with anhydrous ethanol several times and filtered to obtain the product, which was freeze-dried to constant weight at -40 ° C to obtain chitosan oligosaccharide eicosapentaenoate.

[0049] Application example: α-glucosidase is a biological enzyme widely present in organisms. Its main function is to decompose polysaccharides ingested by organisms to obtain glucose, thereby increasing the glucose content in the body and the blood sugar concentration. Therefore, inhibiting the activity of α-glucosidase can reduce the glucose content in the human body and reduce the blood sugar concentration.

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

[0051] First, 80 mg of the sample to be tested was accurately weighed and dissolved in 8 mL of PBS buffer to prepare a sample solution with an initial concentration of 10 mg / mL. The sample solution was then diluted stepwise with PBS buffer to obtain sample solutions with concentrations of 2.0, 4.0, 6.0, 8.0, and 10 mg / mL, respectively.

[0052] Use PBS buffer to prepare 1 U / mL α-glucosidase solution, pNPG solution and 0.1 mol / L sodium carbonate solution respectively and set aside.

[0053] Experimental group: The test solutions were added to 96-well plates, 50 μL / well for each concentration, and then 50 μL of α-glucosidase solution was added to each well and incubated at 37°C for 10 minutes. After the time was up, 50 μL of pNPG solution was added to each well and incubated at 37°C for 20 minutes. Finally, 50 μL of sodium carbonate solution was added, mixed evenly, and 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 amount of PBS buffer, and the other reagents and operations were consistent with those of the experimental group.

[0055] Blank control group: replace the test samples in the experimental group with an equal amount of PBS buffer, and keep the other reagents and operations the same as those of the experimental group.

[0056] Through the above experimental operation, the absorbance of each group is obtained, and then the α-glucosidase inhibition rate of each test compound at the corresponding concentration can be obtained by the following calculation formula:

[0057]

[0058] Among them, A 样品 Refers to the absorbance of the sample solution in the experimental group, A 对照 Refers to the absorbance measured by the negative control group without α-glucosidase solution, A 空白 Refers to the absorbance measured by the blank control group.

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

[0060]

[0061] The following conclusions can be drawn from the data in Table 1: the inhibitory activity of a single chitosan oligosaccharide raw material on α-glucosidase is very low, and when the test concentration is low, there is almost no inhibitory activity. When the highest test concentration is 10 mg / mL, its inhibition rate is only 2.4347%, while the inhibitory activity of the five unsaturated fatty acid salt derivatives prepared in the present invention on α-glucosidase has been significantly improved, and its inhibition rate gradually increases with the increase of concentration, and has concentration dependence, among which the enzyme inhibition rate of chitosan oligosaccharide docosapentaenoate is the strongest, exceeding 30%. It can be considered that the unsaturated fatty acid salt derivatives prepared in the present invention have certain hypoglycemic activity, and as a new 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 only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A chitosan oligosaccharide unsaturated fatty acid salt derivative, characterized in that: The chitosan oligosaccharide unsaturated fatty acid salt derivative is a compound represented by formula (1): Where R is: Among them, the value range of chitosan oligosaccharide n is 2-20.

2. A method for preparing the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 1, characterized in that: The specific steps are as follows: chitosan oligosaccharide and unsaturated fatty acid undergo ion reaction to prepare chitosan oligosaccharide unsaturated fatty acid salt derivatives shown in formula (1), wherein the molar ratio of chitosan oligosaccharide to unsaturated fatty acid is 1:1-1:

3.

3. The method for preparing the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 2, characterized in that: The polymerization degree of the chitosan oligosaccharide raw material used is between 2 and 20, and the applicable range of the molecular weight is 500-5000Da.

4. The method for preparing the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 2, wherein: The raw material of the unsaturated fatty acid is α-linolenic acid, γ-linolenic acid, conjugated linoleic acid, docosahexaenoic acid or eicosapentaenoic acid.

5. Use of the chitosan oligosaccharide unsaturated fatty acid salt derivative as claimed in claim 1 in inhibiting the activity of α-glucosidase.

6. A use of the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 1 in the preparation of a product for reducing blood sugar, characterized in that: The product is a functional food, a health food, a food for special medical purposes or a new drug.

7. A product for lowering blood sugar, characterized in that: The product contains the chitosan oligosaccharide unsaturated fatty acid salt derivative according to claim 1 in a pharmacologically effective concentration.

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