Water-soluble o-acetylated chitooligosaccharides, methods of making and using the same

Abstract

The application discloses water-soluble O-acetylated chitooligosaccharide and a preparation method and application thereof, and the preparation method comprises the following steps: subjecting chitooligosaccharide to an acetylation reaction to obtain fully acetylated chitooligosaccharide; adding the fully acetylated chitooligosaccharide into 10% ammonia water, and stirring for 10-60 min; filtering the reaction solution, and freeze-drying the filtrate to obtain O-acetylated chitooligosaccharide products. The preparation method disclosed by the application has simple steps, the product has good water solubility, and has strong anti-inflammatory activity, thereby providing a new idea for high-value utilization of marine chitin, and also having important significance for promoting the innovative development of new marine drugs.

Description

Technical Field

[0001] This invention relates to the field of marine bioengineering, and in particular to a water-soluble O-acetylated chitosan oligosaccharide, its preparation method, and its application. Background Technology

[0002] Chitin, also known as chitin, is a natural polysaccharide widely found in the shells of marine crustaceans. Chitosan oligosaccharides, degradation products of chitin, are linear oligosaccharides composed of N-acetyl-D-glucosamine linked by β-1,4 glycosidic bonds. Chitosan oligosaccharides have been found to possess various physiological activities, such as anti-tumor, anti-inflammatory, antioxidant, immune-enhancing, gut microbiota-activating, and plant disease-resistant properties.

[0003] Currently, chitosan oligosaccharides prepared from chitosan oligosaccharides via chemical N-acetylation often contain small amounts of O-acetylated chitosan oligosaccharides due to the poor site selectivity of this chemical method. Extensive research has revealed that products containing small amounts of O-acetylated chitosan oligosaccharides exhibit better anti-inflammatory activity, while high-purity chitosan oligosaccharides themselves lack anti-inflammatory activity. Current research focuses on water-insoluble O-acetylated chitosan oligosaccharides; a method for preparing water-soluble O-acetylated chitosan oligosaccharides is still lacking. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a water-soluble O-acetylated chitosan oligosaccharide, its preparation method, and its application, offering a new approach for the high-value utilization of marine chitosan polysaccharides.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A method for preparing a water-soluble O-acetylated chitosan oligosaccharide includes the following steps:

[0007] (1) The chitosan oligosaccharide was acetylated to obtain fully acetylated chitosan oligosaccharide;

[0008] (2) Add the fully acetylated chitosan oligosaccharide to ammonia water with a mass fraction of 10% and stir for 10-60 min;

[0009] (3) Filter the reaction solution and freeze-dry the filtrate to obtain the O-acetylated chitosan oligosaccharide product.

[0010] In the above scheme, the specific method of step (1) is as follows: 10-15 ml of acetic anhydride and 20-30 ml of pyridine are added sequentially to 2-2.2 g of chitosan oligosaccharide, and the reaction is carried out at room temperature for 18-22 h. Then, a 10% sodium acetate solution is added. After the heat dissipation is completed, the mixture is extracted with an organic solvent, washed with a saturated salt solution, dried with anhydrous Na2SO4 or anhydrous MgSO4, and rotary evaporated to obtain the fully acetylated chitosan oligosaccharide product.

[0011] The reaction process is as follows:

[0012]

[0013] Furthermore, the volume ratio of acetic anhydride to pyridine is 1:2.

[0014] Furthermore, the mass-to-volume ratio of the chitosan oligosaccharide to the sodium acetate solution is 1:20-30.

[0015] Furthermore, the saturated salt solution includes a saturated NaCl solution, a saturated NaHCO3 solution, and a saturated NaCl solution, which are used for washing in sequence.

[0016] In the above scheme, the mass-to-volume ratio of the fully acetylated chitosan oligosaccharide to ammonia is 1:0.05-0.1.

[0017] Furthermore, the organic solvent is chloroform or dichloromethane.

[0018] A water-soluble O-acetylated chitosan oligosaccharide prepared by the method described above has the following structural formula:

[0019]

[0020] In the formula, R = H or COCOH3, n = 0, 1, 2, 3, 4.

[0021] In the above scheme, the degree of O-acetylation of the water-soluble O-acetylated chitosan oligosaccharide is 20-150%.

[0022] The application of water-soluble O-acetylated chitosan oligosaccharides as described above in anti-inflammatory drugs or functional foods.

[0023] Through the above technical solution, the water-soluble O-acetylated chitosan oligosaccharide, its preparation method, and its application provided by the present invention have the following beneficial effects:

[0024] This invention uses chitosan oligosaccharide as a raw material to prepare fully acetylated chitosan oligosaccharides with a very high degree of oxygen acetylation. The acetyl groups are then removed using ammonia to obtain water-soluble O-acetylated chitosan oligosaccharides. The prepared O-acetylated chitosan oligosaccharides exhibit good water solubility and are easy to apply in practice. The preparation method is simple and allows for precise control of the O-acetylation degree of chitosan oligosaccharides. This method can be used for further bioactivity screening and large-scale production of O-acetylated chitosan oligosaccharides, which is of great significance for expanding the application fields of chitosan oligosaccharides and the development of novel anti-inflammatory drugs. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0026] Figure 1 This is a flowchart of a method for preparing water-soluble O-acetylated chitosan oligosaccharide disclosed in this invention.

[0027] Figure 2 The infrared spectrum of the fully acetylated chitosan oligosaccharide prepared in the embodiments of the present invention.

[0028] Figure 3 The water-soluble O-acetylated chitosan oligosaccharide prepared in Example 2 of this invention 1 H-NMR analysis diagram.

[0029] Figure 4 This is a comparison of the anti-inflammatory activity of water-soluble O-acetylated chitosan oligosaccharides prepared in the embodiments of the present invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0031] This invention provides a method for preparing water-soluble O-acetylated chitosan oligosaccharides, such as... Figure 1 As shown, the specific implementation is as follows:

[0032] Preparation of fully acetylated chitin oligosaccharides:

[0033] 10 mL of acetic anhydride and 20 mL of pyridine were added sequentially to 2 g of chitosan oligosaccharide, and the reaction was carried out at room temperature for 22 h. Then, 50 mL of sodium acetate (10% by mass) was added. After cooling, the mixture was extracted twice with 40 mL of dichloromethane. The organic phase was washed sequentially with 80 mL each of saturated NaCl solution, saturated NaHCO3 solution, and saturated NaCl solution. After drying with anhydrous Na2SO4, the mixture was filtered, concentrated by rotary evaporation, and the fully acetylated chitosan oligosaccharide was obtained. The obtained sugar is insoluble in water. The infrared spectrum is shown below. Figure 2 As shown, three characteristic absorption peaks appeared at 1655 cm⁻¹. -1 1536cm -1 and 1379cm -1 These correspond to the amide I band, the bending vibration of -NH2, and the amide III band, respectively, at 1737 cm⁻¹. -1 The peak at this location is the O-acetyl absorption peak.

[0034] Example 1

[0035] Take 200 mg of the sugar obtained above and 25 ml of 10% ammonia water, react at room temperature for 50 min under magnetic stirring, filter, and freeze-dry the filtrate to obtain water-soluble O-acetylated chitosan oligosaccharide with an oxyacetyl degree of 20%.

[0036] Example 2

[0037] Take 200 mg of the sugar obtained above and 25 ml of 10% ammonia water, react at room temperature for 30 min under magnetic stirring, filter, and freeze-dry the filtrate to obtain water-soluble O-acetylated chitosan oligosaccharide with an oxyacetyl degree of 93%. 1 H-NMR analysis graph as shown Figure 3 As shown, 1.8-2.0 ppm is the H of N-acetyl, 2.0-2.2 ppm is the H of O-acetyl, and 3.2-5.2 ppm is the H1-H6 on the sugar ring.

[0038] Example 3

[0039] Take 200 mg of the sugar obtained above and 25 ml of 10% ammonia water, react at room temperature for 10 min under magnetic stirring, filter, and freeze-dry the filtrate to obtain water-soluble O-acetylated chitosan oligosaccharide with an oxyacetyl degree of 150%.

[0040] Comparative Example 1

[0041] Take 200 mg of the obtained sugar and 25 ml of 10% ammonia solution, and react at room temperature for 5 minutes with magnetic stirring. Visible insoluble matter still exists, indicating that the product is a water-insoluble O-acetylated chitosan oligosaccharide. This demonstrates that a shorter reaction time results in a higher degree of acetylation, and excessively high acetylation leads to insolubility in water.

[0042] Comparison of anti-inflammatory activities:

[0043] In mouse macrophage experiments, commercially available chitosan oligosaccharides without O-acetyl groups (%) and the products from Examples 1, 2, and 3 (20%, 93%, and 150%) were dissolved in sterile water to obtain a 10 mg / ml stock solution, which was then diluted to 600 μg / ml with cell culture medium. Lipopolysaccharide (LPS) 1 μg / ml was used as a positive control, and the blank group (culture medium) was used as a negative control. After incubation in macrophages for 24 h, the supernatant was collected, and the NO release was determined using the Griess method. Higher NO release indicated more severe inflammation. The results showed that chitosan oligosaccharides with O-acetyl groups had better anti-inflammatory activity than those without O-acetyl groups. Chitosan oligosaccharides with O-acetyl groups (20%-150%) exhibited significant anti-inflammatory activity. Figure 4 As shown.

[0044] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a water-soluble O-acetylated chitosan oligosaccharide, characterized in that, Includes the following steps: (1) The chitosan oligosaccharide was acetylated to obtain fully acetylated chitosan oligosaccharide; (2) Add the fully acetylated chitosan oligosaccharide to ammonia water with a mass fraction of 10% and stir at room temperature for 10-60 min; the mass-volume ratio of the fully acetylated chitosan oligosaccharide to ammonia water is 1:0.05-0.1; (3) Filter the reaction solution and freeze-dry the filtrate to obtain the O-acetylated chitosan oligosaccharide product.

2. The method for preparing a water-soluble O-acetylated chitosan oligosaccharide according to claim 1, characterized in that, The specific method of step (1) is as follows: 10-15 ml of acetic anhydride and 20-30 ml of pyridine are added sequentially to 2-2.2 g of chitosan oligosaccharide. The reaction is carried out at room temperature for 18-22 h. A 10% sodium acetate solution is added. After the heat dissipation is completed, the mixture is extracted with an organic solvent, washed with a saturated salt solution, dried with anhydrous Na2SO4 or anhydrous MgSO4, and rotary evaporated to obtain the fully acetylated chitosan oligosaccharide product.

3. The method for preparing a water-soluble O-acetylated chitosan oligosaccharide according to claim 2, characterized in that, The volume ratio of acetic anhydride to pyridine is 1:

2.

4. The method for preparing a water-soluble O-acetylated chitosan oligosaccharide according to claim 2, characterized in that, The mass-to-volume ratio of the chitosan oligosaccharide to the sodium acetate solution is 1:20-30.

5. The method for preparing a water-soluble O-acetylated chitosan oligosaccharide according to claim 2, characterized in that, The saturated salt solution includes saturated NaCl solution, saturated NaHCO3 solution, and saturated NaCl solution, which are washed sequentially.

6. The method for preparing a water-soluble O-acetylated chitosan oligosaccharide according to claim 2, characterized in that, The organic solvent is chloroform or dichloromethane.

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