阅读说明：本技术 壳寡糖-n-芳樟醇共聚物及其制备方法与应用 (Chitosan oligosaccharide-N-linalool copolymer and preparation method and application thereof ) 是由 刘晓丽 夏文水 毛水芳 于 2020-06-23 设计创作，主要内容包括：本发明公开了一种壳寡糖-N-芳樟醇共聚物及其制备方法与应用,该共聚物的制备方法包括以下步骤：(1)在一定温度下,将芳樟醇与三溴化磷进行溴化反应,得到淡黄色油状液体芳樟基溴；(2)将一定量的芳樟基溴和壳寡糖混合并反应,经过处理得到壳寡糖-N-芳樟醇共聚物。本发明将具有抗炎活性的芳樟醇与壳寡糖的C2位活性氨基发生烷基化反应,不但制备过程和纯化过程简单,而且得到的壳寡糖-N-芳樟醇共聚物具有良好的水溶性、热稳定性和抗炎活性,在食品、医药、化妆品和农业等领域具有很好的应用前景。(The invention discloses a chitosan oligosaccharide-N-linalool copolymer and a preparation method and application thereof, wherein the preparation method of the copolymer comprises the following steps: (1) carrying out bromination reaction on linalool and phosphorus tribromide at a certain temperature to obtain yellowish oily liquid linalyl bromide; (2) mixing a certain amount of linalyl bromide and chitosan oligosaccharide, reacting, and treating to obtain the chitosan oligosaccharide-N-linalool copolymer. According to the invention, linalool with anti-inflammatory activity and C2 active amino of chitosan oligosaccharide are subjected to alkylation reaction, so that the preparation process and the purification process are simple, and the obtained chitosan oligosaccharide-N-linalool copolymer has good water solubility, thermal stability and anti-inflammatory activity, and has good application prospects in the fields of food, medicine, cosmetics, agriculture and the like.)

1. The chitosan oligosaccharide-N-linalool copolymer is characterized by having a structural formula shown as follows:

wherein n is more than or equal to 4 and less than or equal to 20.

2. The method for preparing the copolymer of chitosan oligosaccharide-N-linalool according to claim 1, comprising the steps of:

in the presence of a catalyst, dropwise adding the chitosan oligosaccharide solution into the linalyl bromide solution for reaction; after the reaction is finished, separating the product to obtain the chitosan oligosaccharide-N-linalool copolymer.

3. The method for preparing chitooligosaccharide-N-linalool copolymer according to claim 2, wherein the molecular weight of the chitooligosaccharide is less than or equal to 3000Da, the deacetylation degree is 85% -95%, and the polydispersity index is 0.88.

4. The method for preparing the copolymer of chitosan oligosaccharide-N-linalool as claimed in claim 2, wherein the mass-to-volume ratio of chitosan oligosaccharide to linalyl bromide is 1:1-1:3(g: mL).

5. The method for preparing chitosan oligosaccharide-N-linalool copolymer according to claim 2, wherein the catalyst is triethylamine, and the volume-to-mass ratio of the addition amount of the triethylamine to the chitosan oligosaccharide is 0.6:1-1.8:1(mL: g).

6. The method for preparing chitosan oligosaccharide-N-linalool copolymer according to claim 2, wherein the reaction temperature is 45-60 ℃ and the reaction time is 6-8 h.

7. The method for preparing the copolymer of chitosan oligosaccharide-N-linalool as claimed in claim 2, wherein the isolated product is specifically: after the reaction is finished, centrifuging and collecting the precipitate, performing Soxhlet extraction on the precipitate by using acetone, ethanol or petroleum ether, and performing vacuum drying to obtain light yellow powder; and dialyzing the powder in a dialysis bag with the molecular weight cutoff of 77-100 Da for 24 hours, concentrating and drying to obtain the purified chitosan oligosaccharide-N-linalool copolymer.

8. The method for preparing the copolymer of chitosan oligosaccharide-N-linalool according to claim 2, wherein the method for preparing the linalyl bromide comprises:

in an ice salt bath environment, dropwise adding a phosphorus tribromide solution into a linalool solution containing a catalyst, and stirring to perform bromination reaction; after the reaction is finished, washing the reaction product by using a 5% sodium bicarbonate solution, deionized water and saturated saline solution in sequence, adding a drying agent for drying, filtering, and performing rotary evaporation and concentration to obtain a light yellow oily liquid linalyl bromide.

9. The method for preparing the chitosan oligosaccharide-N-linalool copolymer according to claim 8, wherein the volume ratio of linalool to phosphorus tribromide is 5 (2-3);

the catalyst is pyridine, triethylamine or diisopropylethylamine, and the volume ratio of the addition amount of the catalyst to the linalool is (0.05-0.15) to 1;

the dropping speed is 1.0-1.5mL/min, and the stirring time is 45-90 min;

the drying agent is anhydrous sodium sulfate, anhydrous magnesium sulfate or anhydrous sodium carbonate;

the filter membrane used for filtration is a filter membrane with the diameter of 0.22-0.45 μm;

the time of the rotary evaporation concentration is 30-45min, and the temperature is 30-50 ℃.

10. Use of the chitosan oligosaccharide-N-linalool copolymer of claim 1 as an antibacterial or anti-inflammatory agent.

Technical Field

The invention relates to the technical field of antibacterial agents and anti-inflammatory agents, in particular to a chitosan oligosaccharide-N-linalool copolymer, a preparation method thereof and application of the chitosan oligosaccharide-N-linalool copolymer as an antibacterial agent/anti-inflammatory agent.

Background

There are a large number of natural products produced by living organisms and having particular biological activities in nature. Mainly comprises components or metabolites thereof extracted from animals, plant tissues, marine organisms and microorganisms, and a plurality of endogenous chemical components extracted from human bodies and animal bodies. Such as proteins, polypeptides, amino acids, polysaccharides, brass, terpenes, phenolic quinones, lactones, steroids, tannins, antibiotics, and the like. Since many natural products have some special functional properties, such as activities of anti-oxidation, anti-bacteria, anti-tumor, anti-inflammation, etc., they are widely used in the fields of medicine, food, cosmetics, agriculture, etc., and in recent years, they have received attention from researchers, and have become a research focus.

The chitosan oligosaccharide is low molecular weight alkaline amino oligosaccharide obtained by breaking a chitosan main chain through physical, chemical or enzymatic degradation. It is extracted from the shell of crustacean such as shrimp and crab, has biodegradability, biocompatibility, biological nontoxicity and chemical reaction activity, is often used as a natural antibacterial agent and antioxidant in food industry, besides, chitosan oligosaccharide also shows a certain anti-enteritis activity, so that it can be developed as a potential anti-inflammatory agent. However, since chitosan oligosaccharide is a natural macromolecular product, when used as an anti-inflammatory agent, it has a disadvantage of low activity compared to conventional general anti-inflammatory drugs, and thus, research thereof is currently very limited. Therefore, the structure of the chitosan oligosaccharide can be chemically modified, and due to the amino and hydroxyl groups with chemical reaction activity on the molecular chain, the sites are ideal action sites for chemically modifying the chitosan oligosaccharide, so that the functional activity of the chitosan oligosaccharide can be further improved, and the method is an effective method for a plurality of researches at home and abroad at present. Linalool, with the scientific name 3, 7-dimethyl-1, 6-octadien-3-ol, belongs to the class of chain terpene alcohols, and has both alpha-and beta-isomers. And levorotatory and dextrorotatory optical isomers. In essential oils of different sources, most are isomer mixtures, and have certain antibacterial, anti-inflammatory and antitumor activities.

Disclosure of Invention

The invention aims to solve the technical problem of providing a chitosan oligosaccharide-N-linalool copolymer which has good water solubility, thermal stability and anti-inflammatory activity and has good application prospect in the fields of food, medicine, cosmetics, agriculture and the like.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a chitosan oligosaccharide-N-linalool copolymer, which has the following structural formula:

wherein n is more than or equal to 4 and less than or equal to 20.

The second aspect of the present invention provides the method for preparing the chitosan oligosaccharide-N-linalool copolymer of the first aspect, comprising the following steps:

in the presence of a catalyst, dropwise adding the chitosan oligosaccharide solution into the linalyl bromide solution for reaction; after the reaction is finished, separating the product to obtain the chitosan oligosaccharide-N-linalool copolymer.

There are three chemical reaction sites in the molecular chain of chitosan oligosaccharide: the electron cloud density on the amino nitrogen at the C-2 position is the largest and the nucleophilicity is the strongest, while the electron cloud density on the hydroxyl nitrogen at the C-3 and C-6 positions is smaller than that on the amino nitrogen at the C-2 position, and the alkylation reaction firstly occurs on the amino at the C-2 position due to the influence of steric hindrance effect.

Furthermore, the molecular weight of the chitosan oligosaccharide is less than or equal to 3000Da, and the deacetylation degree is 85-95%; the polydispersity index is preferably 0.88.

Further, the organic solvent used for dissolving the chitosan oligosaccharide and the linalyl bromide can be one or more of N, N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and isopropyl acetate. Preferably, the volume mass ratio of the addition amount of the organic solvent to the chitosan oligosaccharide is 20:1-60:1(mL: g).

Further, the mass-volume ratio of the chitosan oligosaccharide to the linalyl bromide is 1:1-1:3(g: mL).

Further, the catalyst is triethylamine, and the volume mass ratio of the addition amount of the catalyst to the chitosan oligosaccharide is preferably 0.6:1-1.8:1(mL: g).

Further, the reaction temperature is preferably 45-60 ℃, and the reaction time is preferably 6-8 h.

Further, after the reaction is completed, the reaction is terminated by adding an excess amount of an organic solvent, preferably acetone.

Further, the separation product is specifically: after the reaction is finished, centrifuging and collecting the precipitate, performing Soxhlet extraction on the precipitate by using an organic solvent, and performing vacuum drying to obtain light yellow powder; dialyzing the powder in a dialysis bag for a period of time, concentrating and drying to obtain the purified chitosan oligosaccharide-N-linalool copolymer. The organic solvent can be selected from acetone, ethanol or petroleum ether, the dialysis bag is preferably a dialysis bag with the molecular weight cutoff of 77-100 Da, and the dialysis time is preferably 24 hours.

Furthermore, the substitution degree of the synthesized product of the chitosan oligosaccharide-N-linalool copolymer is 0.7-0.9, and the yield is 77-85%.

Further, the preparation method of the linalyl bromide comprises the following steps:

in an ice salt bath environment, dropwise adding a phosphorus tribromide solution into a linalool solution containing a catalyst, and stirring for reaction; after the reaction is finished, washing the reaction product by using a 5% sodium bicarbonate solution, deionized water and saturated saline solution in sequence, adding a drying agent for drying, filtering, and performing rotary evaporation and concentration to obtain a light yellow oily liquid linalyl bromide.

Further, the volume ratio of the linalool to the phosphorus tribromide is preferably 5 (2-3).

Further, the organic solvent for dissolving the linalool and the phosphorus tribromide can be one or more of anhydrous ethyl ether, anhydrous ethanol, propylene glycol and dichloromethane. In the linalool solution, the volume ratio of the organic solvent to the linalool is preferably (6-11): 1; in the phosphorus tribromide solution, the volume ratio of the organic solvent to the phosphorus tribromide is preferably (5-11): 1.

Further, the catalyst can be pyridine, triethylamine or diisopropylethylamine, and the volume ratio of the added amount of the catalyst to the linalool is preferably (0.05-0.15): 1.

Further, the dropping speed is preferably 1.0 to 1.5mL/min, and the stirring time is preferably 45 to 90 min.

Further, the drying agent may be anhydrous sodium sulfate, anhydrous magnesium sulfate, or anhydrous sodium carbonate.

Further, the filtration membrane used for the filtration is preferably a 0.22 to 0.45 μm membrane.

Further, the time of the rotary evaporation concentration is preferably 30-45min, and the temperature is preferably 30-50 ℃.

The third aspect of the invention also provides an application of the chitosan oligosaccharide-N-linalool copolymer of the first aspect as an antibacterial agent or an anti-inflammatory agent.

Compared with the prior art, the invention has the beneficial effects that:

1. based on the idea of substructure connection, linalool molecules with anti-inflammatory activity and C2-site active amino groups of chitosan oligosaccharide are subjected to N alkylation reaction, so that linalool is introduced into the molecular chain of the chitosan oligosaccharide, and the chitosan oligosaccharide-N-linalool copolymer is obtained. The antibacterial experiment result shows that linalool and chitosan oligosaccharide molecules in the chitosan oligosaccharide-N-linalool copolymer can generate a synergistic effect, so that the antibacterial activity of the copolymer is obviously enhanced; in addition, the chitosan oligosaccharide-N-linalool copolymer also has good anti-inflammatory activity.

2. The chitosan oligosaccharide-N-linalool copolymer has simple preparation process and purification process, low toxicity, good water solubility, thermal stability and anti-inflammatory activity, and has good application prospect in the fields of food, medicine, cosmetics, agriculture and the like.

Drawings

FIG. 1 is a synthetic scheme of a chitosan oligosaccharide-N-linalool copolymer;

FIG. 2 is an infrared spectrum of chitosan oligosaccharide, chitosan oligosaccharide-N-linalool copolymer;

FIG. 3 shows the preparation of chitosan oligosaccharide, linalool, and chitosan oligosaccharide-N-linalool copolymer¹H-NMR chart;

FIG. 4 is a thermogram of chitooligosaccharide, chitooligosaccharide-N-linalool copolymer;

FIG. 5 is a graph of the effect of chitooligosaccharide-N-linalool polymer on the proliferation of RAW264.7 cells;

FIG. 6 is a graph of the effect of chitosan oligosaccharide-N-linalool polymer on inflammatory factors.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available.