Acetylated carrageenan with anti-inflammatory activity as well as preparation method and application of acetylated carrageenan

Abstract

The invention relates to the technical field of food processing, in particular to acetylated carrageenan with anti-inflammatory activity and a preparation method and application thereof.The preparation method comprises the following steps that 1, carrageenan is subjected to acidolysis to obtain carrageenan powder with different molecular weights, and the carrageenan powder is added into formamide to obtain a mixed solution; 2, placing the mixed solution in a reaction environment at the temperature of 40-60 DEG C for reaction; 3, reacting to obtain an acetylated carrageenan solution; and 4, adding absolute ethyl alcohol to precipitate, centrifuging to take the precipitate, adding water to redissolve, dialyzing by using distilled water in a 500Da dialysis bag, and freeze-drying to obtain the acetylated carrageenan. The preparation method of the acetylated carrageenan has the beneficial effects that acetylation modification is performed on specific low-molecular-weight carrageenan, so that the acetylated carrageenan has the advantages of reducing inflammation of the low-molecular-weight carrageenan, improving the acetyl substitution degree, promoting the biological activity of the carrageenan and the like, and industrial production can be performed.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to an acetylated carrageenan with anti-inflammatory activity, its preparation method, and its application. Background Technology

[0002] Carrageenan, also known as carrageenan or carrageenan gum, is a linear sulfated polysaccharide extracted from the cell walls of red algae. It is one of the three major seaweed gums in my country (algum gum, carrageenan, and agar). Carrageenan plays an important role in the food industry as a thickener, gelling agent, emulsifier, and stabilizer in the production and processing of products such as jellies, gummies, meat patties, sausages, and ice cream.

[0003] In recent years, the issue of carrageenan products easily inducing intestinal inflammation has attracted widespread attention, becoming a key technological bottleneck affecting public health and the high-quality development of the carrageenan industry. While naturally occurring high-molecular-weight carrageenan in seaweed does not induce intestinal inflammation, some carrageenan degrades during processing, reducing its molecular weight and making it more susceptible to intestinal inflammation. This degradation can lead to intestinal cell apoptosis, damage to the integrity of the intestinal epithelial cell layer, and intestinal immune dysregulation, thus triggering inflammation and making its application in the food industry highly controversial. Sufficiently reducing low-molecular-weight carrageenan will be of great significance for its reuse and improving its comprehensive utilization value.

[0004] Acetylation, sulfonation, carboxymethylation, and phosphorylation are commonly used to modify polysaccharide structures. The introduction of certain functional groups alters molecular weight and other structural features, thereby affecting their processing and biological functional properties. Currently, domestic companies mostly use the acetic anhydride-pyridine method to acetylate polysaccharides. Acetylated polysaccharides exhibit better biological activity. Acetylation has been shown to stretch polysaccharide branches, change the spatial arrangement of polysaccharide chains, and expose hydroxyl groups, thus altering water solubility and hydrophobicity. Simultaneously, acetylation modification of polysaccharides has significant implications for the structure of the gut microbiota and its acetic acid production.

[0005] Chinese invention patent application No. 202011348235.1 discloses a method for preparing a galactose sulfate compound with anti-inflammatory activity. The method is characterized by sequentially subjecting agar extract from laver to dilute acid hydrolysis, enzymatic degradation, and dilute acid degradation, followed by separation and purification to obtain the galactose sulfate compound. This method can efficiently and completely degrade laver polysaccharide polymers into monosaccharide compounds, yielding a galactose sulfate compound, D-galactose-4-sulfatase, with excellent anti-inflammatory properties. This compound is highly suitable for applications in the research and development of anti-inflammatory drugs and skincare and health products with anti-inflammatory effects. While the above technical solution provides a method for degrading polysaccharides to achieve anti-inflammatory effects, it does not address the issue of carrageenan molecular weight reduction potentially inducing intestinal inflammation. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an acetylated carrageenan with anti-inflammatory activity, its preparation method and application.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing acetylated carrageenan with anti-colitis activity, comprising the following steps: Step 1: After acid hydrolysis of carrageenan, a mixed solution of carrageenan fragments is obtained. The different carrageenan fragments are collected by ultrafiltration and freeze-dried to obtain carrageenan powders of different molecular weights. The carrageenan powders are added to formamide to obtain a mixed solution. Step 2: Place the mixed solution obtained in Step 1 in a reaction environment with a temperature of 40℃-60℃ to react; Step 3: Add the reaction solution obtained in Step 2 to a mixed solution of pyridine and acetic anhydride to react and obtain an acetylated carrageenan solution; Step 4: Add 8-12 times the volume of distilled water to the acetylated carrageenan solution obtained in Step 3 to terminate the reaction, concentrate by rotary evaporation, add anhydrous ethanol to precipitate it, let it stand for 12-24 h, centrifuge to collect the precipitate, redissolve it in water, dialyze it with distilled water using a 500 Da dialysis bag, and freeze-dry to obtain acetylated carrageenan.

[0008] Furthermore, in the above-mentioned method for preparing acetylated carrageenan with anti-colitis activity, step 1 specifically involves: using 4M HCl solution to adjust the pH of the carrageenan acid hydrolysis environment to 3, reacting at 50°C for 3 hours, using 3M NaOH to adjust the pH to 7 to terminate the reaction, using an ultrafiltration membrane with a molecular weight of less than 200 kDa to collect the separated sample, then passing it through an ultrafiltration membrane with a molecular weight of greater than 50 kDa to collect the sample with a molecular weight of greater than 50 kDa, and freeze-drying to obtain carrageenan with a molecular weight between 50 kDa and 200 kDa. The carrageenan powder is then added to formamide to obtain a mixed solution.

[0009] Furthermore, in the above-mentioned method for preparing acetylated carrageenan with anti-colitis activity, the concentration of the carrageenan powder in the mixed solution is 50-100 mg / mL.

[0010] Furthermore, in the above-mentioned method for preparing acetylated carrageenan with anti-colitis activity, the volume ratio of pyridine to acetic anhydride in the mixed solution of pyridine and acetic anhydride is 1:1.

[0011] Furthermore, in the above-mentioned method for preparing acetylated carrageenan with anti-colitis activity, step 3 specifically involves: adding the reaction solution obtained in step 2 to a mixed solution of pyridine and acetic anhydride, reacting at 60°C for 4-24 hours to obtain an acetylated carrageenan solution.

[0012] Furthermore, in the above-mentioned method for preparing acetylated carrageenan with anti-colitis activity, in step 4, "adding anhydrous ethanol to precipitate", the mass percentage of ethanol in the solution obtained after adding anhydrous ethanol is 75%.

[0013] Furthermore, in the above-mentioned method for preparing acetylated carrageenan with anti-colitis activity, step 4 specifically involves: adding 10 times the volume of distilled water to the acetylated carrageenan solution obtained in step 3 to terminate the reaction, performing rotary evaporation to concentrate the solution, adding anhydrous ethanol to precipitate the solution, allowing it to stand for 12 hours, centrifuging to collect the precipitate, reconstituted with water, dialyzing with distilled water using a 500 Da dialysis bag for 72 hours, and freeze-drying to obtain acetylated carrageenan.

[0014] Another technical solution provided by the present invention is: to provide a composition with anti-colitis activity, wherein the effective component is acetylated carrageenan prepared by the above preparation method.

[0015] Another technical solution provided by the present invention is: to provide the application of acetylated carrageenan prepared by the above preparation method in the preparation of a composition with anti-colitis activity.

[0016] Furthermore, in the above applications, the colitis is a human colorectal adenocarcinoma Caco-2 cell model or an LPS-induced RAW264.7 macrophage inflammation model.

[0017] The beneficial effects of this invention are as follows: The preparation method of acetylated carrageenan obtained by the above-mentioned technical solution of this invention can acetylate carrageenan of different molecular weights, which has many advantages such as reducing the inflammatory effect of low molecular weight carrageenan, increasing the degree of acetyl substitution, and promoting the biological activity of carrageenan. Utilizing the above characteristics, this application can utilize the waste materials in existing carrageenan production, turning waste into treasure, and can also expand industrial production. Furthermore, the acetylated carrageenan prepared by the method of this invention has good anti-colonic inflammatory activity against human colorectal adenocarcinoma Caco-2 cell model and LPS-induced RAW264.7 macrophage inflammation model, and can be used as an anti-inflammatory composition and food additive. Attached Figure Description

[0018] Figure 1 This figure illustrates the effect of acetylated carrageenan on LPS-induced NO production from RAW264.7 in a specific embodiment of the present invention. Different lowercase letters in the figure represent significant differences (p < 0.05). Detailed Implementation

[0019] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0020] The key concept of this invention is that: existing research on carrageenan mainly focuses on extraction processes and the determination of its physical properties. At the same time, because the molecular weight limit of carrageenan with inflammatory response has not been clearly distinguished, it is impossible to distinguish carrageenan with pro-inflammatory effects, and these pro-inflammatory carrageenan have not been modified for reuse.

[0021] Studies by Premarathna et al. have shown that carrageenan is a polysaccharide sulfate ester composed of galactose and dehydrated galactose, with a molecular weight typically greater than 200 kDa, generally between 400-560 kDa, and semi-refined carrageenan with a molecular weight that can reach 615 kDa.

[0022] This application can separate different segments of carrageenan as needed and modify them by acetylation. It has many advantages, such as reducing the inflammatory effect of low molecular weight carrageenan, increasing the degree of acetyl substitution, and promoting the bioactivity of carrageenan, and can be industrialized.

[0023] The reason for separating different carrageenan fragments is that it is impossible to control the molecular weight of all carrageenan within the range required by the market during the production process. As a result, a lot of carrageenan with non-compliant molecular weights are generated during processing. In order to reduce this loss and reuse the waste generated in production, this application modifies and reuses the unused carrageenan that is not used in production or has inflammatory effects, and obtains carrageenan with beneficial effects.

[0024] Preferably, this application utilizes carrageenan waste generated during the production process to prepare acetylated carrageenan with anti-colitis activity, using carrageenan with a value less than 200 kDa, and further screening for carrageenan with a value greater than 50 kDa. Screening for carrageenan with a value greater than 50 kDa is because the yield of carrageenan with a value less than 50 kDa is relatively low during production, and this fragment has a more significant impact on the performance and inflammatory effect of carrageenan; screening for carrageenan with a value greater than 50 kDa can improve product quality.

[0025] Example 1 A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps: Step 1: Acid hydrolysis of commercially available carrageenan was performed using 4M HCl solution to adjust the pH of the carrageenan hydrolysis environment to 3. The reaction was carried out at 50°C for 3 hours. The pH was then adjusted to 7 using 3M NaOH to terminate the reaction. The sample was collected and separated using an ultrafiltration membrane with a molecular weight greater than 200 kDa, and then freeze-dried to obtain carrageenan with a molecular weight greater than 200 kDa. A mixed solution of carrageenan and formamide was prepared at a concentration of 50 mg / mL. Step 2: Place the mixed solution obtained in Step 1 in a reaction environment at 50°C and react for 30 minutes; Step 3: Add an equal volume of a mixed solution of pyridine and acetic anhydride (1:1) to the reaction solution from step 2, and react at 60°C for 12 hours to obtain an acetylated carrageenan solution. Step 4: Add 10 times the volume of distilled water to the acetylated carrageenan solution from Step 3 to terminate the reaction, concentrate by rotary evaporation, add anhydrous ethanol (final concentration 75%) to precipitate, let it stand overnight (12h), centrifuge to collect the precipitate, redissolve it in water, dialyze it with distilled water using a 500Da dialysis bag for 72h, and freeze-dry to obtain acetylated carrageenan.

[0026] Example 2 A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps: Step 1: Acid hydrolysis of commercially available carrageenan was performed using 4M HCl solution to adjust the pH of the carrageenan hydrolysis environment to 3. The reaction was carried out at 50°C for 3 hours. The pH was then adjusted to 7 using 3M NaOH to terminate the reaction. A separate sample with a molecular weight of less than 200 kDa was collected using an ultrafiltration membrane, followed by another ultrafiltration membrane with a molecular weight greater than 50 kDa to collect the sample with a molecular weight greater than 50 kDa. The sample was then freeze-dried to obtain carrageenan with a molecular weight between 50 kDa and 200 kDa. A mixed solution of carrageenan and formamide was prepared at a concentration of 75 mg / mL. Step 2: Place the mixed solution obtained in Step 1 in a reaction environment at 40℃ and react for 30 minutes; Step 3: Add the reaction solution from Step 2 to an equal volume of a mixed solution of pyridine and acetic anhydride (1:1), and react at 60°C for 12 hours to obtain an acetylated carrageenan solution. Step 4: Add 10 times the volume of distilled water to the acetylated carrageenan solution from Step 3 to terminate the reaction, concentrate by rotary evaporation, add anhydrous ethanol (final concentration 75%) to precipitate, let it stand overnight (12h), centrifuge to collect the precipitate, redissolve it in water, dialyze it with distilled water using a 500Da dialysis bag for 72h, and freeze-dry to obtain acetylated carrageenan.

[0027] Example 3 A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps: Step 1: Acid hydrolysis of commercially available carrageenan was performed using 4M HCl solution to adjust the pH of the carrageenan hydrolysis environment to 3. The reaction was carried out at 50°C for 3 hours. The pH was then adjusted to 7 using 3M NaOH to terminate the reaction. A separate sample with a molecular weight of less than 200 kDa was collected using an ultrafiltration membrane, followed by another sample with a molecular weight greater than 50 kDa using an ultrafiltration membrane. The sample was then freeze-dried to obtain carrageenan with a molecular weight between 50 kDa and 200 kDa. A mixed solution of low molecular weight carrageenan and formamide was prepared at a concentration of 75 mg / mL. Step 2: Place the mixed solution obtained in Step 1 in a reaction environment at 50°C and react for 30 minutes; Step 3: Add the reaction solution from Step 2 to an equal volume of a mixed solution of pyridine and acetic anhydride (1:1), and react at 60°C for 4 hours to obtain an acetylated carrageenan solution. Step 4: Add 10 times the volume of distilled water to the acetylated carrageenan solution from Step 3 to terminate the reaction, concentrate by rotary evaporation, add anhydrous ethanol (final concentration 75%) to precipitate, let it stand overnight (12h), centrifuge to collect the precipitate, redissolve it in water, dialyze it with distilled water using a 500Da dialysis bag for 72h, and freeze-dry to obtain acetylated carrageenan.

[0028] Example 4 A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps: Step 1: Acid hydrolysis of commercially available carrageenan was performed using 4M HCl solution to adjust the pH of the carrageenan hydrolysis environment to 3. The reaction was carried out at 50°C for 3 hours. The pH was then adjusted to 7 using 3M NaOH to terminate the reaction. A separate sample with a molecular weight of less than 200 kDa was collected using an ultrafiltration membrane with a molecular weight greater than 50 kDa. This sample was then passed through an ultrafiltration membrane with a molecular weight greater than 50 kDa to collect the sample with a molecular weight of less than 50 kDa. The resulting carrageenan was freeze-dried to obtain a molecular weight of less than 50 kDa. A mixed solution of low molecular weight carrageenan and formamide was prepared at a concentration of 100 mg / mL. Step 2: Place the mixed solution obtained in Step 1 in a reaction environment at 50°C and react for 30 minutes; Step 3: Add the reaction solution from Step 2 to an equal volume of a mixed solution of pyridine and acetic anhydride (1:1), and react at 60°C for 12 hours to obtain an acetylated carrageenan solution. Step 4: Add 10 times the volume of distilled water to the acetylated carrageenan solution from Step 3 to terminate the reaction, concentrate by rotary evaporation, add anhydrous ethanol (final concentration 75%) to precipitate, let it stand overnight (12h), centrifuge to collect the precipitate, redissolve it in water, dialyze it with distilled water using a 500Da dialysis bag for 72h, and freeze-dry to obtain acetylated carrageenan.

[0029] Example 5 A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps: Step 1: Acid hydrolysis of commercially available carrageenan was performed using 4M HCl solution to adjust the pH of the carrageenan hydrolysis environment to 3. The reaction was carried out at 50°C for 3 hours. The pH was then adjusted to 7 using 3M NaOH to terminate the reaction. A separate sample with a molecular weight of less than 200 kDa was collected using an ultrafiltration membrane with a molecular weight greater than 50 kDa. This sample was then passed through an ultrafiltration membrane with a molecular weight greater than 50 kDa to collect the sample with a molecular weight of less than 50 kDa. The resulting carrageenan was freeze-dried to obtain a molecular weight of less than 50 kDa. A mixed solution of low molecular weight carrageenan and formamide was prepared at a concentration of 75 mg / mL. Step 2: Place the mixed solution obtained in Step 1 in a reaction environment at 60℃ and react for 30 minutes; Step 3: Add the reaction solution from Step 2 to an equal volume of a mixed solution of pyridine and acetic anhydride (1:1), and react at 60°C for 12 hours to obtain an acetylated carrageenan solution. Step 4: Add 10 times the volume of distilled water to the acetylated carrageenan solution from Step 3 to terminate the reaction, concentrate by rotary evaporation, add anhydrous ethanol (final concentration 75%) to precipitate, let it stand overnight (12h), centrifuge to collect the precipitate, redissolve it in water, dialyze it with distilled water using a 500Da dialysis bag for 72h, and freeze-dry to obtain acetylated carrageenan.

[0030] Example 6 A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps: Step 1: Acid hydrolysis of commercially available carrageenan was performed using 4M HCl solution to adjust the pH of the carrageenan hydrolysis environment to 3. The reaction was carried out at 50°C for 3 hours. The pH was then adjusted to 7 using 3M NaOH to terminate the reaction. A separate sample with a molecular weight of less than 200 kDa was collected using an ultrafiltration membrane with a molecular weight greater than 50 kDa. This sample was then passed through an ultrafiltration membrane with a molecular weight greater than 50 kDa to collect the sample with a molecular weight of less than 50 kDa. The resulting carrageenan was freeze-dried to obtain a molecular weight of less than 50 kDa. A mixed solution of low molecular weight carrageenan and formamide was prepared at a concentration of 75 mg / mL. Step 2: Place the mixed solution obtained in Step 1 in a reaction environment at 50°C and react for 30 minutes; Step 3: Add the reaction solution from Step 2 to an equal volume of a mixed solution of pyridine and acetic anhydride (1:1), and react at 60°C for 24 hours to obtain an acetylated carrageenan solution; Step 4: Add 10 times the volume of distilled water to the acetylated carrageenan solution from Step 3 to terminate the reaction, concentrate by rotary evaporation, add anhydrous ethanol (final concentration 75%) to precipitate, let it stand overnight (12h), centrifuge to collect the precipitate, redissolve it in water, dialyze it with distilled water using a 500Da dialysis bag for 72h, and freeze-dry to obtain acetylated carrageenan.

[0031] Comparative Example 1 The difference from the examples is that the carrageenan is not subjected to any acetylation treatment.

[0032] Comparative Example 2 The rest is the same as in Example 1, except that in step 1, the concentration of low molecular weight carrageenan is 200 mg / mL.

[0033] Comparative Example 3 The rest is the same as in Example 1, except that in step 2, the reaction temperature of the mixed solution is 4°C.

[0034] Comparative Example 4 The rest is the same as in Example 1, except that in step 3, the reaction time of the mixed solution is 2 hours.

[0035] Analysis and comparison The physicochemical properties of the acetylated carrageenan prepared in Examples 1-6 and Comparative Examples 1-4 were determined using the following methods: Determination of total sugar, sulfate, 3,6-galactose content and degree of acetyl substitution (1) The total sugar content of acetylated carrageenan from Examples 1-6 and Comparative Examples 1-4 was determined. 1 mg of acetylated carrageenan from different processes, dried to constant weight, was accurately weighed and prepared into a 1 mg / mL acetylated carrageenan solution. The total sugar content in the acetylated carrageenan was determined using the phenol-sulfuric acid method with galactose as the standard. 1 mL of the sample to be tested was placed in a centrifuge tube, 1 mL of 6% phenol solution was added, and then 6 mL of concentrated sulfuric acid was quickly added and mixed thoroughly. The above steps were repeated, and the total sugar content in the sample was determined according to the galactose standard curve.

[0036] (2) Determination of sulfate content in acetylated carrageenan from Examples 1-6 and Comparative Examples 1-4. Accurately weigh 1 mg of acetylated carrageenan from different treatments, dried to constant weight, and prepare a 1 mg / mL acetylated carrageenan solution. Using potassium sulfate as a standard, the sulfate content in the samples was determined by the barium chloride-gelatin turbidimetric method. Take 3 mL of the sample to be tested into a centrifuge tube, add 3.8 mL of 0.2 M HCl, then quickly add 1 mL of gelatin-barium chloride solution and mix thoroughly. Let stand for 30 min. Repeat the above steps, and determine the sulfate content in the samples according to the potassium sulfate standard curve.

[0037] (3) The acetylated carrageenan from Examples 1-3 and Comparative Examples 1-5 was used to determine the 3,6-galactose content. 1 mg of acetylated carrageenan from different treatments, dried to constant weight, was accurately weighed and prepared into a 1 mg / mL acetylated carrageenan solution. Using fructose as a standard, the 3,6-galactose content was determined using the resorcinol method. 1 mL of the acetylated carrageenan solution was placed in an ice-water bath for 5 min. Then, 5.00 mL of resorcinol reagent was added to each test tube, shaken well, and reacted at 80°C for 15 min. After the reaction, the tubes were placed in ice water for 2 min, and the above steps were repeated. The absorbance of the solution in each test tube was measured at 554 nm. The 3,6-galactose content in the sample was determined according to the fructose standard curve.

[0038] (4) The acetylated carrageenan of Examples 1-3 and Comparative Examples 1-5 were used to determine the degree of acetylation. 20 mg (m) of acetylated carrageenan dried to constant weight was accurately weighed and added to 10 mL (V0) of 0.01 M (CO) NaOH solution. The solution was fully dissolved and placed in a 15 mL centrifuge tube. The tube was placed in a constant temperature shaker and reacted for 2 h (250 rpm, 50 °C). After the reaction was completed, 1% phenolphthalein solution was used as an indicator and 0.01 M (Cl) HCl solution was used to titrate until the red color disappeared (pH=7.0). The volume of hydrochloric acid (V1) was recorded and the degree of acetylation was calculated using formula (2).

[0039] (1) (2) Table 1 shows the total sugar content, sulfate content, 3,6-intraether-D-galactose content, and degree of acetylation of acetylated carrageenan in Examples 1-6 and Comparative Examples 1-4 of the present invention.

[0040] Table 1

[0041] Note: "-" in Table 1 indicates that the relevant indicator was not detected.

[0042] As shown in Table 1, the total sugar content of the acetylated carrageenan prepared in Examples 1-6 is comparable to that in Comparative Examples 1-4, but the sulfate content is lower and the 3,6-endoether-D-galactose content is higher. This indicates that the preparation method of the present invention substitutes the sulfate sites with acetyl groups and promotes the formation of 3,6-endoether-D-galactose in the acetylated carrageenan. At the same time, the degree of acetyl substitution is higher than that in Comparative Examples 1-4. Based on the prior art, it is inferred that the acetylated carrageenan prepared by the present invention may have higher biological activity in subsequent applications.

[0043] (8) Anti-colitis test on the acetylated carrageenan prepared in Examples 1-6 The cell models involved include: human colorectal adenocarcinoma Caco-2 cell model and LPS-induced RAW264.7 macrophage inflammation model.

[0044] The testing method is as follows: (1) Human colorectal adenocarcinoma Caco-2 cells were injected with 2×10 5 Cells / mL were seeded in 12-well transwells and cultured for 21 days, forming a tight monolayer with a TEER > 600 Ω•cm. 2 To begin the experiment, acetylated carrageenan was dissolved in sterile PBS to a concentration of 5 mg / mL. After filtration through a 0.22 μM filter, the solution was placed on ice. 500 μL of a mixture of fluorescein and acetylated carrageenan (fluorescein solution concentration 20 μg / mL; sample concentration 100 mg / mL) was added to the AP side, and 1500 mL of HBSS was added to the BL side. After 24 h of incubation, 300 μL samples were taken from the BL side at 0.5, 1, 2, 4, 6, 12, and 24 h. Each time a sample was taken, 300 μL of HBSS was added to the BL side. x 427 nm, λe m The A value was detected at 536 nm. The concentration of fluorescein in the BL-side transport solution was calculated based on the standard curve, and the apparent permeability coefficient Papp was calculated using the following formula to verify the effect of acetylated carrageenan on the integrity of the Caco-2 cell monolayer. The results are shown in Tables 2 and 3.

[0045] Dq / dt represents the amount of fluorescein transported per unit time (μg / s), and A represents the effective membrane area (1.12 cm²). 2 C0 represents the initial concentration of fluorescein (20 μg / mL). Table 2

[0046] Table 3

[0047] (2) RAW264.7 cells in the logarithmic growth phase were injected with 3 × 10⁻⁶ cells. 4Cells were seeded at different concentrations per well in 96-well cell culture plates and incubated. Once cells reached 85% confluence, they were treated with 100 μL of culture medium containing different concentrations of carrageenan fermentation products for 12 h, with six replicates per treatment. The culture medium was then removed, and LPS was added to both the carrageenan fermentation product treatment group and the control group for another 12 h. NO release from RAW264.7 cells was measured using the Griess method. 50 μL of culture supernatant was transferred from each well to a new 96-well cell culture plate, and then 100 μL of Griess reagent (containing 3 mol / L sulfanilamide, 30 mg / L N-1-(naphthyl)ethylenediamine dihydrochloride, and 25% glacial acetic acid) was added to the supernatant. The plates were incubated at room temperature in the dark for 20 min, and the absorbance (OD) at 540 nm was measured. 540 Simultaneously, standard curves were plotted using NaNO2 standard solutions of different concentrations, and the NO concentration in the culture supernatant was calculated. The results are as follows: Figure 1 As shown: The acetylated carrageenan prepared by the extraction method of this invention has anti-inflammatory activity. Because it is derived from natural seaweed, it has good safety and can be used as a biological agent to relieve inflammation. It can also be used in food preparation and can be prepared into anti-inflammatory compositions, etc., showing good application prospects.

[0048] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing acetylated carrageenan having anti-colitis activity, characterized in that: The following steps are involved: Step 1: acid hydrolyze carrageenan to obtain a mixed solution of carrageenan with different fragments, collect the carrageenan with different fragments by ultrafiltration, and freeze-dry to obtain carrageenan powder with different molecular weights, and add the carrageenan powder into formamide to obtain a mixed solution; Step 2: placing the mixed solution obtained in step 1 in a reaction environment at a temperature of 40°C-60°C for reaction; Step 3: adding the reaction solution obtained in step 2 to a mixed solution of pyridine and acetic anhydride to react to obtain an acetylated carrageenan solution; Step 4: Add 8-12 times the volume of distilled water of the formamide to the acetylated carrageenan solution obtained in step 3 to terminate the reaction, perform rotary evaporation concentration, add anhydrous ethanol to precipitate, place for 12-24 hours, centrifuge to obtain the precipitate, add water to redissolve, dialyze with distilled water using a 500Da dialysis bag, and freeze-dry to obtain the acetylated carrageenan.

2. The method for preparing acetylated carrageenan with anti-colitis activity according to claim 1, characterized in that: The step 1 specifically comprises: using 4M HCl solution to adjust the pH value of the carrageenan acid hydrolysis environment to 3, reacting at 50°C for 3 hours, using 3MNaOH to adjust the pH value to 7 to terminate the reaction, using an ultrafiltration membrane less than 200 kDa to collect separation samples less than 200 kDa, then passing through an ultrafiltration membrane greater than 50 kDa to collect samples greater than 50 kDa, freeze-drying to obtain carrageenan with a molecular weight of 50 kDa-200 kDa, and adding the carrageenan powder to formamide to obtain a mixed solution.

3. The method for preparing acetylated carrageenan with anti-colitis activity according to claim 2, characterized in that: The concentration of the carrageenan powder in the mixed solution is 50-100 mg / mL.

4. The method for preparing acetylated carrageenan with anti-colitis activity according to claim 1, characterized in that: The volume ratio of pyridine to acetic anhydride in the mixed solution of pyridine and acetic anhydride is 1:

1.

5. The method for preparing acetylated carrageenan with anti-colitis activity according to claim 1, characterized in that: The step 3 is specifically as follows: adding the reaction solution obtained in step 2 to a mixed solution of pyridine and acetic anhydride, reacting at 60° C. for 4 h to 24 h to obtain an acetylated carrageenan solution.

6. The method for preparing acetylated carrageenan with anti-colitis activity according to claim 1, characterized in that: In step 4, in the step of "adding anhydrous ethanol to precipitate", the mass percentage of ethanol solute in the resulting solution after adding anhydrous ethanol is 75%.

7. The method for preparing acetylated carrageenan with anti-colitis activity according to claim 1, characterized in that: The step 4 is specifically as follows: adding distilled water with a volume of 10 times that of the formamide to the acetylated carrageenan solution obtained in step 3 to terminate the reaction, performing rotary evaporation concentration, adding anhydrous ethanol to precipitate the solution, placing the solution for 12 hours, centrifuging the solution to obtain the precipitate, adding water to redissolve the solution, dialyzing the solution with distilled water using a 500Da dialysis bag for 72 hours, and freeze-drying the solution to obtain the acetylated carrageenan.

8. A composition having anti-colitis activity, characterized in that: The active ingredient is acetylated carrageenan prepared by the preparation method described in any one of claims 1 to 7.

9. Use of the acetylated carrageenan prepared by the preparation method according to any one of claims 1 to 7 in preparing a composition having anti-colitis activity.

10. The use according to claim 9, characterized in that The colitis is a human colorectal adenocarcinoma Caco-2 cell model or an LPS-induced RAW264.7 macrophage inflammation model.

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