An acetylated carrageenan having anti-inflammatory activity, and a method of preparing and using the same

Abstract

The present application relates to the technical field of food processing, and particularly relates to acetylated carrageenan with anti-inflammatory activity and a preparation method and application thereof, the preparation method comprising the following steps: step 1: carrageenan acidolysis, obtaining carrageenan powder with different molecular weights, and adding the carrageenan powder into formamide to obtain a mixed solution; step 2: placing the mixed solution in a reaction environment with a temperature of 40-60 DEG C to react; step 3: obtaining an acetylated carrageenan solution by reaction; step 4: adding anhydrous ethanol to precipitate, centrifuging to obtain the precipitate, resolubilizing with water, using a 500Da dialysis bag to dialyze with distilled water, and freeze-drying to obtain acetylated carrageenan. The present application has the beneficial effect that the preparation method of the acetylated carrageenan obtained by the above technical solution of the present application acetylates and modifies specific low-molecular-weight carrageenan, has the effects of reducing inflammation of low-molecular-weight carrageenan, improving the degree of acetyl substitution, promoting the biological activity of carrageenan and many other advantages, and can be produced industrially.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food processing, and particularly relates to acetylated carrageenan with anti-inflammatory activity and a preparation method and application thereof. BACKGROUND

[0002] Carrageenan, also known as carrageenan or dragon's blood, is a linear sulfated polysaccharide extracted from red algae cell walls, and is one of the three major seaweed gums (carrageenan, carrageenan and agar) in China. Carrageenan can be used as a thickening agent, gelling agent, emulsifier, stabilizer, etc. in the production and processing of jelly, soft candy, meat pie, sausage and ice cream, etc. and plays an important role in the food industry.

[0003] In recent years, the problem that carrageenan products easily induce intestinal inflammation has attracted widespread attention and become a key technical bottleneck affecting people's health and high-quality development of the carrageenan industry. High molecular weight carrageenan naturally exists in seaweed and does not induce intestinal inflammation, but part of the carrageenan is degraded during the processing and preparation of carrageenan, and the low molecular weight carrageenan easily induces intestinal inflammation, causes intestinal cell apoptosis, destroys the integrity of the intestinal epithelial cell layer, causes intestinal immune disorders and induces inflammation, so that its application in the food industry is controversial. It is of great significance to fully reduce low molecular weight carrageenan for reusing low molecular weight carrageenan and improving the comprehensive utilization value of carrageenan.

[0004] Acetylation, sulfonation, carboxymethylation and phosphorylation are often used to modify polysaccharide structure. The introduction of certain functional groups changes the molecular weight and other structural characteristics, thereby affecting their processing and biological functional properties. At present, domestic enterprises often use the acetic anhydride-pyridine method to modify polysaccharides by acetylation. Polysaccharides modified by acetylation exhibit better biological activity, and acetylation is proved to stretch the branches of polysaccharides, change the spatial arrangement of polysaccharide chains, expose hydroxyl groups, and thus change the water solubility and hydrophobicity. At the same time, acetylation modification of polysaccharides has important significance for intestinal flora structure and acetic acid production.

[0005] The Chinese invention patent application with the application number 202011348235.1 discloses a preparation method of a galactose sulfate acetylated compound with anti-inflammatory activity, which is characterized in that the seaweed agar extract is sequentially subjected to dilute acid hydrolysis, enzyme degradation and dilute acid degradation, and a galactose sulfate acetylated compound is obtained after separation and purification. It can efficiently degrade the polysaccharide polymer in seaweed into monosaccharide compounds, and obtain a galactose sulfate acetylated compound D-galactose-4-sulfate with excellent anti-inflammatory effect, which is very suitable for application in the fields of research and development of anti-inflammatory drugs and skin care products, health care products with anti-inflammatory effect, etc. The above technical solution gives the degradation of polysaccharides to obtain products with anti-inflammatory effect, but the above technical solution does not give any solution to the problem that the low molecular weight carrageenan easily induces intestinal inflammation. SUMMARY

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

[0007] To solve the above technical problem, the technical scheme adopted by the present application is as follows: a preparation method of acetylated carrageenan with anti-colitis activity, comprising the following steps:

[0008] Step 1: obtaining a mixed solution of different fragment carrageenan after carrageenan acidolysis, collecting different fragment carrageenan by ultrafiltration, and freeze-drying to obtain carrageenan powder with different molecular weights, and adding the carrageenan powder into formamide to obtain a mixed solution;

[0009] Step 2: placing the mixed solution obtained in step 1 in a reaction environment with a temperature of 40-60°C for reaction;

[0010] Step 3: adding the reaction solution obtained in step 2 into a mixed solution of pyridine and acetic anhydride to obtain an acetylated carrageenan solution by reaction;

[0011] Step 4: adding the acetylated carrageenan solution obtained in step 3 into 8-12 times the volume of distilled water of the formamide to terminate the reaction, performing rotary evaporation concentration, adding anhydrous ethanol to precipitate, placing for 12-24 hours, centrifuging to obtain the precipitate, redissolving with water, using a 500Da dialysis bag to dialyze with distilled water, and freeze-drying to obtain acetylated carrageenan.

[0012] Further, in the preparation method of acetylated carrageenan with anti-colitis activity, step 1 is specifically as follows: using 4M HCl solution to adjust the pH of the carrageenan acidolysis 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 less than 200kDa to collect a sample with a molecular weight less than 200kDa, and then passing the sample through an ultrafiltration membrane with a molecular weight greater than 50kDa to collect a sample with a molecular weight greater than 50kDa, and freeze-drying to obtain carrageenan with a molecular weight of 50-200kDa, and adding the carrageenan powder into formamide to obtain a mixed solution.

[0013] Further, in the preparation method of acetylated carrageenan with anti-colitis activity, the concentration of the carrageenan powder in the mixed solution is 50-100mg / mL.

[0014] Further, in the preparation method of 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.

[0015] Further, in the preparation method of the acetylated carrageenan with anti-colitis activity, the step 3 is specifically: the reaction solution obtained in step 2 is added into a mixed solution of pyridine and acetic anhydride, and reacted at 60℃ for 4h-24h, to obtain an acetylated carrageenan solution.

[0016] Further, in the preparation method of the acetylated carrageenan with anti-colitis activity, the solute mass percentage of ethanol in the solution obtained after adding anhydrous ethanol in the step of "adding anhydrous ethanol to precipitate" in step 4 is 75%.

[0017] Further, in the preparation method of the acetylated carrageenan with anti-colitis activity, the step 4 is specifically: the acetylated carrageenan solution obtained in step 3 is added into 10 times volume of distilled water to terminate the reaction, concentrated by rotary evaporation, precipitated by adding anhydrous ethanol, placed for 12h, centrifuged to obtain the precipitate, redissolved in water, dialyzed in 500Da dialysis bag for 72h, and freeze-dried to obtain the acetylated carrageenan.

[0018] The application provides another technical scheme: providing a composition with anti-colitis activity, and an effective component of the composition is the acetylated carrageenan prepared by the preparation method.

[0019] The application provides still another technical scheme: providing an application of the acetylated carrageenan prepared by the preparation method in preparing a composition with anti-colitis activity.

[0020] Further, in the application, the colitis is a Caco-2 cell model of human colorectal adenocarcinoma cells or an LPS-induced RAW264.7 macrophage inflammation model.

[0021] The application has the advantages that the preparation method of the acetylated carrageenan obtained by the technical scheme has the advantages of acetylation modification on different molecular weight carrageenan, reduction of inflammation effect of low molecular weight carrageenan, improvement of acetyl substitution degree, promotion of biological activity of carrageenan and the like, the existing waste in carrageenan production can be utilized to turn waste into treasure, and the industrial production can be expanded. Further, the acetylated carrageenan prepared by the preparation method has good anti-colitis activity on the Caco-2 cell model of human colorectal adenocarcinoma cells and the LPS-induced RAW264.7 macrophage inflammation model, and can be applied as an anti-inflammatory composition and a food additive. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The figure shows the influence of the acetylated carrageenan on the production of NO by LPS-induced RAW264.7. Different lowercase letters in the figure represent significant differences, and p<0.05. DETAILED DESCRIPTION

[0023] To explain the technical content of the present application, the purposes and effects achieved, the following embodiments are described in conjunction with the accompanying drawings.

[0024] The key idea of the present application is that the research on carrageenan in the prior art mainly focuses on the extraction process and the determination of the physical properties of carrageenan. At the same time, since the molecular weight limit of carrageenan with inflammatory response is not clearly distinguished, it is not possible to distinguish carrageenan with pro-inflammatory effect, and these pro-inflammatory carrageenan are not modified for re-use.

[0025] In the study of Premarathna et al., carrageenan polysaccharide is a polysaccharide sulfate composed of galactose and anhydrosugar, with a molecular weight usually greater than 200 kDa, and the molecular weight is generally between 400-560 kDa, and the molecular weight of semi-refined carrageenan can reach 615 kDa.

[0026] The present application can separate different segments of carrageenan as needed and modify the acetylation of different segments of carrageenan, which has the advantages of reducing the inflammatory effect of low molecular weight carrageenan, improving the degree of acetyl substitution, promoting the biological activity of carrageenan, etc., and can be produced industrially.

[0027] The reason for separating different segments of carrageenan is that it is not possible to control the molecular weight of all carrageenan in the production process to meet the market requirements, and a large amount of carrageenan that does not meet the molecular weight will be produced during processing. To reduce this loss and re-use the waste produced in production, the present application modifies and re-uses this type of unused carrageenan with inflammatory effects that are not used, and obtains beneficial and good carrageenan.

[0028] Preferably, the present application uses waste carrageenan produced during production to prepare acetylated carrageenan with anti-colitis activity, uses carrageenan less than 200 kDa, and further selects carrageenan greater than 50 kDa. The reason for selecting carrageenan greater than 50 kDa is that the yield of carrageenan less than 50 kDa is relatively small during production, and this segment has a more serious impact on the performance and inflammatory effect of carrageenan. Selecting carrageenan greater than 50 kDa can improve product quality.

[0029] Example 1

[0030] A method for preparing acetylated carrageenan with anti-colitis activity, comprising the following steps:

[0031] Step 1: Acid hydrolysis of commercially available carrageenan was performed by adjusting the pH of the carrageenan to 3 using 4M HC1 solution and reacting at 50°C for 3 hours. The reaction was terminated by adjusting the pH to 7 using 3M NaOH. The sample was collected using an ultrafiltration membrane with a molecular weight cut-off of 200 kDa, and freeze-dried to obtain carrageenan with a molecular weight of greater than 200 kDa. A mixed solution of carrageenan and formamide was prepared at a concentration of 50 mg / mL.

[0032] Step 2: The mixed solution obtained in Step 1 was placed in a reaction environment at a temperature of 50°C for 30 minutes.

[0033] Step 3: The reaction solution of Step 2 was added to an equal amount of a mixed solution of pyridine and acetic anhydride (1:1) by volume, and reacted at 60°C for 12 hours to obtain an acetylated carrageenan solution.

[0034] Step 4: The acetylated carrageenan solution of Step 3 was added to 10 times the volume of distilled water to terminate the reaction, and concentrated by rotary evaporation. Anhydrous ethanol was added to precipitate the product, which was left overnight (12 hours). The precipitate was collected by centrifugation, resuspended in water, and dialyzed against distilled water using a 500 Da dialysis bag for 72 hours. The acetylated carrageenan was obtained by freeze-drying.

[0035] Example 2

[0036] A method for preparing an acetylated carrageenan with anti-colitis activity, comprising the following steps:

[0037] Step 1: Acid hydrolysis of commercially available carrageenan was performed by adjusting the pH of the carrageenan to 3 using 4M HC1 solution and reacting at 50°C for 3 hours. The reaction was terminated by adjusting the pH to 7 using 3M NaOH. The sample was collected using an ultrafiltration membrane with a molecular weight cut-off of 200 kDa, and freeze-dried to obtain carrageenan with a molecular weight of greater than 200 kDa. A mixed solution of carrageenan and formamide was prepared at a concentration of 50 mg / mL.

[0038] Step 2: The mixed solution obtained in Step 1 was placed in a reaction environment at a temperature of 50°C for 30 minutes.

[0039] Step 3: The reaction solution of Step 2 was added to an equal amount of a mixed solution of pyridine and acetic anhydride (1:1) by volume, and reacted at 60°C for 12 hours to obtain an acetylated carrageenan solution.

[0040] 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.

[0041] Example 3

[0042] A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps:

[0043] 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.

[0044] Step 2: Place the mixed solution obtained in Step 1 in a reaction environment at 50°C and react for 30 minutes;

[0045] 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.

[0046] 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.

[0047] Example 4

[0048] A method for preparing acetylated carrageenan with anti-colitis activity includes the following steps:

[0049] 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.

[0050] Step 2: The mixed solution obtained in Step 1 is placed in a reaction environment at a temperature of 50°C for 30 min;

[0051] Step 3: The reaction solution of Step 2 is added to an equal amount of a mixed solution of pyridine and acetic anhydride (1:1) by volume ratio, and reacted at 60°C for 12 h to obtain an acetylated carrageenan solution;

[0052] Step 4: The acetylated carrageenan solution of Step 3 is added to 10 times the volume of distilled water to terminate the reaction, concentrated by rotary evaporation, precipitated by adding anhydrous ethanol (final concentration 75%), and allowed to stand overnight (12 h). The precipitate is centrifuged, resuspended in water, dialyzed against distilled water using a 500 Da dialysis bag for 72 h, and freeze-dried to obtain acetylated carrageenan.

[0053] Example 5

[0054] A method for preparing acetylated carrageenan having anti-colitis activity, comprising the following steps:

[0055] Step 1: Acid hydrolysis of commercially available carrageenan is performed by adjusting the pH of the carrageenan acid hydrolysis environment to 3 using a 4M HC1 solution, reacting at 50°C for 3 hours, adjusting the pH to 7 using a 3M NaOH solution to terminate the reaction, collecting a sample of less than 200 kDa using an ultrafiltration membrane with a molecular weight cutoff of less than 200 kDa, and then collecting a sample of less than 50 kDa using an ultrafiltration membrane with a molecular weight cutoff of greater than 50 kDa, and freeze-drying to obtain carrageenan with a molecular weight of less than 50 kDa. A mixed solution of low molecular weight carrageenan and formamide is prepared at a concentration of 75 mg / mL;

[0056] Step 2: The mixed solution obtained in Step 1 is placed in a reaction environment at a temperature of 60°C for 30 min;

[0057] Step 3: The reaction solution of Step 2 is added to an equal amount of a mixed solution of pyridine and acetic anhydride (1:1) by volume ratio, and reacted at 60°C for 12 h to obtain an acetylated carrageenan solution;

[0058] Step 4: The acetylated carrageenan solution of Step 3 is added to 10 times the volume of distilled water to terminate the reaction, concentrated by rotary evaporation, precipitated by adding anhydrous ethanol (final concentration 75%), and allowed to stand overnight (12 h). The precipitate is centrifuged, resuspended in water, dialyzed against distilled water using a 500 Da dialysis bag for 72 h, and freeze-dried to obtain acetylated carrageenan.

[0059] Example 6

[0060] A method for preparing acetylated carrageenan having anti-colitis activity, comprising the following steps:

[0061] Step 1: Acid hydrolysis of commercially available carrageenan was performed by adjusting the pH of carrageenan to 3 using 4M HC1 solution and reacting at 50°C for 3 hours. The reaction was terminated by adjusting the pH to 7 using 3M NaOH. The sample was collected by using a membrane with a cut-off of less than 200 kDa and then by using a membrane with a cut-off of more than 50 kDa. The sample with a molecular weight of less than 50 kDa was collected and freeze-dried to obtain low molecular weight carrageenan. A mixture of low molecular weight carrageenan and formamide was prepared at a concentration of 75 mg / mL.

[0062] Step 2: The mixture obtained in Step 1 was placed in a reaction environment at a temperature of 50°C for 30 min.

[0063] Step 3: The reaction solution of Step 2 was added to an equal amount of a mixed solution of pyridine and acetic anhydride (1:1) by volume ratio, and reacted at 60°C for 24 h to obtain an acetylated carrageenan solution.

[0064] Step 4: The acetylated carrageenan solution of Step 3 was added to 10 times the volume of distilled water to terminate the reaction, concentrated by rotary evaporation, precipitated with anhydrous ethanol (final concentration 75%), and centrifuged to obtain the precipitate. The precipitate was redissolved in water and dialyzed against distilled water for 72 h using a 500 Da dialysis bag. The acetylated carrageenan was obtained by freeze-drying.

[0065] Comparative Example 1

[0066] The difference from the example is that the carrageenan is not acetylated.

[0067] Comparative Example 2

[0068] The same as Example 1, except that in Step 1, the concentration of low molecular weight carrageenan is 200 mg / mL.

[0069] Comparative Example 3

[0070] The same as Example 1, except that in Step 2, the reaction temperature of the mixed solution is 4°C.

[0071] Comparative Example 4

[0072] The same as Example 1, except that in Step 3, the reaction time of the mixed solution is 2 h.

[0073] Analysis Comparison

[0074] The physicochemical properties of the acetylated carrageenan prepared in Examples 1-6 and Comparative Examples 1-4 were determined, and the test methods are as follows:

[0075] Determination of total sugar, sulfate, 3,6-anhydrogalactose content, and acetyl substitution degree

[0076] (1) The acetylated carrageenan of Examples 1-6 and Comparative Examples 1-4 was taken to measure the total sugar content. 1 mg of the acetylated carrageenan of different processes dried to constant weight was accurately weighed and configured into an acetylated carrageenan solution of 1 mg / mL. Galactose was used as a standard, and the total sugar content in the acetylated carrageenan was determined by the phenol-sulfuric acid method. 1 mL of the sample to be tested was taken into a centrifuge tube, 1 mL of 6% phenol solution was added, and 6 mL of concentrated sulfuric acid was quickly added and mixed uniformly. The above steps were repeated, and the total sugar content in the sample was measured according to the galactose standard curve.

[0077] (2) The acetylated carrageenan of Examples 1-6 and Comparative Examples 1-4 was taken to measure the sulfate content. 1 mg of the acetylated carrageenan of different processes dried to constant weight was accurately weighed and configured into an acetylated carrageenan solution of 1 mg / mL. Potassium sulfate was used as a standard, and the sulfate content in the sample was determined by the barium chloride-gelatin turbidimetry. 3 mL of the sample to be tested was taken into a centrifuge tube, 3.8 mL of 0.2M HCl was added, and 1 mL of gelatin-barium chloride was quickly added and mixed uniformly, and then it was placed for 30 min. The above steps were repeated, and the sulfate content in the sample was measured according to the potassium sulfate standard curve.

[0078] (3) The acetylated carrageenan of Examples 1-3 and Comparative Examples 1-5 was taken to measure the 3,6-endo galactose content. 1 mg of the acetylated carrageenan of different processes dried to constant weight was accurately weighed and configured into an acetylated carrageenan solution of 1 mg / mL. Fructose was used as a standard, and the 3,6-endo galactose content was determined by the resorcinol method. 1 mL of the acetylated carrageenan solution was placed in an ice water bath for 5 min, and 5.00 mL of resorcinol reagent was taken into each test tube and shaken uniformly. It was incubated at 80°C for 15 min, then taken out and 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-endo galactose content in the sample was measured according to the fructose standard curve.

[0079] (4) The acetylated carrageenan of Examples 1-3 and Comparative Examples 1-5 was taken to measure the acetyl substitution degree. 20 mg (m) of the acetylated carrageenan dried to constant weight was accurately weighed, 10 mL (V0) of 0.01M (C0) NaOH solution was added, and it was fully dissolved and placed in a 15 mL centrifuge tube. The test tube was placed in a constant temperature shaker for 2 h (250 rpm, 50°C). After the reaction was completed, 1% phenolphthalein solution was used as an indicator, and 0.01 M (C1) HCl solution was used for titration until the red color disappeared (pH=7.0). The volume of hydrochloric acid (V1) was recorded, and the acetyl substitution degree was calculated by formula (2).

[0080] (1)

[0081] (2)

[0082] Table 1 is the total sugar content, sulfate content, 3,6-endo-D-galactose content and acetyl substitution degree of the acetylated carrageenan of Examples 1-6 and Comparative Examples 1-4 of the present application.

[0083] Table 1

[0084]

[0085] Note: "-" in Table 1 indicates that the relevant index is not detected.

[0086] From Table 1, it can be seen that the total sugar content of the acetylated carrageenan prepared in Examples 1-6 is comparable to that of Comparative Examples 1-4, but the sulfate content is lower than that of Comparative Examples 1-4, and the 3,6-endo-D-galactose content is higher than that of Comparative Examples 1-4, indicating that the preparation method of the present application acetylates the position of sulfate, and promotes the formation of 3,6-endo-D-galactose in acetylated carrageenan, and the acetyl substitution degree is higher than that of Comparative Examples 1-4. According to the prior art, it is inferred that the acetylated carrageenan prepared in the present application may have higher biological activity in subsequent applications.

[0087] (8) Anti-colitis inflammation test of the acetylated carrageenan prepared in Examples 1-6

[0088] The cell models involved are: human colorectal adenocarcinoma cell Caco-2 cell model, LPS-induced RAW264.7 macrophage inflammation model.

[0089] The test method is as follows:

[0090] (1) Human colorectal adenocarcinoma cells Caco-2 cells were inoculated in 12-well transwell at a density of 2×10 5 cells / mL, and cultured for 21 days to form a tight monolayer, TEER > 600Ω•cm 2 Start the test, dissolve the acetylated carrageenan in sterilized PBS water to a concentration of 5mg / mL, filter through a 0.22μM filter membrane, and place on ice for standby. Add 500μL of a mixture of fluorescent yellow and acetylated carrageenan solution (fluorescent yellow solution concentration 20μg / mL; sample concentration 100mg / mL) to the AP side, add 1500mL of HBSS to the BL side, and culture for 24h. Take 300μL samples at 0.5, 1, 2, 4, 6, 12, 24h on the BL side, and add 300μL of HBSS after sampling. The fluorescence intensity of the sample was measured at λe x 427 nm, λe mThe A value was detected under 536 nm. The concentration of the fluorescent yellow in the BL side transport liquid was calculated according to the standard curve, and the apparent permeation coefficient Papp was calculated according to the following formula to verify the influence of the acetylated carrageenan on the integrity of the Caco-2 cell monolayer. The results are shown in Tables 2 and 3:

[0091]

[0092] Dq / dt is the fluorescent yellow transport amount per unit time (μg / s), A is the effective membrane area (1.12 cm 2 ), C0 is the initial concentration of the fluorescent yellow (20 μg / mL)

[0093] Table 2

[0094]

[0095] Table 3

[0096]

[0097] (2) RAW264.7 cells in the logarithmic growth phase were inoculated in a 96-well cell culture plate at a concentration of 3×10 4 cells / well, and were incubated in an incubator. After the cells were attached and grew to 85%, the cells were treated with 100 μL of medium containing different concentrations of carrageenan fermentation products for 12 h, and each treatment had 6 replicates. The medium was removed, and LPS was added to the carrageenan fermentation product treatment group and the control group for 12 h. The Griess method was used to determine the NO released by RAW264.7 cells. 50 μL of culture supernatant was taken from each well and placed in a new 96-well cell culture plate. Then 100 μL of Griess reagent (containing 3 mol / L sulfanilic acid, 30 mg / L N-1- (naphthyl) ethylenediamine dihydrochloride, and 25% glacial acetic acid) was added to the supernatant, and the mixture was incubated at room temperature for 20 min in the dark. The absorbance value (OD 540 ) at 540 nm was detected. At the same time, a standard curve was drawn using different concentrations of NaNO2 standard solution, and the NO concentration in the culture supernatant was calculated. The results are shown in Figure 1

[0098] The acetylated carrageenan prepared by the extraction scheme of the present application has anti-inflammatory activity. Since it is derived from natural seaweed, it is safe and can be used as a biological agent to relieve inflammation. It can also be used in the preparation of food and can also be prepared into anti-inflammatory compositions, etc., and has good application prospects.

[0099] ​The above merely illustrates the embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent transformation or direct or indirect application in the related technical field based on the content of the present application specification and drawings is also included in the patent protection scope of the present application.

Claims

1. A method for preparing acetylated carrageenan with anti-colitis activity, characterized in that, Includes 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 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 of 50 kDa-200 kDa. The carrageenan powder is then 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. The carrageenan powder is at a concentration of 50-100 mg / mL in the mixed solution; 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.

2. 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.

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

4. The method for preparing acetylated carrageenan with anti-colitis activity according to claim 1, characterized in that, 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.

5. The use of acetylated carrageenan prepared by any one of claims 1-4 in the preparation of compositions having anti-colitis activity.

6. The application according to claim 5, characterized in that, The colitis mentioned is a human colorectal adenocarcinoma Caco-2 cell model or an LPS-induced RAW264.7 macrophage inflammation model.

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