Carboxymethylated fingered polysaccharide, and preparation method and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine polysaccharides, and particularly relates to carboxymethylized bergamot polysaccharide as well as a preparation method and application thereof. The carboxymethylized bergamot polysaccharide prepared by the preparation method can significantly improve the mRNA expression of tight junction proteins ZO-1 and Occludin in Caco-2 cells in a lipopolysaccharide-induced Caco-2 cell inflammatory injury model, and can relieve Caco-2 cell injury and maintain the integrity of the cell barrier through a mutual transformation pathway of pentose and glucuronic acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine polysaccharides. More specifically, it relates to a carboxymethylized bergamot polysaccharide as well as a preparation method and applications thereof. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is a chronic idiopathic inflammatory bowel disease, mainly divided into ulcerative colitis (UC) and Crohn's disease (CD), and UC is the main type of IBD. Although the pathogenesis of UC is still controversial, severe trauma, infection, malnutrition, chronic inflammation, dysbiosis and environmental factors have been shown to independently or jointly trigger a series of biochemical reactions, ultimately leading to UC. Although a variety of therapeutic drugs (such as aminosalicylic acid, glucocorticoids, immunosuppressants and biological agents) can be used to treat ulcerative colitis, conventional drug therapy has limitations and serious side effects. In addition, for patients with severe disease who are ineffective for drug treatment, surgical treatment (such as total colectomy and ileal storage pouch anastomosis) is required. Therefore, finding a more economical, effective and safe new drug or supplementary therapy has become an urgent need for the treatment of UC disease.

[0003] The intestinal barrier is the first barrier for the intestine to contact with the external environment. When the intestinal barrier is damaged, microorganisms and endotoxins pass through the intestinal barrier, triggering and exacerbating the development of intestinal inflammation. Therefore, maintaining the integrity of the intestinal barrier is crucial for the treatment of UC. The intestinal barrier mainly includes the intestinal epithelial barrier and the intestinal mucosal barrier. In recent years, plant polysaccharides have attracted widespread attention from scholars due to their safety, non-toxicity, low cost, good biocompatibility and other advantages, and can alleviate inflammation by repairing the intestinal barrier. The main way of plant polysaccharides to repair the intestinal barrier is to promote the expression of tight junction (TJ) and mucin (MUC) proteins, and enhance the function of intestinal epithelial cells and mucous barrier. TJ proteins are mainly composed of Ooccludin protein, ZO protein and connecting adhesion molecules, which can tighten the intercellular gap and mediate the specific permeability of the intestine. Abnormal expression of TJ proteins can cause a decrease in intestinal barrier function and permeability, which is a major trigger factor for UC. Mucin is mainly secreted by intestinal goblet cells, with MUC-2 and MUC-3 proteins being the main components. Due to the special carbon-hydrogen structure of MUC proteins, they can competitively bind to the binding sites of intestinal epithelial cells, allowing harmful bacteria to stay in the mucous layer and be cleared during intestinal peristalsis, thereby reducing UC inflammation.

[0004] Fingered citron (Citrus medica 'Fingered') is a traditional medicinal and edible fruit. In addition to the general nutritional ingredients, it also has various functional active ingredients such as volatile oil, flavonoids, coumarins and polysaccharides. At present, the products of fingered citron are mainly primary processed products (such as medicinal materials, fruits, fruit wine and candied fruits, etc.), which have not fully tapped the value of fingered citron, thereby restricting the sustainable development of the industrialization of fingered citron. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the prior art and provide a preparation method of carboxymethylated fingered citron polysaccharide.

[0006] Another object of the present application is to provide carboxymethylated fingered citron polysaccharide prepared by the preparation method.

[0007] Another object of the present application is to provide the use of the carboxymethylated fingered citron polysaccharide in the preparation of drugs for protecting intestinal epithelial cell barrier.

[0008] The above objects of the present application are achieved by the following technical solutions:

[0009] The present application provides a preparation method of carboxymethylated fingered citron polysaccharide, which specifically comprises the following steps:

[0010] S1. Preparation of fingered citron polysaccharide;

[0011] S2. Mixing the fingered citron polysaccharide prepared in S1 with isopropanol and performing alkalization reaction under alkaline conditions;

[0012] S3. Adding isopropanol solution containing monochloroacetic acid, performing etherification reaction, adjusting pH to neutral, and post-treatment to obtain carboxymethylated fingered citron polysaccharide.

[0013] The post-treatment is dialysis, concentration, centrifugal precipitation and vacuum freeze-drying.

[0014] Further, in step S2, the mass-volume ratio of the fingered citron polysaccharide to isopropanol is 0.5g:6-8mL.

[0015] Further, in step S3, the temperature of the etherification reaction is 60-65℃.

[0016] Further, in step S3, the time of the etherification reaction is 3-4h. Further, in step S3, the mass ratio of monochloroacetic acid to fingered citron polysaccharide is 5.5-6:1.

[0017] Further, in step S3, the concentration of monochloroacetic acid in the isopropanol solution containing monochloroacetic acid is 0.14-0.16g / mL.

[0018] Further, in step S1, the preparation of the finger citron polysaccharide is prepared by finger citron pretreatment to remove fat-soluble substances, then the finger citron powder is obtained, water is mixed with the finger citron powder, and then extracted at 85-95°C, concentrated, centrifuged, and the concentrated liquid is obtained, and then the concentrated liquid is subjected to decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II to obtain the finger citron polysaccharide of the present application.

[0019] Further, the alkaline condition is formed by adding an alkaline reagent. Preferably, the alkaline reagent is one or both of sodium hydroxide and potassium hydroxide.

[0020] Further, in step S2, the temperature of the alkalization reaction is 25-35°C.

[0021] Further, in step S2, the time of the alkalization reaction is 1-1.5h.

[0022] Further, the preparation of the finger citron polysaccharide is prepared by finger citron pretreatment to remove fat-soluble substances, then the finger citron powder is obtained, water is mixed with the finger citron powder, and then extracted at 85-95°C, concentrated, centrifuged, and the concentrated liquid is obtained, and then the concentrated liquid is subjected to decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II to obtain the finger citron polysaccharide of the present application.

[0023] Further, the water and the finger citron powder are mixed at a volume-mass ratio of 40-60mL / g.

[0024] Further, the extraction time is 1.5-2h.

[0025] Further, the centrifugation rate is 3000-5000rpm / min, and the centrifugation time is 10-20min.

[0026] Further, the decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II are conventional processes for the extraction of finger citron polysaccharide. More specifically, the present application provides the following methods as a reference:

[0027] Decolorization I: the above concentrated liquid is added to AB-8 macroporous resin for decolorization and centrifugation to obtain an AB-8 treated liquid;

[0028] Deproteinization: Sevag reagent is added to the above AB-8 treated liquid for deproteinization to obtain a deproteinized solution;

[0029] Dialysis: the above deproteinized liquid is dialyzed for 36-48h using a dialysis bag, and then concentrated to obtain a dialyzed solution;

[0030] Alcohol precipitation: anhydrous ethanol is added to the above dialyzed solution for alcohol precipitation, the precipitate is resuspended, and vacuum freeze-drying is performed to obtain finger citron crude polysaccharide;

[0031] Decolorization II: active carbon is added to the above finger citron crude polysaccharide solution for decolorization, the supernatant is obtained by centrifugation, and then concentrated and vacuum freeze-dried to obtain the finger citron polysaccharide.

[0032] Further, in the step of deproteinization, the volume ratio of the Sevag reagent to the AB-8 treated solution is 3-6:1.

[0033] Further, in the step of deproteinization, the volume ratio of the Sevag reagent to the AB-8 treated solution is 3-6:1.

[0034] More specifically, the deproteinization treatment is shaking on a shaker for 20-40 min, centrifugation, and taking the supernatant, i.e. the deproteinized solution.

[0035] Further, the Sevag reagent is a mixed solution of chloroform and n-butanol. More specifically, the volume ratio of chloroform to n-butanol is 3-5:1.

[0036] Preferably, the step of deproteinization is repeated for 6-10 times.

[0037] Preferably, in the step of deproteinization, the centrifugation rate is 3000-5000 rpm / min, and the centrifugation time is 10-20 min.

[0038] Further, the specific conditions of the alcohol precipitation are: adding ethanol to 80-90% of the volume of the ethanol-dialysis solution system, alcohol precipitation at 2-8°C for 24-36 h, taking the precipitate for centrifugation, re-dissolving, and vacuum freeze-drying.

[0039] Further, in the step of decolorization II, the amount of activated carbon added is 0.5-1%.

[0040] Further, the time of decolorization II is 10-30 min.

[0041] Preferably, in the step of decolorization II, the centrifugation rate is 20000-25000 rpm / min, and the centrifugation time is 10-20 min.

[0042] Further, the specific steps of removing fat-soluble substances in the pretreatment are: crushing bergamot, reflux extraction with 85-95% ethanol, filtration, and drying.

[0043] In addition, as a reference, the mass-volume ratio of bergamot to ethanol in the water extraction is 100 g:1-2 L. More preferably, the mass-volume ratio is 100 g:1.5 L.

[0044] Preferably, the molecular weight (Mw) of the dialysis bag is 3.0-3.5 kDa.

[0045] Meanwhile, the present application also protects the carboxymethylated bergamot polysaccharide prepared by the preparation method.

[0046] Further, the degree of substitution of the carboxymethylated finger-polysaccharide is 1.0-1.24.

[0047] Further, the total sugar content of the carboxymethylated finger-polysaccharide is ≥86%.

[0048] Further, the carboxymethylated finger-polysaccharide at least comprises monosaccharide units of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc) and galacturonic acid (GalA) to form a polymer, wherein the molar ratio of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc) and galacturonic acid (GalA) is (0.12-0.16):(0.18-0.22):(0.35-0.42):(0.08-0.13):(99.02-99.18).

[0049] Meanwhile, the application protects the use of the carboxymethylated finger-polysaccharide in the preparation of drugs for protecting the intestinal epithelial cell barrier.

[0050] The application has the following beneficial effects:

[0051] The application discloses a carboxymethylated finger-polysaccharide, a preparation method and application thereof. The carboxymethylated finger-polysaccharide with a degree of substitution of 1.0-1.24 is obtained by carboxymethyl modification of finger-polysaccharide. The carboxymethylated finger-polysaccharide significantly promotes the mRNA expression of ZO-1 and Occludin protein in intestinal epithelial cells, and the effect is better than that of unmodified finger-polysaccharide and sulfated modified finger-polysaccharide. The carboxymethylated finger-polysaccharide can relieve the inflammatory injury of intestinal epithelial cells through the mutual transformation pathway of pentose and glucuronide, and maintain the integrity of the intestinal epithelial cell barrier. Therefore, the carboxymethylated finger-polysaccharide has a wide application prospect in the protection of intestinal health. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 The infrared spectrum of HWE-CP, S-HWE-CP and C-HWE-CP.

[0053] Figure 2 The ion chromatogram of monosaccharide composition. Figure 2 (a) in the figure is the chromatogram of monosaccharide standard; Figure 2 (b) in the figure is the chromatogram of monosaccharide composition of HWE-CP; Figure 2 (c) in the figure is the chromatogram of monosaccharide composition of S-HWE-CP; Figure 2 (d) in the figure is the chromatogram of monosaccharide composition of C-HWE-CP.

[0054] Figure 3Figure of the effect of HWE-CP, S-HWE-CP and C-HWE-CP on Caco-2 cell toxicity.

[0055] wherein, Figure 3 Figure (a) in the above is a figure of the effect of HWE-CP on Caco-2 cell toxicity; Figure 3 Figure (b) in the above is a figure of the effect of S-HWE-CP on Caco-2 cell toxicity; Figure 3 Figure (c) in the above is a figure of the effect of C-HWE-CP on Caco-2 cell toxicity.

[0056] Figure 4 Figure of the effect of HWE-CP, S-HWE-CP and C-HWE-CP on the mRNA expression of ZO-1 and Occludin protein in Caco-2 cells induced by lipopolysaccharide. Wherein, Figure 4 Figure (a) in the above is a figure of the effect of HWE-CP on Caco-2 cell toxicity; Figure 4 Figure (b) in the above is a figure of the effect of S-HWE-CP on Caco-2 cell toxicity;

[0057] Figure 5 Figure of KEGG signal pathway enrichment analysis bubble chart. Wherein, Figure 5 Figure (a) in the above is a figure of KEGG signal pathway enrichment analysis bubble chart of Control VS Model group; Figure 5 Figure (b) in the above is a figure of KEGG signal pathway enrichment analysis bubble chart of C-HWE-CP VS Model group. DETAILED DESCRIPTION

[0058] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0059] (1) Determination of the degree of substitution of carboxymethylized fingered citron polysaccharide by titration method:

[0060] Weigh 10 mg of carboxymethylized fingered citron polysaccharide into a 100 mL triangular flask, add 2 mL of 0.01 mol / L NaOH solution and 30 mL of deionized water, and stir in a 40℃ water bath for 30 min. Dissolve the carboxymethylized fingered citron polysaccharide thoroughly, cool to room temperature, and then add 2 drops of phenolphthalein indicator. Titrate with 0.1 mol / L HCl to neutralize the excess sodium hydroxide solution until the phenolphthalein indicator changes color and the color does not change within 30 seconds. Record the volume of hydrochloric acid consumed. The degree of substitution (DS) is calculated according to formula (1) and formula (2):

[0061]

[0062] wherein: DS is the degree of substitution; A is the carboxymethyl content, mmol; V0 is the volume of sodium hydroxide, mL; C0 is the concentration of sodium hydroxide solution, mol / L; m is the mass of carboxymethylated finger citron polysaccharide sample, g; V1 is the volume of hydrochloric acid consumed; C1 is the concentration of hydrochloric acid solution, mol / L.

[0063] (2) Determination of total sugar content of carboxymethylated finger citron polysaccharide by phenol-sulfuric acid method:

[0064] A mixed sugar (anhydrous L-arabinose: D-galacturonic acid mass ratio of 3:7) was used as a standard. The total sugar content of finger citron polysaccharide was determined by the sulfuric acid-phenol method. Different volumes of mixed sugar solution (0.1 mg / mL) were accurately pipetted, and distilled water was added to 1 mL and shaken well. 6% phenol solution (0.5 mL) and concentrated sulfuric acid (2.5 mL) were added, and carefully mixed, and left to stand for 15 min. The absorbance was measured at 490 nm and the total sugar content standard curve was plotted. The polysaccharide sample solution (0.1 mg / mL) was prepared, and its total sugar content was calculated according to the above operation steps and the total sugar content standard curve.

[0065] (3) Determination of the degree of substitution of sulfated finger citron polysaccharide by barium chloride-gelatin turbidimetry:

[0066] Accurately weigh 73.96 mg of anhydrous sodium sulfate standard and dissolve in HCl solution (1 mol / L), and dilute to 100 mL in a volumetric flask. After mixing, 0.5 mg / mL of sulfate standard solution is obtained. Accurately measure 0.02, 0.04, 0.06, 0.12, 0.16, and 0.20 mL of sulfate standard solution, and add HCl solution (1 mol / L) to 0.20 mL. HCl solution (1 mol / L) is used as a blank control. Add 3.8 mL of TCA (3%) and 1 mL of barium chloride-gelatin solution (1 g of barium chloride dissolved and diluted to 100 mL in 0.5% gelatin solution) and mix well. Plot the sulfate standard curve at an absorbance of 360 nm.

[0067] Weigh 10 mg of sulfated finger citron polysaccharide and dissolve in 5 mL of HCl solution (1 mol / L), seal, and hydrolyze in a boiling water bath at 100°C for 6 h to obtain a hydrolyzed solution of sulfated finger citron polysaccharide, and cool to room temperature. Calculate the mass fraction of sulfate in sulfated finger citron polysaccharide according to the above operation steps and the sulfate content standard curve, and calculate the degree of substitution of sulfated finger citron polysaccharide according to formulas 3 and 4:

[0068]

[0069] wherein, w(SO4 2- ) represents the mass fraction of sulfate (%); w(s) represents the mass fraction of sulfur; and DS represents the degree of substitution.

[0070] Unless otherwise indicated, the reagents and materials used in the following examples are commercially available.

[0071] Example 1 Preparation of carboxymethylated finger citron polysaccharide

[0072] S1. Preparation of finger citron polysaccharide: 200 g of finger citron medicinal material was crushed and added to 3 L of 90% ethanol, extracted by heating reflux for 4 times, filtered, and dried at 50°C to obtain finger citron powder; water and the finger citron powder were mixed at a volume-to-mass ratio of 60 mL / g, and extracted at 95°C for 1 h; the volume of the concentrated extract was reduced to 1 / 4, centrifuged at a speed of 4000 rpm / min for 20 min to obtain a concentrated solution;

[0073] Decolorization I: the concentrated solution was added to AB-8 macroporous resin for decolorization, and the volume ratio of the concentrated solution to AB-8 macroporous resin was 1:10 to obtain an AB-8 treated solution;

[0074] Deproteinization: the AB-8 treated solution was mixed with Sevag reagent (Sevag reagent is a mixture of chloroform and n-butanol at a volume ratio of 4:1) at a volume ratio of 4:1, shaken for 30 min, centrifuged at a speed of 4000 rpm / min for 20 min, and the deproteinization was repeated 10 times;

[0075] Dialysis: the decolorized solution was subjected to water dialysis for 36-48 h using a dialysis bag (Mw 3.5 kDa), and the volume of the dialyzed solution was concentrated to 1 / 4 of the volume of the dialyzed solution to obtain a dialyzed solution;

[0076] Alcohol precipitation: anhydrous ethanol was added to the dialyzed solution to make the volume of ethanol account for 80% of the volume of the ethanol-dialyzed solution system, and the solution was alcohol-precipitated at 4°C for 24 h; the precipitate was redissolved and vacuum freeze-dried to obtain crude finger citron polysaccharide;

[0077] Decolorization II: the crude finger citron polysaccharide was prepared into a solution and added with activated carbon for decolorization treatment, the activated carbon was added in an amount of 0.5%, the decolorization time was 30 min, the high-speed centrifugation speed was 20000 rpm / min, and the centrifugation time was 20 min to obtain a decolorized solution; the decolorized solution was concentrated, centrifuged, and vacuum freeze-dried to obtain the finger citron polysaccharide (HWE-CP);

[0078] S2. 0.5 g of HWE-CP prepared in S1 was dissolved in 6 mL of isopropanol, mechanically stirred for 1.5 h, and 36 mL of a 19% sodium hydroxide solution was slowly added; the mixture was alkalized in a 30°C water bath for 1 h and fully mixed to obtain a mixture;

[0079] S3. Into the reaction solution of step S2, 20 mL of monochloroacetic acid isopropanol solution with a concentration of 0.14 g / mL was added, the system was warmed to 63℃, and etherification reaction was carried out for 3.5 h. Then, the pH was adjusted to 7.0 with glacial acetic acid, and the solution was dialyzed with a 3.5 kDa dialysis bag for 56 h. The dialysate was centrifuged, concentrated, and vacuum freeze-dried to obtain the carboxymethylated finger citron polysaccharide.

[0080] The degree of substitution and total sugar content of the carboxymethylated finger citron polysaccharide were determined by titration and the phenol-sulfuric acid method to be 1.00 and 89.93%, respectively.

[0081] In the present application, the mass fraction of the sodium hydroxide solution is 19%: that is, 19 g of sodium hydroxide is added to 100 mL of water.

[0082] Example 2 Preparation of carboxymethylated finger citron polysaccharide

[0083] S1. Preparation of finger citron polysaccharide: consistent with Example 1;

[0084] S2. 0.5 g of HWE-CP prepared in S1 was dissolved in 6 mL of isopropanol, and mechanical stirring was carried out for 1.5 h. Then, 33 mL of sodium hydroxide solution with a mass fraction of 21% was slowly added, and the mixture was alkalized in a 30℃ water bath for 1 h to obtain a mixture;

[0085] S3. Into the reaction solution of step S2, 20 mL of monochloroacetic acid isopropanol solution with a concentration of 0.14 g / mL was added, the system was warmed to 63℃, and etherification reaction was carried out for 3.5 h. Then, the pH was adjusted to 7.0 with glacial acetic acid, and the solution was dialyzed with a 3.5 kDa dialysis bag for 56 h. The dialysate was centrifuged, concentrated, and vacuum freeze-dried to obtain the carboxymethylated finger citron polysaccharide.

[0086] The degree of substitution and total sugar content of the carboxymethylated finger citron polysaccharide were determined by titration and the phenol-sulfuric acid method to be 1.00 and 89.93%, respectively.

[0087] Example 3 Preparation of carboxymethylated finger citron polysaccharide

[0088] S1. Preparation of finger citron polysaccharide: consistent with Example 1;

[0089] S2. 0.5 g of HWE-CP prepared in S1 was dissolved in 6 mL of isopropanol, and mechanical stirring was carried out for 1.5 h. Then, 33 mL of sodium hydroxide solution with a mass fraction of 21% was slowly added, and the mixture was alkalized in a 30℃ water bath for 1 h to obtain a mixture;

[0090] S3. The reaction solution of step S2 was mixed with 20 mL of isopropyl alcohol solution containing monochloroacetic acid with a concentration of 0.16 g / mL, the system was warmed to 65°C, and etherification was carried out for 3 h. Then, the pH was adjusted to 7.0 with glacial acetic acid, and the solution was dialyzed against water for 48 h using a 3.5 kDa dialysis bag. The dialysate was centrifuged, concentrated, and freeze-dried in vacuum to obtain carboxymethylated finger citron polysaccharide.

[0091] The degree of substitution and total sugar content of the carboxymethylated finger citron polysaccharide were determined by titration and the phenol-sulfuric acid method to be 1.24 and 86.11%, respectively.

[0092] Comparative Example 1: Sulfated modification of finger citron polysaccharide

[0093] Sulfated modification of finger citron polysaccharide was carried out using the concentrated sulfuric acid method. 6 mL of n-butanol was taken in a round-bottom flask, which was placed in an ice water bath, and stirring was started. 18 mL of sulfuric acid was slowly added, and stirring was continued for 5 min. Then, 0.3 g of ammonium sulfate was added, and stirring was continued for another 10 min. Then, 1 g of HWE-CP was slowly added, and the reaction was carried out for 1 h. The reaction solution was neutralized to pH 7.0 using pre-cooled NaOH solution (2 mol / L), and the solution was dialyzed against water for 48 h using a dialysis bag with a molecular weight cut-off of 3.5 kDa. The dialysate was centrifuged, concentrated, and freeze-dried in vacuum to obtain sulfated finger citron polysaccharide.

[0094] The degree of substitution and total sugar content of the sulfated finger citron polysaccharide were determined by gelatin turbidity method and the phenol-sulfuric acid method to be 0.35 and 81.13%, respectively.

[0095] Experimental Example 1: Infrared spectroscopy

[0096] Experimental materials: finger citron polysaccharide (HWE-CP) prepared in step S1 of Example 1, carboxymethylated finger citron polysaccharide (C-HWE-CP) prepared in Example 2, and sulfated finger citron polysaccharide (S-HWE-CP) prepared in Comparative Example 1.

[0097] HWE-CP, S-HWE-CP, and C-HWE-CP were weighed and added to a marble mortar in a mass ratio of 1:50 with dry KBr powder, and were ground and pressed into tablets. The infrared spectra of the tablets were scanned at a wavelength of 4000-400 cm -1 The blank KBr tablet was used as the blank background.

[0098] Experimental results: The infrared spectra of HWE-CP, S-HWE-CP, and C-HWE-CP at a wavelength of 4000-400 cm -1 are shown in Table 1. Figure 1 -1 -1 ​​characteristic peaks at 1614 cm -1 , 1440 cm -1 , 1330 cm -1 are caused by C-O asymmetric vibration, COO symmetric stretching and C-H corner vibration. After the HWE-CP is modified by carboxymethyl, the characteristic peaks of C-HWE-CP at 1608 cm -1 , 1417 cm -1 and 1324 cm -1 are significantly enhanced, indicating the success of carboxymethylation of HWE-CP. At the same time, compared with the characteristic peak of -OH stretching vibration of HWE-CP (3405 cm -1 ), the -OH stretching vibration peak of C-HWE-CP moves to high wavenumber to 3411 cm -1 . After the HWE-CP is modified by sulfation, S-HWE-CP spectrum has two new characteristic peaks at 1238 cm -1 and 820 cm -1 , and the characteristic peaks near 1238 cm -1 and 820 cm -1 are caused by S=O stretching vibration and symmetric C-O-S stretching vibration, and both are characteristic peaks of sulfate, indicating the success of sulfation of HWE-CP. After the HWE-CP is modified by sulfation and carboxymethylation, the characteristic peaks of S-HWE-CP and C-HWE-CP at 1743 cm -1 disappear, and it is possible that during the modification process, the esterified uronic acid structure of the polysaccharide is destroyed under the action of strong acid (concentrated sulfuric acid) and strong base (NaOH).

[0099] Experimental Example 2 Determination of monosaccharide composition

[0100] Experimental materials: finger citron polysaccharide (HWE-CP) prepared in step S1 of Example 1, carboxymethylated finger citron polysaccharide (C-HWE-CP) prepared in Example 2, sulfated finger citron polysaccharide (S-HWE-CP) prepared in Comparative Example 1.

[0101] The ion chromatography method was used for monosaccharide composition determination and analysis. 5.0 mg of HWE-CP, S-HWE-CP and C-HWE-CP were respectively placed in an ampoule, 2 mL of TFA solution (3M) was added, hydrolysis was carried out at 120°C for 4h, the hydrolysis solution was blown dry with a nitrogen blowing instrument, 5 mL of water was added, vortexed, diluted (10 times dilution) and centrifuged (12000 rpm / min, 5 min), the supernatant was filtered through a 0.45 μm water filter and then analyzed by ion chromatography. The chromatographic column was Dionex CarbopacTM PA20 (3 mm x 250 mm), the mobile phase A (H2O), the mobile phase B (15 mM NaOH), the mobile phase C (15 mM NaOH & 100 mM NaAc), the flow rate (0.3 mL / min), the injection volume (25 μL) and the column temperature (30°C); the detector: Thermo Fisher ICS5000, elution according to the elution gradient parameters in Table 1.

[0102] Table 1 Ion chromatography elution conditions

[0103]

[0104]

[0105] Experimental results: Based on 11 kinds of monosaccharide standard, including fucose (Fuc, 1), rhamnose (Rha, 2), arabinose (Ara, 3), galactose (Gal, 4), glucose (Glc, 5), xylose (Xyl, 6), mannose (Man, 7), fructose (Fru, 8), ribose (Rio, 9), galacturonic acid (GalA, 10) and glucuronic acid (GlcA, 11), the monosaccharide composition and proportion were determined by ion chromatography, Figure 2 Figures (a), (b), (c) and (d) are chromatograms of monosaccharide standard mixture, HWE-CP, S-HWE-CP and C-HWE-CP, respectively. As can be seen from the figure, HWE-CP is composed of Rha, Ara, Gal, Glc, Xyl, GalA and GlcA, and the molar percentage is 2.24:10.04:4.93:0.34:0.08:81.94:0.43; S-HWE-CP is composed of Rha, Ara, Gal, Glc, Xyl, GalA and GlcA, and the molar percentage is 3.22, 12.13, 7.56, 0.43, 0.21, 75.44, 1.01; C-HWE-CP is composed of Rha, Ara, Gal, Glc and GalA, and the molar percentage is 0.12, 0.20, 0.41, 0.11, 99.16. Compared with HWE-CP, the types and proportions of monosaccharides in C-HWE-CP are changed, and the proportions of monosaccharides in S-HWE-CP are changed.

[0106] Experimental Example 3 Protection of Intestinal Epithelial Cell Inflammatory Injury

[0107] Experimental materials: Fingered Citron Polysaccharide (HWE-CP) prepared in Step S1 of Example 1, carboxymethylated Fingered Citron Polysaccharide (C-HWE-CP) prepared in Example 2, and sulfated Fingered Citron Polysaccharide (S-HWE-CP) prepared in Comparative Example 1.

[0108] (1) Cell toxicity screening of HWE-CP, S-HWE-CP and C-HWE-CP

[0109] Logarithmic growth phase Caco-2 cells were taken at a density of 5 x 10 3 The cells were plated in a 96-well plate at a density of 5 x 10

[0110] Normal group: The old culture medium was removed, and 100 μL of complete culture medium was added to each well. The cells were cultured in a 37°C cell incubator for 24 h.

[0111] HWE-CP group: The old culture medium was removed, and 100 μL of HWE-CP solution prepared using complete culture medium at concentrations of 50, 100, 200, 400 and 800 μg / mL was added to each well. The cells were cultured in a 37°C cell incubator for 24 h.

[0112] S-HWE-CP group: The old culture medium was removed, and 100 μL of S-HWE-CP solution prepared using complete culture medium at concentrations of 50, 100, 200, 400 and 800 μg / mL was added to each well. The cells were cultured in a 37°C cell incubator for 24 h.

[0113] C-HWE-CP group: The old culture medium was removed, and 100 μL of C-HWE-CP solution prepared using complete culture medium at concentrations of 50, 100, 200, 400 and 800 μg / mL was added to each well. The cells were cultured in a 37°C cell incubator for 24 h.

[0114] After the above culture, the survival rate of Caco-2 cells in each well was determined by CCK-8 method.

[0115] Experimental results: The results of the effects of different doses of HWE-CP, S-HWE-CP and C-HWE-CP on Caco-2 cell toxicity are shown in Table 1. Figure 3As shown, HWE-CP, S-HWE-CP and C-HWE-CP had no toxic effect on Caco-2 cells at a concentration of 50-800 μg / mL (p>0.05), and therefore, 50-800 μg / mL was used as the safe dose range of HWE-CP, S-HWE-CP and C-HWE-CP for subsequent experiments.

[0116] (2) Effect of HWE-CP, S-HWE-CP and C-HWE-CP on mRNA expression of tight junction proteins ZO-1 and Occludin

[0117] ① Cell culture

[0118] Caco-2 cells were seeded at 5×10 5 The cells were seeded at 5×10

[0119] Normal group: 100 μL of complete medium was added;

[0120] LPS group (model group): 100 μL of LPS solution at a concentration of 1 μg / mL (complete medium as solvent, LPS as solute) was added;

[0121] HWE-CP group: 100 μL of HWE-CP solution at a concentration of 400 μg / mL (1 μg / mL of LPS solution as solvent, HWE-CP as solute) was added;

[0122] S-HWE-CP group: 100 μL of S-HWE-CP solution at a concentration of 400 μg / mL (1 μg / mL of LPS solution as solvent, S-HWE-CP as solute) was added;

[0123] C-HWE-CP group: 100 μL of C-HWE-CP solution at a concentration of 400 μg / mL (1 μg / mL of LPS solution as solvent, C-HWE-CP as solute) was added;

[0124] After the above grouping and administration were completed, the cells were cultured for another 24 h, and then cell samples were collected for detection of the expression of target genes.

[0125] ② Extraction of cell RNA

[0126] According to the instructions of the Nuai Zan (FastPure Cell / Tissue Total RNA Isolation Kit V2) kit, the RNA of each group was extracted, and the concentration was measured using an ultramicro UV-visible spectrophotometer.

[0127] ③ RNA reverse transcription to synthesize cDNA

[0128] The extracted RNA was reversely transcribed to prepare cDNA according to the PCR kit instruction of HiScript II Q RT Super Mix for qPCR. The enzyme-free centrifuge tube was placed on ice, and the following system was configured: 5x buffer 4 μL, Total RNA 8 μL, DEPC-H2O 12 μL. After mixing uniformly, the reaction was carried out in PCR instrument according to the program of 50℃ (15 min), 85℃ (5 s), and the product was taken out and placed on ice after the reaction.

[0129] (4) Real-time fluorescent quantitative PCR (qRT-PCR) analysis

[0130] The cDNA was quantified according to the instruction of ChamQ Universal SYBR qPCR Master Mix. After appropriate dilution of the cDNA, the following system was configured: SYBR 10 μL, upstream primer 0.2 μL, downstream primer 0.2 μL, cDNA 2 μL and DEPC water 7.6 μL. The reaction solution was placed in the fluorescent quantitative PCR instrument for amplification, and the amplification program was as follows: pre-denaturation (95℃, 3 min, 1 cycle), denaturation (95℃, 10 s, 1 cycle) and annealing and extension (55℃, 30 s, 40 cycles). The forward and reverse primers were synthesized by Shanghai Sunway Biotech Co., Ltd., and the expression level of the target gene was calculated by 2 -ΔΔCt method (internal reference gene: β-actin), and the primer sequences of the related genes are shown in Table 2.

[0131] Table 2 Primer sequences for qRT-PCR quantification of related genes

[0132]

[0133] Experimental results: The effects of HWE-CP, S-HWE-CP and C-HWE-CP (400 μg / mL) on the mRNA expression of tight junction proteins ZO-1 and Occludin in the LPS (lipopolysaccharide)-induced inflammatory Caco-2 cell injury model are shown in Table 3. Figure 4 HWE-CP, S-HWE-CP and C-HWE-CP can all promote the mRNA expression of ZO-1 (p<0.05), C-HWE-CP can promote the mRNA expression of Occludin (p<0.01), and HWE-CP and S-HWE-CP have no significant effect on the mRNA expression of Occludin (p>0.05), indicating that carboxymethyl modification is more helpful to improve the protective ability of HWE-CP on Caco-2 cell barrier.

[0134] Experimental Example 4 Transcriptome analysis

[0135] Experimental material: carboxymethylated finger polysaccharide (C-HWE-CP) prepared in Example 2.

[0136] The Caco-2 cells in the logarithmic growth phase were inoculated in a 6-well plate at a density of 1 x 10 6 cells / mL, and were cultured in an incubator for 24 h until the cells were completely adherent. The cells were then modeled and administered according to the following scheme: the Control group, the Model group (1 μg / mL LPS), and the C-HWE-CP group (LPS (1 μg / mL) + C-HWE-CP (400 μg / mL)) were continuously cultured for 24 h. The cells were collected using a scraper, quickly frozen in liquid nitrogen for 10 min, and stored in a -80 °C refrigerator. The total RNA of the Caco-2 cells was extracted using TRIzol reagent, and the RNA integrity, RNA purity, RNA concentration, and library construction were verified using an Agilent 2100 bioanalyzer, a nanophotometer, an RNA detection kit, and a Truseq RNA kit, respectively. Sequencing was performed using an Illumina Hiseq platform. In this study, the differentially expressed genes were identified based on the following conditions: log2|fold-change (FC)| > 2. Then, the DEGs were subjected to enrichment analysis based on GO and KEGG to achieve functional annotation and biological description. This work was completed on the platform of Shengong Bioengineering (Shanghai) Co., Ltd. TM RNA extraction, RNA integrity verification, RNA purity evaluation, RNA concentration measurement, and library construction were performed using TRIzol reagent, an Agilent 2100 bioanalyzer, a nanophotometer, an RNA detection kit, and a Truseq RNA kit, respectively. Sequencing was performed using an Illumina Hiseq platform. In this study, the differentially expressed genes were identified based on the following conditions: log2|fold-change (FC)| > 2. Then, the DEGs were subjected to enrichment analysis based on GO and KEGG to achieve functional annotation and biological description. This work was completed on the platform of Shengong Bioengineering (Shanghai) Co., Ltd.

[0137] Experimental results: The DEGs in the LPS-induced Caco-2 cell inflammatory injury model treated with C-HWE-CP were analyzed using the KEGG method to explore the possible involvement of C-HWE-CP in metabolic pathways and signal pathways. In the Control VS Model group (a), there were 313 pathways in which the DEGs were involved, and the significantly changed pathways were Pentose and glucuronate interconversions (ascorbate and aldarate metabolism) and Ascorbate and aldarate metabolism (ascorbate and aldarate metabolism). In the C-HWE-CP VS Model group (b), there were 313 pathways in which the DEGs were involved, and the significantly changed pathways were Pentose and glucuronate interconversions (ascorbate and aldarate metabolism) and Ascorbate and aldarate metabolism (ascorbate and aldarate metabolism). Figure 5 Figure 5 ​​In (b) of the above, there are 134 pathways in which DEGs are involved, among which the pathways with significant changes are Apelin signaling pathway, Bile secretion and Pentose and glucuronate interconversions. By comparing the Control VS Model group and the C-HWE-CP VS Model group, it is found that there is a common significant pathway, Pentose and glucuronate interconversions, which is an important carbohydrate (polysaccharide) metabolic pathway, involved in the interconversion of pentose and glucuronate salt or ester, and glucuronate is synthesized into vitamin C through the above pathway for cell metabolism. C-HWE-CP is an acidic polysaccharide, and it is speculated that C-HWE-CP may alleviate the inflammatory damage of Caco-2 cells induced by LPS through the pentose and glucuronate interconversion pathway, and thus maintain the integrity of the cell barrier.

[0138] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for preparing carboxymethylated bergamot polysaccharide, characterized in that: The specific steps include: S1. Preparation of bergamot polysaccharide; S2. The bergamot polysaccharide prepared in S1 was mixed with isopropyl alcohol and subjected to alkalization reaction under alkaline conditions; S3. After adding an isopropanol solution containing monochloroacetic acid and performing an etherification reaction, the pH was adjusted to neutral and post-treated to obtain carboxymethylated bergamot polysaccharide; In step S1, the bergamot polysaccharide is prepared by subjecting bergamot to water extraction, decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II; In step S3, the mass ratio of monochloroacetic acid to bergamot polysaccharide is 5.5-6:

1.

2. The preparation method according to claim 1, characterized in that In step S2, the mass volume ratio of the bergamot polysaccharide to isopropyl alcohol is 0.5 g: 6-8 mL.

3. The preparation method according to claim 1, characterized in that: In step S3, the temperature of the etherification reaction is 60-65°C.

4. The preparation method according to claim 1, characterized in that In step S3, the concentration of monochloroacetic acid in the isopropanol solution containing monochloroacetic acid is 0.14-16 g / mL.

5. Carboxymethylated bergamot polysaccharide prepared by the preparation method according to any one of claims 1 to 4.

6. The carboxymethylated bergamot polysaccharide according to claim 5, characterized in that The degree of substitution of the carboxymethylated bergamot polysaccharide is 1.0-1.

24.

7. The carboxymethylated bergamot polysaccharide according to claim 5, characterized in that The total sugar content of the carboxymethylated bergamot polysaccharide is ≥86%.

8. Use of the carboxymethylated bergamot polysaccharide according to any one of claims 5 to 7 in the preparation of a drug for protecting the intestinal epithelial cell barrier.

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