Carboxymethylated fingered citron polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine polysaccharide, and particularly relates to carboxymethylated fingered citron polysaccharide as well as a preparation method and application thereof. In a lipopolysaccharide-induced Caco-2 cell inflammation damage model, the carboxymethylated fingered citron polysaccharide prepared by the preparation method provided by the invention can significantly improve the mRNA expression of closely connected proteins ZO-1 and Occludin in Caco-2 cells, and possibly alleviates Caco-2 cell damage through a mutual transformation pathway of pentose and glucuronic acid, and maintains the integrity of a cell barrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine polysaccharides, and more specifically, relates to a carboxymethylated bergamot polysaccharide and a preparation method and application thereof. Background Art

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

[0003] The intestinal barrier is the first barrier between the intestine and the external environment. When the intestinal barrier is damaged, microorganisms and endotoxins pass through the intestinal barrier, triggering and aggravating 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 the attention of many scholars due to their safety, non-toxicity, low cost, good biocompatibility, and their ability to relieve inflammation by repairing the intestinal barrier. Plant polysaccharides repair the intestinal barrier mainly by promoting the expression of tight junctions (TJ) and mucin (MUC) proteins, and enhancing the barrier function of intestinal epithelial cells and mucus. TJ protein is mainly composed of Ooccludin protein, ZO protein and junctional adhesion molecules, which can tighten the intercellular clefts and mediate specific intestinal permeability. Abnormal expression of TJ protein can cause decreased intestinal barrier function and permeability, and is the main triggering factor of UC. Mucin is mainly secreted by intestinal goblet cells, among which MUC-2 and MUC-3 proteins are mainly composed. Due to the special carbon-hydrogen structure of MUC protein, it can competitively bind to the intestinal epithelial cell binding sites, so that harmful bacteria remain in the mucus layer and are cleared during intestinal peristalsis, reducing UC inflammation.

[0004] Buddha's hand (Citrus medica'Fingered') is a traditional fruit that can be used as both medicine and food. In addition to the nutrients of general fruits, it also has a variety of functional active ingredients, such as volatile oils, flavonoids, coumarins and polysaccharides. At present, Buddha's hand products are mainly primary processed products (such as medicinal materials, fruits, fruit wine and candied fruits), and the value of Buddha's hand has not been fully explored, which restricts the sustainable development of Buddha's hand industrialization. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art and provide a method for preparing carboxymethylated bergamot polysaccharide.

[0006] Another object of the present invention is to provide a method for preparing carboxymethylated bergamot polysaccharide.

[0007] Another object of the present invention is to provide the use of the carboxymethylated bergamot polysaccharide in the preparation of a drug for protecting the intestinal epithelial cell barrier.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing carboxymethylated bergamot polysaccharide, which specifically comprises the following steps:

[0010] S1. Preparation of bergamot polysaccharide;

[0011] S2. mixing the bergamot polysaccharide prepared in S1 with isopropanol and performing an alkalization reaction under alkaline conditions;

[0012] S3. Add an isopropanol solution containing monochloroacetic acid to carry out etherification reaction, adjust the pH to neutral, and perform post-treatment to obtain carboxymethylated bergamot polysaccharide.

[0013] The post-treatment comprises dialysis, concentration, centrifugation, vacuum freeze drying of precipitates.

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

[0015] Furthermore, in step S3, the temperature of the etherification reaction is 60-65°C.

[0016] Furthermore, in step S3, the etherification reaction time is 3 to 4 hours. Furthermore, in step S3, the mass ratio of monochloroacetic acid to bergamot polysaccharide is 5.5 to 6:1.

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

[0018] Furthermore, in step S1, the prepared bergamot polysaccharide is prepared by subjecting bergamot to decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II.

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

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

[0021] Furthermore, in step S2, the alkalization reaction time is 1 to 1.5 hours.

[0022] Furthermore, the preparation of bergamot polysaccharide is to pre-treat bergamot to remove fat-soluble substances to obtain bergamot powder, mix water and bergamot powder, extract at 85-95°C, concentrate, and centrifuge to obtain a concentrated solution, and then subject the concentrated solution to decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II to obtain the bergamot polysaccharide of the present invention.

[0023] Furthermore, the water and the bergamot powder are mixed in a volume mass ratio of 40 to 60 mL / g.

[0024] Furthermore, the extraction time is 1.5 to 2 hours.

[0025] Furthermore, the centrifugal speed is 3000-5000 rpm / min, and the centrifugal time is 10-20 min.

[0026] Furthermore, the decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II are conventional processes for extracting bergamot polysaccharides. More specifically, the present invention provides the following methods as reference:

[0027] Decolorization I: Add AB-8 macroporous resin to the above concentrate for decolorization and centrifugation to obtain AB-8 treated solution;

[0028] Deproteinization: Sevag reagent is added to the above AB-8 treatment solution to deproteinize, thereby obtaining a deproteinized solution;

[0029] Dialysis: The above deproteinized solution is dialyzed for 36-48 hours using a dialysis bag and concentrated to obtain a dialysis solution;

[0030] Alcohol precipitation: adding anhydrous ethanol to the above dialysis solution, alcohol precipitation, taking the precipitate to dissolve again, and vacuum freeze-drying to obtain crude bergamot polysaccharide;

[0031] Decolorization II: The above crude bergamot polysaccharide is prepared into a solution and activated carbon is added to decolorize it. The supernatant is obtained by centrifugation, and then concentrated and vacuum-freeze-dried to obtain the bergamot polysaccharide.

[0032] Furthermore, in the decolorization step I, the volume ratio of the concentrate to the AB-8 macroporous resin is 1:10-15, the centrifugal speed is 4000-5000 rpm / min, and the centrifugal time is 10-15 min;

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

[0034] More specifically, the deproteinization treatment is to shake on a shaker for 20 to 40 minutes, centrifuge, and take the supernatant to obtain a deproteinized solution.

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

[0036] Preferably, the deproteinization step is repeated 6 to 10 times.

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

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

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

[0040] Furthermore, the decolorization II time is 10 to 30 minutes.

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

[0042] Furthermore, the specific steps of pre-treatment to remove fat-soluble substances are: crushing the bergamot, using 85-95% ethanol for reflux extraction, filtering, and drying.

[0043] For reference, the mass volume ratio of bergamot to ethanol in water extraction is 100 g: 1 to 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] At the same time, the present invention also protects the carboxymethylated bergamot polysaccharide prepared by the preparation method.

[0046] Furthermore, the degree of substitution of the carboxymethylated bergamot polysaccharide is 1.0 to 1.24.

[0047] Furthermore, the total sugar content of the carboxymethylated bergamot polysaccharide is ≥86%.

[0048] Furthermore, the carboxymethylated bergamot polysaccharide at least comprises a polymer whose monosaccharide units are rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc) and galacturonic acid (GalA). 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] At the same time, the present invention protects the use of the carboxymethylated bergamot polysaccharide in the preparation of drugs for protecting the intestinal epithelial cell barrier.

[0050] The present invention has the following beneficial effects:

[0051] The present invention discloses a carboxymethylated bergamot polysaccharide and a preparation method and application thereof. The present invention performs carboxymethyl modification on bergamot polysaccharide to obtain a carboxymethylated bergamot polysaccharide with a degree of substitution of 1.0 to 1.24. The carboxymethylated bergamot polysaccharide of the present invention significantly promotes the mRNA expression of ZO-1 and Occludin proteins in intestinal epithelial cells, and the effect is better than that of unmodified bergamot polysaccharide and sulfated bergamot polysaccharide. The carboxymethylated bergamot polysaccharide of the present invention may alleviate the inflammatory damage of intestinal epithelial cells through the mutual conversion pathway of pentose and glucuronic acid, and maintain the integrity of the intestinal epithelial cell barrier. Therefore, carboxymethylated bergamot polysaccharide has broad application prospects in intestinal health protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 IR spectra of HWE-CP, S-HWE-CP and C-HWE-CP.

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

[0054] Figure 3This is a graph showing the effects of HWE-CP, S-HWE-CP and C-HWE-CP on the cytotoxicity of Caco-2 cells.

[0055] in, Figure 3 (a) shows the effect of HWE-CP on Caco-2 cell cytotoxicity; Figure 3 (b) shows the effect of S-HWE-CP on the cytotoxicity of Caco-2 cells; Figure 3 (c) is the effect of C-HWE-CP on Caco-2 cell cytotoxicity.

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

[0057] Figure 5 This is a bubble diagram for KEGG signaling pathway enrichment analysis. Figure 5 (a) is the bubble diagram of KEGG signaling pathway enrichment analysis of Control VS Model group; Figure 5 (b) is the bubble chart of KEGG signaling pathway enrichment analysis of the C-HWE-CP VS Model group. DETAILED DESCRIPTION

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

[0059] (1) Titration method for determination of degree of substitution of carboxymethylated bergamot polysaccharide:

[0060] Weigh 10 mg of carboxymethylated bergamot polysaccharide in a 100 mL conical flask, add 2 mL of 0.01 mol / L NaOH solution and 30 mL of deionized water, stir in a 40 °C water bath for 30 min to fully dissolve the carboxymethylated bergamot polysaccharide, cool to room temperature, add 2 drops of phenolphthalein indicator, titrate with 0.1 mol / L HCl, and 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] In the formula: 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 bergamot 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 bergamot polysaccharide by phenol-sulfuric acid method:

[0064] Mixed sugar (mass ratio of anhydrous L-arabinose: D-galacturonic acid is 3:7) is used as the standard. The total sugar content of bergamot polysaccharide is determined by sulfuric acid-phenol method. Accurately aspirate different volumes of mixed sugar solution (0.1 mg / mL), add distilled water to 1 mL, and shake well. Add 6% phenol solution (0.5 mL) and concentrated sulfuric acid (2.5 mL), mix carefully, let stand for 15 minutes, measure the absorbance at 490 nm and draw a standard curve of total sugar content. Prepare polysaccharide sample solution (0.1 mg / mL), and calculate its total sugar content according to the above operation steps and the total sugar content standard curve.

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

[0066] Accurately weigh 73.96 mg of anhydrous sodium sulfate standard and dissolve it in HCl solution (1 mol / L), and dilute to 100 mL of a volumetric flask. After mixing, a 0.5 mg / mL 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 respectively. HCl solution (1 mol / L) is used as a blank control, and 3.8 mL of TCA (3%) and 1 mL of barium chloride-gelatin solution (1 g of barium chloride is dissolved and diluted to 100 mL of 0.5% gelatin solution) are added and mixed. Draw a sulfate standard curve at an absorbance of 360 nm.

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

[0068]

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

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

[0071] Example 1 Preparation of carboxymethylated bergamot polysaccharide

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

[0073] Decolorization I: The above concentrate was added with AB-8 macroporous resin for decolorization, the volume ratio of the concentrate to the AB-8 macroporous resin was 1:10, and AB-8 treatment solution was obtained;

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

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

[0076] Alcohol precipitation: add anhydrous ethanol to the above dialysis solution until the ethanol accounts for 80% of the volume of the ethanol-dialysis solution system, and perform alcohol precipitation at 4°C for 24 hours. Take the precipitate, re-dissolve it, and vacuum freeze-dry it to obtain crude bergamot polysaccharide;

[0077] Decolorization II: The above crude bergamot polysaccharide is prepared into a solution and activated carbon is added for decolorization treatment, the amount of activated carbon added is 0.5%, the decolorization time is 30 min, the high-speed centrifugation rate is 20000 rpm / min and the centrifugation time is 20 min, and a decolorization solution is obtained, and the decolorization solution is concentrated, centrifuged, and vacuum freeze-dried to obtain the bergamot polysaccharide (HWE-CP);

[0078] S2. Dissolve 0.5 g of HWE-CP prepared in S1 in 6 mL of isopropanol, stir mechanically for 1.5 h, slowly add 36 mL of 19% sodium hydroxide solution, alkalize in a 30°C water bath for 1 h, and mix thoroughly to obtain a mixture;

[0079] S3. Add 20 mL of 0.14 g / mL isopropanol solution of monochloroacetic acid to the reaction solution of step S2, heat the system to 63°C, and after etherification reaction for 3.5 hours, adjust the pH to 7.0 with glacial acetic acid, dialyze with flowing water using a 3.5 kDa dialysis bag for 56 hours, and centrifuge, concentrate and freeze-dry the dialysate to obtain carboxymethylated bergamot polysaccharide.

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

[0081] In the present invention, the sodium hydroxide solution with a mass fraction of 19% means that 19 g of sodium hydroxide is added to every 100 mL of water.

[0082] Example 2 Preparation of carboxymethylated bergamot polysaccharide

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

[0084] S2. Dissolve 0.5 g of HWE-CP prepared in S1 in 6 mL of isopropanol, stir mechanically for 1 h, slowly add 35 mL of 20% sodium hydroxide solution, alkalize in a 30°C water bath for 1 h, and mix thoroughly to obtain a mixture;

[0085] S3. Add 20 mL of 0.15 g / mL monochloroacetic acid isopropanol solution to the reaction solution of step S2, heat the system to 60°C, and after etherification reaction for 4 hours, adjust the pH to 7.0 with glacial acetic acid, dialyze with flowing water using a 3.5 kDa dialysis bag for 72 hours, and centrifuge, concentrate and freeze-dry the dialysate to obtain carboxymethylated bergamot polysaccharide.

[0086] The substitution degree and total sugar content of the carboxymethylated bergamot polysaccharide were measured by titration and phenol-sulfuric acid method to be 1.11 and 88.54% respectively.

[0087] Example 3 Preparation of carboxymethylated bergamot polysaccharide

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

[0089] S2. Take 0.5 g of HWE-CP prepared in S1 and dissolve it in 6 mL of isopropanol, stir it mechanically for 1.5 h, slowly add 33 mL of 21% sodium hydroxide solution, alkalize it in a 30°C water bath for 1 h and mix it thoroughly to obtain a mixture;

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

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

[0092] Comparative Example 1 Sulfation modification of bergamot polysaccharide

[0093] The concentrated sulfuric acid method was used to sulfation-modify bergamot polysaccharide. 6 mL of n-butanol was placed in a round-bottom flask, placed in an ice-water bath, stirred, and 18 mL of sulfuric acid was slowly added. After stirring for 5 min, 0.3 g of ammonium sulfate was added, and stirring was continued for 10 min. 1 g of HWE-CP was slowly added and reacted for 1 h. The reaction solution was neutralized to pH 7.0 with a precooled NaOH solution (2 mol / L), and dialyzed with flowing water in a dialysis bag with a molecular weight cutoff of 3.5 kDa for 48 h. The dialyzate was centrifuged, concentrated, and vacuum freeze-dried to obtain sulfated bergamot polysaccharide.

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

[0095] Experimental Example 1 Infrared Spectroscopy Measurement

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

[0097] HWE-CP, S-HWE-CP and C-HWE-CP were weighed and added to dry KBr powder in a mass ratio of 1:50 in an agate mortar. The mixture was fully ground and pressed into tablets at a wavelength of 4000-400 cm -1 The spectrum was scanned and an empty KBr pellet was used as a blank background.

[0098] Experimental results: HWE-CP, S-HWE-CP and C-HWE-CP are in the range of 4000-400 cm -1 The infrared spectral characteristics of Figure 1 All three are between 3405 and 3421 cm -1 2940~2958cm -1The characteristic peak at 1614cm is caused by the stretching vibration of OH and the stretching vibration of CH on -CH2-. -1 、1440cm -1 、1330cm -1 The characteristic peak at 1608cm is caused by asymmetric vibration of CO, symmetric stretching of COO and corner vibration of CH. After HWE-CP is modified with carboxymethyl, the spectrum of C-HWE-CP is -1 、1417cm -1 and 1324cm -1 The characteristic peak intensity of HWE-CP was significantly enhanced, indicating that the carboxylmethylation of HWE-CP was successful. -1 ), the -OH stretching vibration peak of C-HWE-CP shifts to a higher wavenumber of 3411 cm -1 After HWE-CP was modified by sulfation, the spectrum of S-HWE-CP was 1238cm -1 and 820cm -1 Two new characteristic peaks were added at 1238cm -1 and 820cm -1 The characteristic peaks near 1743 cm-1 are caused by S=O stretching vibration and symmetrical COS stretching vibration, and both are characteristic peaks of sulfate esters, indicating that the sulfation of HWE-CP was successful. -1 The characteristic peaks of the polysaccharide disappeared, which may be due to the destruction of the esterified uronic acid structure of the polysaccharide under the action of strong acid (concentrated sulfuric acid) and strong base (NaOH) during the modification process.

[0099] Experimental Example 2 Determination of Monosaccharide Composition

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

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

[0102] Table 1 Ion chromatography elution conditions

[0103]

[0104]

[0105] Experimental results: Based on 11 monosaccharide standards, 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 (a), (b), (c) and (d) are the 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 their molar percentages are 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 their molar percentages are 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 their molar percentages are 0.12, 0.20, 0.41, 0.11, 99.16. Compared with HWE-CP, the types and proportions of monosaccharide composition of C-HWE-CP have changed, and the monosaccharide composition ratio of S-HWE-CP has changed.

[0106] Experimental Example 3 Protection of Intestinal Epithelial Cells from Inflammatory Damage

[0107] Experimental materials: bergamot polysaccharide (HWE-CP) prepared in step S1 of Example 1, carboxymethylated bergamot polysaccharide (C-HWE-CP) prepared in Example 2, and sulfated bergamot polysaccharide (S-HWE-CP) prepared in Comparative Example 1.

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

[0109] Caco-2 cells in the logarithmic growth phase were taken and 5×10 3 The density of cells / well was plated in a 96-well plate, 100 μL of complete medium was added to each well, and the cells were cultured in a 37°C cell culture incubator for 24 h. The experimental cells were grouped and cultured as follows:

[0110] Normal group: remove the old culture medium, add 100 μL complete culture medium to each well, and culture in a cell culture incubator at 37°C for 24 h;

[0111] HWE-CP group: the old culture medium was removed, and HWE-CP solution with concentrations of 50, 100, 200, 400, and 800 μg / mL prepared with complete culture medium was added, 100 μL per well, and cultured in a cell culture incubator at 37°C for 24 h;

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

[0113] C-HWE-CP group: the old culture medium was removed, and C-HWE-CP solution with concentrations of 50, 100, 200, 400, and 800 μg / mL prepared with complete culture medium was added again, 100 μL per well, and each concentration was repeated for 6 wells, and cultured in a cell culture incubator at 37°C for 24 h;

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

[0115] Experimental results: The effects of different doses of HWE-CP, S-HWE-CP and C-HWE-CP on the cytotoxicity of Caco-2 cells are shown in the following table. Figure 3As shown in the data, HWE-CP, S-HWE-CP and C-HWE-CP had no toxic effects on Caco-2 cells at concentrations between 50 and 800 μg / mL (p>0.05). Therefore, 50 to 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) Effects of HWE-CP, S-HWE-CP, and C-HWE-CP on the mRNA expression of tight junction proteins ZO-1 and Occludin

[0117] ①Cell culture

[0118] Caco-2 cells were cultured at 5 × 10 5 Cells / well Cells were seeded in 6-well plates and cultured in a cell culture incubator for 24 hours until the cells were completely attached to the wall. The old culture medium was discarded, and the modeling and drug administration were performed according to the following scheme:

[0119] Normal group: add 100 μL complete medium;

[0120] LPS group (model group): add 100 μL of 1 μg / mL LPS solution (complete culture medium as solvent, LPS as solute);

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

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

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

[0124] After the above group administration, the culture was continued for 24 hours, and then the cell samples were collected to detect the expression of the target gene.

[0125] ②Extraction of cellular RNA

[0126] According to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit V2, RNA from each group was extracted, and its concentration was measured using an ultra-micro UV-visible spectrophotometer.

[0127] ③ RNA reverse transcription to cDNA

[0128] According to the instructions of the HiScript IIQ RT Super Mix for qPCR PCR kit, the extracted RNA was reverse transcribed to prepare cDNA. The enzyme-free centrifuge tube was placed on ice and configured as follows: 5x buffer 4μL, Total RNA 8μL, DEPC-H2O 12μL. After mixing evenly, the reaction was carried out in a PCR instrument according to the program of 50℃ (15min), 85℃ (5s). After the reaction, the product was taken out and placed on ice.

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

[0130] The cDNA was quantified according to the instructions of ChamQ Universal SYBRqPCR Master Mix of Novozyme. After the cDNA was appropriately diluted, the following system was configured: 10 μL SYBR, 0.2 μL upstream primer, 0.2 μL downstream primer, 2 μL cDNA and 7.6 μL DEPC water. The reaction solution was placed in a fluorescent quantitative PCR instrument for amplification. The amplification program was: pre-denaturation (95°C, 3 min, 1 cycle), denaturation (95°C, 10 s, 1 cycle) and annealing and extension (55°C, 30 s, 40 cycles). The forward and reverse primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the expression level of the target gene was measured by 2 -ΔΔCt The primer sequences of 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 were shown in Figure 2. Figure 4 As shown in the data, HWE-CP, S-HWE-CP and C-HWE-CP could promote the mRNA expression of ZO-1 (p<0.05), C-HWE-CP could promote the mRNA expression of Occludin (p<0.01), and HWE-CP and S-HWE-CP had 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 the Caco-2 cell barrier.

[0134] Experimental Example 4 Transcriptome Analysis

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

[0136] Caco-2 cells in logarithmic growth phase were collected and 1×10 6 The cells were inoculated at a density of 100 / mL on a 6-well plate and cultured in an incubator for 24 hours until the cells were completely attached to the wall. The modeling and drug administration were carried out according to the following scheme: the groups were divided into the Control group, the Model group (1μg / mL LPS), and the C-HWE-CP group (LPS (1μg / mL) + C-HWE-CP (400μg / mL)). The cells were cultured for 24 hours, and the cells were collected with a scraper, quickly frozen in liquid nitrogen for 10 minutes, and stored in a -80℃ refrigerator. TRIzol reagent, Agilent 2100 bioanalyzer, nanophotometer spectrophotometer, RNA Detection Kits and Truseq TM RNA kit was used to extract total RNA from Caco-2, verify RNA integrity, evaluate RNA purity, measure RNA concentration, and construct libraries, and sequencing was performed using the Illumina Hiseq platform. In this study, differentially expressed genes were identified based on the following conditions: log2|fold-change(FC)|>2. Then, enrichment analysis of DEGs was performed based on GO and KEGG to achieve their functional annotation and biological description, which was completed on the platform of Sangon Biotech (Shanghai) Co., Ltd.

[0137] Experimental results: KEGG method was used to analyze the DEGs of C-HWE-CP in LPS-induced Caco-2 cell inflammatory injury model to explore the possible involvement of C-HWE-CP in metabolic pathways and signaling pathways. Figure 5 In (a), there were 313 DEGs involved in the pathways, and the pathways with significant changes were Pentose and glucuronate interconversions (pentose and glucuronate interconversion pathway) and Ascorbate and aldarate metabolism (ascorbate and aldarate metabolism pathway); in the C-HWE-CP VS Model group ( Figure 5In (b), there were 134 pathways involved in DEGs, among which the pathways with significant changes were 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 was found that there was a common significant pathway, the interconversion pathway of pentose and glucuronic acid, which is an important carbohydrate (polysaccharide) metabolic pathway that participates in the interconversion of pentose and glucuronic acid salts or esters. Glucuronic acid synthesizes vitamin C through the above pathway for cell metabolism. As an acidic polysaccharide, C-HWE-CP is speculated to alleviate LPS-induced inflammatory damage to Caco-2 cells through the interconversion pathway of pentose and glucuronic acid, thereby maintaining the integrity of the cell barrier.

[0138] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing carboxymethylated bergamot polysaccharide, characterized in that: The specific steps include: S1. Preparation of bergamot polysaccharide; S2. mixing the bergamot polysaccharide prepared in S1 with isopropanol and performing an alkalization reaction under alkaline conditions; S3. Add an isopropanol solution containing monochloroacetic acid to carry out etherification reaction, adjust the pH to neutral, and perform post-treatment to obtain carboxymethylated bergamot polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step S2, the mass volume ratio of the bergamot polysaccharide to isopropanol 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. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of monochloroacetic acid to bergamot polysaccharide is 5.5-6:

1.

6. The preparation method according to claim 1, characterized in that: In step S1, the bergamot polysaccharide is prepared by subjecting bergamot to water extraction, decolorization I, deproteinization, dialysis, alcohol precipitation, and decolorization II.

7. Carboxymethylated bergamot polysaccharide prepared by the preparation method according to any one of claims 1 to 6.

8. The carboxymethylated bergamot polysaccharide according to claim 7, characterized in that: The degree of substitution of the carboxymethylated bergamot polysaccharide is 1.0-1.

24.

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

10. Use of the carboxymethylated bergamot polysaccharide according to any one of claims 7 to 9 in the preparation of a drug for protecting the intestinal epithelial cell barrier.

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