A kind of jujube modified acidic polysaccharide and its preparation method and use

Abstract

The present invention provides a modified acidic polysaccharide from jujube. Per 100 mg of the modified acidic polysaccharide, the polysaccharide contains: 90.26 mg ± 1.39 mg–94.42 mg ± 1.83 mg total polysaccharides; 21.42 mg ± 1.01 mg–50.72 mg ± 0.62 mg total uronic acid; and 0.65 mg GAE ± 0.05 mg GAE–1.21 mg GAE ± 0.03 mg GAE total bound phenols (gallic acid equivalent). The present invention also provides a preparation method and uses of the modified acidic polysaccharide from jujube. The invention modifies jujube acidic polysaccharides through low-temperature alkaline deesterification technology, directional enzyme cutting technology and controllable partial acid hydrolysis technology, and compares the in vivo and in vitro immunoregulatory activities of the jujube acidic polysaccharides before and after modification. The immunoregulatory activity of the modified jujube acidic polysaccharides obtained by the modification is significantly enhanced, especially the modified jujube acidic polysaccharides with the arabinan side chains removed and the galactan side chains reduced, which have better immunoregulatory activity.

Description

Technical Field

[0001] The invention relates to a jujube modified acidic polysaccharide, a preparation method and application thereof, and belongs to the field of health-care foods or medicines. Background Art

[0002] Jujube (Ziziphus jujuba Mill.), a plant of the genus Ziziphus in the family Rhamnaceae, is oval or spherical in shape, 2-3.5 cm long and 1.5-2.5 cm in diameter. Its surface is dark red, slightly shiny, and irregularly wrinkled. The base is concave and has a short stalk. The exocarp is thin, while the mesocarp is brownish-yellow or light brown, fleshy, soft, rich in sugar, and oily. The stone is spindle-shaped, with sharp ends and a hard texture. It has a faint fragrance and a sweet taste. Jujube is widely distributed in my country and is cultivated in many areas, with Xinjiang, Gansu, Ningxia, Shaanxi, Shanxi, Shandong, Hebei, and Henan being the main producing areas. Jujube is a food that can be used as both a food and a medicine. Ancient pharmacology texts considered it an herbal remedy with benefits such as tonifying the spleen and stomach, replenishing qi and promoting body fluid, regulating the body's defense system, and detoxifying drugs. Modern research shows that jujube contains a variety of active substances, including polysaccharides, flavonoids, saponins, phenolic acids, triterpenoid acids, and amino acids. Among them, polysaccharide is one of the main active ingredients of jujube, which has biological activities such as immune regulation, anti-oxidation, anti-tumor, and regulation of intestinal flora.

[0003] Jiao Zhonggao, Molecular Modification and Biological Activity of Jujube Polysaccharides, Northwest Agriculture and Forestry University, 2012, discloses that based on the extraction, separation and purification of jujube polysaccharides, molecular modification technology was applied to the study of jujube polysaccharides. Through ultrasonic treatment, hydrochloric acid degradation and sulfation, carboxymethylation and acetylation modification, the molecules were further modified and modified. In vitro tests were used to study the antioxidant activity, hypoglycemic effect and hyaluronidase inhibition of jujube polysaccharides and their molecular modification products. It was confirmed that jujube polysaccharides have inhibitory effects on α-amylase, α-glucosidase and hyaluronidase and non-enzymatic saccharification reactions. This document discloses that jujube polysaccharides were modified by ultrasonic treatment, hydrochloric acid degradation and sulfation, carboxymethylation and acetylation, and in vitro tests were used to study the antioxidant activity, hypoglycemic effect and hyaluronidase inhibition of jujube polysaccharides and their molecular modification products.

[0004] Zhang Yali et al., "Exploration of the Material Basis of the Blood-Tonifying Effect of Red Dates," Chinese Food and Nutrition, Issue 2, 2005, discloses that red dates have significant immune-promoting activity. Anti-complementary polysaccharides are mostly acidic heteropolysaccharides, the acidic parts of which are mainly galacturonic acid and glucuronic acid. Lin Qinbao, Gao Dawei, Yu Shujuan et al., "Study on the Monosaccharide Composition of Jujube Polysaccharides by High Performance Liquid Chromatography," Journal of Zhengzhou Grain College, 1998, 19(3): 57-60. Polysaccharide components were extracted from jujube to obtain neutral polysaccharide JDP-N and acidic polysaccharide JDP-A. Glycosuric acid analysis of jujube acidic polysaccharides revealed that the uronic acid was D-galacturonic acid, with a content of 40.6%. Pharmacological experiments conducted by the Pharmacology Laboratory of the First Military Medical University showed that it has significant anti-complementary activity and promotes lymphocyte proliferation. The acidic heteropolysaccharides of red date polysaccharides are the intrinsic reason for its anti-complementary activity.

[0005] The immune system is a crucial regulatory system that regulates metabolism, protects against invasion by foreign pathogens, and protects the body from endogenous pathological changes. It is crucial for normal human health. Polysaccharides are macromolecules that bind to pattern recognition receptors on cell membranes, activating various intracellular signal transduction pathways, promoting the production of cytokines or antibodies, and modulating immune function. Previous studies have shown that jujube acidic polysaccharides exhibit potent immunomodulatory activity. The biological activity of polysaccharides is generally closely related to their chemical structure, such as degree of esterification, monosaccharide composition, glycosidic bonds, and molecular weight. Currently, the key effector structures and mechanisms of action underlying the immunomodulatory activity of jujube acidic polysaccharides remain largely unknown. Therefore, modifying jujube acidic polysaccharides and comparing their in vitro and in vivo immunomodulatory activities before and after modification could clarify the key effector structures and mechanisms that influence their in vitro and in vivo immunomodulatory activity. This approach would facilitate the fuller utilization of jujube resources, provide a reference for exploring the immunomodulatory activity and mechanisms of jujube polysaccharides, and offer theoretical and technical support for the development of functional foods derived from jujube polysaccharides. Summary of the Invention

[0006] The present invention provides a modified acidic polysaccharide of jujube, and also provides a preparation method and application of the modified acidic polysaccharide of jujube.

[0007] The present invention provides a modified acidic polysaccharide of jujube. Each 100 mg of the modified acidic polysaccharide of jujube contains:

[0008] Total polysaccharides: 90.26 mg ± 1.39 mg–94.42 mg ± 1.83 mg;

[0009] Total uronic acid: 21.42 mg ± 1.01 mg–50.72 mg ± 0.62 mg;

[0010] Total bound phenolics: 0.65 mg GAE ± 0.05 mg GAE – 1.21 mg GAE ± 0.03 mg GAE

[0011] (gallic acid equivalent);

[0012] The esterification degree of the modified acidic polysaccharide of jujube was 2.55%±0.30%–53.72%±1.01%, and the molecular weight was (1.035±0.040)×10 4 Da–(4.069±0.031)×10 4 Da, the molar proportion of galacturonan (HG) was 12.03%–57.26%, the molar proportion of type I rhamnogalacturonan (RG-I) was 40.43%–81.87%, and the side chain length of RG-I was 0.43–6.63.

[0013] Further preferably, each 100 mg of the modified acidic polysaccharide of jujube contains:

[0014] Total polysaccharides: 90.26mg±1.39mg;

[0015] Total uronic acid: 50.72mg±0.62mg;

[0016] Total bound phenols: 0.65 mg GAE ± 0.05 mg GAE (gallic acid equivalent);

[0017] The esterification degree of the modified acidic polysaccharide of jujube is 2.55%±0.30%, and the molecular weight is (1.035±0.040)×10 4 Da; the molar proportion of galacturonan (HG) is 57.26%, the molar proportion of type I rhamnogalacturonan (RG-I) is 40.43%, and the side chain length of RG-I is 0.43.

[0018] Among them, the modified acidic polysaccharides of jujube mainly contain the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, glucose, glucuronic acid, and mannose. The molar percentage of each monosaccharide is: (21.97%–73.88%): (7.19%–27.14%): (0.00%–38.15%): (9.49%–16.62%): (1.31%–6.23%): (0.44%–1.32%): (0.56%–0.84%).

[0019] Further preferably, the modified jujube acidic polysaccharide mainly contains the following monosaccharides: galacturonic acid, galactose, rhamnose, glucose, glucuronic acid, mannose, and the molar percentage of each monosaccharide is: 73.88%: 7.19%: 16.62%: 1.31%: 0.44%: 0.56%.

[0020] The jujube acidic polysaccharide of the present invention is extracted by using a low eutectic solvent assisted by water bath heating, and then modified by a low-temperature alkaline deesterification technology, a directional enzyme cutting technology or a controllable partial acid hydrolysis technology to obtain the jujube modified acidic polysaccharide.

[0021] The present invention also provides a method for preparing the modified acidic polysaccharide of jujube, which comprises the following steps:

[0022] a. Using water bath heating to assist deep eutectic solvent to extract jujube acidic polysaccharides;

[0023] b. Modify the jujube acidic polysaccharide by using low-temperature alkaline deesterification technology, directional enzyme cutting technology or controlled partial acid hydrolysis technology to obtain the jujube modified acidic polysaccharide.

[0024] The method for extracting jujube acidic polysaccharides using a water bath heating-assisted deep eutectic solvent as described in step a comprises the following steps:

[0025] a. Ultrasonic removal of alcohol-soluble components: Remove the core of the jujube, chop it into pieces, dry it, grind it into powder, and pass it through an 80-mesh sieve; ultrasonically remove the alcohol-soluble compounds in a mixture of jujube powder and 70% ethanol at a ratio of 1:20, using an ultrasonic power of 480 W for 30 minutes; centrifuge after ultrasonication to retain the precipitate;

[0026] b. Deep eutectic solvent-assisted extraction: according to a solid-liquid ratio of 1:30 (w / v), the extraction temperature was 95°C, and the extraction time was 3 hours; wherein the deep eutectic solvent was composed of choline chloride, ethylene glycol, and ultrapure water, and the molar ratio of choline chloride to ethylene glycol was 1:3, and the water content of the deep eutectic solvent was 30%. After the extraction was completed, the supernatant was retained by centrifugation;

[0027] c. Alcohol precipitation: Add 95% ethanol to the extract, let it stand overnight after alcohol precipitation;

[0028] e. Redissolution: Remove the supernatant, retain the precipitate after centrifugation, wash the precipitate with 76% ethanol, and then re-dissolve it in ultrapure water;

[0029] f. Starch removal: Use α-amylase and saccharifying enzymes in sequence to remove starch from the reconstituted solution; g. Inactivation: 95°C for 30 minutes; centrifuge at 4500g for 15 minutes and retain the supernatant;

[0030] h. Ultrafiltration: The supernatant is separated by a membrane with a molecular weight cut-off of 3000 Da to remove small molecules, and then freeze-dried to obtain jujube acidic polysaccharide (JPA).

[0031] Wherein, the modification methods described in step b are respectively:

[0032] a. Low temperature alkaline deesterification technology:

[0033] 0.5% (w / v) of jujube acidic polysaccharide was added to a sodium hydroxide solution with a pH value of 12.0 and stirred at 4°C for 30 minutes; hydrochloric acid (1 M) was added until the pH value was 7.0, and the solution was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and then freeze-dried to obtain a jujube modified acidic polysaccharide (JPA-AD) with deesterification;

[0034] b. Directed enzyme digestion technology:

[0035] 0.5% (w / v) jujube acidic polysaccharide was added to a 0.3U / mL pectinase solution, reacted at 40°C for 6 hours, inactivated, centrifuged, and the supernatant was collected; the supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and then freeze-dried to obtain a jujube modified acidic polysaccharide (JPA-E) with a reduced proportion of galacturonic acid polysaccharide.

[0036] c. Modified acidic polysaccharides of jujube with arabinose side chains removed and galactose side chains reduced by controlled partial acid hydrolysis technology:

[0037] 0.5% (w / v) jujube acidic polysaccharide was added to 0.5M trifluoroacetic acid solution to make the final concentration of trifluoroacetic acid in the system 0.25M, and the mixture was reacted at 95°C for 1.5h and 4h respectively; sodium hydroxide solution (1M) was added to the solution until the pH value reached 7.0, and the mixture was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000Da and freeze-dried to obtain jujube modified acidic polysaccharide (JPA-AH1) with arabinose side chains removed and jujube modified acidic polysaccharide (JPA-AH2) with arabinose side chains removed and galactose side chains reduced, respectively; among them, under the condition of reaction for 1.5h, the jujube modified acidic polysaccharide (JPA-AH1) with arabinose side chains removed was prepared; under the condition of reaction for 4h, the jujube modified acidic polysaccharide (JPA-AH2) with arabinose side chains removed and galactose side chains reduced was prepared.

[0038] The present invention also provides use of the modified jujube acidic polysaccharide in preparing health-care food that helps to increase immunity.

[0039] The invention also provides the use of the modified jujube acidic polysaccharide in preparing a medicine with immunomodulatory activity.

[0040] The invention modifies jujube acidic polysaccharides through low-temperature alkaline deesterification technology, directional enzyme cutting technology and controllable partial acid hydrolysis technology, and compares the in vivo and in vitro immunoregulatory activities of the jujube acidic polysaccharides before and after modification. The immunoregulatory activity of the modified jujube acidic polysaccharides obtained by the modification is significantly enhanced, especially the modified jujube acidic polysaccharides with the arabinan side chains removed and the galactan side chains reduced, which have better immunoregulatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 High-performance gel exclusion chromatography of jujube acidic polysaccharides and their modified products (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharides, jujube modified acidic polysaccharides with ester removal, jujube modified acidic polysaccharides with reduced galacturonic acid polysaccharide ratio, jujube modified acidic polysaccharides with arabinose side chains removed, and jujube modified acidic polysaccharides with arabinose side chains removed and galactose side chains reduced, respectively);

[0042] Figure 2 Chromatograms of monosaccharide composition of jujube acidic polysaccharide and its modified products (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharide, jujube modified acidic polysaccharide with ester removal, jujube modified acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, jujube modified acidic polysaccharide with arabinose side chains removed, and jujube modified acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, respectively);

[0043] Figure 3 Fourier transform infrared spectra of jujube acidic polysaccharide and its modified products (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharide, jujube modified acidic polysaccharide with esterification removed, jujube modified acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, jujube modified acidic polysaccharide with arabinose side chains removed, and jujube modified acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, respectively);

[0044] Figure 4 Jujube acidic polysaccharide and its modified products 1 H NMR spectra (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharide, jujube modified acidic polysaccharide with ester removal, jujube modified acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, jujube modified acidic polysaccharide with arabinose side chains removed, and jujube modified acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, respectively);

[0045] Figure 5 Jujube acidic polysaccharide and its modified products 13 C NMR spectra (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharide, jujube modified acidic polysaccharide with ester removal, jujube modified acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, jujube modified acidic polysaccharide with arabinose side chains removed, and jujube modified acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, respectively);

[0046] Figure 6Effects of jujube acidic polysaccharides and their modified products on the cytotoxicity (A), nitric oxide (NO) production (B), tumor necrosis factor-α (TNF-α) production (C), and interleukin-6 (IL-6) production (D) of RAW264.7 macrophages (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharides, modified jujube acidic polysaccharides with deesterification, modified jujube acidic polysaccharides with reduced galacturonic acid polysaccharides, modified jujube acidic polysaccharides with de-arabinose side chains, and modified jujube acidic polysaccharides with de-arabinose side chains and reduced galactose side chains, respectively; error bars represent standard deviations; significant (p < 0.05) differences between different samples are indicated by different letters; significant differences between the tested samples and the blank control are indicated by *p < 0.05 and **p < 0.01);

[0047] Figure 7 Effects of jujube acidic polysaccharides and their modified products on the production of nitric oxide (NO) (A), tumor necrosis factor-α (TNF-α) (B), and interleukin-6 (IL-6) (C) in RAW 264.7 macrophages treated with C29 or TAK-242, as well as the effects of modified jujube acidic polysaccharides on protein expression (D) (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharides, modified jujube acidic polysaccharides with deesterification, modified jujube acidic polysaccharides with reduced galacturonic acid polysaccharides, modified jujube acidic polysaccharides with de-arabinose side chains, and modified jujube acidic polysaccharides with de-arabinose side chains and reduced galactose side chains, respectively; error bars represent standard deviations; significant differences between the tested samples and the blank control are indicated by *p < 0.05 and **p < 0.01).

[0048] Figure 8 Effects of jujube acidic polysaccharides and their modified products on the body weight (A) and organ indexes (B and C) of mice (Note: NC, MC, PC, GJPA, GJPA-AD, GJPA-E, and GJPA-AH2 represent the blank control group, model group, positive group, jujube acidic polysaccharide administration group, jujube modified acidic polysaccharide administration group with deesterification, jujube modified acidic polysaccharide administration group with reduced galacturonan ratio, and jujube modified acidic polysaccharide administration group with de-arabinose side chains and reduced galactose side chains, respectively; error bars represent standard deviations; significant (p < 0.05) differences between different samples are indicated by data with different letters; significant differences are indicated by ns p > 0.05, *p < 0.05, and **p < 0.01);

[0049] Figure 9Histopathological observation of the spleen (A) and liver (B) of mice (Note: NC, MC, PC, GJPA, GJPA-AD, GJPA-E, and GJPA-AH2 represent the blank control group, model group, positive group, group treated with jujube acidic polysaccharide, group treated with jujube modified acidic polysaccharide with esterification removed, group treated with jujube modified acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, and group treated with jujube modified acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, respectively);

[0050] Figure 10 Effects of jujube acidic polysaccharides and their modified products on cytokines and immunoglobulins in serum (Note: NC, MC, PC, GJPA, GJPA-AD, GJPA-E, and GJPA-AH2 represent blank control group, model group, positive group, and group receiving jujube acidic polysaccharide, group receiving modified jujube acidic polysaccharide with esterification removed, group receiving modified jujube acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, and group receiving modified jujube acidic polysaccharide with arabinose side chains removed and galactose side chains reduced; error bars represent standard deviations; significant (p < 0.05) differences between different samples are represented by data with different letters). DETAILED DESCRIPTION

[0051] Example 1 Preparation method of modified acidic polysaccharide from jujube of the present invention

[0052] 1. Preparation of jujube acidic polysaccharide:

[0053] a. Ultrasonic Removal of Alcohol-Soluble Components: Remove the core of the jujube, chop it, dry it at 55°C, grind it into a powder, and pass it through an 80-mesh sieve. Ultrasonicate the mixture at a ratio of 1:20 (jujube powder): 70% ethanol at 480W for 30 minutes to remove the alcohol-soluble compounds. After ultrasonic extraction, centrifuge at 5000g for 10 minutes and retain the precipitate.

[0054] b. Deep eutectic solvent-assisted extraction: according to a solid-liquid ratio of 1:30 (w / v), the extraction temperature was 95°C, and the extraction time was 3 hours; wherein the deep eutectic solvent was composed of choline chloride, ethylene glycol, and ultrapure water, and the molar ratio of choline chloride to ethylene glycol was 1:3, and the water content of the deep eutectic solvent was 30%. After the extraction was completed, the supernatant was retained by centrifugation;

[0055] c. Alcohol precipitation: Add 4 times the volume of 95% ethanol to the extract, and let it stand overnight after alcohol precipitation.

[0056] e. Redissolution: Remove the supernatant, centrifuge at 5000g for 10 min, retain the precipitate, wash the precipitate with 76% ethanol, and then re-dissolve in ultrapure water.

[0057] f. Starch removal: Use 10 U / mL of α-amylase at 90°C for 8 h, and 10 U / mL of saccharifying enzyme at 59°C for 12 h to remove starch from the reconstituted solution.

[0058] g. Inactivation: 95℃ for 30min; centrifuge at 4500g for 15min and retain the supernatant.

[0059] h. Ultrafiltration: The supernatant is separated by a membrane to remove small molecules (molecular weight cut-off is 3000 Da) and freeze-dried to obtain jujube acidic polysaccharide (JPA).

[0060] 2. Preparation of deesterified jujube modified acidic polysaccharide using low-temperature alkaline deesterification technology:

[0061] a. Take 0.5% (w / v) of the jujube acidic polysaccharide described in 1 and add it to a sodium hydroxide solution with a pH value of 12.0, and stir at 4°C for 30 minutes;

[0062] b. Add hydrochloric acid (1 M) to the solution in step a until the pH value is 7.0, ultrafilter through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and then freeze-dry to obtain the jujube modified acidic polysaccharide (JPA-AD) with deesterification.

[0063] 3. Preparation of modified acidic polysaccharides of jujube with reduced galacturonan ratio using directional enzyme digestion technology

[0064] a. Take 0.5% (w / v) of the jujube acidic polysaccharide described in 1 and add it to 0.3U / mL pectinase solution, react at 40°C for 6h, inactivate, centrifuge, and take the supernatant;

[0065] b. The supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and then freeze-dried to obtain a jujube modified acidic polysaccharide (JPA-E) with a reduced ratio of galacturonic acid polysaccharide.

[0066] 4. Preparation of jujube modified acidic polysaccharides with removed arabinose side chains or with reduced galactose side chains by controlled partial acid hydrolysis technology:

[0067] a. Take 0.5% (w / v) of the jujube acidic polysaccharide described in 1 and add 0.5M trifluoroacetic acid solution to make the final concentration of trifluoroacetic acid in the system 0.25M, and react at 95°C for 1.5h and 4h respectively;

[0068] b. Add sodium hydroxide solution (1M) to the solution in step a until the pH value is 7.0, ultrafilter through an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and then freeze-dry to obtain a modified acidic polysaccharide of jujube with arabinose side chains removed (JPA-AH1) and a modified acidic polysaccharide of jujube with arabinose side chains removed and galactose side chains reduced (JPA-AH2). Wherein, under the condition of reaction for 1.5h, the modified acidic polysaccharide of jujube with arabinose side chains removed (JPA-AH1) was prepared; under the condition of reaction for 4h, the modified acidic polysaccharide of jujube with arabinose side chains removed and galactose side chains reduced (JPA-AH2) was prepared.

[0069] Example 2 Characterization of the physicochemical properties of modified acidic polysaccharides from jujube

[0070] 1 Experimental methods

[0071] The total polysaccharide, total uronic acid, total bound phenolic, and total protein contents of the modified acidic polysaccharide from jujube were determined by colorimetry. The molecular weight and dispersity of the modified acidic polysaccharide from jujube were determined by gel size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS-RID, Wyatt Technology Co., Santa Barbara, CA, USA). The monosaccharide composition of the modified acidic polysaccharide from jujube was determined by high-performance liquid chromatography (L-20A, Shimadzu, Japan) coupled with pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The functional groups and degree of esterification of the modified acidic polysaccharide from jujube were analyzed by Fourier transform infrared spectroscopy (PerkinElmer, Waltham, MA, USA). The glycosidic bonds of the modified acidic polysaccharide from jujube were analyzed by nuclear magnetic resonance spectroscopy (Bruker, Rheinstetten, Germany). Methods reference: Authors: Ding-Tao Wu, Meng-Xi Fu, Huan Guo, Yi-Chen Hu, Xiao-Qin Zheng, Ren-You Gan and Liang Zou; Title: Microwave-assisted deep eutectic solvent extraction, structural characteristics, and biological functions of polysaccharides from sweet tea (Lithocarpus litseifolius) leaves; Journal: ANTIOXIDANTS, Volume 11, Issue 8; DOI: 10.3390 / antiox11081578.

[0072] 2 Chemical composition of modified acidic polysaccharides from jujube

[0073] The chemical compositions of jujube acidic polysaccharides (JPA) and modified jujube acidic polysaccharides (JPA-AD, JPA-E, JPA-AH1, and JPA-AH2) are shown in Table 1. The results showed that the acidic and modified jujube acidic polysaccharides were primarily composed of polysaccharide compounds. The total polysaccharide contents of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 were 92.20 mg / 100 mg, 92.04 mg / 100 mg, 94.42 mg / 100 mg, 90.32 mg / 100 mg, and 90.26 mg / 100 mg, respectively. The results showed that structural modification of jujube polysaccharides by low-temperature alkaline deesterification, directed enzymatic cleavage, and controlled partial acid hydrolysis did not affect the total polysaccharide content.

[0074] The uronic acid content of the modified acidic polysaccharide (JPA-AD) prepared by low-temperature alkaline deesterification decreased from 27.57 mg / 100 mg to 21.42 mg / 100 mg, which may be due to the β-elimination reaction that can hydrolyze the pectin polysaccharide by cleaving the main chain. The uronic acid content of the modified acidic polysaccharide (JPA-E) prepared by directed enzymatic cleavage decreased to 22.19%, which may be due to the destruction of the main chain by pectinase. The uronic acid content of the modified acidic polysaccharides (JPA-AH1 and JPA-AH2) prepared by controlled partial acid hydrolysis increased to 41.66 mg / 100 mg and 50.72 mg / 100 mg, respectively, which is due to the removal of the side chains arabinose and galactose of RG-I.

[0075] In addition, the total protein and total bound phenolic contents of JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 were all lower than those of JPA. The total protein contents of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 were 1.54 mg / 100 mg, 1.30 mg / 100 mg, 0.40 mg / 100 mg, 0.68 mg / 100 mg, and 0.56 mg / 100 mg, respectively. The total bound phenolic contents of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 were 1.65 mg GAE / 100 mg, 1.21 mg GAE / 100 mg, 1.03 mg GAE / 100 mg, 0.85 mg GAE / 100 mg, and 0.65 mg GAE / 100 mg, respectively.

[0076] Table 1 Chemical composition of jujube acidic polysaccharides and their modified products

[0077]

[0078] Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharide, modified jujube acidic polysaccharide with deesterification, modified jujube acidic polysaccharide with reduced galacturonan ratio, modified jujube acidic polysaccharide with arabinose side chains removed, and modified jujube acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, respectively. Results are expressed as (mean ± SD); superscript ae indicates significant differences (p < 0.05); statistical significance was assessed using ANOVA.

[0079] 3 Structural characteristics of modified acidic polysaccharides from jujube

[0080] In order to further determine the primary structural characteristics of jujube acidic polysaccharides and jujube modified acidic polysaccharides (JPA, JPA-AD, JPA-E, JPA-AH1, JPA-AH2), their molecular weight, monosaccharide composition, functional groups and glycosidic bonds were studied by high-performance size exclusion chromatography, high-performance liquid chromatography, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectrometry, and thus determine whether their modification was successful.

[0081] (1) Molecular weight distribution

[0082] The biological activity of polysaccharides is related to their molecular weight. Generally, polysaccharides with lower molecular weight have better activity. Figure 1 Gel exclusion chromatograms of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 are shown. The results show that JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 all exhibit symmetrical elution profiles. The gel exclusion chromatograms of JPA and JPA-AD are extremely similar, almost completely overlapping (with essentially identical retention times), indicating that the molecular weight of jujube acidic polysaccharides remains largely stable after low-temperature alkaline deesterification. The molecular weights of JPA and JPA-AD are 4.080×10 4 Da and 4.069×10 4 Da. However, the gel exclusion chromatography of JPA-E, JPA-AH1, and JPA-AH2 shifted significantly to the right, and the retention time prolonged as the molecular weight decreased. The molecular weights of JPA-E, JPA-AH1, and JPA-AH2 decreased significantly to 1.12×10 4 Da, 1.186×10 4 Da and 1.035×10 4 In addition, the polydispersity (M w / M n ) is 1.186-1.430.

[0083] Table 2 Molecular weight of jujube acidic polysaccharide and its modified products (M w ), dispersion coefficient (M w / M n ), monosaccharide composition molar ratio

[0084]

[0085] Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharide, modified jujube acidic polysaccharide with ester removal, modified jujube acidic polysaccharide with reduced galacturonic acid polysaccharide ratio, modified jujube acidic polysaccharide with arabinose side chains removed, and modified jujube acidic polysaccharide with arabinose side chains removed and galactose side chains reduced, respectively; HG, galacturonic acid polysaccharide, HG (%) = GalA (%) - Rha (%); RG-I, type I rhamnogalacturonic acid polysaccharide, RG-I (%) = 2*Rha (%) + Gal (%) + Ara (%); the results in the table are expressed as (mean ± SD); the superscript ac indicates significant difference (p < 0.05); statistical significance was tested by ANOVA test.

[0086] (2) Monosaccharide composition analysis

[0087] In order to reveal the effects of different modification methods on the chemical structure of jujube acidic polysaccharides, the monosaccharide composition, chemical groups and glycosidic bonds of jujube modified acidic polysaccharides were systematically studied. The HPLC profiles of the constituent sugars of JPA, JPA-AD, JPA-E, JPA-AH1 and JPA-AH2 were obtained by PMP pre-column derivatization and HPLC analysis ( Figure 2). The results showed that the monosaccharide composition of JPA, JPA-AD, JPA-E, JPA-AH1 and JPA-AH2 was consistent, namely galacturonic acid (GalA), galactose (Gal), arabinose (Ara), rhamnose (Rha), glucose (Glc), glucuronic acid (GlcA) and mannose (Man). The results showed that after the jujube acidic polysaccharide was modified by low-temperature alkaline deesterification technology and directed enzymatic cleavage technology, its constituent sugar types did not change, but the molar percentage of its constituent sugars changed, the uronic acid content ratio decreased slightly, resulting in a slight increase in the neutral sugar ratio. The HG content of JPA-E was significantly reduced to 14.23%, and the RG-I side chain length increased to 6.63. Pectinase destroyed the HG domain of the jujube acidic polysaccharide. The results showed that the jujube modified acidic polysaccharide with low HG content was successfully prepared by directed enzymatic cleavage technology. Furthermore, modified jujube acidic polysaccharides through controlled partial acid hydrolysis significantly altered the types and proportions of their constituent sugars, particularly arabinose (Ara) and galactose (Gal) located in the RG-I side chains. Specifically, the arabinose side chains in JPA-AH1 were largely removed; further degradation nearly eliminated the arabinose side chains in JPA-AH2, while the galactose side chain content was also significantly reduced.

[0088] (3) Infrared spectral characteristics

[0089] FT-IR spectroscopy was used to further explore the changes in the structural characteristics of jujube acidic polysaccharides before and after modification. Figure 3 As shown in the figure, the infrared spectra of JPA, JPA-AD, JPA-E, JPA-AH1 and JPA-AH2 are similar, and all have typical pectin acidic polysaccharide absorption bands, which are 3410.6 cm -1 、2949.9cm -1 、1745.1cm -1 、1623.4cm -1 、1447.3cm -1 、1245.5cm -1 、1101.4cm -1 and 1018.1cm -1 In addition, after modification by low-temperature alkaline deesterification technology and controlled different acid hydrolysis technology, the esterification functional groups of JPA, JPA-AD, JPA-AH1, and JPA-AH2 were 1745.1 cm -1 The absorption band peak area at 1745.1cm -1 and 1623.4cm -1The peak areas of the nearby absorption bands were used to calculate the esterification degrees of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2, which were 58.10%, 11.79%, 53.72%, 11.25%, and 2.55%, respectively. This indicated that the modified acidic polysaccharides with low esterification degree of jujube were successfully prepared. The directed enzymatic cleavage technology had little effect on the esterification degree, and the controlled partial acid hydrolysis technology also significantly affected the esterification degree.

[0090] (4) Nuclear magnetic resonance spectroscopy

[0091] In order to further reveal the primary structural characteristics of the modified acidic polysaccharide of jujube, nuclear magnetic resonance analysis was performed. Figure 4 and Figure 5 As shown, JPA, JPA-AD, JPA-E, JPA-AH1, JPA-AH2 1 H and 13 The C NMR spectra showed similar characteristic signals, indicating that JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 shared similar primary chemical structures. Specifically, signals of sugar residues in both α and β configurations were detected in JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2.

[0092] In JPA, signals at 1.25 ppm, 5.24 ppm, and 16.47 ppm are assigned to H-6, H-1, and C-6 of 1,2,4-α-L-Rhap. Signals at 4.96 ppm, 100.35 ppm, and 170.61 ppm are assigned to H-1, C-1, and C-6 of 1,4-α-D-GalAMep. Signals at 5.02 ppm and 99.48 ppm are assigned to H-1 and C-1 of 1,4-α-D-GalAp. Signals at 5.09 ppm and 107.42 ppm are associated with H-1 and C-1 of 1,5-α-L-Araf. Signals at 5.15 ppm and 109.16 ppm are associated with H-1 and C-1 of T-α-L-Araf, respectively. Signals at 3.80 ppm and 52.81 ppm are attributed to the methyl ester of 6-O GalA (GalA-OCH3). The signal at 2.07 ppm represents the O-acetyl group of GalAp. The signal at 20.08 ppm also represents the O-acetyl group. The signal at 4.46 ppm belongs to the H-1 of 1,3,6-β-D-Galp. The signal at 4.53 ppm belongs to the H-1 of 1,3-β-D-Galp. The signal at 4.64 ppm belongs to the H-1 of 1,4-β-D-Galp.

[0093] In JPA-AD, the signal intensities at 2.07 ppm, 2.18 ppm, 3.80 ppm, 20.08 ppm, 20.50 ppm, 52.81 ppm, and 170.61 ppm weakened or even disappeared, indicating that low-temperature alkaline deesterification can successfully reduce or remove the methylation and acetylation levels of GalAp residues in jujube acidic polysaccharides. In JPA-E, the signal intensities at 4.96 ppm and 5.02 ppm weakened or even disappeared, indicating that the targeted enzymatic digestion technique can reduce the GalAp residues in jujube acidic polysaccharides. In JPA-AH1 and JPA-AH2, the signal intensities at 2.07ppm, 2.18ppm, 3.80ppm, 4.46ppm, 4.53ppm, 4.64ppm, 5.09ppm, 5.15ppm, 5.18ppm, 20.08ppm, 20.50ppm, 52.81ppm, 103.12ppm, 107.09ppm, 107.42ppm, 109.16ppm, and 170.61ppm weakened or even disappeared, and 1,4-α-D-GalAp residues were observed at 5.07ppm (1H) in JPA-AH1 and JPA-AH2. This indicates that the Araf and GalAp residues of jujube acidic polysaccharides can be successfully reduced or removed through controlled partial acid hydrolysis technology, and the methylation and acetylation levels of GalAp residues can be partially reduced.

[0094] The beneficial effects of the present invention are demonstrated by the following efficacy tests.

[0095] Test Example 1 Determination of in vitro immune activity and mechanism of action of jujube acidic polysaccharides and their modified products

[0096] 1. Experimental methods:

[0097] 1. Materials and Reagents

[0098] Table 3 Materials and reagents for in vitro immunoassay

[0099]

[0100]

[0101] 2. Cytotoxicity assay:

[0102] RAW 264.7 macrophages were cultured at a volume of 5 × 10 3Cells were plated at a concentration of 100 μL / well in a 96-well microplate and cultured overnight in an incubator. The supernatant was aspirated and 100 μL of JPA, JPA-AD, JPA-E, JPA-AH1, or JPA-AH2 (25, 50, 100, 200, or 400 μg / mL) was added to the wells and incubated for 24 hours. A blank culture medium was used as a blank control, and LPS (1 μg / mL) was used as a positive control. The supernatant was aspirated and 100 μL of MTT solution (1 mg / mL) was added to the wells and incubated for 4 hours. The supernatant was aspirated and 100 μL of DMSO was added to the wells. The OD was measured at 570 nm. The cell proliferation rate was calculated using the following formula:

[0103] Cell proliferation rate (%) = 1-(A1-A2) / A1×100%

[0104] Among them, A1 is the absorbance value of the blank group, and A2 is the absorbance value of the sample group.

[0105] 3. Determination of nitric oxide and cytokine content:

[0106] RAW 264.7 cells 1×10 5 Cells were cultured overnight in a 24-well plate at a concentration of 100 cells / well. The supernatant was aspirated, and 1 mL of JPA, JPA-AD, JPA-E, JPA-AH1, or JPA-AH2 polysaccharides (100, 200, or 400 μg / mL) was added to the wells and incubated for 48 hours. Culture medium served as a blank control, and LPS (1 μg / mL) served as a positive control. Subsequently, the supernatant was collected and nitric oxide (NO) and cytokine levels (TNF-α and IL-6) were determined according to the kit instructions.

[0107] 4. Cellular immune mechanism of modified acidic polysaccharides from jujube

[0108] 4.1 Inhibition of TLR2 / 4 signaling

[0109] RAW 264.7 cells were plated at 1 × 10 5 Cells were cultured overnight in 24-well plates at 1 mL / well. After removing the culture medium, 1 mL of culture medium containing or not TAK-242 (1 μM) or C29 (30 μM) was added to the wells and incubated for 4 hours. The cells were then treated with JPA, JPA-AD, JPA-E, JPA-AH1, JPA-AH2 (400 μg / mL), or LPS (1 μg / mL) for 48 hours. Total NO content was measured by the Griess method, and TNF-α and IL-6 levels were measured using ELISA kits.

[0110] 4.2 Western blot analysis

[0111] RAW 264.7 cells were plated at 4 × 10 5Cells were cultured in 6-well plates overnight. After removing the culture medium, 2 mL of JPA-AH1 (400 μg / mL) was added to the wells and incubated for 48 hours. The culture medium was used as a blank control and LPS (1 μg / mL) was used as a positive control. The supernatant was removed and the well plates were rinsed twice with PBS. 1 mL of PBS was added to each well, the cells were gently blown off and transferred to a 1.5 mL centrifuge tube, and the cells were collected after centrifugation. RIPA cell lysis buffer (containing 0.1% PMSF and protein phosphatase inhibitors) was added and lysed on ice for 30 minutes. Capillary protein immunoblot analysis was performed on the Protein Simple Wes system using a 12-230 kDa separation module (SM-W004) and an anti-rabbit detection module (DM-001) according to the manufacturer's instructions. Data were analyzed using Compass software, and the appropriate exposure time was set to ensure maximum signal.

[0112] 2. Experimental results:

[0113] 1. In vitro immunological activity and structure-activity relationship of jujube acidic polysaccharides and their modified products

[0114] Figure 6 A shows the cytotoxic effects of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 on RAW 264.7 cells. The results showed that JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 had no toxic effects on cell viability at concentrations of 25 μg / mL–400 μg / mL. Figure 6 As shown in Figures B, C, and D, different concentrations (100 μg / mL–400 μg / mL) of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 can significantly increase the levels of NO, IL-6, and TNF-α produced by RAW 264.7 cells, indicating that modified acidic polysaccharides from jujube have significant immunostimulatory effects in vitro, and modification can enhance the in vitro immunostimulatory effects of acidic polysaccharides from jujube.

[0115] Among them, the in vitro immunostimulatory effect of JPA-AD was significantly enhanced, indicating that the immunostimulatory effect of jujube acidic polysaccharides was enhanced after the esterification degree of jujube acidic polysaccharides was reduced by low-temperature alkaline deesterification. JPA-E showed a slight decrease in esterification, molecular weight, and HG fraction, but also a stronger immunostimulatory effect, suggesting that enzymatic hydrolysis can enhance the immunostimulatory effect of jujube acidic polysaccharides by reducing molecular weight and HG fraction, thereby increasing the RG-I fraction. The immunostimulatory effects of JPA-AH1 and JPA-AH2 were significantly enhanced, exceeding those of the other groups, and the immunostimulatory effect of JPA-AH2 was higher than that of JPA-AH1. Among them, JPA-AH1 removed the arabinose side chain, and JPA-AH2 further reduced the length of the galactose side chain on the basis of removing the arabinose side chain. This shows that acid hydrolysis can reduce the side chain length of jujube acidic polysaccharide RG-Ⅰ, exposing more main chains and enhancing the immune stimulating effect. In addition, the molecular weight and esterification degree of JPA-AH1 and JPA-AH2 were significantly reduced, and the uronic acid content was significantly increased, which may also lead to further enhancement of the immune stimulating effect of JPA-AH1 and JPA-AH2.

[0116] The results showed that the esterification degree, HG and RG-Ⅰ ratio, RG-Ⅰ side chain length, uronic acid and molecular weight of jujube acid polysaccharide all affected the in vitro immune effect of jujube acid polysaccharide. Among them, the esterification degree, RG-Ⅰ side chain length and HG ratio of jujube acid polysaccharide were negatively correlated with its immunostimulatory effect, while the main chain exposure degree, uronic acid content and RG-Ⅰ ratio were positively correlated with its immunostimulatory effect. In addition, the RG-Ⅰ side chain length, i.e. the main chain exposure degree and uronic acid content, had a more significant effect on the in vitro immune activity of jujube acid polysaccharide.

[0117] 2. Potential cellular immune mechanism of modified acidic polysaccharides from jujube

[0118] 2.1 Inhibition of TLR2 / 4 signaling

[0119] To further reveal the potential mechanism of macrophage activation mediated by JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2, RAW 264.7 macrophages were treated with TAK-242 and C29. TAK-242 and C29 are small molecule specific inhibitors of TLR 4 and TLR 2, respectively, which can selectively bind to TLR 4 and TLR 2 and interfere with the interaction between TLR 4 and TLR 2 and their adaptor molecules. Figure 7As shown in A, B, and C, the addition of TAK-242 and C29 significantly inhibited the release of NO, TNF-α, and IL-6 by JPA, JPA-AD, JPA-E, JPA-AH1, JPA-AH2 (400 μg / mL), and positive LPS (1 μg / mL). These results indicate that JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 immunomodulate RAW 264.7 macrophages through TLR4 and TLR2 receptors, and the inhibitory rate of TAK-242 was significantly higher than that of C29.

[0120] These results confirmed that JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 can all activate RAW 264.7 cells and exert immune stimulation by interacting with TLR2 or TLR4 receptors on the surface of macrophages, and that TLR4 receptors have a greater influence on the immune effects of modified acidic polysaccharides from jujube.

[0121] 2.2 Western blot analysis

[0122] Polysaccharides are natural macromolecules that bind to pattern recognition receptors on the cell membrane, activating various intracellular signal transduction pathways, promoting the production of cytokines or antibodies, and regulating immune function. As mentioned above, jujube acidic polysaccharides can participate in the activation of RAW 264.7 macrophages through TLR4 and TLR2 receptors. Nuclear factor-κB (NF-κB) is a major immunoregulatory signaling pathway in RAW 264.7 cells. NF-κB protein is typically a dimer formed by the p65 / p50 subunits. Under normal circumstances, NF-κB protein is inactivated due to binding to the inhibitory protein IκB. However, when cells are stimulated and signaling is transmitted, the IKK kinase in the cell is activated and phosphorylates the IκB protein. This causes the IκB protein to detach from the trimer. This allows the p65 subunit to rapidly enter the cell nucleus and activate gene transcription, leading to the activation of the intracellular NF-κB pathway.

[0123] In order to explore the regulatory effect of jujube acidic polysaccharides on the NF-κB signaling pathway, JPA-AH1 was selected to investigate it. The key proteins p65 and phosphorylated p65 (p-p65) involved in the NF-κB signaling pathway were detected by Western blot. Figure 7As shown in Figure D, compared with the control group, p-p65 expression levels were significantly upregulated in the positive group treated with 1 μg / mL LPS and in the sample group treated with 400 μg / mL JPA-AH1. These results suggest that JPA-AH1 may enhance immune function by regulating the NF-κB signaling pathway. In summary, the immunomodulatory mechanism of jujube acidic polysaccharides is closely related to the NF-κB signaling pathway mediated by TLR2 and TLR4 receptors.

[0124] Test Example 2 Determination of in vivo immune activity of jujube acidic polysaccharides and their modified products

[0125] 1. Experimental Methods

[0126] 1. Materials and Reagents

[0127] Table 4 Materials and reagents for in vivo immunoassay

[0128]

[0129] 2. Animal Experiment Design

[0130] Thirty-six SPF male BALA / c mice were randomly divided into seven groups (n=6) after six days of adaptive feeding: blank control (NC), model group (MC), GJPA group, GJPA-AD group, GJPA-E group, GJPA-AH2 group, and positive control group (PC). The dosing regimen for each group is shown in Table 5. Mouse body weights were recorded daily during the experiment. Mice were sacrificed at the end of the experiment, and blood and organs were collected.

[0131] Table 5 Dosage regimen for each group of mice

[0132]

[0133] 3. Body weight and organ index measurement

[0134] Weigh the mice daily. After the experiment, sacrifice the mice and remove the thymus and spleen to weigh them. Calculate the immune organ index using the following formula:

[0135]

[0136] 4. Histopathological Observation

[0137] The spleen and liver of mice were obtained, and tissues from the same parts of each organ were fixed in 4% paraformaldehyde and sent to Sewell Biotechnology Co., Ltd. for paraffin embedding and sectioning. The tissues were then stained with hematoxylin and eosin (H&E) and finally photographed and analyzed under a microscope.

[0138] 5. Determination of cytokine and immunoglobulin levels in serum

[0139] Blood was collected from the mouse orbital cavity and allowed to stand at room temperature for two hours before centrifugation to obtain the upper serum layer. Serum cytokine (TNF-α, IL-6, IFN-γ) and immunoglobulin (IgA and IgG) levels were measured using ELISA kits.

[0140] 2. Experimental Results

[0141] 1. Effects on body weight and organ indices of immunosuppressed mice

[0142] In this study, except for the blank control group (NC group), mice in the other groups were intraperitoneally injected with cyclophosphamide for 3 consecutive days. Figure 8 A. After modeling, the weight of mice in the other groups decreased significantly compared with the NC group, which preliminarily proved that the modeling was successful. After 10 days of gavage, the weight of mice in all groups increased significantly, and compared with the model group (MC group), the weight of mice in the other groups increased significantly. On the last day of the experiment, the weight of mice in the positive drug group (PC group) and the polysaccharide group (GJPA, GJPA-AD, GJPA-E, GJPA-AH2) was not significantly different from that in the NC group. The results show that jujube acidic polysaccharides and their modified products have a good effect on restoring the weight of immunosuppressed mice.

[0143] The thymus and spleen are the main organs of the immune system that coordinate adaptive immune responses and are considered important immune organs of the body. Figure 8 As shown in Figures B and C, the thymus index and spleen index of mice in each group were measured. Compared with the NC group, the organ index of mice in the MC group was significantly decreased, indicating that the immunosuppressive model was successfully established. Compared with the MC group, the organ index of mice in the PC group and polysaccharide groups (GJPA, GJPA-AD, GJPA-E, and GJPA-AH2) was significantly increased, indicating that jujube acidic polysaccharides and their modified products can promote the regeneration and development of damaged cells in immunosuppressed mice, thereby improving the immunity of immunosuppressed mice to a certain extent.

[0144] 2. Effects on spleen and liver tissue morphology in immunosuppressed mice

[0145] In order to further understand the effects of jujube acidic polysaccharides and their modified products on immunosuppressive mice, HE staining was used to observe the tissue morphology of the spleen and liver of each group of mice.

[0146] like Figure 9As shown in A, the boundary between the red and white pulp of the spleen of mice in the NC group was clear and arranged in an orderly manner. The boundary between the white and red pulp of the spleen of mice in the MC group was blurred, the white pulp area was reduced, the spleen was congested, and cells were necrotic. Compared with the MC group, the red and white pulp of the spleen of mice in the PC group and polysaccharide groups (GJPA, GJPA-AD, GJPA-E, GJPA-AH2) were more evenly distributed, with a relatively clear boundary, a larger white pulp area, and more neatly arranged cells. In addition, compared with the PC group, the boundary between the white and red pulp of the spleen of mice in the polysaccharide group was more obvious, and the white pulp area was larger. The study showed that positive drugs and jujube acidic polysaccharides and their modified products all had a significant effect on the recovery of the spleen in mice with CTX-induced immunosuppression.

[0147] like Figure 9 As shown in Figure 2, the hepatic lobule structure of the NC group mice was clear and intact, with hepatic cords arranged radially around the central vein, and the hepatocytes were morphologically intact and neatly arranged. In the MC group, hepatocytes were disorganized, with missing nuclei and necrosis. Inflammatory infiltration was observed near the central vein, indicating that intraperitoneal injection of CTX damages the mouse liver tissue. Compared with the MC group, the hepatic cords around the central vein in the PC group and the polysaccharide groups (GJPA, GJPA-AD, GJPA-E, and GJPA-AH2) restored their radial morphology and hepatocyte structure. GJPA-AD, GJPA-E, and GJPA-AH2 were more effective than GJPA, with the liver tissue morphology of the GJPA-AH2 group being closer to that of the NC group. These results indicate that jujube acidic polysaccharides and their modified products can effectively reduce CTX-induced liver tissue damage in immunosuppressed mice, with the modified jujube acidic polysaccharide GJPA-AH2 exhibiting a more pronounced effect.

[0148] 3. Effects on cytokines and immunoglobulins in serum

[0149] like Figure 10 As shown in the figure, compared with the NC group, the levels of cytokines (TNF-α, IL-6, IFN-γ) and immunoglobulins (IgA, IgG) in the serum of immunosuppressed mice in the MC group were significantly reduced, indicating that the injection of CTX led to a decrease in the immunity of the mice. After the administration of positive drugs and GJPA, GJPA-AD, GJPA-E, and GJPA-AH2, the levels of cytokines and immunoglobulins in the serum were significantly increased, and the effects of GJPA-AD, GJPA-E, and GJPA-AH2 were better than those of GJPA.

Claims

1. A modified acidic polysaccharide of jujube, characterized by: Each 100 mg of modified acidic polysaccharide from jujube contains: Total polysaccharides: 90.26 mg – 94.42 mg; Total uronic acid: 22.19 mg – 50.72 mg; Total bound phenolics: 0.65 mg GAE – 1.03 mg GAE gallic acid equivalents; The esterification degree of the modified acidic polysaccharide from jujube is 2.55%–53.72%, and the molecular weight is 1.035×10 4 Da –1.186× 10 4 Da, the molar proportion of galacturonan HG is 14.23% – 57.26%, the molar proportion of type I rhamnogalacturonan RG-I is 40.43% – 81.87%, and the side chain length of RG-I is 0.43 – 6.63; it mainly contains the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, glucose, glucuronic acid, and mannose. The molar percentage of each monosaccharide is: (23.72% – 73.88%): (7.19% – 27.14%): (0.00% – 35.75%): (9.49% – 16.62%): (1.31% –4.64%): (0.44% – 1.32%): (0.56% – 0.84%); The modified jujube acidic polysaccharide is obtained by extracting the jujube acidic polysaccharide using a water bath heating-assisted low eutectic solvent, and then modifying the jujube acidic polysaccharide using a pectinase directional enzymatic digestion technology or a controllable partial acid hydrolysis technology.

2. The modified acidic polysaccharide of jujube according to claim 1, characterized in that: Each 100 mg of modified acidic polysaccharide from jujube contains: Total polysaccharides: 90.26 mg; Total uronic acid: 50.72 mg; Total bound phenols: 0.65 mg GAE gallic acid equivalents; The esterification degree of the modified acidic polysaccharide from jujube is 2.55%, and the molecular weight is 1.035×10 4 Da, the molar proportion of galacturonan HG is 57.26%, the molar proportion of type I rhamnogalacturonan RG-Ⅰ is 40.43%, and the side chain length of RG-I is 0.

43.

3. The modified acidic polysaccharide of jujube according to claim 1, characterized in that: It mainly contains the following monosaccharides: galacturonic acid, galactose, rhamnose, glucose, glucuronic acid, and mannose. The molar percentage of each monosaccharide is: 73.88%, 7.19%, 16.62%, 1.31%, 0.44%, and 0.56%.

4. A method for preparing the modified acidic polysaccharide of jujube according to any one of claims 1 to 3, characterized in that: It includes the following steps: a. Using water bath heating to assist deep eutectic solvent to extract jujube acidic polysaccharides; b. Modify the jujube acidic polysaccharide by using pectinase directional enzymatic digestion technology or controlled partial acid hydrolysis technology to obtain the jujube modified acidic polysaccharide.

5. The method for preparing modified acidic polysaccharide of jujube according to claim 4, characterized in that: The method for extracting jujube acidic polysaccharides using a deep eutectic solvent assisted by water bath heating in step a comprises the following steps: a. Ultrasonic removal of alcohol-soluble components: Remove the core of the jujube, chop it into pieces, dry it, grind it into powder, and pass it through an 80-mesh sieve; ultrasonically remove the alcohol-soluble compounds in a mixture of jujube powder and 70% ethanol at a ratio of 1:20, with an ultrasonic power of 480 W and a sonication time of 30 min; after sonication, centrifuge and retain the precipitate; b. Deep eutectic solvent assisted extraction: according to 1:30 w / v The solid-liquid ratio was 95°C, and the extraction time was 3 h. The deep eutectic solvent consisted of choline chloride, ethylene glycol, and ultrapure water. The molar ratio of choline chloride to ethylene glycol was 1:3, and the water content of the deep eutectic solvent was 30%. After the extraction was completed, the supernatant was retained by centrifugation. c. Alcohol precipitation: Add 95% ethanol to the extract, let it stand overnight after alcohol precipitation; e. Redissolution: Remove the supernatant, retain the precipitate after centrifugation, wash the precipitate with 76% ethanol, and then re-dissolve it in ultrapure water; f. Starch removal: Use α-amylase and saccharifying enzymes in sequence to remove starch from the re-dissolved solution; g. Inactivation: 95°C for 30 min; centrifugation at 4500 g for 15 min and retain the supernatant; h. Ultrafiltration: Take the supernatant and separate it through a membrane to remove small molecules with a molecular weight cutoff of 3000 Da. Then freeze-dry it to obtain jujube acidic polysaccharide JPA.

6. The method for preparing modified acidic polysaccharide from jujube according to claim 4, characterized in that: The modification methods described in step b are: a. Directed enzyme digestion technology: Take 0.5% jujube acidic polysaccharide w / v 0.3 U / mL pectinase solution was added, reacted at 40°C for 6 h, inactivated, centrifuged, and the supernatant was collected. The supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da and then freeze-dried to obtain the modified acidic polysaccharide JPA-E with reduced galacturonic acid polysaccharide content. b. Modified acidic polysaccharides of jujube with arabinose side chains removed and galactose side chains reduced by controlled partial acid hydrolysis technology: Take 0.5% jujube acidic polysaccharide w / v 0.5 M trifluoroacetic acid solution was added to make the final concentration of trifluoroacetic acid in the system 0.25 M, and the reaction was carried out at 95 °C for 1.5 h and 4 h, respectively; 1 M sodium hydroxide solution was added to the solution until the pH value was 7.0, and the solution was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and then freeze-dried to obtain jujube modified acidic polysaccharide JPA-AH1 with removed arabinose side chains and jujube modified acidic polysaccharide JPA-AH2 with removed arabinose side chains and reduced galactose side chains, respectively; among them, the jujube modified acidic polysaccharide JPA-AH1 with removed arabinose side chains was prepared under the reaction condition of 1.5 h; the jujube modified acidic polysaccharide JPA-AH2 with removed arabinose side chains and reduced galactose side chains was prepared under the reaction condition of 4 h.

7. Use of the modified acidic polysaccharide of jujube according to any one of claims 1 to 3 in the preparation of health food that helps to increase immunity.

8. Use of the modified acidic polysaccharide of jujube according to any one of claims 1 to 3 in the preparation of a drug having immunomodulatory activity.

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