Jujube modified acidic polysaccharide as well as preparation method and application thereof

Abstract

The invention provides a Chinese date modified acidic polysaccharide. Each 100 mg of the Chinese date modified acidic polysaccharide contains 90.26 mg + / -1.39 mg to 94.42 mg + / -1.83 mg of total polysaccharide, the total uronic acid content is 21.42 mg + / -1.01 mg to 50.72 mg + / -0.62 mg; total bound phenol: 0.65 mg of GAE + / -0.05 mg of GAE-1.21 mg of GAE + / -0.03 mg of GAE (equivalent weight of gallic acid). The invention also provides a preparation method and application of the jujube modified acidic polysaccharide. The jujube acidic polysaccharide is modified through a low-temperature alkaline deesterification technology, a directional enzyme digestion technology and a controllable partial acid hydrolysis technology, the in-vivo and in-vitro immunomodulatory activity of the jujube acidic polysaccharide before and after modification is compared, and the immunomodulatory activity of the jujube modified acidic polysaccharide obtained through modification is remarkably enhanced; especially, the jujube modified acidic polysaccharide with araban side chains removed and galactan side chains reduced has better immunocompetence.

Description

Technical Field

[0001] The invention relates to a jujube modified acidic polysaccharide and a preparation method and application thereof, belonging to the field of health food or medicine. Background Art

[0002] Jujube (Ziziphus jujuba Mill.), a plant of the genus Ziziphus in the family Rhamnaceae. The fruit is oval or spherical, 2-3.5cm long and 1.5-2.5cm in diameter. The surface is dark red, slightly shiny, and has irregular wrinkles. The base is sunken and has a short fruit stalk. The exocarp is thin, the mesocarp is brown or light brown, fleshy, soft, rich in sugar and oily. The core is spindle-shaped, with sharp points at both ends and a hard texture. It has a slight fragrance and tastes sweet. Jujube is widely distributed in my country and is planted in many places. Among them, Xinjiang, Gansu, Ningxia, Shaanxi, Shanxi, Shandong, Hebei, Henan and other places are the main production areas of jujube. Jujube is a food that has both food and medicine. In ancient pharmacology books, it was considered a herbal medicine with the effects of 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 such as polysaccharides, flavonoids, saponins, phenolic acids, triterpene acids, and amino acids. Among them, polysaccharides are one of the main active ingredients of jujube, which has biological activities such as immunomodulation, 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, disclosed that on the basis of jujube polysaccharide extraction and separation and purification, molecular modification technology was applied to jujube polysaccharide research, and further molecular modification and modification were performed by ultrasonic treatment, hydrochloric acid degradation and sulfation, carboxylmethylation and acetylation, and the antioxidant activity, hypoglycemic effect and inhibitory effect on hyaluronidase of jujube polysaccharides and their molecular modification products were studied by in vitro test methods, and 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, carboxylmethylation and acetylation, and the antioxidant activity, hypoglycemic effect and inhibitory effect on hyaluronidase of jujube polysaccharides and their molecular modification products were studied by in vitro test methods.

[0004] Zhang Yali et al., Discussion on the Material Basis of the Blood-tonifying Effect of Red Dates, Chinese Food and Nutrition, Issue 2, 2005, disclosed that red dates have significant immune-promoting activity. Anti-complement active polysaccharides are mostly acidic heteropolysaccharides, and their acidic parts 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 jujube neutral polysaccharide JDP-N and acidic polysaccharide JDP-A. The uronic acid analysis of jujube acidic polysaccharide showed that its uronic acid was D-galacturonic acid with a content of 40.6%. According to the pharmacological experiment of the Pharmacology Research Laboratory of the First Military Medical University, it has significant anti-complement activity and lymphocyte proliferation-promoting effects. The acidic heteropolysaccharide of red date polysaccharide is the intrinsic reason for its anti-complement activity.

[0005] The immune system is an important regulatory system that regulates the body's metabolism, prevents invasion by foreign pathogens and the body's own pathological changes, and is of indispensable importance to the normal physiological health of the human body. Polysaccharides are macromolecules that can bind to pattern recognition receptors on the cell membrane to activate various signal transduction pathways in the cell, promote the production of cytokines or antibodies, and regulate the body's immune function. Previous studies have shown that jujube acid polysaccharides have good immunomodulatory activity. Generally, the biological activity of polysaccharides is closely related to their chemical structure, such as degree of esterification, monosaccharide composition, glycosidic bonds, molecular weight, etc. At present, the key effector structure and mechanism of the immunomodulatory activity of jujube acid polysaccharides are still unclear. Therefore, by modifying jujube acid polysaccharides and comparing the in vivo and in vitro immunomodulatory activity of jujube acid polysaccharides before and after modification, the key effector structure and mechanism of action that affect the in vivo and in vitro immunomodulatory activity of jujube acid polysaccharides can be clarified, which is conducive to making fuller use of jujube resources, and providing a reference for the exploration of the immunomodulatory activity and mechanism of jujube polysaccharides, and providing theoretical and technical support for the development of jujube polysaccharide-related functional foods. Summary of the invention

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

[0007] The present invention provides a modified jujube acidic polysaccharide, wherein each 100 mg of the modified jujube acidic polysaccharide contains:

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

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

[0010] Total bound phenol: 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 modified jujube acidic polysaccharide 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 acid polysaccharide of the invention is extracted by using a low eutectic solvent assisted by water bath heating, and then modified by low temperature alkaline deesterification technology, directional enzyme cutting technology or controllable partial acid hydrolysis technology to obtain the jujube modified acid 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. Use low-temperature alkaline deesterification technology, directional enzyme cutting technology or controlled partial acid hydrolysis technology to modify the jujube acid polysaccharide to obtain the jujube modified acid polysaccharide.

[0024] The method for extracting jujube acidic polysaccharides by 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, cut it into pieces, dry it, grind it into powder, and pass it through an 80-mesh sieve; ultrasonically remove the alcohol-soluble compounds according to the ratio of jujube powder to 70% ethanol = 1:20, with an ultrasonic power of 480W and an ultrasonic time of 30 minutes; after ultrasonication, centrifuge and retain the precipitate;

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

[0027] c. Alcohol precipitation: Add 95% ethanol to the extract and 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 add ultrapure water to redissolve;

[0029] f. Starch removal: Use α-amylase and saccharifying enzyme to remove starch in the reconstituted solution; g. Inactivation: 95℃ for 30min; centrifuge at 4500g for 15min and retain the supernatant;

[0030] h. Ultrafiltration: The supernatant is separated by a membrane to remove small molecules. The membrane has a molecular weight cutoff of 3000Da, 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 (1M) was added until the pH value was 7.0, and ultrafiltration was performed through an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and then freeze-dried to obtain jujube modified acidic polysaccharide (JPA-AD) without esterification;

[0034] b. Directed enzyme cutting technology:

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

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

[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 solution 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; wherein, 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 invention also provides the use of the modified jujube acidic polysaccharide in preparing health 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 by 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 arabinan side chains removed and galactan side chains reduced have better immunoregulatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 High performance gel exclusion chromatograms of jujube acidic polysaccharide and its modified products (Note: JPA, JPA-AD, JPA-E, JPA-AH1, 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 chain removed, jujube modified acidic polysaccharide with arabinose side chain removed and galactose side chain reduced, respectively);

[0042] Figure 2 Chromatogram of monosaccharide composition of jujube acidic polysaccharide and its modified products (Note: JPA, JPA-AD, JPA-E, JPA-AH1, 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 chain removed, jujube modified acidic polysaccharide with arabinose side chain removed and galactose side chain 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, 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 chain removed, jujube modified acidic polysaccharide with arabinose side chain removed and galactose side chain 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 polysaccharides, jujube modified acidic polysaccharides with esterification removed, jujube modified acidic polysaccharides with reduced galacturonic acid polysaccharides, 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);

[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 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);

[0046] Figure 6Effects of jujube acidic polysaccharides and their modified products on cytotoxicity (A), production of nitric oxide (NO) (B), production of tumor necrosis factor-α (TNF-α) (C), and production of interleukin-6 (IL-6) (D) in RAW264.7 macrophages (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharides, jujube modified acidic polysaccharides with esterification removed, jujube modified acidic polysaccharides with reduced galacturonic acid polysaccharides, 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; error bars represent standard deviations; significant (p<0.05) differences between different samples are represented by data with different letters; significant differences between the tested samples and the blank control are represented 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, and the effects of jujube modified acidic polysaccharides on protein expression (D) (Note: JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 represent jujube acidic polysaccharides, jujube modified acidic polysaccharides with esterification removed, jujube modified acidic polysaccharides with reduced galacturonic acid polysaccharides, 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; 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, GJPA-AH2 represent blank control group, model group, positive group, group treated with jujube acidic polysaccharides, group treated with jujube modified acidic polysaccharides with esterification removed, group treated with jujube modified acidic polysaccharides with reduced galacturonic acid polysaccharides, group treated with jujube modified acidic polysaccharides with arabinose side chains removed and galactose side chains reduced; 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] Fig. 9Histopathological observation of mouse spleen (A) and liver (B) (Note: NC, MC, PC, GJPA, GJPA-AD, GJPA-E, GJPA-AH2 represent blank control group, model group, positive group, group treated with jujube acid polysaccharide, group treated with jujube modified acid polysaccharide with esterification removed, group treated with jujube modified acid polysaccharide with reduced galacturonic acid polysaccharide ratio, group treated with jujube modified acid polysaccharide with arabinose side chain removed and galactose side chain reduced, respectively);

[0050] Fig.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, 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; 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 of 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, cut it into pieces, dry it at 55°C, grind it into powder, and pass it through an 80-mesh sieve; ultrasonically remove the alcohol-soluble compounds at a ratio of jujube powder to 70% ethanol = 1:20, with an ultrasonic power of 480W and an ultrasonic time of 30 minutes. After ultrasonic extraction, centrifuge at 5000g for 10 minutes and retain the precipitate.

[0054] b. Low eutectic solvent-assisted extraction: according to a solid-liquid ratio of 1:30 (w / v), an extraction temperature of 95°C, and an extraction time of 3h; wherein the low eutectic solvent is composed of choline chloride, ethylene glycol and ultrapure water, and the molar ratio of choline chloride to ethylene glycol is 1:3, and the water content of the low eutectic solvent is 30%. After the extraction is completed, the supernatant is 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 add ultrapure water to redissolve.

[0057] f. Starch removal: Use 10U / mL of α-amylase, the hydrolysis temperature is 90℃, the hydrolysis time is 8h; and 10U / mL of saccharifying enzyme, the hydrolysis temperature is 59℃, and the hydrolysis time is 12h in sequence to remove the starch in 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 3000Da). Freeze-drying is performed to obtain jujube acidic polysaccharide (JPA).

[0060] 2. Preparation of jujube modified acidic polysaccharides with deesterification by 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 a jujube modified acidic polysaccharide (JPA-AD) without esterification.

[0063] 3. Preparation of modified acidic polysaccharides of jujube with reduced galacturonic acid polysaccharide ratio by 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 galacturonic acid polysaccharide ratio.

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

[0067] a. Take 0.5% (w / v) of the jujube acidic polysaccharide described in 1, 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 of step a until the pH value is 7.0, ultrafilter through an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and freeze-dry to obtain the modified acidic polysaccharide (JPA-AH1) of jujube with arabinose side chains removed and the modified acidic polysaccharide (JPA-AH2) of jujube with arabinose side chains removed and galactose side chains reduced. Wherein, under the condition of 1.5h reaction, the modified acidic polysaccharide (JPA-AH1) of jujube with arabinose side chains removed was prepared; under the condition of 4h reaction, the modified acidic polysaccharide (JPA-AH2) of jujube with arabinose side chains removed and galactose side chains reduced 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 phenol and total protein contents of modified acidic polysaccharides of jujube were determined by colorimetry. The molecular weight and dispersity of modified acidic polysaccharides of jujube were determined by gel size exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS-RID, Wyatt Technology Co., Santa Barbara, CA, USA). The monosaccharide composition of modified acidic polysaccharides of jujube was determined by high performance liquid chromatography (L-20A, Shimadzu, Japan) combined with 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization. The functional groups and degree of esterification of modified acidic polysaccharides of jujube were analyzed by Fourier transform infrared spectroscopy (PerkinElmer, Waltham, MA, USA). The glycosidic bonds of modified acidic polysaccharides of jujube were analyzed by nuclear magnetic resonance spectroscopy (Bruker, Rheinstetten, Germany). Specific method reference: Author: 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 name: 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 polysaccharide (JPA) and jujube modified acidic polysaccharide (JPA-AD, JPA-E, JPA-AH1, JPA-AH2) are shown in Table 1. The results show that jujube acidic polysaccharide and jujube modified acidic polysaccharide are mainly polysaccharide compounds. The total polysaccharide contents of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 are 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 show that the structure of jujube polysaccharide is modified by low-temperature alkaline deesterification technology, directional enzyme cutting technology, and controlled partial acid hydrolysis technology, and none of the three methods has an effect on the total polysaccharide content of jujube polysaccharide.

[0074] The uronic acid content in the modified acidic polysaccharide (JPA-AD) of jujube prepared by low-temperature alkaline deesterification technology 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 its main chain. The uronic acid content in the modified acidic polysaccharide (JPA-E) of jujube prepared by directional enzyme cleavage technology decreased to 22.19%, which may be caused by the destruction of the main chain by pectinase. The uronic acid content in the modified acidic polysaccharides (JPA-AH1 and JPA-AH2) of jujube prepared by controlled partial acid hydrolysis technology increased to 41.66 mg / 100 mg and 50.72 mg / 100 mg, respectively, which was due to the removal of the side chain arabinose and galactose of RG-I.

[0075] In addition, the total protein and total bound phenol contents of JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 were 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 phenol 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 acid polysaccharide, jujube modified acid polysaccharide with esterification removed, jujube modified acid polysaccharide with reduced galacturonic acid polysaccharide ratio, jujube modified acid polysaccharide with arabinose side chain removed, and jujube modified acid polysaccharide with arabinose side chain removed and galactose side chain reduced, respectively. The results in the table are expressed as (mean ± standard deviation); superscript ae indicates significant difference (p<0.05); statistical significance was tested by 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 spectrometer and nuclear magnetic resonance spectrometer to determine whether their modification is successful.

[0081] (1) Molecular weight distribution

[0082] The biological activity of polysaccharides is related to their molecular weight. Generally, polysaccharides with lower molecular weights have better activity. Figure 1 The gel exclusion chromatograms of JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 were shown. The results showed that JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 all presented symmetrical elution curves. The gel exclusion chromatograms of JPA and JPA-AD were extremely similar and almost completely overlapped (the retention times were basically the same), indicating that the molecular weight of jujube acidic polysaccharides was basically stable after being modified by low-temperature alkaline deesterification technology. The molecular weights of JPA and JPA-AD were 4.080×10 4 Da and 4.069×10 4 Da. However, the gel exclusion chromatograms of JPA-E, JPA-AH1, and JPA-AH2 shifted significantly to the right, and the retention time increased with the decrease of molecular weight. 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 (M) of jujube acidic polysaccharides and their modified products 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 acid polysaccharide, jujube modified acid polysaccharide with esterification removed, jujube modified acid polysaccharide with reduced galacturonic acid polysaccharide ratio, jujube modified acid polysaccharide with arabinose side chain removed, and jujube modified acid polysaccharide with arabinose side chain removed and galactose side chain 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.

[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 PMP pre-column derivatization method and HPLC analysis were used to obtain the HPLC profiles of the constituent sugars of JPA, JPA-AD, JPA-E, JPA-AH1 and JPA-AH2 ( 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 acidic polysaccharide of jujube was modified by low-temperature alkaline deesterification technology and directional enzyme cutting technology, the type of its constituent sugars did not change, but the molar percentage of its constituent sugars changed, the proportion of uronic acid content decreased slightly, resulting in a slight increase in the proportion of neutral sugars. The HG content of JPA-E was significantly reduced to 14.23%, the length of the RG-I side chain increased to 6.63, and pectinase destroyed the HG domain of the acidic polysaccharide of jujube. The results showed that the modified acidic polysaccharide of jujube with low HG content was successfully prepared by directional enzyme cutting technology. In addition, after the acidic polysaccharide of jujube was modified by controlled partial acid hydrolysis technology, the types and proportions of its constituent sugars changed significantly, especially arabinose (Ara) and galactose (Gal) located in the side chain of RG-I. Specifically, the arabinose side chain in JPA-AH1 was largely removed; after further degradation, the arabinose side chain in JPA-AH2 was almost removed, and the content of galactose side chain 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 acid 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 being modified 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, indicating that the jujube modified acidic polysaccharide with low esterification degree was successfully prepared, while the directional enzymatic cutting technology had little effect on its esterification degree, and the controlled partial acid hydrolysis technology would also significantly affect its esterification degree.

[0090] (4) Nuclear magnetic resonance spectroscopy

[0091] In order to further reveal the primary structural characteristics of jujube modified acidic polysaccharides, 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 had similar primary chemical structures. Specifically, α- and β-configuration sugar residue signals were detected in JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2.

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

[0093] In JPA-AD, the signal intensities at 2.07ppm, 2.18ppm, 3.80ppm, 20.08ppm, 20.50ppm, 52.81ppm and 170.61ppm weakened or even disappeared, indicating that the methylation and acetylation of GalAp residues in jujube acidic polysaccharides can be successfully reduced or removed by low-temperature alkaline deesterification technology. In JPA-E, the signal intensities at 4.96ppm and 5.02ppm weakened or even disappeared, indicating that the directional enzyme cleavage technology 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, 107.42, 109.16ppm and 170.61ppm weakened or even disappeared, and the 1,4-α-D-GalAp residues of JPA-AH1 and JPA-AH2 were observed at 5.07ppm (1H). This indicates that the Araf and Galp residues of jujube acidic polysaccharides can be successfully reduced or removed by controlled partial acid hydrolysis technology in this study, and the methylation and acetylation degrees 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 5 × 10 3The cells were placed in a 96-well microplate at a concentration of 100 μL / well and cultured overnight in an incubator. The supernatant was aspirated and 100 μL JPA, JPA-AD, JPA-E, JPA-AH1, JPA-AH2 (25, 50, 100, 200, 400 μg / mL) was added to the wells and incubated for 24 h. The 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 MTT solution (1 mg / mL) was added to the wells and cultured for 4 h. The supernatant was aspirated and 100 μL DMSO was added to the wells and the OD was measured at 570 nm. The cell proliferation rate was calculated by 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 The 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, and JPA-AH2 polysaccharides (100, 200, and 400 μg / mL) were added to the wells for incubation for 48 h. The culture medium was used as a blank control and LPS (1 μg / mL) was used as a positive control. Subsequently, the supernatant in the wells was taken and the contents of nitric oxide (NO) and cytokines (TNF-α and IL-6) were determined according to the instructions of the kit.

[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 The cells were cultured in 24-well plates overnight. After the culture medium was removed, 1 mL of culture medium containing or not containing TAK-242 (1 μM) and containing or not containing C29 (30 μM) was added to the wells and cultured for 4 h. Then, the cells were treated with JPA, JPA-AD, JPA-E, JPA-AH1, JPA-AH2 (400 μg / mL) or LPS (1 μg / mL) for 48 h, and the total NO content was determined by the Griess method, and the levels of TNF-α and IL-6 were determined by ELISA kits.

[0110] 4.2 Western blot analysis

[0111] RAW 264.7 cells were plated at 4 × 10 5Each well was cultured overnight in a 6-well plate. After removing the culture medium, 2 mL of JPA-AH1 (400 μg / mL) was added to the well and incubated for 48 h. 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 plate was 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. The data was 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 at concentrations of 25 μg / mL–400 μg / mL, JPA, JPA-AD, JPA-E, JPA-AH1, and JPA-AH2 had no toxic effects on cell viability. Figure 6 As shown in 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 improve 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. The esterification of JPA-E was slightly reduced, the molecular weight and HG ratio were reduced, and the immunostimulatory effect was enhanced, indicating that enzymatic hydrolysis can reduce the molecular weight and HG ratio, that is, increase the RG-Ⅰ ratio to improve the immunostimulatory effect of jujube acidic polysaccharides. The immunostimulatory effects of JPA-AH1 and JPA-AH2 were significantly enhanced, significantly higher than those of 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, expose more main chains, and enhance the immunostimulatory effect. 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 immunostimulatory 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. 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 side chain length of RG-Ⅰ, 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] In order 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 in 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 can immunomodulate RAW 264.7 macrophages through TLR4 and TLR 2 receptors, and the inhibition rate of adding TAK-242 is 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 to exert immunostimulatory effects by interacting with TLR2 or TLR4 receptors on the surface of macrophages, and that TLR4 receptors have a greater impact on the immune effects of modified acidic polysaccharides from jujube.

[0121] 2.2 Western blot analysis

[0122] Polysaccharide is a natural macromolecular substance that can bind to pattern recognition receptors on the cell membrane to activate various signal transduction pathways in the cell, promote the production of cytokines or antibodies, and regulate the body's 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 immune regulation signal pathway in RAW 264.7 cells. NF-κB protein is generally a dimeric protein formed by p65 / p50 subunits. In most cases, NF-κB protein is inactive due to binding to the inhibitory protein IκB. When the cell is stimulated to transmit a signal, the IKK kinase in the cell is activated and the IκB protein is phosphorylated. At this time, the IκB protein will fall off from the trimer. As a result, the p65 subunit quickly enters the cell nucleus and activates gene transcription, and then the NF-κB pathway in the cell is activated.

[0123] In order to explore the regulatory effect of jujube acidic polysaccharides on the NF-κB signaling pathway, JPA-AH1 was selected to explore it. The key protein p65 and phosphorylated p65 (p-p65) involved in the NF-κB signaling pathway were detected by Western blot. Figure 7As shown in D, compared with the control group, the expression level of p-p65 was significantly upregulated after the positive group was treated with 1 μg / mL LPS and the sample group was treated with 400 μg / mL JPA-AH1. The results showed that JPA-AH1 can enhance the body's 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 TLR 2 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] After 6 days of adaptive feeding, 36 SPF male BALA / c mice were randomly divided into 7 groups: blank control group (NC), model group (MC), GJPA group, GJPA-AD group, GJPA-E group, GJPA-AH2 group and positive control group (PC), with n=6 mice in each group. The dosing regimen for each group of mice is shown in Table 5. The body weight of mice was recorded every day during the experiment. After the experiment, the mice were killed and their blood and organs were collected.

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

[0132]

[0133] 3. Body weight and organ index determination

[0134] The mice were weighed every day. After the experiment, the mice were killed and their thymus and spleen were removed and weighed to calculate the immune organ index. The formula is as follows:

[0135]

[0136] 4. Histopathological Observation

[0137] The spleen and liver of mice were taken, and the same parts of tissues 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 filmed and analyzed under a microscope.

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

[0139] Blood was collected from the eye sockets of mice and allowed to stand for two hours at room temperature before centrifugation to obtain the upper serum. ELISA kits were used to measure the cytokines (TNF-α, IL-6, IFN-γ) and immunoglobulin levels (IgA and IgG) in the serum.

[0140] 2. Experimental Results

[0141] 1. Effects on body weight and organ indexes 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 restorative effect on 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 B and C, the thymus index and spleen index of each group of mice were measured. Compared with the NC group, the organ index of the MC group mice decreased significantly, indicating that the immunosuppressive model was successfully established. Compared with the MC group, the organ index of the mice in the PC group and the polysaccharide group (GJPA, GJPA-AD, GJPA-E, 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 immunosuppressed mice, HE staining was used to observe the tissue morphology of spleen and liver of each group of mice.

[0146] like Fig. 9As shown in A, the boundary between the red pulp 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 pulp and red pulp of the spleen of mice in the MC group was blurred, the area of ​​the white pulp became smaller, the spleen was congested, and the cells were necrotic. Compared with the MC group, the red pulp 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, the boundary was relatively obvious, the area of ​​the white pulp became larger, and the cells were arranged more neatly. In addition, compared with the PC group, the boundary between the white pulp and red pulp of the spleen of mice in the polysaccharide group was more obvious, and the area of ​​the white pulp 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 CTX-induced immunosuppressive mice.

[0147] like Fig. 9 As shown in B, the liver lobule structure of mice in the NC group was clear and complete, the hepatic cords were arranged radially around the central vein, and the hepatocytes were morphologically complete and neatly arranged. In the MC group, the hepatocytes were arranged in disorder, the nuclei were missing, the cells were necrotic, and there was inflammatory infiltration near the central vein, indicating that intraperitoneal injection of CTX would cause damage to the liver tissue of mice. Compared with the MC group, the hepatic cords around the central vein of the liver of mice in the PC group and polysaccharide groups (GJPA, GJPA-AD, GJPA-E, GJPA-AH2) restored the radial morphology, and the hepatocyte morphology and structure were restored intact. The effects of GJPA-AD, GJPA-E, and GJPA-AH2 were better than those of GJPA, and the liver tissue morphology of the GJPA-AH2 group was closer to that of the NC group. The results showed that jujube acidic polysaccharides and their modified products can effectively reduce the liver tissue damage of immunosuppressed mice induced by CTX, and GJPA-AH2 jujube modified acidic polysaccharides have a more obvious effect.

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

[0149] like Fig.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 immunity of mice decreased after injection of CTX. After the administration of positive drugs and GJPA, GJPA-AD, GJPA-E, and GJPA-AH2, the levels of cytokines and immunoglobulins in the serum increased significantly, 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 in that: Each 100mg of modified acid polysaccharide from jujube contains: Total polysaccharides: 90.26mg±1.39mg–94.42mg±1.83mg; Total uronic acid: 21.42mg±1.01mg–50.72mg±0.62mg; Total bound phenols: 0.65 mg GAE ± 0.05 mg GAE–1.21 mg GAE ± 0.03 mg GAE (gallic acid equivalent); 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.

2. The modified acidic polysaccharide of jujube according to claim 1, characterized in that: Each 100mg of modified acid polysaccharide from jujube contains: Total polysaccharides: 90.26mg±1.39mg; Total uronic acid: 50.72mg±0.62mg; Total bound phenols: 0.65 mg GAE ± 0.05 mg GAE (gallic acid equivalent); 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.

3. The modified acidic polysaccharide of jujube according to claim 1, characterized in that: It mainly contains 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%).

4. The modified acidic polysaccharide of jujube according to claim 3, 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%.

5. The modified acidic polysaccharide of jujube according to any one of claims 1 to 4, characterized in that: The method adopts water bath heating to assist low eutectic solvent to extract jujube acidic polysaccharides, and then modifies the jujube acidic polysaccharides through low temperature alkaline deesterification technology, directional enzyme cutting technology or controlled partial acid hydrolysis technology to obtain jujube modified acidic polysaccharides.

6. A method for preparing the modified acidic polysaccharide of jujube according to any one of claims 1 to 5, characterized in that: It includes the following steps: a. Using water bath heating to assist deep eutectic solvent to extract jujube acidic polysaccharides; b. Use low-temperature alkaline deesterification technology, directional enzyme cutting technology or controlled partial acid hydrolysis technology to modify the jujube acid polysaccharide to obtain the jujube modified acid polysaccharide.

7. The method for preparing modified acidic polysaccharides from jujube according to claim 6, characterized in that: The method for extracting jujube acidic polysaccharides by using a water bath heating-assisted deep eutectic solvent as described in step a comprises the following steps: a. Ultrasonic removal of alcohol-soluble components: remove the core of the jujube, cut it into pieces, dry it, grind it into powder, and pass it through an 80-mesh sieve; ultrasonically remove the alcohol-soluble compounds according to the ratio of jujube powder to 70% ethanol = 1:20, with an ultrasonic power of 480W and an ultrasonic time of 30 minutes; after ultrasonication, centrifuge and retain the precipitate; b. Low eutectic solvent-assisted extraction: according to a solid-liquid ratio of 1:30 (w / v), an extraction temperature of 95°C, and an extraction time of 3h; wherein the low eutectic solvent is composed of choline chloride, ethylene glycol and ultrapure water, and the molar ratio of choline chloride to ethylene glycol is 1:3, and the water content of the low eutectic solvent is 30%. After the extraction is completed, the supernatant is retained by centrifugation; c. Alcohol precipitation: Add 95% ethanol to the extract and 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 add ultrapure water to redissolve; f. Starch removal: Use α-amylase and saccharifying enzymes to remove starch from the reconstituted solution; g. Inactivation: 95°C for 30 min; centrifuge at 4500g for 15 min and retain the supernatant; h. Ultrafiltration: The supernatant is separated by a membrane to remove small molecules with a molecular weight cutoff of 3000Da, and then freeze-dried to obtain jujube acidic polysaccharide (JPA).

8. The method for preparing modified acidic polysaccharides from jujube according to claim 6, characterized in that: The modification methods described in step b are: a. Low temperature alkaline deesterification technology: 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 (1M) was added until the pH value was 7.0, and ultrafiltration was performed through an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and then freeze-dried to obtain jujube modified acidic polysaccharide (JPA-AD) without esterification; b. Directed enzyme cutting technology: 0.5% (w / v) of jujube acidic polysaccharide was added to 0.3U / mL pectinase solution, reacted at 40°C for 6h, 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 jujube modified acidic polysaccharide (JPA-E) with reduced galacturonic acid polysaccharide ratio; c. Modified acidic polysaccharides of jujube with arabinose side chains removed by controlled partial acid hydrolysis and arabinose side chains reduced: 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 solution was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and then 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; wherein, 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.

9. Use of the modified acidic polysaccharide of jujube according to any one of claims 1 to 5 in the preparation of health food that helps to increase immunity.

10. Use of the modified acidic polysaccharide of jujube according to any one of claims 1 to 5 in the preparation of a drug having immunomodulatory activity.

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