Wild jujube pulp crude polysaccharide, polysaccharide composition and preparation method and application thereof

Abstract

The invention relates to the technical field of biological purification, and provides wild jujube pulp crude polysaccharide, a polysaccharide composition as well as a preparation method and application thereof. The crude polysaccharide is obtained by performing enzymolysis extraction and alcohol precipitation treatment on wild jujube pulp, and the polysaccharide composition is obtained by separating and purifying the crude polysaccharide. The wild jujube pulp crude polysaccharide provided by the invention has good effects of regulating blood sugar and regulating blood fat and anti-oxidation and anti-inflammatory effects, and the polysaccharide composition also has obvious anti-oxidation and anti-inflammatory effects and has a wide market prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biological purification, and in particular to a crude polysaccharide of wild jujube pulp, a polysaccharide composition, and a preparation method and application thereof. Background Art

[0002] The sour jujube, also known as mountain jujube, kudzu needle, wild jujube, and thorn, is a plant of the genus Ziziphus of the family Rhamnaceae. It is native to China and mostly grows in mountainous or semi-mountainous areas. It has strong adaptability and is mainly distributed in northern my country. It is an important wild economic plant and has been listed as a food and medicine by the Ministry of Health. Wild sour jujube has extremely high nutritional value. The pulp of sour jujube is rich in a variety of nutrients, mainly including a variety of minerals, soluble sugars, organic acids, polyphenols, sterols, flavonoids, as well as a variety of trace elements and a large number of vitamins. It has antioxidant, immunomodulatory, anti-cancer, sleep-inducing and lipid-lowering effects. In addition, sour jujube seeds also have anti-depression, brain-tonifying and calming properties.

[0003] The sour jujube is the only wild fruit resource in my country that has been industrially developed on a large scale. However, there are many restrictive problems in the development of the sour jujube industry. First, the production methods in the production areas are relatively backward, with low production efficiency and low product added value; second, the facilities and equipment in the production areas are backward. After the sour jujube seeds are used as Chinese medicinal materials, the sour jujube pulp and peel are not fully utilized. There is a lack of research on deep processing technology, resulting in serious waste of resources.

[0004] Polysaccharides are widely distributed in nature and have extremely important biological functions. Many studies have shown that polysaccharides extracted from the pulp of wild jujube have anti-aging, immunomodulatory, and anti-myocardial ischemia effects. In recent years, a variety of technologies have been used to extract polysaccharides, such as alkaline extraction, microwave-assisted extraction, ultrasound-assisted extraction, supercritical fluid extraction, etc. These extraction technologies have their own advantages, but they also have disadvantages, such as affecting the flavor of the finished product, destroying the polysaccharide structure, polluting the environment, and high cost.

[0005] Therefore, the present invention uses the wild jujube pulp as raw material, and studies the key technologies for extracting wild jujube functional substances and preparing functional products to solve the problem of waste of wild jujube resources and provide technical support for the sustainable development and utilization of the wild jujube industry. Summary of the invention

[0006] The purpose of the present invention is to provide a crude polysaccharide from wild jujube pulp, a polysaccharide composition, and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0009] S1. Take the raw material of sour jujube pulp, add water at a material-liquid ratio of 1:4-12 g / mL, adjust the pH value of the mixed liquid to 6.5-7.5, add papain in an amount of 0.01%-0.10% of the mixed liquid, extract by enzymolysis at 30-70°C for 1-1.5h, heat to kill the enzyme, centrifuge and filter to obtain an enzymolysis solution;

[0010] S2. Concentrate the obtained enzymatic hydrolysate to a relative density of 1.05-1.06, precipitate with alcohol, centrifuge, and collect the precipitate to obtain crude polysaccharide.

[0011] Furthermore, the sour jujube pulp raw material is powder obtained by crushing and sieving dried sour jujube pulp.

[0012] Furthermore, the alcohol precipitation method is: adding 4 times the volume fraction of 95% ethanol to the enzymatic hydrolyzate concentrate, alcohol precipitation at 4°C for 24 hours, and centrifugation at 4000 rpm for 15 minutes.

[0013] The present invention also provides a polysaccharide composition of wild jujube pulp, which is obtained by separating and purifying the above-mentioned crude polysaccharide of wild jujube pulp. The polysaccharide composition of wild jujube pulp comprises a neutral polysaccharide component (WPS-a) and an acidic polysaccharide component (SPS-3a).

[0014] Furthermore, the neutral polysaccharide component is composed of monosaccharides fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, and the molar ratio of each monosaccharide is 0.75:16.81:19.97:17.54:33.04:2.52:1.66:6.86:0.86; the acidic polysaccharide component is composed of monosaccharides rhamnose, arabinose, galactose, glucose and galacturonic acid, and the molar ratio of each monosaccharide is 2.54:2.76:2.31:91.75:0.65.

[0015] Furthermore, the weight average molecular weight of the neutral polysaccharide component is 3.73×10 5 ±0.01×10 5 Da; the weight average molecular weight of the acidic polysaccharide component is 1.36×10 7 ±0.01×10 7 Da.

[0016] Furthermore, the molecular configurations of the neutral polysaccharide component and the acidic polysaccharide component are spherical.

[0017] Furthermore, the separation and purification process includes protein removal; macroporous resin adsorption; dialysis, freeze drying; anion exchange chromatography purification and gel chromatography purification.

[0018] Furthermore, the papain-Sevag method is used to remove protein.

[0019] Furthermore, the anion exchange chromatography purification includes the following steps: taking a crude polysaccharide sample and dissolving it in pure water to prepare a polysaccharide mother solution with a concentration of 10 mg / mL, centrifuging, taking the supernatant, and purifying it with an anion exchange chromatography column, using pure water, 0.1M NaCl solution, 0.2M NaCl solution and 0.3M NaCl solution in sequence for isogradient elution, with a flow rate of 4mL / min, 15mL per tube, collecting all the eluates, and vacuum freeze-drying to obtain ion exchange purified polysaccharides.

[0020] Furthermore, the gel column chromatography purification includes the following steps: taking the above-mentioned ion exchange purified polysaccharide sample, adding pure water to prepare a solution with a concentration of 15 mg / mL, centrifuging, taking the supernatant, purifying it with a gel chromatography column, eluting with pure water, with a flow rate of 1 mL / min, collecting all the eluents, and vacuum freeze-drying to obtain gel-purified polysaccharides.

[0021] In addition, the present invention also provides the use of the above-mentioned wild jujube pulp crude polysaccharide or the above-mentioned wild jujube pulp polysaccharide composition in the preparation of medicines, health foods or foods with blood lipid and blood sugar regulating, anti-oxidation and anti-inflammatory effects.

[0022] Furthermore, the dosage form of the medicine includes one or more of capsules, tablets, granules, pills, powders, granules or oral liquids; the dosage form of the health food includes one or more of capsules, tablets, granules, powders, granules, oral liquids, teas, wines, beverages, biscuits or candies; the dosage form of the food includes one or more of tablets, granules, candies, jellies, beverages, solid beverages, biscuits, noodles or sticks.

[0023] The present invention also provides a pharmaceutical composition, comprising the above-mentioned crude polysaccharide from the fruit pulp of Ziziphus jujuba or the above-mentioned polysaccharide composition from the fruit pulp of Ziziphus jujuba.

[0024] The present invention also provides a health food, comprising the above-mentioned crude polysaccharide from the fruit pulp of Ziziphus jujuba or the above-mentioned polysaccharide composition from the fruit pulp of Ziziphus jujuba.

[0025] The present invention also provides a food, comprising the above-mentioned crude polysaccharide from the fruit pulp of Ziziphus jujuba or the above-mentioned polysaccharide composition from the fruit pulp of Ziziphus jujuba.

[0026] Compared with the prior art, the crude polysaccharide from the fruit pulp of the sour jujube provided by the present invention is extracted by enzymatic hydrolysis, an appropriate amount of papain is added during the extraction of the polysaccharide from the sour jujube fruit pulp, and the polysaccharide extraction rate is improved by regulating specific process parameters, and the polysaccharide extraction rate is as high as more than 7%. The crude polysaccharide from the sour jujube fruit pulp has good effects of regulating blood sugar and blood lipids, and has antioxidant and anti-inflammatory effects. The present invention separates and purifies the crude polysaccharide to obtain a polysaccharide composition including a neutral polysaccharide component and an acidic polysaccharide component, and the neutral and acidic polysaccharide components in the polysaccharide composition both have significant antioxidant, anti-inflammatory and immunomodulatory effects. The crude polysaccharide or polysaccharide composition provided by the present invention can be applied to the preparation of medicines, health foods or foods, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the elution curve of polysaccharide ion purification in the test example of the present invention;

[0028] Figure 2 This is the elution curve of polysaccharide WPS gel purification in the test example of the present invention;

[0029] Figure 3 This is the elution curve of the polysaccharide SPS-3 gel purification in the test example of the present invention;

[0030] Figure 4 This is an absolute molecular weight analysis diagram of the polysaccharide WPS-a in the test example of the present invention;

[0031] Figure 5 This is an absolute molecular weight analysis diagram of the polysaccharide SPS-3a in the test example of the present invention;

[0032] Figure 6 This is a molecular configuration analysis diagram of the polysaccharide WPS-a in the test example of the present invention;

[0033] Figure 7 It is a molecular configuration analysis diagram of the polysaccharide SPS-3a in the test example of the present invention;

[0034] Figure 8 is the infrared scanning spectrum of the polysaccharide WPS-a in the test example of the present invention;

[0035] Fig. 9 is the infrared scanning spectrum of the polysaccharide SPS-3a in the test example of the present invention;

[0036] Fig.10 1H NMR spectrum of polysaccharide WPS-a in the test example of the present invention;

[0037] Fig.11 The COSY spectrum of polysaccharide WPS-a in the test example of the present invention;

[0038] Fig.12HSQC spectrum of polysaccharide WPS-a in the test example of the present invention;

[0039] Fig.13 is the 13C NMR spectrum of the polysaccharide WPS-a in the test example of the present invention;

[0040] Fig.14 is the HMBC spectrum of the polysaccharide WPS-a in the test example of the present invention;

[0041] Fig.15 is the NOESY spectrum of polysaccharide WPS-a in the test example of the present invention;

[0042] Fig.16 1H NMR spectrum of polysaccharide SPS-3a in the test example of the present invention;

[0043] Fig.17 The COSY spectrum of polysaccharide SPS-3a in the test example of the present invention;

[0044] Fig.18 HSQC spectrum of polysaccharide SPS-3a in the test example of the present invention;

[0045] Fig.19 13C NMR spectrum of polysaccharide SPS-3a in the test example of the present invention;

[0046] Fig. 20 is the HMBC spectrum of the polysaccharide SPS-3a in the test example of the present invention;

[0047] Fig.21 It is the NOESY spectrum of polysaccharide SPS-3a in the test example of the present invention;

[0048] Fig. 22 This is a scanning electron microscope image of the polysaccharide WPS-a in the test example of the present invention (2K);

[0049] Fig.23 This is a scanning electron microscope image (5K) of the polysaccharide WPS-a in the test example of the present invention;

[0050] Fig.24 This is a scanning electron microscope image (20K) of the polysaccharide SPS-3a in the test example of the present invention;

[0051] Fig.25 This is a scanning electron microscope image (5K) of the polysaccharide SPS-3a in the test example of the present invention;

[0052] Fig.26 The results of the scavenging ability of polysaccharides on different free radicals in the test examples of the present invention;

[0053] Fig. 27 The results of the effects of polysaccharides on macrophage proliferation activity in the test examples of the present invention;

[0054] Fig.28 The results of the effects of polysaccharides on the contents of various cytokines in the test examples of the present invention;

[0055] Fig.29 The results of changes in body weight, food intake and energy intake in the test examples of the present invention;

[0056] Fig.30 The results of changes in the white adipose tissue index, brown adipose tissue index and liver tissue index in the test examples of the present invention;

[0057] Fig.31 HE staining results of liver tissue in the test example of the present invention;

[0058] Fig.32 This is the HE staining result of white adipose tissue in the test example of the present invention;

[0059] Fig.33 This is the HE staining result of jejunum tissue in the test example of the present invention;

[0060] Fig.34 This is the result of oil red staining of white fat in the test example of the present invention;

[0061] Fig.35 The test results of IL-6, IL-1β, TNF-α, MCP1, adiponectin, leptin, serum insulin, glycosylated hemoglobin and blood glucose in the test examples of the present invention;

[0062] Fig.36 The results of GSH-PX enzyme activity, SOD enzyme activity and MDA content test in the test examples of the present invention;

[0063] Fig.37 These are the four blood lipid test results in the test example of the present invention. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0065] Example 1

[0066] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0067] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:8 g / mL, adjust the pH value of the mixed liquid to 7, add papain in an amount of 0.05% of the mixed liquid, extract by enzymolysis at 50°C for 1h, heat to inactivate the enzyme, and centrifuge to obtain an enzymolysis solution;

[0068] S2. Concentrate the obtained enzymatic hydrolysate to a relative density of 1.05-1.06, add 4 times the volume fraction of 95% ethanol, precipitate at 4°C for 24 hours, centrifuge at 4000 rpm for 15 minutes, collect the precipitate, and obtain crude polysaccharide.

[0069] Example 2

[0070] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0071] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:8 g / mL, adjust the pH value of the mixed liquid to 6.5, add papain in an amount of 0.01% of the mixed liquid, extract by enzymolysis at 50°C for 1h, heat to inactivate the enzyme, and centrifuge to obtain an enzymolysis solution;

[0072] S2. Concentrate the obtained enzymatic hydrolysate to a relative density of 1.05-1.06, add 4 times the volume fraction of 95% ethanol, precipitate at 4°C for 24 hours, centrifuge at 4000 rpm for 15 minutes, collect the precipitate, and obtain crude polysaccharide.

[0073] Example 3

[0074] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0075] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:8 g / mL, adjust the pH value of the mixed liquid to 7, add papain in an amount of 0.10% of the mixed liquid, extract by enzymolysis at 50°C for 1h, heat to inactivate the enzyme, and centrifuge to obtain an enzymolysis solution;

[0076] S2. Concentrate the obtained enzymatic hydrolysate to a relative density of 1.05-1.06, add 4 times the volume fraction of 95% ethanol, precipitate at 4°C for 24 hours, centrifuge at 4000 rpm for 15 minutes, collect the precipitate, and obtain crude polysaccharide.

[0077] Example 4

[0078] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0079] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:4 g / mL, adjust the pH value of the mixed liquid to 7, add papain in an amount of 0.10% of the mixed liquid, extract by enzymolysis at 50°C for 1h, heat to inactivate the enzyme, and centrifuge to obtain an enzymolysis solution;

[0080] S2. The obtained enzymatic hydrolysate was concentrated to a relative density of 1.05-1.06, 4 times the volume fraction of 95% ethanol was added, and the mixture was precipitated at 4°C for 24 hours. The mixture was centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected to obtain crude polysaccharide.

[0081] Example 5

[0082] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0083] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:12 g / mL, adjust the pH value of the mixed liquid to 7, add papain in an amount of 0.10% of the mixed liquid, extract by enzymolysis at 50°C for 1h, heat to inactivate the enzyme, and centrifuge to obtain an enzymatic solution;

[0084] S2. The obtained enzymatic hydrolysate was concentrated to a relative density of 1.05-1.06, 4 times the volume fraction of 95% ethanol was added, and the mixture was precipitated at 4°C for 24 hours. The mixture was centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected to obtain crude polysaccharide.

[0085] Example 6

[0086] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0087] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:8 g / mL, adjust the pH value of the mixed liquid to 7, add papain in an amount of 0.05% of the mixed liquid, extract by enzymolysis at 50°C for 1.5h, heat to inactivate the enzyme, and centrifuge to obtain an enzymatic solution;

[0088] S2. The obtained enzymatic hydrolysate was concentrated to a relative density of 1.05-1.06, 4 times the volume fraction of 95% ethanol was added, and the mixture was precipitated at 4°C for 24 hours. The mixture was centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected to obtain crude polysaccharide.

[0089] Example 7

[0090] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0091] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:8 g / mL, adjust the pH value of the mixed liquid to 7.5, add papain in an amount of 0.05% of the mixed liquid, extract by enzymolysis at 70°C for 1h, heat to inactivate the enzyme, and centrifuge to obtain an enzymatic solution;

[0092] S2. The obtained enzymatic hydrolysate was concentrated to a relative density of 1.05-1.06, 4 times the volume fraction of 95% ethanol was added, and the mixture was precipitated at 4°C for 24 hours. The mixture was centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected to obtain crude polysaccharide.

[0093] Example 8

[0094] A crude polysaccharide of wild jujube pulp is prepared by the following preparation method:

[0095] S1. Take the dried sour jujube pulp, crush it, pass it through a 60-mesh sieve, add water at a solid-liquid ratio of 1:8 g / mL, adjust the pH value of the mixed liquid to 7, add papain in an amount of 0.05% of the mixed liquid, extract by enzymolysis at 30°C for 1h, heat to inactivate the enzyme, and centrifuge to obtain an enzymatic solution;

[0096] S2. The obtained enzymatic hydrolysate was concentrated to a relative density of 1.05-1.06, 4 times the volume fraction of 95% ethanol was added, and the mixture was precipitated at 4°C for 24 hours. The mixture was centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected to obtain crude polysaccharide.

[0097] The crude polysaccharide obtained above is taken and then conventional preparation processes and corresponding auxiliary materials are used to prepare corresponding preparations, such as candies, solid beverages, biscuits, noodles, sticks, etc.

[0098] Example 9

[0099] A polysaccharide composition of jujube pulp is obtained by separating and purifying the crude polysaccharide obtained in Example 8, and specifically comprises the following steps:

[0100] Dissolve the crude polysaccharide in an appropriate amount of distilled water, add a small amount of papain, and enzymolyze overnight. Mix the Sevage reagent (chloroform: n-butanol = 5:1, V / V) with the polysaccharide solution at a ratio of 1:4 (V / V), remove the protein three times using the Sevage method, remove the organic layer with a separatory funnel, and collect the upper aqueous phase. Add petroleum ether to the solution, mix thoroughly, and collect the lower aqueous phase. Add macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight. Collect the liquid, dialyze it with a 3000Da dialysis bag for 48 hours to remove small molecular components, and freeze-dry to obtain the jujube pulp polysaccharide.

[0101] The freeze-dried polysaccharide sample was dissolved in pure water to prepare a polysaccharide mother solution with a concentration of 10 mg / mL. The mixture was centrifuged and the supernatant was taken for purification using an anion exchange chromatography column. Pure water, 0.1 M NaCl solution, 0.2 M NaCl solution and 0.3 M NaCl solution were used in sequence for isocratic elution at a flow rate of 4 mL / min, 15 mL per tube, and all the eluates were collected and vacuum freeze-dried to obtain ion exchange purified polysaccharides.

[0102] Take the above ion exchange purified polysaccharide sample and add pure water to make a solution with a concentration of 15 mg / mL. Centrifuge, take the supernatant, purify it with a gel chromatography column, elute with pure water, the flow rate is 1 mL / min, collect all the eluate, and vacuum freeze-dry to obtain gel-purified polysaccharide.

[0103] The polysaccharide obtained above is taken, and then conventional preparation technology and corresponding auxiliary materials are used to prepare corresponding preparations.

[0104] Comparative Example 1

[0105] A jujube pulp polysaccharide composition, compared with Example 1, papain is replaced by pectinase, and the rest is the same as Example 1.

[0106] Comparative Example 2

[0107] A jujube pulp polysaccharide composition, compared with Example 1, papain is replaced by cellulase, and the rest is the same as Example 1.

[0108] Comparative Example 3

[0109] A jujube pulp polysaccharide composition, compared with Example 1, papain is replaced by hemicellulase, and the rest is the same as Example 1.

[0110] Comparative Example 4

[0111] A jujube pulp polysaccharide composition, compared with Example 1, papain is replaced by laccase, and the rest is the same as Example 1.

[0112] Comparative Example 5

[0113] A jujube pulp polysaccharide composition, compared with Example 1, the amount of papain added is changed to 0.20%, and the rest is the same as Example 1.

[0114] Test Example 1

[0115] The phenol-sulfuric acid method was used to determine the soluble total sugar content and purity of the crude polysaccharides obtained in Examples 1, 6, 8 and Comparative Examples 1 to 5, and the polysaccharide extraction rate was calculated. The specific operation was: accurately weigh a small amount of crude polysaccharide sample, add water to dissolve it, add sulfuric acid-phenol reagent, react in the dark for 10 minutes, measure the absorbance at 490nm, draw a standard curve Y=390.61x-20.792 (fitting degree 0.9977), and calculate the extraction rate. The results are shown in Table 1.

[0116] Table 1 Polysaccharide extraction rate

[0117] project Example 1 Example 6 Example 8 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Polysaccharide extraction rate 7.09％ 7.19％ 7.33％ 5.12％ 5.95％ 5.36％ 5.22％ 6.59％

[0118] It can be seen from the above data that, compared with the comparative example, the polysaccharide extraction rate of the crude polysaccharide of jujube pulp provided by the embodiment of the present invention is higher, which can reach more than 7%, which is conducive to the full utilization of jujube pulp resources.

[0119] Test Example 2

[0120] The crude polysaccharide of Solanum jujuba was separated, purified and analyzed. The crude polysaccharide obtained in Example 8 was purified according to Example 9 of the present invention, as follows:

[0121] Dissolve the crude polysaccharide in an appropriate amount of distilled water, add a small amount of papain, and enzymolyze overnight. Mix the Sevage reagent (chloroform: n-butanol = 5:1, V / V) with the polysaccharide solution at a ratio of 1:4 (V / V), remove the protein three times using the Sevage method, remove the organic layer with a separatory funnel, and collect the upper aqueous phase. Add petroleum ether to the solution, mix thoroughly, and collect the lower aqueous phase. Add macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight. Collect the liquid, dialyze it with a 3000Da dialysis bag for 48 hours to remove small molecular components, and freeze-dry to obtain the jujube pulp polysaccharide.

[0122] The polysaccharide sample was dissolved in pure water to prepare a polysaccharide mother solution with a concentration of 10 mg / mL. The mixture was centrifuged and the supernatant was purified using an anion exchange chromatography column (DEAE seplife FF). Pure water, 0.1 M NaCl solution, 0.2 M NaCl solution and 0.3 M NaCl solution were used in sequence for isocratic elution at a flow rate of 4 mL / min. 15 mL was used in each tube and all the eluate was collected.

[0123] The total sugar content of the eluate in each collection tube was determined by the sulfuric acid-phenol method, and the ion purification elution curve was drawn. The main peak was selected to merge the eluates of each collection tube corresponding to the same elution peak, and the volume was evaporated to 1 / 5 of the original volume. The 3000Da dialysis bag was dialyzed for 48 to 72 hours to remove salt. The sulfuric acid-phenol method was used to identify the polysaccharide content and purity after ion purification.

[0124] Take the above ion exchange purified polysaccharide sample and add pure water to make a solution with a concentration of 15 mg / mL. Centrifuge, take the supernatant, and purify it with a gel chromatography column (Sephacryl S-400HR). Use pure water to elute 1.5 times the column volume at a flow rate of 1 mL / min. Collect one tube for every 12 mL and collect all the eluate.

[0125] Determine the total sugar content of the eluate in each collection tube, draw the gel purification elution curve, select the main peak and merge the eluates from each collection tube corresponding to the same elution peak, concentrate by rotary evaporation to 1 / 5 of the original volume, freeze-dry, and identify the content and purity of the polysaccharide after gel purification.

[0126] Purification results of crude polysaccharide from Ziziphus jujuba

[0127] (1) Ion purification results

[0128] The ion purification elution curve of the sample was plotted with the number of eluent tubes as the horizontal axis and the total sugar content and the NaCl concentration in the eluent as the vertical axis, as shown in Table 2 and Figure 1As shown. This ion purification obtained 5 elution peak components, including a water-eluted polysaccharide (WPS) peak and four salt-eluted polysaccharide peaks (SPS-1, SPS-2, SPS-3 and SPS-4). Considering that the elution peaks of SPS-1, SPS-2 and SPS-4 are relatively small and difficult to collect, only WPS and SPS-3 were collected for gel purification analysis. The polysaccharide purities of the collected WPS and SPS-3 components were 71.1% and 87.7%, respectively.

[0129] Table 2 Ion purification elution results

[0130]

[0131]

[0132] (2) Gel purification results

[0133] The gel purification elution curves of the samples were plotted with the number of eluent tubes as the horizontal axis and the total sugar content as the vertical axis. Figure 2 As shown in Table 4 and Figure 3 As shown, the gel purification of SPS-3 mainly obtained two elution peak components, SPS-3a and SPS-3b. The purities of the collected polysaccharides of the four components were WPS-a 86.8%, WPS-b 75.7%, SPS-3a 91.6%, and SPS-3b 70.0%; the purification yields of the polysaccharides of the four components were WPS-a 1.5%, WPS-b 1.5%, SPS-3a 3.5%, and SPS-3b 11.52%.

[0134] Table 3 WPS gel purification results

[0135]

[0136] Table 4 SPS-3 gel purification results

[0137]

[0138] Test Example 3

[0139] The structure of the polysaccharide obtained from the wild jujube pulp of the present invention is analyzed

[0140] 1. Purpose of the experiment

[0141] Explore the structural characteristics of polysaccharide molecules in jujube pulp.

[0142] 2. Test Materials

[0143] Jujube polysaccharides WPS-a and SPS-3a.

[0144] 3. Test methods

[0145] 3.1 Determination of molecular weight of jujube polysaccharide

[0146] (1) Sample pretreatment

[0147] Inorganic salt phase: The sample was dissolved in 0.1 M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) with a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before testing on an instrument.

[0148] DMSO phase: The samples were dissolved in DMSO solution at a concentration of 1 mg / mL and filtered through a filter with a pore size of 0.45 μm before detection.

[0149] (2) Instrument parameters

[0150] The chromatographic system was a gel chromatography-differential-multi-angle laser light scattering system, the liquid phase system was U3000, the differential detector was Optilab T-rEX, and the laser light scattering detector was DAWN HELEOSⅡ. Gel exclusion chromatography columns (OhpakSB-805HQ (300×8mm), Ohpak SB-804HQ (300×8mm) and Ohpak SB-803HQ (300×8mm) were used in series.

[0151] (3) Detection parameters

[0152] Inorganic salt phase: column temperature 45°C, injection volume 100 μL, mobile phase A (0.02% NaN3, 0.1 M NaNO3), flow rate 0.4 mL / min, elution gradient: isocratic 100 min.

[0153] DMSO phase: column temperature 60°C; injection volume 100 μL; mobile phase B (0.5% LiBr, DMSO), flow rate 0.3 mL / min, elution gradient: isocratic 120 min.

[0154] (4) Qualitative and quantitative analysis methods

[0155] The concentration information of the sample is detected by a differential detector according to its refractive intensity, the light scattering information of the macromolecules is detected by a multi-angle laser light scattering instrument, and the molecular weight corresponding to each component is calculated according to the Mark-Houwink equation.

[0156] 3.2 Determination of monosaccharide composition of jujube polysaccharide

[0157] (1) Preparation of standard products

[0158] After accurately weighing each standard, add water to prepare a 10 mg / mL standard solution stock solution, then take an appropriate amount of the standard stock solution and mix it to prepare a standard mixed standard with a maximum index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL, and prepare the series of standards required for the instrument according to the concentration gradient in the following Table 5.

[0159] Table 5 Monosaccharide mixed standard gradient concentration information

[0160]

[0161]

[0162] (2) Sample pretreatment

[0163] Take a clean chromatographic bottle, weigh an appropriate amount of polysaccharide sample, add 1mL 2M TFA acid solution, and heat at 121℃ for 2h. Pass nitrogen and blow dry. Add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add sterile water to dissolve, and transfer to the chromatographic bottle for testing.

[0164] (3) Chromatographic conditions

[0165] The chromatographic system used was the Thermo ICS5000 ion chromatography system (ICS5000, Thermo Fisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector.

[0166] Using Dionex TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume 5μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 mL / min; column temperature was 30°C; elution gradient: 0 min phase A / phase B / phase C (95:5:0, V / V), 26 min phase A / phase B / phase C (85:5:10, V / V), 42 min phase A / phase B / phase C (85:5:10, V / V), 42.1 min phase A / phase B / phase C (60:0:40, V / V), 52 min phase A / phase B / phase C (60:40:0, V / V), 52.1 min phase A / phase B / phase C (95:5:0, V / V), 60 min phase A / phase B / phase C (95:5:0, V / V).

[0167] 3.3 Analysis of the bonding structure of jujube polysaccharide (methylation)

[0168] (1) Sample pretreatment

[0169] Take a small amount of sample (2-3 mg), add 500 μL DMSO to dissolve. Add 1 mg NaOH and incubate for 30 min. Add 50 μL iodomethane solution and react for 1 h. Add 1 mL water and 2 mL dichloromethane, vortex to mix, centrifuge and discard the aqueous phase, repeat water washing 3 times. Take the lower dichloromethane phase and blow it dry with nitrogen. Add 100 μL 2M TFA, react at 121℃ for 90 min, evaporate to dryness at 30℃. Add 50 μL 2M ammonia water, 50 μL 1M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL methanol, blow dry with nitrogen. Add 250 μL acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h.

[0170] Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex to mix, centrifuge and discard the aqueous phase, repeat the water washing 3 times. Take the lower dichloromethane phase and detect it on GC-MS.

[0171] (2) Chromatographic conditions

[0172] The chromatographic system was an Agilent gas chromatograph system (Agilent 7890A), and the chromatographic column was BPX70 (30m×0.25mm×0.25μm, SGE, Australia). The injection volume was 1μL, the split ratio was 10:1, and the carrier gas was high-purity helium; the initial temperature of the column oven was 140°C for 2.0 min, and the temperature was raised to 230°C at 3°C / min and maintained for 3 min.

[0173] (3) Mass spectrometry conditions

[0174] The mass spectrometry system is a quadrupole mass spectrometry detection system from Aiglent, USA, equipped with an electron impact ion source and a MassHunter workstation. Using an electron impact ion source, the analytes were detected in full scan mode with a mass scan range (m / z): 30-600.

[0175] 3.4 Determination of jujube polysaccharide by infrared scanning

[0176] (1) Sample pretreatment

[0177] Weigh a small amount of polysaccharide sample and mix it with 200 mg of potassium bromide, press it into a sheet with a thickness of 1 mm, and then test it on a machine.

[0178] (2) Scanning conditions

[0179] Scanning and analysis were performed using a Nicolet iZ-10 Fourier transform infrared spectrometer with an instrument resolution of 4.00 cm -1 , scanning range is 4000-400cm -1, Scan times: 32 times. Sampling gain: 8.0; Moving mirror speed: 0.4747; Aperture: 80.00; DTGSKBr detector; KBr beam splitter; Infrared light source.

[0180] 3.5 NMR scanning analysis of jujube polysaccharide

[0181] (1) Sample pretreatment

[0182] Take an appropriate amount of purified polysaccharide and fully dissolve it to D 2 O, prepare a polysaccharide solution with a concentration of greater than or equal to 40 mg / mL. Transfer the dissolved solution to an NMR tube and add 0.5 mL. Place the NMR tube in an NMR spectrometer and scan the one-dimensional 1 H spectrum, 13 C spectrum, two-dimensional COSY, HSQC, HMBC, and NOESY spectra.

[0183] (2) NMR conditions

[0184] A Bruker (Germany) 600MHz NMR spectrometer was used to quantitatively analyze the target, with a scanning temperature of 25°C. Liquid probe QXI 1H / 31P / 13C / 15N 5mm quadruple resonance reverse detection probe, technical parameters: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating) BBFO 1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe technical parameters: signal-to-noise ratio (1H): 798; resolution (Hz): 0.26 (rotating); signal-to-noise ratio (13C): 328; resolution (Hz): 0.1.

[0185] 3.6 Electron microscopy analysis of jujube polysaccharides

[0186] (1) Sample pretreatment

[0187] The polysaccharide sample was passed through a 100-mesh sieve, and a small amount was placed on a conductive carbon tape. After gold spraying, it was scanned and photographed using an electron microscope with a magnification of 100-4000 times.

[0188] (2) Scanning conditions

[0189] High-resolution field emission scanning electron microscope, resolution: 1.0nm@15kV; 1.9nm@1kV; 0.8nm@30kV (STEM mode); acceleration voltage: 0.02-30kV; beam current: 12pA to 100nA; magnification: 500-5,000; electron gun: thermal field emission Schottky electron gun, beam stability better than 0.2% / h; detector: efficient in-lens Duo, Everhart Thornley secondary electron detector; sample chamber: 330mm(ф×270mm(h); image processing: storage resolution up to 32768×24576 pixels; multiple integration and averaging modes.

[0190] 4 Test results

[0191] 4.1 Results of molecular weight determination of jujube polysaccharide

[0192] The chromatographic data were processed using the ASTRA 6.1 software. The following figure shows the absolute molecular weight analysis graph and molecular configuration analysis graph of the sample. The absolute molecular weight analysis graph uses the retention time of the detection as the horizontal axis and the molar mass as the vertical axis; the molecular configuration analysis graph uses the molar mass as the horizontal axis and the root mean square radius as the vertical axis. The absolute molecular weight analysis graphs of the two polysaccharides WPS-a and SPS-3a are shown in Figure 1. Figure 4 , 5 As shown in the figure, the molecular configuration analysis diagrams of the two polysaccharides WPS-a and SPS-3a are respectively as follows Figure 6 , 7 The molecular weight, polydispersity and root mean square are shown in Table 6:

[0193] Table 6 Molecular weight, polydispersity and RMS of WPS-a and SPS-3a

[0194]

[0195] From the table above, it can be seen that the molecular weight of SPS-3a is larger among the two polysaccharide components, and the uniformity of the two polysaccharide components is better. From the molecular configuration analysis diagram, it can be seen that the slopes of the configuration diagrams of WPS-a and SPS-3a are 0.09 and 0.10 respectively, indicating that the molecular configurations of the two polysaccharide components are spherical.

[0196] 4.2 Results of monosaccharide composition determination of jujube polysaccharide

[0197] The external standard method was used for quantification, and the standard curve was prepared by preparing standard samples of different concentrations. The concentration of the standard was used as the horizontal axis, and the peak area of ​​the standard was used as the vertical axis to draw a graph. The content of each component in the solid sample (μg / mg) = C*V*F / M, where C is the measured concentration in μg / mL; V is the volume of the sample solution in mL; F is the dilution factor; and M is the total amount of the sample weighed in mg. The results are shown in Table 7.

[0198] Table 7 Quantitative detection results of WPS-a and SPS-3a

[0199]

[0200]

[0201] WPS-a is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, and the molar ratio of monosaccharide composition (%) is 0.75:16.81:19.97:17.54:33.04:2.52:1.66:6.86:0.86; SPS-3a is composed of rhamnose, arabinose, galactose, glucose and galacturonic acid, and the molar ratio of monosaccharide composition (%) is 2.54:2.76:2.31:91.75:0.65.

[0202] 4.3 Results of structural analysis (methylation) of jujube polysaccharide bonds

[0203] The bond structure analysis results of the jujube polysaccharides WPS-a and SPS-3a after methylation treatment are shown in Table 8, which are basically consistent with the analysis results of the monosaccharide composition.

[0204] In WPS-a, →4)-Galp-(1→ and →3,6)Galp-(1→ accounted for 30.5%; →6)-Glcp-(1→ and →4,6)-Glcp-(1→ accounted for 30.6%; Araf-(1→ and →5)-Araf-(1→ accounted for 21%; →2,4)Rhap-(1→ accounted for 8.9%.

[0205] In SPS-a, →4)-Glcp-(1→, Glcp-(1→, →4,6)-Glcp-(1→, →6)-Glcp-(1→, →3,4)-Glcp-(1→ and →2,4)-Glcp-(1→ accounted for 95.7%; →6)-Galp-(1→ and →3,4,6)-Galp-(1→ accounted for 1.8%; →2,4)-Rhap-(1→ and →4)-Manp-(1→ accounted for 2.5%.

[0206] Table 8 Bonding structure analysis results of polysaccharide samples

[0207]

[0208] 4.4 Results of infrared scanning determination of jujube polysaccharide

[0209] The infrared scanning spectra of jujube polysaccharides WPS-a and SPS-3a are shown in Figure 8 , 9 shown.

[0210] The infrared spectrum of WPS-a shows that the absorption band is at 3600-3200cm -1 It is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugars. The specific peaks are as follows: 3435.54cm -1 It is the stretching vibration absorption peak of OH, showing the strong intramolecular and intermolecular interactions between polysaccharide chains. -1 The absorption peak at 1626.92cm is attributed to CH stretching vibration, which is the characteristic peak of polysaccharides. -1 The absorption peak is attributed to C=O stretching vibration. -1 There is an absorption peak at , which is attributed to the stretching vibration of CO.

[0211] The infrared spectrum of SPS-3a shows that 3344.13 cm -1 It is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. -1 The absorption peak at 1638.99 cm is attributed to CH stretching vibration. -1 The absorption peak is attributed to C=O stretching vibration. -1 There is an absorption peak at 930.66cm, which is attributed to the stretching vibration of CO. -1 There is an absorption peak at 848.52cm -1 There is an absorption peak at , indicating the existence of α-type glycosidic bond.

[0212] 4.5 Results of NMR scanning analysis of jujube polysaccharide

[0213] NMR data processing is based on the polysaccharide bonding structure analysis data, combined with the NMR spectrum to analyze the configuration, correlation and other data of each bonding structure, and finally obtain the main structural information of the polysaccharide.

[0214] (1) WPS-a NMR analysis results

[0215] One-dimensional hydrogen spectrum mainly solves the configuration problem of glycosidic bonds in polysaccharide structure. Fig.10 ) are concentrated at 3-6ppm. Usually, the anomeric hydrogen signals of β-glycosidic bond configuration are mainly distributed at δ4.3-4.8ppm, and the anomeric hydrogen signals of α-glycosidic bond configuration are mainly distributed at δ4.8-5.8ppm. The hydrogen spectrum signals of this sample are mainly concentrated between δ3.0-5.5ppm, and multiple coupled signal peaks are identified in the anomeric signal region of δ4.3-5.8ppm, indicating that this sample contains multiple sugar residues. Due to signal overlap, COSY (such as Fig.11 ) and HSQC (such as Fig.12 ) spectrum analysis, five anomeric hydrogen signals were identified, with chemical shifts of δ4.8, 5.18, 5.24, 5.38 and 5.44 ppm, and were recorded as residues A, B, C, D and E, respectively. The non-anomeric hydrogen signals are mainly concentrated in the δ3.2-4.2 ppm region. Most of the signals overlap severely, so it is necessary to combine COSY and HSQC spectra to assign the H2-H6 chemical shifts of sugar residues. Among them, the strong signal peak near δ4.71 ppm is the solvent peak. The strong signal peaks near δ3.75 and 4.02 ppm are O-CH 3 The signal of hydrogen.

[0216] Compared with 1H NMR, polysaccharides have a higher Fig.13 ) has a wide distribution of chemical shift signals. The anomeric carbon signals of polysaccharides in 13CNMR are concentrated at 95-110ppm. Multiple signal peaks were identified in the anomeric carbon region of this sample. Combining the cross peaks in the anomeric region of the 13CNMR spectrum and the HSQC spectrum, the anomeric carbon signals of residues A, B, C, D and E were determined to be δ97.9, 101.39 and 97.74ppm respectively. Combining the sample bonding structure (methylation) information, anomeric signals and literature reports, it is inferred that sugar residue A is →4)-β-D-Galp-(1→, residue B is →6)-α-D-Glcp-(1→, residue C is →2,4)-α-L-Rhap-(1→, residue D is α-L-Arap-(1→, residue E is →5)-α-L-Arap-(1→, and their 1H and 13C chemical shifts are assigned. The results are shown in Table 9. Combined with the HSQC spectrum, the signal peaks near the cross peaks δ3.75 / 56.99ppm and δ4.02 / 56.26ppm are O-CH 3 However, due to the serious overlap of spectral signals, it is not possible to distinguish O-CH 3 The specific position of the signal within the sugar residue.

[0217] Table 9 Sugar residues of WPS-a 1 H and 13 Chemical shift of C

[0218]

[0219] Combining one-dimensional NMR and two-dimensional NMR spectra, the sugar residue signals of the samples were assigned according to the chemical shifts of 13C and 1H of the sugar residues in the samples, combined with HMBC (such as Fig.14) spectrum analysis showed that there were structures and connection modes in the polysaccharide: sugar residue A-H1 and residue B-C6 had a cross peak of δ4.8 / 68.69ppm, and sugar residue A-C1 and residue B-H6 had a cross peak of δ102.97 / 4.14ppm. Sugar residue D-H1 and residue E-C5 had a cross peak of δ5.38 / 66.24ppm. Sugar residue E-H1 and residue C-C4 had a cross peak of δ5.44 / 83.79ppm. Combined with NOESY ( Fig.15 ) spectrum further verified the connection order of each residue in the polysaccharide, and there was a cross peak δ5.18 / 3.76ppm between the sugar residue B-H1 and the residue C-H2.

[0220] Therefore, based on the analysis of one-dimensional and two-dimensional NMR information, it was inferred that the polysaccharide is mainly composed of →4)-β-D-Galp-(1→ and →6)-α-D-Glcp-(1→ connected to each other to form the main chain, and the side chain is mainly composed of α-L-Arap-(1→5)-α-L-Arap-(1→ connected to the O-4 position of the sugar residue →2,4)-α-L-Rhap-(1→. The possible structure of the polysaccharide chain is as follows.

[0221]

[0222] (2) SPS-3a NMR analysis results

[0223] SPS-3a 1H NMR (such as Fig.16 ) signals are mainly concentrated between δ3.0 and 5.5 ppm. In the anomeric signal region of δ4.3-5.8 ppm, a strong coupling signal peak is mainly identified, namely δ5.29 ppm, which is recorded as residue A. The non-anomeric hydrogen signals are mainly concentrated in the region of δ3.2-4.2 ppm. Most of the signals overlap severely, so they need to be combined with COSY (such as Fig.17 ) and HSQC (such as Fig.18 ) spectrum to assign the H2-H6 chemical shifts of each sugar residue. Among them, the strong signal peak near δ4.71ppm is the solvent peak.

[0224] SPS-3a also mainly identified a strong signal peak in the anomeric carbon region, namely δ99.62ppm. Combined with the 13C NMR spectrum (such as Fig.19) and the cross peaks in the anomeric region of the HSQC spectrum, and three cross peak signals were identified: δ5.29 / 99.62ppm, δ4.87 / 97.55ppm and δ5.23 / 99.78ppm, which were recorded as sugar residues A, B and C, respectively. Combining the SPS-3a bonding structure (methylation) information, the anomeric signal and the comprehensive literature reports, it was inferred that sugar residue A was →4)-α-D-Glcp-(1→, residue B was α-D-Glcp-(1→, and residue C was →4,6)-α-D-Glcp-(1→, and their 1H and 13C chemical shifts were assigned. The results are shown in Table 10.

[0225] Table 10 Sugar residues of SPS-3a 1 H and 13 Chemical shift of C

[0226]

[0227] Combining the one-dimensional NMR and two-dimensional NMR spectra, the sugar residue signals of SPS-3a were assigned according to the chemical shifts of 13C and 1H of each sugar residue in SPS-3a, combined with HMBC (such as Fig. 20 ) spectrum analysis of the structure and connection mode of the polysaccharide: sugar residue A-H1 and residue A-C4 have a cross peak of δ5.29 / 76.6ppm, and residue C-C4 has a cross peak of δ5.29 / 76.46ppm. Sugar residue C-H1 and residue A-H4 have a cross peak of δ5.23 / 76.6ppm. Since the rest of the signals are weak, combined with NOESY (such as Fig.21 ) spectrum further verifies the connection order of each residue in the polysaccharide. Sugar residue A-H1 has a cross peak with residue A-H4 at δ5.29 / 3.84ppm, and with residue C-H4 at δ5.29 / 3.9ppm. Sugar residue B-H1 has a cross peak with residue C-H6 at δ4.87 / 3.75ppm. Sugar residue C-H1 has a cross peak with residue A-H4 at δ5.23 / 3.84ppm.

[0228] Based on the analysis of one-dimensional and two-dimensional NMR information, it was inferred that the polysaccharide was mainly composed of →4)-α-D-Glcp-(1→ interconnected to form the main chain, and the side chain was mainly composed of α-D-Glcp-(1→ connected to the O-6 position of the sugar residue →4,6)-α-D-Glcp-(1→.

[0229] Therefore, the possible structure of the polysaccharide chain is as follows.

[0230]

[0231] 4.6 Results of electron microscopy analysis of jujube polysaccharide

[0232] WPS-a electron microscope scanning image Fig. 22 , 23 As shown in the figure, under the condition of 2K magnification, it is irregular flake and rod-shaped, with different sizes, and the flake structure is the main body, which may be related to the light and thin flocculent layer after freeze-drying. Under the condition of 5K magnification, it can be clearly seen that the flake structure is connected by many branches, interlaced and aggregated with each other, and the surface of the polysaccharide is loose and slightly rough.

[0233] SPS-3a electron microscope scanning image Fig.24 , 25 As shown, under the conditions of magnification of 20K and 5K, it was observed that the polysaccharide sample was relatively compact and smooth on the surface, with a large number of spherical and ellipsoidal particles distributed therein.

[0234] Test Example 4

[0235] In order to better verify the characteristics of the jujube polysaccharide of the present invention, the polysaccharide components purified above were subjected to in vitro antioxidant and anti-inflammatory activities studies.

[0236] 1. Purpose of the experiment

[0237] To explore the antioxidant and anti-inflammatory activities of jujube polysaccharides and provide a basis for the application of jujube polysaccharides.

[0238] 2 Test materials

[0239] The samples of WPS-a and SPS-3a were separated and purified from jujube polysaccharide.

[0240] 3 Test methods

[0241] 3.1 In vitro antioxidant test

[0242] The antioxidant capacity was comprehensively analyzed and compared by the scavenging ability of DPPH free radicals, ABTS free radicals, hydroxyl free radicals and superoxide anion free radicals, with Vc as the positive control. The samples were prepared into a polysaccharide solution with an original concentration of 1600 μg / mL, and gradient dilution was performed to select 6 concentrations, namely 800, 400, 200, 100, 50, and 25 μg / mL. The scavenging ability of polysaccharides on DPPH free radicals, ABTS free radicals, hydroxyl free radicals and superoxide anion free radicals was determined respectively. Three parallels were made for each group.

[0243] 3.2 Cell assay

[0244] (1) Cell culture

[0245] Mouse mononuclear macrophage leukemia cells (RAW264.7) were used, and the culture conditions were: 90% DMEM, 10% FBS, 1% double antibody, 37°C, 5% CO2.

[0246] (2) Preparation of polysaccharide solution

[0247] LPS was prepared with DMEM basal medium and filtered for sterilization. Polysaccharide samples were diluted with DMEM basal medium from 1600 μg / mL to 25 μg / mL. The original concentration solution was prepared one day in advance and allowed to stand at 4°C overnight before gradient dilution.

[0248] (3) Effect of Jujube Polysaccharide on Macrophage Proliferation Activity

[0249] Take the cells growing in the logarithmic phase at a density of 5×105 / mL, and inoculate 100μL per well in a 96-well plate. After culturing in a cell culture incubator at 37°C and 5% CO2 for 24h, observe the cell state. After the cells adhere to the wall and grow, discard the old culture medium, add 100μL DMEM basal culture medium (without serum) to the blank control group, add LPS (1μg / mL, 5μg / mL and 10μg / mL, prepared with DMEM basal culture medium) to the positive control group, add polysaccharide sample solutions of different concentration gradients (prepared with DMEM basal culture medium) to the test group, and do not inoculate cells in the zeroing group. The rest of the operations are the same. Set three replicate wells for each group and continue to culture at 37°C and 5% CO2 for 24h. Finally, add 10μL CCK-8 reagent to each well and incubate for another 2h. Measure the absorbance at 450nm.

[0250] (4) Effects of Jujube Polysaccharide on the Content of IL-6, IL-1β, COX-2 and TNF-α in LPS-induced Macrophages

[0251] Take RAW264.7 cells in the logarithmic growth phase at a density of 5×105 / mL, inoculate 100μL per well in a 96-well plate, culture at 37°C, 5% CO2 for 24h, discard the old culture medium, add DMEM basal culture medium (without serum) to the blank control group, and treat the experimental group with polysaccharide samples of different concentrations for 1h, then add 5ug / mL lipopolysaccharide (LPS). Set up three replicate wells for each group, and collect the cell culture supernatant into a sterile centrifuge tube for testing after another 24h of culture. The ELISA detection steps are strictly tested in accordance with the instructions of the kit.

[0252] 4 Test results

[0253] 4.1 In vitro antioxidant test results

[0254] The results of the scavenging ability of polysaccharide samples on different free radicals are shown in Tables 11, 12, 13, 14 and Fig.26As shown, D1 represents the different concentration gradients of WPS-a samples, and D2 represents the different concentration gradients of SPS-3a samples. It can be seen from the table and the figure that with the increase of polysaccharide treatment concentration, the ABTS free radical, DPPH free radical and hydroxyl free radical scavenging ability of WPS-a showed a gradual increasing trend, and there was no significant change in anti-superoxide anion activity. With the increase of polysaccharide treatment concentration, the hydroxyl free radical scavenging ability of sample SPS-3a showed a gradual increasing trend, and there was no significant change in other indicators. WPS-a has strong ABTS free radical and DPPH free radical scavenging ability, and SPS-3a has strong hydroxyl free radical scavenging ability.

[0255] Table 11 ABTS free radical scavenging energy (ug Trolox / mL)

[0256] sample 25 μg / mL 50 μg / mL 10 μg / mL 200 μg / mL 400 μg / mL 800 μg / mL Vc 0.16 0.30 0.58 0.91 0.91 0.91 D1 -0.077 -0.065 -0.033 0.022 0.0573 0.22 D2 -0.077 -0.085 -0.081 -0.048 -0.071 -0.060

[0257] Table 12 DPPH free radical scavenging ability (ug Trolox / mL)

[0258] sample 25 μg / mL 50 μg / mL 100 μg / mL 200 μg / mL 400 μg / mL 800 μg / mL Vc 33.99 33.99 33.99 33.99 33.99 33.99 D1 2.38 4.95 7.24 10.88 17.60 24.29 D2 0.51 -0.05 -0.39 0.13 -1.21 -0.57

[0259] Table 13 Hydroxyl radical scavenging ability (U / mL)

[0260] sample 25 μg / mL 50 μg / mL 100 μg / mL 200 μg / mL 400 μg / mL 800 μg / mL Vc 50.19 68.07 77.47 91.22 95.12 96.72 D1 33.39 39.88 43.47 50.65 52.94 55.16 D2 47.37 52.33 56.92 61.12 64.63 67.54

[0261] Table 14 Superoxide anion scavenging capacity (U / L)

[0262] sample 25 μg / mL 50 μg / mL 100 μg / mL 200 μg / mL 400 μg / mL 800 μg / mL Vc 24.76 43.16 54.48 94.81 162.03 166.27 D1 2.83 11.08 9.91 8.02 5.90 12.50 D2 12.97 8.96 9.43 9.20 12.50 7.31

[0263] 4.2 Cell test results

[0264] (1) Effect of Jujube Polysaccharide on Macrophage Proliferation Activity

[0265] The proliferation ability of macrophages reflects their immune function to a certain extent. Activated macrophages have strong proliferation ability and can more effectively play an immunoregulatory role.

[0266] The RAW264.7-CCK8 test was performed with 12 groups of wells, each group was repeated 3 times, and the OD value at 450nm was measured (cell proliferation index = [(As-Ab) / (Ac-Ab)]×100%). The test results are shown in Table 15 and Fig. 27 As shown, 1-N1 represents WPS-a; 2-N1 represents SPS-3a. From the test results, each concentration gradient of WPS-a and SPS-3a can significantly promote the proliferation of activated macrophages. When WPS-a is 25μg / mL, the proliferation index of macrophages is 146.98%, and when SPS-3a is 25μg / mL, the proliferation index of macrophages is 153.39%.

[0267] Table 15 Effects on macrophage proliferation activity

[0268]

[0269] (2) Effects of polysaccharides on the levels of IL-6, IL-1β, COX-2, and TNF-α in LPS-induced macrophages

[0270] As shown in Table 16 and Fig.28 As shown in the figure, 1-N1 represents WPS-a, 2-N1 represents SPS-3a, and a certain concentration of WPS-a can significantly reduce the levels of IL-6, IL-1β, COX-2 and TNF-α, while SPS-3a has no obvious inhibitory effect on the macrophage factor IL-1β. Overall, WPS-a is better than SPS-3a in inhibiting the level of macrophage inflammatory factors.

[0271] Table 16 Effects on the levels of various cytokines

[0272]

[0273] When the concentration of WPS-a was 100 μg / mL, the inhibitory effect on macrophage cytokines IL-1β, IL-6, TNF-α and COX-2 was the best. Compared with the model group, the levels of each cytokine decreased by 16.79%, 24.57%, 24.38% and 29.91%, respectively.

[0274] When the concentration of SPS-3a was 200 μg / mL, the inhibitory effect on TNF-α and COX-2 was the best, and the levels of each cytokine decreased by 18.16% and 10.96% respectively compared with the model group; when the concentration of SPS-3a was 50 μg / mL, the inhibitory effect on IL-6 was the best, and the level of IL-6 decreased by 17.11% compared with the model group.

[0275] From the above data, it can be seen that the jujube polysaccharides WPS-a and SPS-3a obtained in the present invention have good antioxidant, anti-inflammatory and immune regulation effects.

[0276] Test Example 5

[0277] The crude polysaccharide of jujube pulp obtained in Example 8 of the present invention was subjected to an animal test on the function of regulating blood lipids and blood sugar

[0278] 1. Purpose of the experiment

[0279] To explore the effects of crude polysaccharides from Solanum jujuba on blood glucose and lipid metabolism.

[0280] 2 Test materials

[0281] The crude polysaccharide after extraction and alcohol precipitation of the sour jujube pulp was prepared according to Example 8.

[0282] 3 Test methods

[0283] 3.1 Animal model construction

[0284] A high-fat model was established using 5-6 week-old SD rats. After 7 days of adaptation, rats in other groups except the control group were fed with a Western high-fat diet, and animals were gavage-treated for 8 weeks. Blood lipid changes in animals were monitored every 2 weeks. Animal body weight, food intake and energy intake were tested weekly. Glucose tolerance test was performed in the 7th week of the experiment. The liver, white fat and brown fat were weighed when the samples were collected, and the tissue index was calculated. Liver tissue, white fat and jejunum tissue were taken for HE staining, and liver tissue was taken for oil red staining. Blood was collected for blood biochemistry and ELISA tests.

[0285] The experiment set up 5 groups, namely control group, high-fat model group, high-dose test group (1000 mg / kg), medium-dose test group (500 mg / kg), and low-dose test group (250 mg / kg), with 10 mice in each group.

[0286] 3.2 Analysis of body weight, food intake, energy intake and tissue index

[0287] Animal body weight, food intake and energy intake were measured weekly. Liver, white fat and brown fat were weighed when samples were collected, and tissue index was calculated. Tissue index = tissue weight / total animal weight × 100%

[0288] 3.3 Histopathological analysis

[0289] (1) Paraffin sections and HE staining

[0290] Tissue Embedding:

[0291] ① Ethanol dehydration: The tissue was dehydrated step by step with ethanol solutions of different concentrations: 70%, 80%, 90%, 95%, 100%, each level of ethanol for 40 minutes;

[0292] ② Transparent: The tissue is immersed in three xylene tanks in sequence, each tank for 1 hour;

[0293] ③ Wax immersion: The tissue is immersed in three paraffin tanks in turn, each tank for 1 hour;

[0294] ④ Embedding: Pour liquid paraffin into the mold box, and then place the wax-soaked tissue block flat on the bottom, with the cut surface facing downward. After the paraffin solidifies, remove the embedding frame. Trim the wax block after it has completely cooled and hardened. Keep an appropriate amount of paraffin on the outside of the tissue for slicing.

[0295] Slice making: Fix the pre-cooled wax block on the paraffin slicer so that the cut surface of the wax block is parallel to the blade. The inclination of the knife is usually 15°. Turn the rotary propeller to adjust the slice thickness to 4μm and cut into slices of uniform thickness. Hold the brush in your left hand and rotate the slicer handle with your right hand. After the slice is brought out, gently lift it with the brush, and then use tweezers to gently tweeze the wax slice and put it into the slide box with the front side. The water temperature is about 45°C. After it is spread flat, pick up the slice. Attach the slice. Hold one end of the slide with your left hand and vertically put it into the water to attach the slice. Use tweezers in your right hand to help push it and attach it to two-thirds of the slide. After the slice is attached, let it dry slightly in the air, bake it on a 65°C slice baking machine for 1 hour, and then bake it in an oven for 2 hours.

[0296] Dewaxing of paraffin sections: Place the paraffin sections in xylene I for 10 min - xylene II for 10 min - xylene III for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 90% alcohol for 5 min - 80% alcohol for 5 min - 70% alcohol for 5 min - 50% alcohol for 5 min for gradient dewaxing.

[0297] HE staining: paraffin sections were stained with hematoxylin for 0.5-1 min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, then blued with 1% ammonia aqueous solution for 1 min, rinsed with running water for a few seconds, stained with eosin solution for a few seconds, and rinsed with running water.

[0298] Dehydration and sealing: Put the paraffin sections into 75% ethanol for 2 min, 85% ethanol for 2 min, anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, and xylene for 5 min to make them transparent. Take the sections out of xylene and seal them with neutral gum.

[0299] (2) Oil red staining

[0300] The supernatant was removed by aspiration, and the cells were washed three times with PBS, fixed with frozen 4% paraformaldehyde for 5 minutes, washed once with PBS, and fixed in propylene glycol for 5 minutes. The propylene glycol was gently removed by aspiration, and a 0.5% oil red O propylene glycol solution was added. The cells were placed in a 60°C oven for staining for 15 minutes, rinsed with 85% propylene glycol for 5 minutes, and then washed three times with PBS, washed once with distilled water, and sealed. The intracellular fat particles were observed under a microscope to detect the amount of intracellular lipid droplets.

[0301] 3.4 Enzyme-linked immunosorbent assay (ELISA)

[0302] (1) Sample addition: Set up standard wells, test sample wells, and blank wells. Set up 3 standard wells, and add 100 μL of standards of different concentrations (see reagent preparation 2) in sequence. Add 100 μL of standard diluent (see the last tube of the second step of reagent preparation) to the blank well, and add 100 μL of the test sample to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 1 hour.

[0303] (2) Discard the liquid, spin dry, and do not wash.

[0304] (3) Add 100 μL of detection solution A working solution (prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 1 h.

[0305] (4) Discard the liquid in the wells, wash each well with 350 μL of washing buffer, soak for 1-2 minutes, and tap the plate on absorbent paper to remove all the liquid in the wells. Repeat the plate wash three times. After the last wash, aspirate or pour out the remaining washing buffer, turn the plate upside down on absorbent paper, and absorb all the liquid remaining in the wells. This process can also be completed using a spray bottle, multichannel pipette, or automatic plate washer.

[0306] (5) Add 100 μL of detection solution B working solution (prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 30 min.

[0307] (6) Discard the liquid in the wells, spin dry, and wash the plate 5 times using the same method as step 4.

[0308] (7) Add 90 μL of TMB substrate solution to each well, cover the ELISA plate, and develop color at 37°C in the dark (the reaction time should be controlled within 10-20 min and should not exceed 30 min. The reaction can be terminated when the first 3-4 wells of the standard wells have a clear blue gradient and the gradient in the last 3-4 wells is not obvious).

[0309] (8) Add 50 μL of stop solution to each well to stop the reaction. The blue color will immediately turn yellow. The order of adding the stop solution should be the same as that of adding the substrate solution. If the color is uneven, gently shake the ELISA plate to mix the solution evenly.

[0310] (9) After ensuring that there are no water droplets on the bottom of the ELISA plate and no bubbles in the wells, immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0311] 3.5 Blood biochemical analysis

[0312] Use a fully automatic biochemical analyzer for testing, add reagents and samples to be tested, the serum sample is no less than 200ul, and the reagent requires about 300ul; test and export the test data.

[0313] 4 Test results

[0314] 4.1 Results of analysis of body weight, food intake, energy intake and tissue index

[0315] Changes in body weight, food intake, and energy intake are shown in Tables 17, 18, 19 and Fig.29As shown in the figure, during the whole test period, the weight and food intake of the experimental animals continued to increase, among which the control group grew the slowest and the model group grew the fastest. Compared with the model group, each dose group had an inhibitory effect on the growth of body weight, among which the high dose group had the most significant effect. High-fat feeding and each dose group had significant differences in the food intake of the experimental animals.

[0316] Table 17 Body weight change (g)

[0317] Grouping Blank group Model Group High dose group Medium dose group Low dose group 0W 230.60 238.00 230.70 229.00 232.30 1W 266.30 282.30 278.60 272.10 267.20 2W 285.70 329.10 306.50 326.60 328.60 3W 338.60 385.60 361.50 368.40 379.10 4W 376.50 435.10 400.90 405.20 409.00 5W 418.30 462.90 410.60 435.60 436.00 6W 436.00 499.60 413.90 462.20 464.70 7W 461.50 532.80 430.89 484.50 490.60 8W 485.10 555.70 457.56 506.40 517.30

[0318] Table 18 Changes in food intake (g)

[0319]

[0320]

[0321] Table 19 Changes in energy intake (kcal)

[0322] Grouping Blank group Model Group High dose group Medium dose group Low dose group 0W 727.35 1105.65 964.68 962.05 962.58 1W 695.65 1126.17 1016.76 1069.88 998.87 2W 813.14 1346.03 1272.92 1261.87 1217.69 3W 909.00 1576.95 1402.84 1369.70 1387.06 4W 935.48 1594.31 1431.25 1404.42 1412.31 5W 925.04 1595.36 1500.15 1555.38 1555.38 6W 976.89 1641.12 1551.17 1574.32 1553.80 7W 1082.45 1673.21 1633.23 1677.94 1652.69 8W 1148.84 1708.97 1637.96 1655.85 1677.94

[0323] After energy conversion, the energy value of the high-fat feeding group was significantly higher than that of the control group. There was no significant difference between the model group and the low, medium and high dose groups.

[0324] The changes of white adipose tissue index, brown adipose tissue index and liver tissue index are shown in Table 20 and Fig.30 As shown, compared with the model group, the white adipose tissue index of the low, medium and high dose groups decreased, while the brown adipose tissue increased. The changes in the medium and high dose groups were relatively obvious, indicating that the jujube polysaccharide can promote the transformation of white fat to gray fat to a certain extent. From the perspective of liver index changes, compared with the model group, the liver index of the low, medium and high doses decreased, among which the medium dose group could significantly reduce the liver index of high-fat rats.

[0325] Table 20 Changes in white adipose tissue index, brown adipose tissue index and liver tissue index (%)

[0326] Grouping Blank group Model Group High dose group Medium dose group Low dose group White adipose tissue index 1.28 1.94 1.71 1.46 1.46 Brown adipose tissue index 0.084 0.095 0.11 0.10 0.092 Liver tissue index 2.72 3.10 2.95 2.33 2.83

[0327] 4.2 Pathological HE and Oil Red Staining Results

[0328] After modeling and 8 weeks of drug treatment, the results of pathological HE and white fat oil red staining of liver tissue, white adipose tissue and jejunum tissue were as follows: Fig.31 , 32 As shown in Figures 33 and 34, compared with the control group, the liver tissue, white fat and jejunal tissue in the model group were severely damaged, the intertissue gaps increased, and the improvement was more obvious in the medium-dose group.

[0329] 4.3 Enzyme-linked immunosorbent assay (ELISA) results

[0330] IL-6, IL-1β, TNF-α, MCP1, adiponectin, leptin, serum insulin, glycosylated hemoglobin and blood glucose levels were determined by ELISA test. The test results are shown in Table 21 and Fig.35 shown.

[0331] Table 21

[0332] Grouping Control group Model Group High dose group Medium dose group Low dose group IL-6 (ng / L) 134.65 182.29 159.13 153.39 170.95 IL-1β (ng / L) 3.92 10.73 7.84 7.49 8.92 TNF-α(ng / L) 10.17 19.08 15.27 14.08 16.65 MCP1(ng / L) 27.38 64.92 45.03 50.49 55.22 Adiponectin (mg / L) 14.13 6.38 8.47 9.21 7.01 Leptin (ng / mL) 2.99 2.61 2.81 2.71 2.73 Serum insulin (mIU / L) 28.76 42.40 34.08 36.57 39.38 Glycated hemoglobin (mmol / L) 16.22 33.78 28.40 23.95 30.33 blood sugar 9.22 31.07 19.49 23.54 27.06

[0333] Compared with the control group, the levels of IL-6, IL-1β, TNF-α, MCP1, adiponectin, leptin, serum insulin, glycosylated hemoglobin and blood glucose in the model group were significantly changed (P<0.01), indicating that the high-fat model was successfully established.

[0334] Compared with the model group, the levels of inflammation-related indicators L-6, IL-1β, TNF-α and MCP1 in the low, medium and high dose groups were significantly reduced (P<0.01), indicating that jujube polysaccharide has a positive anti-inflammatory effect.

[0335] Compared with the model group, the levels of adiponectin (ADPN) in the medium and high-dose groups, and the levels of leptin (LEP) in the high-dose group were significantly increased (P<0.01), indicating that jujube polysaccharides can regulate fat metabolism and reduce fat content. At the same time, the increase in adiponectin content can also improve insulin resistance and have a positive effect on blood sugar regulation.

[0336] Compared with the model group, the medium and high dose contents of glycated hemoglobin (HbAc1), serum insulin and blood glucose, which are related indicators of blood glucose metabolism, were significantly reduced (P<0.01), indicating that jujube polysaccharide has a positive effect on blood glucose regulation.

[0337] 4.4 Blood biochemical analysis results

[0338] The results of the four tests of glutathione peroxidase (GSH-PX) activity, SOD activity enzyme, malondialdehyde (MDA) and blood lipids are shown in Tables 22, 23, 24, 25, 26 and Fig.36 , 37 shown.

[0339] Table 22 GSH-PX enzyme activity, SOD enzyme activity and MDA content test results

[0340] Grouping Control group Model Group High dose group Medium dose group Low dose group MDA (nmol / mL) 5.52 6.96 5.53 5.98 6.10 SOD activity (U / mgprot) 44.99 29.65 37.11 33.34 29.26 GSH-PX activity 1929.52 1175.24 1358.73 1458.10 1259.05

[0341] Table 23 TG test results (mmol / L)

[0342] Grouping Control group Model Group High dose group Medium dose group Low dose group 0 Weeks 0.72 0.72 0.67 0.73 0.70 2 weeks 0.72 0.91 0.84 1.01 0.89 4 weeks 0.90 2.22 1.73 1.65 2.04 6 weeks 0.69 3.10 2.41 2.26 2.76 8 weeks 0.70 3.12 2.37 2.24 2.77

[0343] Table 24 CHOL test results (mmol / L)

[0344] Grouping Control group Model Group High dose group Medium dose group Low dose group 0 Weeks 1.59 1.49 1.52 1.50 1.50 2 weeks 1.41 2.66 2.29 2.11 2.38 4 weeks 1.49 3.54 2.82 2.61 3.35 6 weeks 1.54 3.92 3.46 3.20 3.55 8 weeks 1.57 3.97 3.42 3.23 3.65

[0345] Table 25 HDL-C test results (mmol / L)

[0346] Grouping Control group Model Group High dose group Medium dose group Low dose group 0 Weeks 1.53 1.50 1.48 1.65 1.58 2 weeks 1.55 1.33 1.35 1.39 1.30 4 weeks 1.58 1.05 1.19 1.26 1.12 6 weeks 1.47 0.84 0.96 1.12 0.81 8 weeks 1.54 0.74 0.89 1.04 0.73

[0347] Table 26 LDL-C test results (mmol / L)

[0348] Grouping Control group Model Group High dose group Medium dose group Low dose group 0 Weeks 0.37 0.40 0.39 0.42 0.40 2 weeks 0.39 0.61 0.58 0.53 0.56 4 weeks 0.38 0.89 0.69 0.80 0.82 6 weeks 0.36 1.25 0.88 0.93 1.08 8 weeks 0.40 1.32 0.98 1.01 1.19

[0349] The results of four blood lipid tests showed that jujube polysaccharides can prevent the increase of serum TC, CHOL, and LDL-C levels induced by a high-fat diet. A decrease in HDL-C is an independent risk factor for the occurrence and development of atherosclerotic cardiovascular disease. A high-fat diet can significantly reduce the HDL-C level of mice, and the increase in HDL-C levels slowed down after treatment with jujube polysaccharides. This shows that jujube polysaccharides can slow down the increase of TC, TG, LDL-C and the decrease of HDL-C in mice induced by a high-fat diet, improve lipid metabolism disorders, and have the effect of regulating blood lipids, which is consistent with previous research results.

[0350] The results of the antioxidant capacity test showed that compared with the model group, the GSH-PX and SOD enzyme activities in each dose group increased, and the MDA content decreased, and there was a significant difference between the high dose and the medium dose (P<0.01), indicating that jujube polysaccharide can improve antioxidant capacity and reduce cell oxidative damage.

[0351] The wild jujube crude polysaccharide and polysaccharide components provided by the invention have significant effects on regulating blood lipids and blood sugar, antioxidant activity, anti-inflammatory effect and immunomodulatory effect, and can be used in preparing medicines, health foods or foods with relevant functional effects.

[0352] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A crude polysaccharide from wild jujube pulp, characterized in that: The crude polysaccharide of sour jujube pulp is prepared by the following preparation method: S1. Take the raw material of sour jujube pulp, add water at a material-liquid ratio of 1:4-12 g / mL, adjust the pH value of the mixed liquid to 6.5-7.5, add papain in an amount of 0.01%-0.10% of the mixed liquid, extract by enzymolysis at 30-70°C for 1-1.5h, heat to kill the enzyme, centrifuge and filter to obtain an enzymolysis solution; S2. Concentrate the obtained enzymatic hydrolysate to a relative density of 1.05-1.06, precipitate with alcohol, centrifuge, and collect the precipitate to obtain crude polysaccharide.

2. The crude polysaccharide from wild jujube pulp as claimed in claim 1, characterized in that The sour jujube pulp raw material is powder obtained by crushing and sieving dried sour jujube pulp.

3. The crude polysaccharide from wild jujube pulp as claimed in claim 1, characterized in that The alcohol precipitation method comprises: adding 4 times the volume fraction of 95% ethanol to the enzymatic hydrolysate concentrate, alcohol precipitation at 4° C. for 24 hours, and centrifugation at 4000 revolutions for 15 minutes.

4. A polysaccharide composition of wild jujube pulp, characterized in that: The polysaccharide composition is obtained by separating and purifying the crude polysaccharide from the jujube pulp as claimed in any one of claims 1 to 3, wherein the polysaccharide composition comprises a neutral polysaccharide component and an acidic polysaccharide component.

5. The sour jujube pulp polysaccharide composition according to claim 4, characterized in that The neutral polysaccharide component is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, and the molar ratio is 0.75:16.81:19.97:17.54:33.04:2.52:1.66:6.86:0.86; the acidic polysaccharide component is composed of rhamnose, arabinose, galactose, glucose and galacturonic acid, and the molar ratio is 2.54:2.76:2.31:91.75:0.

65.

6. The polysaccharide composition of wild jujube pulp according to claim 5, characterized in that The weight average molecular weight of the neutral polysaccharide component is 3.73×10 5 ±0.01×10 5 Da; the weight average molecular weight of the acidic polysaccharide component is 1.36×10 7 ±0.01×10 7 Da.

7. The sour jujube pulp polysaccharide composition according to claim 5, characterized in that The molecular configurations of the neutral polysaccharide component and the acidic polysaccharide component are spherical.

8. The polysaccharide composition of wild jujube pulp as claimed in claim 4, characterized in that The separation and purification process includes protein removal; macroporous resin adsorption; dialysis, freeze drying; anion exchange chromatography purification and gel chromatography purification.

9. The sour jujube pulp polysaccharide composition according to claim 8, characterized in that The anion exchange chromatography purification comprises the following steps: taking a crude polysaccharide sample after freeze-drying, dissolving it in pure water to prepare a polysaccharide mother solution with a concentration of 10 mg / mL, centrifuging, taking the supernatant, and purifying it by anion exchange column chromatography, using pure water, 0.1M NaCl solution, 0.2M NaCl solution and 0.3M NaCl solution in sequence for isogradient elution, with a flow rate of 4mL / min, 15mL per tube, collecting all the eluates, and vacuum freeze-drying to obtain ion exchange purified polysaccharides.

10. The polysaccharide composition of wild jujube pulp according to claim 9, characterized in that The gel chromatography purification comprises the following steps: taking the above-mentioned ion exchange purified polysaccharide sample, adding pure water to prepare a solution with a concentration of 15 mg / mL, centrifuging, taking the supernatant, purifying it with gel separation column chromatography, eluting with pure water at a flow rate of 1 mL / min, collecting all the eluents, and vacuum freeze-drying to obtain gel-purified polysaccharides.

11. Use of the crude polysaccharide of jujube pulp as claimed in any one of claims 1 to 3 or the polysaccharide composition of jujube pulp as claimed in any one of claims 4 to 10 in the preparation of medicines, health foods or foods having lipid and blood sugar regulating, anti-oxidation and anti-inflammatory effects.

12. The use according to claim 11, characterized in that The dosage form of the medicine includes one or more of capsules, tablets, granules, pills, powders, granules or oral liquids; the dosage form of the health food includes one or more of capsules, tablets, granules, powders, granules, oral liquids, teas, wines, beverages, biscuits or candies; the dosage form of the food includes one or more of tablets, granules, candies, jellies, beverages, solid beverages, biscuits, noodles or sticks.

13. A pharmaceutical composition, characterized in that The invention comprises the crude polysaccharide of wild jujube pulp according to any one of claims 1 to 3 or the polysaccharide composition of wild jujube pulp according to any one of claims 4 to 10.

14. A health food, characterized in that: The invention comprises the crude polysaccharide of wild jujube pulp according to any one of claims 1 to 3 or the polysaccharide composition of wild jujube pulp according to any one of claims 4 to 10.

15. A food, characterized in that The invention comprises the crude polysaccharide of wild jujube pulp according to any one of claims 1 to 3 or the polysaccharide composition of wild jujube pulp according to any one of claims 4 to 10.

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