Preparation method of jujube polysaccharide with anti-oxidation and nerve cell protection activity

Abstract

The invention discloses a preparation method of jujube polysaccharide with antioxidant and nerve cell protection activity, and relates to a preparation method of jujube polysaccharide, which comprises the following steps of: firstly, obtaining extract from jujube by using a petroleum ether, 90% ethanol and distilled water condensation reflux extraction method, deproteinizing and decolorizing, separating by using DEAE-52 cellulose column chromatography, and separating by using ethanol to obtain the jujube polysaccharide with antioxidant and nerve cell protection activity. And finally, further purifying by using SephadexG-200 gel, so as to finally obtain the jujube polysaccharide ZJP-1. And the molecular weight of the ZJP-1 is 9.04 kDa. In-vitro DPPH, ABTS and hydroxyl radical scavenging activity results show that the jujube polysaccharide ZJP-1 has antioxidant activity. Meanwhile, the activity of T-AOC, SOD and GSH-Px in damaged nerve cells can be improved, the content of MDA is reduced, and oxidative damage of the nerve cells is effectively relieved.

Description

Technical Field

[0001] The invention relates to a method for extracting, separating and purifying plant polysaccharides, and in particular to a method for preparing jujube polysaccharides with antioxidant and nerve cell protective activities. Background Art

[0002] The jujube (Zizyphus jujuba Mill.), a plant of the Rhamnaceae family, is a plant with both medicinal and edible properties. It is known as one of the "five fruits" and is as famous as plum, apricot, chestnut and peach. Jujube usually refers to its dried and mature fruit, which shows the effects of replenishing qi and blood, improving sleep, regulating the digestive system and delaying aging. Modern pharmacological studies have shown that jujube has antioxidant, anti-fatigue, immunomodulatory and anti-aging activities. Its main components include flavonoids, triterpene acids, amino acids, cerebrosides, minerals and phenolic acids, among which the content of polysaccharides exceeds 50%, which is an important active substance of jujube. Polysaccharides belong to a class of biological macromolecules with diverse structures. They are polymers composed of monosaccharide residues and connected to each other by glycosidic bonds. Polysaccharides have a variety of biological activities, and more and more studies have shown that most of the biological activities and functions of polysaccharides are related to antioxidants. Although the research on polysaccharides started later than that of biological macromolecules such as proteins and nucleic acids, as their important physiological functions are continuously discovered, polysaccharides have become an important part of the research and development of natural source drugs and health products.

[0003] As a traditional Chinese medicinal and edible plant, jujube has been widely used for thousands of years, and its biological activity is recognized by both traditional Chinese medicine theory and modern pharmacology. However, research on jujube polysaccharides is still limited. Summary of the invention

[0004] The present invention aims to provide a method for preparing jujube polysaccharides with antioxidant and neuron cell protective activities. The method adopts a hot water reflux extraction method to obtain an extract from jujube fruits, removes small molecules, proteins and pigments, separates the extract using a DEAE-52 cellulose column, and elutes the extract using deionized water and a 0.1 mol / L to 1 mol / L NaCl solution. The present invention extracts, separates, purifies, characterizes the structure and studies the antioxidant activity of jujube polysaccharides, laying a theoretical foundation for the further development and utilization of jujube.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] A method for preparing jujube polysaccharides having antioxidant and neuron-protective activities, the method comprising the following preparation process:

[0007] (1) taking dried jujube fruits, sequentially performing reflux extraction with petroleum ether to remove fat, performing reflux extraction with ethanol to remove small molecules, adding distilled water to condense and reflux extract 2-6 times, and combining the filtrates; after the extract is concentrated, adding 5 times the volume of 95% ethanol, precipitating at 4° C. for 12 hours, and drying at 65° C. to obtain a crude polysaccharide extract;

[0008] (2) Enzymatic hydrolysis combined with Sevage method to remove protein: add 1 / 4 volume of Sevage reagent (dichloromethane: n-butanol = 4:1) and react for 30 minutes, then centrifuge and collect the supernatant. Repeat this operation three times. The lower layer precipitates as denatured protein colloid. Collect the upper water layer and dry it to obtain deproteinized crude polysaccharide. Weigh 1 mg of deproteinized crude polysaccharide to determine the protein content. Repeat this operation until the determined protein content is stable.

[0009] (3) decolorizing the deproteinized polysaccharide using AB-8 macroporous resin to obtain a depigmented polysaccharide;

[0010] (4) The deproteinized and depigmented polysaccharide was further purified by DEAE-52 cellulose column chromatography and Sephadex G-200 column chromatography, and then dialyzed and dried to obtain a new pure jujube polysaccharide ZJP-1; the total sugar content was 95.33±2.02%, the molecular weight was 9.04 kDa, and it was composed of arabinose, rhamnose, galactose, glucose and galacturonic acid, with the mass percentages being 4.30%, 12.39%, 5.84%, 0.91% and 76.55%, respectively.

[0011] The method for preparing a jujube polysaccharide with antioxidant and neuron cell protective activity is disclosed. The prepared ZJP-1 has a main chain structure of: (1→4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap-(1→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap-(1→4)-α-D-GalpA.

[0012] The method for preparing a jujube polysaccharide with antioxidant and neuron cell protective activity, wherein the prepared uniform jujube fruit polysaccharide is evaluated for its in vitro antioxidant activity by scavenging DPPH, ABTS and hydroxyl free radicals in vitro; and by detecting the levels of T-AOC, SOD, GSH-Px and MDA in model cells, ZJP-1 inhibits the generation of ROS by increasing the activity of endogenous antioxidant enzymes.

[0013] Advantages and effects of the present invention:

[0014] 1. The results of in vitro antioxidant activity showed that jujube polysaccharides have good antioxidant activity. When the concentration of polysaccharide sample ZJP-1 reached 5.0 mg / mL, the scavenging effect of DPPH free radicals was 78.67%; at this dosing concentration, the scavenging effect of ZJP-1 on ABTS free radicals was 53.35%. At the same time, ZJP-1 also has a certain hydroxyl free radical scavenging activity, and the hydroxyl free radical scavenging ability can reach 80.07% at the maximum concentration.

[0015] 2. The results of the neuronal cell protective activity test showed that ZJP-1 had a certain protective effect on SH-SY5Y cells, and the effect increased in a dose-dependent manner. When the cells were pretreated with ZJP-1 at a concentration of 250 μg / mL, the survival rate of SH-SY5Y cells increased to 80.08%. At the same time, the antioxidant activity mechanism of jujube polysaccharide ZJP-1 was explored. The study found that ZJP-1 could slow down the oxidative damage caused by H2O2 to SH-SY5Y cells, and at 250 μg / mL, it could restore the T-AOC capacity to 0.099 mmol / g, increase the SOD and GSH-Px enzyme activities to 33.59 U / mg and 18.69 mU / mg, and reduce the MDA content to 7.69 μmol / mg, and it was dose-dependent. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result diagram of the monosaccharide composition of ZJP-1;

[0017] Figure 2 is the methylation profile of ZJP-1;

[0018] Figure 3 is the infrared spectrum of ZJP-1;

[0019] Figure 4 It is ZJP-1 1 HNMR and 13 CNMR spectrum;

[0020] Figure 5 It is ZJP-1 1 H- 1 HCOSY, HSQC, HMBC and TOCSY spectra;

[0021] Figure 6 This is the result of ZJP-1 scavenging DPPH, ABTS and hydroxyl free radicals;

[0022] Figure 7 This is a graph showing the protective activity of ZJP-1 on H2O2-induced SH-SY5Y neuronal cells and oxidative stress indicators. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings and examples. The experimental methods described in the following examples are conventional methods unless otherwise specified. The reagents and biological materials described can be obtained from commercial sources unless otherwise specified.

[0024] (1) Raw material processing and preliminary extraction

[0025] The jujube fruit polysaccharides were extracted by reflux distilled water. The jujube was pitted and sliced, and refluxed and extracted twice with 10 times the volume of petroleum ether (1:10, w / v), each time for 2 hours for defatting. After drying, the residue was refluxed and extracted 3 times with 15 times the volume of 90% ethanol (1:15, w / v), each time for 2 hours to remove small molecular components such as flavonoids and saponins. Finally, hot water was used to extract for 2 hours at a solid-liquid ratio of 1:15 (g / mL), and repeated 3 times. After the extract was concentrated, 5 times the volume of 95% ethanol was added, precipitated at 4°C for 12 hours, and dried at 65°C to obtain the jujube crude polysaccharide extract.

[0026] (2) Deproteinization

[0027] Deproteinize by enzymatic hydrolysis combined with Sevage method. Prepare the crude polysaccharide extract to a concentration of 0.08g / mL, add 2.5% neutral protease, react at 50°C for 1 hour, react at 90°C for 10 minutes to inactivate the enzyme, and collect the supernatant by centrifugation. Add 1 / 4 volume of Sevage reagent (dichloromethane: n-butanol = 4:1), react for 30 minutes, centrifuge, collect the supernatant, repeat the operation three times, and obtain deproteinized crude polysaccharide. Repeat the measurement of protein content until it meets the standard.

[0028] (3) Depigmentation

[0029] Use AB-8 macroporous resin to remove pigments. Soak the resin in 95% ethanol, wash until the resin has no alcohol smell, then load it into the chromatographic column, use a small amount of distilled water to dissolve the deproteinized crude polysaccharide, and evenly load it onto the surface of the chromatographic column. Elute with distilled water until the eluent is colorless, collect the eluent, concentrate it under reduced pressure, dialyze it with a dialysis bag (3500kDa) for 48 hours, and vacuum dry it to obtain the depigmented jujube polysaccharide.

[0030] (4) Separation and purification

[0031] The crude polysaccharide was separated and purified using a DEAE-52 cellulose column. DEAE-52 cellulose was loaded into the chromatographic column and loaded after precipitation overnight. Weigh 1.0g of crude polysaccharide, dissolve it in distilled water, and load it. Hang the column for 12 hours. Gradient elution was performed with distilled water and 0.1mol / L~1.0mol / L NaCl. The eluate was collected at a speed of 1mL / min using an automatic sampler. The phenol-sulfuric acid method was used to monitor the polysaccharide content and draw an absorbance-tube number elution curve. The eluate of the symmetrical peak was combined, concentrated under reduced pressure, dialyzed, and dried to obtain jujube polysaccharides of different components. The obtained polysaccharide components were purified using a Sephadex G-200 column. Weigh 100mg of each gradient jujube polysaccharide, dissolve it in 200μL water, hang the sample for 2 hours, and elute with distilled water. The phenol-sulfuric acid method was used to determine and draw the elution curve, collect the symmetrical peak, and concentrate and dry to obtain the jujube pure polysaccharide ZJP-1.

[0032] (5) Structural characterization

[0033] The obtained jujube polysaccharide ZJP-1 has a molecular weight of 9.04 kDa and is mainly composed of arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc) and galacturonic acid (GalA), with mass percentages of 4.30%, 12.39%, 5.84%, 0.91% and 76.55%, respectively. Combined with methylation, infrared spectroscopy, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy data analysis, it can be seen that ZJP-1 contains 8 kinds of sugar residues and contains structural fragments →4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap -(1→4)-α-D-GalpA, containing branched α-D-GalpA-(1→6)-β-D-Galp-(1→4)-β-L-Rhap-(1→ and α-D-GalpA-(1→4)-β-D-Galp-(1→4)-β-L-Rhap-(1→. At the same time, it was found that the structure of jujube polysaccharide ZJP-1 became more compact with the continuous increase of molecular weight, and had a triple helix structure.

[0034] (6) Biological activity

[0035] In vitro antioxidant tests showed that ZJP-1 had a certain ability to scavenge DPPH, ABTS and hydroxyl free radicals. ZJP-1 could effectively alleviate the oxidative damage caused by H2O2 to SH-SY5Y cells. At a concentration of 250 μg / mL, it could restore T-AOC capacity, increase SOD and GSH-Px enzyme activities, and reduce MDA content in a dose-dependent manner.

[0036] The examples are as follows:

[0037] 1) Extraction, separation and purification of jujube polysaccharides

[0038] The jujube polysaccharide was extracted by hot water reflux extraction. The jujube was pitted and sliced, dried in an oven at 65°C for 48 hours, and then refluxed with petroleum ether at a solid-liquid ratio of 1:10 (g / mL) for 2 times, each time for 2 hours to remove lipids. After drying, the residue was extracted with 90% ethanol 1:15 (g / mL) 3 times, each time for 2 hours to remove small molecular components such as flavonoids and saponins. Distilled water 1:15 (g / mL) was extracted 3 times, each time for 2 hours. After the extract was appropriately concentrated, 5 times the volume of 95% ethanol was added, precipitated at 4°C for 12 hours, and dried at 65°C to obtain a total extract. The above total extract was prepared into a 0.08g / mL aqueous solution, 2.5% neutral protease was added, reacted at 50°C for 1 hour, and then reacted at 90°C for 10 minutes to inactivate the enzyme, centrifuged and the supernatant was collected. 1 / 4 volume of Sevage reagent (dichloromethane: n-butanol = 4:1) was added and reacted for 30 minutes, followed by centrifugation, and the supernatant was collected. The water layer was dried to obtain deproteinized crude polysaccharide.

[0039] The crude polysaccharide of jujube was decolorized by AB-8 macroporous resin chromatography column. 10g of crude polysaccharide of jujube after protein removal was weighed and dissolved in 15mL distilled water. The outlet valve was opened to make the liquid level and the filler level, and the crude polysaccharide solution was spread on the surface of the filler and hung for 6 hours. Distilled water was used as the eluent and 10 column volumes were collected. After the eluent was appropriately concentrated, it was placed in a dialysis bag (3500Da) and dialyzed for 48 hours. After vacuum drying, the depigmented crude polysaccharide of jujube was obtained.

[0040] The decolorized jujube crude polysaccharide was separated by DEAE-52 cellulose anion exchange chromatography column, and gradient elution was performed with deionized water, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0M NaCl solution in turn. The sugar content in each test tube was determined by the phenol-sulfuric acid method, and an elution curve with the tube number as the horizontal coordinate and the absorbance as the vertical coordinate was drawn. The eluate in the symmetrical peak shape was collected according to the elution curve, and the eluate was concentrated and dialyzed (3500Da) for 48 hours, and the crude polysaccharide was obtained after drying. The different components obtained were purified by SephadexG-200 column, concentrated under reduced pressure, dialyzed and dried, and finally a jujube polysaccharide (ZJP-1) was obtained.

[0041] 2) Determination of molecular weight of jujube polysaccharide (ZJP-1)

[0042] The molecular weight of jujube polysaccharide was determined by high performance gel permeation chromatography using dextran of different molecular weights (T4, T10, T20, T40, T100, T200, T500) as standard. ZJP-1 and standard were prepared into a 10 mg / mL solution using mobile phase and filtered through a 0.22 μm filter membrane.

[0043] The measurement conditions are as follows:

[0044] Mobile phase: 0.02M potassium dihydrogen phosphate (KHPO) solution

[0045] Flow rate: 0.6mL / min

[0046] Injection volume: 40 μL

[0047] Chromatographic column: TSKgelG5000PW column (ID = 7.5nm, L = 300mm)

[0048] Detector: Differential refractive index detector

[0049] The standard curve was established with the logarithm of the molecular weight of the standard (lgMw) as the abscissa and the retention time (RT, min) as the ordinate. According to the pseudo formula y = -2.4286x + 25.24 (R 2 =0.9922), calculated the logarithm of the molecular weight of sample ZJP-1, and finally obtained its relative molecular weight as 9.04 kDa.

[0050] 3) Determination of monosaccharide composition of jujube polysaccharide (ZJP-1)

[0051] Take 5 mg of jujube polysaccharide, add 1 mL of 2M trifluoroacetic acid (TFA) solution, and heat at 121°C for 2 hours. Ventilate, blow dry, add anhydrous methanol to wash, and repeat three times. Add sterile water to dissolve and place in a chromatographic bottle for testing. Use an ion chromatograph for determination, equipped with an electrochemical detector and Dionex TM CarboPac TM PA20 chromatographic column (150×3.0 mm, 10 μm).

[0052] The chromatographic conditions were as follows:

[0053] Mobile phase A: water (H2O)

[0054] Mobile phase B: 0.1 M NaOH

[0055] Mobile phase C: 0.1M NaOH and 0.2M sodium acetate (NaAc) solution

[0056] Elution gradient:

[0057] 0min: A / B / C=95:5:0 (V / V)

[0058] 26min: A / B / C=85:5:10 (V / V)

[0059] 42min: A / B / C=85:5:10 (V / V)

[0060] 42.1min:A / B / C=60:0:40(V / V)

[0061] 52min: A / B / C=60:40:0 (V / V)

[0062] 52.1min: A / B / C=95:5:0 (V / V)

[0063] 60min: A / B / C=95:5:0 (V / V)

[0064] Flow rate: 0.5mL / min

[0065] Column temperature: 30°C

[0066] Injection volume: 5 μL

[0067] By comparing the chromatograms of ZJP-1 and mixed standard samples (such as Figure 1 The results showed that ZJP-1 was composed of arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc) and galacturonic acid (GalA), with mass percentages of 4.30%, 12.39%, 5.84%, 0.91%, 15.78% and 76.55%, respectively.

[0068] 4) Analysis of methylation results of jujube polysaccharide (ZJP-1)

[0069] Take 10 mg of jujube polysaccharide and dissolve it in 2 mL of 75% THF-0.1 M morpholineethanesulfonic acid aqueous solution, adjust pH to 4.75 with 10% Et3N aqueous solution, add 20 mg of carbodiimide respectively, stir at room temperature for 1 hour, heat to 50°C, add 2 mL of 2M NaBD4 (or NaBH4) twice at intervals of 30 minutes, and continue heating and stirring at 50°C for 1 hour. Use 2M acetic acid to terminate the reaction, dialyze the reaction solution through a 3.5kD dialysis bag for 24 hours, and then vacuum concentrate to remove the solvent. Dissolve the fully dried sample in 1 mL of DMSO, pass nitrogen, seal, and ultrasonic (40-50°C) until fully dissolved. Add 1 mL of NaOH-DMSO suspension and ultrasonic for 30 minutes. Slowly add 1 mL of iodomethane with a syringe in an ice bath, ultrasonic for 20 minutes (18-20°C), and repeat 3 times. Add 1 mL of water to terminate the reaction, let it stand for 20 minutes, and then put it into a 3.5kD dialysis bag for dialysis for 72 hours. After concentration and drying, the methylated polysaccharide was obtained, and infrared detection was used to observe whether the characteristic peak of the hydroxyl group disappeared. The above sample was hydrolyzed with 2mL trifluoroacetic acid (2M) in a stoppered test tube at 110℃ for 6 hours. After the reaction, an appropriate amount of anhydrous methanol was added to evaporate the solvent. This process was repeated until the excess trifluoroacetic acid was completely removed. The hydrolyzed sample was obtained after drying with nitrogen. The hydrolyzed sample was dissolved in 1mL of a newly prepared NaBD4 aqueous solution (10mg / mL) and reacted at 60℃ for 2 hours. The reaction solution was neutralized with 2M acetic acid, and methanol was added to evaporate the solvent, and repeated three times. 1mL of a newly prepared pyridine-acetic anhydride (1:1) was added to the above reactants, reacted at 120℃ for 30 minutes, cooled to room temperature, and passed through a 0.22μm filter membrane to wait for injection detection. The determination was carried out using a gas chromatography-mass spectrometry chromatograph (7890A / 5975C) equipped with an FID detector and an AgilentHP-1MSUI capillary column (30m×0.320mm, 0.25μm).

[0070] The detection conditions are set as:

[0071] Nitrogen flow rate: 1.0mL / min

[0072] Split ratio: 10:1

[0073] Initial temperature: 50°C, hold for 1 minute

[0074] Heating rate: 50℃ / min to 130℃, 3℃ / min to 230℃, hold for 2 minutes

[0075] Mass scan range: 40-500amu

[0076] Solvent delay: 3.6 minutes

[0077] Injection volume: 1.0 μL

[0078] like Figure 2As shown in the figure, the glycosidic bond connection mode between monosaccharides in the jujube polysaccharide ZJP-1 was studied by methylation analysis. The glycosidic bond type of ZJP-1 was identified based on the fragment ion mass spectrum of partially methylated alditol acetates (PMAAs). At the same time, the glycosidic bond connection mode and the acidic sugar ratio were determined by comparing the mass spectrometry characteristic ion fragments after the reduction of carboxyl groups by different reducing agents (NaBH4 or NaBD4) in the same sample. By comparing the characteristic ion fragments of 2,3,4,6-Me4-GalpA with the database, it was found that the fragments at 161 and 205 were all replaced by [M+2] peaks 163 and 207. Therefore, it was judged that only GalpA-(1→) was contained in this connection mode, but not Galp-(1→. Similarly, by comparing the relative ion intensities of fragments 233:235, it was inferred that the content of →4)-Galp-(1→:→4)-GalpA-(1→) was 1:8. In summary, galacturonic acid in ZJP-1 is mainly expressed as →4)-Gal The ZJP-1 protein exists in the form of pA-(1→ and GalpA-(1→), accounting for 77.59% by molar percentage. Rhamnose exists in the form of →3,4)-Rhap-(1→, and contains a small amount of galactose and arabinose in the form of →4)-Galp-(1→, →2,4,6)-Galp-(1→ and →3,5)-Araf-(1→. Multiple sites are substituted, indicating that ZJP-1 contains a branched structure. Since the content of glucose in ZJP-1 is low, less than 1%, the connection mode of glucose could not be detected.

[0079] 5) Spectral analysis of jujube polysaccharide (ZJP-1)

[0080] like Figure 3 As shown in the infrared spectrum, 3424cm -1 There is an obvious broad peak at 2936cm, which is the strong absorption peak of -OH stretching vibration. -1 The strong absorption peaks at 1420 cm -1 The absorption peak at 1621cm- is the CH stretching vibration peak. All of the above are infrared characteristic absorption peaks of polysaccharides. 1 The typical carboxyl (-COOH) absorption peak is at 1749 cm -1 The characteristic aldehyde group (-CHO) absorption peak at 1260cm indicates the presence of uronic acid, indicating that ZJP-1 is an acidic sugar. -1 The absorption peak at 1104-1016cm is attributed to the stretching vibration of COC in the jujube polysaccharide sample. -1 The absorption peaks in the region are mainly caused by the stretching vibration of the (C-OH) side group and the ring vibration of the (COC) glycosidic bond. Three absorption peaks appear in this region, indicating that the sample contains pyranoside, and 638cm -1The absorption peaks near the polysaccharide are the symmetrical stretching vibration signals of the pyranose ring skeleton. According to the analysis of monosaccharide composition and methylation results, the structure of the jujube polysaccharide ZJP-1 was further elucidated by 1D and 2D NMR spectra.

[0081] like Figures 4 to 5 It can be seen that there are 8 obvious terminal sugar signals in the HSQC spectrum of ZJP-1. 1 H and 13 CNMR spectrum, the chemical shifts of the terminal group signals of each sugar residue can be determined as δ5.18 / 98.5, δ5.22 / 92.2, δ4.53 / 96.1, δ4.93 / 97.5, δ5.03 / 99.5, δ4.45 / 103.2, δ4.88 / 100.4 and δ5.01 / 107.3. 1 HNMR and HSQC spectra, 1 H- 1 H COSY spectrum and TOCSY spectrum were analyzed. Taking the sugar residue end group signal δ5.18 / 98.5 as an example, a cross peak δ5.18 / 4.12 can be observed in the TOCSY spectrum, indicating that the signal δ4.12 belongs to the sugar residue, and cross peaks δ4.12 / 4.49, δ4.12 / 3.60, and δ4.12 / 3.86 were observed, indicating that the above hydrogen signals belong to the same spin coupling system. Combined with the HSQC spectrum, 5 groups of cross peaks δ3.60 / 70.0, δ3.86 / 73.6, δ4.12 / 70.0 and δ4.49 / 71.7 were obtained. 1 H- 1 The signal δ3.86 / 4.12 was observed in the HCOSY spectrum, indicating that δ3.86 and δ4.12 are hydrogen signals connected by three bonds, that is, the carbons with chemical shift values ​​of δ73.6 and δ70.0 are directly connected. A cross peak δ5.18 / 171.0 was observed in the HMBC spectrum, that is, the chemical shift value of the sugar residue C6 was δ171.0, indicating that the sugar residue was an acidic sugar. Combined with relevant references, it is speculated that δ3.65 / 69.6, δ3.86 / 73.6, δ4.12 / 70.0 and δ4.49 / 71.7 are H2 / C2-H5 / C5 signals, respectively, and this group of signals is attributed to α-D-GalpA-(1→(A). When observing the signal related to the end group signal δ5.22 / 92.2, 1 H- 1A cross peak of δ5.22 / 3.74 was observed in the HCOSY spectrum, which means that δ3.74 is the H2 signal of the sugar residue. Two groups of cross peaks of δ3.74 / 4.02 and δ3.74 / 4.38 were observed in the TOCSY spectrum, which were speculated to be H2 / H3 and H2 / H4 signals. A cross peak of δ3.74 / 71.2 was observed in the HMBC spectrum, which was speculated to be C5 signal. According to the HSQC spectrum, the H2 / C2-H5 / C5 signals of the sugar residue were determined to be δ3.74 / 71.9, δ4.02 / 76.2, δ4.38 / 78.9 and δ4.63 / 71.2. Combined with the methylation results and references, this group of signals was attributed to →4)-α-D-GalpA(B). When searching for signals related to the terminal group signal δ4.53 / 96.1, 1 H- 1A cross peak of δ4.53 / 3.41 was observed in the HCOSY spectrum, cross peaks of δ4.53 / 3.41, δ4.53 / 3.68 and δ4.53 / 4.28 were observed in the TOCSY spectrum, and a cross peak of δ3.68 / 70.7 was observed in the HMBC spectrum. Combined with the HSQC spectrum, the H2 / C2-H5 / C5 signals of the sugar residues were determined to be δ3.41 / 71.4, δ3.68 / 72.1, δ4.28 / 78.4 and δ4.60 / 70.7. Combined with the methylation results and references, this group of signals was attributed to →4)-β-D-GalpA(C). The remaining five sugar residues of ZJP-1 were analyzed by the same method, and δ4.93 / 97.5, δ5.07 / 99.5, δ4.45 / 103.2, δ4.88 / 100.4, and δ5.01 / 107.3 were assigned to →4)-α-D-Galp-(1→(D), →4)-α-DGalpA-(1→(E), →2,4,6)-β-D-Galp-(1→(F), →3,4)-β-L-Rhap-(1→(G), and →3,5)-αL-Araf-(1→(H) terminal group signal. In addition, the signals δ5.07 / 170.9 and δ3.73 / 170.9 in the HMBC spectrum and the signal δ3.73 / 52.9 in the HSQC spectrum indicated that the 6-carboxyl group of →4)-α-DGalpA-(1→(E) contained a methyl ester substitution. The signals δ2.10 / 174.0 and δ2.00 / 173.6 in the HMBC spectrum and the signals δ2.10 / 20.5 and δ2.00 / 2 0.2 indicates that ZJP-1 contains acetyl substitutions at two positions. The signal δ3.39 / 59.7 in the HSQC spectrum indicates that ZJP-1 contains methoxy substitutions. A cross peak δ5.07 / 83.6 was observed in the HMBC spectrum, indicating that the H1 signal of →4)-α-D-GalpA-(1→(E) is remotely correlated with the C3 signal of →3,5)-α-L-Araf-(1→(H), indicating that the 1 position of →4)-α-D-GalpA-(1→(E) is remotely correlated with the C3 signal of →3,5)-α-L-Araf-(1→(H). ,5)-α-L-Araf-(1→(H) at the 3-position. Another cross peak δ5.01 / 81.7 in the HMBC spectrum indicated that the 1-position of →3,5)-α-L-Araf-(1→(H) was connected to the 3-position of →3,4)-β-L-Rhap-(1→(G). Combining the signals in the two groups of HMBC spectra, it was shown that the fragment →4)-α-D-GalpA-(1→3)-α-LAraf-(1→3)-β-L-Rhap-(1→) existed in ZJP-1.Cross peaks δ4.88 / 79.2 and δ4.38 / 100.4 were observed in the HMBC spectrum, indicating the presence of signals related to GH1 / EC4 and B H4 / GC1, that is, the fragments →3,4)-β-L-Rhap-(1→4)-α-D-GalpA-(1→ and →3,4)-β-L-Rhap-(1→4)-α-D-GalpA were present in ZJP-1. Combined with the above-mentioned E H1 / HC3- and H H1 / GC3-related signals, this showed the presence of the ZJP-1 backbone (E → 4)-α-D-GalpA-(1 → 3)-α-L-Araf-(1 → 3)-β-L-Rhap-(1 → 4)-α-D-GalpA-(1 → and → 4)-αD-GalpA-(1 → 3)-α-L-Araf-(1 → 3)-β-L-Rhap-(1 → 4)-α-D-GalpA. The cross peak δ3.65 / 103.2 in the HMBC spectrum indicates that the 4-position of →3,4)-β-L-Rhap-(1→(G) is connected to the 1-position of →2,4,6)-β-D-Galp-(1→(F), that is, there is a branched chain →2,4,6)-β-D-Galp-(1→4)-β-L-Rhap-(1→. Cross peaks δ5.18 / 69.3 and δ5. .18 / 77.5, that is, there is a correlation between AH1 / FC6 and AH1 / FC4, indicating that the 4th and 6th positions of →2,4,6)-β-D-Galp-(1→(F) are replaced by α-D-GalpA-(1→(A), that is, there are α-D-GalpA-(1→6)-β-D-Galp-(1→ and α-D-GalpA-(1→4)-β-D-Galp-(1→.

[0082] 6) DPPH free radical scavenging ability

[0083] After dissolving the jujube polysaccharide with pure water, add an equal volume of anhydrous ethanol to prepare a 10 mg / mL sample stock solution, and then dilute it into different concentrations of sample test solutions ranging from 0.05 mg / mL to 5 mg / mL. Use anhydrous ethanol to prepare a 0.2 mM solution of DPPH powder. Take ascorbic acid (Vc) as the positive control, add 0.1 mL of the sample to be tested and 0.1 mL of DPPH solution to the sample group (A1), add 0.1 mL of the sample to be tested and anhydrous ethanol to the background group (A2), and add 0.1 mL of 50% ethanol and 0.1 mL of DPPH solution to the blank group (A0). After the solution is added to a 96-well plate, keep it away from light for 30 minutes, measure the absorbance at 517 nm, and calculate the DPPH free radical scavenging rate R according to the formula R% = (1-(A1-A2) / A0) × 100%. Figure 6 As shown in the figure, as the concentration of BCPR increases, the DPPH free radical scavenging rate also increases. When the concentration reaches 5.0 mg / mL, the scavenging capacity of ZJP-1 is 78.67%.

[0084] 7) ABTS free radical scavenging ability

[0085] Mix equal volumes of 7mM ABTS solution and 2.45mM K2S2O8 solution, and react in the dark for 12-16 hours to form an ABTS stock solution. Dilute it with anhydrous ethanol before use to an absorbance of 0.70±0.02 at 734nm. Take ascorbic acid (Vc) as the positive control. In a 96-well plate, add 10μL of the sample to be tested and 190μL of ABTS solution to the sample group (A1), add 10μL of the sample to be tested and 190μL of anhydrous ethanol to the background group (A2), and add 10μL of 50% ethanol and 190μL of ABTS solution to the blank group (A0). React in the dark for 10 minutes, measure the absorbance at 734nm, and calculate the ABTS free radical scavenging rate R according to the formula R%=(1-(A1-A2) / A0)×100%. Figure 5 It can be seen that when the concentration reaches 5 mg / mL, the free radical scavenging ability of ZJP-1 is 53.35%.

[0086] 8) Hydroxyl radical scavenging ability

[0087] Prepare 9.0mM FeSO4, 9.0mM salicylic acid and 8.8mM H2O2 solutions. Take ascorbic acid (Vc) as the positive control. Sample preparation A0 is 50μL of FeSO4 solution, 50μL of salicylic acid solution, and 50μL of H2O2 solution; A1 is 50μL of FeSO4 solution, 50μL of salicylic acid solution, 50μL of H2O2 solution, and 50μL of sample solution; A2 is 50μL of FeSO4 solution, 50μL of salicylic acid solution, 50μL of sample solution, and 50μL of purified water. After 10 minutes of reaction, measure the absorbance at 510nm, and calculate the hydroxyl radical scavenging rate R according to the formula R%=(1-(A1-A2) / A0)×100%. The results are as follows: Figure 5 As shown, ZJP-1 has a good scavenging ability in the range of 2.0-5.0 mg / mL.

[0088] 9) Protective effect of jujube polysaccharide (ZJP-1) on H2O2-induced oxidative damage in SH-SY5Y cells

[0089] (1) MTT assay for the effect of polysaccharides on cell viability

[0090] Take out the frozen SH-SY5Y human neuroblastoma cells from the -80℃ refrigerator, quickly put them into a water bath, and take them out after they are completely thawed. Transfer the cells into a 5mL EP tube and centrifuge at 1000rpm for 5 minutes. Aspirate the cell freezing solution, add 1mL of culture solution and mix gently, transfer to a culture bottle, and culture in a 37℃, 5% CO2 incubator. Inoculate the cultured cells in a 96-well plate until the cells adhere to the wall. After culturing the cells for 24 hours, aspirate the culture solution, add 100μL of different concentrations of jujube polysaccharide sample solutions (31.25, 62.5, 125, 250, 500, 1000μg / mL) to the drug-added group, and add 100μL of culture solution to the blank group. After culturing for 48 hours, add 20μL MTT solution and continue to place at 37℃ for 4 hours. After aspirating the liquid in the well, add 150μL DMSO, shake for 10 minutes, and measure the absorbance at 490nm. According to the formula, cell survival rate (%) = (OD 样品 / OD 空白 )×100%to calculate the cell survival rate. After SH-SY5Y cells were treated with different concentrations of ZJP-1 polysaccharide for 48 hours, the cell viability was not affected.

[0091] (2) Establishment of cell oxidative damage model

[0092] After 24 hours of cell culture, the 96-well plate was taken out, the old culture medium was aspirated and 50 μL of new culture medium was added for 24 hours, and 50 μL of different concentrations of H2O2 (250-650 μM) were added for 4 hours. MTT was added to detect cell viability. Different concentrations of H2O2 caused damage to SH-SY5Y cells, but at a concentration of 500 μM, the cell survival rate was stable at about 60%. Therefore, 500 μM H2O2 was selected to establish the damage model and used in subsequent experiments.

[0093] (3) Protective effect of jujube polysaccharides on H2O2-induced oxidative damage in SH-SY5Y cells

[0094] After 24 hours of cell culture, the old culture medium in the 96-well plate was removed, and 50 μL of the jujube polysaccharide sample solution prepared with culture medium was added to the drug group, and 50 μL of culture medium was added to the blank group and the model group. After 24 hours of culture, 50 μL of H2O2 was added to the drug group and the model group, and 50 μL of culture medium was added to the blank group for further 4 hours. MTT was added to detect cell viability. The oxidative damage model was established using the above selected H2O2 concentration. The results are shown in Figure 7As shown. Under the action of 500μM H2O2, the survival rate of the model group was 58.98%, while the survival rates of SH-SY5Y cells pretreated with different concentrations of jujube polysaccharides increased. In the range of 62.5-250μg / mL, the survival rate of SH-SY5Y cells reached 80.08% at a concentration of 250μg / mL of ZJP-1, indicating that jujube polysaccharide ZJP-1 has a certain protective effect on H2O2-induced oxidative damage of SH-SY5Y cells.

[0095] 10) Effects of jujube polysaccharide (ZJP-1) on oxidative stress indicators

[0096] SH-SY5Y cells were used as an in vitro model to detect the effects of ZJP-1 on oxidative stress indicators GSH-Px, MDA, SOD, and T-AOC. Figure 7 As shown in the figure, compared with the H2O2 group (0.067mmol / g), 125μg / mL and 250μg / mL ZJP-1 pretreatment significantly increased the total antioxidant capacity of SH-SY5Y cells, which were 0.011mmol / g and 0.033mmol / g, respectively. Superoxide dismutase (SOD) can catalyze the dismutation reaction of superoxide anions to generate H2O2 and O2, and is an important antioxidant enzyme in the body. Figure 7 As shown in the figure, compared with the control group (37.39U / mg), the SOD activity of the model group (21.15U / mg) was significantly reduced. After pretreatment with different concentrations of ZJP-1, the SOD activity in SH-SY5Y cells increased to 27.51U / mg, 29.84U / mg and 33.59U / mg, respectively, indicating that ZJP-1 can restore the decrease in SOD activity caused by H2O2 in cells, and under 250μg / mL ZJP-1 pretreatment, the SOD activity in cells was close to that of the control group. For GSH-Px, the activity in cells pretreated with ZJP-1 was also significantly restored, and 250μg / mL of ZJP-1 restored to 18.69mU / mg, which was dose-dependent. In addition, the MDA content reflects the degree of cellular oxidative damage. ZJP-1 can effectively reduce the MDA content induced by H2O2, especially at a concentration of 250μg / mL, the MDA content dropped to 7.69μmol / mg, which was lower than the control group level, showing its antioxidant potential.

Claims

1. A method for preparing jujube polysaccharide with antioxidant and neuron protective activity, characterized in that: The method comprises the following preparation process: (1) taking dried jujube fruits, sequentially performing reflux extraction with petroleum ether to remove fat, performing reflux extraction with ethanol to remove small molecules, adding distilled water to condense and reflux extract 2-6 times, and combining the filtrates; after the extract is concentrated, adding 5 times the volume of 95% ethanol, precipitating at 4° C. for 12 hours, and drying at 65° C. to obtain a crude polysaccharide extract; (2) Enzymatic hydrolysis combined with Sevage method to remove protein: add 1 / 4 volume of Sevage reagent (dichloromethane: n-butanol = 4:1) and react for 30 minutes, then centrifuge and collect the supernatant. Repeat this operation three times. The lower layer precipitates as denatured protein colloid. Collect the upper water layer and dry it to obtain deproteinized crude polysaccharide. Weigh 1 mg of deproteinized crude polysaccharide to determine the protein content. Repeat this operation until the determined protein content is stable. (3) decolorizing the deproteinized polysaccharide using AB-8 macroporous resin to obtain a depigmented polysaccharide; (4) The deproteinized and depigmented polysaccharide was further purified by DEAE-52 cellulose column chromatography and Sephadex G-200 column chromatography, and then dialyzed and dried to obtain a new pure jujube polysaccharide ZJP-1; the total sugar content was 95.33±2.02%, the molecular weight was 9.04 kDa, and it was composed of arabinose, rhamnose, galactose, glucose and galacturonic acid, with the mass percentages being 4.30%, 12.39%, 5.84%, 0.91% and 76.55%, respectively.

2. The method for preparing jujube polysaccharide with antioxidant and neuron-protective activity according to claim 1, characterized in that: The prepared ZJP-1 has a main chain structure of: →4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap-(1→4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-(1→3)-α-L-Araf-(1→3)-β-L-Rhap-(1→4)-α-D-GalpA.

3. The method for preparing jujube polysaccharide with antioxidant and neuron-protective activity according to claim 1, characterized in that: The prepared uniform jujube fruit polysaccharide was evaluated for its in vitro antioxidant activity by scavenging DPPH, ABTS and hydroxyl free radicals in vitro; and by detecting the levels of T-AOC, SOD, GSH-Px and MDA in model cells, ZJP-1 inhibited the generation of ROS by increasing the activity of endogenous antioxidant enzymes.

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