A method for preparing jujube polysaccharide with uric acid-lowering effect and its application

Abstract

This invention discloses a method for preparing jujube polysaccharides with uric acid-lowering effects and their applications. The invention employs a method for preparing jujube polysaccharides using a combination of fractional alcohol precipitation and single-step column chromatography purification. This method first removes small molecule impurities and oligosaccharides through fractional alcohol precipitation, enriching polysaccharides within a specific molecular weight range. Then, it undergoes one-step DEAE column chromatography purification, thus obtaining high-purity, highly uniform jujube polysaccharides with simplified steps and low cost. The obtained polysaccharides have well-defined structural characteristics, belonging to the pectin polysaccharide family, and are rich in arabinose, galactose, and rhamnose. They possess typical rhamnose-galacturonic acid polysaccharide domains, providing a clear material basis for their functional research in regulating uric acid metabolism and improving hyperuricemia. In vitro experiments inhibiting xanthine oxidase activity and experiments in a hyperuricemic animal model verified that the jujube polysaccharides with specific structures extracted by this method have significant uric acid-lowering effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing and applying jujube polysaccharide with uric acid-lowering effects. Background Technology

[0002] Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorders, characterized by elevated serum uric acid levels. If not addressed promptly and effectively, it can not only progress to gout but also induce or exacerbate endotoxemia, chronic low-grade inflammation, gastrointestinal barrier dysfunction, and kidney damage, seriously threatening human health. In recent years, increasing research has confirmed that increasing the proportion of plant-based foods in the diet helps regulate uric acid metabolism disorders and reduce the risk of hyperuricemia and its related complications. However, polysaccharides from different plant sources exhibit significant differences in their core physiological activities. To date, no research has demonstrated whether jujube polysaccharides can improve hyperuricemia or specifically...

[0003] In the preparation of jujube polysaccharides, existing extraction technologies mainly include hot water extraction, alkaline extraction, enzymatic extraction, and microwave / ultrasound-assisted extraction. Hot water extraction typically requires prolonged high-temperature treatment, which not only consumes a lot of energy and has low extraction efficiency, but also easily causes partial degradation of polysaccharides and the dissolution of a large number of coexisting impurities (such as proteins and pigments), increasing the difficulty of subsequent purification. Although alkaline extraction can improve the yield to some extent, the strong alkaline environment easily breaks the glycosidic bonds of polysaccharides, destroying their natural structure, and the subsequent neutralization and desalting steps are cumbersome and easily introduce salt contamination. Enzymatic extraction suffers from problems such as high cost of enzyme preparations, harsh reaction conditions, and easy enzyme inactivation, making it difficult to balance extraction efficiency and process economy. Although microwave and ultrasound-assisted methods can enhance the extraction process through physical effects, they are mostly used as auxiliary pretreatment methods and still need to be combined with other methods. When scaled up to large-scale production, they have inherent defects such as large equipment investment and poor process reproducibility. More importantly, the crude jujube polysaccharides obtained by the above-mentioned existing technologies generally have prominent problems such as wide molecular weight distribution, low purity, and unclear structure. To obtain homogeneous and high-purity polysaccharides, repeated column chromatography (such as the combination of ion exchange chromatography and gel filtration chromatography) is often required, resulting in an extremely cumbersome operation process, long time consumption, high consumption of organic reagents, low yield, and high production costs. This seriously restricts the fine structural analysis of jujube polysaccharides, the study of structure-activity relationship, and the subsequent development of functional products. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0006] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing jujube polysaccharides with uric acid-lowering effects.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing jujube polysaccharide with uric acid-lowering effect, characterized in that it includes: After the red dates are dried and pulverized, water is added for extraction. The extracts are combined and concentrated, and then precipitated with alcohol to obtain crude polysaccharide. The crude polysaccharide was defatted and deproteinized, decolorized and neutralized with alkaline solution, and purified by dialysis to obtain defatted, deproteinized and decolorized crude polysaccharide. The defatted, deproteinized, and decolorized crude polysaccharide was fractionated and precipitated with different volume fractions of ethanol. One component obtained by fractional alcohol precipitation was eluted by gradient elution on a DEAE-cellulose ion exchange chromatography column. The target eluent was collected, dialyzed, and dried to obtain jujube polysaccharide.

[0008] As a preferred embodiment of the preparation method described in this invention, the jujubes are dried, pulverized, and then extracted with water. The extraction conditions are: material-to-liquid ratio of 1:8 to 1:10, extraction temperature of 80 to 90°C, extraction time of 1 to 1.5 hours, and extraction is repeated 1 to 2 times.

[0009] As a preferred embodiment of the preparation method described in this invention, the defatting is performed using petroleum ether, with a volume ratio of crude polysaccharide to petroleum ether of 1:1 to 1:1.5, and the defatting is repeated 3 to 4 times; the protein removal is performed using Sevage reagent, which is a mixture of chloroform and n-butanol in a volume ratio of 4:1, and the mixture is shaken for 25 to 35 minutes, and the protein removal is repeated 5 to 6 times.

[0010] As a preferred embodiment of the preparation method described in this invention, the alkaline solution is used for decolorization and neutralization, wherein the pH of the polysaccharide solution is adjusted to 9-10 with 3-4 mol / L NaOH solution, 30%-35% NaOH solution is added and the reaction is stirred for 1-1.5 hours, and the reaction is repeated 1-3 times before neutralization.

[0011] In a preferred embodiment of the preparation method described in this invention, the molecular rejection capacity of the dialysis bag is 500–1000 Da, and the dialysis time is 48–50 h.

[0012] As a preferred embodiment of the preparation method described in this invention, the fractional alcohol precipitation is as follows: anhydrous ethanol is added to the crude polysaccharide solution to make the final volume fraction of ethanol reach 40% and the precipitate is collected. The supernatant is taken and anhydrous ethanol is added to 60% and the precipitate is collected. The supernatant is taken and anhydrous ethanol is added to 80% and the precipitate is collected. The precipitate is collected by centrifugation after standing at 4°C for 20-24 h at each concentration.

[0013] As a preferred embodiment of the preparation method described in this invention, the elution conditions of the DEAE-cellulose ion exchange chromatography column are as follows: gradient elution with 0, 0.1, 0.3, and 0.5 mol / L NaCl solutions sequentially, with an elution volume of 2 column volumes and a flow rate of 1.0–1.2 mL / min; and collection of the eluent under the 0.3 mol / L NaCl gradient.

[0014] Another objective of this invention is to overcome the shortcomings of the prior art and provide a jujube polysaccharide, characterized in that the monosaccharide molar composition of the jujube polysaccharide is: mannose 7.5%, rhamnose 11.5%, galacturonic acid 12.4%, glucose 0.9%, galactose 20.6%, and arabinose 47.1%.

[0015] As a preferred embodiment of the jujube polysaccharide of the present invention, the jujube polysaccharide has the structure of a pectin-type polysaccharide, the main chain of which contains 1,4-α-D-GalpA and 1,2-α-L-Rhap residues to form the smooth region HG and the hair region RG-I, and the side chains are mainly composed of arabinose and galactose residues.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of jujube polysaccharide in the preparation of drugs, health foods, or special medical purpose formula foods for lowering uric acid levels.

[0017] Beneficial effects of this invention: (1) The fractional alcohol precipitation combined with single column chromatography, combined with multi-step impurity removal, results in jujube polysaccharides with narrow molecular weight distribution, high purity and good uniformity. No repeated chromatographic separation is required, and the process is simple and low in cost.

[0018] (2) The gentle processing preserves the natural pectin structure, which is rich in characteristic monosaccharides. The structure is well-defined, ensuring reliable activity and providing a basis for structure-activity relationship research and quality control.

[0019] (3) It has a clear uric acid-lowering activity and can be used as a functional factor in uric acid-lowering drugs, health foods or special medical purpose formula foods. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a scanning electron microscope image of a polysaccharide sample.

[0021] Figure 2 This figure shows the comparison results of polysaccharide samples inhibiting xanthine oxidase activity in vitro.

[0022] Figure 3 This is a diagram showing the monosaccharide composition of the jujube polysaccharide obtained by this invention.

[0023] Figure 4 The image shows the NMR analysis results of the jujube polysaccharide obtained by this invention.

[0024] Figure 5 This is a schematic diagram of the repeating unit structure of the jujube polysaccharide obtained by the present invention.

[0025] Figure 6 The jujube polysaccharide prepared in this invention reduces serum uric acid levels in hyperuricemic mice and inhibits renal xanthine oxidase.

[0026] Figure 7 The present invention provides a method for regulating the expression of uric acid transporter protein in the kidneys of hyperuricemic mice using jujube polysaccharides prepared in this invention. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] The red dates used in this invention are oil dates ( Zizyphus jujubaAll dates (mill) are from Jiaxian County, Yulin City, Shaanxi Province, and all dates are supplied by Jiaxian Taiyangsheng Date Technology Co., Ltd.

[0031] The instruments used in this invention embodiment are as follows: Multiskan Go full-wavelength microplate reader (Thermal Instruments, USA); GTR216C high-speed benchtop refrigerated centrifuge (Hunan Kecheng Instrument Equipment Co., Ltd.); Vortex-Genie 2 vortex mixer (Scientific Industries, USA); FD-1A-50 freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd.); LC-2050 high-performance liquid chromatograph (Shimadzu Corporation, Japan); 8890-5977B gas chromatograph-mass spectrometer (Agilent Technologies, USA); Bruker AV NEO 600 nuclear magnetic resonance spectrometer (Bruker AG, Switzerland); Axio Imager. M2 fully automated upright fluorescence microscope (Carl Zeiss, Germany); ACQUITY gel permeation chromatography system (Waters Corporation, USA); and HITACHI TM3000 scanning electron microscope (Hitachi High Technology Corporation, Japan).

[0032] Example 1 This embodiment provides a method for preparing jujube polysaccharides with uric acid-lowering effects, specifically including the following steps: (1) Crush the red dates and sieve them. Add deionized water at a ratio of 1:9 and extract at 85℃ for 1.5h. Repeat the extraction twice with the filter residue. Collect and combine the extracts and concentrate the volume to 1 / 4 of the original volume. (2) Add anhydrous ethanol to the above extract until the final volume fraction of ethanol is 85%, let stand at 4°C for 20 h, collect the precipitate and freeze dry to obtain crude polysaccharide of jujube; (3) Mix the crude polysaccharide with petroleum ether at a volume ratio of 1:1, defatting 4 times, then shake the crude polysaccharide solution with Sevage reagent (chloroform: n-butanol = 4:1, v / v) for 35 min to remove protein, repeat 5 times to obtain polysaccharide solution; (4) Adjust the pH of the above polysaccharide solution to 9-10 with 4 mol / L NaOH, add 30% NaOH solution at room temperature and stir for 1.5 h. Repeat twice, then neutralize and concentrate. (5) The above concentrated solution was placed in a dialysis bag (1000 Da) and dialyzed for 48 hours. The dialysate was collected and freeze-dried to obtain defatted, deproteinized and decolorized crude polysaccharide of jujube. (6) Add deionized water to the above crude jujube polysaccharide and heat to dissolve. Stir and add anhydrous ethanol until the final volume fraction of ethanol is 40%. Let stand at 4°C for 24 hours. After centrifugation at 3500 r / min for 5 minutes, collect the precipitate and remove the residual ethanol to obtain jujube polysaccharide alcohol precipitation 40 fraction. (7) Dissolve the above alcohol-precipitated fraction in deionized water, centrifuge at 8000 r / min for 10 min, take the supernatant and filter it with a 45 μm filter membrane, seal with deionized water, and elute the filtrate with a DEAE-52 cellulose ion exchange chromatography column using a gradient elution of 0, 0.1, 0.3, and 0.5 mol / L NaCl for 2 column volumes at a flow rate of 1.0 mL / min. Concentrate and dialyze for 48 h, and freeze dry to obtain the purified jujube polysaccharide fraction.

[0033] Example 2 This embodiment provides a method for preparing jujube polysaccharides with uric acid-lowering effects, specifically including the following steps: (1) Crush the red dates and sieve them. Add deionized water at a ratio of 1:10 and extract at 80℃ for 1.5h. Repeat the extraction once with the filter residue. Collect and combine the extracts and concentrate the volume to 1 / 5 of the original volume. (2) Add anhydrous ethanol to the above extract until the final volume fraction of ethanol is 90%, let stand at 4°C for 24 h, collect the precipitate and freeze dry to obtain crude polysaccharide of jujube. (3) Mix the crude polysaccharide with petroleum ether at a volume ratio of 1:1.5, defatting is repeated 3 times, and then the crude polysaccharide solution is shaken thoroughly with Sevage reagent (chloroform: n-butanol = 4:1, v / v) for 25 min to remove protein. Repeat 6 times to obtain polysaccharide solution. (4) Adjust the pH of the above polysaccharide solution to 9 with 3 mol / L NaOH, add 35% NaOH solution at room temperature and stir for 1 h. Repeat 3 times, then neutralize and concentrate. (5) The above concentrated solution was placed in a dialysis bag (500 Da) and dialyzed for 50 h. The dialysate was collected and freeze-dried to obtain defatted, deproteinized and decolorized crude polysaccharide of jujube. (6) Add deionized water to the above crude jujube polysaccharide and heat to dissolve. Stir and add anhydrous ethanol until the final volume fraction of ethanol is 45%. Let stand at 4°C for 20 h. After centrifugation at 3000 r / min for 6 min, collect the precipitate and remove the residual ethanol to obtain the jujube polysaccharide alcohol precipitation component. (7) Dissolve the above alcohol-precipitated fraction in deionized water, centrifuge at 7500 r / min for 15 min, take the supernatant and filter it with a 45 μm filter membrane, use deionized water liquid seal, and elute the filtrate with a DEAE cellulose ion exchange chromatography column using a gradient elution of 0, 0.1, 0.3, and 0.5 mol / L NaCl for 2 column volumes at a flow rate of 1.2 mL / min. Concentrate and dialyze for 48 h, and freeze dry to obtain the purified jujube polysaccharide fraction.

[0034] Example 3 The difference between this embodiment and embodiment 1 is that the final volume fraction of ethanol in step (6) is adjusted to 60% and 80% respectively, while the remaining steps are the same as in embodiment 1, to obtain jujube polysaccharide component 60 and jujube polysaccharide component 80.

[0035] Example 4 This embodiment is a comparative analysis of the crude polysaccharides of jujubes obtained in the previous embodiment, and specifically includes the following steps: (1) Monosaccharide composition analysis: Take 20 mg of jujube polysaccharide sample into a 10 mL hydrolysis tube, add 2 mL of 3 mol / L trifluoroacetic acid, and hydrolyze at 110 °C for 3 h. After cooling, centrifuge, take the supernatant, evaporate to dryness, and redissolve in 1 mL of deionized water to obtain jujube polysaccharide hydrolysate. Take 100 μL of the standard mixture or jujube polysaccharide hydrolysate, add 200 μL of 0.5 mol / L PMP and 300 μL of 0.3 mol / L NaOH, mix well, and react in a 70 °C water bath for 2 h. After the reaction, cool to room temperature and add 300 μL of 0.3 mol / L HCl solution to neutralize. Add 1 mL of chloroform to the mixed solution, vortex for 30 s, centrifuge, discard the chloroform layer, and continue extraction until excess PMP is removed. Filter through a 0.22 μm filter membrane for later use.

[0036] The chromatographic column was an Agilent 5TC-C18 column (250 mm × 4.6 mm).

[0037] The method is as follows: Mobile phase A consists of 0.45 g KH2PO4, 0.5 mL triethylamine, 100 mL acetonitrile and 900 mL deionized water (pH 6.8); Mobile phase B is pure acetonitrile, the detection wavelength is 250 nm, the injection volume is 10 μL, the flow rate is 1 mL / min, and the column oven is 35 ℃.

[0038] Gradient elution program: 0-4 min, 6% A; 4-9 min, 6-10% A; 9-60 min, 10% A; 60-65 min, 10-6% A.

[0039] Table 1 shows the monosaccharide composition and molar percentage of each component. The crude jujube polysaccharide is the crude polysaccharide obtained after water extraction and alcohol precipitation, followed by defatting, decolorization, and deproteinization; that is, the crude jujube polysaccharide before fractional alcohol precipitation. Jujube polysaccharide component 40 is the alcohol-precipitated component with a 40% (v / v) ethanol concentration. Jujube polysaccharide component 60 is the alcohol-precipitated component obtained by adding anhydrous ethanol to the supernatant after 40% (v / v) ethanol precipitation, bringing the concentration to 60% (v / v). Jujube polysaccharide component 80 is the alcohol-precipitated component obtained by adding anhydrous ethanol to the supernatant after 60% (v / v) ethanol precipitation, bringing the concentration to 80% (v / v). The jujube polysaccharide is the final polysaccharide. The crude jujube polysaccharide consists of arabinose, glucose, xylose, galacturonic acid, mannose, ribose, rhamnose, and galactose, with arabinose having the highest content at 33.9%. The monosaccharide compositions of the three fractional alcohol-precipitated jujube polysaccharide components differ, but all are mainly composed of arabinose, xylose, and glucose. Among the polysaccharides, fraction 40 of jujube polysaccharides had the highest glucose content (59.4%), fraction 60 had the highest galactose content (40.3%), and fraction 80 had the highest arabinose content (53.5%). In addition to these three main monosaccharides, fractions 40 and 60 of jujube polysaccharides also contained small amounts of rhamnose, galacturonic acid, mannose, xylose, and ribose, while fraction 80 did not contain any galacturonic acid or xylose. Jujube polysaccharides are mainly composed of arabinose, galactose, rhamnose, galacturonic acid, mannose, and trace amounts of glucose, with arabinose being the most abundant at 47.1% and glucose at only 0.9%.

[0040] Table 1. Monosaccharide composition and molar percentage of each polysaccharide component.

[0041] (2) Molecular weight analysis The mobile phase was prepared with a sample concentration of 5 mg / mL, centrifuged at 14000 rpm for 10 min, and then filtered through a 0.22 μm filter before being tested.

[0042] Analysis was performed using a high-performance size exclusion chromatography-multi-angle laser light scattering-differential refractive index detector (HPSEC-MALLS-RID) system.

[0043] The mobile phase was 0.1 mol / L sodium nitrate (containing 0.03% sodium azide).

[0044] Table 2 shows the molecular weight distribution of each component. The crude jujube polysaccharide is the crude polysaccharide obtained after water extraction and alcohol precipitation, followed by defatting, decolorization, and deproteinization; that is, the crude jujube polysaccharide before fractional alcohol precipitation. Jujube polysaccharide fraction 40 is the alcohol precipitation fraction with a volume fraction of 40% ethanol. Jujube polysaccharide fraction 60 is the alcohol precipitation fraction obtained after adding anhydrous ethanol to the supernatant following alcohol precipitation with a volume fraction of 40% ethanol, bringing the volume fraction to 60%. Jujube polysaccharide fraction 80 is the alcohol precipitation fraction obtained after adding anhydrous ethanol to the supernatant following alcohol precipitation with a volume fraction of 60% ethanol, bringing the volume fraction to 80%. The final jujube polysaccharide is shown. All the obtained crude polysaccharides exhibited multiple peaks, indicating the presence of multiple components. The main peak accounted for over 70% of the mass, indicating that this main peak is the major component, while the other peaks had lower mass percentages, presumably representing other impurities. Furthermore, the weight-average molecular weight decreased with increasing ethanol volume fraction. The jujube polysaccharide exhibited a single peak with a uniform chromatographic distribution, indicating high purity.

[0045] Table 2. Molecular weight distribution of each polysaccharide component

[0046] (3) Scanning electron microscopy analysis A suitable amount of dried jujube crude polysaccharide samples were taken, gold-plated, and then observed and photographed using a scanning electron microscope (SEM) at magnifications of 500 and 1000 times.

[0047] Scanning electron microscopy analysis images such as Figure 1 As shown, the crude polysaccharide of jujube is the crude polysaccharide obtained after water extraction and alcohol precipitation, followed by defatting, decolorization, and deproteinization; that is, the crude polysaccharide of jujube before fractional alcohol precipitation. Jujube polysaccharide fraction 40 is the alcohol precipitation fraction with a volume fraction of 40% ethanol. Jujube polysaccharide fraction 60 is the alcohol precipitation fraction obtained by adding anhydrous ethanol to the supernatant after alcohol precipitation with a volume fraction of 40% ethanol, bringing the volume fraction to 60%. Jujube polysaccharide fraction 80 is the alcohol precipitation fraction obtained by adding anhydrous ethanol to the supernatant after alcohol precipitation with a volume fraction of 60% ethanol, bringing the volume fraction to 80%. The jujube polysaccharide is the final polysaccharide. The surfaces of the crude jujube polysaccharide and jujube polysaccharide fraction 40 are rough, exhibiting scaly and irregularly plate-like structures, respectively, containing numerous large particles and honeycomb structures. Jujube polysaccharide fraction 60 also exhibits a relatively rough plate-like structure, but overall it is not as rough as the crude jujube polysaccharide and jujube polysaccharide. The surface of jujube polysaccharide fraction 80 is the smoothest, containing a small amount of porous structures. The irregular sheet-like structure combined with abundant honeycomb pores creates a high specific surface area and a good spatial network, which is beneficial for improving the dissolution rate, adsorption performance, and loading capacity of polysaccharides and bioactive substances. Jujube polysaccharides have smooth sheet-like and filamentous structures, accompanied by a small amount of honeycomb porous structure, and the overall structure is softer and looser than the unpurified state. Due to different non-covalent interactions such as hydrogen bonds and van der Waals forces, crude polysaccharide molecules tend to aggregate into large clumps, while the purified polysaccharide components have more regular molecular chains and weaker aggregation, thus exhibiting a looser and softer state.

[0048] (4) Total sugar content analysis Total sugar content was determined using the phenol-sulfuric acid method. 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 0.1 mg / mL glucose standard solution were pipetted into separate test tubes, and water was added to bring the volume to 1 mL. 1 mL of 5% (w / v) phenol solution was added to each test tube, followed immediately by 5 mL of concentrated sulfuric acid. The mixture was thoroughly mixed and allowed to stand for 10 min. The test tubes were then placed in a 30°C water bath for 20 min. The absorbance was measured at 490 nm after the reaction.

[0049] A standard curve was plotted using absorbance values ​​and corresponding glucose contents, yielding the linear regression equation: y = 0.0087x + 0.0936, R² = 0.995.

[0050] Pipette 1 mL of 250 μg / mL sample solution into a 10 mL test tube and measure the absorbance according to the steps above.

[0051] The formula for total sugar content is as follows: Polysaccharide content (%) = (m1V1 / m2V2) × f × 100.

[0052] Where m1 is the calculated glucose concentration (μg / mL); V1 is the sample volume (mL); m2 is the sample mass (g); V2 is the volume of sample solution transferred for testing (mL); and f is the correction factor of 0.9 for converting glucose to dextran.

[0053] Table 3 shows the yield and total sugar content of the polysaccharides. The crude jujube polysaccharide was obtained after water extraction and alcohol precipitation, followed by defatting, decolorization, and deproteinization; that is, the crude jujube polysaccharide before fractional alcohol precipitation. Jujube polysaccharide fraction 40 was the alcohol-precipitated fraction with 40% ethanol (v / v). Jujube polysaccharide fraction 60 was the alcohol-precipitated fraction obtained by adding anhydrous ethanol to the supernatant after 40% ethanol precipitation to a volume of 60%. Jujube polysaccharide fraction 80 was the alcohol-precipitated fraction obtained by adding anhydrous ethanol to the supernatant after 60% ethanol precipitation to a volume of 80%. The jujube polysaccharide was the final polysaccharide. The yield of crude jujube polysaccharide was approximately 4.17%, but the purity was low, only 51.65%. After fractional alcohol precipitation, the yields of jujube polysaccharide fractions 40, 60, and 80 were approximately 2.08%, 0.55%, and 0.70%, respectively, with purities of 57.86%, 67.23%, and 41.31%, respectively. Among the polysaccharides, fraction 80 of jujube polysaccharides had the lowest purity. This is because the first two alcohol precipitation processes (40% and 60%) precipitated most of the larger molecular weight polysaccharides, while smaller molecular weight components and some impurities only precipitated at the 80% alcohol precipitation concentration. Furthermore, although the purity of the crude jujube polysaccharide was relatively high, the content of components with molecular weights within the 60% alcohol precipitation range was low, resulting in a low yield for this component. Ultimately, the jujube polysaccharides obtained through the entire fractional purification process had a yield of 0.66% and a purity as high as 90.08%, achieving a good balance between yield and purity, controllable quality, a clearly defined molecular weight range, and an efficient and cost-effective purification process. Different letters (ae) in this table indicate significant differences in data (P < 0.05).

[0054] Table 3. Yield and total sugar content of each polysaccharide component.

[0055] (5) Analysis of xanthine oxidase activity inhibition rate The polysaccharide sample was thoroughly mixed with 50 μL of a 100 U / L xanthine oxidase solution and incubated at 37°C for 30 min. Then, 100 μL of xanthine solution was added to initiate the enzymatic reaction, and the absorbance value A was recorded in real time.

[0056] PBS was used as a blank control and allopurinol as a positive control during the experiment. The inhibition rate of the sample against xanthine oxidase was calculated using the following formula: Inhibition rate % = (1 - (Ai - Aj) / (A1 - A0)) × 100, where: Ai, absorbance value of the sample solution after enzymatic reaction; Aj, absorbance value of the sample solution without enzyme in the system; A0, absorbance value of the enzyme after reaction with the substrate without sample solution in the system; A1, absorbance value of the system without sample and without enzyme in the system.

[0057] Figure 2This image shows the comparison results of the in vitro inhibition of xanthine oxidase activity by polysaccharide samples. A represents the comparison results of each crude polysaccharide sample, and B represents the comparison of jujube polysaccharide before and after purification. The crude jujube polysaccharide is the crude polysaccharide obtained after water extraction and alcohol precipitation, followed by defatting, decolorization, and deproteinization; that is, the crude jujube polysaccharide before fractional alcohol precipitation. Jujube polysaccharide fraction 40 is the alcohol precipitation fraction with a volume fraction of 40% ethanol. Jujube polysaccharide fraction 60 is the alcohol precipitation fraction obtained by adding anhydrous ethanol to the supernatant after alcohol precipitation with a volume fraction of 40% ethanol, bringing the volume fraction to 60%. Jujube polysaccharide fraction 80 is the alcohol precipitation fraction obtained by adding anhydrous ethanol to the supernatant after alcohol precipitation with a volume fraction of 60% ethanol, bringing the volume fraction to 80%. Jujube polysaccharide is the final polysaccharide, and allopurinol is the positive control. Figure 1 It was found that among the crude polysaccharides, jujube polysaccharide fractions 40 and 60 showed the best inhibitory effect on xanthine oxidase activity, with little difference between them, both significantly better than crude jujube polysaccharide and jujube polysaccharide fraction 80. Further purified jujube polysaccharide showed the best inhibitory effect on xanthine oxidase activity, similar to that of the positive control drug allopurinol. In this figure, data with different letter labels (ac) indicate significant differences (P < 0.05).

[0058] Example 5 This embodiment involves structural analysis of the jujube polysaccharide obtained in Example 1, specifically including the following steps: (1) Monosaccharide composition analysis: Take 20 mg of jujube polysaccharide sample into a 10 mL hydrolysis tube, add 2 mL of 3 mol / L trifluoroacetic acid, and hydrolyze at 110 °C for 3 h. After cooling, centrifuge, take the supernatant, evaporate to dryness, and redissolve in 1 mL of deionized water to obtain jujube polysaccharide hydrolysate. Take 100 μL of the standard mixture or jujube polysaccharide hydrolysate, add 200 μL of 0.5 mol / L PMP and 300 μL of 0.3 mol / L NaOH, mix well, and react in a 70 °C water bath for 2 h. After the reaction, cool to room temperature and add 300 μL of 0.3 mol / L HCl solution to neutralize. Add 1 mL of chloroform to the mixed solution, vortex for 30 s, centrifuge, discard the chloroform layer, and continue extraction until excess PMP is removed. Filter through a 0.22 μm filter membrane for later use.

[0059] The chromatographic column was an Agilent 5TC-C18 column (250 mm × 4.6 mm).

[0060] The method is as follows: Mobile phase A consists of 0.45 g KH2PO4, 0.5 mL triethylamine, 100 mL acetonitrile and 900 mL deionized water (pH 6.8); Mobile phase B is pure acetonitrile, the detection wavelength is 250 nm, the injection volume is 10 μL, the flow rate is 1 mL / min, and the column oven is 35 ℃.

[0061] Gradient elution program: 0-4 min, 6% A; 4-9 min, 6-10% A; 9-60 min, 10% A; 60-65 min, 10-6% A.

[0062] Figure 3 The image shows a high-performance liquid chromatogram (HPLC) of the monosaccharide composition of jujube polysaccharides. The vertical axis represents the absorbance measured by a UV detector, and the horizontal axis represents the elution time. Figure 2 It is known that jujube polysaccharides are mainly composed of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose. The molar percentages of monosaccharides in jujube polysaccharides were calculated based on the standard curve: mannose 7.5%, rhamnose 11.5%, galacturonic acid 12.4%, glucose 0.9%, galactose 20.6%, and arabinose 47.1%. Therefore, the jujube polysaccharides prepared in this invention have clearly defined monosaccharide components.

[0063] (2) Methylation analysis: 5 mg of jujube polysaccharide sample was dissolved in 1 mL of DMSO. Following the recommended procedure of the methylation kit, methylation reagent was added to the system, and the mixture was reacted with the polysaccharide at 30 °C for 60 min. The methylated polysaccharide was hydrolyzed with 2 mol / L TFA for 90 min and then reduced with NaBH4 for 8 h. After neutralization with glacial acetic acid, the reducing solution was reacted with acetic anhydride at 100 °C for 1 h, followed by washing with toluene 4 to 5 times to remove excess acetic anhydride.

[0064] The obtained monosaccharides were extracted four times with CH2Cl2, dried, and identified by gas chromatography-mass spectrometry using an RXI-5 SILMS column (30m×0.25mm×0.25μm).

[0065] The temperature program was as follows: initial temperature 120℃, increased to 250℃ at a rate of 3℃ / min. The injection port and detector temperatures were both 250℃, and the carrier gas was helium at a flow rate of 1 mL / min.

[0066] As shown in Table 4, based on the structural characteristics of pectin-type polysaccharides, it can be preliminarily determined that the chemical structure of jujube polysaccharides is likely composed of four residues: 1,4-D-GalpA, 1,3,4-D-GalpA, 1,2-L-Rhap, and 1,2,4-L-Rhap, forming its main chain. Other sugar residues may be present in the side chains. Based on the proportions of each residue in the main chain during methylation, the preliminary calculation yields a ratio of approximately 47.42:3.51:15.45 = 13.5:1:4.4 for the HG smooth region, type II pectin region, and type I pectin region, indicating that jujube polysaccharides possess a pectin-type structure with numerous "hairs."

[0067] Table 4. Results of methylation test of jujube polysaccharides

[0068] (3) NMR analysis: 30 mg of purified jujube polysaccharide sample was dissolved in 0.5 mL of D2O, and the sample was analyzed using a high-resolution nuclear magnetic resonance spectrometer at room temperature.

[0069] The structural characteristics of jujube polysaccharides were analyzed using one-dimensional (¹H, ¹³C) and two-dimensional (DEPT¹³⁵, COSY, HSQC, HMBC, NOESY) NMR spectroscopy. The NMR analysis of jujube polysaccharides is as follows: Figure 4 The AG images are NMR 1H, 13C, DEPT135, COSY, HSQC, HMBC and NOESY images, respectively. The polysaccharide exhibits multiple anomeric (H1) proton signals in the δ 4.3~5.9 ppm range. Combined with methylation information, it indicates that the glycosidic bonds of this polysaccharide are both α- and β-type, which is consistent with the structural characteristics of typical pectin-type polysaccharides.

[0070] In this spectrum, the anomeric carbon signals were identified as superimposed positions around δ99.13, 100.59, 104.35, and 107.39 ppm. In 13C NMR, the low-field signal in the δ170–180 ppm range indicates the presence of a carboxyl or acetyl substituent in the uronic acid. Combined with the weak signal near 2.0 ppm in the 1H NMR and the 1.98 / 175.68 ratio in the HMBC, this confirms the presence of a very small amount of acetyl substituent. The high-field weak signal in the δ16–25 ppm range indicates the presence of a methyl group at the 6-position deoxyglucose (rhamnose). The δ16.51 ppm signal peak corresponds to δ1.17 ppm in the 1H NMR, and a corresponding C / H cross signal peak at δ16.51 / 1.17 ppm appears in the HSQC NMR spectrum, indicating the presence of a certain amount of rhamnose residues.

[0071] Based on the anomeric proton signal from 1H NMR, the 1H-13C crossover signal in the HSQC two-dimensional spectrum was used in conjunction with relevant data to confirm the location of each residue. The methylation and NMR results yielded the structural repeating units of jujube polysaccharide, such as... Figure 5 It can be seen that its overall structure is dominated by the HG structural unit of →4-α-D-GalpA-(1→4)-α-D-GalpA→, and secondarily by the RG hair region of →4-α-D-GalpA-(1→2)-α-L-Rhap→, with arabinose and galactose as the main side chains. Therefore, the jujube polysaccharide prepared in this invention has clearly defined repeating structural units.

[0072] Comparative Example 1 The difference between this comparative example and Example 1 is that step (6) is omitted, and the crude polysaccharide after defatting, deproteinizing and decolorizing is directly subjected to DEAE column chromatography to obtain jujube polysaccharide. The obtained polysaccharide was found to have a wide molecular weight distribution and poor activity.

[0073] Example 6 This embodiment illustrates the application of the jujube polysaccharide obtained in Example 1 in lowering uric acid, specifically including the following steps: Forty healthy male C57BL / 6J mice (5 weeks old) were randomly divided into five groups of eight each after a one-week acclimatization period: a control group, a hyperuricemia group, a low-dose jujube polysaccharide group, a medium-dose jujube polysaccharide group, and a high-dose jujube polysaccharide group. Potassium oxonate is a uricase inhibitor that can block hepatic uric acid metabolism; hypoxanthine is an intermediate product of purine metabolism, which is ultimately converted into uric acid. The combined intake of both can induce hyperuricemia.

[0074] The control group was administered 0.5% CMC-Na daily by gavage, while the hyperuricemia group, low-dose jujube polysaccharide group, medium-dose jujube polysaccharide group, and high-dose jujube polysaccharide group were administered a suspension of potassium oxonate (250 mg / kg) and hypoxanthine (250 mg / kg) daily by gavage. One hour later, mice in the low-dose, medium-dose, and high-dose jujube polysaccharide groups were administered jujube polysaccharide solution at doses of 50 mg / kg·bw, 100 mg / kg·bw, and 200 mg / kg·bw, respectively, by gavage. The control group and the hyperuricemia group were given the same volume of 0.5% CMC-Na. During the 8-week treatment period, all mice had free access to standard rodent feed. After the last gavage administration, the mice were fasted for 12 hours. The mice were then euthanized under anesthesia, and blood, kidney, and colon contents were collected.

[0075] Mouse blood was centrifuged at 3000×g for 15 min, and the supernatant serum was collected. The serum uric acid level was determined using a biochemical analyzer (BTS-3700, Biosystems Co., Ltd., Madrid, Spain); kidney tissue was homogenized in pre-cooled physiological saline, centrifuged, and the supernatant was collected. The xanthine oxidase activity was determined using a commercial colorimetric kit.

[0076] Mouse kidney tissue was fixed with paraformaldehyde solution, embedded in paraffin, and then sectioned. The tissue sections were first incubated overnight with primary antibodies ABCG2, GLUT9, and URAT1 (Wuhan Sanying), then washed with PBS, and then incubated with the corresponding fluorescent secondary antibody (Wuhan Sanying) for 2 hours in the dark. The sections were then mounted with mounting medium containing DAPI, and finally photographed using an upright fluorescence microscope (Zeiss, Germany), and the fluorescence intensity was quantified.

[0077] Figure 6Jujube polysaccharide was used to inhibit uric acid metabolism disorder in mice with hyperuricemia. In this study, A represents the serum uric acid level of mice in each group, and B represents the renal xanthine oxidase activity of mice in each group.

[0078] The control group was administered 0.5% CMC-Na by gavage. The hyperuricemia group, the low-dose jujube polysaccharide group, the medium-dose jujube polysaccharide group, and the high-dose jujube polysaccharide group were administered a suspension of potassium oxonate (250 mg / kg) and hypoxanthine (250 mg / kg) by gavage. One hour later, the mice in the low-dose, medium-dose, and high-dose jujube polysaccharide groups were administered jujube polysaccharide solution at doses of 50 mg / kg·bw, 100 mg / kg·bw, and 200 mg / kg·bw, respectively. The control group and the hyperuricemia group were given the same volume of 0.5% CMC-Na. In this figure, data with different letters (ac) indicate significant differences (P < 0.05).

[0079] from Figure 6 It was found that long-term intake of potassium oxonate and hypoxanthine in mice led to a significant increase in serum uric acid and renal xanthine oxidase activity. However, gavage administration of low, medium, and high doses of jujube polysaccharide to hyperuricemic mice reduced serum uric acid levels and renal xanthine oxidase activity. In particular, high-dose jujube polysaccharide treatment effectively reduced serum uric acid levels and renal xanthine oxidase activity in hyperuricemic mice. These results indicate that jujube polysaccharide can effectively inhibit uric acid metabolism disorders induced by potassium oxonate and hypoxanthine, and has a uric acid-lowering effect.

[0080] Figure 7 This study investigated the regulatory effect of jujube polysaccharides on uric acid transport proteins. In the images, A shows the immunofluorescence intensity of GLUT9, a uric acid reabsorption protein in the kidneys of mice in each group; B shows the immunofluorescence intensity of URAT1, a uric acid reabsorption protein in the kidneys of mice in each group; and C shows the immunofluorescence intensity of ABCG2, a uric acid transport protein in the kidneys of mice in each group.

[0081] The control group was administered 0.5% CMC-Na by gavage. The hyperuricemia group, the low-dose jujube polysaccharide group, the medium-dose jujube polysaccharide group, and the high-dose jujube polysaccharide group were administered a suspension of potassium oxonate (250 mg / kg) and hypoxanthine (250 mg / kg) by gavage. One hour later, the mice in the low-dose, medium-dose, and high-dose jujube polysaccharide groups were administered jujube polysaccharide solution at doses of 50 mg / kg·bw, 100 mg / kg·bw, and 200 mg / kg·bw, respectively. The control group and the hyperuricemia group were given the same volume of 0.5% CMC-Na. In this figure, data with different letters (ac) indicate significant differences (P < 0.05).

[0082] from Figure 7It was found that long-term intake of potassium oxonate and hypoxanthine in mice significantly increased the expression of renal reabsorption proteins GLUT9 and URAT1, and significantly decreased the expression of uric acid transporter ABCG2, hindering normal uric acid excretion, leading to uric acid accumulation and elevated serum uric acid levels. However, gavage administration of low, medium, and high doses of jujube polysaccharide to hyperuricemic mice reduced the expression of renal reabsorption proteins GLUT9 and URAT1, and increased the expression of uric acid transporter ABCG2. In particular, high-dose jujube polysaccharide treatment effectively reduced the expression of renal reabsorption proteins GLUT9 and URAT1 and significantly increased the expression of uric acid transporter ABCG2. These results indicate that jujube polysaccharide can effectively inhibit the abnormal expression of uric acid transporters induced by potassium oxonate and hypoxanthine, promote uric acid excretion, and thus achieve uric acid-lowering function.

[0083] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing jujube polysaccharide with uric acid-lowering effect, characterized in that: include, After drying and pulverizing red dates, water was added for extraction. The extracts were combined and concentrated, and crude polysaccharide was obtained by alcohol precipitation. The crude polysaccharide was defatted and deproteinized, decolorized and neutralized with alkaline solution, and purified by dialysis to obtain defatted, deproteinized and decolorized crude polysaccharide. The defatted, deproteinized, and decolorized crude polysaccharide was fractionated and precipitated with different volume fractions of ethanol. The fraction obtained by fractional alcohol precipitation was eluted by gradient elution on a DEAE-cellulose ion exchange chromatography column. The target eluent was collected, dialyzed, and dried to obtain jujube polysaccharide.

2. The preparation method according to claim 1, characterized in that: The jujubes are dried, pulverized, and then extracted with water. The extraction conditions are: material-to-liquid ratio of 1:8 to 1:10, extraction temperature of 80 to 90°C, extraction time of 1 to 1.5 hours, and repeated extraction 1 to 2 times.

3. The preparation method according to claim 1, characterized in that: The defatting was performed using petroleum ether, with a volume ratio of crude polysaccharide to petroleum ether of 1:1 to 1:1.5, and the defatting was repeated 3 to 4 times. The protein was removed using Sevage reagent, which is a mixture of chloroform and n-butanol in a volume ratio of 4:

1. The mixture was shaken for 25 to 35 minutes and the protein was removed 5 to 6 times.

4. The preparation method according to claim 1, characterized in that: The alkaline solution is used for decolorization and neutralization. The pH of the polysaccharide solution is adjusted to 9-10 with 3-4 mol / L NaOH solution, and 30%-35% NaOH solution is added and stirred for 1-1.5 hours. This process is repeated 1-3 times before neutralization.

5. The preparation method according to claim 1, characterized in that: The molecular rejection capacity of the dialysis bag is 500–1000 Da, and the dialysis time is 48–50 h.

6. The preparation method according to claim 1, characterized in that: The fractional alcohol precipitation is as follows: anhydrous ethanol is added to the crude polysaccharide solution to make the final volume fraction of ethanol reach 40% and the precipitate is collected. The supernatant is taken and anhydrous ethanol is added to 60% and the precipitate is collected. The supernatant is taken and anhydrous ethanol is added to 80% and the precipitate is collected. The precipitate is collected by centrifugation after standing at 4°C for 20-24 h at each concentration.

7. The preparation method according to claim 1, characterized in that: The elution conditions for the DEAE-cellulose ion exchange chromatography column are as follows: sequential elution with NaCl solutions of 0, 0.1, 0.3, and 0.5 mol / L, with an elution volume of 2 column volumes and a flow rate of 1.0–1.2 mL / min; and collection of the eluent under the 0.3 mol / L NaCl gradient.

8. The jujube polysaccharide prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The monosaccharide molar composition of the jujube polysaccharide is as follows: mannose 7.5%, rhamnose 11.5%, galacturonic acid 12.4%, glucose 0.9%, galactose 20.6%, and arabinose 47.1%.

9. The jujube polysaccharide as described in claim 8, characterized in that: The structure of the jujube polysaccharide is a pectin-type polysaccharide. The main chain contains 1,4-α-D-GalpA and 1,2-α-L-Rhap residues, forming the smooth region HG and the hair region RG-I. The side chains are mainly composed of arabinose and galactose residues.

10. The use of the jujube polysaccharide as described in claim 8 or 9 in the preparation of a drug for lowering uric acid levels.

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