Degradation polysaccharide of red jujube, degradation polysaccharide component, and preparation method and application thereof

Abstract

The application belongs to the technical field of food deep processing, and particularly relates to a red date degraded polysaccharide, a degraded polysaccharide component, and a preparation method and application thereof. The application provides a preparation method of the red date degraded polysaccharide, in which the red date polysaccharide is degraded by using an ultrasonic wave assisted method combined with H2O2-Vc, and the red date degraded polysaccharide is separated and purified by using a DEAE-Sepharose Fast Flow gel column and a Sephacryl S-100 chromatographic column. The red date degraded polysaccharide and the polysaccharide component have the following characteristics: after the degradation treatment, the fragmentation degree of the polysaccharide surface is deepened, the degradation does not change the basic structure of the polysaccharide, and the apparent morphological characteristics change to a certain extent; the degradation treatment can improve the antioxidant capacity of the polysaccharide and is inversely proportional to the polysaccharide molecular weight.

Description

Technical Field

[0001] This invention belongs to the field of food deep processing technology, specifically relating to a jujube degradable polysaccharide, degradable polysaccharide components, their preparation method and application. Background Technology

[0002] Jujube (Ziziphus jujube Mill.) is the fruit of a plant belonging to the genus Ziziphus in the family Rhamnaceae. It is widely distributed in subtropical and tropical regions, and in my country, it is mainly found in provinces along the Yellow River and the Aksu region of Xinjiang. my country is the world's largest producer and sole exporter of jujubes, accounting for over 90% of global production; therefore, my country possesses unique advantages for the development and in-depth research of jujubes. Jujubes are rich in various bioactive components, such as polyphenols, flavonoids, vitamins, polysaccharides, and cyclic nucleotides. Studies have shown that polysaccharides are one of the main functionally active components of jujubes, exhibiting various biological activities including antioxidant, immunomodulatory, antitumor, hypoglycemic, hypolipidemic, and intestinal flora regulation. This indicates that jujube polysaccharides have high application value in the functional food field. Currently, research on jujube polysaccharides mainly focuses on structural characterization and activity studies. Due to the large molecular weight, high viscosity, and poor solubility of natural jujube polysaccharides, their product development is limited. Therefore, finding effective methods to degrade jujube polysaccharides is fundamental to studying the degradation of jujube polysaccharide structures and developing new products.

[0003] Currently, polysaccharide degradation methods can be categorized into physical degradation, chemical degradation, biodegradation, and combined degradation methods. Chemical degradation is simple to operate but may cause environmental pollution; physical degradation can reduce environmental pollution but suffers from incomplete degradation and high equipment requirements; biodegradation has mild reaction conditions but high production costs and is difficult to screen for enzymes and microorganisms. Summary of the Invention

[0004] This invention utilizes ultrasound-assisted H2O2-Vc co-degradation of jujube polysaccharides. The degradation conditions are optimized using response surface methodology with DPPH free radical scavenging rate as an indicator, in order to prepare polysaccharides with high antioxidant activity, thereby laying a theoretical foundation for the development and utilization of jujube polysaccharides.

[0005] This invention employs an ultrasound-assisted H2O2-Vc combined method to degrade jujube polysaccharides. Degradation conditions were optimized, and the degradation products were separated and purified using DEAE Sepharose Fast Flow and Sephacryl S-100 chromatography columns. The purified polysaccharide components were then subjected to structural analysis and in vitro antioxidant activity studies.

[0006] 1. Optimization of degradation conditions for jujube polysaccharides

[0007] The traditional water extraction and alcohol precipitation method was used to extract ziziphus jujuba polysaccharide (ZMP) from red dates, and then the ultrasonic-assisted H2O2-Vc combined method was used to degrade the ziziphus jujuba polysaccharide. Taking the DPPH free radical scavenging rate as the index, according to the single factor experiment and response surface optimization, the optimal degradation conditions were obtained as follows: ultrasonic time was 65 min, the concentration of degradation agent H2O2-Vc was 10 mmol / L, the degradation temperature was 51 °C, the yield of degraded polysaccharide (DZMP) was 72.16%, and the DPPH free radical scavenging rate of DZMP under the optimal conditions was 81.76%. After degradation, the total sugar content increased to 81.78%, and the protein content decreased slightly.

[0008] 2. Isolation, purification and structural analysis of degraded polysaccharides from red dates

[0009] DEAE Sepharose Fast Flow chromatography column and Sephacryl S-100 gel column were used to isolate and purify DZMP, and a neutral polysaccharide component (DPZMP1) and three acidic polysaccharide components (DPZMP2, DPZMP3 and DPZMP4) were obtained. DPZMP3 was selected for structural analysis according to the purity of polysaccharide components. Monosaccharide composition analysis showed that DPZMP3 was mainly composed of rhamnose, arabinose, galactose and galacturonic acid, and its molar ratio was 1:1.4875:1.6:7.675, and the average molecular weight was 34.275 kDa; combined methylation and NMR analysis showed that DPZMP3 belonged to HG-type pectin; infrared spectroscopy analysis showed that DPZMP3 had typical characteristic absorption peaks of carbohydrates and contained characteristic absorption peaks of uronic acid, further proving that DPZMP3 was an acidic sugar. The absorption peak at 840 cm -1 corresponded to the α configuration, which was consistent with the NMR results; ultraviolet spectroscopy analysis showed that the DPZMP3 component did not contain proteins and nucleic acids, which was consistent with the chemical test results; SEM results showed that the degree of fragmentation of the polysaccharide surface increased after degradation treatment. Polysaccharide structure analysis showed that degradation did not change the basic structure of the polysaccharide, but the apparent morphological characteristics changed to a certain extent.

[0010] 3. Study on in vitro antioxidant activity of degraded polysaccharide components from red dates

[0011] The results of studying the in vitro antioxidant activities of ziziphus jujuba polysaccharide, degraded ziziphus jujuba polysaccharide and its purified components showed that the DPPH free radical scavenging ability, hydroxyl free radical scavenging ability, superoxide anion free radical scavenging ability, reducing power and total antioxidant ability of ZMP, DZMP and DPZMP3 were dose-dependent. After degradation, the antioxidant ability of ziziphus jujuba polysaccharide was significantly improved, and the antioxidant abilities of the three were in the order of ZMP < DZMP < DPZMP3. Especially for the DPPH free radical scavenging activity, when the concentration was 2.0 mg / mL, IC 50The value decreased from 0.4432 mg / mL to 0.2174 mg / mL. Therefore, degradation treatment can improve the antioxidant capacity of polysaccharides in a way that is inversely proportional to the molecular weight of the polysaccharides. Attached Figure Description

[0012] Figure 1 The effect of ultrasound time on the scavenging of DPPH free radicals by DZMP;

[0013] Figure 2 The effect of H2O2-Vc concentration on the scavenging of DPPH free radicals by DZMP;

[0014] Figure 3 The effect of degradation temperature on the DPPH radical scavenging effect of DZMP;

[0015] Figure 4 Elution curves for polysaccharide degradation in jujubes;

[0016] Figure 5 Elution curve of Sephacryl S-100;

[0017] Figure 6 Chromatogram of HPGPC for the detection of degraded polysaccharide components in jujube;

[0018] Figure 7 The ion chromatograms are for a mixture of monosaccharide standards and the degradation of polysaccharide components from jujube. The monosaccharide mixture standards are: fucose, galactosamine hydrochloride (GalN), rhamnose (Rha), arabinose (Ara), glucosamine hydrochloride (GlcN), galactose (Gal), glucose (Glc), N-acetyl-D-glucosamine (GlcNAc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (GalA), guluronic acid (GulA), glucuronic acid (GlcA), and mannouronic acid (ManA).

[0019] Figure 8 NMR spectrum of DPZMP3 polysaccharide fraction (A: 1 H-NMR spectrum, B: 13 C-NMR spectrum, C:HSQC spectrum);

[0020] Figure 9 The ultraviolet absorption spectrum of the degraded polysaccharide components of jujube;

[0021] Figure 10 The infrared absorption spectrum of DPZMP3;

[0022] Figure 11 This is a scanning electron microscope (SEM) image of DPZMP3.

[0023] Figure 12The DPPH free radical scavenging activity of ZMP, DZMP, and DPZMP3;

[0024] Figure 13 The hydroxyl radical scavenging activities of ZMP, DZMP, and DPZMP3;

[0025] Figure 14 The ability to restore ZMP, DZMP, and DPZMP3;

[0026] Figure 15 The superoxide anion free radical scavenging activity of ZMP, DZMP, and DPZMP3;

[0027] Figure 16 The total antioxidant capacity of ZMP, DZMP, and DPZMP3. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments and accompanying drawings. Unless otherwise specified, the reagents and materials used in the embodiments of the present invention are all purchased from conventional commercial stores in the art, and are of analytical purity or even spectral grade. Furthermore, when performing statistical analysis of experimental data, each experiment was repeated three times, and the results are expressed as mean ± standard deviation; analysis of variance and data processing were performed using Design Expert and SPSS software; and graphs were generated using Origin 2018 software.

[0029] Example 1

[0030] 1.1 Preparation of Jujube Polysaccharides

[0031] Fresh red dates were pitted, dried, and pulverized to obtain date powder. Several times the volume of ethanol was added and refluxed to defatt the powder, removing fat-soluble substances. Excess ethanol was evaporated at low temperature to obtain defatted date powder. Deionized water was added at a ratio of 1:20 (m / V), and the mixture was extracted at 80℃ for 1 hour. The supernatant was collected by centrifugation, and the residue was re-extracted with water. The concentrated extracts were combined, and four times the volume of anhydrous ethanol solution was added. The mixture was allowed to stand overnight at 4℃, and the precipitate was collected and reconstituted with water to obtain a polysaccharide solution. This solution was then decolorized, deproteinized, concentrated, and freeze-dried to obtain jujube polysaccharide (ZMP).

[0032] 1.2 Single-factor experimental design

[0033] Using DPPH free radical scavenging rate as an indicator, a single-factor experiment was conducted. The scavenging rate of DPPH free radicals by degradation products obtained under different ultrasonic times, H2O2-Vc (1:1) concentrations, and degradation temperatures was studied. The determination method of DPPH free radical scavenging rate is as follows. 50 mL of 4 mg / mL polysaccharide solution was degraded under different conditions, and then concentrated, dialyzed, precipitated with alcohol, and freeze-dried to obtain degraded polysaccharides under different conditions. 2 mL of 1 mg / mL degraded polysaccharide solution under different conditions was mixed thoroughly with 2 mL of DPPH-ethanol (0.1 mmol / L) solution, reacted at 37℃ in the dark for 30 min, and the absorbance of the solution was measured at a wavelength of 517 nm to calculate the scavenging rate of DPPH free radicals by the degradation products. The DPPH free radical scavenging rate was calculated according to formula (1):

[0034]

[0035] In the formula: A1 is the absorbance of the sample group, A2 is the absorbance of the blank group (anhydrous ethanol instead of DPPH), and A0 is the absorbance of the control group (deionized water instead of the sample).

[0036] Effect of sonication time: 4 mg / mL polysaccharide solution was used, and the effects of different sonication times (20, 40, 60, 80 and 100 min) on the DPPH free radical scavenging rate were investigated under the conditions of H2O2-Vc concentration of 15 mmol / L and degradation temperature of 50℃.

[0037] Effect of H2O2-Vc concentration: A 4 mg / mL polysaccharide solution was used, and the effects of different H2O2-Vc concentrations (5, 10, 15, 20, 25 mmol / L) on the DPPH free radical scavenging rate were investigated under the conditions of ultrasonic time of 60 min and degradation temperature of 50℃.

[0038] Effect of degradation temperature: 4 mg / mL polysaccharide solution was used, and the effects of different degradation temperatures (30, 40, 50, 60, 70 °C) on the DPPH free radical scavenging rate were investigated under the conditions of H2O2-Vc concentration of 10 mmol / L and ultrasonic time of 60 min.

[0039] 1.2.1 Effect of sonication time on DZMP scavenging of DPPH free radicals

[0040] like Figure 1 As shown, the DPPH radical scavenging rate of DZMP initially increased and then plateaued with increasing ultrasonic degradation time. This may be because the molecular weight of jujube polysaccharides reaches the defined chain length with increasing time, leading to a plateauing of the DPPH radical scavenging rate. Therefore, a degradation time of 60 min was selected for response surface methodology optimization.

[0041] 1.2.2 Effect of H2O2-Vc concentration on the scavenging of DPPH free radicals by DZMP

[0042] like Figure 2 As shown, when the H2O2-Vc concentration is between 5 and 10 mmol / L, the DPPH radical scavenging rate increases with increasing H2O2-Vc concentration. This may be because the molecular weight of the polysaccharide decreases with increasing H2O2-Vc concentration, leading to a gradual increase in active groups. When the H2O2-Vc concentration exceeds 10 mmol / L, the DPPH radical scavenging rate of DZMP gradually decreases, possibly due to excessive degradation of jujube polysaccharides, resulting in reduced polysaccharide activity. Therefore, a response surface methodology optimization experiment was conducted with an H2O2-Vc concentration of 10 mmol / L.

[0043] 1.2.3 Effect of degradation temperature on the DPPH radical scavenging effect of DZMP

[0044] like Figure 3 As shown, the DPPH radical scavenging rate of DZMP first increases and then decreases with increasing degradation temperature. This is because appropriately increasing the degradation temperature accelerates molecular motion, thus increasing the degradation rate of jujube polysaccharides; however, excessively high temperatures lead to H2O2 decomposition and a decrease in vitamin C activity, resulting in a decrease in the degradation rate. Therefore, a degradation temperature of 50℃ was selected for response surface methodology.

[0045] 1.3 Response Surface Experimental Design

[0046] Based on single-factor experiments and following the Box-Behnken experimental design principle, with ultrasonic time (A), H2O2-Vc concentration (B), and degradation temperature (C) as independent variables, and DPPH free radical scavenging rate as the response value, a response surface methodology was designed to optimize the degradation process of jujube polysaccharides. The factors and levels are shown in Table 1.

[0047] Table 1 Factors and levels in response surface methodology

[0048]

[0049] 1.3.1 Model Building and Analysis of Variance

[0050] The results of the response surface methodology experiment are shown in Table 2. Using Design Expert software, Table 2 was fitted to obtain the regression equation:

[0051] Y=82.06+1.17A+0.28B+0.57C-0.50AB-0.39AC-0.22BC-2.03A 2 -2.52B 2 -1.77C 2

[0052] The analysis of variance for the regression model is shown in Table 3. The p-value for this regression model is less than 0.0001, indicating that the model is highly significant. The lack-of-fit term p>0.05 is not significant, and the coefficient of determination is R0.05. 2 =0.9857, indicating that the model has a good fit; the corrected coefficient of determination R0 Adj 2 =0.9674, and R 2 The close proximity indicates high model accuracy, making it suitable for predicting response values. The F-value shows that ultrasonic time has the greatest impact on polysaccharide degradation, while H2O2-Vc concentration has the least. The interaction term AB and the quadratic term A... 2 B 2 C 2 The effect of DZMP on DPPH radical scavenging was extremely significant (P<0.01), the interaction term BC had a significant effect on DZMP on DPPH radical scavenging (P<0.05), and the effects of other factors were not significant.

[0053] Table 2. Response Surface Experimental Design and Results

[0054]

[0055]

[0056] Table 3. Analysis of Variance for Regression Models

[0057]

[0058]

[0059] Note: * indicates a significant difference (P<0.05); ** indicates an extremely significant difference (P<0.01).

[0060] 1.3.2 Interaction

[0061] Response surface methodology and contour plots can visually reflect the influence of various factors on the DPPH radical scavenging effect of degraded polysaccharides and whether the interactions between these factors are significant. A steeper response surface and a contour plot that deviates more from a circle indicate a more significant interaction. It can be seen that with changes in ultrasonic time and H2O2-Vc concentration, the DPPH radical scavenging effect of degraded polysaccharides initially increases and then decreases. The DPPH radical scavenging rate of degraded polysaccharides reaches its maximum when the ultrasonic time is 65.48 min and the H2O2-Vc concentration reaches 10.18 mmol / L. Simultaneously, it can be seen that the interaction between ultrasonic time and H2O2-Vc concentration is significant, consistent with the results of the analysis of variance. It can also be seen that with increasing degradation temperature, the DPPH radical scavenging effect of degraded polysaccharides initially increases and then decreases; simultaneously, with increasing degradation time, the DPPH radical scavenging effect of degraded polysaccharides initially increases and then slowly decreases, and the interaction is not significant. It can be seen that with the increase of degradation temperature and H2O2-Vc concentration, the scavenging of DPPH free radicals by the degraded polysaccharide increases first. When the degradation temperature reaches 51℃ and the H2O2-Vc concentration reaches 10.18mmol / L, the scavenging rate of DPPH free radicals by the degraded polysaccharide reaches its peak, and the interaction between the two is significant.

[0062] 1.4 Preparation of degraded polysaccharides

[0063] Based on the optimal degradation conditions obtained by single-factor and response surface methodology, jujube polysaccharides were degraded under these conditions, and then degraded jujube polysaccharides (DZMP) were prepared through processes such as concentration, dialysis, and freeze-drying.

[0064] Analysis using Design Expert software revealed the optimal degradation conditions for jujube polysaccharides to be: ultrasonic time 65.48 min, H2O2-Vc concentration 10.11 mmol / L, and degradation temperature 51.31℃. Theoretically, the predicted DPPH free radical scavenging rate was 82.255%. Considering experimental feasibility, the above conditions were modified, and the final optimized degradation conditions were: ultrasonic time 65 min, H2O2-Vc concentration 10 mmol / L, and degradation temperature 51℃. Under these conditions, the DPPH free radical scavenging rate was 81.76%, with an error of 0.60% between the result and the predicted value. This indicates that the model can effectively predict the degradation of jujube polysaccharides and has a certain degree of reliability.

[0065] 1.5 Physicochemical Properties of Polysaccharides

[0066] The total sugar content of ZMP and DZMP was determined by the phenol-sulfuric acid method; the protein content of ZMP and DZMP was determined by the Coomassie brilliant blue method.

[0067] As shown in Table 4, the total sugar content of jujube polysaccharides increased from 75.47% to 81.78% after degradation, while the protein content decreased from 2.35% to 1.14%. The results indicate that degradation has little effect on the polysaccharide and protein content.

[0068] Table 4. Main components of degradation products

[0069]

[0070] Note: Different letters indicate significant differences (P<0.05).

[0071] This embodiment establishes a method for the degradation of jujube polysaccharides using ultrasound-assisted H2O2-Vc, with DPPH radical scavenging rate as the indicator. Response surface methodology was used to optimize the conditions for ultrasound-assisted H2O2-Vc degradation of jujube polysaccharides: ultrasound time of 65 min, H2O2-Vc concentration of 10 mmol / L, and degradation temperature of 51℃. Under these optimal conditions, the DPPH radical scavenging rate of DZMP was 81.76%, and the yield was 72.16%. After degradation, the total sugar content increased from 75.47% to 81.78%, while the protein content decreased slightly.

[0072] Example 2

[0073] 2.1 Isolation and purification of degraded polysaccharides

[0074] Column packing and equilibration: Pack the column material into a chromatographic column with dimensions of 100cm × Φ2.6cm, equilibrate the column with deionized water at a flow rate of 1.5mL / min, and elute for 3 column volumes.

[0075] DEAE-Sepharose Fast Flow Anion Exchange Chromatography: 300 mg of jujube-degraded polysaccharide was weighed and dissolved in 10 mL of deionized water. The solution was centrifuged at 8000 r / min for 15 min, and the supernatant was collected and filtered through a 0.45 μm filter membrane before loading onto a column. The eluent was filtered through a 0.45 μm filter membrane and then sonicated for 5 min to remove impurities and air bubbles. Eluent was first eluted with deionized water, then sequentially eluted with 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions. The absorbance of the eluent at 490 nm was measured every 5 tubes using the phenol-sulfuric acid method, and elution curves were plotted. The same fractions were combined and concentrated under reduced pressure, dialyzed for 72 h, with deionized water replaced every 2 h. Finally, the dialysates of each fraction were combined, concentrated under reduced pressure, and freeze-dried to obtain the fractions of jujube-degraded polysaccharide, named DZMP1, DZMP2, DZMP3, and DZMP4.

[0076] Sephacryl S-100 gel column chromatography: 50 mg of jujube-degraded polysaccharides (DZMP1, DZMP2, DZMP3, DZMP4) purified by DEAE-Sepharose FastFlow chromatography were reconstituted, centrifuged at 8000 r / min for 15 min, and the supernatant was collected, filtered through a 0.45 μm filter membrane, and loaded onto the column. Before elution with deionized water, the solution was filtered through a 0.45 μm filter membrane and sonicated for 5 min to remove impurities and air bubbles. The elution flow rate was 0.8 mL / min, and 10 mL of eluent was collected from each tube. The absorbance of the eluent at 490 nm was measured every 5 tubes using the phenol-sulfuric acid method, and elution curves were plotted. Identical fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain purified fractions, named DPZMP1, DPZMP2, DPZMP3, and DPZMP4.

[0077] 2.1.1 Isolation and purification of jujube-degraded polysaccharides

[0078] The DEAE-Sepharose FastFlow chromatography column was used to separate the jujube-degraded polysaccharides. The elution curve is shown below. Figure 4 As shown in the figure, four sugar peaks were obtained by elution with 0, 0.1, 0.2, and 0.3 mol / L NaCl solutions, respectively. These peaks were enriched, concentrated, dialyzed, and lyophilized, and named DZMP1, DZMP2, DZMP3, and DZMP4. Based on the elution characteristics of the DEAE-Sepharose Fast Flow chromatography column, DZMP1 is a neutral polysaccharide, while the other three jujube degradation polysaccharide components, DZMP2, DZMP3, and DZMP4, are acidic polysaccharides, with DZMP1 and DZMP4 showing the lowest yields.

[0079] Further purification was performed using a Sephacryl S-100 gel column, and the elution curve is shown below. Figure 5 As shown. The corresponding sugar peaks were collected, enriched, concentrated, and freeze-dried to obtain purified polysaccharides, which were named DPZMP1, DPZMP2, DPZMP3, and DPZMP4.

[0080] 2.2 Physicochemical property analysis of purified components

[0081] Determination of total sugar content: The total sugar content of the purified polysaccharides (DPZMP1, DPZMP2, DPZMP3, DPZMP4) was determined using the method described in Example 1.

[0082] Protein content determination: The protein content of the purified polysaccharides (DPZMP1, DPZMP2, DPZMP3, DPZMP4) was determined using the method described in Example 1.

[0083] Determination of total phenol content: The total phenol content of purified polysaccharides (DPZMP1, DPZMP2, DPZMP3, DPZMP4) was determined by the Folin-Ciocalteu colorimetric method.

[0084] Samples with higher total sugar content and purity are easier to characterize structurally and have higher reliability. Table 5 shows the physicochemical properties of the four purified components. The acidic polysaccharide components DPZMP2, DPZMP3, and DPZMP4 have total sugar contents exceeding 90%, with DPZMP3 having the highest total sugar content at 95.81%. The neutral polysaccharide component DPZMP1 has a total sugar content of only 89.41%. Compared to DZMP, the polysaccharide content significantly increased after purification, with most impurities removed, resulting in higher polysaccharide purity. Degraded polysaccharide components with higher sugar content and purity are easier to characterize structurally. Therefore, DPZMP3 was selected for structural analysis and activity studies.

[0085] The protein content of the degraded polysaccharide components from jujubes was determined using the Coomassie Brilliant Blue method. The neutral polysaccharide component DPZMP1 had a protein content of 1.05%, while the acidic polysaccharide components DPZMP2, DPZMP3, and DPZMP4 all had protein contents below 1.0%. Furthermore, the polyphenol content of each component was also low, and their impact on the polysaccharide structure was negligible. Therefore, considering the sugar content and purity of each component, DPZMP3 was selected for structural analysis and activity studies.

[0086] Table 5. Basic physicochemical properties of jujube degraded polysaccharide components

[0087]

[0088]

[0089] Note: Different letters indicate significant differences (P<0.05).

[0090] 2.3 Determination of molecular weight

[0091] The purity and molecular weight of the jujube degraded polysaccharide component (DPZMP3) were analyzed by high-performance liquid chromatography-gel chromatography (HPGPC). The conditions were as follows: detector: RI-10A differential detector; column: BRT105-104-102 tandem gel column (8×300 mm); column temperature: 40℃; mobile phase: 0.05 mol / L NaCl solution; flow rate: 0.6 mL / min; injection volume: 20 μL.

[0092] Sample preparation: The sample was prepared into a 5 mg / mL solution, filtered through a 0.22 μm filter membrane, and then transferred to a sample vial for instrumental testing.

[0093] The standard curve was obtained by using Dextran Standards 1152 with different molecular weights as standards for detection. The standard curve was plotted using the retention time of the chromatographic peak of the standard and the logarithm of its molecular weight, and the molecular weight of DPZMP3 was calculated.

[0094] The molecular weight and purity of DPZMP3, a purified polysaccharide fraction from jujube degradation, were determined using the HPGPC method. Based on dextran standards of different molecular weights, the fitted lgMw-RT calibration curve equation was: y = -0.1924x + 12.2, with a correlation coefficient R0. 2 The value is 0.9943, indicating a good linear relationship, and it can be used to determine the molecular weight and purity of DPZMP3. For example... Figure 6 As shown, the purified polysaccharide fraction DPZMP3 from jujube degradation has only one symmetrical chromatographic peak (39.839 min) in addition to the peak in the mobile phase (46.6 min), indicating that the DPZMP3 fraction is homogeneous and has high purity, and can be used for structural characterization. The molecular weight of DPZMP3 was calculated from the standard curve as lgMw 4.5, Mw / Da 34275.

[0095] 2.4 Monosaccharide Composition Analysis

[0096] The monosaccharide composition of the jujube degraded polysaccharide component (DPZMP3) was determined by ion chromatography. The conditions were as follows: detector: electrochemical detector; column: Dionex Carbopac™ PA20 (3×150 mm); column temperature: 30℃; mobile phase: A: H2O; B: 15 mmol / L NaOH, C: 100 mmol / L NaOAC; flow rate: 0.3 mL / min; injection volume: 5 μL.

[0097] For sample preparation, 5 mg of DPZMP3 was weighed and placed in an ampoule. 2 mL of TFA (3 mol / L) was added and dissolved thoroughly. The ampoule was sealed and placed in an oven at 120°C for 3 hours for hydrolysis. The hydrolysate was then transferred to a tube, dried under nitrogen, and methanol was added to the residue. The mixture was dried further to remove TFA completely. 5 mL of deionized water was added and vortexed. 50 μL of the mixture was added to 950 μL of deionized water, centrifuged, filtered through a 0.22 μm filter, and transferred to a sample vial for ion chromatography analysis.

[0098] For the determination of standards, 16 monosaccharide standards were weighed and prepared into a mixed standard solution. Ion chromatography analysis was performed using the same processing method as for the samples.

[0099] Monosaccharide composition analysis is the foundation of polysaccharide structure characterization. After hydrolysis of DPZMP3, the repeating units in the sugar chain were analyzed by ion chromatography. Figure 7 Image A shows the ion chromatograms of 16 monosaccharide standards. Figure 7Image B shows the ion chromatogram of the monosaccharide composition of the DPZMP3 polysaccharide component. Comparing the retention times of each monosaccharide standard, it can be seen that the DPZMP3 degraded polysaccharide component from jujube is mainly composed of rhamnose, arabinose, galactose, and galacturonic acid, with a molar ratio of 1:1.4875:1.6:7.675, indicating that degradation does not change the basic structure of the polysaccharide. After degradation, the contents of rhamnose, arabinose, and xylose decrease, while galactose and galacturonic acid increase, indicating that ultrasound-assisted H2O2-Vc preferentially acts on the glycosidic bonds near rhamnose, arabinose, and xylose, while the glycosidic bonds near other monosaccharides are more stable. Based on the monosaccharide composition and ratio, the DPZMP3 component can be identified as a pectin polysaccharide, with the main chain primarily composed of galacturonic acid.

[0100] 2.5 Methylation Analysis

[0101] The methylation method for the DPZMP3 component of jujube degradation is as follows: Weigh 2-3 mg of DPZMP3 sample into a reaction flask, add 1 mL of DMSO and methylation reagent A solution (anhydrous alkaline solution) to dissolve it completely. After vacuum dehumidification and deoxygenation, add N2 protective gas and seal the flask, protecting it from light. Then add methylation reagent B solution (iodomethane solution) and react in a water bath at 30℃ with magnetic stirring for 60 min in the dark. Finally, add 2 mL of ultrapure water to terminate the reaction. Dialyze the above mixture in a dialysis bag for 2-3 days until the contents of the dialysis bag are clear. Freeze-dry the solution and determine whether methylation is complete.

[0102] The lyophilized methylated polysaccharide was added to 1 mL of 2 mol / L TFA and hydrolyzed under sealed conditions for 90 min. The mixture was then evaporated to dryness under reduced pressure. Deionized water was then added, and the mixture was evaporated to dryness under reduced pressure. This process was repeated 4-5 times to remove TFA.

[0103] The hydrolyzed monosaccharides were derivatized to determine the positions of the glycosidic bonds. The hydrolysate was added to 2 mL of double-distilled water and reacted with 60 mg of sodium borohydride at room temperature for 8 h. Then, glacial acetic acid was added dropwise for neutralization, and methanol was added in small, repeated additions until the borates were removed by evaporation. Next, 1 mL of acetic anhydride was added, and the mixture was reacted at 100 °C for 1 h to acetylate. After cooling to room temperature, toluene was added to remove excess acetic anhydride. Finally, the acetylated product was extracted with CH₂Cl₂, and the organic layer was collected, filtered through a 0.45 μm filter, and brought to a final volume of 10 mL. The resulting solution was then transferred to a sample vial for instrumental analysis.

[0104] GC-MS chromatographic conditions were as follows: detector: RXI-5SILMS; column: 30m×0.25mm×0.25μm; carrier gas flow rate: helium, flow rate: 1mL / min; injector temperature: 250℃; detector temperature: 250℃ / min; temperature program: initial temperature 120℃, increased to 250℃ / min at 3℃ / min; hold for 5min.

[0105] The purified polysaccharide component DPZMP3 was subjected to methylation, acid hydrolysis, and derivatization, and the glycosidic bond type and linkage site were determined by GC-MS.

[0106] The complete methylation of DPZMP3 components was determined by Fourier transform infrared spectroscopy. The fully methylated DPZMP3 components underwent a series of processing steps and were analyzed by GC-MS to obtain the total ion chromatogram for methylation analysis. DPZMP3 mainly contained six chromatographic peaks. The mass spectra corresponding to the six methylated sugar peaks were compared with a standard spectral library (standard spectral library: https: / / www.ccrc.uga.edu / ) to determine the types of partially methylated sugars.

[0107] As shown in Table 6, the DPZMP3 component mainly contains six methylated sugar residues: non-reducing terminal residues T-Araf and 1,5-Araf glycosidic bonds mainly composed of arabinose; 1,2,4-Rhap glycosidic bonds mainly composed of rhamnose; 1,2,4-Rhap, 1,4,6-Galp, and 1,4-Galp glycosidic bonds mainly composed of galactose; and 1,4-GalpA glycosidic bonds mainly composed of galacturonic acid. In the methylation analysis, the molar ratios of rhamnose, arabinose, galactose, and galacturonic acid were close to those of the monosaccharide composition, indirectly proving that the methylation of the DPZMP3 component was essentially complete. Combined with the monosaccharide composition analysis, it can be inferred that the backbone of DPZMP3 is a pectin-type polysaccharide linked by galacturonic acid through 1,4-GalpA, and linked to other neutral sugars through 1,2,4-Rhap.

[0108] Table 6. DPZMP3 methylation analysis results

[0109]

[0110] 2.6 Nuclear Magnetic Resonance Analysis

[0111] 50 mg of the purified polysaccharide fraction DPZMP3 from jujube was weighed, dissolved repeatedly in heavy water, and then lyophilized. Finally, the lyophilized sample was dissolved in 500 μL of heavy water, sonicated until completely dissolved, and placed in an NMR tube. The sample was scanned using an NMR spectrometer at 303 K. 1 H NMR spectrum, 13 C NMR and HSQC spectra were obtained, and the NMR spectra were analyzed.

[0112] Figure 8 (A) is a polysaccharide component of DPZMP3. 1 H-NMR spectrum, (B) is the DPZMP3 polysaccharide component. 13C-NMR spectroscopy, through one-dimensional spectra, can deduce basic information about polysaccharides, such as glycosidic bond type and linkage mode. Studies have shown that a chemical shift of terminal hydrogens greater than 5.0 ppm indicates the α-configuration, while a shift less than 5.0 ppm indicates the β-configuration. Based on... 1 The H-NMR spectrum reveals three distinct hydrogen signal peaks in the terminal hydrogen-related region, with chemical shifts of 5.16 ppm, 4.54 ppm, and 4.54 ppm, respectively. Analysis of the chemical shifts of the terminal hydrogens in DPZMP3 confirms that sugar residue A is in the α configuration, while residues B and C are in the β configuration. HSQC spectroscopy is used to analyze the interaction between hydrogen and carbon nuclei in sugar residues. Through HSQC spectroscopy and... 13 Analysis of the C-NMR spectrum determined the chemical shifts of the terminal hydrogen and terminal carbon of the sugar residues to be 5.16 / 99.13 ppm, 4.54 / 103.15 ppm, and 4.54 / 103.15 ppm, respectively. Based on... 13 C-NMR and HSQC spectra determined the chemical shifts of C2-C6 in sugar residue A to be 67.83 ppm, 175.53 ppm, 68.03 ppm, 77.88 ppm, and 70.61 ppm, respectively, with corresponding hydrogen signal chemical shifts of 3.70 ppm, 4.04 ppm, 4.34 ppm, and 4.70 ppm. Combining the methylation results with data from relevant literature, sugar residue A can be inferred to be galacturonic acid, specifically 1,4-GalpA. Furthermore, the absorption peak at carbon 6 at 175.53 ppm also indicates the presence of galacturonic acid. By combining the methylation analysis results with the chemical shifts of sugar residues from relevant literature, sugar residues B and C can be identified as 1,4-Galp and 1,4,6-Galp, respectively, indicating that the degradation treatment does not destroy the basic structure of the polysaccharide.

[0113] Table 7. Assignment of C and H chemical shifts of DPZMP3 monosaccharide residues.

[0114]

[0115] 2.7 Ultraviolet Spectroscopy Scan

[0116] Prepare a 0.5 mg / mL DPZMP3 solution, using deionized water as a blank control, and perform ultraviolet spectral scanning in the range of 200–400 nm.

[0117] like Figure 9 As shown, no absorption peaks were found at 280 nm and 260 nm in the DPZMP3 component, indicating that no proteins or nucleic acids were found in the DPZMP3 component, which is consistent with the results of chemical testing.

[0118] 2.8 Infrared Spectroscopy Scan

[0119] Weigh 3 mg of fully dried DPZMP3 and 300 mg of potassium bromide (dried in an oven at 105℃ for 4-6 hours), mix, grind, and compress into tablets. Set the resolution to 0.4 cm⁻¹. -1 , in the range of 400 to 4000 cm -1 Infrared spectral scanning was performed within the range.

[0120] Infrared spectrum of DPZMP3 component of degraded polysaccharide from jujube, as shown in... Figure 10 As shown, at 3413cm -1 The broad and strong absorption peak at 2933 cm⁻¹ is due to the hydroxyl stretching vibration (OH); -1 The absorption peak at 1612 cm⁻¹ corresponds to the hydrocarbon stretching vibration (CH₂); the peak at 1612 cm⁻¹ corresponds to the hydrocarbon stretching vibration (CH₂). -1 The absorption peak at 1419 cm⁻¹ is due to the stretching vibration of the aldehyde group (C=O). -1 The absorption peak at 1200-1000 cm⁻¹ corresponds to the CH-angle vibration, indicating that DPZMP₃ contains uronic acid and is an acidic polysaccharide. -1 The absorption peaks on the left and right sides correspond to the stretching vibrations of COC and C-OH-, confirming the presence of the pyranose ring. At 894 cm⁻¹... -1 The presence of a strong absorption peak indicates that DPZMP3 is a β-pyranose, suggesting that the degradation did not alter the main functional group structure of the polysaccharide.

[0121] 2.9 Scanning electron microscopy analysis

[0122] The microstructure of the polysaccharide component DPZMP3 was characterized using SEM. A suitable amount of dried sample was adhered to conductive adhesive and gold-plated under vacuum. The test voltage was 3.0 kV, and the observation and imaging were performed using a scanning electron microscope.

[0123] Scanning electron microscopy images of DPZMP3 polysaccharide components at different magnifications are as follows: Figure 11 As shown in the figure, the DPZMP3 polysaccharide component consists of many irregularly sized flakes and blocks. At 10,000x magnification, the polysaccharide surface exhibits a porous honeycomb structure and a sponge-like texture. This indicates that degradation alters the spatial structure of the polysaccharide, causing changes in its surface morphology. This may be due to the cavitation and shearing effects generated by ultrasound during degradation, as well as the formation of hydroxyl radicals that disrupt the glycosidic bonds between polysaccharides, reducing molecular aggregation and dispersing molecular distribution, thus altering the polysaccharide's spatial structure.

[0124] Example 3

[0125] 3.1 Determination of DPPH free radical scavenging rate

[0126] The DPPH free radical scavenging rate was determined according to the method in Example 1.

[0127] like Figure 12As shown, the scavenging activity of jujube polysaccharide on DPPH free radicals is concentration-dependent. At a concentration of 2.0 mg / mL, the scavenging rates of ZMP, DZMP, DPZMP3, and Vc reached 81.55%, 89.64%, 92.89%, and 96.93% respectively, and the scavenging abilities were in the order of ZMP < DZMP < DPZMP3 < Vc. Calculated by SPSS analysis, the IC 50 values of the polysaccharides were 0.4432 mg / mL, 0.3153 mg / mL, and 0.2174 mg / mL respectively. The results showed that the scavenging activity of DZMP and DPZMP3 on DPPH free radicals was significantly stronger than that of ZMP, and was close to the DPPH free radical scavenging activity of the positive control Vc. This indicates that the degradation treatment can improve the DPPH free radical scavenging ability of polysaccharides, and the polysaccharide components obtained by separation and purification have higher purity and stronger DPPH free radical scavenging ability.

[0128] 3.2 Determination of Hydroxyl Radical Scavenging Ability

[0129] Add 1 mL of 6 mmol / L FeSO4 solution, 1 mL of 6 mmol / L salicylic acid-ethanol solution, and 1 mL of 6 mmol / L H2O2 solution to 1 mL of a sample solution with a certain concentration (0.2, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0 mg / mL). After shaking well, react in the dark at 37 °C for 30 min, and measure the absorbance of the solution at a wavelength of 517 nm, with Vc of the same concentration as the positive control. The hydroxyl radical scavenging rate is calculated according to formula (2):

[0130]

[0131] In the formula: A1 is the absorbance of the sample group, A2 is the absorbance of the blank group (deionized water replaces the H2O2 solution), and A0 is the absorbance of the control group (deionized water replaces the sample).

[0132] As Figure 13 shown, at a concentration of 0.2 - 2.0 mg / mL, all samples had a certain degree of scavenging effect on hydroxyl radicals, and the scavenging effect increased with the increase of the sample concentration. At a concentration of 2.0 mg / mL, the scavenging rates of ZMP, DZMP, DPZMP3, and Vc on hydroxyl radicals were 29.83%, 33.46%, 41.82%, and 92.65% respectively, and the scavenging abilities were in the order of ZMP < DZMP < DPZMP3 < Vc. The results showed that ZMP, DZMP, and DPZMP3 all exhibited a certain degree of hydroxyl radical scavenging activity, and were weaker than the positive control. This may be because the degradation caused the cleavage of polysaccharide glycosidic bonds, the decrease in molecular weight, the decrease in intermolecular forces, and the exposure of more groups, making it easier for small molecular polysaccharides to combine with hydroxyl radicals.

[0133] 3.3 Reducing Power

[0134] Add 2 mL of sample solution of a certain concentration (0.2, 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 mg / mL), 2 mL of 1% [K3Fe(CN)6] solution, and 2 mL of 0.2 mmol / L PBS buffer solution (pH 6.6) to a 15 mL centrifuge tube, mix thoroughly, react at 50 °C in the dark for 20 min, then terminate the reaction by adding 2 mL of 10% TCA, and then centrifuge at 3000 rpm for 10 min. Take 2 mL of supernatant, 2 mL of deionized water, and 0.4 mL of 0.1% FeCl3, mix well, react at room temperature for 10 min, and measure the absorbance of the solution at a wavelength of 700 nm. Use the same concentration of Vc as a positive control. The reducing power is calculated according to formula (3):

[0135]

[0136] In the formula: A1 is the absorbance of the sample group, and A0 is the absorbance of the control group (deionized water replaces the sample).

[0137] like Figure 14 As shown, within the concentration range of 0.2–2.0 mg / mL, the reducing power of all samples increased with increasing solution concentration. At a concentration of 2.0 mg / mL, the reducing powers of ZMP, DZMP, and DPZMP3 were 0.280, 0.428, and 0.507, respectively. At the same concentration, the reducing power of the degraded polysaccharides was 1.8 times that of the untreated ones, and the reducing power of all polysaccharide samples was weaker than that of vitamin C. Based on these results, degradation leads to the breakage of glycosidic bonds in polysaccharides, reducing their molecular weight and generating more free hydrogen-donating atoms to exert their antioxidant effect.

[0138] 3.4 Determination of superoxide radical scavenging capacity

[0139] Add 0.4 mL of sample solution of a certain concentration (0.2, 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 mg / mL) and 2 mL of 50 mmol / L Tris-HCl solution (pH 8.2) to a colorimetric tube, mix thoroughly, incubate at 25°C for 20 min, add 2 mL of 7 mmol / L pyrogallol, mix thoroughly, incubate at 25°C in the dark for 5 min, and finally add 0.4 mL of HCl (10 mmol / L) to terminate the reaction. Measure the absorbance of the solution at a wavelength of 420 nm, using the same concentration of Vc as a positive control. The reducing power is calculated according to formula (4):

[0140]

[0141] In the formula: A1 is the absorbance of the sample group, A2 is the absorbance of the blank group (deionized water instead of pyrogallol solution), and A0 is the absorbance of the control group (deionized water instead of sample).

[0142] As Figure 15 shown, the scavenging effects of ZMP, DZMP, DPZMP3 and Vc on superoxide anion radicals were dose-dependent. The scavenging abilities for superoxide radicals were in the order of ZMP < DZMP < DPZMP3 < Vc. The IC 50 values of ZMP, DZMP and DPZMP3 were 6.35 mg / mL, 2.09 mg / mL and 1.34 mg / mL, respectively. At a concentration of 2.0 mg / mL, the scavenging rates of ZMP, DZMP and DPZMP3 on superoxide anion radicals were 38.48%, 49.57% and 55.96%, respectively, all lower than that of the positive control group Vc. In addition, degradation may also lead to the exposure of active groups (such as sulfate groups) in the polysaccharide, enhancing the antioxidant ability of the polysaccharide.

[0143] 3.5 Determination of total antioxidant capacity

[0144] The total antioxidant capacity was determined by the FRAP microplate method. The determination method is shown in the table. The reaction solution was thoroughly mixed, reacted at 37 °C in the dark for 30 min, and the absorbance of the solution was measured at 593 nm. Vc with the same concentration was used as the positive control, and the total antioxidant capacity was expressed in terms of Fe 2+ concentration.

[0145] Table 8 Determination method of total antioxidant capacity

[0146]

[0147] Note: The FRAP working solution was prepared by mixing the FRAP detection buffer, TPTZ solution and FeCl3 solution in a ratio of 10:1:1.

[0148] As Figure 16 shown, in the concentration range of 0.2 - 2.0 mg / mL, the total antioxidant capacities of ZMP, DZMP, DPZMP and Vc were dose-dependent. When the sample concentration was 2.0 mg / mL, the total antioxidant capacities of ZMP, DZMP and DPZMP3 were equivalent to 0.363 mmol / L FeSO4, 0.561 mmol / L FeSO4 and 0.725 mmol / L FeSO4, respectively, much lower than that of the positive control Vc. The results showed that the degraded polysaccharide DZMP and the purified polysaccharide DPZMP had more reducing groups and specific surface areas, resulting in enhanced total antioxidant capacity.

[0149] In vitro antioxidant experiments showed that the DPPH radical scavenging ability, hydroxyl radical scavenging ability, superoxide anion radical scavenging ability, reducing power, and total antioxidant capacity of ZMP, DZMP, and DPZMP3 were dose-dependent. After degradation, the antioxidant capacity of jujube polysaccharide was significantly improved, and the antioxidant capacities of the three were in the order of ZMP < DZMP < DPZMP3. When the concentration was 2.0 mg / mL, the DPPH radical scavenging rates of DZMP and DPZMP3 were close to that of Vc, and their IC 50 values were 0.3153 mg / mL and 0.2174 mg / mL, respectively; the hydroxyl radical scavenging abilities were 33.46% and 41.82%; the superoxide anion radical scavenging abilities were 49.57% and 55.96%; the reducing powers were 0.428 and 0.507; the total antioxidant capacities were equivalent to 0.561 mmol / L FeSO4 and 0.725 mmol / L FeSO4. This indicates that the degradation treatment can improve the in vitro antioxidant capacity of polysaccharides. This may be because the degradation causes the cleavage of polysaccharide glycosidic bonds and the decrease of intermolecular forces, resulting in the degradation of polysaccharides into small molecular fragments, the reduction of molecular weight, the enhancement of solubility, the decrease of viscosity, and the exposure of more active groups, making the contact between active groups and free radicals easier, thus improving the antioxidant capacity of polysaccharides. The polysaccharide components obtained by separation and purification have higher purity and stronger in vitro antioxidant capacity.

[0150] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing jujube-degraded polysaccharide DPZMP3, characterized in that, Includes the following steps: Jujube polysaccharides extracted from jujubes using a water extraction and alcohol precipitation method were degraded using an ultrasound-assisted method combined with H2O2-Vc to obtain degraded jujube polysaccharides. The ultrasound-assisted method combined with H2O2-Vc included: an ultrasound time of 40–80 min, a H2O2-Vc concentration of 5–20 mmol / L, and a degradation temperature of 40–60 °C. The polysaccharide component DPZMP3 was isolated and purified from the degraded jujube polysaccharides. DPZMP3 was obtained by separating and purifying the degraded jujube polysaccharides using a DEAE-Sepharose Fast Flow gel column and a Sephacryl S-100 chromatography column. The separation and purification using the DEAE-Sepharose Fast Flow gel column included elution with deionized water, followed by sequential elution with 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions to obtain a 0.2 mol / L... The sugar peaks eluted with NaCl solution were enriched, concentrated, dialyzed, and lyophilized to obtain the jujube-degraded polysaccharide DZMP3; the separation and purification using a Sephacryl S-100 chromatography column included further purification of DZMP3 using a Sephacryl S-100 gel column to obtain DPZMP3.

2. The jujube degraded polysaccharide DPZMP3 obtained by the preparation method according to claim 1, characterized in that, The DPZMP3 is an acidic polysaccharide, mainly composed of rhamnose, arabinose, galactose, and galacturonic acid, with an average molecular weight of 34.275 kDa.

3. The jujube-degraded polysaccharide DPZMP3 according to claim 2, characterized in that, The molar ratio of rhamnose, arabinose, galactose, and galacturonic acid is 1:1.4875:1.6:7.

675.

4. The application of the jujube degraded polysaccharide DPZMP3 obtained by the preparation method according to claim 1 or the jujube degraded polysaccharide DPZMP3 according to any one of claims 2-3 in the preparation of formulations with high antioxidant activity.

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