Polysaccharide for preventing or treating diabetes and preparation method thereof

Abstract

A polysaccharide for preventing or treating diabetes, characterized in that the polysaccharide is lemon peel polysaccharide, the average average molecular weight is 195.26 kDa, and the polysaccharide is composed of D-mannose (Man), D-galacturonic acid (GalA), D-glucose (Glc), D-galactose (Gal), L-rhamnose (Rha), D-glucuronic acid (GlcA) and L-arabinose (Ara). The lemon peel polysaccharide prepared in the application has uniform molecular weight distribution, the average molecular weight is 195.26 kDa, the purity is high and reaches 95.22%, and the yield is as high as 77.5%. The polysaccharide can effectively inhibit the activity of alpha-glucosidase, can promote the absorption of MIN6 cells to glucose, can improve the glucose uptake amount of MIN6 cells, can improve the secretion of insulin, can achieve the purpose of regulating blood glucose, and can effectively prevent and treat diabetes.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation technology, specifically to a polysaccharide for the prevention or treatment of diabetes and its preparation method. Background Technology

[0002] Polysaccharides ( polysaccharide Polysaccharides are high-molecular-weight carbohydrates composed of at least 10 monosaccharides linked by glycosidic bonds. Over the years, research on polysaccharides has deepened, attracting considerable attention due to the significant differences in structure, function, and activity among different types of polysaccharides. An increasing number of plant polysaccharide compounds possess various biological activities such as anti-tumor, hypoglycemic, anti-liver damage, antiviral, and antioxidant effects. Therefore, their safety, high efficiency, and low toxicity have made them a hot topic in the pharmaceutical and food industries. Among citrus polysaccharides, those from orange peel and grapefruit peel are relatively well-studied. Reports indicate that both grapefruit peel and orange peel polysaccharides exhibit strong antioxidant activity and significant immune activity against cells.

[0003] Lemon peel is a residue generated during lemon processing, accounting for up to 50% of fresh fruit waste and representing a potential source of value-added byproducts. While some of the byproducts and waste generated by the food industry are recycled, other residues are still disposed of through landfills, composting, anaerobic digestion, incineration, thermal decomposition, and gasification. From both environmental and economic perspectives, this not only causes environmental pollution but also leads to resource waste. Studies have shown that lemon peel contains more polyphenolic compounds and dietary fiber than the pulp. For these reasons, researchers are increasingly focusing on the development and utilization of lemon peel, striving to develop various processing methods. This will not only improve the level of lemon industrialization but also significantly reduce ecological pressure, achieving high-value comprehensive utilization of lemon peel.

[0004] The first step in obtaining lemon peel polysaccharides is extraction. Researching the extraction process aims to obtain polysaccharides with a high yield. The second step is separation and purification. Obtaining high-purity polysaccharides allows for precise structural analysis and in-depth exploration of their potential physiological functions. The analysis of polysaccharide structures mainly includes examining the purity of the compound, the types of functional groups, monosaccharide composition, glycosidic bond types, and molecular weight. Understanding the structure of polysaccharides is crucial for their biological activity and related pharmaceutical product research. Polysaccharides obtained using different preparation methods exhibit significant differences in structure, and their functional activities, such as hypoglycemic efficiency, also vary considerably.

[0005] Existing reports on lemon peel polysaccharides only focus on the optimization of extraction methods and the determination of functional activities, without in-depth research on their structural analysis and functional effects. Therefore, it is of great significance to study the structural characterization of lemon peel polysaccharide components with hypoglycemic capabilities obtained by different extraction methods and to explore their hypoglycemic mechanisms. Summary of the Invention

[0006] The purpose of this invention is to provide a polysaccharide for the prevention or treatment of diabetes.

[0007] The second objective of this invention is to provide a method for preparing the aforementioned polysaccharide. The polysaccharide prepared by this method has high purity, uniform molecular weight distribution, and excellent hypoglycemic activity.

[0008] The objective of this invention is achieved through the following technical solution: A polysaccharide for the prevention or treatment of diabetes, characterized in that: the polysaccharide is lemon peel polysaccharide with an average molecular weight of 195.26 kDa, comprising D-mannose (Man), D-galacturonic acid (GalA), D-glucose (Glc), D-galactose (Gal), L-rhamnose (Rha), D-glucuronic acid (GlcA) and L-arabinose (Ara).

[0009] Furthermore, in the lemon peel polysaccharide, Man, Rha, GlcA, GalA, Glc, Gal, and Ara are present in the following molar percentages: 0.85~0.92: 4.02~4.08: 0.74~0.95: 54.94~57.19: 1.12~1.63: 11.62~12.15: 23.87~25.89.

[0010] Preferably, the molar percentages of Man, Rha, GlcA, GalA, Glc, Gal, and Ara are 0.88:4.06:0.84:56.53:1.32:11.96:24.41.

[0011] The method for preparing the above-mentioned lemon peel polysaccharide is characterized by comprising the following steps: (1) Take lemon peel, wash it, take the white part of the inner peel, dry it to constant weight, and pulverize it through an 80-mesh sieve to obtain lemon peel powder; (2) Take lemon peel powder and add it to distilled water. Adjust the pH to 9.0 with sodium hydroxide, add cellulase for enzymatic hydrolysis, and sonicate at the same time. After treatment, centrifuge, take the supernatant, add 5 times its volume of anhydrous ethanol for alcohol precipitation, let stand at 4℃ for 12h, then centrifuge, remove the supernatant, freeze-dry the precipitate to obtain crude lemon peel polysaccharide. (3) Separation and purification Lemon peel polysaccharide was obtained by sequentially performing dialysis, separation and purification on a DEAE-52 cellulose chromatography column, and separation and purification on a dextran gel G-100 chromatography column.

[0012] Furthermore, in step (2), the ratio of lemon peel powder to distilled water is 1:20~30, with units of g / mL, the amount of cellulase added is 1.0~2.0%, the power of ultrasonic treatment is 250~350 W, the ultrasonic enzymatic hydrolysis temperature is 50~55 ℃, and the ultrasonic enzymatic hydrolysis time is 35~45 min.

[0013] Furthermore, in step (3), the dialysis treatment involves removing the protein from the crude polysaccharide of lemon peel using the Sevage method to prepare a polysaccharide solution of 10 mg / mL, which is then dialyzed in distilled water using a dialysis bag with a molecular weight of 2000 Da. The residue in the bag is then freeze-dried to obtain the dialysis polysaccharide.

[0014] Furthermore, the DEAE-52 cellulose chromatography column separation and purification involves preparing a 10 mg / L polysaccharide solution from the dialyzed polysaccharide, filtering it through a 0.45 μm filter membrane, and then loading it onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm). The solution is eluted with a salt solution of 0–0.5 mol / L to obtain polysaccharide A.

[0015] Furthermore, the dextran gel chromatography separation and purification involves preparing a 20 mg / mL polysaccharide solution of polysaccharide A, filtering it through a 0.45 μm filter membrane, and then loading it onto a Sephadex G-100 dextran gel chromatography column (2.6 cm × 30 cm). Ultrapure water is used for elution at a flow rate of 0.5 mL / min, 10 mL / tube. The absorbance of each tube is detected using the phenol-sulfuric acid method. The eluents from the absorption peaks are combined, concentrated, dialyzed, and freeze-dried to obtain polysaccharide B.

[0016] Most specifically, a method for preparing a polysaccharide for the prevention or treatment of diabetes is characterized by comprising the following steps: (1) Take lemon peel, wash it, remove the yellow part of the outer peel, cut the remaining white pulp into small pieces, dry it at 60°C to constant weight, pulverize it into ultrafine powder, and pass it through an 80-mesh sieve to obtain lemon peel powder; (2) Add distilled water to the lemon peel powder. Lemon peel powder was added to distilled water at a ratio of 1:20-30 (g / mL). The pH was adjusted to 9.0 with sodium hydroxide. Cellulase was added for enzymatic hydrolysis, and the mixture was simultaneously sonicated. After treatment, the mixture was centrifuged, and the supernatant was collected. Five times its volume of anhydrous ethanol was added for alcohol precipitation. The mixture was allowed to stand at 4°C for 12 hours, then centrifuged again. The supernatant was removed, and the precipitate was freeze-dried to obtain crude lemon peel polysaccharide. The amount of cellulase added was 1.0-2.0%, the power of sonication was 250-350W, the temperature of sonication was 50-55°C, and the time of sonication was 35-45 min. The crude polysaccharide from lemon peel was prepared into a 10 mg / mL polysaccharide solution by removing protein using the Sevage method. The solution was then dialyzed in distilled water using a dialysis bag with a molecular weight of 2000 Da. The residue inside the bag was freeze-dried to obtain the dialyzed polysaccharide. The DEAE-52 cellulose chromatography column is used to prepare a 10 mg / L polysaccharide solution by dialyzing polysaccharide, filtering it through a 0.45 μm filter membrane, and then loading it onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm). The solution is eluted with a salt solution of 0–0.5 mol / L to obtain polysaccharide A. Polysaccharide A was prepared into a 20 mg / mL polysaccharide solution, filtered through a 0.45 μm filter membrane, and then loaded onto a Sephadex G-100 dextran gel chromatography column (2.6 cm × 30 cm). Ultrapure water was used for elution at a flow rate of 0.5 mL / min, 10 mL / tube. The absorbance of each tube was measured using the phenol-sulfuric acid method. The eluents from the absorption peaks were combined, concentrated, dialyzed, and freeze-dried to obtain polysaccharide B, which is lemon peel polysaccharide.

[0017] The present invention has the following technical effects: The lemon peel polysaccharide prepared by this invention has a uniform molecular weight distribution, with an average molecular weight of 195.26 kDa, high purity (95.22%), and a yield as high as 77.5%. This polysaccharide can effectively inhibit the activity of α-glucosidase, promote glucose uptake by MIN6 cells, increase glucose uptake by MIN6 cells, and increase insulin secretion, thereby regulating blood sugar and effectively preventing and treating diabetes. Attached Figure Description

[0018] Figure 1 The effects of different factors on the yield of crude polysaccharides from lemon peel.

[0019] Figure 2 Infrared spectrum of lemon peel polysaccharide LPs-1a.

[0020] Figure 3 Lemon peel polysaccharide LPs-1a 1 H-NMR (A), 13 C-NMR (B), DEPT-135° (C), and COSY (D) analytical spectra.

[0021] Figure 4 : HSQC (A), HMBC (B), NOSEY (C) analytical spectra of lemon peel polysaccharide LPs-1a and the inferred structural diagram of the repeating unit of LPs-1a (D).

[0022] Figure 5 Scanning electron microscopy image of lemon peel polysaccharide LPs-1a.

[0023] Figure 6 HPLC chromatograms of monosaccharide standards and LPs-1a.

[0024] Figure 7 HPGPC chromatogram of lemon peel polysaccharide LPs-1a.

[0025] Figure 8 IC50 of different polysaccharide components on the inhibitory activity against α-glucosidase 50 value.

[0026] Figure 9 Effects of different polysaccharide components on MIN6 cell survival.

[0027] Figure 10 Effects of different polysaccharide components on glucose uptake in MIN6 cells.

[0028] Figure 11 Effects of different polysaccharide components on insulin secretion in MIN6 cells.

[0029] Figure 12 Volcano plot of differential metabolites between Control and Model groups (A) and LPs-1a and Model groups (B). Detailed Implementation

[0030] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0031] Example 1 A method for preparing a polysaccharide for the prevention or treatment of diabetes, characterized by comprising the following steps: (1) Take lemon peel, wash it, remove the yellow part of the outer peel, cut the remaining white pulp into small pieces, dry it at 60°C to constant weight, pulverize it into ultrafine powder, and pass it through an 80-mesh sieve to obtain lemon peel powder; (2) Add distilled water to the lemon peel powder. Lemon peel powder was added to distilled water at a ratio of 1:20 (g / mL). The pH was adjusted to 9.0 with sodium hydroxide. Cellulase was added for enzymatic hydrolysis, and the mixture was simultaneously sonicated. After treatment, the mixture was centrifuged, and the supernatant was collected. Five times its volume of anhydrous ethanol was added for alcohol precipitation. The mixture was allowed to stand at 4°C for 12 hours, then centrifuged again. The supernatant was removed, and the precipitate was freeze-dried to obtain crude lemon peel polysaccharide. The amount of cellulase added was 1.1%, the power of sonication was 300 W, the sonication temperature was 55°C, and the sonication time was 44 min. This was denoted as LPs. The crude polysaccharide from lemon peel was prepared into a 10 mg / mL polysaccharide solution by removing protein using the Sevage method. The solution was then dialyzed in distilled water using a dialysis bag with a molecular weight of 2000 Da. The residue in the bag was freeze-dried to obtain the dialyzed polysaccharide, denoted as LPS-T. The DEAE-52 cellulose chromatography column was used to prepare a 10 mg / L polysaccharide solution by dialyzing polysaccharides. After passing the solution through a 0.45 μm filter membrane, the solution was loaded onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm). The solution was divided into three groups and eluted with a gradient using ultrapure water, 0.1 mol / L NaCl, and 0.3 mol / L NaCl as eluents, respectively. The resulting polysaccharides were designated as LPs-0, LPs-1, and LPs-2. Because different polysaccharides contain different charges, anion exchange chromatography was used to separate them. The polysaccharides showed peaks in ultrapure water, 0.1 mol / L NaCl solution, and 0.3 mol / L NaCl solution, with symmetrical and sharp peaks. Three fractions were collected and named the water-eluted fraction (LPs-0), the 0.1 mol / L NaCl-eluted fraction (LPs-1), and the 0.3 mol / L NaCl-eluted fraction (LPs-2), with purified yields of 17.60±1.06%, 68.80±1.45%, and 13.20±0.55%, respectively.

[0032] Since LPs-1 had the highest yield, the highest-yielding fraction was further purified using a Sephadex G-100 dextran gel chromatography column. Specifically, the polysaccharide LPs-0 was prepared into a 20 mg / mL polysaccharide solution, filtered through a 0.45 μm filter membrane, and then loaded onto a Sephadex G-100 dextran gel chromatography column (2.6 cm × 30 cm). Ultrapure water was used for elution at a flow rate of 0.5 mL / min, 10 mL / tube. The absorbance of each tube was measured using the phenol-sulfuric acid method. The eluents from the absorption peaks were combined, concentrated, dialyzed, and freeze-dried to obtain the polysaccharide, which was designated as LPs-1a.

[0033] In the extraction of crude polysaccharide LPs from lemon peel, the process was optimized, and preliminary structural characterization and antioxidant assays were performed. Results are as follows: Figure 1 As shown. From Figure 1As shown in Figure A, when the ultrasonic time is <40 min, the yield of lemon peel polysaccharides continuously increases with time (P < 0.05). With 40 min as the critical point, the yield of lemon peel polysaccharides begins to decrease. This is because if the ultrasonic time is further extended, the polysaccharides will be destroyed and decomposed by the cavitation and mechanical shearing of the ultrasound waves, impurities will dissolve, and the released heat will raise the temperature of the reaction system, potentially causing polysaccharide decomposition. If the ultrasonic time is too short, it is not conducive to breaking down the cell walls of the lemon peel, affecting the dissolution of polysaccharides. Therefore, 40 min was chosen as the zero level for optimizing the ultrasonic time in the response surface methodology. Figure 1 As shown in B, the polysaccharide yield reached its highest value of 6.39% when the ultrasonic extraction temperature of lemon peel polysaccharide reached 50 ℃, which was significantly different from other levels (P < 0.05). However, the polysaccharide yield tended to decrease at higher temperatures. This is because: firstly, higher temperatures lead to more vigorous diffusion of solvent molecules, making the polysaccharide more susceptible to degradation; secondly, excessively high temperatures can cause denaturation of enzyme proteins, thereby destroying cellulase activity and reducing the reaction rate, thus resulting in a lower crude polysaccharide yield. Therefore, selecting ultrasonic temperatures of 40, 50, and 60 ℃ as the response surface conditions is more appropriate.

[0034] The effect of the material-to-liquid ratio on polysaccharide yield is as follows: Figure 1 As shown in Figure C, the polysaccharide yield initially increased and then decreased with decreasing material-to-liquid ratio, exhibiting a significant difference (P < 0.05), and peaked at 1:30 (g / mL). This is mainly because the concentration difference between the inside and outside of plant cell walls promotes polysaccharide diffusion into the solvent. Higher concentrations result in greater polysaccharide dissolution in the solvent. Once the solute concentration in the solvent reaches equilibrium, further increases in solvent volume do not increase the solute concentration. Furthermore, the enzyme concentration decreases with increasing solvent volume, thus affecting the structure of the plant cell membrane and reducing the polysaccharide yield. Therefore, a material-to-liquid ratio of 1:30 (g / mL) is preferable.

[0035] like Figure 1 As shown in Figure D, when the enzyme dosage is <1%, the yield of lemon peel polysaccharides significantly increases with increasing enzyme dosage (P < 0.05). This is because the contact probability between the enzyme and cellulose molecules increases with increasing enzyme dosage, leading to a higher rate of cellulose hydrolysis and consequently, a higher polysaccharide yield. Further increasing the enzyme dosage beyond 1% does not increase the yield of lemon peel polysaccharides, indicating that at this concentration, the enzyme concentration is almost saturated, and further addition of cellulase cannot further affect the polysaccharide yield; there is no statistically significant difference. A 1% enzyme dosage was used as the zero level for response surface optimization.

[0036] Infrared spectral results of lemon peel polysaccharides are as follows Figure 2 As shown. At 3426 cm-1 The presence of an OH stretching vibration absorption peak at 2929 cm⁻¹ indicates the presence of intramolecular and intermolecular hydrogen bonds in LPs-1a. -1 The peak represents the CH stretching vibration on the methylene group, and is a characteristic absorption peak of polysaccharides, at 1747 cm⁻¹. -1 The absorption peak nearby is likely due to the stretching vibration of the C=O group in the methyl esterified carboxyl group of uronic acid; 1633 cm⁻¹ -1 The absorption peak is either due to the stretching vibration of C=O or caused by bound water; 1232 cm⁻¹ -1 The absorption peak is due to the stretching vibration of OH; 1103 cm⁻¹ -1 For the stretching vibration of COC, 10¹⁸ cm -1 The strong absorption peak indicates the presence of pyranose; 919 cm⁻¹ -1 and 831 cm -1 The presence of absorption peaks indicates the linkage between β-glycosidic and α-glycosidic bonds. Therefore, it is further speculated that lemon peel polysaccharide LPs-1a is an acidic polysaccharide with a pyranose ring linked by partially carboxymethylated β-glycosidic and α-glycosidic bonds. Furthermore, by calculating the 1747 cm⁻¹... -1 The peak area is 1747 cm⁻¹ -1 and 1633cm -1 The ratio of the sum of the peak areas is closely related to the degree of esterification (DE). The calculated DE = 44.38% < 50%, which belongs to low methoxyl pectin.

[0037] Methylation experiment: The LPs-1a bond structure was analyzed by GC / MS using an Agilent gas chromatograph. The system (Agilent 7890A; Agilent Technologies, USA), HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), used high-purity helium as the carrier gas. The mass spectrometry system employed an Aigilent quadrupole mass spectrometer (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The scanning mode was full scan (SCAN), with a mass scan range (m / z) of 30–600.

[0038] Methylation results showed that the backbone structure of LPs-1a is linked by →4)-GalpA-(1→ and →4)-GalpA-6-O-methyl-(1→), with AG-I side chains. At the same time, GalpA-(1→ and →2,4)-Rhap-(1→) linkage structures exist, with Galp and Araf forming side chains, which are typical HG and RG-I domains that constitute pectin.

[0039] Nuclear magnetic resonance: Weigh out LPs-1a sample, dissolve it completely in D2O, transfer it to an NMR tube, and perform NMR. 1 H, 13 C. NMR spectra were measured using DEPT-135°, HSQC, HMBC, COSY, and NOSEY, and the NMR data were processed using MestReNova software.

[0040] One-dimensional NMR spectrum of LPs-1a 1 H-NMR, 13 C-NMR, DEPT135° such Figure 3 As shown. From 1 H-NMR spectrum ( Figure 3 As can be seen from A), the chemical shift δ using the terminal matrix quantum signal is... H Whether the concentration is greater than 5 ppm determines the α / β configuration of the residues. Generally, δH > 5 ppm indicates the α configuration, and δ H <5 ppm, β configuration. From 1 The H-NMR spectrum revealed seven terminal hydrogen signals (H-1) at δ5.17, δ5.10, δ5.07, δ5.03, δ5.00, δ4.88, and δ4.56 ppm for LPs-1a, named residues A through G, indicating the presence of α / β glycosidic bonds. This result is consistent with the infrared spectrum. The corresponding chemical shifts at δ3.2–δ4.0 ppm correspond to proton H-2 to H-5 or H-6 of the sugar ring. Correspondingly, in 13 C-NMR ( Figure 3 Anomaly carbon signal peaks were found in (B), with values ​​of δ107.42, δ107.32, δ106.78, δ106.31, δ104.28, δ100.38, and δ99.5 ppm, respectively. DEPT135° ( Figure 3C) is also a type of carbon spectrum used to distinguish primary carbon (-CH3-), secondary carbon (-CH2-), tertiary carbon (-CH-), and quaternary carbon (-C-). Secondary carbon peaks point downwards, quaternary carbon peaks are absent, and the remaining peaks point upwards. Combining COSY, HSQC, HMBC, NOSEY two-dimensional spectra with reported residue chemical shifts further confirmed all C and H signals of LPs-1a. COSY picture( Figure 3 D) Provides hydrogen-related signals from hydrogen atoms in carbons adjacent to the compound's structure. Specific analysis follows: Residue A: δc 107.42, δH 5.00 ppm is a typical terminal C-H signal of T-α-L-Araf-(1→). H-1 (δ 5.0 ppm) and δ 4.05 ppm show a significant correlation, confirming δ 4.05 ppm as the H-2 signal, corresponding to HSQC ( Figure 4 A) The carbon chemical shift signal C-2 corresponding to δ4.05 ppm was found to be δ80.92 ppm. The H-2 signal was correlated with δ3.88 ppm, thus identifying δ3.88 ppm as the H-3 signal. Similarly, the H-4 signal was found to be δ3.99 ppm, and the H-5 signal to be δ3.74 / 3.64 ppm. Checking the HSQC spectrum, the C-3, C-4, and C-5 signals of residue A were identified as δ76.46, δ83.94, and δ61.02 ppm, respectively. Observing the DEPT 135° spectrum, the C-5 (A5) signal peak was downward, indicating it was a secondary carbon in α-L-Araf. Literature review and matching showed that the carbon-hydrogen signal of this residue was basically consistent with T-α-L-Araf-(1→). Combining the monosaccharide composition and methylation analysis, the presence of the Ara group in LPs-1a confirmed that residue A was present. It is T-α-L-Araf-(1→).

[0041] Residues B and C: For residues B and C, the C-1 / H-1 signal of residue B corresponds to δ 107.32 / 5.03 ppm, and the C-1 / H-1 signal of residue C corresponds to δ 106.78 / 5.10 ppm. Based on the coupling signals in the COSY and HSQC spectra, the C-2 / H-2, C-3 / H-3, C-4 / H-4, and C-5 / H-5 signals of residue B are assigned as δ 81.29 / 4.21, δ 78.05 / 3.61, δ 82.26 / 4.13, and δ 66.77 / 3.80 / 3.72 ppm, respectively, and the C-2 / H-2, C-3 / H-3, C-4 / H-4, and C-5 / H-5 signals of residue C are assigned as δ 106.78 / 5.10 ppm. The signals were δ68.54 / 4.05, δ83.96 / 3.96, δ76.63 / 3.88, and δ66.77 / 3.72 ppm. Observing the DEPT 135° spectrum, the C-5 (B5 / C5) signal peak was downward, which is the secondary carbon in α-L-Araf. Since the chemical shifts of the C-5 signal of residue B and the C-3 and C-5 signals of residue C shifted to the lower field, it indicates that substitution glycosylation occurred at C-5 of residue B and C-3 and C-5 of residue C. Combined with monosaccharide composition and methylation analysis, residue B was determined to be →5)-α-L-Araf-(1→, and residue C was →3,5)-α-L-Araf-(1→).

[0042] Residue D: The signal at δ 16.47 ppm is the methyl group of Rhap, associated with C-6 of Rhap. According to the HSQC spectrum, the corresponding H-6 signal is 1.17 ppm. According to the COSY spectrum, the H-1, H-2, H-3, H-4, and H-5 signals associated with residue D are δ 5.03, δ 4.21, δ 3.61, δ 4.17, and δ 3.94 ppm, respectively. According to the HSQC spectrum coupling signal, the signals associated with C-1, C-2, C-3, C-4, and C-5 are δ 99.5, δ 81.29, δ 71.82, δ 80.72, and δ 68.48 ppm, respectively. The C-2 and C-4 signals show a chemical shift towards the lower field, indicating glycosylation between these two carbons. Therefore, according to relevant literature, residue D is determined to be →2,4)-α-L-Rhap-(1→).

[0043] Residue E: δc 104.28, δH 4.56 ppm is a typical terminal C-H signal of →4)-β-D-Galp-(1→, δH < 5 ppm, indicating this residue is in the β configuration. According to the COSY cross-signal, δ4.56 / δ3.61, δ3.61 / δ3.61, δ3.69 / 4.09, δ4.09 / δ3.72, δ3.72 / δ3.64 ppm are cross-correlated. Therefore, H-2, H-3, H-4, H-5, and H-6 of residue E are δ3.61, δ3.61, δ4.09, δ3.72, and δ3.64 ppm, respectively. According to the HSQC spectrum, C-2, C-3, C-4, C-5, and C-6 of residue E are... The signals were δ72.79, δ73.22, δ77.58, δ71.73, and δ60.66 ppm, respectively. The C-4 signal shifted to a lower field due to substitution, indicating that the carbon was glycoside. Based on relevant literature, the residue E was determined to be →4)-β-D-Galp-(1→).

[0044] Residues F and G: The chemical shift values ​​of the carboxyl signal peaks of uronic acids are generally attributed to δ 170-180 ppm. (Observation...) 13The C-NMR spectrum clearly shows a peak around δ170 ppm, thus confirming that the peaks at δ170.58 / δ170.69 ppm are related to the C-6 carboxyl group signal of GalpA, further confirming that LPs-1a is an acidic polysaccharide. The signal at δ21.38 / 1.42 ppm corresponds to the -CH3 group of the acetyl group on O-2 / 3 of GalpA, and the signal at δ52.79 / 3.72 ppm indicates that the C-6 carboxyl group of GalpA (residue F) has undergone methyl ester substitution (OMe). According to literature review, the typical terminal C-H signals of →4)-α-D-GalpA(OMe)-(1→ are δ100.37 / 4.88 ppm. Based on the coupling signals of the COSY spectrum cross, the H-2 to H-5 signals corresponding to residue F are δ3.63, δ3.93, δ4.37, δ5.00 / 5.07 ppm, and the H-1 to H-5 signals corresponding to residue G are δ5.07, δ4.05, δ3.63, δ3.92, δ4.12 ppm. Combining with the HSQC spectrum, the C-2 to C-5 signals of residue F are δ67.70, δ69.21, δ78.95, δ70.5 ppm, and the C-1 to C-6 signals of residue G are δ67.70, δ69.21, δ78.95, δ70.5 ppm. The signals were δ99.5, δ68.41, δ74.44, 79.55, δ70.23 and δ170.69 ppm. The chemical shifts of the signals at C-4 of residues F and G were shifted to the lower field. After the C-4 position was substituted, the carbon was glycoside. Based on literature comparison, residue F was determined to be →4)-α-D-GalpA(OMe)-(1→, and residue G was →4)-β-D-GalpA-(1→).

[0045] Residue H: Due to partial signal overlap or weak related signals, the chemical signal of residue H could not be determined. However, combined with monosaccharide composition and methylation analysis, it was determined that Rhap sugar residues exist in LPs-1a. Therefore, by comparing with relevant literature, the terminal C-H signal of residue H was assigned to δ106.31 / 5.17 ppm, and the C-6 / H-6 signal was assigned to δ16.47 / 1.16 ppm. It is speculated that residue H is →2)-α-L-Rhap-(1→).

[0046] HMBC ( Figure 4B) The coupling signals between long-range carbons and protons can be linked. NOSEY (Figure 4C) can determine the spatial proximity between protons in LPs-1a. These two spectra can be used to determine the main linkage sequence of sugar residues in LPs-1a. The weak signal can be observed in Figure 4B, which may be due to the large molecular weight of LPs-1a, resulting in an indistinct signal. The HMBC spectrum shows a correlation signal at δ66.77 / 5.0 ppm, indicating that the H-1 of sugar residue A is coupled with the C-5 of sugar residues B and C, indicating the presence of the T-α-L-Araf-(1→5)-α-L-Araf-(1→) structure. T-α-L-Araf-(1→) is linked at the O-5 position to →3,5)-α-L-Araf-(1→. The signal at δ83.96 / 5.0 ppm indicates the presence of T-α-L-Araf-(1→) at the O-3 position. The structure is linked at position →3,5)-α-L-Araf-(1→). In the NOSEY spectrum, the signal at δ5.0 / 3.69 ppm indicates that the H-1 signal of residue A is coupled to the H-3 signal of residue E, linked by a 1,3-glycosidic bond; the signal at δ5.03 / 3.72 ppm indicates that the H-1 signal of residue B is coupled to the H-5 signal of residue C, linked by a 1,5-glycosidic bond; the signal at δ3.92 / 4.17 ppm indicates that the H-1 signal of residue C is coupled to the H-4 signal of residue D, linked by a 1,4-glycosidic bond; the signal at δ5.10 / 4.09 ppm indicates that the H-1 signal of residue C is coupled to the H-4 signal of residue E, linked by a 1,4-glycosidic bond; and the signal at δ5.03 / 3.92 ppm indicates that the H-1 signal of residue D is coupled to the H-4 signal of residue G. The signals exhibit coupling signals linked by 1,4-glycosidic bonds. Signals at δ 5.03 / 4.37 ppm indicate coupling between H-1 of residue D and H-4 of residue F, linked by 1,4-glycosidic bonds. Signals at δ 4.56 / 3.61 ppm indicate coupling between H-1 of residue E and H-3 of residue D, linked by 1,3-glycosidic bonds. Signals at δ 5.07 / 4.21 ppm indicate coupling between H-1 of residue G and H-2 of residue D, linked by 1,2-glycosidic bonds. Based on this information, the possible repeating unit structure of LPs-1a can be inferred as follows: Figure 4 As shown in D.

[0047] LPs-1a surface morphology as follows Figure 5As shown, LPs-1a exhibits a significantly different morphology compared to crude polysaccharide. At 100x magnification (a), LPs-1a clearly displays a velvety texture with numerous small pores, indicating the influence of ultrasound and the porous network resulting from purification of the crude polysaccharide. At 300x magnification (b), the polysaccharide surface shows abundant fibers, some exhibiting a sheet-like structure. At 500x (c) and 1000x (d), the polysaccharide surface is not entirely smooth with small particles, exhibiting an overall loose and porous structure. At 2000x (e) and 3000x (f), the polysaccharide fibers have numerous fuzzy, hair-like structures, which contribute to the polysaccharide's enhanced bioactivity.

[0048] Different raw materials, extraction methods, and raw material processing can all lead to different compositions of extracted monosaccharides. Figure 6 The HPLC chromatograms of LPs-1a and monosaccharide standards are shown, with monosaccharides used as the reference. The standards contain: 1-mannose (Man); 2-glucosamine hydrochloride (GlcN); 3-rhamnose (Rha); 4-glucuronic acid (GlcA); 5-galacturonic acid (GalA); 6-galactosamine hydrochloride (GalN); 7-glucose (Glc); 8-galactose (Gal); 9-xylose (Xyl); 10-arabinose (Ara); and 11-fucose (Fuc). LPs-1a was found to consist of seven monosaccharides: D-galacturonic acid (GalA), D-mannose (Man), D-glucose (Glc), D-galactose (Gal), L-rhamnose (Rha), D-glucuronic acid (GlcA), and L-arabinose (Ara). The molar ratios of each monosaccharide are shown in Table 1.

[0049] Table 1: Molar percentage of each monosaccharide in LPs-1a (%)

[0050] It can be seen that galacturonic acid, arabinose, and galactose constitute a large proportion of the monosaccharide content in LPs-1a. Compared to LPs, the content of galacturonic acid increased significantly and the content of arabinose decreased after purification, indicating a reduction in the side chain content.

[0051] Calculated using Table 1: ; R1 reflects the proportion of linear pectin domains, such as the HG type, in the polysaccharide. LPs-1a has a relatively high R1 of 1.40, suggesting it is a linear polysaccharide polygalacturonic acid (HG type domain). R2 reflects the proportion of rhamnogalacturonic acid polysaccharide (RG-I domain), which consists of a main chain of GalA and Rha, with branches of Gal and Ara. A low R2 value indicates a lower content, suggesting the main chain is rich in HG rather than RG-I type. R3 reflects the proportion of RG-I type side chains. An R3 of 8.95 indicates that LPs-1a has a high proportion of side chains and more branches. The type and degree of branching of pectin affect its biological activity; for example, pectin rich in RG-I structures can enhance its activity in preventing cancer, cardiovascular disease, and fibrosis. High content of RG-I type side chains can significantly inhibit α-glucosidase activity and promote the expression of PI3K-AKT signaling pathway, thus exhibiting strong hypoglycemic activity.

[0052] Different extraction methods and purification techniques all affect the molecular weight and molecular weight distribution uniformity of polysaccharides. Although citron ( C.medica ), pomelo C.maxima ),lime( C. ×aurantium ),lemon( C.× limon ),Tangerine( C.reticulate All of these belong to the genus Citrus in the Rutaceae family, but the molecular weight of the lemon peel polysaccharide prepared in this invention differs from existing studies on the molecular weight of Citrus polysaccharides. For example, existing techniques use microwave extraction, DEAE-Sepharose FastFlow anion exchange chromatography, and Sephadex G-75 gel filtration chromatography to purify the polysaccharide component TPPs-2-1, which has a molecular weight of 17.8 kDa. Alternatively, a combination of enzyme and ultrasound, combined with microwave and DEAE-Sepharose CL-6B gel chromatography, and Sephadex G-150 gel chromatography can be used to purify lemon polysaccharides, yielding polysaccharide components LPUE-1, LPUE-2, LPME-1, and LPME-2, with molecular weights of 5.80, 43.27, 7.26, and 48.79 kDa, respectively. Figure 7 The HPGPC spectrum of LPs-1a was obtained. Based on the calibration curve, the Mp of lemon peel polysaccharide LPs-1a was calculated to be 145.13 kDa, Mw to be 195.26 kDa, and Mn to be 119.73 kDa. The distribution coefficient Mw / Mn was calculated to be 1.63. Based on the peak area, the purity of LPs-1a was calculated to be 95.22%, which is relatively high, indicating that the lemon peel polysaccharide is relatively homogeneous. The peak at 47.76 min is the mobile phase salt peak.

[0053] α-glucosidase inhibition test: like Figure 8 As shown, α-glucosidase is a key enzyme that hydrolyzes the α-1,4 glycosidic bonds in carbohydrates to release glucose. Therefore, inhibiting its activity can lower postprandial blood glucose and achieve the goal of preventing diabetes and its complications. The hypoglycemic activity of polysaccharides can be demonstrated by testing their inhibitory effect on α-glucosidase. The IC50 values ​​of the inhibitory abilities of each component of LPs, LPs-T, LPs-1, and LPs-1a on α-glucosidase were tested. 50 The values ​​showed significant differences among the components. P <0.05). IC50 of each component 50 The IC50 value ranged from 1.22 to 6.37 mg / mL. Compared with other plant polysaccharides, LPs-1a had a higher IC50 value. 50 The value decreased significantly, and the inhibitory activity against α-glucosidase was more significant. Furthermore, the yield of LPs-1a was the highest among all components, at 77.5 ± 0.74%. This result indicates that, after screening, the LPs-1a component is the main hypoglycemic component of lemon peel polysaccharides.

[0054] In vitro blood glucose lowering capacity test: (1) Effect of LPs-1a on MIN6 cell survival: Logarithmic growth phase MIN6 cells were counted and seeded into 96-well plates at a density of 2 × 10⁴ cells / mL, 100 μL per well. The wells were filled with PBS or sterile water and cultured for 24 h. Different concentrations (25, 50, 100, 200, 400, 800 μg / mL) of polysaccharide fractions (LPs, LPs-T, LPs-0, LPs-1, LPs-2, LPs-1a) were added to each well as the treatment group, and normal cultured cells served as the control group. Each group was configured in triplicate. After 24 h of treatment, 10 μL of MTT reagent (5 mg / mL) was added to each well, and the cells were incubated for another 4 h. The culture medium in the wells was discarded, and 110 μL of Formazan solution was added to each well. The cells were shaken for 10 min, and the absorbance of each well was measured at 490 nm using a microplate reader.

[0055] The effect of the isolated and purified components on the survival rate of MIN6 cells was investigated using the MTT assay. The results are shown in Figure 9. The figure shows the toxic effects of LPs-1a on MIN6 cells for 24 h in the concentration range of 25-800 μg / mL. Statistical analysis showed that there was no significant difference between LPs-1a and the control group in the concentration range of 25-800 μg / mL (P>0.05), indicating that LPs-1a does not have a toxic effect on MIN6 cells at 800 μg / mL. Although there was no significant difference in cell survival rate between LPs-1a and the control group at 800 μg / mL (P>0.05), the cell survival rate of LPs-1a was significantly lower than that of the control group, with a survival rate of only 83.57±11.96%. Therefore, subsequent experiments were conducted at concentrations of 100, 200, and 400 μg / mL.

[0056] (2) Effect of LPs-1a on glucose uptake in MIN6 cells MIN6 cells were seeded into 96-well plates at a density of 2 × 10⁵ cells / mL. After cell adhesion, the original culture medium was discarded, and the cells were washed twice with KRBH buffer. The high glucose injury group (HG) was treated with fresh KRBH buffer containing 25 mmol / L glucose solution. The drug treatment groups (LPs, LPs-T, LPs-0, LPs-1, LPs-2, LPs-1a) and the positive control group (Met) were incubated with fresh KRB buffer of different concentrations (100, 200, 400 μg / mL). After 2 h, 25 mmol / L glucose solution was added and incubation continued for 20 min. The glucose content of the supernatant was measured using a glucose oxidase assay kit.

[0057] like Figure 10As shown, compared with the HG group treated with high glucose KRBH buffer, the glucose content of the positive control group treated with metformin hydrochloride (Met) was significantly reduced at concentrations of 100, 200, and 400 μg / mL (P < 0.05), while the glucose content of the LPs-1a group treated with Metformin hydrochloride was significantly reduced at concentrations of 100 and 200 μg / mL (P < 0.05). No statistically significant differences were found in the glucose content of other components at any concentration. This suggests that metformin hydrochloride and LPs-1a can promote glucose uptake by MIN6 cells, increasing glucose uptake. MIN6 cells, the main site of insulin secretion and synthesis in mouse pancreatic β-cells, are susceptible to insulin secretion defects when exposed to a high glucose environment for extended periods, affecting the body's absorption of blood glucose. The experimental results showed that LPs-1a could increase glucose uptake in cells. Among the LPs-1a concentrations of 100, 200, and 400 μg / mL, the 100 μg / mL concentration showed the lowest glucose uptake (11.85 ± 1.79 mmol / L) and was the most effective in promoting glucose uptake in MIN6 cells.

[0058] (3) Effect of LPs-1a on insulin secretion in MIN6 cells MIN6 cells were seeded into 96-well plates at a density of 2 × 10⁵ cells / mL. After cell adhesion, the original culture medium was discarded, and the cells were washed twice with KRBH buffer and starved in KRBH buffer for 2 h. The HG group was given fresh KRBH buffer containing 25 mmol / L glucose solution. The drug administration groups (LPs, LPs-T, LPs-0, LPs-1, LPs-2, LPs-1a) and the Met group were given fresh KRBH buffer containing 25 mmol / L glucose solution and different concentrations (100, 200, 400 μg / mL) of each component. The supernatant was used to detect insulin content using a mouse insulin ELISA kit.

[0059] like Figure 11As shown, at drug concentrations of 100, 200, and 400 μg / mL, compared with the HG group, the insulin secretion of MIN6 cells treated with the Met group and the LPs-1a group was significantly increased (P < 0.05). At a drug concentration of 200 μg / mL, the LPs-T group and the LPs-1 group significantly stimulated insulin secretion of MIN6 cells compared with the HG group (P < 0.05). At a drug concentration of 400 μg / mL, the LPs-0 group and the LPs-1 group significantly stimulated insulin secretion of MIN6 cells compared with the HG group (P < 0.05), while the other components showed no statistical significance (P > 0.05). Combined with the results of glucose uptake, the polysaccharide components can promote glucose uptake by mouse pancreatic β cells, thereby promoting increased insulin secretion and achieving the purpose of regulating blood glucose. LPs-1a promoted insulin secretion at all three dosage concentrations. Based on the comprehensive evaluation of glucose uptake results, 100 μg / mL LPs-1a was selected as the cellular mechanism for elucidating the hypoglycemic effect of LPs-1a using metabolomics.

[0060] Lemon peel polysaccharide (LPs-1a) metabolomics analysis based on MIN6 cells: Three groups were set up: the drug administration group, the model group, and the blank group.

[0061] The drug administration group (LPs-1a group): The cell culture medium was lemon peel polysaccharide. The LPs-1a component, which had the lowest glucose uptake in MIN6 cells and stimulated insulin secretion, was selected and dissolved in fresh KRBH buffer containing 25 mmol / L glucose solution to make a final concentration of 100 μg / mL.

[0062] Model group: The cell culture medium was fresh KRBH buffer containing 25 mmol / L glucose solution, which induced MIN6 cells to form a high glucose injury model.

[0063] Control group: Cells were cultured normally in RPMI-1640 complete medium containing 10% fetal bovine serum.

[0064] Nine MIN6 cell culture flasks were divided into three groups, with three replicates per group. The cell density in each flask was controlled at 1 × 10⁷ cells / mL. After cell adhesion, the original culture medium was discarded, and the cells were washed twice with KRBH buffer and starved in KRBH buffer for 2 h. The corresponding culture medium was added according to 5.2.1, and the cells were incubated at 37 ℃ for 2 h. The cells were carefully scraped from the culture flasks with a cell scraper, centrifuged at 1000 r / min for 5 min, and the cell pellet was flash-frozen in liquid nitrogen for 15 min and stored at -80 ℃. Metabolomics testing was performed by Shanghai Ling'en Biotechnology Co., Ltd. (www.biozeron.com).

[0065] Screening and identification of differential metabolites: To identify differentially expressed metabolites associated with LPS-1a intervention, potential differentially expressed metabolites were screened based on a VIP ≥ 1 in the OPLS-DA model and a P ≤ 0.05 in the independent samples t-test. The results of these differentially expressed metabolites were then visualized, as shown below. Figure 12As shown in Figure A, there are 466 differentially regulated metabolites between the Control group and the Model group. Among them, red indicates 193 significantly upregulated metabolites and blue indicates 273 significantly downregulated metabolites. Figure 12B shows that there are 30 differentially regulated metabolites between the LPs-1a group and the Model group, with 13 significantly upregulated metabolites and 17 significantly downregulated metabolites. Identification was performed using online databases of standards such as KEGG and HMDB. 125 pathological metabolites were screened from the Control and Model groups. Among them, 60 metabolites, including succinic acid, glutamic acid, xanthine, tryptophan, pilocarpine, 2-piperidinic acid, ornithine, taurine, p-hydroxybenzoic acid, 2'-deoxyuridine-5'-monophosphate, folic acid, ribosyl kinin, 3-hydroxy-2-octylglutaric acid, glutamic acid, bile acids, N-(4-chlorophenylethyl)-N'-(4-chlorophenyl)urea, and L-threonic acid, were significantly upregulated. 65 metabolites, including 1-methyladenosine, phosphatidylcholine, azelaic acid, 19(R)-hydroxyprostaglandin F1α, erythrose-4-phosphate, LO-phosphoserine, trans-cinnamic acid, taurine, aspartic acid, N-acetyl-D-glucosamine, prostaglandins, glutathione disulfide, and nicotinic acid, were also significantly upregulated. Several metabolites were significantly downregulated; the table lists some of the differentially regulated metabolites. After LPs-1a intervention in MIN6 cells, 11 differentially regulated metabolites were found to have regulatory effects on MIN6 cells. Among them, 1-methyladenosine, phosphatidylcholine, azelaic acid, and 19(R)-hydroxyprostaglandin F1α were significantly upregulated, while pilocarpine, glutamate, taurine, chenodeoxycholic acid, kinin nucleoside, 3-hydroxy-2-octylglutaric acid, N-(4-chlorophenylethyl)-N'-(4-chlorophenyl)urea, and L-threonic acid were significantly downregulated. These results indicate that LPs-1a intervention has a certain impact on cell metabolism and can restore some differentially regulated metabolites in the Control and Model groups.

[0066] Metabolic pathway analysis: The mechanism by which LPs-1a treats high glucose-induced MIN6 cell damage involves significantly upregulating 1-methyladenosine via the purine metabolism pathway, significantly downregulating phosphatidylcholine PC (O-16:2) via the glycerophospholipid metabolism pathway, significantly downregulating taurine and chenodeoxycholic acid and azelaic acid in the cholesterol metabolism pathway, respectively, significantly downregulating glutamate in the histidine metabolism pathway, and significantly upregulating 19(R)-hydroxyprostaglandin F1α, the ω-1 hydroxylase metabolite of the arachidonic acid derivative PGF1α, in the arachidonic acid metabolism pathway, with the histidine metabolism pathway being the main pathway. It is speculated that LPs-1a regulates the corresponding metabolic pathways based on the above metabolites, thereby protecting MIN6 damaged cells under high glucose conditions, increasing MIN6 glucose uptake, and promoting insulin secretion to further achieve the purpose of lowering blood glucose.

[0067] Example 2 A method for preparing a polysaccharide for the prevention or treatment of diabetes, comprising the following steps: (1) Take lemon peel, wash it, remove the yellow part of the outer peel, cut the remaining white pulp into small pieces, dry it at 60°C to constant weight, pulverize it into ultrafine powder, and pass it through an 80-mesh sieve to obtain lemon peel powder; (2) Add distilled water to the lemon peel powder. Lemon peel powder was added to distilled water at a ratio of 1:30 (g / mL). The pH was adjusted to 9.0 with sodium hydroxide. Cellulase was added for enzymatic hydrolysis, and the mixture was simultaneously sonicated. After treatment, the mixture was centrifuged, and the supernatant was collected. Five times its volume of anhydrous ethanol was added for alcohol precipitation. The mixture was allowed to stand at 4°C for 12 hours, then centrifuged again. The supernatant was removed, and the precipitate was freeze-dried to obtain crude lemon peel polysaccharide. The amount of cellulase added was 2.0%, the power of the sonication was 350W, the sonication temperature was 50°C, and the sonication time was 35 minutes. The crude polysaccharide from lemon peel was prepared into a 10 mg / mL polysaccharide solution by removing protein using the Sevage method. The solution was then dialyzed in distilled water using a dialysis bag with a molecular weight of 2000 Da. The residue inside the bag was freeze-dried to obtain the dialyzed polysaccharide. The DEAE-52 cellulose chromatography column is used to prepare a 10 mg / L polysaccharide solution by dialyzing polysaccharide, filtering it through a 0.45 μm filter membrane, and then loading it onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm). The solution is eluted with a 0.1 mol / L salt solution to obtain polysaccharide A. Polysaccharide A was prepared into a 20 mg / mL polysaccharide solution, filtered through a 0.45 μm filter membrane, and then loaded onto a Sephadex G-100 dextran gel chromatography column (2.6 cm × 30 cm). Ultrapure water was used for elution at a flow rate of 0.5 mL / min, 10 mL / tube. The absorbance of each tube was measured using the phenol-sulfuric acid method. The eluents from the absorption peaks were combined, concentrated, dialyzed, and freeze-dried to obtain polysaccharide B, which is lemon peel polysaccharide.

[0068] The molar percentages of Man, Rha, GlcA, GalA, Glc, Gal, and Ara in the lemon peel polysaccharide prepared in this embodiment were 0.92:4.02:0.95:54.94:1.63:11.62:25.89. The yield of this lemon polysaccharide reached 76.94±0.52%, exhibiting excellent inhibitory effects on α-glucosidase. At a concentration of 100 μg / mL, the lowest glucose uptake was 11.52±1.54 mmol / L. It significantly promoted glucose uptake by MIN6 cells and simultaneously increased insulin secretion, thus achieving the goal of regulating blood glucose.

[0069] Example 3 A method for preparing a polysaccharide for the prevention or treatment of diabetes, comprising the following steps: (1) Take lemon peel, wash it, remove the yellow part of the outer peel, cut the remaining white pulp into small pieces, dry it at 60°C to constant weight, pulverize it into ultrafine powder, and pass it through an 80-mesh sieve to obtain lemon peel powder; (2) Add distilled water to the lemon peel powder. Lemon peel powder was added to distilled water at a ratio of 1:25 (g / mL). The pH was adjusted to 9.0 with sodium hydroxide. Cellulase was added for enzymatic hydrolysis, and the mixture was simultaneously sonicated. After treatment, the mixture was centrifuged, and the supernatant was collected. Five times its volume of anhydrous ethanol was added for alcohol precipitation. The mixture was allowed to stand at 4°C for 12 hours, then centrifuged again. The supernatant was removed, and the precipitate was freeze-dried to obtain crude lemon peel polysaccharide. The amount of cellulase added was 1.0-2.0%, the power of the sonication was 250 W, the sonication temperature was 54°C, and the sonication time was 45 min. The crude polysaccharide from lemon peel was prepared into a 10 mg / mL polysaccharide solution by removing protein using the Sevage method. The solution was then dialyzed in distilled water using a dialysis bag with a molecular weight of 2000 Da. The residue inside the bag was freeze-dried to obtain the dialyzed polysaccharide. The DEAE-52 cellulose chromatography column is used to prepare a 10 mg / L polysaccharide solution by dialyzing polysaccharide, filtering it through a 0.45 μm filter membrane, and then loading it onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 30 cm). The solution is eluted with a salt solution of 0–0.5 mol / L to obtain polysaccharide A. Polysaccharide A was prepared into a 20 mg / mL polysaccharide solution, filtered through a 0.45 μm filter membrane, and then loaded onto a Sephadex G-100 dextran gel chromatography column (2.6 cm × 30 cm). Ultrapure water was used for elution at a flow rate of 0.5 mL / min, 10 mL / tube. The absorbance of each tube was measured using the phenol-sulfuric acid method. The eluents from the absorption peaks were combined, concentrated, dialyzed, and freeze-dried to obtain polysaccharide B, which was lemon peel polysaccharide.

[0070] The molar percentages of Man, Rha, GlcA, GalA, Glc, Gal, and Ara in the lemon peel polysaccharide prepared in this embodiment were 0.85:4.08:0.74:57.19:1.12:12.15:23.87. The yield of this lemon polysaccharide reached 76.69±0.47%, exhibiting excellent inhibitory effects on α-glucosidase. At a concentration of 100 μg / mL, the lowest glucose uptake was 11.69±1.39 mmol / L. It significantly promoted glucose uptake by MIN6 cells and simultaneously increased insulin secretion, thus achieving the goal of regulating blood glucose.

Claims

1. A method for preparing a polysaccharide for the prevention or treatment of diabetes, characterized in that, Includes the following steps: (1) Take lemon peel, wash it, remove the yellow part of the outer peel, cut the remaining white pulp into small pieces, dry it at 60°C to constant weight, pulverize it into ultrafine powder, and pass it through an 80-mesh sieve to obtain lemon peel powder; (2) Add distilled water to lemon peel powder, with a material-to-liquid ratio of 1:20~30, in g / mL. Adjust the pH to 9.0 with sodium hydroxide, add cellulase for enzymatic hydrolysis, and simultaneously perform ultrasonic treatment. After treatment, centrifuge, take the supernatant, add 5 times its volume of anhydrous ethanol for alcohol precipitation, let stand at 4℃ for 12h, then centrifuge, remove the supernatant, freeze-dry the precipitate to obtain crude lemon peel polysaccharide. The amount of cellulase added is 1.0~2.0%, the ultrasonic treatment power is 250~350W, the ultrasonic enzymatic hydrolysis temperature is 50~55℃, and the ultrasonic enzymatic hydrolysis time is 35~45 min to obtain crude lemon peel polysaccharide, denoted as LPs; After removing proteins from the crude polysaccharide of lemon peel using the Sevage method, a polysaccharide solution of 10 mg / mL was prepared. The solution was then dialyzed in distilled water using a dialysis bag with a molecular weight of 2000 Da. The residue in the bag was freeze-dried to obtain the dialyzed polysaccharide, denoted as LPS-T. The DEAE-52 cellulose chromatography column is used to prepare a 10 mg / L polysaccharide solution by dialyzing polysaccharide, filtering it through a 0.45 μm filter membrane, and then loading it onto the DEAE-52 cellulose anion exchange chromatography column. The solution is eluted with a 0.1 mol / L salt solution to obtain polysaccharide A, denoted as LPs-1. Polysaccharide A was prepared into a 20 mg / mL polysaccharide solution, filtered through a 0.45 μm filter membrane, and then loaded onto a Sephadex G-100 dextran gel chromatography column (2.6 cm × 30 cm). Ultrapure water was used for elution at a flow rate of 0.5 mL / min, 10 mL / tube. The absorbance of each tube was measured using the phenol-sulfuric acid method. The eluents from the absorption peaks were combined, concentrated, dialyzed, and freeze-dried to obtain polysaccharide B, which is lemon peel polysaccharide, denoted as LPs-1a.

2. A polysaccharide for the prevention or treatment of diabetes prepared by the method described in claim 1, characterized in that: The polysaccharide is lemon peel polysaccharide with an average molecular weight of 195.26 kDa, and consists of D-mannose (Man), D-galacturonic acid (GalA), D-glucose (Glc), D-galactose (Gal), L-rhamnose (Rha), D-glucuronic acid (GlcA), and L-arabinose (Ara).

3. The polysaccharide for preventing or treating diabetes as described in claim 2, characterized in that: In the lemon peel polysaccharide, Man, Rha, GlcA, GalA, Glc, Gal, and Ara are present in the following molar percentages: 0.85~0.92: 4.02~4.08: 0.74~0.95: 54.94~57.19: 1.12~1.63: 11.62~12.15: 23.87~25.89.

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