Peach fruit thinning oligosaccharide with blood sugar reducing activity, and extraction method and application thereof

Abstract

The application discloses a peach fruit-thinning oligosaccharide with blood sugar reducing activity, and an extraction method and application thereof, characterized in that W1-2 oligosaccharide is mainly composed of the following monosaccharides: glucose 29.73%, arabinose 23.47%, glucosamine 18.43%, galactose 11.1%, fructose 7.91%, mannose 6.82% and galactosamine 2.54%; and W2-2 oligosaccharide is mainly composed of the following monosaccharides: glucose 86.95%, arabinose 7.39%, galactose 3.53%, mannose 1.28% and fructose 0.85%. The preparation method comprises the following steps: adding peach fruit-thinning polysaccharide crude extract into pure water for extraction, and then alcohol precipitation overnight to obtain peach fruit-thinning crude polysaccharide; and the peach fruit-thinning polysaccharide is separated and purified, and then separated by an ultrafiltration tube to obtain two peach fruit-thinning oligosaccharides with high activity. The peach fruit-thinning oligosaccharides have the advantages of low molecular weight, strong water solubility, high purity and high blood sugar reducing activity.

Description

Technical Field

[0001] This invention relates to a polysaccharide with hypoglycemic activity, and more particularly to a peach sucrose oligosaccharide with hypoglycemic activity, its extraction method, and its application. Background Technology

[0002] Peaches are generally considered to nourish the skin and cleanse the digestive system, while also exhibiting effects such as nourishing yin and moistening the lungs, relieving coughs and phlegm. However, peach trees have a high fruit set rate, and excessively concentrated fruit can compete for nutrients from the tree, negatively impacting fruit quality and yield. Therefore, it is necessary to thin out some young fruit to rationally regulate nutrient competition among the fruits and ensure the overall yield and quality of the peaches. Currently, the utilization of thinned peach fruit in actual production is minimal; only a small portion is processed into food or animal feed. Most thinned peach fruit is discarded in the field, wasting resources and causing biological pollution, even breeding pathogens and accelerating the spread of fruit tree diseases. In fact, thinned peach fruit is a valuable plant resource that can be utilized, rich in polyphenols, polysaccharides, and other functional substances. Effective utilization of thinned peach fruit can not only reduce the waste of biological resources but also expand the development of the peach industry.

[0003] Polysaccharides are a class of substances with high polymerization degree and large molecular weight, widely found in animals, plants, and microbial cells. With complex structures, polysaccharides are not only one of the three major nutrients for life but also participate in various life activities, possessing diverse physiological activities such as antioxidation, anti-aging, hypoglycemia, hypolipidemia, antitumor, immunomodulation, anti-inflammation, and intestinal protection. Many studies have confirmed that plant polysaccharides participate in regulating glucose metabolism in the body. They can lower blood sugar by regulating the activity of glucose-metabolizing enzymes, promoting insulin secretion, increasing insulin sensitivity, antagonizing glucagon, regulating glycogen synthesis and degradation, and repairing damaged pancreatic islet cells. Furthermore, as natural drugs, plant polysaccharides have fewer toxic side effects and higher safety compared to currently available hypoglycemic drugs, making them an ideal alternative to synthetic drugs. Therefore, discovering bioactive polysaccharides from plants and using them in the development of functional foods and new clinical drugs has become a current hot topic in food and medical research.

[0004] Oligosaccharides, also known as oligosaccharides, are a general term for low-degree polymeric sugars formed by 2-10 monosaccharides linked by glycosidic bonds. Based on their health benefits, oligosaccharides can be divided into functional oligosaccharides and ordinary oligosaccharides. Functional oligosaccharides mainly possess bioactive functions such as antioxidation, hypoglycemia, hypolipidemia, and regulation of gut microbiota. Type II diabetes is a common chronic disease among middle-aged and elderly people, primarily caused by low insulin levels or insulin resistance. The side effects of commonly used drugs greatly limit their effectiveness in clinical treatment, necessitating the development of hypoglycemic drugs with fewer side effects. Functional oligosaccharides have various anti-diabetic mechanisms of action, such as inhibiting α-glucosidase, reducing insulin resistance, and regulating hormones and gut microbiota. Currently, the development and utilization of peach fruits mainly focuses on the extraction of polyphenols and polysaccharides. While the bioactivity of peach fruit polysaccharides has been studied, the large molecular weight of polysaccharides results in high solution viscosity, making it difficult to enter the body and limiting their application range. Research on peach fruit oligosaccharides and their bioactive functions has not yet been reported. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a peach fructooligosaccharide with low molecular weight, strong water solubility, high purity and high hypoglycemic activity, as well as its extraction method and application.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: a peach-fruit oligosaccharide with hypoglycemic activity, wherein the peach-fruit oligosaccharide includes W1-2 oligosaccharide and W2-2 oligosaccharide. The W1-2 oligosaccharide is mainly composed of the following monosaccharides and their mass percentages: glucose 25-33%, arabinose 23-27%, glucosamine 15-21%, galactose 9-14%, fructose 4-12%, mannose 3-9%, and glucosamine 1-4%. The W2-2 oligosaccharide is mainly composed of the following monosaccharides and their mass percentages: glucose 81-93%, arabinose 4-11%, galactose 2-5%, mannose 0.5-2.1%, and fructose 0.2-1.5%.

[0007] Preferably, the W1-2 oligosaccharide is mainly composed of the following monosaccharides and their mass percentages: glucose 29.73%, arabinose 23.47%, glucosamine 18.43%, galactose 11.1%, fructose 7.91%, mannose 6.82%, and glucosamine 2.54%, and the W2-2 oligosaccharide is mainly composed of the following monosaccharides and their mass percentages: glucose 86.95%, arabinose 7.39%, galactose 3.53%, mannose 1.28%, and fructose 0.85%.

[0008] Furthermore, the monosaccharides of the W1-2 oligosaccharides are linked by at least one of the following glycosidic bonds: 1-arabinose, 1,5-arabinose, 1-glucose, 1-galactose, 1,3,5-arabinose, 1,4-glucose, 1,4-galactose, and 1,4,6-galactose.

[0009] Furthermore, the monosaccharides of the W2-2 oligosaccharide are linked by at least one of the following glycosidic bonds: 1-arabinose, 1,3-galactose, 1,5-arabinose, 1-glucose, 1-galactose, 1,3,5-arabinose, 1,2,5-arabinose, 1,2-arabinose, 1,3-glucose, 1,4-mannose, and 1,6-mannose.

[0010] This invention also provides a method for preparing the above-mentioned peach fructooligosaccharide with hypoglycemic activity, comprising the following steps:

[0011] Step 1, Raw material pretreatment: Peach fruit powder is added to an ethanol aqueous solution and ultrasonically removed to remove peach fruit polyphenols, resulting in crude extract of peach fruit polysaccharide.

[0012] Step 2, Extraction of crude polysaccharides from peach and fruit: Add the crude polysaccharide extract from peach and fruit obtained in Step 1 to pure water at a mass ratio of 1:30 and extract for 35-40 minutes at 100-130℃ and pH 4-6. After extraction, filter while hot, centrifuge the filtrate, concentrate by rotary evaporation, add 3 times the volume of ethanol to the concentrate, precipitate overnight at 4℃. After precipitation, dissolve the precipitate in water, remove the residual ethanol by rotary evaporation, pour into a plate, and freeze-dry to obtain crude polysaccharides from peach and fruit.

[0013] Step 3: Isolation and purification of peach and fruit polysaccharides: Dissolve the crude peach and fruit polysaccharides in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL. Vortex to promote dissolution, centrifuge to remove the insoluble matter at the bottom, and then filter the supernatant through a 0.45 μm filter membrane to obtain the loading solution. Slowly drop all the loading solution into an anion exchange resin chromatography column using a dropper, using pure water as the eluent, and collect the second and third elution peak components, which both contain peach and fruit oligosaccharides.

[0014] Step 4: Ultrafiltration separation of peach and fruit oligosaccharides: The two components containing peach and fruit oligosaccharides obtained in Step 3 were separated using a 3kDa ultrafiltration tube, retaining the smaller molecular weight fractions to obtain two highly active peach and fruit oligosaccharides, named W1-2 oligosaccharide and W2-2 oligosaccharide, respectively.

[0015] Further, step 1 specifically involves: freeze-drying peach fruits for 48 hours, crushing them with a grinder, passing them through a 60-mesh sieve to obtain peach fruit powder, adding the peach fruit powder to a 70% ethanol aqueous solution at a mass-volume ratio of 1g:50mL, sonicating for 25 minutes at a power of 610W and a temperature of 50℃, filtering the residue, and air-drying it at room temperature to obtain crude extract of peach fruit polysaccharides.

[0016] Further, step 2 specifically involves: adding the crude peach and fruit polysaccharide extract obtained in step 1 to pure water at a mass ratio of 1:30, and extracting for 40 min at 110℃ and pH 5.0; after extraction, filtering while hot, centrifuging the filtrate at 9000 rpm for 10 min, then concentrating by rotary evaporation, adding 3 times the volume of ethanol to the concentrate, and precipitating overnight at 4℃; after precipitation, dissolving the precipitate in water, removing residual ethanol by rotary evaporation, then pouring into plates and freeze-drying to obtain the crude peach and fruit polysaccharide. Under optimal extraction conditions, the product yield was 10.63%, and the inhibition rate against α-glucosidase was 76.58%.

[0017] Further, step 3 specifically involves: dissolving the crude polysaccharide of peach and succulent in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; vortexing to promote dissolution; centrifuging at 8000 r / min for 10 min to remove the insoluble matter at the bottom; and then filtering the supernatant through a 0.45 μm filter membrane to obtain the loading solution; taking 10 mL of the loading solution and slowly dropping it onto a DEAE anion exchange resin chromatography column using a dropper; eluting with pure water; controlling the eluent flow rate at 1.0 mL / min; collecting 5.0 mL of eluent from each tube; collecting 45 tubes; and collecting tubes 16-22 and 23-35 separately to obtain two components containing peach and succulent oligosaccharides.

[0018] The present invention also provides the application of the above-mentioned peach fruit oligosaccharide in the preparation of hypoglycemic drugs.

[0019] The present invention also provides the application of the above-mentioned peach and fruit oligosaccharides in the preparation of α-glucosidase inhibitors.

[0020] Compared with the prior art, the advantages of the present invention are as follows:

[0021] 1. This invention uses ultrasonic extraction with 70% ethanol aqueous solution to remove polyphenols from peach fruits and vegetables, thus avoiding browning during subsequent polysaccharide extraction from peach fruits and vegetables;

[0022] 2. By using a grinder to pulverize and classifying the fruit, a peach powder with uniform, small, and stable particles is obtained, which increases the specific surface area and thus improves the yield of peach polysaccharides.

[0023] 3. Peach fruit polysaccharides were extracted using hot water extraction. The effects of temperature, pH, and time on the yield and α-glucosidase inhibition rate were investigated. The optimal process was found to be 110℃, pH 5.0, and 40 min, at which the yield was 10.63% and the inhibition rate of α-glucosidase was 76.58%.

[0024] 4. Two peach fructooligosaccharides were obtained by ion exchange chromatography and ultrafiltration separation. The molecular weight of W1-2 was 724 Da and the molecular weight of W2-2 was 427 Da. They showed good hypoglycemic activity and their inhibition rates against α-glucosidase reached 88.38% and 94.82%, respectively.

[0025] In summary, this invention discloses for the first time a peach fruit oligosaccharide with hypoglycemic activity, its extraction method, and its application. Utilizing peach fruit byproducts from peach processing as raw materials, the method extracts and purifies peach fruit polysaccharides. The purified components are then separated by ultrafiltration to obtain a peach fruit oligosaccharide with low molecular weight, high water solubility, high purity, and high hypoglycemic activity. This method fully leverages the advantages of large quantities of peach fruit waste, low cost, and near-natural properties, solving the problem of efficient utilization of processing waste and providing a reference for the further development and utilization of peach fruit resources. Attached Figure Description

[0026] Figure 1 The pure water elution curve of peach fruit polysaccharide extracted in Specific Example 1;

[0027] Figure 2 The molecular weight results of the peach fructooligosaccharides isolated in Specific Example 1;

[0028] Figure 3 The effect of different extraction conditions on the yield of peach fruit polysaccharides and the inhibition rate of α-glucosidase in Specific Example 2 is shown, where A is the extraction temperature, B is the extraction pH, and C is the extraction time.

[0029] Figure 4 The monosaccharide composition of peach fructooligosaccharides W1-2 in specific embodiment three;

[0030] Figure 5 The monosaccharide composition of peach fructooligosaccharide W2-2 in specific embodiment three;

[0031] Figure 6 The inhibitory effect of peach sucrose oligosaccharides on α-glucosidase in specific embodiment four;

[0032] Figure 7 The effect of different concentrations of W1-2 on cellular glucose uptake is illustrated in Specific Example 4.

[0033] Figure 8The effect of different concentrations of W2-2 on cellular glucose uptake is illustrated in Specific Example 4.

[0034] Figure 9 The effect of peach fruit polysaccharide on body weight in type II diabetic mice, as shown in Example 5.

[0035] Figure 10 The effect of peach fruit polysaccharide on fasting blood glucose in type II diabetic mice, as shown in Specific Example 5;

[0036] Figure 11 The linear relationship between blood glucose and time in mice of different experimental groups after oral administration of glucose solution in Specific Example 5;

[0037] Figure 12 The effect of peach fruit polysaccharide on glucose tolerance in specific embodiment five. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Specific Embodiment 1: A method for preparing highly active peach fructooligosaccharides, comprising the following steps:

[0040] Step 1, Raw material pretreatment: Freeze-dry peach fruits for 48 h, grind them in a grinder, and pass them through a 60-mesh sieve to obtain peach fruit powder. Add the peach fruit powder to a 70 vt% ethanol aqueous solution at a mass-volume ratio of 1 g (dry basis): 50 mL. Under the conditions of 610 W power, 50 °C temperature and 25 min time, ultrasonically remove the peach fruit polyphenols, filter the residue, and air dry at room temperature to obtain crude extract of peach fruit polysaccharides.

[0041] Step 2, Extraction of crude polysaccharides from peach and fruit: The crude polysaccharide extract from peach and fruit obtained in Step 1 was added to pure water at a mass ratio of 1:30 and extracted at 110℃ and pH 5.0 for 40 min. After extraction, the extract was filtered through a silk screen while hot, and the filtrate was centrifuged at 9000 rpm for 10 min and concentrated by rotary evaporation. Three times the volume of ethanol was added to the concentrate, and the mixture was precipitated overnight at 4℃. After precipitation, the precipitate was dissolved in water, and the residual ethanol was removed by rotary evaporation. The precipitate was then poured into a plate and freeze-dried to obtain crude polysaccharides from peach and fruit.

[0042] Step 3: Isolation and purification of peach and fruit polysaccharides: Weigh 100 mg of crude peach and fruit polysaccharides into a 50 mL centrifuge tube, add 10 mL of distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, vortex to dissolve, centrifuge at 8000 r / min for 10 min, remove the insoluble matter at the bottom, and then filter the supernatant through a 0.45 μm filter membrane to obtain the loading solution; slowly drop the entire loading solution onto a DEAE anion exchange resin chromatography column using a dropper, elute with pure water, control the eluent flow rate at 1.0 mL / min, collect 5.0 mL of eluent from each tube, and collect 45 tubes. The pure water elution curve of peach and fruit polysaccharides is shown below. Figure 1 As shown, tubes 16-22 and 23-35 were collected and named W1 and W2 respectively;

[0043] Step 4: Ultrafiltration separation of peach fruit polysaccharides: W1 and W2 obtained in step 3 were separated using a 3kDa ultrafiltration tube, retaining the smaller molecular weight fractions to obtain two highly active peach fruit oligosaccharides, named W1-2 and W2-2 respectively.

[0044] The homogeneity of the components was assessed by determining their molecular weights. The chromatographic conditions were as follows: High-performance liquid chromatography (Agilent 1260) equipped with a 2414 differential detector, and an Ultrahidedrogel column. TM The column (7.8 mm × 300 mm) was used with a mobile phase of 0.1 mol / L sodium nitrate solution (NaNO3). The column temperature and detector temperature were both 45 ℃, the mobile phase flow rate was 0.5 mL / min, and the injection volume was 15 μL. A series of pullulan polysaccharides with known molecular weights of 6.4, 9.8, 21.9, 49.4, 107, 218, 354, and 692 kDa were used as standards (concentration 10 mg / mL). The logarithm of the molecular weight was plotted on the x-axis as retention time. 10 Using Mw as the ordinate, a standard curve was constructed. The results are as follows: Figure 2 As shown, the molecular weight of W1-2 is 724 Da, and the molecular weight of W2-2 is 451 Da.

[0045] Specific Implementation Example 2 and Specific Implementation Example 1: Optimization of the method for extracting oligosaccharides from peach fruit.

[0046] Method for determining the α-glucosidase inhibition rate: Using a pipette, pipette 80 μL of 1 mg / mL, 2 mg / mL, 4 mg / mL, and 8 mg / mL peach fruit polysaccharide solution and 60 μL of α-glucosidase solution, respectively, mix well, vortex, and incubate at 37℃ for 10 min; then add 100 μL of 4-nitrophenyl-β-D-galactopyranoside, and continue incubation at 37℃ for 15 min; finally, add 1 mL of 0.1 mol / L Na₂CO₃ solution to terminate the reaction, and measure the absorbance at 400 nm using a microplate reader. Calculate the inhibition rate according to the formula.

[0047] In the formula, As is the absorbance of the mixed solution of the sample solution and the 4-nitrophenyl-β-D-galactopyranoside solution and the α-glucosidase solution; Ac is the absorbance of the mixed solution in which PBS buffer solution replaces the α-glucosidase solution; and Ab is the absorbance of the mixed solution in which PBS buffer solution replaces the sample solution.

[0048] 1. The yield and α-glucosidase inhibition rate of the polysaccharides extracted from each group in the single-factor experiment were evaluated.

[0049] Extraction temperature optimization: The extraction method for peach sucrose oligosaccharides is the same as step 2 of the specific embodiment above, except that the extraction pH is fixed at 5, the extraction time is 60 min, and different extraction temperatures are set at 80℃, 90℃, 100℃, 110℃, and 120℃. The results are as follows... Figure 3 As shown in Figure A, the inhibitory effect of α-glucosidase gradually increases with increasing extraction temperature. When the temperature rises to 100℃, the inhibition rate reaches its maximum. Further increases in temperature will decrease the inhibition rate. Although the polysaccharide yield is highest at 110℃, its inhibition rate is significantly lower than that at 100℃. Therefore, 100℃ is chosen as the optimal extraction temperature.

[0050] pH optimization for extraction: The extraction method for peach sucrose oligosaccharides is the same as step 2 of the specific embodiment above, except that the extraction temperature is fixed at 100℃, the extraction time is 60 min, and different extraction pH values ​​are set at 3, 4, 5, 6, and 7. The results are as follows... Figure 3 As shown in Figure B, except for pH=5, there was no significant difference in polysaccharide yield at other pH levels. The α-glucosidase inhibition rate was highest at pH=6, so pH=6 was selected as the optimal extraction pH.

[0051] Extraction time optimization: The extraction method for peach sucrose oligosaccharides is the same as step 2 of the specific embodiment above, except that the extraction temperature is fixed at 100℃, the extraction pH is 6, and different extraction times are set at 30, 40, 50, 60, and 70 min. The results are as follows... Figure 3As shown in Figure C, the polysaccharide extracted for 40 min exhibited a significantly higher α-glucosidase inhibition rate than other groups, and 40 min was selected as the optimal extraction time.

[0052] 2. Orthogonal experimental design

[0053] Based on the single-factor experiments, extraction temperature (90℃, 100℃, 110℃), pH (5, 6, 7), and time (30, 40, 50) were selected as the factors to be investigated. Orthogonal experiments were used to optimize the extraction process. The orthogonal experimental design and results are shown in Table 1.

[0054] Table 1. Results of the orthogonal experiment

[0055]

[0056] In the orthogonal experiment, a comprehensive scoring method was used to analyze the experimental results. The results showed that the order of factors was A>C>B, namely temperature, time, and pH. The optimal combination was A3C2B1, under which the polysaccharide yield was 10.63% and the α-glucosidase inhibition rate was 76.58%. Therefore, the optimal extraction process for peach fruit polysaccharides was determined to be an extraction temperature of 110 ℃, an extraction pH of 5.0, and an extraction time of 40 min.

[0057] Structural analysis of peach fructooligosaccharides W1-2 and W2-2 extracted by the method of specific embodiment 3 and specific embodiment 1.

[0058] 1. Monosaccharide Composition Analysis: Solutions of W1-2 and W2-2 were prepared at 2 mg / mL. 1 mL of each solution was placed in a test tube, and 0.17 mL of concentrated trifluoroacetic acid was added. The solutions were hydrolyzed at 121℃ for 120 min. After hydrolysis, the solutions were dried under nitrogen, reconstituted with 1 mL of water, filtered through a 0.22 μm filter, and diluted 20-fold to a concentration of 100 ppm. The monosaccharide composition of the samples was determined by ion-exchange chromatography. A high-performance ion chromatography system (Dionex ICS-5000, Dionex Corporation, USA) equipped with a pulsed amperometric detector was used for detection. The column was a CarboPac PA20 (2×250 mm, id, 5 μm). The mobile phases were ultrapure water, 0.25 mol / L NaOH solution, and 1.0 mol / L sodium acetate (NaAc) solution. The flow rate was 0.5 mL / min.

[0059] Figure 4 It is composed of monosaccharides of W1-2, which is mainly composed of 9 monosaccharides, including: glucose (Glc) p 29.73%, arabinose (Ara) f 23.47%, glucosamine 18.43%, galactose (Gal) p) 11.1%, fructose 7.91%, mannose 6.82%, aminogalactose 2.54%, and the molecular weight of W1-2 is 724 Da. Figure 5 It is composed of monosaccharides of W2-2, which is mainly composed of glucose (86.95%), arabinose (7.39%), galactose (3.53%), mannose (1.28%), and fructose (0.85%). The molecular weight of W2-2 is 451 Da.

[0060] 2. Methylation Analysis: Dissolve W1-2 and W2-2 separately in water to prepare solutions of 1 mg / mL. Take 100 μL and place it in a threaded reaction tube, air dry under nitrogen, add 165 μL of anhydrous dimethyl sulfoxide (DMSO), seal, and sonicate for 30 min. Under infrared light, add NaOH to the anhydrous DMSO, grind, and prepare a slurry of 120 mg / mL. Then add 150 μL-300 μL of the slurry to the reaction tube, sonicate under ice bath for 50 min, and after complete dissolution, add 50 μL-100 μL of iodomethane, sonicate for 30 min. Repeat this operation 3 times to ensure complete methylation. Extract the methylated polysaccharide repeatedly with dichloromethane, combine the dichloromethane layers and wash with water 2-3 times to remove incompletely methylated polysaccharides. Discard the aqueous layer, add anhydrous sodium sulfate to the dichloromethane layer, and remove the dichloromethane by nitrogen blowing to obtain the methylated polysaccharide.

[0061] 1.0 mL of 2.0 mol / L trichloroacetic acid was added to a reaction tube containing dried methylated polysaccharide. The tube was sealed and placed in an oil bath at 120 °C for 90 min for hydrolysis. After cooling, inositol (0.1 mg / mL) was added as an internal standard, and the mixture was dried under nitrogen. The polysaccharide was then reconstituted with water, and sodium borodeide was added for reduction until no more bubbles were produced upon contact with acetic acid, indicating complete reduction. Methanol was added to the reduced reaction solution, and the mixture was purged with nitrogen twice. Then, 625 μL of acetic anhydride was added, and the reaction was carried out at 100 °C for 150 min. After acetylation, the mixture was dried under nitrogen, reconstituted with dichloromethane, and dried over anhydrous sodium sulfate to obtain a partially methylated sugar alcohol acetate derivative (PMAA), which was then analyzed by GC-MS. The results are shown in Tables 2 and 3 below.

[0062] Table 2. Methylation analysis results of W1-2

[0063]

[0064] GC-MS was used to analyze the methylated W1-2 and compared with a standard database to determine the glycosidic bond types. The results are shown in Table 2. After methylation, W1-2 produced glucose, galactose, and arabinose residues, with corresponding proportions of 41.63%, 32.21%, and 26.16%, respectively, consistent with the monosaccharide composition results. The methylation results of W1-2 indicated the presence of eight glycosidic bonds: 1-arabinose (1-Araf), 1,5-arabinose (1,5-Araf), 1-glucose (1-Glcp), 1-galactose (1-Galp), 1,3,5-arabinose (1,3,5-Araf), 1,4-glucose (1,4-Glcp), 1,4-galactose (1,4-Galp), and 1,4,6-galactose (1,4,6-Galp).

[0065] Table 3. Methylation analysis results of W2-2

[0066]

[0067] As shown in Table 3, after methylation, the glycosidic bonds of W2-2 are broken, generating glucose residues, arabinose residues, galactose residues and mannose residues, with corresponding proportions of 69.87%, 10.77%, 11.92% and 7.44%, respectively, which is consistent with the results of monosaccharide composition. Methylation results of W2-2 showed that there are 11 glycosidic bonds in W2-2, namely 1-arabinose (1-Araf), 1,3-galactose (1,3-Galp), 1,5-arabinose (1,5-Araf), 1-glucose (1-Glcp), 1-galactose (1-Galp), 1,3,5-arabinose (1,3,5-Araf), 1,2,5-arabinose (1,2,5-Araf), 1,2-arabinose (1,2-Araf), 1,3-glucose (1,3-Glcp), 1,4-mannose (1,4-Manp), and 1,6-mannose (1,6-Manp).

[0068] Activity analysis of peach sucrose oligosaccharides W1-2 and W2-2 extracted by the method of specific embodiment 4 and specific embodiment 1.

[0069] 1. The inhibitory effect of W1-2 and W2-2 on α-glucosidase.

[0070] Method for determining α-glucosidase inhibition rate: Using a pipette, pipette 80 μL of 1.0, 2.0, 4.0, and 8.0 mg / mL peach fruit polysaccharide solution and 60 μL of α-glucosidase solution, respectively, mix well, vortex, and incubate at 37℃ for 10 min; then add 100 μL of 4-nitrophenyl-β-D-galactopyranoside, and continue incubation at 37℃ for 15 min; finally, add 1 mL of 0.1 mol / L Na₂CO₃ solution to terminate the reaction, and measure the absorbance at 400 nm using a microplate reader. Calculate the inhibition rate according to the formula.

[0071] In the formula, As is the absorbance of the mixed solution of the sample solution and the 4-nitrophenyl-β-D-pyranogalactopyranoside solution and the α-glucosidase solution; Ac is the absorbance of the mixed solution in which PBS buffer solution replaces the α-glucosidase solution; and Ab is the absorbance of the mixed solution in which PBS buffer solution replaces the sample solution.

[0072] like Figure 6 As shown, within the concentration range of 1.0–8.0 mg / mL, the inhibition rates of α-glucosidase by W1-2 and W2-2 increased with increasing concentration. At a polysaccharide concentration of 8 mg / mL, the inhibitory abilities of different components on α-glucosidase activity were in the following order: W2-2 (94.82%) > W1-2 (88.38%) > peach fruit crude polysaccharide (60.12%, abbreviated as PPS). The inhibition rates of α-glucosidase by W2-2 and W1-2 were significantly higher than those by peach fruit crude polysaccharide. The IC50 values ​​for inhibiting α-glucosidase by W1-2 and W2-2 were calculated. 50 The values ​​were 0.57 mg / mL and 0.37 mg / mL, respectively, which were significantly lower than PPS's 5.46 mg / mL, indicating that W1-2 and W2-2 had better hypoglycemic activity than PPS.

[0073] 2. Effects of W1-2 and W2-2 on glucose uptake in insulin-resistant HepG2 cells.

[0074] Experimental methods: A model group (IR-HepG2 cells + sample solutions of different concentrations), a control group (IR-HepG2 cells + cell culture medium, Model), and a blank group (HepG2 cells + cell culture medium, CK) were set up, with 6 replicates per group. HepG2 cells in the logarithmic growth phase were diluted with culture medium to a concentration of 2 × 10⁻⁶. 5Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h until adherence. After cell attachment, the old culture medium was removed, and the cells were treated with 12 mM glucosamine for 16 h to construct an IR-HepG2 model (IR-HepG2 refers to a cell model exhibiting insulin resistance; HepG2 refers to cells in a normal state). After successful construction, the culture medium in each well was discarded, and the cells were washed twice with PBS. 100 μL of cell culture medium was added to the blank group and the control group. 100 μL of W1-2 (0.25, 0.5, 1, 2 mg / mL) and W2-2 (0.125, 0.25, 0.5, 1, 2 mg / mL) were added to the model group, respectively. After culturing in a 37℃, 5% CO2 environment for 24 h, the culture medium was discarded. The cells were washed twice with PBS solution to remove residual drug solution. 100 μL of high-glucose DMEM culture medium was added to each well of each group and cultured for 24 h. The glucose content of each well was measured using a glucose detection kit (GOD-POD method). The glucose uptake of the model group, control group, and blank group was calculated.

[0075] Figure 7 and Figure 8 The effects of different concentrations of W1-2 and W2-2 on glucose uptake in IR-HepG2 cells were compared. The selected concentrations had no effect on proliferation. Results are as follows: Figure 7 As shown, compared with the model group, W1-2 at concentrations of 0.5–2 mg / mL significantly increased intracellular glucose consumption in HepG2 cells, with increases of 1.47-fold, 2.29-fold, and 2.46-fold, respectively. The results are as follows: Figure 8 As shown, W2-2 at concentrations of 0.25–1 mg / mL significantly increased glucose consumption in HepG2 cells, with increases of 1.60-fold, 2.19-fold, and 2.16-fold, respectively. These results indicate that both W1-2 and W2-2 have the potential to improve insulin resistance in HepG2 cells.

[0076] Specific Example 5: In vivo antidiabetic effect of crude polysaccharide from peach fruit prepared by the method of Specific Example 1.

[0077] 1. Animal Experiment Design: Twenty-four 6-week-old SPF-grade male C57BL / 6 mice, weighing (20 ± 2 g), were purchased from Shanghai Silex Laboratory Animal Co., Ltd. All mice were housed at the Ningbo University Animal Experiment Center under a relative humidity of 50 ± 10% and a temperature of 23 ± 1 ℃, with a 12-hour light-dark cycle. Three mice were housed per cage with free access to food and water. All experimental procedures were strictly conducted in accordance with the "Regulations on the Management of Laboratory Animals in China" and approved by the Welfare and Ethics Committee of the Ningbo University Laboratory Animal Center.

[0078] After one week of acclimatization, six mice were randomly selected as the normal control group (NC) and fed a normal diet. The remaining 18 mice were used as the model group and fed a high-fat diet (HFD) to induce obesity and insulin resistance. After six weeks of feeding, all mice were fasted overnight but allowed water for 12 hours. Mice in the model group were intraperitoneally injected with a 50 mg / kg BW solution of 1% freshly prepared low-temperature streptozotocin (STZ) (dissolved in 0.1 mol / L citrate buffer, pH=4.5), while mice in the NC group were intraperitoneally injected with the same dose of citrate buffer. 72 hours after the first intraperitoneal injection, all mice were again fasted overnight but allowed water for 12 hours. Mice in the model group and NC group were intraperitoneally injected with a 50 mg / kg BW solution of freshly prepared low-temperature STZ and an equal dose of citrate buffer, respectively. 72 hours after the second intraperitoneal injection, fasting blood glucose levels were rapidly measured in each group using an ACCU-CHEK blood glucose meter via tail clipping. A fasting blood glucose level greater than or equal to 11.1 mmol / L was considered a successful establishment of the T2DM mouse model. Successfully constructed type 2 diabetes mellitus (T2DM) mice were randomly divided into three groups of six mice each: a diabetes model group (DM group), a low-dose peach and fruit crude polysaccharide intervention group (LPP group, 200 mg / kg BW), and a high-dose peach and fruit crude polysaccharide intervention group (HPP group, 400 mg / kg BW). Mice in the LPP and HPP groups were administered different doses of peach and fruit crude polysaccharide solution dissolved in 0.9% sterile saline via gavage once daily, while mice in the NC and DM groups were administered the same dose of 0.9% sterile saline via gavage. The intervention lasted for four weeks, and the body weight and fasting blood glucose (FBG) of each group were recorded weekly. After the experiment, all mice were fasted overnight, euthanized by cervical dislocation under isoflurane anesthesia, and biological samples were collected for further analysis.

[0079] 2. Measurement of fasting blood glucose and glucose tolerance: All mice were fasted overnight for 12 hours with free access to water on a fixed day each week. Fasting blood glucose was measured in each group of mice the following day by tail clipping. Oral glucose tolerance test was performed in the last week of the peach fructose intervention; the area under the curve (AUC) of the time-blood glucose curve was calculated using GraphPad Prism 9.0 according to the trapezoidal rule.

[0080] 3. Experimental results: Effects of peach fruit polysaccharide on body weight and fasting blood glucose.

[0081] Effects of peach fruit polysaccharide on body weight in type II diabetic mice Figure 9 As shown, during the experiment, the body weight of mice in the DM group continuously decreased as their diabetes worsened, while the body weight of mice in the NC group steadily increased. At the beginning of the experiment, there was no significant difference in body weight among the LPP, HPP, and DM groups. p >0.05), after 2 weeks of intervention with peach fruit crude polysaccharide, the weight loss of mice in the LPP and HPP groups was significantly lower than that in the DM group ( p<0.05); After 4 weeks of intervention with peach fruit polysaccharide, the body weight of mice in both the LPP and HPP groups increased, a trend consistent with that of the NC group; at the end of the experiment, there was no significant difference in body weight between the LPP and HPP groups and the NC group. p >0.05). These results indicate that peach fruit polysaccharide can alleviate the degree of weight loss in diabetic mice in the early stage of the experiment; and can reverse the trend of weight loss and bring the weight back to normal levels in the later stage of the experiment.

[0082] The effect of peach fruit polysaccharide on hyperglycemia in type 2 diabetes mellitus (T2DM) mice was evaluated by measuring fasting blood glucose (FBG) weekly during the experiment. Figure 10 As shown, at week 0, there was no significant difference in fasting blood glucose levels among the DM, LPP, and HPP groups ( p >0.05), and significantly higher than the NC group mice ( p <0.01 indicates successful model construction, with no significant difference in FBG among the groups. One week after intervention with peach fruit crude polysaccharide, FBG in the LPP and HPP groups began to decrease, showing a significant difference compared to the DM group. p <0.05). As diabetes worsened, fasting blood glucose (FBG) in the DM group mice gradually increased from week 1 to week 3. Intervention with peach and fruit crude polysaccharide significantly inhibited the increase in FBG and alleviated hyperglycemia. After 4 weeks of peach and fruit crude polysaccharide intervention, compared with the DM group, the FBG in the LPP and HPP groups was significantly reduced (p<0.01), and high-dose peach and fruit crude polysaccharide showed a better hypoglycemic effect.

[0083] 4. Experimental results: Effect of peach fruit polysaccharide on sugar tolerance.

[0084] like Figure 11 As shown, blood glucose levels in all experimental groups of mice rose rapidly after oral administration of glucose solution, reaching a peak at 30 min, and then gradually decreased over the next 90 min. Compared with the other three groups, the NC group mice were more sensitive to changes in glucose, and their blood glucose returned to normal within 120 min. However, the blood glucose levels in the DM group mice remained high after 120 min, indicating that glucose tolerance in untreated T2DM mice was severely impaired. Peach and fruit crude polysaccharide intervention significantly reduced the peak blood glucose level in T2DM mice. At the end of the oral glucose tolerance test (OGTT), the blood glucose levels in the high- and low-dose peach and fruit crude polysaccharide intervention groups were significantly lower than those in the DM group (p<0.05). Further quantification of the mice's glucose tolerance was achieved using the area under the curve (AUC), such as... Figure 12As shown, the AUC values ​​of all diabetic mice were significantly higher than those of the NC group (p<0.01). Meanwhile, the AUC values ​​of diabetic mice decreased sequentially in the DM, LPP, and HPP groups, indicating that all doses of peach fruit polysaccharide significantly improved glucose tolerance in diabetic mice in a dose-dependent manner.

[0085] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A peach fructooligosaccharide, characterized in that... W1-2 oligosaccharides are mainly composed of the following monosaccharides and their mass percentages: glucose 29.73%, arabinose 23.47%, glucosamine 18.43%, galactose 11.1%, fructose 7.91%, mannose 6.82%, and glucosamine 2.54%. W2-2 oligosaccharides are mainly composed of the following monosaccharides and their mass percentages: glucose 86.95%, arabinose 7.39%, galactose 3.53%, mannose 1.28%, and fructose 0.85%. The monosaccharides in W1-2 oligosaccharides are linked by at least one of the following glycosidic bonds: 1-arabinose. The W2-2 oligosaccharide comprises 1,5-arabinose, 1-glucose, 1-galactose, 1,3,5-arabinose, 1,4-glucose, 1,4-galactose, and 1,4,6-galactose, wherein the monosaccharides are linked by at least one of the following glycosidic bonds: 1-arabinose, 1,3-galactose, 1,5-arabinose, 1-glucose, 1,3,5-arabinose, 1,2,5-arabinose, 1,2-arabinose, 1,3-glucose, 1,4-mannose, and 1,6-mannose. The W1-2 oligosaccharide has a molecular weight of 724 Da, and the W2-2 oligosaccharide has a molecular weight of 451 Da.

2. A method for preparing peach sucrose oligosaccharide according to claim 1, characterized in that... Includes the following steps: Step 1, Raw material pretreatment: Peach fruit powder is added to an ethanol aqueous solution and ultrasonically removed to remove peach fruit polyphenols, resulting in crude extract of peach fruit polysaccharide. Step 2, Extraction of crude polysaccharides from peach and fruit: Add the crude polysaccharide extract from peach and fruit obtained in Step 1 to pure water at a mass ratio of 1:30 and extract for 35-40 minutes at 100-130℃ and pH 4-6. After extraction, filter while hot, centrifuge the filtrate, concentrate by rotary evaporation, add 3 times the volume of ethanol to the concentrate, precipitate overnight at 4℃. After precipitation, dissolve the precipitate in water, remove the residual ethanol by rotary evaporation, pour into a plate, and freeze-dry to obtain crude polysaccharides from peach and fruit. Step 3: Isolation and purification of peach and fruit polysaccharides: Dissolve the crude peach and fruit polysaccharides in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL. Vortex to promote dissolution, centrifuge to remove the insoluble matter at the bottom, and then filter the supernatant through a 0.45 μm filter membrane to obtain the loading solution. Slowly drop all the loading solution into an anion exchange resin chromatography column using a dropper, using pure water as the eluent, and collect the second and third elution peak components, which both contain peach and fruit oligosaccharides. Step 4: Ultrafiltration separation of peach and fruit oligosaccharides: The two components containing peach and fruit oligosaccharides obtained in Step 3 were separated using a 3kDa ultrafiltration tube, retaining the smaller molecular weight fractions to obtain two highly active peach and fruit oligosaccharides, named W1-2 oligosaccharide and W2-2 oligosaccharide, respectively.

3. The method for preparing peach fructooligosaccharides according to claim 2, characterized in that... Step 1 is as follows: freeze-dry the peach fruit for 48 hours, grind it in a grinder, pass it through a 60-mesh sieve to obtain peach fruit powder, add the peach fruit powder to a 70% ethanol aqueous solution at a mass-volume ratio of 1g:50mL, sonicate for 25 minutes at a power of 610W and a temperature of 50℃, filter the residue, and air-dry it at room temperature to obtain crude extract of peach fruit polysaccharides.

4. The method for preparing peach fructooligosaccharides according to claim 2, characterized in that... Step 2 is as follows: The crude extract of peach fruit polysaccharide obtained in Step 1 is added to pure water at a mass ratio of 1:30 and extracted at 110℃ and pH 5.0 for 40 min. After extraction, the extract is filtered while hot, and the filtrate is centrifuged at 9000 rpm for 10 min and concentrated by rotary evaporation. Three times the volume of ethanol is added to the concentrate, and the extract is precipitated overnight at 4℃. After precipitation, the precipitate is dissolved in water, and the residual ethanol is removed by rotary evaporation. The precipitate is then poured into a plate and freeze-dried to obtain the crude peach fruit polysaccharide.

5. The method for preparing peach fructooligosaccharides according to claim 2, characterized in that... Step 3 is as follows: Dissolve the crude polysaccharide of peach fruit in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL. Vortex to promote dissolution. Centrifuge at 8000 r / min for 10 min to remove the insoluble matter at the bottom. Then filter the supernatant through a 0.45 μm filter membrane to obtain the loading solution. Take 10 mL of the loading solution and slowly drop it into a DEAE anion exchange resin chromatography column. Elute with pure water, controlling the eluent flow rate at 1.0 mL / min. Collect 5.0 mL of eluent from each tube. Collect 45 tubes. Collect tubes 16-22 and 23-35 separately to obtain two components containing peach fruit oligosaccharides.

6. The use of the peach folio oligosaccharide according to claim 1 in the preparation of hypoglycemic drugs.

7. The use of the peach folio oligosaccharide according to claim 1 in the preparation of an α-glucosidase inhibitor.

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