Peach fruit oligosaccharide with hypoglycemic activity as well as extraction method and application of peach fruit oligosaccharide

Abstract

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

Description

Technical Field

[0001] The invention relates to a polysaccharide with blood sugar lowering activity, in particular to a peach fructooligosaccharide with blood sugar lowering activity and an extraction method and application thereof. Background Art

[0002] Peach fruit is generally believed to nourish the skin, clean the stomach and intestines, and also show the effects of nourishing yin and moistening the lungs, relieving cough and removing phlegm. However, the fruit setting rate of peach trees is relatively high, and excessively enriched fruits will compete with each other for nutrients on the fruit trees, which will affect the quality and yield of the fruit. Therefore, it is necessary to additionally thin out some young fruits to reasonably adjust the nutritional competition between fruits and ensure the overall yield and quality of peach fruits. At present, the utilization of peach thinning in actual production is extremely small, and only a small part of it will be made into food or feed. Most of the peach thinning fruits are discarded in the field, which not only wastes resources, but also causes biological pollution, and even breeds pathogens, accelerating the spread of fruit tree diseases. In fact, peach thinning is a plant resource that can be utilized as a resource and has a high added value. It is rich in functional substances such as polyphenols and polysaccharides. If peach thinning can be effectively utilized, it 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, which are widely present in animals, plants and microbial cells. The structure of polysaccharides is relatively complex. It is not only one of the three major nutrients that constitute life, but also participates in various life activities in the body. It has multiple physiological activities such as anti-oxidation, anti-aging, hypoglycemic, hypolipidemic, anti-tumor, immunomodulatory, anti-inflammatory and intestinal protection. Many studies have confirmed that plant polysaccharides participate in regulating the sugar metabolism process in the body. It can achieve the purpose of lowering blood sugar by regulating the activity of sugar metabolism enzymes, promoting insulin secretion, increasing insulin sensitivity, antagonizing glucagon, regulating glycogen synthesis and degradation, and repairing damaged pancreatic cells. In addition, plant polysaccharides, as natural medicines, have the characteristics of less toxic side effects and higher safety compared with the hypoglycemic drugs currently on the market, and are considered to be an ideal substitute for synthetic drugs. Therefore, discovering polysaccharides with biological activity from plants and using them in the development of functional foods and new clinical drugs has become a hot spot in current food and medical research.

[0004] Oligosaccharides, also known as oligosaccharides, are a general term for low-polymer sugars formed by 2-10 monosaccharides connected by glycosidic bonds. Oligosaccharides can be divided into functional oligosaccharides and ordinary oligosaccharides based on whether they have health functions. Functional oligosaccharides mainly have biological activities such as anti-oxidation, hypoglycemic, hypolipidemic and intestinal flora regulation. Type II diabetes is a common chronic disease in the middle-aged and elderly, mainly caused by low insulin levels or insulin resistance. The side effects of commonly used drugs greatly limit the effect of drugs in clinical treatment, and it is urgent to develop hypoglycemic drugs with fewer side effects. Functional oligosaccharides have a variety of anti-diabetic mechanisms of action, such as inhibiting α-glucosidase, reducing insulin resistance, regulating hormones and intestinal microbiota, etc. At present, the development and utilization of peach fruit mainly focuses on the extraction of polyphenols and polysaccharides. The biological activity of peach fruit polysaccharides has been studied, but the molecular weight of polysaccharides is large, resulting in high viscosity of their solutions, making it difficult to enter the body, limiting the scope of application of polysaccharides. There are no reports on the research of peach fruit oligosaccharides and their biological activity functions. 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 blood sugar lowering activity and an extraction method and application thereof.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a peach fructooligosaccharide with hypoglycemic activity, wherein the peach fructooligosaccharide comprises W1-2 oligosaccharide and W2-2 oligosaccharide, wherein 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 aminogalactose 1-4%, and 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 aminogalactose 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 oligosaccharide are connected 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 connected 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] The present invention also provides a method for preparing the peach fructooligosaccharide having hypoglycemic activity, comprising the following steps: Step 1, raw material pretreatment: adding peach thinning fruit powder to ethanol aqueous solution to remove peach thinning fruit polyphenols by ultrasonication to obtain a crude extract of peach thinning fruit polysaccharide; Step 2, extracting crude polysaccharides from peach thinning fruit: adding the crude polysaccharide extract of peach thinning fruit obtained in step 1 to pure water at a mass ratio of 1:30, and extracting for 35 to 40 minutes at 100 to 130° C. and pH 4 to 6; after the extraction is completed, filtering while hot, centrifuging the filtrate and concentrating it by rotary evaporation, adding ethanol 3 times the volume of the concentrate, and precipitating it at 4° C. overnight, after the precipitation is completed, dissolving the precipitate in water, removing the residual ethanol by rotary evaporation, and then pouring it into a plate and freeze-drying it to obtain crude polysaccharides from peach thinning fruit; Step 3, separation and purification of peach sparse fruit polysaccharide: dissolve the crude peach sparse fruit polysaccharide in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, vortex oscillation to promote its dissolution, centrifuge to remove the insoluble matter at the bottom, and then filter the supernatant through a 0.45 μm filter membrane to obtain a loading solution; slowly drop all the loading solution into an anion exchange resin chromatography column with a dropper, use pure water as an eluent, and collect the second elution peak component and the third elution peak component both containing peach sparse fruit oligosaccharide; Step 4, ultrafiltration separation of peach fructo-oligosaccharide: The two components containing peach fructo-oligosaccharide obtained in step 3 are separated by 3 kDa ultrafiltration tubes, and the small molecular weight fraction is retained to obtain two highly active peach fructo-oligosaccharides, which are named W1-2 oligosaccharide and W2-2 oligosaccharide, respectively.

[0011] Furthermore, step 1 is specifically as follows: freeze-dry the peach thinned fruit for 48 hours, crush it with a grinder, pass it through a 60-mesh sieve to obtain peach thinned fruit powder, add the peach thinned fruit powder into 70vt% ethanol aqueous solution at a mass volume ratio of 1g:50mL, ultrasonicate for 25min at a power of 610W and a temperature of 50°C, filter the residue by suction, and dry it at room temperature to obtain a crude extract of peach thinned fruit polysaccharide.

[0012] Further, step 2 is specifically 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°C and pH 5.0 for 40 min; after the extraction is completed, the product is filtered while hot, the filtrate is centrifuged at 9000rpm for 10 min, and then concentrated by rotary evaporation, 3 times the volume of ethanol of the concentrate is added, and the alcohol precipitation is carried out at 4°C overnight. After the precipitation is completed, the alcohol precipitate is dissolved in water, the residual ethanol is removed by rotary evaporation, and then poured into a plate and freeze-dried to obtain the crude peach fruit polysaccharide. Under the optimal extraction conditions, the product yield is 10.63%, and the inhibition rate of α-glucosidase is 76.58%.

[0013] Furthermore, step 3 is specifically as follows: dissolving the crude polysaccharide of peach sparse fruit in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, vortexing to promote its dissolution, centrifuging at 8000 r / min for 10 min, removing the insoluble matter at the bottom, and then filtering the supernatant through a 0.45 μm filter membrane to obtain a loading solution; taking 10 mL of the loading solution and slowly dripping it into a DEAE anion exchange resin chromatography column with a dropper, eluting with pure water, controlling the eluent flow rate to 1.0 mL / min, collecting 5.0 mL of eluent in each tube, collecting 45 tubes, and collecting tubes 16-22 and 23-35 respectively to obtain two components containing peach sparse fruit oligosaccharides.

[0014] The present invention also provides application of the peach fructooligosaccharide in preparing blood sugar lowering drugs.

[0015] The present invention also provides application of the peach fructooligosaccharide in preparing alpha-glucosidase inhibitor.

[0016] Compared with the prior art, the advantages of the present invention are: 1. The present invention uses 70vt% ethanol aqueous solution to ultrasonically extract and remove polyphenols in peach thinning fruits, thereby avoiding browning during the subsequent extraction of polysaccharides from peach thinning fruits; 2. The powder is crushed by a grinder and subjected to classification treatment to obtain peach thinning fruit powder with uniform, small particles and stable properties, thereby increasing the specific surface area and improving the yield of peach thinning fruit polysaccharide; 3. The hot water extraction method was used to extract peach polysaccharides, and the effects of temperature, pH and time on the yield and α-glucosidase inhibition rate were investigated. The optimal process was 110℃, pH 5.0 and 40 min, at which the yield was 10.63% and the inhibition rate of α-glucosidase was 76.58%; 4. Two peach fructooligosaccharides were obtained by ion exchange chromatography and ultrafiltration separation. The molecular weight of W1-2 is 724 Da, and the molecular weight of W2-2 is 427 Da. Their inhibition rates on α-glucosidase can reach 88.38% and 94.82%, respectively, showing good hypoglycemic activity.

[0017] In summary, the present invention discloses for the first time a peach thinning oligosaccharide with hypoglycemic activity and its extraction method and application. Peach thinning fruit, a byproduct of peach processing, is used as a raw material to extract and purify peach thinning fruit polysaccharide, and the purified components are subjected to ultrafiltration separation to obtain a peach thinning fruit oligosaccharide with low molecular weight, strong water solubility, high purity and high hypoglycemic activity. The method makes full use of the advantages of large amount of peach thinning fruit waste, low cost, close to nature, etc., solves the problem of efficient utilization of processing waste, and provides a reference basis for the further development and utilization of peach thinning fruit resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The pure water elution curve of the peach sucrose polysaccharide extracted in the specific embodiment 1; Figure 2 The molecular weight results of peach fructooligosaccharides separated in specific example 1; Figure 3 The effects of different extraction conditions on the yield of peach polysaccharide and α-glucosidase inhibition rate in specific example 2, wherein A is the extraction temperature, B is the extraction pH, and C is the extraction time; Figure 4 is the monosaccharide composition of peach fructooligosaccharide W1-2 in specific embodiment 3; Figure 5 is the monosaccharide composition of peach fructooligosaccharide W2-2 in specific embodiment 3; Figure 6 The inhibitory effect of peach fructooligosaccharide on α-glucosidase in specific example 4; Figure 7 The effect of different concentrations of W1-2 on cellular glucose uptake in specific example 4; Figure 8 The effect of different concentrations of W2-2 on cellular glucose uptake in specific example 4; Fig. 9 The effect of the crude polysaccharide of peach fruit on the body weight of type 2 diabetic mice in Example 5; Fig.10 The effect of the crude polysaccharide of peach fruit on fasting blood glucose in type 2 diabetic mice in Example 5; Fig.11 The linear relationship between blood glucose and time after oral administration of glucose solution to mice in different experimental groups in Specific Example 5; Fig.12 This is the effect of the crude polysaccharide from peach fruit on glucose tolerance in the specific example 5. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to the accompanying drawings.

[0020] Specific embodiment 1, a method for preparing high-activity peach fructooligosaccharide, comprising the following steps: Step 1, raw material pretreatment: freeze-dry the peach fruit for 48 hours, crush it with a grinder, pass it through a 60-mesh sieve to obtain peach fruit powder, add the peach fruit powder into a 70vt% ethanol aqueous solution at a mass volume ratio of 1g (dry basis): 50mL, remove the peach fruit polyphenols by ultrasonication at a power of 610W, a temperature of 50°C, and a time of 25min, filter the residue by suction, and dry it at room temperature to obtain a crude extract of peach fruit polysaccharide; Step 2, extracting crude polysaccharides from peach fruit: adding the crude polysaccharide extract of peach fruit obtained in step 1 to pure water at a mass ratio of 1:30, and extracting at 110°C and pH 5.0 for 40 min; after the extraction is completed, filtering the silk mesh while hot, suction filtering, taking the filtrate and centrifuging it at 9000 rpm for 10 min, and then concentrating it by rotary evaporation, adding ethanol 3 times the volume of the concentrate, and precipitating it at 4°C overnight. After the precipitation is completed, dissolving the alcohol precipitate in water, rotary evaporating to remove the residual ethanol, and then pouring it into a plate and freeze-drying it to obtain crude polysaccharides from peach fruit; Step 3, separation and purification of peach sparse fruit polysaccharide: weigh 100 mg of peach sparse fruit crude polysaccharide in a 50 mL centrifuge tube, add 10 mL of distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, vortex and oscillate to promote its dissolution, 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 a sample solution; slowly drop all the sample solution into a DEAE anion exchange resin chromatography column with a dropper, elute with pure water, control the eluent flow rate to 1.0 mL / min, collect 5.0 mL of eluent in each tube, collect 45 tubes, and the pure water elution curve of peach sparse fruit polysaccharide is as shown in Figure 1 As shown, tubes 16-22 and 23-35 were collected and named W1 and W2, respectively; Step 4, ultrafiltration separation of peach fritter polysaccharide: W1 and W2 obtained in step 3 are separated using 3 kDa ultrafiltration tubes, respectively, and the small molecular weight fraction is retained to obtain two highly active peach fritter oligosaccharides, which are named W1-2 and W2-2, respectively.

[0021] The molecular weights of the different components were determined to assess whether they were homogeneous components. The chromatographic conditions were as follows: HPLC (Agilent 1260) equipped with a 2414 differential detector and an Ultrahxdrogel column. TMLinear (7.8 mm × 300 mm), the mobile phase was 0.1 mol / L sodium nitrate solution (NaNO3). The column temperature and detector temperature were both 45 °C, 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 of 10 mg / mL), with retention time as the horizontal axis and the logarithm of molecular weight (Log 10 Mw) as the ordinate, and a standard curve was drawn. Figure 2 As shown, the molecular weight of W1-2 is 724 Da, and the molecular weight of W2-2 is 451 Da.

[0022] Specific embodiment 2: Optimization of the method for extracting peach fructooligosaccharides in specific embodiment 1.

[0023] Method for determining the inhibition rate of α-glucosidase: Use a pipette to take 80 μL of 1 mg / mL, 2 mg / mL, 4 mg / mL and 8 mg / mL of peach fruit crude polysaccharide solution and 60 μL of α-glucosidase solution, mix them, vortex and incubate at 37°C for 10 min; then add 100 μL of 4-nitrophenyl-β-D-pyranogalactoside, and continue to incubate at 37°C for 15 min; finally, add 1 mL of 0.1 mol / L Na2CO3 solution to terminate the reaction, and use an enzyme reader to measure its absorbance at a wavelength of 400 nm. Calculate the inhibition rate according to the formula,

[0024] Where As is the absorbance of the mixed solution of the sample solution, 4-nitrophenyl-β-D-galactopyranoside solution and α-glucosidase solution; Ac is the absorbance of the mixed solution of PBS buffer solution instead of α-glucosidase solution; Ab is the absorbance of the mixed solution of PBS buffer solution instead of the sample solution.

[0025] 1. The yield and α-glucosidase inhibition rate of the polysaccharides extracted from each group in the single-factor experiment were evaluated.

[0026] Optimization of extraction temperature: The method for extracting peach oligosaccharides was the same as step 2 of the above-mentioned specific embodiment 1, except that the extraction pH was fixed at 5, the extraction time was 60 min, and different extraction temperatures were set at 80°C, 90°C, 100°C, 110°C, and 120°C. Figure 3As shown in Figure A, as the extraction temperature increases, the α-glucosidase inhibition ability gradually increases. When the temperature rises to 100°C, the inhibition rate reaches the maximum, and if the temperature continues to rise, the inhibition rate will decrease. Although the polysaccharide yield is the highest at 110°C, its inhibition rate is significantly lower than 100°C, so 100°C is selected as the optimal extraction temperature.

[0027] Extraction pH optimization: The extraction method of peach fructooligosaccharide was the same as step 2 of the above specific embodiment 1, except that the extraction temperature was fixed at 100°C, the extraction time was 60 min, and different extraction pH values ​​were set at 3, 4, 5, 6, and 7. Figure 3 As shown in B, except for pH=5, there is no significant difference in the polysaccharide yield at other pH values. When pH is 6, the α-glucosidase inhibition rate is the highest, so pH=6 is selected as the optimal extraction pH.

[0028] Optimization of extraction time: The method for extracting peach fructooligosaccharides is the same as step 2 of the above specific embodiment 1, except that the extraction temperature is fixed at 100°C, the extraction pH is 6, and different extraction times are set at 30, 40, 50, 60, and 70 min. Figure 3 As shown in C, the polysaccharide extracted for 40 min showed a much higher α-glucosidase inhibition rate than other groups, and 40 min was selected as the optimal extraction time.

[0029] 2. Orthogonal Experimental Design On the basis of single factor experiment, extraction temperature (90℃, 100℃, 110℃), pH (5, 6, 7), and time (30, 40, 50) were selected as investigation factors, and orthogonal experiment was used to optimize the extraction process. The orthogonal experiment design table and results are shown in Table 1.

[0030] Table 1 Orthogonal experiment results

[0031] In the orthogonal experiment, the comprehensive scoring method was used to analyze the experimental results. From the test results, it can be seen that the order of each factor is A>C>B, temperature, time, pH. The best combination is A3C2B1. Under this condition, the polysaccharide yield is 10.63%, and the α-glucosidase inhibition rate is 76.58%. Therefore, the optimal extraction process of peach fruit polysaccharide is finally determined to be extraction temperature 110 ℃, extraction pH 5.0, and extraction time 40 min.

[0032] Specific Example 3: Structural analysis of peach fructooligosaccharides W1-2 and W2-2 extracted by the method of Specific Example 1.

[0033] 1. Analysis of monosaccharide composition: W1-2 and W2-2 were prepared into 2 mg / mL solutions, 1 mL was taken into a test tube, 0.17 mL of concentrated trifluoroacetic acid was added, and hydrolysis was carried out at 121°C for 120 min. After the hydrolysis was completed, nitrogen was blown to dryness, 1 mL of water was added for re-dissolution, filtered through a 0.22 μm filter membrane, and diluted 20 times to a concentration of 100 ppm. The monosaccharide composition of the sample was determined by ion exchange chromatography. The detection was carried out using a high-performance ion chromatography system (Dionex ICS-5000, Dionex, USA) equipped with a pulsed amperometric detector. The chromatographic column was CarboPac PA20 (2×250 mm, id, 5 μm), and the mobile phases were ultrapure water, 0.25 mol / L NaOH solution, and 1.0 mol / L sodium acetate (NaAc) solution. The mobile phase flow rate was 0.5 mL / min.

[0034] Figure 4 It is composed of monosaccharides of W1-2. W1-2 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%, amino galactose 2.54%, and the molecular weight of W1-2 is 724 Da. Figure 5 It is the monosaccharide composition of W2-2. W2-2 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.

[0035] 2. Methylation analysis: W1-2 and W2-2 were dissolved in water to prepare a 1 mg / mL solution. Take 100 μL and place it in a screw-mouthed reaction tube, air-dry with nitrogen, add 165 μL of anhydrous dimethyl sulfoxide (DMSO), seal, and ultrasonically dissolve for 30 min. Under an infrared lamp, add NaOH to anhydrous DMSO, grind, and prepare a 120 mg / mL slurry. Then add 150 μL-300 μL of slurry to the reaction tube, ultrasonicate for 50 min in an ice bath, add 50 μL-100 μL of iodomethane after complete dissolution, and ultrasonicate for 30 min. Repeat this operation 3 times to ensure complete methylation. Use dichloromethane to repeatedly extract the methylated polysaccharide, combine the dichloromethane layers and wash with water 2 to 3 times to remove the incompletely methylated polysaccharide. Discard the water layer, add anhydrous sodium sulfate to the dichloromethane layer, blow nitrogen to remove dichloromethane, and obtain the methylated polysaccharide.

[0036] Add 1.0 mL 2.0 mol / L trichloroacetic acid to the reaction tube containing the dried methylated polysaccharide, seal it and place it in a 120℃ oil bath for hydrolysis for 90 min, cool it, add inositol as an internal standard (0.1 mg / mL), blow it dry with nitrogen, re-dissolve it with water, add sodium borodeuteride to reduce it until no bubbles are generated when it encounters acetic acid, which means that the reduction is complete. Add methanol to the reduced reaction solution, blow it with nitrogen, repeat it twice, then add 625 μL acetic anhydride, react at 100℃ for 150 min, blow it dry with nitrogen, re-dissolve it with dichloromethane, dry it with anhydrous sodium sulfate, and obtain a partially methylated sugar alcohol acetate derivative (PMAA), which is determined by GC-MS. The results are shown in Tables 2 and 3 below.

[0037] Table 2 Methylation analysis results of W1-2

[0038] The methylated W1-2 was analyzed by GC-MS and compared with the standard information library to analyze the glycosidic bond type of W1-2. The results are shown in Table 2. After methylation, W1-2 generated glucose residues, galactose residues and arabinose residues, with corresponding proportions of 41.63%, 32.21% and 26.16%, respectively, which was consistent with the monosaccharide composition results. The methylation results of W1-2 showed that there were 8 glycosidic bonds in W1-2, namely 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).

[0039] Table 3 Methylation analysis results of W2-2

[0040] As shown in Table 3, after methylation treatment, the glycosidic bond of W2-2 was broken to generate glucose residues, arabinose residues, galactose residues and mannose residues, with corresponding proportions of 69.87%, 10.77%, 11.92% and 7.44%, respectively, which was consistent with the monosaccharide composition results. The methylation results of W2-2 showed that there were 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).

[0041] Specific Example 4: Activity analysis of peach fructooligosaccharides W1-2 and W2-2 extracted by the method of Specific Example 1.

[0042] 1. Inhibitory effect of W1-2 and W2-2 on α-glucosidase.

[0043] Method for determining the inhibition rate of α-glucosidase: Use a pipette to take 80 μL of 1.0, 2.0, 4.0 and 8.0 mg / mL of peach fruit crude polysaccharide solution and 60 μL of α-glucosidase solution, mix them, vortex and incubate at 37°C for 10 min; then add 100 μL of 4-nitrophenyl-β-D-pyranogalactoside, and continue to incubate at 37°C for 15 min; finally, add 1 mL of 0.1 mol / L Na2CO3 solution to terminate the reaction, and use an enzyme reader to measure its absorbance at a wavelength of 400 nm. Calculate the inhibition rate according to the formula,

[0044] Wherein, As is the absorbance of the mixed solution of the sample solution, 4-nitrophenyl-β-D-galactopyranoside solution and α-glucosidase solution; Ac is the absorbance of the mixed solution of PBS buffer solution instead of α-glucosidase solution; Ab is the absorbance of the mixed solution of PBS buffer solution instead of the sample solution.

[0045] like Figure 6As shown in the figure, in the concentration range of 1.0~8.0 mg / mL, the inhibition rate of W1-2 and W2-2 on α-glucosidase increased with the increase of concentration. When the polysaccharide concentration was 8 mg / mL, the inhibitory ability of different components on α-glucosidase activity was as follows: W2-2 (94.82%)>W1-2 (88.38%)>peach fruit polysaccharide (60.12%, abbreviated as PPS). The inhibition rate of W2-2 and W1-2 on α-glucosidase was significantly higher than that of peach fruit polysaccharide. It was calculated that the IC values ​​of W1-2 and W2-2 for inhibiting α-glucosidase were 50 The values ​​were 0.57 mg / mL and 0.37 mg / mL, respectively, which were significantly lower than 5.46 mg / mL of PPS, indicating that the hypoglycemic activity of W1-2 and W2-2 was better than that of PPS.

[0046] 2. Effects of W1-2 and W2-2 on glucose uptake in insulin-resistant HepG2 cells.

[0047] Experimental method: Model group (IR-HepG2 cells + sample solutions of different concentrations), control group (IR-HepG2 cells + cell culture medium, Model), and blank group (HepG2 cells + cell culture medium, CK) were set up, with 6 replicates in each group. HepG2 cells in the logarithmic growth phase were diluted to 2×10 5 / mL, plated in 96-well plates at 100 μL / well, cultured at 37°C, 5% CO2 for 24 h. After the cells adhered to the wall, the old culture medium was removed, and 12 mM glucosamine was used for 16 h to construct the IR-HepG2 model (IR-HepG2 refers to a cell model that produces insulin resistance; HepG2 is a cell in a normal state). After successful construction, the culture medium of each well was discarded and washed twice with PBS solution. 100 μL of cell culture medium was added to the blank group and the control group, and 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 for 24 h at 37°C and 5% CO2, the culture medium was discarded and the cells were washed twice with PBS solution to remove the 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 the culture medium from each well was determined using a glucose detection kit (GOD-POD method), and the glucose uptake of the model group, control group, and blank group was calculated.

[0048] Figure 7 and Figure 8 The effects of different concentrations of W1-2 and W2-2 on glucose uptake by IR-HepG2 cells were compared, and none of the selected concentrations had an effect on proliferation. Figure 7As shown in the figure, compared with the model group, W1-2 at a concentration of 0.5-2 mg / mL could significantly increase the glucose consumption in HepG2 cells, with the glucose consumption increased by 1.47 times, 2.29 times, and 2.46 times, respectively. Figure 8 As shown in the results, W2-2 at concentrations of 0.25-1 mg / mL can significantly increase the glucose consumption in HepG2 cells, with glucose consumption increased by 1.60 times, 2.19 times, and 2.16 times, respectively. The above results show that both W1-2 and W2-2 have the potential to improve insulin resistance in HepG2 cells.

[0049] Specific Example 5: Anti-diabetic effect of the crude polysaccharide of peach fruit prepared by the method of Specific Example 1 in vivo.

[0050] 1. Animal Experiment Design: 24 6-week-old SPF male C57BL / 6 mice, weighing (20 ± 2 g), were purchased from Shanghai Slake Experimental Animal Co., Ltd. All mice were housed in the Animal Experiment Center of Ningbo University, with a relative humidity of 50 ± 10%, a temperature of 23 ± 1 ℃, and a 12-h light-dark cycle. During the feeding period, there were 3 mice per cage and free access to food and water. All animal experimental procedures were carried out in strict accordance with the "Regulations on the Administration of Animals in Animal Experiments in China" and approved by the Welfare and Ethics Committee of the Experimental Animal Center of Ningbo University.

[0051] After one week of adaptive feeding, 6 mice were randomly selected as the normal group (NC) and fed with normal diet. The remaining 18 mice were used as the modeling group and fed with a high-fat diet (HFD) to produce obesity and insulin resistance. After 6 weeks of feeding, all mice were fasted overnight but not water for 12 hours. The mice in the modeling group were intraperitoneally injected with 50 mg / kg·BW 1% freshly prepared low-temperature streptozotocin (STZ) (dissolved in 0.1 mol / L citrate buffer, pH=4.5), and the mice in the NC group were intraperitoneally injected with an equal dose of citrate buffer. 72 hours after the first intraperitoneal injection, all mice were fasted overnight again but not water for 12 hours. The mice in the modeling group and the NC group were intraperitoneally injected with 50 mg / kg·BW freshly prepared low-temperature STZ solution and an equal dose of citrate buffer, respectively. 72 hours after the second intraperitoneal injection, the fasting blood glucose of each group of mice was quickly measured by cutting the tail and collecting blood using an ACCU-CHEK blood glucose meter. The T2DM mouse model was successfully established when the fasting blood glucose value was greater than or equal to 11.1 mmol / L. The successfully constructed T2DM mice were randomly divided into three groups, with 6 mice in each group: diabetes model group (DM group), low-dose peach fruit polysaccharide intervention group (LPP group, 200 mg / kg·BW) and high-dose peach fruit polysaccharide intervention group (HPP group, 400 mg / kg·BW). The mice in the LPP and HPP groups were gavaged with different doses of peach fruit polysaccharide solution dissolved in 0.9% sterile saline once a day, while the mice in the NC and DM groups were gavaged with the same dose of 0.9% sterile saline. The intervention period was 4 weeks, and the body weight and FBG of mice in each group were recorded weekly. After the experiment, all mice were fasted overnight and killed by cervical dislocation after isoflurane anesthesia, and biological samples were collected for further analysis.

[0052] 2. Determination of fasting blood glucose and glucose tolerance: All mice were fasted overnight for 12 hours on a fixed day each week, and blood was collected by tail cutting the next day to determine the fasting blood glucose of each group of mice. Oral glucose tolerance test was performed in the last week of 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.

[0053] 3. Experimental results: Effects of peach fruit crude polysaccharides on body weight and fasting blood sugar.

[0054] Effects of crude polysaccharide from peach fruit on body weight of type Ⅱ diabetic mice Fig. 9 As shown in the figure, during the experiment, the weight of mice in the DM group continued to decrease as the diabetes worsened, while the weight of mice in the NC group increased steadily. At the beginning of the experiment, there was no significant difference in the weight of mice in 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 crude polysaccharide, the body weight of mice in the LPP and HPP groups increased, and the trend was consistent with that of mice in the NC group; at the end of the experiment, there was no significant difference in the body weight of mice in the LPP and HPP groups compared with those in the NC group ( p >0.05). These results indicate that the crude polysaccharide from peach fruit can alleviate the degree of weight loss in diabetic mice in the early stage of the experiment; in the later stage of the experiment, it can reverse the trend of weight loss and return to normal levels.

[0055] Fasting blood glucose (FBG) was measured once a week during the experiment to evaluate the effect of peach fruit crude polysaccharide on hyperglycemia in T2DM mice. Fig.10 As shown, at week 0, there was no significant difference in fasting blood glucose among mice in the DM, LPP, and HPP groups ( p >0.05, and was significantly higher than that of mice in the NC group ( p <0.01), indicating that the model was successfully constructed, and there was no significant difference in FBG among the mice in each group. After one week of intervention with peach fruit crude polysaccharide, the FBG of mice in the LPP group and HPP group began to decrease, which was significantly different from that in the DM group ( p <0.05). As the condition of diabetes worsened, the FBG of mice in the DM group gradually increased from 1 to 3 weeks. The intervention of P. persica polysaccharide significantly inhibited the increase of FBG and alleviated hyperglycemia. After 4 weeks of intervention with P. persica polysaccharide, the FBG of mice in the LPP and HPP groups was significantly reduced compared with the DM group (p<0.01), and high-dose P. persica polysaccharide showed a better hypoglycemic effect.

[0056] 4. Experimental results: Effects of crude polysaccharides from peach fruit on glucose tolerance.

[0057] like Fig.11 As shown in the figure, the blood glucose levels of mice in all experimental groups rose rapidly after oral administration of glucose solution and reached a peak blood glucose level at 30 minutes, and then gradually decreased within the next 90 minutes. Compared with the other three groups, mice in the NC group were more sensitive to changes in glucose, and their blood glucose returned to normal within 120 minutes. However, the blood glucose of mice in the DM group remained at a high level after 120 minutes, indicating that the glucose tolerance of untreated T2DM mice was severely deteriorated. Intervention with peach fruit crude polysaccharide can significantly reduce the peak blood glucose level of T2DM mice. At the end of the oral glucose tolerance test (OGTT) experiment, the blood glucose levels of mice in the high- and low-dose peach fruit crude polysaccharide intervention groups were significantly lower than those in the DM group (p<0.05). The area under the curve was further used to quantify the glucose tolerance of mice, as shown in the figure. Fig.12As shown in the figure, the AUC values ​​of all diabetic mice were significantly higher than those of the NC group (p<0.01). At the same time, the AUC values ​​of diabetic mice decreased in the DM group, LPP group, and HPP group, indicating that all doses of peach fruit crude polysaccharides can significantly improve the glucose tolerance of diabetic mice in a dose-dependent manner.

[0058] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A peach fructooligosaccharide with hypoglycemic activity, characterized in that The peach fructooligosaccharide 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 aminogalactose 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%.

2. The peach fructooligosaccharide with hypoglycemic activity according to claim 1, characterized in that 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 aminogalactose 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%.

3. The peach fructooligosaccharide with hypoglycemic activity according to claim 1, characterized in that The monosaccharides of the W1-2 oligosaccharide are connected 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.

4. The peach fructooligosaccharide with hypoglycemic activity according to claim 1, characterized in that The monosaccharides of the W2-2 oligosaccharide are connected 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.

5. A method for preparing peach fructooligosaccharide with hypoglycemic activity according to claim 1, characterized in that The following steps are involved: Step 1, raw material pretreatment: adding peach thinning fruit powder to ethanol aqueous solution to remove peach thinning fruit polyphenols by ultrasonication to obtain a crude extract of peach thinning fruit polysaccharide; Step 2, extracting crude polysaccharides from peach thinning fruit: adding the crude polysaccharide extract of peach thinning fruit obtained in step 1 to pure water at a mass ratio of 1:30, and extracting for 35 to 40 minutes at 100 to 130° C. and pH 4 to 6; after the extraction is completed, filtering while hot, centrifuging the filtrate and concentrating it by rotary evaporation, adding ethanol 3 times the volume of the concentrate, and precipitating it at 4° C. overnight, after the precipitation is completed, dissolving the precipitate in water, removing the residual ethanol by rotary evaporation, and then pouring it into a plate and freeze-drying it to obtain crude polysaccharides from peach thinning fruit; Step 3, separation and purification of peach sparse fruit polysaccharide: dissolve the crude peach sparse fruit polysaccharide in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, vortex and oscillate to promote its dissolution, centrifuge to remove the insoluble matter at the bottom, and then filter the supernatant through a 0.45 μm filter membrane to obtain a loading solution; slowly drop all the loading solution into an anion exchange resin chromatography column with a dropper, use pure water as an eluent, and collect the second elution peak component and the third elution peak component both containing peach sparse fruit oligosaccharide; Step 4, ultrafiltration separation of peach fructo-oligosaccharide: The two components containing peach fructo-oligosaccharide obtained in step 3 are separated by 3 kDa ultrafiltration tubes, and the small molecular weight fraction is retained to obtain two highly active peach fructo-oligosaccharides, which are named W1-2 oligosaccharide and W2-2 oligosaccharide, respectively.

6. The method for preparing peach fructooligosaccharide with hypoglycemic activity according to claim 5, characterized in that Step 1 is specifically as follows: freeze-dry the peach fruit for 48 h, crush it with a grinder, and pass it through a 60-mesh sieve to obtain peach fruit powder, add the peach fruit powder into 70vt% ethanol aqueous solution at a mass volume ratio of 1g:50mL, ultrasonicate for 25min at a power of 610W and a temperature of 50℃, filter the residue by suction, and dry it at room temperature to obtain a crude extract of peach fruit polysaccharide.

7. The method for preparing peach fructooligosaccharide with hypoglycemic activity according to claim 5, characterized in that Step 2 is specifically as follows: the crude extract of peach sparse fruit polysaccharide obtained in step 1 is added to pure water at a mass ratio of 1:30, and extracted at 110°C and pH 5.0 for 40 min; after the extraction is completed, it is filtered while hot, the filtrate is centrifuged at 9000 rpm for 10 min, and then concentrated by rotary evaporation, ethanol 3 times the volume of the concentrated solution is added, and alcohol precipitation is carried out at 4°C overnight. After the precipitation is completed, the alcohol precipitate is dissolved in water, the residual ethanol is removed by rotary evaporation, and then poured into a plate and freeze-dried to obtain the crude peach sparse fruit polysaccharide.

8. The method for preparing peach fructooligosaccharide with hypoglycemic activity according to claim 5, characterized in that Step 3 is specifically as follows: dissolving the crude polysaccharide of peach fritters in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL, vortexing to promote its dissolution, centrifuging at 8000 r / min for 10 min, removing the insoluble matter at the bottom, and then filtering the supernatant through a 0.45 μm filter membrane to obtain a loading solution; taking 10 mL of the loading solution and slowly dropping it into a DEAE anion exchange resin chromatography column with a dropper, eluting with pure water, controlling the eluent flow rate to 1.0 mL / min, collecting 5.0 mL of the eluent in each tube, collecting 45 tubes, and collecting tubes 16-22 and 23-35 respectively to obtain two components containing peach fritters oligosaccharides.

9. Use of the peach fructooligosaccharide according to claim 1 in the preparation of hypoglycemic drugs.

10. Use of the peach fructooligosaccharide according to claim 1 in the preparation of α-glucosidase inhibitors.

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