Preparation of gleditsia sinensis polysaccharide and application of gleditsia sinensis polysaccharide in product with antioxidant and / or hypoglycemic functions

Abstract

The invention discloses preparation of gleditsia sinensis polysaccharide and application of the gleditsia sinensis polysaccharide in products with functions of resisting oxidation and / or reducing blood sugar. According to the invention, Gleditsia sinensis Lam. Endosperm is used as a raw material, a green and efficient extraction technology is adopted to obtain purified Gleditsia sinensis Lam. Polysaccharide GSP-1, and the structure of the Gleditsia sinensis Lam. Polysaccharide GSP-1 is analyzed through high performance gel permeation chromatography, infrared spectroscopy, ion chromatography, gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The GSP-1 has excellent antioxidation and hypoglycemic effects, promotes glucose consumption, promotes glucose metabolism, effectively improves insulin resistance, and has a positive promotion effect on regulating blood sugar and preventing and treating diabetes mellitus; the method has wide application prospects in the aspects of food processing, health care product development, functional food addition and the like for assisting in treating diabetes and controlling blood sugar, and has important significance in improving the added value of gleditsia sinensis, improving the scientific and technological innovation ability of agricultural products and driving industrial transformation and upgrading.

Description

Technical Field

[0001] This invention relates to the field of natural active ingredient extraction and pharmaceutical technology, specifically to the preparation of a saponin polysaccharide and its application in products with antioxidant and / or hypoglycemic functions. Background Technology

[0002] Diabetes mellitus is a chronic disease characterized by hyperglycemia, caused by absolute or relative insulin deficiency and impaired insulin utilization. This includes decreased insulin secretion due to pancreatic islet cell dysfunction, or insulin resistance due to the body's insensitivity to insulin, preventing the effective utilization and storage of glucose in the blood. Oxidative stress is closely related to insulin resistance. Prolonged hyperglycemia leads to excessive production of reactive oxygen species (ROS), triggering oxidative stress, which in turn exacerbates insulin resistance and β-cell dysfunction. Therefore, active ingredients with both antioxidant and hypoglycemic activities may break this vicious cycle, providing more comprehensive metabolic regulation and thus achieving the goal of treating diabetes. Currently, clinically used drugs for diabetes mainly include insulin secretagogues (such as sulfonylureas), insulin sensitizers (such as metformin), and α-glucosidase inhibitors (such as acarbose). However, long-term use of these drugs can easily cause side effects such as hypoglycemia, gastrointestinal reactions, and liver and kidney damage. Therefore, developing naturally derived, low-toxicity active ingredients with both antioxidant and hypoglycemic functions is a current research hotspot in this field.

[0003] Gleditsia sinensis is an important member of the legume family, widely distributed in Southeast Asia and North and South America. It is used as a traditional medicine to treat scabies, skin diseases, urinary tract infections, and phlegm. Phytochemical studies have identified the main chemical components of Gleditsia sinensis plants as alkaloids, galactomannan, flavonoids, and other active ingredients. Gleditsia sinensis polysaccharide, a natural plant polysaccharide extracted from Gleditsia sinensis seeds, has been proven to possess antioxidant capabilities. For example, Liu Fang found that saponin polysaccharides have good antioxidant capacity (Liu Fang. Extraction, separation, preliminary structural analysis and some bioactivity studies of saponin polysaccharides [D]. Kunming University of Science and Technology, 2012.); Fu Jin and Gao Jie found that saponin polysaccharides have good DPPH scavenging capacity and hydroxyl radical scavenging capacity (Fu Jin, Yao Qiuping, Deng Shuixiu, et al. Study on extraction kinetics and antioxidant activity of saponin polysaccharides from Guizhou [J]. Food Industry Technology, 2021, 42 (01): 8-14.); (Gao Jie, Dong Wenbin, Wang Yong, et al. Optimization of ultrasonic extraction process and in vitro antioxidant activity study of saponin polysaccharides [J]. Food Industry Technology, 2019, 40 (21): 124-129+135.). However, the saponin polysaccharides extracted by the current technology are mainly crude extracts, and the fine structure of purified saponin polysaccharides has not yet been resolved, and their bioactivity has not been systematically and deeply explored. Summary of the Invention

[0004] To address the problems in the prior art, this invention provides a method for preparing saponin and its application in products with antioxidant and / or hypoglycemic functions.

[0005] The first objective of this invention is to provide a saponin polysaccharide.

[0006] A second objective of this invention is to provide the application of the aforementioned saponin in the preparation of products with antioxidant and / or hypoglycemic functions.

[0007] A third objective of this invention is to provide the use of the said saponin polysaccharide in the preparation of products that improve insulin resistance.

[0008] A fourth objective of this invention is to provide the use of the said saponin polysaccharide in the preparation of products for the prevention and / or treatment of diabetes.

[0009] To achieve the above objectives, the present invention is implemented through the following solution: A saponin polysaccharide, the preparation method of which includes the following steps: S1. The aqueous extract of saponin endosperm is subjected to alcohol precipitation, solid-liquid separation, and the solid is collected to obtain crude product 1. The crude product 1 is dissolved in water to obtain saponin polysaccharide extract; wherein the saponin is... Gleditsia sinensis Lam. S2. Remove the protein from the saponin extract, remove the solvent, collect the precipitate to obtain crude product 2, dissolve the crude product 2 in water to obtain a saponin crude polysaccharide solution; S3. Purify the saponin crude polysaccharide solution by ion exchange chromatography, elute with water, collect the first elution peak, remove the solvent, and obtain the final product.

[0010] Preferably, in step S1, the aqueous extract of the soapberry endosperm is obtained by ultrasound-assisted hot water extraction. More preferably, in step S1, the ultrasound conditions for the ultrasound-assisted hot water extraction include: power 300W-500W and time 10min-30min. Even more preferably, in step S1, the ultrasound conditions for the ultrasound-assisted hot water extraction include: power 350W and time 20min.

[0011] More preferably, in step S1, the ratio of soapberry endosperm to water is 1g:(20-40)mL. Even more preferably, in step S1, the ratio of soapberry endosperm to water is 1g:35mL.

[0012] More preferably, in step S1, the ultrasonic-assisted hot water extraction method includes the following steps: S11. Extracting the pulverized soapberry endosperm with water using ultrasonic extraction and collecting the ultrasonic product; S12. Heating the obtained ultrasonic product in a water bath at 60℃~90℃ for 1h~5h to obtain the water extract of the soapberry endosperm.

[0013] More preferably, in step S11, the pulverized soapberry endosperm is obtained by grinding dried soapberry endosperm. Even more preferably, in step S11, the pulverized soapberry endosperm is obtained by grinding dried soapberry endosperm and passing it through a 60-mesh sieve.

[0014] More preferably, in step S11, the mass-to-volume ratio of the pulverized soapberry endosperm to water is (1-3) g: (15-50) mL. Even more preferably, in step S11, the mass-to-volume ratio of the pulverized soapberry endosperm to water is 1 g: 35 mL.

[0015] More preferably, in step S11, the water is deionized water.

[0016] More preferably, in step S12, the obtained ultrasonic product is heated in a water bath at 80°C for 4 hours to obtain the aqueous extract of the soapberry endosperm.

[0017] Preferably, in step S1, the aqueous extract of the soapberry endosperm is first centrifuged, the supernatant is collected and concentrated to one-tenth to three-tenths of its original volume to obtain concentrate 1, and then concentrate 1 is subjected to alcohol precipitation. More preferably, in step S1, the aqueous extract of the soapberry endosperm is first centrifuged, the supernatant is collected and concentrated to one-tenth of its original volume to obtain concentrate 1, and then concentrate 1 is subjected to alcohol precipitation. More preferably, in step S1, the concentration method includes rotary evaporation under reduced pressure. More preferably, in step S1, the centrifugation conditions for the aqueous extract of the soapberry endosperm include: centrifugation at 3000 rpm to 5000 rpm for 10 min to 20 min. Even more preferably, in step S1, the centrifugation conditions for the aqueous extract of the soapberry endosperm include: centrifugation at 3000 rpm for 10 min.

[0018] Preferably, in step S1, alcohol precipitation is performed using 90% v / v to 95% v / v ethanol. More preferably, in step S1, alcohol precipitation is performed using 95% v / v ethanol.

[0019] Preferably, in step S1, the conditions for alcohol precipitation include: standing at 4°C to 28°C for 12 to 14 hours. More preferably, in step S1, the conditions for alcohol precipitation include: standing at 4°C for 12 hours. Further preferably, in step S1, the volume ratio of the concentrated solution 1 to 95% V / V ethanol is 1:(90 to 95). Even more preferably, in step S1, the volume ratio of the concentrated solution 1 to 95% V / V ethanol is 1:4.

[0020] Preferably, in step S1, the solid-liquid separation method includes centrifugation. More preferably, in step S1, the centrifugation conditions for solid-liquid separation include: centrifugation at 3000 rpm to 5000 rpm for 10 min to 20 min. Even more preferably, in step S1, the centrifugation conditions for solid-liquid separation include: centrifugation at 3000 rpm for 10 min.

[0021] Preferably, in step S1, the concentration of the saponin extract is 200 mg / mL to 300 mg / mL. More preferably, in step S1, the concentration of the saponin extract is 250 mg / mL.

[0022] Preferably, in step S2, the protein in the saponin extract is removed using the Sevage method.

[0023] More preferably, in step S2, the Sevage method includes the following steps: S21. The saponin extract and Sevage reagent are thoroughly mixed at a volume ratio of 1:(3-4), centrifuged, and separated into three layers. The solution is divided into three layers: the upper layer is a clear liquid containing saponin, the middle layer is protein, and the lower layer is an organic phase. The uppermost clear liquid is collected. S22. The obtained clear liquid is thoroughly mixed with Sevage reagent at a volume ratio of 1:(3-4), centrifuged, and separated into three layers. The uppermost clear liquid is collected. This step is repeated until there is no middle layer after separation, that is, the protein is completely removed. All the obtained clear liquids are combined.

[0024] More preferably, in step S21, the saponin extract and Sevage reagent are thoroughly mixed at a volume ratio of 1:4.

[0025] More preferably, in step S21, the centrifugation conditions include: centrifugation at 2000 rpm to 3000 rpm for 2 to 5 minutes. Even more preferably, in step S21, the centrifugation conditions include: centrifugation at 2500 rpm for 2 minutes.

[0026] More preferably, in step S22, the centrifugation conditions include: centrifugation at 2000 rpm to 3000 rpm for 2 to 5 minutes. Even more preferably, in step S22, the centrifugation conditions include: centrifugation at 2500 rpm for 2 minutes.

[0027] More preferably, in step S22, after combining all the obtained clear liquids, the solution is concentrated to one-half to one-quarter of its original volume to obtain concentrated solution 2. Even more preferably, in step S22, after combining all the obtained clear liquids, the solution is concentrated to one-third of its original volume to obtain concentrated solution 2.

[0028] Preferably, in step S2, the method for removing the solvent includes freeze drying.

[0029] Preferably, in step S2, the crude product 2 is dissolved in ultrapure water to obtain a crude saponin polysaccharide solution.

[0030] Preferably, in step S2, the concentration of the crude saponin solution is 70 mg / mL to 120 mg / mL.

[0031] Preferably, in step S2, the concentration of the crude saponin solution is 100 mg / mL.

[0032] Preferably, in step S3, the crude polysaccharide solution of the soapberry is purified using a DEAE-52 cellulose anion exchange chromatography column.

[0033] Preferably, in step S3, water is used for elution at a flow rate of 0.6 mL / min to 1.2 mL / min. More preferably, in step S3, water is used for elution at a flow rate of 1 mL / min.

[0034] Preferably, in step S3, the elution peak is determined using the phenol-sulfuric acid method. More preferably, in step S3, the absorption light is measured at a wavelength of 490 nm.

[0035] Preferably, in step S3, the ion exchange chromatography method includes the following steps: S31. Centrifuge the crude polysaccharide solution of Gleditsia sinensis, collect the supernatant, filter, and collect the filtrate; S32. Elute the product obtained in step S31 sequentially with water and a NaCl solution with a concentration of 0.1 M to 0.3 M at a flow rate of 0.6 mL / min to 1.2 mL / min, collect the first elution peak, remove the solvent, and obtain the final product.

[0036] More preferably, in step S31, the centrifugation conditions include: centrifugation at 2500 rpm to 4000 rpm for 5 to 10 minutes. Even more preferably, in step S31, the centrifugation conditions include: centrifugation at 3000 rpm for 5 minutes.

[0037] More preferably, in step S31, filtration is performed using a 0.45 μm aqueous filter membrane.

[0038] More preferably, in step S32, the water is deionized water.

[0039] Preferably, in step S3, the eluent obtained by elution with deionized water is collected in tubes 1 to 45 at a rate of 6 mL to 9 mL per tube. More preferably, in step S3, the eluent obtained by elution with deionized water is collected in tubes 16 to 42 at a rate of 8 mL per tube.

[0040] Preferably, in step S3, the solvent removal method includes sequentially performing vacuum concentration, dialysis, and freeze-drying. More preferably, in step S3, the dialysis method includes dialysis using a 3000 Da dialysis bag with water as the solvent for 36 h to 48 h. Even more preferably, in step S3, the dialysis method includes dialysis using a 3000 Da dialysis bag with water as the solvent for 48 h.

[0041] Preferably, the method further includes: S4. Purifying the product obtained in step S3 using dextran gel chromatography, collecting the eluent obtained by elution with water, removing the solvent, and obtaining the final product. More preferably, in step S4, the product obtained in step S3 is purified using a Sephadex G-150 dextran gel chromatography column.

[0042] More preferably, in step S4, the dextran gel chromatography method includes the following steps: S41. Dissolve the product obtained in step S3 in water to obtain a saponin solution with a concentration of 10 mg / mL to 30 mg / mL, centrifuge, collect the supernatant, filter, and collect the filtrate; S42. Elute the product obtained in step S41 with water at a flow rate of 0.5 mL / min to 0.7 mL / min.

[0043] More preferably, in step S41, the water is deionized water.

[0044] More preferably, in step S41, a saponin solution with a concentration of 20 mg / mL is obtained. Even more preferably, in step S41, the centrifugation conditions include: centrifugation at 2500 rpm to 4000 rpm for 5 to 10 minutes. Still more preferably, in step S41, the centrifugation conditions include: centrifugation at 3000 rpm for 5 minutes. Even more preferably, in step S41, filtration is performed using a 0.45 μm aqueous filter membrane.

[0045] More preferably, in step S42, the water is ultrapure water.

[0046] More preferably, in step S42, water is used for elution at a flow rate of 0.6 mL / min.

[0047] Preferably, in step S4, the eluent obtained by elution with ultrapure water is collected in 5 mL to 7 mL increments per tube, and tubes 1 to 20 are collected. More preferably, in step S4, the eluent obtained by elution with ultrapure water is collected in 6 mL increments per tube, and tubes 3 to 17 are collected.

[0048] Preferably, in step S4, the method for removing the solvent includes: sequentially performing vacuum concentration and freeze drying.

[0049] Preferably, the saponin contains mannose, galactose and glucose in a molar ratio of 76.24:22.57:1.2.

[0050] Preferably, the main structure of the saponin polysaccharide is based on β-(1-4)-d-mannan as the main chain and α-(1-6)-linked d-galactose as the side chain.

[0051] Preferably, the weight-average molecular weight of the saponin is 695.548 kDa.

[0052] The application of the saponin in the preparation of products with antioxidant and / or hypoglycemic functions, the application of the saponin in the preparation of products that improve insulin resistance, and the application of the saponin in the preparation of products that prevent and / or treat diabetes should also be within the scope of protection of this invention.

[0053] Compared with the prior art, the present invention has the following beneficial effects: This invention uses soapberry ( Gleditsia sinensis Lam. Using the endosperm of *Gleditsia sinensis* as raw material, a green and efficient extraction technology was employed to obtain purified *Gleditsia sinensis* polysaccharide GSP-1. Its structure was then analyzed and purified using gel permeation chromatography, methylation, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. *Gleditsia sinensis* polysaccharide GSP-1 exhibits excellent antioxidant and hypoglycemic effects, promoting glucose consumption and metabolism, effectively improving insulin resistance, and playing a positive role in regulating blood sugar and preventing diabetes. It has broad application prospects in the development of food processing for adjuvant treatment of diabetes and blood sugar control, the development of health products, and the addition of functional foods. It is of great significance for increasing the added value of *Gleditsia sinensis*, enhancing the technological innovation capabilities of agricultural products, and driving industrial transformation and upgrading. Attached Figure Description

[0054] Figure 1 The image shows the elution curve of crude saponin from Example 1 on a DEA2-52 cellulose ion chromatography column.

[0055] Figure 2 The results show the inhibitory capacity of GSP-1, GSP-2, and GSP-3 on starch digestive enzymes in Example 1; a represents the inhibition rate of α-glucosidase; b represents the inhibition rate of α-amylase.

[0056] Figure 3 The image shows the elution curve of GSP-1 on a Sephadex G-150 dextran gel exchange chromatography column in Example 1.

[0057] Figure 4 The image shows the HPSEC chromatogram of GSP-1 in Example 2.

[0058] Figure 5 The image shows the infrared spectrum analysis of GSP-1 in Example 2.

[0059] Figure 6 This is the gas chromatogram of GSP-1 in Example 2.

[0060] Figure 7 The total ion chromatogram is shown for polymethacrylic acid (PMAA), the methylated product of GSP-1 in Example 2.

[0061] Figure 8 This is the PMAA secondary ion mass spectrum of GSP-1 in Example 2.

[0062] Figure 9 This is the PMAA secondary ion mass spectrum of GSP-1 in Example 2.

[0063] Figure 10 This is the PMAA secondary ion mass spectrum of GSP-1 in Example 2.

[0064] Figure 11 This is the PMAA secondary ion mass spectrum of GSP-1 in Example 2.

[0065] Figure 12 This is the PMAA secondary ion mass spectrum of GSP-1 in Example 2.

[0066] Figure 13 For GSP-1 in Example 2 1 H NMR spectrum.

[0067] Figure 14 For GSP-1 in Example 2 13 CNMR spectrum.

[0068] Figure 15 The image shows the HSQC NMR spectrum of GSP-1 in Example 2.

[0069] Figure 16 The image shows the COSYNMR spectrum of GSP-1 in Example 2.

[0070] Figure 17 The NOESY NMR spectrum of GSP-1 in Example 2 is shown.

[0071] Figure 18 This is a predicted structure diagram of GSP-1 in Example 2.

[0072] Figure 19 The results are the reduction capacity test results of GSP-1 in Example 3.

[0073] Figure 20 The following are the evaluation results of the free radical scavenging ability of GSP-1 in Example 3; a is the result of the superoxide anion scavenging ability test; b is the result of the hydroxyl radical scavenging ability test.

[0074] Figure 21 The effect of different concentrations of GSP-1 on the viability of RAW.64 cells in Example 3.

[0075] Figure 22 The effect of different concentrations of GSP-1 on the antioxidant capacity of RAW.64 cells under H2O2-induced oxidative stress in Example 3 is shown in Figure a. Cell ROS activity assay results are shown in Figure b. Cell SOD enzyme activity assay results are shown in Figure b.

[0076] Figure 23 The figure shows the effect of different concentrations of GSP-1 on glucose uptake in IR-HepG2 cells in Example 4.

[0077] Figure 24 The graph shows the effect of different concentrations of GSP-1 on hexokinase (HK) and pyruvate kinase (PK) in IR-HepG2 cells in Example 4; a is the hexokinase (HK) graph; b is the pyruvate kinase (PK) graph. Detailed Implementation

[0078] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0079] Example 1: Preparation method of saponin 1. Determination of raw materials This embodiment first uses soapberry ( Gleditsia sinensis Lam. ), American soapberry ( Gleditsia triacanthos Linn ) and Yunnan soapberry ( Gleditsia japonica Miq Using the endosperm as raw material, the endosperm was collected, dried, ground into powder, and passed through a 60-mesh sieve. Then, a hot water extraction method was used, with the following extraction conditions: a material-to-liquid ratio of 1g:20mL, and a water bath at 80℃ for 4 hours. The water extract was collected to obtain crude polysaccharides from three types of soapberries.

[0080] Subsequently, a preliminary evaluation of the crude polysaccharide extraction efficiency and in vitro hypoglycemic activity was conducted. Experimental data showed that there were significant differences in the crude polysaccharide yield among the three types of Gleditsia sinensis (p<0.05). Gleditsia sinensis Lam. ), American soapberry ( Gleditsia triacanthos Linn ) and Yunnan soapberry ( Gleditsia japonica Miq The extraction rates of crude polysaccharides were 74.2±1.8%, 28.89±1.5%, and 28.89±1.5%, respectively. Furthermore, the in vitro hypoglycemic activities of the crude polysaccharides from the three Gleditsia sinensis species also showed significant differences (p<0.05). Gleditsia sinensis Lam. ), American soapberry ( Gleditsia triacanthos Linn ) and Yunnan soapberry ( Gleditsia japonica Miq The crude polysaccharide solution (2 mg / mL) showed the following inhibition rates against α-glucosidase: 54.35±1.2%, 42.19±0.8%, and 37.23±0.9%, respectively, and against α-amylase: 38.19±0.7%, 24.35±0.5%, and 20.19±0.6%, respectively.

[0081] According to statistical analysis, soapberry ( Gleditsia sinensis Lam. The extraction efficiency and in vitro hypoglycemic activity of the crude polysaccharide were significantly better than those of the other two soap pods (p<0.01), so it was used as a raw material for further extraction, separation and purification.

[0082] 2. Preparation of Gleditsia sinensis polysaccharide extract Dry soap pods ( Gleditsia sinensis Lam. The endosperm was ground into powder and passed through a 60-mesh sieve. 50 g of powder was taken and added to 1.75 L of deionized water at a material-to-liquid ratio of 1 g:35 mL. Extraction was performed using an ultrasonic extractor at 350 W for 20 min, followed by a water bath at 80 °C for 4 h. After cooling to room temperature (30 °C), the extract was centrifuged at 3500 rpm for 10 min. The collected supernatant was then concentrated to one-tenth of its original volume using rotary evaporation under reduced pressure to obtain concentrate 1. Four times the volume of 95% V / V ethanol was added to concentrate 1, and the mixture was stirred briefly. The mixture was then allowed to stand at 4 °C for alcohol precipitation overnight (12 h). After centrifugation at 3000 rpm for 10 min, the precipitate was collected and dissolved in deionized water at a mass-to-volume ratio of precipitate to deionized water of 1 g:4 mL to obtain the saponin extract.

[0083] 3. Soapberry polysaccharides remove protein The saponin extract obtained in the previous step was mixed with Sevage's reagent at a volume ratio of 1:4, vortexed, and centrifuged at 2500 rpm for 2 min. The solution separated into three layers: the upper layer was a clear liquid containing saponin, the middle layer was protein, and the lower layer was an organic phase. The clear liquid of the upper layer was collected. The addition of Sevage's reagent and centrifugation were repeated until no protein appeared in the middle layer. All the collected clear liquids were combined and concentrated under reduced pressure at 45°C to one-third of the original volume to obtain concentrate 2. The concentrate was freeze-dried to obtain crude saponin polysaccharide.

[0084] 4. Isolation of crude polysaccharides from soapberry The crude polysaccharide from the previous step was dissolved in ultrapure water to obtain a 100 mg / mL solution. The solution was centrifuged at 3000 rpm for 5 min, and the supernatant was collected and filtered through a 0.45 μm aqueous filter membrane. The filtrate was collected. After the DEAE-52 ion chromatography column system stabilized, the filtrate was loaded onto the column. Elution was performed sequentially with deionized water and 0.1–0.3 M NaCl solutions (i.e., 0.1 M, 0.2 M, and 0.3 M NaCl aqueous solutions) at a flow rate of 1 mL / min. 8 mL of eluent was collected per tube, with 40 tubes collected for each concentration gradient. The absorbance of each eluent tube at 490 nm was determined using the phenol-sulfuric acid method. An elution curve was established based on the absorbance. Figure 1 As shown, the three fractions eluted by deionized water (tubes 16-42), 0.1 M Nacl solution (tubes 63-70), and 0.2 M Nacl solution (tubes 92-96) were collected and labeled as GSP-1, GSP-2, and GSP-3, respectively.

[0085] The eluents containing the three components were concentrated under reduced pressure and transferred to a 3000 Da dialysis bag. Dialysis was performed in distilled water at 4°C for 48 h, with the distilled water changed every 6 h. After dialysis, the eluents were freeze-dried under vacuum. This yielded lyophilized powders of GSP-1 (eluted with distilled water), GSP-2 (eluted with 0.1 M NaCl solution), and GSP-3 (eluted with 0.2 M NaCl solution). The polysaccharide yields were calculated to be 70.38 ± 1.38% for GSP-1, 4.04 ± 1.12% for GSP-2, and 1.36 ± 0.95% for GSP-3.

[0086] 5. Determination of the inhibitory effect of crude polysaccharide from Gleditsia sinensis on amylase. The α-glucosidase and α-amylase activities of GSP-1, GSP-2, and GSP-3 were measured, and the fraction with the highest activity was selected for further purification.

[0087] (1) Preparation of the sample solution to be tested Dissolve the lyophilized powders of GSP-1, GSP-2, and GSP-3 obtained in the previous step in deionized water to obtain GSP-1 solutions with concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 2.0 mg / mL; GSP-2 solutions with concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 2.0 mg / mL; and GSP-3 solutions with concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 2.0 mg / mL.

[0088] (1) Assay of α-glucosidase activity Prepare α-glucosidase solution with a working concentration of 0.2 U / mL and PNPG solution with a working concentration of 5 mmol / L using phosphate buffer (pH=6.8).

[0089] Mix 20 μL of each concentration of GSP-1, GSP-2 and GSP-3 solution with 40 μL of α-glucosidase solution; incubate at 37℃ for 10 min; then add 100 μL of PNPG solution to each mixture and continue incubating at 37℃ for 30 min; finally, add 100 μL of 0.1 M Na2CO3 solution to end the reaction, measure the absorbance at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and set up a blank control (i.e., phosphate buffer instead of saponin) and a background control (i.e., phosphate buffer instead of α-glucosidase solution). Calculate the α-glucosidase inhibition rate according to formula (1).

[0090] Formula (1): ; Where As is the absorbance of the reaction solution of saponin, pNPG and α-glucosidase; Ac is the absorbance of the blank control; and Ab is the absorbance of the background control.

[0091] The α-glucosidase inhibition rates of GSP-1, GSP-2, and GSP-3 are as follows: Figure 2 As shown in a, GSP-1 exhibited the strongest inhibitory effect on α-glucosidase activity within the concentration range of 0.2–2.0 mg / mL. At a concentration of 2.0 mg / mL, the inhibition rates of GSP-1, GSP-2, and GSP-3 on α-glucosidase activity were 66.35±0.40%, 27.24±0.53%, and 22.49±0.49%, respectively, and the inhibition rates tended to plateau.

[0092] (2) Assay of α-amylase activity An α-amylase solution with a working concentration of 0.4 U / mL and a corn starch solution with a working concentration of 1% (%wt) were prepared using phosphate buffer (pH=6.8).

[0093] Mix 100 μL of each concentration of GSP-1, GSP-2, and GSP-3 solution with 100 µL of α-amylase solution and incubate at 37°C for 10 min. Then add 100 μL of corn starch solution (1%, m / v) to each mixture to start the reaction and continue incubation for 5 min. Then add 750 μL of DNS (3,5-dinitrosalicylic acid) to complete the reaction and immediately transfer to a 100°C water bath for 10 min to inactivate α-amylase. Measure the absorbance at 405 nm using a microplate reader. Set up a blank control (i.e., phosphate buffer instead of saponin) and a background control (i.e., phosphate buffer instead of α-amylase solution). Calculate the α-amylase inhibition rate according to formula (2).

[0094] Formula (2): ; Where Es is the absorbance of the reaction solution of saponin, corn starch, and α-amylase; Eb is the absorbance of the background control; and Ec is the absorbance of the blank control. The α-amylase inhibition rates of GSP-1, GSP-2, and GSP-3 are as follows: Figure 2 As shown in b, within the concentration range of 0.2–2.0 mg / mL, GSP-1 exhibited the strongest inhibitory effect on α-amylase activity, with the inhibition rate increasing from 14.06±1.24% to 48.20±1.18%. In contrast, the inhibition rate of GSP-2 increased from 6.06±0.24% to 25.20±1.22%, and the inhibition rate of GSP-3 increased from 3.06±0.13% to 20.18±1.23%.

[0095] The above results indicate that GSP-1 of the saponin polysaccharide has a stronger inhibitory effect on amylase than the other two components, so GSP-1 was purified.

[0096] 6. GSP-1 purification 100 mg of the lyophilized GSP-1 powder obtained in step three was reconstituted in 5 mL of deionized water to prepare a 20 mg / mL GSP-1 solution. The solution was centrifuged at 3000 rpm for 5 min, and the supernatant was collected. The solution was filtered through a 0.45 μm aqueous filter membrane, and the filtrate was collected. Further purification was performed using a Sephadex G-150 dextran gel chromatography column with ultrapure water as the eluent at a flow rate of 0.6 mL / min. 6 mL was collected from each of 20 tubes. The absorbance of each eluent tube at 490 nm was measured using the phenol-sulfuric acid method. The elution curve is shown below. Figure 3 As shown, the eluent from tubes 3 to 17 was collected, concentrated, and freeze-dried to obtain purified GSP-1 powder.

[0097] Example 2: Structural Identification of Gaposa Polysaccharide GSP-1 1. Molecular weight determination 1.0 mg of the purified GSP-1 powder prepared in Example 1 was weighed and dissolved in 1 mL of deionized water to obtain the sample for testing. The weight-average molecular weight (Mw) of GSP-1 was determined using high-performance liquid chromatography (HPLC) with a differential detector and high-performance size exclusion chromatography (HPSEC). Chromatographic conditions: Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ tandem columns (300×8 mm); column temperature 45℃; mobile phase 0.1 M NaNO3 solution; flow rate 0.6 mL / min; injection volume 10 μL. Standard curves were plotted using dextran standards with different molecular weights (6 kDa–2500 kDa), and the weight-average molecular weight of GSP-1 was calculated based on the fitting equation of the standard curve and the retention time of the sample. The HPSEC chromatogram of GSP-1 is shown below. Figure 4 As shown, the single symmetrical peak indicates that GSP-1 is a highly homogeneous polysaccharide. The calculated weight-average molecular weight of GSP-1 is 695.548 kDa.

[0098] 2. Infrared spectral analysis of GSP-1 1 mg of purified GSP-1 powder obtained in Example 1 was mixed with 100 mg of KBr powder and extruded into tablets. Fourier transform infrared spectroscopy (FT-IR) analysis was performed, and the concentrations in the range of 4000–400 cm⁻¹ were measured. -1 The transmittance within the specified range was used to obtain FT-IR spectra, which were then analyzed.

[0099] The FT-IR spectrum of GSP-1 is as follows: Figure 5 As shown, at 3391.76 cm -1 (OH) and 2923.40 cm -1The typical absorption band of polysaccharides can be clearly observed at (CH); at 1643.28 cm⁻¹ -1 The peak observed at 1200–1000 cm⁻¹ may be attributed to the deviation vibration of C=O bonds or the vibration of bound water; in addition, the peak observed at 1200–1000 cm⁻¹ may also be attributed to the vibration of C=O bonds or bound water. -1 The wavelength range at which GSP-1 is located is a unique characteristic absorption region of polysaccharides; GSP-1 exhibits this characteristic at 1022 cm⁻¹. -1 The presence of an absorption peak near 872.98 cm⁻¹ indicates the presence of a pyranose ring in GSP-1; -1 and 814.43cm -1 The absorption peak at that location indicates that GSP contains β - Configuration and α - Configurational glycosidic bonds are present.

[0100] 3. Monosaccharide composition analysis of GSP-1 Accurately weigh 5 mg ± 0.05 mg of the purified GSP-1 powder obtained in Example 1, add 1 mL of 2M TFA solution, heat at 121°C for 2 hours; purge with nitrogen and dry; add methanol to wash, then dry again, repeat the methanol washing twice; add sterile water to dissolve, pass through a 0.22 μm organic membrane, collect the filtrate and transfer it to a chromatographic bottle for analysis.

[0101] Thirteen monosaccharide standards (fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), and mannuronic acid (Man-UA)) were accurately weighed, dissolved in 2M TFA, and prepared into a monosaccharide standard mixture with a total concentration of 100 μg / mL. The mixture was then filtered through a 0.22 μm organic membrane and subjected to gas chromatography analysis.

[0102] Gas chromatograms of 13 standards are shown below. Figure 6 As shown in a, the gas chromatogram of GSP-1 is as follows: Figure 6 As shown in b, by comparison, GSP-1 is a neutral polysaccharide, mainly composed of mannose, galactose and glucose in a molar ratio of 76.24:22.57:1.2.

[0103] 4. Analysis of the glycosidic bond linkage mode of GSP-1 To elucidate the glycosidic bond linkage mode of GSP-1, methylation analysis was performed. The specific method is as follows: 1 mg of purified GSP-1 powder prepared in Example 1 was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) and 1 mg of NaOH and reacted for 30 min to obtain the test solution. 50 μL of iodomethane reagent was slowly added, and the mixture was incubated for 60 min. The reaction was then terminated by adding 2 mL of distilled water. Extraction was performed with 1 mL of chloroform (CHCl3), and the organic phase was collected. This extraction was repeated three times. The extracts were combined, concentrated by rotary evaporation, and freeze-dried. Subsequently, 100 μL of 2M TFA solution was added, and the mixture was reacted at 121 °C for 90 min, then evaporated to dryness at 30 °C. 50 μL of 2M ammonia and 50 μL of 1M NaBD4 reducing solution were added, and the mixture was allowed to stand for 2.5 h. 20 μL of acetic acid was added to stop the reaction. 250 μL of acetic anhydride was added to the reactants, and the mixture was thoroughly mixed and reacted in an oil bath at 100 °C for 2.5 h. After cooling to room temperature (37 °C), 1 mL of deionized water was added, and the mixture was allowed to stand for 10 min. Extract with 500 μL of dichloromethane, centrifuge, remove the aqueous phase, and collect the dichloromethane phase. Repeat the extraction twice, combine the dichloromethane phases, and analyze by GC-MS.

[0104] The total ion spectrum of GSP-1 is as follows: Figure 7 As shown, the mass spectrum of GSP-1 is as follows: Figures 8-12 As shown, there are 5 different types of glycosidic bonds, and the specific information is shown in Table 1.

[0105] Table 1. Methylation analysis results of GSP-1 glycosidic bond linkage.

[0106] 5. Nuclear magnetic resonance analysis of the GSP-1 structure Further analysis of the configuration, substitution sites, and sequence of sugar residues in GSP-1 was performed using nuclear magnetic resonance (NMR). The specific method was as follows: 50 mg of purified GSP-1 powder obtained in Example 1 was dissolved thoroughly in 550 μL of D2O and then freeze-dried under vacuum. The D2O dissolution and freeze-drying steps were repeated three times. The sample after dissolution and drying in 550 μL of D2O was centrifuged at 8000 rpm for 10 min. The supernatant was passed through a 0.22 μm organic membrane, and the filtrate was collected and transferred to an NMR tube. The sample was then analyzed using NMR. 1 H NMR, 13 C NMR, 1 H- 1 H COSY NMR, (CH)HSQC NMR, and NOESY NMR. Glycosidic bonds. 1 H and 13 The signal assignments for C are shown in Table 2, and they are labeled as A, B, and C according to their glycosidic bonds.

[0107] GSP-1 1 H NMR spectrum as follows Figure 13 As shown, anomeric hydrogen signals were observed in the range of 4.4–5.2 ppm, indicating that GSP-1 contains… α - Glucoside bond (δ>5.00 ppm) and β -Glucoside bond, which is consistent with the FT-IR spectroscopy results. GSP-1 13 C NMR spectrum as follows Figure 14 As shown, three sets of anomaly carbon signals were found in the 90–100 ppm anomaly carbon region, located at δ 99.51, 99.96 and 100.17 ppm, respectively.

[0108] The structure of GSP-1 was analyzed using two-dimensional NMR spectra. Firstly, combined with... Figure 14 shown 13 C NMR and Figure 15 The (CH)HSQC NMR spectrum shown identifies three sets of H1 / C1 related signals in the anodic region, namely 4.64 / 100.91, 4.64 / 99.96, and 5.03 / 99.51 ppm; among them, 4.64 / 100.91 is the H1 / C1 signal of →4)-Man-(1→, 4.64 / 99.96 is the H1 / C1 signal of →4,6)-Man-(1→, and 5.03 / 99.51 is the H1 / C1 signal of Gal-(1→).

[0109] Based on the previously speculated 4.64 ppm, which belongs to the anolyte H1 in the residual sugar A→4)-Man-(1→, combined with... Figure 16 shown 1 H- 1 The H COSY NMR spectrum revealed a signal of 4.14 ppm associated with the residual sugar, confirming the positions of H1 and H2; further, combined with... Figure 15 The (CH)HSQC spectrum shown indicates that the signal for H2 / C2 is 4.14 / 69.55 ppm. Using the same method, the positions of H3 / C3, H4 / C4, H5 / C5, and H6 / C6 are determined to be 3.78 / 71.33, 3.84 / 76.37, 3.59 / 75.47, and 3.92 / 61.20, respectively. Following this method, the signal positions of residues B and C were further determined, as shown in Table 2.

[0110] Table 2 GSP-1 1 H and 13 C NMR chemical shift

[0111] Through such Figure 17The NOESY spectrum shown verifies the presence of glycosidic bonds in GSP-1. The signal of 4.64 / 100.91 confirms that residue A is →4)-β-D-Man-(1→); the signal of 4.64 / 99.96 confirms that residue B is →4,6)-β-D-Manp-(1→); and the signal of 5.03 / 99.51 confirms that residue C is α-D-Galp-(1→).

[0112] Therefore, based on the results of the above tests, such as Figure 18 As shown, the main structure of GSP-1 is based on β-(1-4)-d-mannan as the main chain and α-(1-6)-linked d-galactose as the side chain.

[0113] Example 3 Evaluation of the antioxidant activity of GSP-1 1. GSP-1's reducing power and free radical scavenging ability The purified GSP-1 powder obtained in Example 1 was prepared into GSP-1 solutions of different concentrations (0 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL) using deionized water.

[0114] Vitamin C (Vc) was used as a positive control. Vc solutions of different concentrations (0 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL) were prepared with deionized water.

[0115] The reducing power of GSP-1 was determined using the Total Antioxidant Kit (FRAP method) developed in Nanjing. The superoxide anion scavenging capacity of GSP-1 was determined using the Solarbiot superoxide anion scavenging capacity assay kit. The hydroxyl radical scavenging capacity of GSP-1 was determined using the Solarbiot hydroxyl radical scavenging capacity assay kit.

[0116] The results of the GSP-1 reducing power test are as follows: Figure 19 As shown, within the concentration range of 0 mg / mL to 5 mg / mL, the reducing power of GSP-1 gradually increased with increasing concentration, approaching that of the Vc group; when the concentration of GSP-1 reached 2 mg / mL, the absorbance at 700 nm was 1.48, which was greater than the absorbance of saponin polysaccharides GSP-GSP-4 at the same concentration at 700 nm (Liu Fang. Extraction, separation, preliminary analysis of structure and study of some bioactivity of saponin polysaccharides [D]. Kunming University of Science and Technology, 2012.).

[0117] Superoxide anion radicals have a longer lifetime than other free radicals and can further participate in the formation of more oxygen-containing substances. For example... Figure 20 As shown in a, the superoxide radical scavenging ability of GSP-1 is positively correlated with its concentration. When the concentration of GSP-1 is 0.5 mg / mL, the superoxide radical scavenging rate of GSP-1 reaches 67.21%, which is significantly higher than that of the existing saponin polysaccharides GSP-GSP-4 (33.38%~66.42%) (Liu Fang. Extraction, separation, preliminary structural analysis and some bioactivity studies of saponin polysaccharides [D]. Kunming University of Science and Technology, 2012.) Hydroxyl radicals (OH·) are among the most harmful reactive oxygen species in living organisms. They participate in cellular activities through dehydrogenation and electron transfer, causing damage to nucleic acids, proteins, and lipids, resulting in severe damage to biomolecules, tissue necrosis, and even the death of living cells. For example... Figure 20 As shown in b, the scavenging ability of GSP-1 against hydroxyl radicals is positively correlated with its concentration. When the concentration of GSP-1 is 0.5 mg / mL, the hydroxyl radical scavenging rate of GSP-1 is 73.05%, which is significantly higher than that of the existing saponin polysaccharides GSP-GSP-4 (3.19% to 72.87%) (Liu Fang. Extraction, separation, preliminary analysis of structure and study of some bioactivity of saponin polysaccharides [D]. Kunming University of Science and Technology, 2012).

[0118] 2. Effects of GSP-1 on RAW.64 cell viability The purified GSP-1 powder obtained in Example 1 was prepared into GSP-1 solutions of different concentrations (200 μg / mL, 400 μg / mL and 600 μg / mL) using DMEM complete medium (i.e. DMEM containing 10% v / v fetal bovine serum and 1% v / v penicillin and streptomycin antibiotics).

[0119] After four passages of revived RAW.64 cells, RAW 264.7 cells in logarithmic growth phase were selected and formulated into a density of 3 × 10⁻⁶ cells / cells. 4 Cell suspension at 100 μL / mL was rapidly and evenly seeded into 96-well plates and incubated at 37°C with 5% CO2. After 24 h of culture, the old culture medium was discarded, and different concentrations of GSP-1 solution were added. RAW 264.7 cells without GSP-1 were used as the control group (i.e., Normal). Cells were cultured for another 24 h. Cell viability was assessed using a CCK-8 assay kit to determine the cytotoxicity of GSP-1.

[0120] like Figure 21As shown, compared with the control group, GSP-1 had no obvious toxic side effects on RAW.64 cells in the concentration range of 200-600 μg / mL. The cell viability increased with the increase of GSP-1 concentration, indicating that GSP-1 is safe for cells in this concentration range and promotes cell proliferation.

[0121] 3. Effects of GSP-1 on the antioxidant capacity of RAW.64 cells (1) Effect of GSP-1 on ROS content in RAW.64 cells The purified GSP-1 powder obtained in Example 1 was prepared into GSP-1 solutions of different concentrations (200 μg / mL, 400 μg / mL, 600 μg / mL) using DMEM complete medium.

[0122] RAW.64 cells in the logarithmic growth phase were formulated into 3×10⁻⁶ cells. 4 Cell suspensions of 1 cell / mL were seeded into black 96-well plates and incubated at 37°C with 5% CO2 for 24 h. After the cells adhered, they were grouped and processed.

[0123] Control, blank (Normal), model, experimental (different concentrations of GSP-1), and positive (200 μg / mL Vc) groups were set up. The control group was added to 100 μL of DMEM complete medium; the experimental groups were added to 100 μL of GSP-1 solution containing different concentrations; the positive group was added to 100 μL of DMEM complete medium containing Vc; and the blank and model groups were added to 100 μL of DMEM complete medium. Cells were cultured for 24 h, then the old medium was removed, and cells were washed with PBS. Except for the blank group, all other groups were treated with 100 μL of DMEM complete medium containing 0.45 mM H2O2 for 1 h. After washing twice with PBS buffer, cells were treated with a reactive oxygen species (ROS) assay kit developed by Nanjing Jiancheng. Fluorescence intensity was measured using a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm). The ROS production rate (%) was calculated based on the experimental group. The formula for calculating the ROS production rate (%) is as follows: .

[0124] like Figure 22As shown in Figure a, compared with the control group, the ROS production rate in RAW264.7 cells of the model group was significantly increased after H2O2 stimulation (P < 0.05), indicating that the intracellular ROS content in cells treated with H2O2 was abnormally increased, and the RAW264.7 cells were already in a state of oxidative damage. When RAW264.7 cells were treated with different concentrations of GSP-1, the intracellular ROS production rate decreased significantly, showing a negative correlation with the concentration of GSP-1. This indicates that GSP-1 inhibits the ROS activity of cells and enhances the antioxidant capacity of cells.

[0125] (2) Effect of GSP-1 on SOD enzyme activity in RAW.64 cells RAW 264.7 cells in the logarithmic growth phase were formulated to a density of 1×10⁻⁶ cells / cells. 6 Cell suspension of 2 mL / mL was seeded into six-well plates and incubated at 37°C with 5% CO2 for 24 h. After cell attachment, the cells were grouped and processed.

[0126] Cells were divided into a normal control group, a model group, an experimental group (different concentrations of GSP-1), and a positive control group (200 μg / mL of vitamin C). The experimental group received 100 μL of GSP-1 solution at different concentrations; the positive control group received 100 μL of DMEM complete medium containing vitamin C; and the normal control and model groups received 100 μL of DMEM complete medium. Cells were cultured for 24 h, after which the old medium was removed and cells were washed with PBS. Except for the normal control group, all other groups received 100 μL of complete medium containing 0.45 mM H2O2 and were treated for 1 h. Cells were then washed twice with PBS buffer, and 100 μL of cell lysis buffer (Triton X-100 containing 1 mM PMSF) was added to each well and aspirated. This process was performed on ice for 40 min at low temperature, followed by centrifugation to collect the supernatant. The SOD content in the cells was measured using a SOD assay kit developed by Nanjing Jiancheng.

[0127] like Figure 22As shown in b, the SOD activity in the model group cells treated with H2O2 for 1 h was significantly lower than that in the control group cells (P < 0.05), indicating that the cells were already under oxidative stress. After treatment with different concentrations of GSP-1, the intracellular SOD activity significantly increased (P < 0.05) and showed a positive correlation with the GSP-1 concentration. When the GSP-1 concentration reached 600 μg / mL, the intracellular SOD activity increased to 25.06 U / mgprot, comparable to that of vitamin C. This indicates that GSP-1 enhances the SOD enzyme activity of cells, strengthens the antioxidant capacity of cells, and helps cells repair themselves under oxidative stress.

[0128] Example 4 Evaluation of the hypoglycemic effect of GSP-1 1. Effect of GSP-1 on glucose uptake in insulin-resistant HepG2 cells The purified GSP-1 powder obtained in Example 1 was prepared into GSP-1 solutions of different concentrations (0.2 mg / mL, 0.6 mg / mL, 1 mg / mL) using DMEM complete medium.

[0129] HepG2 cells in the logarithmic growth phase were formulated to a density of 3 × 10⁻⁶. 4 Cell suspension of cells / mL was evenly seeded at 100 μL / well in 96-well plates and cultured for 24 h. After cell adhesion, cells were grouped and treated.

[0130] The experiment included a normal group, a model group, an experimental group, and a positive control group (Met). Except for the normal group, all other groups were treated with a final concentration of 10... -6 Insulin resistance was induced in cells by treatment with mol / L insulin for 36 h, thus constructing an insulin-resistant HepG2 cell model (i.e., IR-HepG2). Afterwards, the old culture medium from each group was discarded, and the cells were washed twice with PBS solution.

[0131] 100 μL of GSP-1 solution containing different concentrations was added to each well of the experimental group; 100 μL of DMEM complete medium containing 0.1 mg / mL metformin hydrochloride was added to each well of the positive group; and 100 μL of DMEM complete medium was added to each well of the normal and model groups. Culture was continued for 24 h. After culture, glucose consumption in each group was measured using a glucose kit developed in Nanjing.

[0132] like Figure 23As shown, the glucose consumption in the model group was significantly lower than that in the normal group, indicating that HepG2 cells were already in a state of insulin resistance. After treatment with different concentrations (0.2 mg / mL, 0.6 mg / mL, and 1 mg / mL) of GSP-1, the glucose consumption in the experimental groups was significantly higher than that in the model group, increasing by 1.05 times, 1.45 times, and 2 times respectively. This indicates that GSP-1 promotes glucose uptake in insulin-resistant cells.

[0133] 2. Effects of GSP-1 on glycolysis in insulin-resistant HepG2 cells The purified GSP-1 powder obtained in Example 1 was prepared into GSP-1 solutions of different concentrations (0.2 mg / mL, 0.6 mg / mL, 1 mg / mL) using DMEM complete medium.

[0134] HepG2 cells in the logarithmic growth phase were formulated to a density of 3 × 10⁻⁶. 4 Cell suspension of cells / mL was evenly seeded at 100 μL / well in 96-well plates and cultured for 24 h. After cell adhesion, cells were grouped and treated.

[0135] The experiment included a normal group, a model group, an experimental group, and a positive control group (Met). Except for the normal group, all other groups were treated with a final concentration of 10... -6 Insulin resistance was induced in cells by treatment with mol / L insulin for 36 h, thus constructing an insulin-resistant HepG2 cell model (i.e., IR-HepG2). Afterwards, the old culture medium from each group was discarded, and the cells were washed twice with PBS solution.

[0136] 100 μL of GSP-1 solution with different concentrations was added to each well of the experimental group; 100 μL of DMEM complete medium containing 0.1 mg / mL metformin hydrochloride was added to each well of the positive group; and 100 μL of DMEM complete medium was added to each well of the normal group and the model group.

[0137] Afterwards, discard the old culture medium from each group, wash twice with PBS solution, and then digest the cells with trypsin and collect them into 1.5 mL centrifuge tubes. After centrifugation, resuspend the cells in 200 μL PBS and count them. Analyze the cells using a hexokinase kit and a pyruvate kinase kit at cell counts (10⁻⁶). 4 Cells were added to the extract at a ratio of 500:1 (volume volume: mL); then, the cells were disrupted using an ultrasonic disruptor under ice-water bath conditions (ultrasound 3 s, 10 s interval, power 150 W, 30 cycles); the supernatant was collected by centrifugation, and the contents of hexokinase (HK) and pyruvate kinase (PK) in each group of cells were measured.

[0138] like Figure 24 As shown in figures a and b, the levels of hexokinase and pyruvate kinase in the model group were significantly lower than those in the control group, indicating that HepG2 cells were already in a state of insulin resistance. After treatment with different concentrations (0.2 mg / mL, 0.6 mg / mL, and 1 mg / mL) of GSP-1, the activities of hexokinase and pyruvate kinase in the experimental group were significantly increased compared to the model group. When the concentration of GSP-1 was 1 mg / mL, the activities of HK and PK in the experimental group increased by 64.59% and 125.04%, respectively, compared to the model group. These experimental results indicate that GSP-1 promotes glycolysis in insulin-resistant cells.

[0139] The above results indicate that GSP-1 possesses both excellent antioxidant capacity and hypoglycemic effect, promotes glucose consumption and metabolism, effectively improves insulin resistance, and has a positive promoting effect on regulating blood sugar and preventing and treating diabetes. It can be widely used in food processing, health product development, and functional food additives for the adjuvant treatment of diabetes and blood sugar control.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A saponin polysaccharide, characterized in that, Its preparation method includes the following steps: S1. The aqueous extract of Gleditsia sinensis endosperm is subjected to alcohol precipitation, solid-liquid separation, and solid collection to obtain crude product 1. Crude product 1 is dissolved in water to obtain a Gleditsia sinensis polysaccharide extract; the Gleditsia sinensis is... Gleditsia sinensis Lam. ; S2. Remove the protein from the saponin extract, remove the solvent, collect the precipitate to obtain crude product 2, dissolve the crude product 2 in water to obtain saponin crude polysaccharide solution; S3. Purify the crude polysaccharide solution of Gleditsia sinensis by ion exchange chromatography, elute with water, collect the first elution peak, remove the solvent, and obtain the product.

2. The saponin according to claim 1, characterized in that, In step S1, the aqueous extract of the soapberry endosperm is obtained by ultrasonic-assisted hot water extraction.

3. The saponin according to claim 1, characterized in that, In step S3, water is used for elution at a flow rate of 0.6 mL / min to 1.2 mL / min.

4. The saponin according to claim 1, characterized in that, The preparation method further includes: S4. Purify the product obtained in step S3 by dextran gel chromatography, collect the eluent obtained by elution with water, remove the solvent, and obtain the final product.

5. The saponin according to claim 1, characterized in that, It contains mannose, galactose and glucose in a molar ratio of 76.24:22.57:1.

2.

6. The saponin according to claim 1, characterized in that, Its main structure consists of β-(1-4)-d-mannan as the main chain and α-(1-6)-linked d-galactose as the side chain.

7. The saponin according to claim 1, characterized in that, Its weight-average molecular weight is 695.548 kDa.

8. The use of the saponin according to any one of claims 1 to 7 in the preparation of products with antioxidant and / or hypoglycemic functions.

9. The use of the saponin according to any one of claims 1 to 7 in the preparation of products that improve insulin resistance.

10. The use of the saponin according to any one of claims 1 to 7 in the preparation of products for the prevention and / or treatment of diabetes.

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