Wine stewed corni fructus oligosaccharide, and preparation method and application thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine processing, in particular to a kind of wine stewed fructus corni oligosaccharide and its preparation method and application.The preparation method comprises: after yellow rice wine is used to moisten fructus corni, it is stewed by water separation, total sugar extract is obtained by defatting, water extraction, protein removal and decolorization purification, then alcohol precipitation grading, freeze-drying is carried out, and wine stewed fructus corni oligosaccharide is obtained.Experimental results show that wine stewing process can significantly improve the proportion of oligosaccharide in fructus corni total sugar, and the obtained oligosaccharide can significantly improve the insulin resistance state of IR-HepG2 cells, and the effect is equivalent to that of metformin hydrochloride.The present application also discloses the application of the oligosaccharide in preparing drugs for improving insulin resistance and treating type 2 diabetes.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine processing technology, specifically to a wine-stewed Cornus officinalis oligosaccharide, its preparation method, and its application. Background Technology

[0002] After processing, Cornus officinalis has significantly enhanced liver and kidney tonifying effects. However, the current processing standards are not uniform, and the effects of different processing techniques on its active ingredients and the mechanism of synergistic effects are not yet clear, which restricts the standardized clinical application of Cornus officinalis.

[0003] Type 2 diabetes is a metabolic disease characterized by insulin resistance. While existing first-line treatments such as metformin hydrochloride, glimepiride, and acarbose have some efficacy, they all have significant limitations: metformin hydrochloride causes severe gastrointestinal reactions, commonly including bloating and diarrhea; glimepiride easily induces hypoglycemia and weight gain; acarbose has limited hypoglycemic capacity and significant gastrointestinal side effects; and insulin requires injection, making it inconvenient to use. All of these drugs pose safety risks or the risk of drug resistance, making them unsuitable for long-term use. The active ingredients of naturally derived traditional Chinese medicine have significant research value in the treatment of type 2 diabetes due to their multi-target mechanism of action, high safety, and lack of drug resistance. However, current research lacks a systematic elucidation of the material basis and key active components of processed Cornus officinalis. Summary of the Invention

[0004] This invention provides a wine-stewed Cornus officinalis oligosaccharide, its preparation method, and its application, in order to solve the problems in the prior art such as inconsistent processing standards for Cornus officinalis, unclear material basis for enhanced efficacy of processing, and potential safety hazards in drugs for the treatment of type 2 diabetes.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a method for preparing oligosaccharides from stewed Cornus officinalis, comprising: S1: Take the raw Cornus officinalis, remove the fruit stalk and kernel, add rice wine at a ratio of 15% to 25% of the weight of the medicinal material, mix thoroughly, place in a covered container and let it soak for 2 to 4 hours. After the rice wine is absorbed, place it in a stewing pot and stew it in water for 6 to 8 hours until the Cornus officinalis flesh turns black. After taking it out, dry it at 60℃ to obtain stewed Cornus officinalis. S2: Take the wine-stewed Cornus officinalis, grind it into powder and pass it through a 60-mesh sieve. Using anhydrous ethanol as a solvent, add 8-12 mL of anhydrous ethanol per gram of medicinal material and reflux at 75-85℃ for 6-10 h until the ethanol extract is colorless. Dry it to obtain defatted medicinal powder. S3: Take defatted medicinal powder, add 32-40 mL of water per gram of medicinal material and soak at room temperature for 8-12 h, heat in a 100℃ water bath for 2.5-3.5 h, filter, concentrate the filtrate under reduced pressure and directly remove protein and decolorize without alcohol precipitation to obtain total sugar extract; S4: Take the total sugar extract, redissolve it in water, add 3 to 5 times the volume of anhydrous ethanol to the redissolved solution, stir well, let it stand at 4℃ for 8 to 12 h, centrifuge, take the supernatant, concentrate under reduced pressure to remove ethanol, and freeze dry to obtain the wine-stewed Cornus officinalis oligosaccharide.

[0006] In a preferred embodiment of the present invention, the proportion of rice wine added is 20% of the weight of the medicinal material.

[0007] In a preferred embodiment of the present invention, the volume of anhydrous ethanol added per gram of medicinal material is 10 mL, the reflux extraction temperature is 80℃, and the reflux extraction time is 8 h.

[0008] In a preferred embodiment of the present invention, the volume of water added per gram of medicinal material is 36 mL, and the water bath heating time is 3 hours.

[0009] In a preferred embodiment of the present invention, the protein removal is performed using the Sevag method, and the decolorization is performed using S-8 macroporous resin.

[0010] In a preferred embodiment of the present invention, the amount of water added for reconstitution is 10 mL of water per gram of total sugar extract, and the amount of anhydrous ethanol added is 4 times the volume of the reconstitution solution.

[0011] The present invention also proposes a wine-stewed Cornus officinalis oligosaccharide, which is prepared by the above-mentioned method for preparing wine-stewed Cornus officinalis oligosaccharide.

[0012] This invention also proposes the application of wine-stewed Cornus officinalis oligosaccharide in the preparation of drugs to improve insulin resistance.

[0013] This invention also proposes the application of wine-stewed Cornus officinalis oligosaccharide in the preparation of drugs for treating type 2 diabetes.

[0014] The beneficial effects of this invention are: 1. This invention systematically compares various processing techniques for Cornus officinalis and identifies wine-stewing as the optimal processing method. The resulting product has a significantly higher oligosaccharide content than other processing techniques and the raw product, providing a scientific basis for the standardization of Cornus officinalis processing.

[0015] 2. This invention clarifies for the first time that the material basis for the enhanced efficacy of wine-stewed Cornus officinalis is the oligosaccharide fraction, and reveals the mechanism by which acid and heat conditions during the processing promote the degradation of polysaccharides into highly active short-chain oligosaccharides, providing a new approach for the study of the processing mechanism of traditional Chinese medicine.

[0016] 3. The wine-stewed Cornus officinalis oligosaccharide prepared in this invention has significant activity in improving insulin resistance, with effects comparable to the first-line Western medicine metformin hydrochloride. Moreover, it is naturally derived, has no risk of hypoglycemia, and is liver and kidney friendly, providing a new candidate drug for the treatment of type 2 diabetes. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The figure shows the CCK-8 assay results of the effect of total sugar in raw and processed Cornus officinalis on the survival rate of HepG2 cells. In the figure, A is raw Cornus officinalis, B is processed Cornus officinalis steamed with wine, C is processed Cornus officinalis stewed with wine, D is processed Cornus officinalis steamed with wine under high pressure, and E is processed Cornus officinalis steamed.

[0018] Figure 2 The graph shows the effect of different concentrations of insulin on glucose consumption and cell survival rate of HepG2 cells, where A represents the result of glucose consumption and B represents the result of cell survival rate.

[0019] Figure 3 The image shows the results of Oil Red O staining, where A is the normal control group and B is the insulin resistance model group, magnified 400 times.

[0020] Figure 4 The images show the appearance of raw Cornus officinalis and its various processed products. A represents raw Cornus officinalis, B represents Cornus officinalis steamed in wine, C represents Cornus officinalis stewed in wine, D represents Cornus officinalis steamed in wine under high pressure, and E represents Cornus officinalis steamed in plain water.

[0021] Figure 5 A comparative graph showing the effect of total sugar from raw Cornus officinalis and different processed products on glucose consumption in insulin-resistant HepG2 cells.

[0022] Figure 6 This is a comparative graph showing the effects of different sugar components and polysaccharide degradation products of Cornus officinalis on glucose consumption in insulin-resistant HepG2 cells.

[0023] Figure 7 Capillary zone electrophoresis spectra of oligosaccharides from raw Cornus officinalis and four processed products.

[0024] Figure 8 This is a calibration curve for gel permeation chromatography.

[0025] Figure 9 This is a molecular weight distribution diagram of polysaccharides in raw and wine-stewed Cornus officinalis. Detailed Implementation

[0026] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0027] Example 1: S1: Take the raw Cornus officinalis, remove the fruit stalks, kernels and other impurities, add rice wine at a ratio of 20% of the weight of the medicinal material, mix thoroughly and place in a covered container to seal and moisten for 2 to 4 hours. After the rice wine is fully absorbed by the medicinal material, transfer the medicinal material to a stewing pot and stew for 6 to 8 hours until the Cornus officinalis flesh turns black. After taking it out, dry it at 60℃ to obtain stewed Cornus officinalis.

[0028] S2: Take the stewed Cornus officinalis, grind it into powder, and pass it through a 60-mesh sieve. Using anhydrous ethanol as a solvent, add 10 mL of anhydrous ethanol per gram of medicinal material and reflux at 80°C for 8 hours until the ethanol extract becomes colorless. After taking it out, dry it in a fume hood to obtain defatted medicinal powder.

[0029] S3: Take defatted medicinal powder, add 36 mL of water per gram of medicinal material, soak at room temperature for 12 hours, then heat in a 100℃ water bath for 3 hours, filter while hot, collect the filtrate, concentrate the filtrate to an appropriate volume by rotary evaporation under reduced pressure, remove protein impurities by Sevag method without alcohol precipitation, and then decolorize and purify using S-8 macroporous resin. The decolorization conditions are: temperature 60℃, sample concentration 10 mg / mL, solution pH 5, time 4 h, polysaccharide retention rate is above 90%, and the total sugar extract is obtained.

[0030] S4: Take the total sugar extract and redissolve it in 10 mL of pure water per gram of total sugar extract. Slowly add 4 times the volume of anhydrous ethanol to the redissolved solution, stir well, and let it stand at 4°C for 12 hours. Centrifuge and collect the supernatant. Concentrate the supernatant under reduced pressure to remove the ethanol. Freeze-dry the obtained concentrate to obtain the wine-stewed Cornus officinalis oligosaccharide, and store it in a dry and sealed container for later use.

[0031] Example 2: S1: Take the raw Cornus officinalis, remove the fruit stalks, kernels and other impurities, add rice wine at a ratio of 20% of the weight of the medicinal material, mix thoroughly and place in a covered container to seal and moisten for 1 hour, spread the medicinal material out on a steamer lined with filter cloth, steam over water for about 6 hours until the flesh of the Cornus officinalis turns black, take it out and dry at 60℃ to obtain wine-steamed Cornus officinalis.

[0032] S2~S4: Same as Example 1.

[0033] Example 3: S1: Take the raw Cornus officinalis, remove the fruit stalks, kernels and other impurities, add rice wine at a ratio of 20% of the weight of the medicinal material, mix thoroughly and place in a covered container to seal and moisten for 1 hour, place in a high pressure sterilizer and steam at 115℃ for 1 hour, take it out and dry at 60℃ to obtain high pressure steamed Cornus officinalis.

[0034] S2~S4: Same as Example 1.

[0035] Example 4: S1: Take the raw Cornus officinalis, remove the fruit stalks, kernels and other impurities, place it in a steamer, heat it over high heat first, and after the steam rises, turn to low heat and steam until the outer skin turns purplish-black. After turning off the heat, let it sit overnight, take it out and dry it at 60℃ to obtain steamed Cornus officinalis.

[0036] S2~S4: Same as Example 1.

[0037] Compare with Example 1: Take the raw Cornus officinalis, remove impurities such as fruit stalks and kernels, add 36 mL of water per gram of herb and soak at room temperature for 12 hours, heat in a 100℃ water bath for 3 hours, filter while hot, collect the filtrate, concentrate under reduced pressure, add 4 times the volume of anhydrous ethanol, let stand at 4℃ for 12 hours, centrifuge, collect the precipitate, redissolve in water to obtain raw Cornus officinalis polysaccharide. Take the above raw Cornus officinalis polysaccharide and prepare a 15 mg / mL solution. Take 20 mL, add 2.5 g of pretreated D303 macroporous resin, place in a constant temperature shaker and shake at 50℃ for 7 h for decolorization, filter to remove the resin, and collect the decolorized polysaccharide solution. Take the above decolorized polysaccharide solution, add hydrochloric acid to adjust the concentration to 1.8 mol / L, degrade in an 80℃ water bath for 4 h to obtain the degraded oligosaccharide of raw Cornus officinalis polysaccharide, store in a dry and sealed container for later use.

[0038] Experimental Example 1: This experiment used the CCK-8 assay to investigate the effect of total sugar extracts from samples obtained from Examples 1 to 4 and unprocessed Cornus officinalis at different concentrations on the survival rate of HepG2 cells, in order to determine the safe dosage concentration range for subsequent experiments.

[0039] The processed products obtained in Examples 1 to 4 and the unprocessed raw Cornus officinalis were extracted according to steps S2 to S3 in Example 1 to obtain total sugar extracts for each sample. HepG2 cells in the logarithmic growth phase were digested with trypsin, centrifuged at 1000 r / min for 5 minutes to collect the cells, added fresh culture medium, and resuspended. The cells were then centrifuged at 8 × 10⁻⁶ cm⁻¹. 3 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. Total sugar solutions of each sample with concentrations of 50, 100, 200, 400, 800, and 1600 μg / mL were added, with a 0 μg / mL blank control group. After another 24 hours of incubation, the culture medium was discarded, and 100 μL of medium containing 10% CCK-8 was added to each well. The plates were incubated in the dark for 0.5 hours. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0040] in Figure 1 A represents raw Cornus officinalis that has not undergone any processing. Figure 1 B is wine-steamed Cornus officinalis, i.e., Example 2. Figure 1 C represents Cornus officinalis stewed in wine, as described in Example 1. Figure 1 D represents high-pressure steamed Cornus officinalis, i.e., Example 3. Figure 1 E represents the CCK-8 assay results for total sugar in each sample of steamed Cornus officinalis (i.e., in Example 4). The vertical axis represents cell viability, the horizontal axis represents drug concentration, error bars represent standard deviation, and asterisks indicate statistically significant differences compared to the 0 μg / mL blank control group. This indicates that P is less than 0.05. This indicates that P is less than 0.01. This indicates that P < 0.001. All samples showed no significant inhibitory effect on HepG2 cell growth within the low concentration range, and cell morphology remained normal. Cell viability decreased to varying degrees with increasing drug concentration. Figure 1 As shown in Figure A, the cell viability of unprocessed Cornus officinalis raw product decreased significantly when the total sugar concentration reached 1600 μg / mL (P < 0.001), indicating a certain degree of cytotoxicity. Figure 1 B to Figure 1 In Examples E, the cell viability of each processed product group (1-4) decreased relatively little at the same concentration, indicating lower toxicity, suggesting that processing helps reduce the toxicity of the medicinal material. At a concentration of 800 μg / mL, the cell viability of all samples was higher than 80%, and 800 μg / mL was determined as the dosage concentration for subsequent efficacy experiments.

[0041] Experimental Example 2: This experiment established a HepG2 insulin-resistant cell model through continuous induction with high concentrations of insulin, and validated the model by measuring glucose consumption and Oil Red O staining.

[0042] HepG2 cells in logarithmic growth phase were harvested and cultured at a concentration of 8 × 10⁻⁶ cells / cells. 3 Seeds were planted at a density of 10 cells / well in 96-well plates. After 24 hours of routine incubation, the culture medium was discarded, and the plates were subjected to serum-free starvation for 12 hours. Subsequently, 10 cells / well were added to each plate. -9 10 -8 10 -7 10 -6 10 -5 Cells were induced and cultured in medium containing different concentrations of insulin (mol / L) for 30 hours. After induction, the medium was replaced with insulin-free medium and cultured for another 24 hours. The supernatant from each well was aspirated, and the glucose content was determined using the glucose oxidase method to calculate the glucose consumption of each group. Simultaneously, medium containing 10% CCK-8 was added to each well to detect cell viability.

[0043] See results Figure 2 ,in Figure 2A represents the glucose consumption of HepG2 cells after treatment with different concentrations of insulin. The vertical axis represents glucose consumption, and the horizontal axis represents insulin concentration. The leftmost column with a concentration of 0 represents the normal control group, i.e., the group without insulin treatment. Figure 2 B represents the cell viability results for each concentration group, with the vertical axis representing cell viability and the horizontal axis representing insulin concentration; error bars represent standard deviation, and asterisks indicate statistically significant differences compared to the normal control group. This indicates that P is less than 0.05. This indicates that P is less than 0.01. This indicates that P is less than 0.001. (From...) Figure 2 As can be seen from Figure A, with increasing insulin-induced concentration, glucose consumption in HepG2 cells decreased in a dose-dependent manner. When the insulin concentration reached 10... -7 At a concentration of 10 mol / L, cellular glucose uptake was significantly inhibited (P < 0.05), indicating that the cells had developed significant insulin resistance. This resistance persisted with further increases in concentration to 10 mol / L. -5 The inhibitory effect was more significant at mol / L, with P < 0.001. Figure 2 B shows that in 10 -9 Up to 10 -5 Within the mol / L concentration range, the cell survival rate in each group was higher than 100% and significantly higher than that in the normal control group (P < 0.01 or P < 0.001). This indicates that insulin has no cytotoxicity within this range and exerts its growth-proliferating effect, confirming that the reduced glucose consumption is due to decreased cell sensitivity to insulin rather than a reduction in cell number. Although 10 -5 The inhibitory effect on glucose consumption was more significant at a concentration of 10 mol / L, but cell viability showed a downward trend. To avoid non-specific cell damage or over-modeling that could mask the true efficacy of the drug due to excessively high concentrations, a concentration of 10 mol / L was selected. -7 mol / L was used as the modeling concentration for subsequent experiments.

[0044] After modeling, discard the culture medium from each well, gently rinse the cells 2 to 3 times with phosphate buffer, fix with tissue fixative for 20 to 30 minutes, discard the fixative, and wash twice more with phosphate buffer. Add 60% isopropanol and wash for 5 to 10 seconds to remove excess water, then immediately add prepared Oil Red O working solution and stain in the dark for 30 minutes. After removing the staining solution, add 60% isopropanol for a quick 3 to 5 seconds to remove excess dye, and rinse 2 to 3 times with sterile water. Then add hematoxylin staining solution and stain for 3 to 5 minutes, rinse with normal water, and let stand for about 10 minutes until the cell nuclei turn blue. Observe the accumulation of lipid droplets in the cytoplasm under a microscope.

[0045] Oil Red O staining results are shown in Figure 3,in Figure 3 A represents the normal control group that did not receive insulin treatment. Figure 3 B is 10 - 7 In the insulin resistance model group induced by continuous insulin concentration of mol / L, all magnified 400 times, red lipid droplets represent Oil Red O staining positive signals, and blue represents hematoxylin-stained cell nuclei. Figure 3 Compared with the normal control group Figure 3 The number of red lipid droplets in the cytoplasm of HepG2 cells in model B was significantly increased. The appearance of this typical abnormal lipid metabolism phenotype confirms that glucose metabolism is blocked under insulin resistance, leading to hyperlipidemic synthesis and a large accumulation of triglycerides. This indicates that the insulin resistance cell model has been successfully constructed and can be used for subsequent pharmacodynamic and mechanistic studies.

[0046] Experimental Example 3: This experiment systematically compares the oligosaccharide content and insulin resistance improvement efficacy of products obtained from Examples 1 to 4 and unprocessed Cornus officinalis to screen for the optimal processing technology.

[0047] Take the total sugar extracts of raw Cornus officinalis from Examples 1 to 4 and without processing, accurately weigh 0.5 g of the purified total sugar, add 10 mL of pure water to redissolve each, add 4 times the volume of anhydrous ethanol and stir evenly, let stand overnight at 4℃, centrifuge, take the supernatant and concentrate under reduced pressure to remove ethanol, redissolve the precipitate with water and freeze dry to obtain the small molecule oligosaccharide fraction and the large molecule polysaccharide fraction of each sample, calculate the ratio of oligosaccharides to polysaccharides, and the results are shown in Table 1.

[0048] Table 1. Proportion of oligosaccharides and polysaccharides in the total sugar content of raw and processed Cornus officinalis products.

[0049] Compared with the unprocessed raw Cornus officinalis, the proportion of oligosaccharides in the total sugar of each processed product in Examples 1 to 4 was increased. Among them, the product obtained by the wine stewing process in Example 1 had the highest proportion of oligosaccharides, which increased from 39.26% in the raw product to 52.74%, showing the most significant increase. This suggests that the wine stewing process is most conducive to promoting the degradation of polysaccharides into oligosaccharides.

[0050] The appearance of each sample of Cornus officinalis is shown in the figure. Figure 4 . Figure 4 The Cornus officinalis shown in A is a red fruit with a wrinkled and glossy surface; Figure 4 B to Figure 4 The four processed products shown in E—wine-steamed, wine-stewed, high-pressure wine-steamed, and clear-steamed—are all black in appearance, moist in texture, and glossy. This conforms to the traditional processing theory that processing for kidney-tonifying purposes and achieving a black color is the ideal characteristic, suggesting that the internal chemical components of the medicinal material underwent significant transformation during the processing.

[0051] HepG2 cells were loaded at 8 × 103 Seeds were planted at a density of 10 cells / well in 96-well plates. After 24 hours of routine incubation, the old medium was discarded, and the plates were subjected to serum-free starvation for 12 hours. Except for the normal control group, all other groups were treated with a solution containing 10... -7 An insulin resistance model was established by incubation in medium containing mol / L insulin for 30 hours. After modeling, each group was administered the following drugs: the normal control group and model group were given blank MEM medium, the positive control group was given MEM medium containing 0.01 mg / mL metformin hydrochloride, and each drug administration group was given MEM medium containing 800 μg / mL of the corresponding sample's total sugar extract. The raw product group corresponded to unprocessed Cornus officinalis, the wine-steamed group corresponded to Example 2, the wine-stewed group corresponded to Example 1, the high-pressure group corresponded to Example 3, and the steamed group corresponded to Example 4. After culturing for another 24 hours, the supernatant from each well was collected, and the glucose content was determined using the glucose oxidase method to calculate the glucose consumption of each group.

[0052] See results Figure 5 The vertical axis represents glucose consumption, the horizontal axis represents each experimental group, and the error bars represent the standard deviation. This indicates that P is less than 0.01 compared to the model group. A p-value of <0.001 indicates a statistically significant difference compared to the model group; ## indicates a statistically significant difference compared to the normal control group; when both an asterisk and a hash mark are marked above the bar, it indicates that the group has statistically significant differences compared to both the model group and the normal control group. Compared to the normal control group, the model group showed a significant decrease in cellular glucose consumption (p <0.001), indicating successful modeling. All treatment groups increased cellular glucose consumption to varying degrees, with significant differences in efficacy among groups. The wine-stewed group showed the best effect on increasing glucose consumption, comparable to the positive control drug metformin hydrochloride group (p >0.05); the high-pressure group and the wine-steaming group were next; the steaming group was next; and the raw product group had the weakest effect. Considering the oligosaccharide content ratio and efficacy results, the wine-stewed process (Example 1) was confirmed as the optimal processing method for subsequent studies.

[0053] Experiment Example 4: In this experiment, the total sugars of stewed Cornus officinalis were further classified into oligosaccharide and polysaccharide fractions. At the same time, a group of degradation products of the polysaccharide fraction of the raw product was set up for comparison to clarify the material basis for the effect of stewed Cornus officinalis in improving insulin resistance.

[0054] The oligosaccharides degraded from the raw Cornus officinalis polysaccharide obtained in Control Example 1 were used as the raw-degradation group drug sample for later use. The preparation of the oligosaccharide and polysaccharide fractions of Cornus officinalis stewed in wine was the same as the alcohol precipitation fractionation procedure in Experimental Example 3.

[0055] The methods for seeding, culturing, and establishing the insulin resistance model of HepG2 cells were the same as in Experiment 2. After modeling, the cells were randomly divided into 7 groups: the normal control group and the model group were given blank MEM medium, the positive control group was given MEM medium containing 0.01 mg / mL metformin hydrochloride, the wine-stewed oligosaccharide group was given MEM medium containing 800 μg / mL wine-stewed Cornus officinalis oligosaccharide fraction, the wine-stewed polysaccharide group was given MEM medium containing 800 μg / mL wine-stewed Cornus officinalis polysaccharide fraction, the raw product-degradation group was given MEM medium containing 800 μg / mL raw product polysaccharide degradation products, and the wine-stewed group was given MEM medium containing 800 μg / mL wine-stewed Cornus officinalis total sugar extract. After culturing for 24 hours, the supernatant of each group was collected, and the glucose content was determined by glucose oxidase method to calculate the glucose consumption of each group.

[0056] See results Figure 6 The vertical axis represents glucose consumption, the horizontal axis represents each experimental group, and the error bars represent the standard deviation. A p-value of <0.01 indicates a statistically significant difference compared to the model group; # indicates a statistically significant difference compared to the normal control group; ## indicates a statistically significant difference compared to the normal control group; when both an asterisk and a hash mark are marked above the bar, it indicates that the group has statistically significant differences compared to both the model group and the normal control group. All treatment groups showed varying degrees of activity in improving insulin resistance. The wine-stewed oligosaccharide group had the highest glucose consumption, significantly better than the wine-stewed polysaccharide group (p <0.05), indicating that small molecule oligosaccharides are the main active site for wine-stewed Cornus officinalis to improve insulin resistance. The activity of the raw product-degraded group was significantly higher than that of the wine-stewed polysaccharide group, consistent with the trend of the wine-stewed oligosaccharide group, suggesting that the degradation of polysaccharides into oligosaccharides is the key mechanism for the enhanced efficacy of wine-stewed processing. During processing, heat treatment and an acidic environment cause the breakage of some glycosidic bonds in the polysaccharide chains, generating highly active short-chain oligosaccharides, which are the core material basis for wine-stewed Cornus officinalis to improve insulin resistance. The above results further confirm that the activity of the wine-oligosaccharide group in improving insulin resistance is comparable to that of the positive control drug metformin hydrochloride (P > 0.05), which provides a pharmacodynamic basis for its use in the treatment of type 2 diabetes.

[0057] Experimental Example 5: The oligosaccharide composition of raw Cornus officinalis and four processed products was analyzed using capillary zone electrophoresis. The electrophoresis conditions were as follows: uncoated elastic quartz capillary column, total length 55 cm, inner diameter 50 μm, effective length 46 cm; running buffer: 40 mmol / L borax buffer, pH 10.1; detection wavelength: 245 nm; operating voltage: 13.0 kV; injection method: gravity injection 15 cm × 10 s; column temperature: room temperature.

[0058] Oligosaccharides from each sample were derivatized by PMP and then analyzed by electrophoresis. The spectral results are shown below. Figure 7 Multiple oligosaccharide characteristic peaks were detected in the oligosaccharide electrophoresis spectra of the raw product and four processed products (distilled with alcohol, high-pressure distilled with alcohol, steamed, and stewed with alcohol). Monosaccharide components such as xylose, glucose, mannose, fucose, galactose, glucuronic acid, and galacturonic acid were also detected. The number and peak area of ​​oligosaccharide peaks in the four processed products showed high consistency, and the migration time of common peaks was roughly the same, indicating that different processing techniques did not change the chemical nature of the oligosaccharide components of Cornus officinalis. The main components exhibited good chemical stability under heat treatment and the action of excipients. Combined with the quantitative results in Table 1, it can be seen that the effect of processing on the oligosaccharides of Cornus officinalis is mainly reflected in changes in content. While maintaining the stability of the types of chemical components, the processing enhances the effect by adjusting the proportion of each component, exhibiting characteristics of stable quality and quantitative change.

[0059] Experimental Example 6: The molecular weight distribution of polysaccharides from raw and wine-processed Cornus officinalis was determined by gel permeation chromatography (GPC). The chromatographic conditions were as follows: Ultrahydrogel columns (7.8 × 50 mm, 10 μm and 7.8 × 300 mm, 10 μm) were used in series; a differential refractive index detector was used; the column temperature was 45℃; the flow rate was 1 mL / min; and the mobile phase was 0.1 mol / L NaNO3 solution. The GPC calibration curves are shown below. Figure 8 The retention time showed a good linear relationship with molecular weight, and the standard curve equation was y = -0.0336049x. 3 +0.969277x 2 -9.92014x+39.8602, R 2 =0.9994.

[0060] The molecular weight parameters of polysaccharides from raw and wine-stewed Cornus officinalis products are shown in Table 2, and the molecular weight distribution diagram is shown in [the original text]. Figure 9 .

[0061] Table 2. Molecular weight parameters of polysaccharides in raw and wine-stewed Cornus officinalis products.

[0062] Where Mp represents the peak molecular weight, Mn represents the number-average molecular weight, Mw represents the weight-average molecular weight, and PDI is the ratio of Mw to Mn, which is an indicator of the molecular weight distribution width of polymers. The closer the PDI is to 1, the better the uniformity of polysaccharide molecules.

[0063] Compared to the raw product, the weight-average molecular weight of the polysaccharides after wine stewing decreased sharply from approximately 158 kDa to approximately 50 kDa, a reduction of about 68%. The number-average molecular weight and peak molecular weight also decreased significantly, and the main chromatographic peak shifted significantly towards the lower molecular weight range. Figure 9Meanwhile, the polydispersity index decreased from 2.119 to 1.651, indicating that its molecular weight distribution narrowed significantly and its uniformity improved. The main reason for these changes is that the acid and heat conditions during the wine-stewing process cause some glycosidic bonds in the polysaccharide chains to break, cutting the originally high-molecular-weight macromolecular polysaccharides into smaller, more uniform medium- and low-molecular-weight polysaccharide fragments. These fragments are then transformed into highly active short-chain oligosaccharides during the alcohol precipitation and fractionation process. This provides molecular weight-level evidence for the enhanced efficacy of wine-stewing Cornus officinalis.

[0064] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing oligosaccharides from stewed Cornus officinalis, characterized in that, include: S1: Take the raw Cornus officinalis, remove the fruit stalk and kernel, add rice wine at a ratio of 15% to 25% of the weight of the medicinal material, mix thoroughly, place in a covered container and let it soak for 2 to 4 hours. After the rice wine is absorbed, place it in a stewing pot and stew it in water for 6 to 8 hours until the Cornus officinalis flesh turns black. After taking it out, dry it at 60℃ to obtain stewed Cornus officinalis. S2: Take the wine-stewed Cornus officinalis, grind it into powder and pass it through a 60-mesh sieve. Using anhydrous ethanol as a solvent, add 8-12 mL of anhydrous ethanol per gram of medicinal material and reflux at 75-85℃ for 6-10 h until the ethanol extract is colorless. Dry it to obtain defatted medicinal powder. S3: Take defatted medicinal powder, add 32-40 mL of water per gram of medicinal material and soak at room temperature for 8-12 h, heat in a 100℃ water bath for 2.5-3.5 h, filter, concentrate the filtrate under reduced pressure and directly remove protein and decolorize without alcohol precipitation to obtain total sugar extract; S4: Take the total sugar extract, redissolve it in water, add 3 to 5 times the volume of anhydrous ethanol to the redissolved solution, stir well, let it stand at 4℃ for 8 to 12 h, centrifuge, take the supernatant, concentrate under reduced pressure to remove ethanol, and freeze dry to obtain the wine-stewed Cornus officinalis oligosaccharide.

2. The method for preparing oligosaccharides from stewed Cornus officinalis according to claim 1, characterized in that, In S1, the proportion of rice wine added is 20% of the weight of the medicinal material.

3. The method for preparing oligosaccharides from stewed Cornus officinalis according to claim 1, characterized in that, In S2, the volume of anhydrous ethanol added per gram of medicinal material is 10 mL, the reflux extraction temperature is 80℃, and the reflux extraction time is 8 h.

4. The method for preparing oligosaccharides from stewed Cornus officinalis according to claim 1, characterized in that, In S3, the volume of water added per gram of medicinal material is 36 mL, and the water bath heating time is 3 h.

5. The method for preparing oligosaccharides from stewed Cornus officinalis according to claim 1, characterized in that, In S3, the protein removal is performed using the Sevag method, and the decolorization is performed using S-8 macroporous resin.

6. The method for preparing oligosaccharides from stewed Cornus officinalis according to claim 1, characterized in that, In S4, the amount of water added for reconstitution is 10 mL of water per gram of total sugar extract, and the amount of anhydrous ethanol added is 4 times the volume of the reconstitution solution.

7. A wine-stewed Cornus officinalis oligosaccharide, characterized in that, The wine-stewed Cornus officinalis oligosaccharide is prepared by the preparation method described in any one of claims 1 to 6.

8. The use of the wine-stewed Cornus officinalis oligosaccharide as described in claim 7 in the preparation of a drug to improve insulin resistance.

9. The use of a wine-stewed Cornus officinalis oligosaccharide as described in claim 7 in the preparation of a drug for treating type 2 diabetes.

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