Xanthan oligosaccharides having glp-1 agonist activity and uses thereof

Abstract

The application provides a Huangdache oligosaccharide with GLP-1 agonist activity and application thereof, and belongs to the technical field of medicines. 3 The oligosaccharide component ELYP-3 has a molecular weight of 3.2*10 3 Da, and the molar ratio of monosaccharides is rhamnose:galacturonic acid:glucose:galactose:arabinose = 1:32.42:45.37:9.11:6.54. The Huangdache oligosaccharide fragment obtained by glycosidase degradation has significant hypoglycemic and weight loss activities, the preparation process is simple, specific and green, and provides scientific basis and theoretical support for the development of natural GLP-1 agonists. Meanwhile, the functional oligosaccharide provides a wide application prospect for the development of medicines with blood glucose regulation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food and medicine, and particularly relates to a Huangdache oligosaccharide with GLP-1 agonist activity and application thereof. BACKGROUND

[0002] Tea generally refers to a beverage prepared from the buds and leaves of Camellia sinensis (L.) O. Kuntze, a perennial evergreen shrub of the Camellia family, and is considered a healthy food that can maintain health and prevent human diseases. In China, there is a record of using coarse old tea to treat diabetes. According to the textual research of herbal medicine, the pharmacological components of tea leaves for treating diabetes are tea polysaccharides. Modern pharmacological studies have shown that, as the main active ingredient in tea leaves, tea polysaccharides have a wide range of activities, such as reducing blood sugar, lowering blood lipids, anti-coagulation, anti-thrombosis, enhancing immunity, protecting the cardiovascular system, anti-cancer, anti-oxidation, and radiation protection, in addition to the effect of lowering blood sugar. Tea polysaccharides are a class of complex and functional acidic protein heteropolysaccharides, and the rich structural characteristics of tea polysaccharides are the basis for the various activities of polysaccharides. Due to the microscopic heterogeneity and the diversity of monosaccharide composition of tea polysaccharides, and the fact that they are flexible in solution and difficult to crystallize, the targeted research of tea polysaccharides is quite lagging behind, which is a key reason hindering the application of functional polysaccharides in clinical research.

[0003] The core problem of polysaccharide targeting research is to solve the recognition domain with sugar or the active site of interaction, so it is a new idea to break through the bottleneck of the research on the structure and function mechanism of tea polysaccharides by degrading tea polysaccharides into oligosaccharides and studying the active center of polysaccharides at the level of oligosaccharides. At present, the methods for degrading polysaccharides include physical degradation, chemical degradation, and enzymatic degradation. The physical degradation methods include ultrasonic, radiation, microwave, and heat treatment, among which the ultrasonic method is time-consuming, low in efficiency, and high in energy consumption and noise; the radiation method requires specific equipment and technology, and is high in requirement for equipment, and is easy to cause harm to the operator; the microwave and heat treatment methods may change the physical and chemical properties of polysaccharides; the acid hydrolysis and oxidation degradation in the chemical degradation method are poor in reaction regularity, difficult to control, and poor in reproducibility, and have problems such as difficult control of the composition of degradation products, environmental pollution, etc. Compared with physical and chemical degradation, the enzymatic degradation method can cut specific glycosidic bonds, the reaction is easy to control, and the degradation process is mild without by-product generation, so it is an ideal degradation method. Therefore, the application of the enzymatic degradation method for preparing functional oligosaccharides from polysaccharides shows a good prospect for industrialization.

[0004] At present, there are few reports on the degradation of Huangdache polysaccharides, and there is no report on Huangdache polysaccharides with GLP-1 agonist activity. SUMMARY

[0005] The technical problem solved by the present application is how to provide a Huangdache oligosaccharide with GLP-1 agonist activity.

[0006] The present application solves the above technical problems by the following technical means:

[0007] The first aspect of the present application provides a Huangdache oligosaccharide with GLP-1 agonist activity, wherein the Huangdache oligosaccharide has a molecular weight of 3.2 ×10 3 The oligosaccharide component ELYP-3 of Da has a monosaccharide composition and molar ratio of rhamnose: galacturonic acid: glucose: galactose: arabinose = 1: 32.42: 45.37: 9.11: 6.54.

[0008] Preferably, the carbohydrate content in the ELYP-3 is 93.59%.

[0009] Preferably, the ELYP-3 contains β-configuration glycosidic bonds and α-configuration glycosidic bonds.

[0010] Preferably, the particle size of the ELYP-3 is mainly concentrated in the range of 10-1000 nm.

[0011] Preferably, the average particle size of the ELYP-3 is 132.2 nm.

[0012] The second aspect of the present application provides the Huangdache oligosaccharide described above for any one of the following applications:

[0013] (1) for preparing a product for promoting GLP-1 secretion of intestinal endocrine cells STC-1;

[0014] (2) for preparing a product for promoting GLP-1 secretion for preventing and / or treating diabetes.

[0015] Preferably, in (2)-(3), the product is a drug.

[0016] The third aspect of the present application provides a drug for preventing and / or treating diabetes, wherein the effective component comprises the Huangdache oligosaccharide described above.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable salt, specifically including an organic salt or an inorganic salt.

[0018] Preferably, the drug further comprises a pharmaceutically acceptable pharmaceutical excipient.

[0019] Preferably, the pharmaceutical excipient is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, and a lubricant.

[0020] The present application has the following beneficial effects:

[0021] 1. The present application provides a yellow tea oligosaccharide ELYP-3 with GLP-1 agonist activity, which is composed of rhamnose: galacturonic acid: glucose: galactose: arabinose = 1: 32.42: 45.37: 9.11: 6.54 (molar ratio). The yellow tea oligosaccharide fragment has good effect in in vitro cell experiment, and the oligosaccharide fragment can promote the secretion of GLP-1 in intestinal endocrine cells STC-1, and significantly up-regulate the expression level of key genes regulating GLP-1 synthesis and secretion.

[0022] 2. The yellow tea oligosaccharide ELYP-3 provided by the present application also has excellent effect in a type 2 diabetes mouse model, and the oligosaccharide fragment significantly improves the secretion level of GLP-1 in the blood, ileum and colon of the mouse, and has obvious effect on reducing blood sugar, reducing blood lipid, reducing lipid accumulation and improving insulin resistance.

[0023] 3. The yellow tea oligosaccharide ELYP-3 provided by the present application lays a reliable material foundation for developing natural oligosaccharide source with GLP-1 agonist activity of blood sugar lowering drugs, or drugs with blood sugar regulating effect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The influence of different yellow tea oligosaccharide fragments in Example 1 of the present application on the secretion of GLP-1 in intestinal endocrine L cells;

[0025] Figure 2 The ultraviolet spectrum (A) and Fourier infrared spectrum (B) of the yellow tea oligosaccharide ELYP-3 in Example 2 of the present application;

[0026] Figure 3 The molecular weight (A), particle size (B) and triple helix structure spectrum (C) of the yellow tea oligosaccharide ELYP-3 in Example 2 of the present application;

[0027] Figure 4 The SEM graph of the yellow tea oligosaccharide ELYP-3 in Example 2 of the present application; A: LYP, B: ELYP-3;

[0028] Figure 5 The monosaccharide composition spectrum of the yellow tea oligosaccharide ELYP-3 in Example 2 of the present application; A: standard, B: ELYP;

[0029] Figure 6 The influence of the yellow tea oligosaccharide ELYP-3 in Example 3 of the present application on the synthesis and secretion of GLP-1 in intestinal endocrine L cells STC-1;

[0030] Figure 7Effect of Huangdache oligosaccharide ELYP-3 on GLP-1 content in portal vein plasma when directly administered to jejunum (A) or ileum (B) of rats in the present application embodiment 4;

[0031] Figure 8 Effect of Huangdache oligosaccharide ELYP-3 on GLP-1 content in portal vein plasma when directly administered to jejunum (A) or ileum (B) of rats in the present application embodiment 4;

[0032] Figure 9 Effect of Huangdache oligosaccharide ELYP-3 on GLP-1 content in portal vein plasma when directly administered to jejunum (A) or ileum (B) of rats in the present application embodiment 4; DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the following will combine the embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0034] In the following examples, the test materials and reagents used, etc. can be obtained from commercial channels if not specifically stated.

[0035] If not specifically stated, the specific techniques or conditions in the embodiments can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. If not specifically stated, the quantitative tests in the following embodiments are set up with more than three repeated experiments, and the results are averaged.

[0036] Example 1: Preparation of Huangdache oligosaccharide fragments

[0037] (1) Preparation of Huangdache polysaccharide Huangdache was dried and crushed, and then an appropriate amount of Huangdache powder was weighed out. 95% ethanol was added at a solid-liquid ratio of 1:20 g / mL, and stirring was carried out at 25°C and a rotation speed of 50 rpm / min for 24 h to remove fat and pigment impurities. The residue was collected by suction filtration, and then the solid-liquid ratio was 1:20 g / mL. The sample was stirred and extracted at 90°C water bath for 2.5 h, and the supernatant was collected by filtration. Four times the volume of anhydrous ethanol (the final concentration of ethanol was 80%) was added, and the sample was placed at 4°C for 12 h. The sample was then centrifuged to collect the precipitate. The Sevage method was used to remove the protein in the precipitate. The protein-removed sugar solution was rotary evaporated, dialyzed, freeze-dried, and further purified by DEAE cellulose DE-52 anion exchange chromatography column and Sephadex G-100 gel chromatography column to obtain a uniform polysaccharide component LYP from Huangdache.

[0038] (2) Preparation of Huangdache polysaccharide oligosaccharide fragments Huangdache polysaccharide was dissolved in 50 mM pH=4.0 acetic acid-sodium acetate buffer solution to prepare a 1 mg / mL Huangdache polysaccharide solution, 200 U / mg galacturonanase was added, and enzymatic reaction was performed at 50°C for 12 h. After the reaction, the enzyme activity was terminated by boiling. The reaction solution was centrifuged (5700 rpm, 10 min) to remove the denatured protease, and the supernatant was collected. After desalting and impurity removal by ultrafiltration membrane with a molecular weight cut-off of 0.5 KDa, the membrane cut-off solution and the permeate were collected, concentrated, and freeze-dried to obtain M g . W The 0.5 KDa ~1 KDa, 1~3KDa, and 3 KDa ~5 KDa oligosaccharide components were named ELYP-1, ELYP-2, and ELYP-3, respectively.

[0039] (3) Activity screening of Huangdache oligosaccharide fragments for promoting GLP-1 secretion by intestinal endocrine L cells The different polymeric degree Huangdache oligosaccharide fragments ELYP-1, ELYP-2, and ELYP-3 obtained were administered to intestinal endocrine L cells STC-1, respectively, for activity screening evaluation of promoting GLP-1 secretion. Well-grown STC-1 cells were inoculated in a 48-well plate (cell density was 1×10 6 6 / mL). Incubation was performed in a 5% CO2 incubator for 24 h. The culture solution was aspirated, and a solution of ELYP-1, ELYP-2, and ELYP-3 with a final concentration of 100 μg / mL was added, respectively, and incubation was continued for 2 h. The blank control group (Control) was complete culture solution, and a Huangdache uniform polysaccharide component LYP group was set. After incubation, the culture solution was collected, centrifuged (400 g 4°C, 15 min) to obtain the supernatant, and the content of GLP-1 in the supernatant was determined.

[0040] The results are shown in Table 1. Figure 1 Compared with the control group, LYP and the different polymeric degree oligosaccharide fragments ELYP-1, ELYP-2, and ELYP-3 degraded by galacturonanase significantly increased the level of GLP-1 secreted by STC-1 cells, and the content was 1.76, 2.05, 2.14, and 2.44 times that of the control group, respectively. It can be seen that ELYP-3 has the best activity for promoting GLP-1 secretion by STC-1 cells. The GLP-1 secretion amount of ELYP-3 acting on STC-1 cells was 1.39 times that of LYP, 1.19 times that of ELYP-1, and 1.14 times that of ELYP-2. Therefore, ELYP-3 is the best active fragment for promoting GLP-1 secretion by intestinal epithelial cells.

[0041] Example 2: Purification and structure identification of Huangdache oligosaccharide fragment ELYP-3

[0042] (1) Purification of oligosaccharide fragments of Huangdache:

[0043] The ELYP-3 component in Example 1 was dissolved in double distilled water and loaded onto a polyacrylamide gel Bio gel P-2 column at a concentration of 40 mg / mL. Elution was performed with deionized water at a flow rate of 10 mL / h. The polysaccharide content in the eluate was detected by the phenol-sulfuric acid colorimetric method. The sugar-containing eluate was collected, vacuum-concentrated, and freeze-dried to obtain purified oligosaccharide ELYP-3 of Huangdache.

[0044] (2) Determination of the neutral carbohydrate content of oligosaccharide ELYP-3 of Huangdache:

[0045] The phenol-sulfuric acid method was used to determine the carbohydrate content of ELYP-3 obtained in (1) above. Spectrophotometric detection at 490 nm showed that the neutral carbohydrate content of ELYP-3 was 93.59%.

[0046] (3) UV spectrum analysis of ELYP-3:

[0047] ELYP-3 was scanned using a full-wavelength scanning enzyme marker at a wavelength range of 190-400 nm. The results are shown in Figure 2 A, which shows that ELYP-3 has no absorption peaks at 260 nm and 280 nm, indicating that the purified enzymatic oligosaccharide contains almost no pigments, proteins, and nucleic acids.

[0048] (4) Infrared spectrum analysis of ELYP-3:

[0049] The characteristic groups of ELYP-3 were scanned and analyzed using a Nicolette is50 Fourier infrared spectrometer. The results are shown in Figure 2 B, which shows that ELYP-3 has typical sugar characteristic peaks. The signal peak at 3440 cm -1 is attributed to the O-H stretching vibration within or between sugar molecules; the signal peak at 2930 cm -1 is attributed to the stretching of C-H; the signal peaks at 1630 cm -1 and 1414 cm -1 are respectively attributed to the stretching vibration of the carbonyl group (C=O) in COOH and the C-H variable angle vibration; the signal peak at 1149 cm -1 is attributed to the stretching vibration of C-O-C and C-O-H in the pyranose ring; and the signal peak at 946 cm -1 indicates that ELYP-3 contains β-configuration glycosidic bonds and α-configuration glycosidic bonds.

[0050] (5) Determination of the molecular weight of oligosaccharide ELYP-3 of Huangdache:

[0051] The molecular weight distribution of ELYP-3 was determined by high performance gel permeation chromatography-differential-multiple angle laser light scattering method. The results are shown in Figure 3 A. The molecular weight of ELYP-3 is: 3.2 × 10 3 Da. The detection conditions: Waters Arc HPLC liquid phase system, Brookhaven BI-MwA multiple angle laser light scattering detector, Waters 2414 RI differential detector, TSK G2500PWXL analysis column (7.8 mm × 300 mm), mobile phase 0.1 M NaNO3 solution, flow rate 0.6 mL / min, column temperature 35 °C, sample size 100 μL.

[0052] (6) Determination of the particle size of the yellow tea oligosaccharide ELYP-3:

[0053] The particle size of ELYP-3 was determined by Zetasizer Pro nanoparticle size analyzer. A 1 mg / mL solution of ELYP-3 was prepared, filtered through a 0.22 μm water system membrane, and the particle size was measured at 25 °C, 633 nm wavelength, and 90° scattering angle. As shown in Figure 3 B, the particle size of ELYP-3 is mainly concentrated in the range of 10-1000 nm, and the average particle size is 132.2 nm.

[0054] (7) Determination of the triple helix structure in the yellow tea oligosaccharide ELYP-3:

[0055] The presence of a triple helix structure in ELYP-3 was determined by Congo red experiment. A 2 mg / mL solution of ELYP-3 was prepared, and Congo red solution was added at a ratio of 1:1 v / v. After mixing, different concentrations of NaOH solution (0-0.5 mol / mL) were added, and a mixture solution without ELYP-3 was used as a control. The mixture was allowed to stand at room temperature for 10 min, and the maximum absorption wavelength of the Congo red-yellow tea polysaccharide complex was determined by ultraviolet full wavelength scanning. As shown in Figure 3 C, with increasing concentration of NaOH, the maximum absorption wavelength of the Congo red-ELYP-3 complex decreases and finally stabilizes, indicating that ELYP-3 does not have a triple helix structure.

[0056] (8) Analysis of the microstructure of the yellow tea oligosaccharide ELYP-3:

[0057] The apparent morphology of ELYP-3 was analyzed by Hitachi SU1000 scanning electron microscope. 2 mg of dried ELYP-3 was weighed and adhered to a metal iron block with conductive glue. After spraying a layer of conductive film on the surface of the sample, the apparent morphology was observed. The results are shown in Figure 4As shown in FIG. B, the surface of ELYP-3 presents broken irregular granular fragments.

[0058] (9) Monosaccharide composition analysis of the yellow tea oligosaccharide ELYP-3:

[0059] The monosaccharide composition of ELYP-3 was determined by high performance liquid chromatography (HPLC) with pre-column derivatization of 1-phenyl-3-methyl-5-P pyrazolone (PMP). 5 mg of dried ELYP-3 was placed in a 10 mL ampoule, 3 mL of 3M hydrochloric acid solution was added, the tube was sealed, hydrolysis was performed at 110°C for 1 h, and then the solution was allowed to cool. The pH was adjusted to neutral with 3.0 mol / L sodium hydroxide solution, and the oligosaccharide hydrolysate was obtained. 100 μL of 0.6 M NaOH solution and an equal volume of 0.6 M PMP methanol solution were added to the above hydrolysate, and the mixture was reacted at 70°C for 100 min. After the reaction was completed, 50 μL of 0.3 M HCl solution was added, and the solution was rotary evaporated at 50°C. Chloroform was added for washing three times, and the supernatant was filtered and subjected to HPLC detection. The results are shown in FIG. C. Figure 5 As shown in FIG. C, by comparing the chromatogram with the standard monosaccharide, it was found that ELYP-3 was mainly composed of five kinds of monosaccharides, and the molar ratio was rhamnose: galacturonic acid: glucose: galactose: arabinose = 1:32.42:45.37:9.11:6.54.

[0060] Detection conditions: Waters Arc HPLC liquid chromatograph; Waters-C18 column (250 mm × 4.6 mm), ultraviolet detector, column temperature 30°C; mobile phase 0.1 mol / L phosphate buffer (PBS, pH 6.7): acetonitrile = 83:17 (v / v), flow rate 1.0 mL / min, detection wavelength 245 nm.

[0061] Example 3: Effect of yellow tea oligosaccharide fragments on the synthesis and secretion of GLP-1 by intestinal endocrine L cells in vitro

[0062] The effect of ELYP-3 prepared in Example 1 on the promotion of the synthesis and secretion of GLP-1 by intestinal endocrine L cells was studied using STC-1 cells as a model. The logarithmic growth phase STC-1 cells were adjusted to a concentration of 1 × 10 6 6-well cell culture plates, and incubated at 37°C in a 5% CO2 incubator for 24 h. Then, 25 and 100 μg / mL of ELYP-3 were added, respectively, and incubation was continued for 2 h. The blank control group was complete culture medium. After incubation, the cells and cell supernatant of each group were collected, and RT-qPCR was used to detect the key genes in the process of GLP-1 precursor synthesis, processing, and extracellular release. gcg, pcsk1, α-gustducin, plcb2, gipr, glp1r, t1r2 / 3 ) mRNA expression level, ELISA to detect GLP-1 content in cell supernatant.

[0063] The results are shown in Figure 6 Figure 2, ELYP-3 can significantly promote the high secretion of GLP-1 in intestinal endocrine cells STC-1, and up-regulate the mRNA expression level of key genes encoding GLP-1 precursor synthesis gcg, pcsk1 ), processing maturity (α-gustducin, plcb2 ), extracellular release glp1r, t1r2 / 3 ) process, with a dose effect. The above results show that ELYP-3 acts on STC-1 cells, not only effectively increasing the intracellular synthesis of GLP-1, but also promoting the release of GLP-1 from intracellular to extracellular at a high level.

[0064] Example 4: Effect of Huangdache oligosaccharide fragments in vivo on GLP-1 synthesis and secretion in normal mice and T2DM mice

[0065] (1) In situ detection of GLP-1

[0066] 5-week-old male SD rats were selected in the SPF level animal room at a temperature of 23±2℃, relative humidity of 50%-60%, 12h light and 12h dark conditions, and were adaptively fed for 1 week. Then, the rats were randomly divided into 3 groups (n=6): control group (normal saline in situ administration), low concentration ELYP-3 in situ administration (ELYP-3L, 0.5g / kg), and high concentration ELYP-3 in situ administration (ELYP-3H, 2g / kg). After the rats in each group were anesthetized on the operating table, the abdominal cavity was opened, the hepatic portal vein was cannulated, and then the jejunum or ileum was ligated. Direct administration was performed on the ligated jejunum or ileum. At 15, 30, 60, 90, and 120 min after administration, blood was collected from the portal vein using a syringe containing EDTA-2Na (1 mg / mL), aprotinin (500 klU / mL), and DPP-IV inhibitor (100 mM). The blood was centrifuged at 3500 rpm at 4℃ for 15 min, and the plasma was collected. The GLP-1 secretion level in the plasma at different administration times was determined.

[0067] To further study whether ELYP-3 can directly act on intestinal endocrine L cells to promote GLP-1 secretion in vivo, an in situ administration experiment of ELYP-3 in the intestinal tract in vivo was performed. The plasma GLP-1 level after direct in situ administration of ELYP-3 in the jejunum or ileum was detected. The results are shown in Figure 7As shown, compared with the control group, the GLP-1 in the plasma rapidly increased within 15 min after the jejunum or ileum was directly administered with ELYP-3, reached the highest value at 30 min, and the increase of the GLP-1 content in the plasma gradually tended to be flat after 30 min. The results show that ELYP-3 can directly act on the intestinal endocrine L cells to promote the GLP-1 secretion in vivo.

[0068] (2) Determination of GLP-1 synthesis and secretion levels in T2DM mice in vivo

[0069] Construction of T2DM mouse model and administration intervention Four-week-old male C57 / 6J mice were adaptively fed in a SPF level animal room under the conditions of a temperature of 23±2℃, a relative humidity of 50%-60%, 12h light and 12h darkness, and after one week, the mice were randomly divided into a normal group (ND, fed with ordinary feed + intragastrically administered with normal saline), a high-fat diet model group (HFD, fed with high-fat feed + intragastrically administered with normal saline), a positive group (Positive, fed with high-fat feed + intragastrically administered with metformin 50 mg / kg / d), a low-dose ELYP-3 group (ELYP-3L, fed with high-fat feed + intragastrically administered with ELYP-3 50 mg / kg / d), and a high-dose ELYP-3 group (ELYP-3H, fed with high-fat feed + intragastrically administered with ELYP-3 200 mg / kg / d), with 12 mice in each group. After being fed with high-fat feed for 8 weeks, the mice were once intraperitoneally injected with 100 mg / kg BW streptozotocin (STZ), and the fasting blood glucose was measured 72h after the STZ injection, and the mice with a blood glucose value of more than 11.1 mmol / L were selected as the diabetic model mice. The corresponding intervention substances were continuously administered by intragastric administration until the end of the experiment at the 12th week, the mice were killed by CO2 after being fasted overnight, the blood was taken from the heart, and the colon and ileum tissues were collected, frozen in liquid nitrogen, and stored at -80℃ for standby use.

[0070] The T2DM mice were constructed through HFD feeding and STZ injection, and the influence of the application example of the present application on the GLP-1 synthesis and secretion content in the T2DM model mice in vivo was explored. The results are shown in Figure 8 As shown in A, compared with the ND group, the GLP-1 content in the plasma of the T2DM model mice was significantly reduced, and the administration of different doses of ELYP-3 significantly increased the GLP-1 content in the plasma of the T2DM mice. In order to further study the influence of ELYP-3 on the synthesis and secretion of GLP-1 by the intestinal endocrine L cells in the T2DM model mice in vivo, the mRNA expression levels of the key genes (Gcg, Pcsk1, Pcsk2, and Gcg) encoding the GLP-1 synthesis in the precursor, processing into mature, and extracellular release in the ileum tissues were detected by RT-qPCR. gcg, pcsk1, α-gustducin , plcb2, trpm5, glp1r, t1r2 / 3 )mRNA expression level. As shown in B, compared with the ND group, the mRNA expression levels of Gcg, Pcsk1, Pcsk2, and Gcg in the ileum tissues of the T2DM model mice were significantly reduced, and the administration of different doses of ELYP-3 significantly increased the mRNA expression levels of Gcg, Pcsk1, Pcsk2, and Gcg in the ileum tissues of the T2DM mice. Figure 8As shown in BI, ELYP-3 intervention can significantly upregulate the synthesis of GLP-1 precursor in the ileum of T2DM mice ( gcg, pcsk1 ), processed and mature (α-gustducin, plcb2 ), extracellular release ( trpm5, glp1r, t1r2 / 3 These results indicate that ELYP-3 activates the GLP-1 receptor in T2DM mice, effectively increasing the intracellular synthesis of GLP-1 and promoting its release from the cell to the extracellular space.

[0071] Example 5 Effects of Huangdacha Oligosaccharide Fragments on Blood Glucose, Blood Lipids and Insulin Resistance in T2DM Mice

[0072] During the experiment, fasting blood glucose levels in T2DM mice were monitored weekly using a Roche blood glucose meter. Following the experiment, plasma lipid profiles, including total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDLc), and high-density lipoprotein (HDL-c), were measured using biochemical assays. Fasting insulin levels were measured using an ELISA kit. The insulin resistance index (HOMA-IR) and beta-cell function index (HOMA-β) were calculated using the following formulas: HOMA-IR = fasting blood glucose (FBG) (mmol / L) × fasting insulin level (FINS) (mU / L) / 22.5; HOMA-β = [20 × FINS (mU / L)] / [FBG (mmol / L) - 3.5]. The aim was to investigate the efficacy of the present invention in improving blood glucose, blood lipids, and insulin resistance in T2DM mice.

[0073] like Figure 9 As shown in AB, compared with the ND group, the fasting blood glucose and fasting insulin levels of mice in the T2DM model group were significantly increased. This shows that T2DM mice have typical hyperglycemia and insulin resistance. After intervention with different doses of ELYP-3, the fasting blood glucose and fasting insulin levels of T2DM mice were significantly reduced, indicating that ELYP-3 intervention has an auxiliary blood glucose lowering effect. Further calculation of the insulin resistance index (HOMA-IR) and β-cell function index of each group of mice showed the following results: Figure 9 As shown in CD, ELYP-3 intervention can significantly reduce HOMA-IR and increase β-cell function index in T2DM mice, indicating that ELYP-3 can significantly improve insulin resistance and enhance insulin sensitivity in T2DM mice.

[0074] The blood lipid levels of the mice were characterized by determining the total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) in the serum of the T2DM mice. As shown in Table 1, the blood lipid levels of the T2DM mice were significantly improved after the ELYP-3 intervention. Figure 9 As shown in Table 2, the ELYP-3 intervention at different doses reduced the TC, TG and LDL-c levels in the plasma of the T2DM mice, while increasing the HDL-c level, indicating that the ELYP-3 intervention significantly improved the hyperlipidemia of the T2DM mice and had the effect of assisting in reducing blood lipids.

[0075] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the same; although the present application has been described in detail with reference to the foregoing examples, it should be understood by those skilled in the art that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A yellow tea oligosaccharide having GLP-1 agonist activity, characterized in that: The yellow tea oligosaccharide has a molecular weight of 3.2 × 10 3 The oligosaccharide component ELYP-3 of Da has a monosaccharide composition and molar ratio of rhamnose: galacturonic acid: glucose: galactose: arabinose = 1:32.42:45.37:9.11:6.54; the average particle size of the ELYP-3 is 132.2 nm; The ELYP-3 is obtained by the following process: (1) Preparation of polysaccharide from Camellia sinensis: After drying and crushing the Camellia sinensis, an appropriate amount of Camellia sinensis powder was weighed, and 95% ethanol was added at a solid-liquid ratio of 1:20 g / mL. The mixture was stirred at 25°C and 50 rpm / min for 24 h to remove fat and pigment impurities. The residue was collected by filtration, and the mixture was stirred and extracted at a solid-liquid ratio of 1:20 g / mL in a water bath at 90°C for 2.5 h. The supernatant was collected by filtration, and 4 times the volume of anhydrous ethanol was added to the mixture to make the final concentration of anhydrous ethanol 80%. The mixture was allowed to stand at 4°C for 12 h and then centrifuged to collect the precipitate. The protein in the precipitate was removed by the Sevage method. The deproteinized sugar solution was concentrated by rotary evaporation, dialyzed, and freeze-dried. It was further purified by DEAE cellulose DE-52 anion exchange chromatography column and Sephadex G-100 gel chromatography column to obtain the uniform polysaccharide component LYP from Camellia sinensis. (2) Preparation of oligosaccharide fragments of E. coli polysaccharide: The purified E. coli polysaccharide was dissolved in 50 mM acetic acid-sodium acetate buffer solution with a pH of 4.0 to prepare a 1 mg / mL E. coli polysaccharide solution. 200 U / mg galacturonidase was added and the enzymatic hydrolysis reaction was carried out at 50 °C for 12 h. After the reaction, the enzyme activity was terminated by boiling. The reaction solution was centrifuged at 5700 g for 10 min to remove the denatured protease. The supernatant was collected and desalted and impurities were removed by ultrafiltration membrane with a molecular weight cutoff of 0.5 KDa. The supernatant was then passed through 1 KDa, 3 KDa, and 5 KDa membrane packs in sequence. The membrane retentate and permeate were collected, concentrated, and freeze-dried to obtain oligosaccharide components with MW ranging from 0.5 KDa to 1 KDa, 1 to 3 KDa, and 3 KDa to 5 KDa, respectively. They were named ELYP-1, ELYP-2, and ELYP-3.

2. Any use of the yellow tea oligosaccharide according to claim 1 in the following: (1) Application in the preparation of products that promote the secretion of GLP-1 by enteroendocrine cells STC-1; (2) Use in the preparation of products for the prevention and / or treatment of diabetes by promoting GLP-1 secretion; The product is a drug.

3. A drug for preventing and / or treating diabetes, characterized in that: The effective ingredients include the yellow tea oligosaccharide according to claim 1.

4. The drug for preventing and / or treating diabetes according to claim 3, characterized in that The medicine also includes pharmaceutically acceptable excipients.

5. The drug for preventing and / or treating diabetes according to claim 4, characterized in that The pharmaceutical excipient is selected from one or more of a diluent, a filler, a binder, a wetting agent, a disintegrant, an absorption accelerator, a surfactant, and a lubricant.

6. The drug for preventing and / or treating diabetes according to claim 5, characterized in that The pharmaceutical excipient is a diluent.

7. The drug for preventing and / or treating diabetes according to claim 5, characterized in that The pharmaceutical excipient is a filler.

8. The drug for preventing and / or treating diabetes according to claim 5, characterized in that The pharmaceutical excipient is a binder.

9. The drug for preventing and / or treating diabetes according to claim 5, characterized in that The pharmaceutical excipient is a wetting agent.

10. The drug for preventing and / or treating diabetes according to claim 5, characterized in that The pharmaceutical excipient is a disintegrant.

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