Huangdai tea oligosaccharide with GLP-1 agonist activity and application of Huangdai tea oligosaccharide

Abstract

The invention provides large yellow tea oligosaccharide with GLP-1 agonist activity and application of the large yellow tea oligosaccharide, and belongs to the technical field of food and medicine. The yellow tea oligosaccharide is an oligosaccharide component ELYP-3 with the molecular weight of 3.2 * 10 < 3 > Da, and the monosaccharide composition and the molar ratio of rhamnose to galacturonic acid to glucose to galactose to arabinose are 1: 32.42: 45.37: 9.11: 6.54. The preparation method has the beneficial effects that the large yellow tea oligosaccharide fragment obtained through glycosidase degradation has remarkable blood glucose reducing and weight losing activity, and the preparation process is simple to operate, high in specificity and environmentally friendly. Scientific basis and theoretical support are provided for development of natural GLP-1 agonists, and meanwhile, wide application prospects are provided for development of functional oligosaccharides for food, medicine or health care products with the blood sugar regulating effect.

Description

Technical Field

[0001] The invention belongs to the field of food and medicine, and specifically relates to yellow tea oligosaccharide with GLP-1 agonist activity and application thereof. Background Art

[0002] Tea usually refers to a beverage made from the buds and leaves of the tea tree (Camellia sinensis (L.) O. Kuntze, a perennial evergreen shrub of the Theaceae family), which is considered a healthy food that can maintain health and prevent human diseases. There are records of people in my country drinking rough old tea to treat diabetes. According to the textual research of Materia Medica, the pharmacological component of tea for treating diabetes is tea polysaccharides. Modern pharmacological research has shown that tea polysaccharides, as the main active ingredient in tea, have a wide range of active functions such as lowering blood sugar, anti-coagulation, anti-thrombosis, enhancing body immunity, protecting cardiovascular, anti-cancer, anti-oxidation, and radiation protection. Tea polysaccharides are a class of acidic protein heteropolysaccharides with complex structures and diverse functions. Their rich and diverse structural characteristics are the basis for polysaccharides to exert various activities. Due to the microscopic heterogeneity of tea polysaccharide structure and the diversity of monosaccharide composition, as well as its high flexibility and difficulty in crystallization in solution state, its targeted research is quite lagging, which is the 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 of sugar or the active site of interaction. Therefore, tea polysaccharides are degraded into oligosaccharides, and the active center of polysaccharides is studied from the oligosaccharide level, which provides a new idea for breaking through the bottleneck of tea polysaccharide structure and function mechanism research. At present, there are physical degradation, chemical degradation and enzymatic degradation methods for degrading polysaccharides. Physical degradation methods include ultrasound, radiation, microwave, heat treatment and other methods. Among them, ultrasound method takes a long time, has low efficiency, high energy consumption and noise; radiation method requires specific equipment and technology, has high requirements for equipment, and is easy to cause harm to operators; microwave and heat treatment methods may change the physical and chemical properties of polysaccharides; acid hydrolysis and oxidative degradation in chemical degradation methods have poor reaction regularity, difficult process control, poor reproducibility, and problems such as difficult control of degradation product composition and environmental pollution. Compared with physical and chemical degradation, enzymatic hydrolysis can cut specific glycosidic bonds, the reaction is easy to control, and the conditions are mild during the degradation process, and no by-products are generated. It is an ideal degradation method. Therefore, the application of enzymatic degradation of polysaccharides to prepare functional oligosaccharides shows good industrial prospects.

[0004] Currently, there are few reports on the degradation of Camellia ternata polysaccharides, and there are no reports on Camellia ternata polysaccharides with GLP-1 agonist activity. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a yellow tea oligosaccharide with GLP-1 agonist activity.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention provides a yellow tea oligosaccharide having GLP-1 agonist activity, wherein 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.

[0008] Preferably, the carbohydrate content of 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 to 1000 nm.

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

[0012] The second aspect of the present invention proposes any of the following uses of the above-mentioned yellow tea oligosaccharide:

[0013] (1) Application in the preparation of products that promote the secretion of GLP-1 by enteroendocrine cells STC-1;

[0014] (2) Use in the preparation of products for promoting GLP-1 secretion to prevent and / or treat diabetes.

[0015] Preferably, in (2) to (3), the product includes food, medicine or health care product.

[0016] The third aspect of the present invention provides a drug for preventing and / or treating diabetes, the active ingredient of which includes the above-mentioned yellow tea oligosaccharide.

[0017] Preferably, the drug also includes a pharmaceutically acceptable salt, specifically an organic salt or an inorganic salt.

[0018] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[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 beneficial effects of the present invention are:

[0021] 1. The present invention provides a huangdacha 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). This huangdacha oligosaccharide fragment has good effects in in vitro cell experiments. The oligosaccharide fragment can promote the secretion of GLP-1 by intestinal endocrine cells STC-1 and significantly up-regulate the expression levels of key genes regulating GLP-1 synthesis and secretion.

[0022] 2. The huangdacha oligosaccharide ELYP-3 provided by the present invention also has excellent effects in a type II diabetes mouse model. The oligosaccharide fragment significantly increases the secretion levels of GLP-1 in the blood, ileum and colon of mice, and at the same time has obvious effects on reducing blood sugar, reducing blood lipids, reducing lipid accumulation and improving insulin resistance.

[0023] 3. The huangdacha oligosaccharide ELYP-3 provided by the present invention lays a reliable material foundation for the development of hypoglycemic drugs with GLP-1 agonist activity from natural oligosaccharide sources, or foods, drugs or health products with blood sugar-regulating effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shows the effect of different huangdacha oligosaccharide fragments on the secretion of GLP-1 by intestinal endocrine L cells in Example 1 of the present invention;

[0025] Figure 2 Shows the ultraviolet spectrum (A) and Fourier transform infrared spectrum (B) of huangdacha oligosaccharide ELYP-3 in Example 2 of the present invention;

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

[0027] Figure 4 Shows the SEM images of huangdacha oligosaccharide ELYP-3 in Example 2 of the present invention; A: LYP, B: ELYP-3;

[0028] Figure 5 Shows the monosaccharide composition spectrum of huangdacha oligosaccharide ELYP-3 in Example 2 of the present invention; A: standard, B: ELYP;

[0029] Figure 6 Shows the effect of huangdacha oligosaccharide ELYP-3 on promoting the synthesis and secretion of GLP-1 by intestinal endocrine L cells STC-1 in Example 3 of the present invention;

[0030] Figure 7Effect of yellow tea oligosaccharide ELYP-3 in Example 4 of the present invention on the content of GLP-1 in portal vein plasma when it is directly administered to the jejunum (A) or ileum (B) of rats;

[0031] Figure 8 Effects of the yellow tea oligosaccharide ELYP-3 in Example 4 of the present invention on plasma GLP-1 (A) and ileal GLP-1 synthesis and secretion (BI) in type 2 diabetic mice;

[0032] Fig. 9 Effects of the yellow tea oligosaccharide ELYP-3 in Example 5 of the present invention on blood glucose (A), insulin resistance (BD) and blood lipids (EH) in type 2 diabetic mice. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0035] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0036] Example 1: Preparation of Yellow Tea Oligosaccharide Fragments

[0037] (1) Preparation of Camellia sinensis polysaccharide After drying and crushing, 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 then 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 filtered and collected. Four volumes of anhydrous ethanol (final ethanol concentration of 80%) were added thereto. 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 Camellia sinensis polysaccharide component LYP.

[0038] (2) Preparation of oligosaccharide fragments of yellow tea polysaccharide The purified yellow tea polysaccharide was dissolved in 50 mM acetic acid-sodium acetate buffer solution with a pH of 4.0 to prepare a 1 mg / mL yellow tea polysaccharide solution. 200 U / mg galacturonidase was added and the enzymatic reaction was carried out at 50°C for 12 h. After the reaction was completed, the enzyme activity was terminated by boiling. The reaction solution was centrifuged (5700 g, 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 packages in sequence. The membrane retentate and permeate were collected, concentrated, and freeze-dried to obtain M W The oligosaccharide components at 0.5KDa~1KDa, 1~3KDa, and 3KDa~5KDa were named ELYP-1, ELYP-2, and ELYP-3, respectively.

[0039] (3) Screening of the activity of yellow tea oligosaccharide fragments in promoting the secretion of GLP-1 by enteroendocrine L cells The obtained yellow tea oligosaccharide fragments ELYP-1, ELYP-2, and ELYP-3 with different polymerization degrees were respectively administered to enteroendocrine L cells STC-1 to screen and evaluate the activity of promoting GLP-1 secretion. STC-1 cells with good growth status were taken and inoculated in 48-well plates (cell density was 1×10 6 / mL). Place in 5% CO 2 Incubate in an incubator for 24 hours. Remove the culture medium, add ELYP-1, ELYP-2, and ELYP-3 solutions with a final concentration of 100 μg / mL, and continue incubating for 2 hours. The blank control group (Control) is a complete culture medium, and a yellow tea uniform polysaccharide component LYP group is set up. After the incubation, the culture medium is collected, and the supernatant is centrifuged (400g, 4°C, 15min), and the content of GLP-1 in the supernatant is determined.

[0040] The results are as follows Figure 1 As shown in the figure, compared with the control group, LYP and oligosaccharide fragments ELYP-1, ELYP-2, and ELYP-3 with different polymerization degrees degraded by galacturonidase significantly increased the level of GLP-1 secretion by STC-1 cells, and their contents were 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 in promoting the secretion of GLP-1 by STC-1 cells. Among them, the amount of GLP-1 secreted by ELYP-3 on STC-1 cells is 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 to promote the secretion of GLP-1 by intestinal epithelial cells.

[0041] Example 2: Purification and structural identification of the yellow tea oligosaccharide fragment ELYP-3

[0042] (1) Purification of oligosaccharide fragments of yellow tea:

[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 loading concentration of 40 mg / mL. The column was eluted with deionized water at a flow rate of 10 mL / h. The polysaccharide content in the eluate was detected by phenol-sulfuric acid colorimetry. The sugar-containing eluate was collected, vacuum concentrated, and freeze-dried to obtain purified yellow tea oligosaccharide ELYP-3.

[0044] (2) Determination of carbohydrate content in yellow tea oligosaccharide ELYP-3:

[0045] The carbohydrate content of ELYP-3 obtained in the above (1) was determined by the phenol-sulfuric acid method. The carbohydrate content of ELYP-3 was 93.59% as measured by a spectrophotometer at 490 nm.

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

[0047] The full wavelength scanning microplate reader was used to scan ELYP-3 in the wavelength range of 190 to 400 nm. Figure 2 As shown in A, ELYP-3 has no absorption peaks at 260nm and 280nm in the scanning spectrum, indicating that the purified enzymatic oligosaccharide contains almost no pigment, protein and nucleic acid.

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

[0049] The characteristic groups of ELYP-3 were analyzed by using Nicolette is50 Fourier transform infrared spectrometer. Figure 2 As shown in B, ELYP-3 has a typical sugar characteristic peak, 3440 cm -1 The signal peak at 2930cm is attributed to the OH stretching vibration within or between sugar molecules; -1 The signal peak at 1630 cm is attributed to the stretching vibration of CH; -1 and 1414cm -1 The signal peaks at 1149 cm-1 are attributed to the stretching vibration of the carbonyl (C=O) in COOH and the angular vibration of CH; -1 The signal peak at 946 cm is attributed to the stretching vibration of COC and COH in the pyranose ring; -1 The signal peak at indicated that ELYP-3 contained β-configuration glycosidic bonds and α-configuration glycosidic bonds.

[0050] (5) Molecular weight determination of yellow tea oligosaccharide ELYP-3:

[0051] The molecular weight distribution of ELYP-3 was determined by high performance gel permeation chromatography-differential-multi-angle laser light scattering. Figure 3 As shown in A, the molecular weight of ELYP-3 is: 3.2×10 3 Da. Detection conditions: Waters Arc HPLC system, Brookhaven BI-MwA multi-angle laser light scattering detector, Waters 2414RI differential detector, TSK G2500PWXL analytical column (7.8 mm × 300 mm), mobile phase 0.1 M NaNO 3 solution, flow rate 0.6mL / min, column temperature 35℃, injection volume 100μL.

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

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

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

[0055] The Congo red experiment was used to determine whether there was a triple helix structure in ELYP-3. Prepare 2 mg / mL ELYP-3 solution, add Congo red solution at a ratio of 1:1 v / v, mix well, add NaOH solution of different final concentrations (0-0.5 mol / mL), use the mixed solution without ELYP-3 solution as a control, let it stand at room temperature for 10 minutes, and use a UV full wavelength scanner to determine the maximum absorption wavelength of the Congo red-yellow tea polysaccharide complex. Figure 3 As shown in Figure C, with the increase of NaOH concentration, the maximum absorption wavelength of the complex of Congo red and ELYP-3 decreases and finally tends to be stable, indicating that ELYP-3 does not have a triple helix structure.

[0056] (8) Microstructure analysis of yellow tea oligosaccharide ELYP-3:

[0057] The surface morphology of ELYP-3 was analyzed using a Hitachi SU1000 scanning electron microscope. Weigh 2 mg of dry ELYP-3 and adhere it to a metal iron block with conductive glue. After spraying a conductive film on the surface of the sample, its surface morphology was observed. Figure 4 As shown in B, the surface of ELYP-3 showed broken irregular particle fragments.

[0058] (9) Analysis of monosaccharide composition of yellow tea oligosaccharide ELYP-3:

[0059] The monosaccharide composition of ELYP-3 was determined by high performance liquid chromatography with 1-phenyl-3-methyl-5-P pyrazolone (PMP) pre-column derivatization. 5 mg of dry ELYP-3 was placed in a 10 mL ampoule, 3 mL of 3 M hydrochloric acid solution was added, the tube was sealed, and hydrolyzed at 110 ° C for 1 hour. After cooling, the pH was adjusted to neutral with 3.0 mol / L sodium hydroxide solution to obtain an oligosaccharide hydrolyzate. 100 μL of 0.6 M NaOH solution and an equal volume ratio of 0.6 M PMP methanol solution were added to the above hydrolyzate, and the reaction was carried out at 70 ° C for 100 minutes. After the reaction, 50 μL of 0.3 M HCl solution was added, and after rotary evaporation at 50 ° C, chloroform was added for washing 3 times. The supernatant was filtered and then tested by HPLC. The results are as follows Figure 5 As shown, by comparing the spectrum with the monosaccharide standards, ELYP-3 is mainly composed of five monosaccharides, and the molar ratio of 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), UV 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: Effects of Camellia ternata oligosaccharide fragments on the synthesis and secretion of GLP-1 by enteroendocrine L cells in vitro

[0062] Using enteroendocrine L cells STC-1 as a model, the effect of ELYP-3 prepared in Example 1 on promoting the synthesis and secretion of GLP-1 by enteroendocrine L cells was studied. STC-1 cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 1×10 6 Cells were inoculated into 6-well cell culture plates at 37°C and 5% CO 2 After the cells were attached to the culture medium for 24 hours, ELYP-3 was added at a final concentration of 25 and 100 μg / mL, respectively, and the cells were incubated for another 2 hours. The blank control group was filled with complete culture medium. After the incubation, the cells and cell supernatants of each group were collected, and the RT-qPCR method was used to detect the mRNA expression levels of key genes (gcg, pcsk1, α-gustducin, plcb2, gipr, glp1r, t1r2 / 3) encoding GLP-1 in the process of precursor synthesis → processing and maturation → extracellular release in the cells, and the ELISA method was used to detect the content of GLP-1 in the cell supernatant.

[0063] The results are as follows Figure 6 shown. ELYP-3 can significantly promote the efficient secretion of GLP-1 by intestinal endocrine cells STC-1, and up-regulate the mRNA expression levels of key genes encoding GLP-1 precursor synthesis (gcg, pcsk1), processing and maturation (α-gustducin, plcb2), and extracellular release (glp1r, t1r2 / 3), showing a dose effect. The above results indicate that ELYP-3 acts on STC-1 cells, not only effectively increasing the intracellular synthesis of GLP-1, but also highly promoting the release of GLP-1 from intracellular to extracellular

[0064] Example 4: Effects of Oligosaccharide Fragments of Huangdacha Tea on the Synthesis and Secretion of GLP-1 in Normal Mice and T2DM Mice in Vivo

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

[0066] Male SD rats at 5 weeks of age were adaptively raised in an SPF-class animal room at a temperature of 23±2°C, relative humidity of 50%-60%, and 12h light and 12h dark conditions. After 1 week, the rats were randomly divided into 3 groups (n = 6): control group (in-situ administration of normal saline), low-concentration ELYP-3 in-situ administration (ELYP-3L, 0.5 g / kg), and high-concentration ELYP-3 in-situ administration (ELYP-3H, 2 g / kg). Each group of rats was anesthetized on the operating table, the abdominal cavity was opened, the hepatic portal vein was cannulated, and then the jejunum or ileum was ligated. The drug was directly administered on the ligated jejunum or ileum. After 15, 30, 60, 90, and 120 min of drug 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°C for 15 min, and the plasma was collected to measure the secretion level of GLP-1 in the plasma at different drug administration times

[0067] To further study whether ELYP-3 can directly act on intestinal endocrine L cells in vivo to promote GLP-1 secretion, an in-situ intestinal administration experiment of ELYP-3 in vivo was used to detect the plasma GLP-1 level after direct in-situ administration of ELYP-3 in the jejunum or ileum. The results are as follows Figure 7 shown. Compared with the control group, the plasma GLP-1 increased rapidly within 15 min after direct administration of ELYP-3 in the jejunum or ileum, reached the highest value at 30 min, and the increase in plasma GLP-1 content gradually leveled off after 30 min. The results indicate that ELYP-3 can directly act on intestinal endocrine L cells in vivo to promote GLP-1 secretion

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

[0069] Construction of T2DM mouse model and drug intervention Four-week-old male C57 / 6J mice were selected and adaptively raised in an SPF animal room at a temperature of 23±2℃, a relative humidity of 50%-60%, and 12 h of light and 12 h of darkness. After one week, the mice were randomly divided into a normal group (ND, fed with ordinary feed + intragastric administration of normal saline), a high-fat diet model group (HFD, fed with high-fat feed + intragastric administration of normal saline), a positive group (Positive, fed with high-fat feed + intragastric administration of metformin 50 mg / kg / d), a low-dose ELYP-3 group (ELYP-3L, fed with high-fat feed + intragastric administration of ELYP-3 50 mg / kg / d), and a high-dose ELYP-3 group (ELYP-3H, fed with high-fat feed + intragastric administration of ELYP-3 200mg / kg / d), 12 mice in each group, after 8 weeks of high-fat feeding, a single intraperitoneal injection of 100mg / kgBW streptozotocin (STZ), fasting blood glucose was measured 72h after STZ injection, and mice with blood glucose values ​​above 11.1mmol / L were selected as diabetic model mice. The corresponding intervention substances were continuously gavaged until the end of the experiment after 12 weeks, and the mice were fasted overnight, CO 2 The animals were sacrificed, blood was collected from the heart, and colon and ileum tissues were collected, frozen in liquid nitrogen, and stored at -80°C for later use.

[0070] T2DM mice were established by HFD feeding and STZ injection, and the effects of the application examples of the present invention on the synthesis and secretion of GLP-1 content in T2DM model mice were investigated. Figure 8 As shown in A, compared with the ND group, the GLP-1 content in the plasma of T2DM model mice was significantly reduced, and intervention with different doses of ELYP-3 significantly increased the GLP-1 content in the plasma of T2DM mice. To further study the effect of ELYP-3 on the synthesis and secretion of GLP-1 by intestinal endocrine L cells in T2DM model mice, the mRNA expression levels of key genes (gcg, pcsk1, α-gustducin, plcb2, trpm5, glp1r, t1r2 / 3) encoding GLP-1 in the process of precursor synthesis → processing and maturation → extracellular release in ileal tissue were detected by RT-qPCR. Figure 8As shown in BI, ELYP-3 intervention can significantly upregulate the mRNA expression levels of key genes encoding GLP-1 precursor synthesis (gcg, pcsk1), processing and maturation (α-gustducin, plcb2), and extracellular release (trpm5, glp1r, t1r2 / 3) in the ileum of T2DM mice, with a dose effect. The above results show that ELYP-3 activates the GLP-1 receptor in T2DM mice, and also effectively increases the intracellular synthesis of GLP-1, and also promotes the release of GLP-1 from intracellular to extracellular at a high level.

[0071] Example 5 Effect of Huangdacha oligosaccharide fragments on improving blood glucose, blood lipids and insulin resistance in T2DM mice

[0072] During the experiment, the fasting blood glucose of T2DM mice was monitored weekly using a Roche blood glucose meter. After the experiment, the total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDLc) and low-density lipoprotein (HDL-c) and other blood lipid indicators in the plasma of each group of T2DM mice were detected using a biochemical kit, and the fasting insulin of T2DM mice was measured by an ELISA kit. The insulin resistance index (HOMA-IR) and β-cell function index (HOMA-β) were calculated according to the following formulas: HOMA-IR = fasting blood glucose value (FBG) (mmol / L) × fasting insulin content (FINS) (mU / L) / 22.5; (HOMA-β) = [20 × FINS (mU / L)] / [FBG (mmol / L)-3.5]. The improvement effect of the application example of the present invention on blood glucose, blood lipids and insulin resistance in T2DM mice was explored.

[0073] like Fig. 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 increased significantly. 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 can be significantly reduced, indicating that ELYP-3 intervention has the effect of assisting in lowering blood sugar. Further calculation of the insulin resistance index (HOMA-IR) and β-cell function index of each group of mice, the results are as follows Fig. 9 As shown in CD, ELYP-3 intervention can significantly reduce the HOMA-IR of T2DM mice and increase the β-cell function index, indicating that ELYP-3 can significantly improve the insulin resistance of T2DM mice and enhance insulin sensitivity.

[0074] The blood lipid levels of T2DM mice were characterized by measuring total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) in the serum. Fig. 9As shown in EH, different doses of ELYP-3 intervention reduced the levels of TC, TG, and LDL-c in the plasma of T2DM mice, while increasing the level of HDL-c, indicating that ELYP-3 intervention significantly improved hyperlipidemia in T2DM mice and had an auxiliary lipid-lowering effect.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

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.

2. The yellow tea oligosaccharide with GLP-1 agonist activity according to claim 1, characterized in that: The carbohydrate content of the ELYP-3 is 93.59%.

3. The yellow tea oligosaccharide with GLP-1 agonist activity according to claim 1, characterized in that: The ELYP-3 contains β-configuration glycosidic bonds and α-configuration glycosidic bonds.

4. The yellow tea oligosaccharide with GLP-1 agonist activity according to claim 1, characterized in that: The particle size of the ELYP-3 is mainly concentrated in the range of 10 to 1000 nm.

5. The yellow tea oligosaccharide with GLP-1 agonist activity according to claim 1, characterized in that: The average particle size of the ELYP-3 is 132.2 nm.

6. The use of the yellow tea oligosaccharide according to any one of claims 1 to 5 in any of 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 products include food, medicine or health products.

7. A drug for preventing and / or treating diabetes, characterized in that: The effective ingredients include the yellow tea oligosaccharide described in any one of claims 1 to 5.

8. The drug for preventing and / or treating diabetes according to claim 7, characterized in that: The drug also includes pharmaceutically acceptable salts, specifically organic salts or inorganic salts.

9. The drug for preventing and / or treating diabetes according to claim 7, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

10. The drug for preventing and / or treating diabetes according to claim 9, characterized in that: 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.

Images