Preparation method and application of large yellow tea oligosaccharide fragment ELYP

Abstract

The invention provides a preparation method and application of a large yellow tea oligosaccharide fragment ELYP, and belongs to the field of food and medicine. The preparation process comprises the following steps: 1, dissolving the large yellow tea polysaccharide in a buffer solution system for enzymolysis reaction; 2, galacturonide glycosidase is added for an enzymatic hydrolysis reaction; 3, terminating the enzymolysis reaction, and centrifuging, desalting, membrane separating and freeze-drying reaction liquid to obtain oligosaccharide fragments with different polymerization degrees, and 4, retaining oligosaccharide fragment components with MW of 0.5 KDa to 5KDa, so as to obtain the oligosaccharides with the MW of 0.5 KDa to 5KDa. The method has the beneficial effects that the yellow large tea oligosaccharide fragment obtained through glycosidase degradation proves that the yellow large tea oligosaccharide can activate a GLP-1 receptor and promote synthesis and secretion of GLP-1, and has the effects of remarkably reducing blood sugar, blood fat and body weight. And the preparation process is simple to operate, high in specificity and environment-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 foods, medicines or health care products with blood sugar regulating or weight losing effects.

Description

Technical Field

[0001] The present invention belongs to the field of food and medicine, and particularly relates to a preparation method and application of a low molecular weight oligosaccharide fragment ELYP from Huangdacha tea. Background Art

[0002] Tea generally refers to a drink made from the buds and leaves of the tea tree (Camellia sinensis (L.) O. Kuntze, a perennial evergreen shrub of the genus Camellia in the family Theaceae), and is considered a healthy food that can maintain health and prevent human diseases. In China, there are records of using coarse and old tea to treat diabetes. According to textual research on Chinese herbal medicines, the pharmacological component of tea for treating diabetes is tea polysaccharide. Modern pharmacological studies have shown that in addition to its hypoglycemic effect, tea polysaccharide, as the main active ingredient in tea, also has a wide range of active functions such as reducing blood lipid, anti-coagulation, anti-thrombosis, enhancing the body's immunity, protecting the cardiovascular system, anti-cancer, anti-oxidation, and anti-radiation. Tea polysaccharide is a type of acidic protein heteropolysaccharide with a complex structure and diverse functions, and its rich and diverse structural characteristics are the basis for the polysaccharide to exert various activities. Due to the microscopic heterogeneity of the tea polysaccharide structure and the diversity of monosaccharide composition, and its large flexibility and difficulty in crystallization in the solution state, the research on its targeting lags far behind, which is the key reason hindering the application of functional polysaccharides in clinical research.

[0003] The core issue in the research on polysaccharide targeting is to solve the sugar recognition domain or the active site for interaction. Therefore, degrading tea polysaccharide into oligosaccharides and studying the active center of polysaccharides at the oligosaccharide level provides a new idea for breaking through the bottleneck in the research on the structural and functional mechanism of tea polysaccharide. Currently, the methods for degrading polysaccharides include physical degradation, chemical degradation, and enzymatic degradation. Physical degradation methods include ultrasonic, radiation, microwave, heat treatment, etc. Among them, the ultrasonic method has a long time, low efficiency, high energy consumption, and high noise; the radiation method requires specific equipment and technology, has high requirements for equipment, and is prone to harm operators; microwave and heat treatment methods may change the physical and chemical properties of polysaccharides; in chemical degradation methods such as acid hydrolysis and oxidative degradation, the reaction regularity is poor, the process is difficult to control, the reproducibility is not good, and there are problems such as difficult control of degradation product components and environmental pollution. Compared with physical and chemical degradation, enzymatic hydrolysis can cleave specific glycosidic bonds, the reaction is easy to control, and the degradation process is mild with no by-products generated, which is an ideal degradation method. Therefore, the application of preparing functional oligosaccharides by enzymatic degradation of polysaccharides shows good industrial prospects.

[0004] Currently, there are few research reports on the degradation of Huangdacha tea polysaccharide. To discover the target molecule of tea polysaccharide, there is no report on how to provide a method to screen high-activity oligosaccharide fragments with GLP-1 secretion-promoting activity from tea polysaccharide. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to provide a preparation method of highly active oligosaccharide fragments with GLP-1 secretion promoting effect.

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

[0007] The first aspect of the present invention provides a preparation method of oligosaccharide fragment ELYP from large yellow tea, comprising the following steps:

[0008] S1: Dissolve large yellow tea polysaccharide (LYP) in the buffer solution for enzymatic hydrolysis reaction;

[0009] S2: Add glycosidase for enzymatic hydrolysis reaction;

[0010] S3: Terminate the enzymatic hydrolysis reaction, centrifuge the reaction solution, desalt, perform membrane separation, and freeze-dry to obtain different oligosaccharide fragments;

[0011] S4: Retain the oligosaccharide fragment fraction with a molecular weight (M W between 0.5 KDa and 5 KDa, and that's it.

[0012] Preferably, the buffer solution in step S1 is 50 mM acetic acid-sodium acetate buffer solution (pH 4.0); more preferably, dissolve large yellow tea polysaccharide in sodium acetate buffer solution to a final concentration of 0.5 - 3 mg / mL, and more preferably 1 mg / mL.

[0013] Preferably, the glycosidase in step S2 is endo-galacturonosidase, and the addition amount is 150 - 250 U / mg, the enzymatic hydrolysis temperature is 40 - 60 °C, and the enzymatic hydrolysis time is 10 - 14 h. More preferably, it is 200 U / mg, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 12 h.

[0014] Preferably, for the centrifugation in step S3, the specific conditions are 5000 - 6000 g, 25 - 35 min, and more preferably 5700 g, 30 min.

[0015] Preferably, in step S3, the desalting is specifically: take the supernatant after centrifugation, perform desalting treatment through a 500 Da ultrafiltration membrane, and collect the fraction with a molecular weight (M W ) > 500 Da; the membrane separation is specifically: use 1 KDa, 3 KDa, and 5 KDa ultrafiltration membrane cartridges (Minimate TM TFF Capsule) in the tangential flow ultrafiltration device Masterflex L / S system, specifically: take the fraction after desalting, pass through the 1 KDa, 3 KDa, and 5 KDa membrane cartridges in sequence, and collect the membrane retentate and permeate to obtain oligosaccharide components with M W between 0.5 KDa and 1 KDa, 1 - 3 KDa, and 3 KDa - 5 KDa.

[0016] Preferably, in step S4, M is retained. W The oligosaccharide fragment component with a molecular weight of 3 KDa to 5 KDa.

[0017] In the second aspect of the present invention, the yellow big tea oligosaccharide fragment ELYP prepared by the above preparation method is proposed.

[0018] In the third aspect of the present invention, the following any application of the yellow big tea oligosaccharide fragment ELYP prepared by the above preparation method is proposed:

[0019] (1) Use in the preparation of a reagent for promoting the synthesis and / or secretion of GLP-1;

[0020] (2) Use in the preparation of a product for preventing and / or treating hyperlipidemia;

[0021] (3) Use in the preparation of a product for preventing and / or treating obesity.

[0022] Preferably, in (2) to (3), the product includes one or more of food, medicine, and health products.

[0023] In the fourth aspect of the present invention, a drug for preventing and / or treating hyperlipidemia and / or obesity is proposed, and its active ingredient includes the yellow big tea oligosaccharide fragment ELYP prepared by the above preparation method.

[0024] Preferably, the drug is made into a pharmaceutically acceptable dosage form.

[0025] Preferably, the dosage form is tablets, pills, ointments, oral liquids or granules.

[0026] On the other hand, the present invention provides a preparation method of a yellow big tea oligosaccharide fragment for promoting GLP-1 secretion degraded by endo-galacturonase, or the use of the above method in the preparation of a drug for preventing and / or treating diabetes or hyperlipidemia or obesity that promotes GLP-1 secretion.

[0027] On the other hand, the present invention provides the use of the yellow big tea oligosaccharide fragment described in the above technical solution or the yellow big tea polysaccharide fragment prepared by the preparation method described in the above technical solution in the preparation of a health product or functional food for assisting in improving insulin resistance or reducing blood lipid levels or losing weight.

[0028] The beneficial effects of the present invention are as follows:

[0029] 1. The present invention is activity-guided by promoting the synthesis and secretion of glucagon-like peptide-1 (GLP-1). Through enzymatic hydrolysis, membrane separation, and polyacrylamide gel chromatography purification techniques, highly active oligosaccharide fragments are prepared from Huangdacha polysaccharide. The process method is simple, highly specific, and environmentally friendly, and is suitable for large-scale production. This 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 glucose-regulating effects.

[0030] 2. The Huangdacha oligosaccharide fragments provided by the present invention have good effects in in vitro cell experiments. The oligosaccharide fragments 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.

[0031] 3. The Huangdacha oligosaccharide fragments provided by the present invention also have excellent effects in a type II diabetic mouse model. The oligosaccharide fragments significantly increase the secretion levels of GLP-1 in the blood, ileum, and colon of mice, and at the same time have obvious effects on lowering blood glucose, reducing blood lipids, decreasing lipid accumulation, and improving insulin resistance. Description of the Drawings

[0032] Figure 1 Shows the effects of different Huangdacha oligosaccharide fragments on the secretion of GLP-1 by intestinal endocrine L cells in Example 1 of the present invention;

[0033] 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;

[0034] 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;

[0035] Figure 4 Shows the SEM images of Huangdacha oligosaccharide ELYP-3 in Example 2 of the present invention; A: LYP, B: ELYP-3;

[0036] Figure 5 Shows the monosaccharide composition spectrum of Huangdacha oligosaccharide ELYP-3 in Example 2 of the present invention; A: standard product, B: ELYP;

[0037] Figure 6 Shows the effects of Huangdacha oligosaccharide ELYP-3 on the synthesis and secretion of GLP-1 by intestinal endocrine L cells STC-1 in Example 3 of the present invention;

[0038] Figure 7 Shows the effects of directly administering Huangdacha oligosaccharide ELYP-3 to the jejunum (A) or ileum (B) of rats on the content of GLP-1 in portal vein plasma in Example 4 of the present invention;

[0039] Figure 8 Effect of oligosaccharides ELYP-3 from Huangdacha tea on plasma GLP-1 (A) and GLP-1 synthesis and secretion in ileum (B-I) of type Ⅱ diabetic mice in Example 4 of the present invention;

[0040] Figure 9 Effect of oligosaccharides ELYP-3 from Huangdacha tea on blood glucose (A), insulin resistance (B-D) and blood lipids (E-H) of type Ⅱ diabetic mice in Example 5 of the present invention. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] The test materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0043] For those not specifying specific techniques or conditions in the embodiments, they can all be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. Without special instructions, the quantitative tests in the following embodiments are all set with more than three repeated experiments, and the results are averaged.

[0044] Example 1: Preparation of oligosaccharide fragments from Huangdacha tea

[0045] (1) Preparation of Huangdacha polysaccharide: After Huangdacha tea was dried and crushed, an appropriate amount of Huangdacha tea powder was weighed and added with 95% ethanol at a solid-liquid ratio of 1:20 g / mL, and stirred at 25 °C and 50 rpm / min for 24 h to remove fat and pigment impurities. After collecting the residue by suction filtration, it was stirred and extracted in a water bath at 90 °C for 2.5 h at a solid-liquid ratio of 1:20 g / mL, and the supernatant was collected by filtration. Four volumes of absolute ethanol (final ethanol concentration 80%) were added thereto, and after standing at 4 °C for 12 h, the precipitate was collected by centrifugation. The Sevage method was used to remove the protein in the precipitate. The deproteinized sugar solution was rotary evaporated, dialyzed, and freeze-dried, and further purified by DEAE cellulose DE-52 anion exchange chromatography column and Sephadex G-100 gel chromatography column to obtain the homogeneous polysaccharide component LYP of Huangdacha tea.

[0046] (2) Preparation of Oligosaccharide Fragments of Huangda Tea Polysaccharide The purified Huangda tea polysaccharide was dissolved in 50 mM acetic acid - sodium acetate buffer solution with pH = 4.0 to prepare a 1 mg / mL Huangda tea polysaccharide solution. 200 U / mg galacturonide glycosidase was added, and the enzymatic hydrolysis reaction was carried out at 50 °C for 12 h. After the reaction, boiling was used to terminate the enzyme activity. The reaction solution was centrifuged (5700 g, 10 min) to remove the denatured protease, and the supernatant was collected. After desalting and impurity removal through an ultrafiltration membrane with a molecular weight cut-off of 0.5 KDa, it was 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 Oligosaccharide components in the range of 0.5 KDa - 1 KDa, 1 - 3 KDa, and 3 KDa - 5 KDa were respectively named ELYP-1, ELYP-2, and ELYP-3.

[0047] (3) Activity Screening of Huangda Tea Oligosaccharide Fragments in Promoting GLP-1 Secretion by Enteroendocrine L Cells The obtained Huangda tea oligosaccharide fragments ELYP-1, ELYP-2, and ELYP-3 with different degrees of polymerization were respectively administered to the enteroendocrine L cell line STC-1 for the screening and evaluation of the activity of promoting GLP-1 secretion. STC-1 cells in good growth state were seeded in a 48-well plate (cell density of 1×10 6 cells / mL). It was incubated in a 5% CO 2 2 incubator for 24 h. The culture medium was aspirated, and solutions of ELYP-1, ELYP-2, and ELYP-3 with a final concentration of 100 μg / mL were added respectively, and incubation was continued for 2 h. The blank control group (Control) was complete culture medium, and a group of the homogeneous polysaccharide component LYP of Huangda tea was set. After incubation, the culture medium was collected, centrifuged to obtain the supernatant (400 g, 4 °C, 15 min), and the content of GLP-1 in the supernatant was measured.

[0048] The results were as Figure 1 shown. Compared with the control group, LYP and the oligosaccharide fragments ELYP-1, ELYP-2, and ELYP-3 with different degrees of polymerization degraded by galacturonide glycosidase all significantly increased the level of GLP-1 secreted 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 GLP-1 secretion by STC-1 cells. Among them, the amount of GLP-1 secreted by 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.

[0049] Example 2: Purification and Structural Identification of Huangda Tea Oligosaccharide Fragment ELYP-3

[0050] (1) Purification of Oligosaccharide Fragments from Huangda Tea:

[0051] Dissolve the ELYP-3 component in Example 1 in double-distilled water, load it onto a polyacrylamide gel Bio gel P-2 column, with a sample loading concentration of 40 mg / mL, and elute it with deionized water at a flow rate of 10 mL / h. Detect the polysaccharide content in the eluate by the phenol-sulfuric acid colorimetric method, collect the sugar-containing eluate, and after vacuum concentration and freeze-drying, obtain the purified oligosaccharide ELYP-3 from Huangda Tea.

[0052] (2) Determination of Carbohydrate Content in Oligosaccharide ELYP-3 from Huangda Tea:

[0053] Use the phenol-sulfuric acid method to determine the carbohydrate content of ELYP-3 obtained in (1) above. After detection by a spectrophotometer at 490 nm, the carbohydrate content in ELYP-3 is 93.59%.

[0054] (3) Ultraviolet Spectrum Analysis of ELYP-3:

[0055] Scan ELYP-3 with a full-wavelength scanning microplate reader in the wavelength range of 190 - 400 nm. The results are as Figure 2 shown in Figure A. At 260 nm and 280 nm in the scanning spectrum of ELYP-3, there are no absorption peaks, indicating that the enzymatically hydrolyzed oligosaccharides after purification contain almost no pigments, proteins, and nucleic acids.

[0056] (4) Infrared Spectrum Analysis of ELYP-3:

[0057] Use a Nicolette is50 Fourier transform infrared spectrometer to scan and analyze the characteristic groups of ELYP-3. The results are as Figure 2 shown in Figure B. ELYP-3 has typical sugar characteristic peaks. The signal peak at 3440 cm -1 is attributed to the stretching vibration of O-H within or between sugar molecules; the signal peak at 2930 cm -1 is attributed to the stretching vibration 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 bending vibration of C-H; 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; the signal peak at 946 cm -1 indicates that ELYP-3 contains β-configuration glycosidic bonds and α-configuration glycosidic bonds.

[0058] (5) Determination of the Molecular Weight of Oligosaccharide ELYP-3 from Huangda Tea:

[0059] The molecular weight distribution of ELYP-3 was determined by high performance gel permeation chromatography - differential refractive index - multi-angle laser light scattering method. The results are as follows Figure 3 shown in A. The molecular weight of ELYP-3 was 3.2×10 3 Da. Detection conditions: Waters Arc HPLC liquid phase system, Brookhaven BI-MwA multi-angle laser light scattering detector, Waters 2414RI differential refractive index detector, TSK G2500PWXL analytical column (7.8mm×300mm), mobile phase 0.1M NaNO 3 solution, flow rate 0.6mL / min, column temperature 35℃, injection volume 100μL.

[0060] (6) Determination of the particle size of ELYP-3, an oligosaccharide from Huangdacha tea:

[0061] The particle size of ELYP-3 was measured using a Zetasizer Pro nano particle size analyzer. A 1mg / mL ELYP-3 solution was prepared, filtered through a 0.22μm aqueous membrane, and its particle size was measured at 25℃, 633nm wavelength, and 90° scattering angle. As shown in Figure 3 B, the particle size of ELYP-3 mainly concentrated in the range of 10 - 1000nm, and the average particle size was 132.2nm.

[0062] (7) Determination of the triple helix structure in ELYP-3, an oligosaccharide from Huangdacha tea:

[0063] The presence of a triple helix structure in ELYP-3 was determined using the Congo red experiment. A 2mg / mL ELYP-3 solution was prepared, and Congo red solution was added at a volume ratio of 1:1 v / v. After mixing, different final concentrations of NaOH solution (0 - 0.5mol / mL) were added. A mixed solution without ELYP-3 was used as a control. The mixture was allowed to stand at room temperature for 10min, and the maximum absorption wavelength of the Congo red - Huangdacha tea polysaccharide complex was measured using an ultraviolet full wavelength scanner. As shown in Figure 3 C, as the concentration of NaOH increased, the maximum absorption wavelength of the Congo red - ELYP-3 complex decreased and finally stabilized, indicating that ELYP-3 does not have a triple helix structure.

[0064] (8) Microstructural analysis of ELYP-3, an oligosaccharide from Huangdacha tea:

[0065] The apparent morphology of ELYP-3 was analyzed using a Hitachi SU1000 scanning electron microscope. 2mg of dried ELYP-3 was weighed and adhered to a metal iron block using conductive glue. After spraying a conductive film on the sample surface, its apparent morphology was observed. The results are as shown in Figure 4 B. The surface of ELYP-3 presented fractured irregular particle fragments.

[0066] (9) Analysis of monosaccharide composition of huangdacha oligosaccharide ELYP-3:

[0067] The monosaccharide composition of ELYP-3 was determined by pre-column derivation high performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone (PMP). Take 5 mg of dried ELYP-3 and place it in a 10 mL ampoule. Add 3 mL of 3M hydrochloric acid solution, seal the tube, hydrolyze at 110 °C for 1 h, cool it down, and adjust the pH to neutral with 3.0 mol / L sodium hydroxide solution to obtain the oligosaccharide hydrolysis solution. Add 100 μL of 0.6M NaOH solution and an equal volume ratio of 0.6M PMP methanol solution to the above hydrolysis solution, react at 70 °C for 100 min. After the reaction, add 50 μL of 0.3M HCl solution, rotary evaporate to dryness at 50 °C, wash with chloroform three times, and filter the supernatant for HPLC detection. The results are as Figure 5 shown. By comparing the chromatograms with monosaccharide standards, ELYP-3 is mainly composed of 5 monosaccharides, and their molar ratio is rhamnose:galacturonic acid:glucose:galactose:arabinose = 1:32.42:45.37:9.11:6.54.

[0068] 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 solution (PBS, pH 6.7): acetonitrile = 83:17 (v / v), flow rate 1.0 mL / min, detection wavelength 245 nm.

[0069] Example 3: Effect of huangdacha oligosaccharide fragments on the synthesis and secretion of GLP-1 by intestinal endocrine L cells in vitro

[0070] Using intestinal endocrine L cell STC-1 as a model, the promoting effect of ELYP-3 prepared in Example 1 on the synthesis and secretion of GLP-1 by intestinal endocrine L cells was studied. Take STC-1 cells in the logarithmic growth phase, adjust the cell concentration to 1×10 6 cells / mL and inoculate them into a 6-well cell culture plate. After adherent culture in an incubator at 37 °C and 5% CO 2 for 24 h, add ELYP-3 with final concentrations of 25 and 100 μg / mL respectively, and continue to incubate for 2 h. The blank control group is complete culture medium. After the incubation, collect the cells and cell supernatants of each group respectively. The mRNA expression levels of key genes (gcg, pcsk1, α-gustducin, plcb2, gipr, glp1r, t1r2 / 3) during the process of GLP-1 precursor synthesis → processing and maturation → extracellular release in the cells are detected by RT-qPCR method, and the content of GLP-1 in the cell supernatant is detected by ELISA.

[0071] 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.

[0072] Example 4: Effects of Oligosaccharide Fragments of Huangda Tea on the Synthesis and Secretion of GLP-1 in Normal Mice and T2DM Mice in Vivo

[0073] (1) In-situ Detection of GLP-1

[0074] Male SD rats at 5 weeks of age were adaptively raised in an SPF-class animal house at a temperature of 23 ± 2 °C, relative humidity of 50%-60%, and 12 h of light and 12 h of darkness. 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). After anesthetizing each group of rats on the operating table, the abdominal cavity was opened, a cannula was inserted into the hepatic portal vein, 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). After centrifugation at 3500 rpm at 4 °C for 15 min, plasma was collected to measure the secretion level of GLP-1 in plasma at different drug administration times.

[0075] To further study whether ELYP-3 can directly act on intestinal endocrine L cells to promote GLP-1 secretion in vivo, an in-situ intestinal drug 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 to promote GLP-1 secretion in vivo.

[0076] (2) Determination of GLP-1 synthesis and secretion levels in T2DM mice

[0077] Construction of T2DM mouse model and administration intervention: Male C57 / 6J mice at 4 weeks of age were adaptively raised in a SPF-class animal room at a temperature of 23 ± 2 °C, relative humidity of 50%-60%, and 12 h of light and 12 h of darkness. After 1 week, the mice were randomly divided into a normal group (ND, fed with normal diet + intragastric administration of normal saline), a high-fat diet model group (HFD, fed with high-fat diet + intragastric administration of normal saline), a positive group (Positive, fed with high-fat diet + intragastric administration of metformin 50 mg / kg / d), a low-dose ELYP-3 group (ELYP-3L, fed with high-fat diet + intragastric administration of ELYP-3 50 mg / kg / d), and a high-dose ELYP-3 group (ELYP-3H, fed with high-fat diet + intragastric administration of ELYP-3 200 mg / kg / d), with 12 mice in each group. After 8 weeks of high-fat feeding, streptozotocin (STZ) at 100 mg / kg BW was injected intraperitoneally once. The fasting blood glucose was measured 72 h after STZ injection, and the mice with blood glucose values above 11.1 mmol / L were selected as diabetic model mice. The corresponding intervention substances were continuously administered by gavage until the end of the 12th week. After fasting overnight, CO 2 The mice were sacrificed, blood was collected from the heart, and colon and ileum tissues were collected. After freezing in liquid nitrogen, they were stored at -80 °C for later use.

[0078] T2DM mice were constructed by HFD feeding and STZ injection to explore the effect of the application example of the present invention on the synthesis and secretion of GLP-1 content in T2DM model mice. The results are as Figure 8 shown in Figure A. Compared with the ND group, the GLP-1 content in the plasma of T2DM model mice was significantly decreased, and the administration of 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) in the ileum tissue during the process of GLP-1 precursor synthesis → processing and maturation → extracellular release were detected by RT-qPCR method. As Figure 8As shown in B-I, ELYP-3 intervention can significantly 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 (trpm5, glp1r, t1r2 / 3) in the ileum of T2DM mice, showing a dose effect. The above results indicate that ELYP-3 activates the GLP-1 receptor in T2DM mice, which not only effectively increases the intracellular synthesis of GLP-1, but also highly promotes the release of GLP-1 from intracellular to extracellular

[0079] Example 5 Improvement effect of Huangdacha oligosaccharide fragments on blood glucose, blood lipids and insulin resistance in T2DM mice

[0080] During the experiment, the fasting blood glucose of T2DM mice was monitored weekly using a Roche blood glucose meter. After the experiment, blood lipid indicators such as total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDLc) and high-density lipoprotein cholesterol (HDL-c) in the plasma of each group of T2DM mice were detected using a biochemical kit, and the fasting insulin of T2DM mice was measured using 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]. To explore the improvement effect of the application examples of the present invention on blood glucose, blood lipids and insulin resistance in T2DM mice.

[0081] As Figure 9 As shown in A-B, compared with the ND group, the fasting blood glucose and fasting insulin levels of the T2DM model group mice were significantly increased. This indicates 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 an auxiliary hypoglycemic effect. Further, by calculating the insulin resistance index (HOMA-IR) and β-cell function index of each group of mice, the results are as Figure 9 As shown in C-D, after ELYP-3 intervention, the HOMA-IR of T2DM mice can be significantly reduced and the β-cell function index can be increased, indicating that ELYP-3 can significantly improve the insulin resistance of T2DM mice and enhance insulin sensitivity.

[0082] By measuring the total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) in the serum of T2DM mice, the blood lipid levels of the mice were characterized. As Figure 9As shown in E-H, intervention with different doses of ELYP-3 decreased 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 the effect of assisting in reducing blood lipid levels.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing yellow tea oligosaccharide fragment ELYP, characterized in that: The following steps are involved: S1: dissolving yellow tea polysaccharide in a buffer solution for enzymatic hydrolysis; S2: adding glycosidase to carry out enzymolysis reaction; S3: terminating the enzymatic hydrolysis reaction, centrifuging the reaction solution, desalting, membrane separation, and freeze-drying to obtain different oligosaccharide fragments; S4: Keep M W The oligosaccharide fragment components between 0.5KDa and 5KDa are obtained.

2. The preparation method according to claim 1, characterized in that: The buffer solution in step S1 is a 50 mM acetic acid-sodium acetate buffer solution (pH 4.0); more preferably, the yellow tea polysaccharide is dissolved in the sodium acetate buffer solution to a final concentration of 0.5 to 3 mg / mL.

3. The preparation method according to claim 1, characterized in that: The glycosidase described in step S2 is endogalacturonidase, the addition amount is 150-250 U / mg, the enzymolysis temperature is 40-60° C., and the enzymolysis time is 10-14 h.

4. The preparation method according to claim 1, characterized in that: The centrifugation in step S3 is carried out under the conditions of 5000-6000 g for 25-35 min, more preferably 5700 g for 30 min.

5. The preparation method according to claim 1, characterized in that: In step S3, the desalting process is as follows: taking the supernatant after centrifugation, passing it through a 500Da ultrafiltration membrane for desalting, and collecting the molecular weight (M W )>500Da components; the membrane separation is specifically, using 1KDa, 3KDa, 5KDa ultrafiltration membrane packages (Minimate TM TFF Capsule, specifically: take the desalted components, pass them through 1KDa, 3KDa, and 5KDa membrane packages in sequence, collect the membrane retentate and permeate, and obtain M W Oligosaccharide components ranging from 0.5KDa to 1KDa, 1 to 3KDa, and 3KDa to 5KDa.

6. The preparation method according to claim 1, characterized in that: In step S4, M is retained W Oligosaccharide fragment components between 3KDa and 5KDa.

7. The yellow tea oligosaccharide fragment ELYP obtained by the preparation method according to any one of claims 1 to 6.

8. Any use of the yellow tea oligosaccharide fragment ELYP according to claim 7 in the following: (1) Use in the preparation of an agent for promoting the synthesis and / or secretion of GLP-1; (2) Use in the preparation of products for the prevention and / or treatment of hyperlipidemia; (3) Use in the preparation of products for preventing and / or treating obesity; The product includes one or more of food, medicine, and health care products.

9. A drug for preventing and treating hyperlipidemia and / or obesity, characterized in that: The active ingredient comprises the yellow tea oligosaccharide fragment ELYP as claimed in claim 7.

10. The drug according to claim 9, characterized in that The drug is prepared into pharmaceutically acceptable dosage forms, such as tablets, pills, ointments, oral liquids or granules.

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