Enteromorpha oligosaccharide and its preparation method and application

Abstract

The invention discloses a Enteromorpha oligosaccharide and its preparation method and application. The Enteromorpha oligosaccharide is prepared by degradation of Enteromorpha polysaccharide, has a degree of polymerization of 2-10, contains rhamnose, glucose, glucuronic acid and xylose, and has a simple preparation method. The present invention verifies for the first time that Enteromorpha oligosaccharide can significantly reduce the fasting blood sugar level of Type 2 diabetic mice, reduce the insulin content and glycosylated serum protein level in serum, improve the blood lipid metabolism ability of Type 2 diabetic mice, improve the glycogen synthesis ability in the liver of mice, improve the liver function abnormality of Type 2 diabetic mice, and Enteromorpha oligosaccharide has high safety, and has good prospects in developing food or medicine with the function of controlling Type 2 diabetes.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and particularly relates to enteromorpha oligosaccharide and a preparation method and application thereof. Background Art

[0002] Enteromorpha belongs to the Chlorophyceae, Ulvales, and Ulvaceae. It is a common seaweed along the coast of my country and has a long history of being used as food and medicine. Traditional Chinese medicine believes that Enteromorpha is sweet and salty, slightly cold, and has the effects of cooling blood, promoting diuresis, clearing away heat and detoxifying. Seaweed contains a variety of nutrients necessary for the human body, such as protein, fat, carbohydrates, vitamins, etc. Among them, polysaccharides have biological activities such as anticoagulation, immunomodulation, lowering blood sugar, lowering blood lipids, and antiviral. Therefore, in addition to being eaten directly, seaweed can also be processed and purified for use in the research and development of health foods and medical drugs. Rhamnose sulfate is a sulfated polysaccharide mainly composed of rhamnose. In nature, it mainly exists in green algae such as Enteromorpha, reef film, and Ulva.

[0003] Sulfated rhamnan in Enteromorpha is composed of highly sulfated rhamnose molecules and various highly branched polysaccharides, including xylose, glucose, galactose and glucuronic acid. Although the study of the structure of Enteromorpha polysaccharides and oligosaccharides began very early, there are few studies on the fine structure. Ray extracted and separated and purified a water-soluble sulfated polysaccharide from Enteromorpha flatulosa using oxalate. The structural analysis results of the polysaccharide showed that it was mainly composed of (1→4)-Rha, (1→2,4)-Rha, (1→4)-Xyl, (1→4)-GlcA and terminal ClcA, and the sulfate group was located at the C-3 position of (1→4)-Rha and the C-2 position of (1→4)-Xyl. Chattopadhyay studied the sulfated polysaccharides extracted from Enteromorpha indica and found that the polysaccharide is composed of (1→4)-Rha and (1→2,4)-Rha partially sulfated at C-3 and (1→4)-Xyl, (1→4)-Glc, (1→3)-Gal, (1→6)-Gal, (1→4)-GlcA partially sulfated at C-2 and the terminal ClcA. Therefore, the structures of different Enteromorpha polysaccharides have slight differences in connection mode and sulfation position.

[0004] Diabetes is a type of endocrine metabolic disorder with complex etiology, with hyperglycemia as the main sign. The treatment of diabetes mainly includes diet control, exercise therapy, drug therapy, etc. Commonly used oral medications include metformin, gliclazide, acarbose, rosiglitazone or injection of insulin and insulin analogs to control blood sugar levels. Studies in recent years have shown that natural seaweed oligosaccharides have the potential to lower blood sugar and can act through multiple mechanisms. Seaweed oligosaccharides can inhibit the activity of α-glucosidase, thereby slowing down the rise of blood sugar. In addition, seaweed oligosaccharides can promote insulin secretion, improve insulin resistance, and protect pancreatic beta cells through antioxidant and anti-inflammatory effects, thereby lowering blood sugar levels. Enteromorpha is a large green algae widely distributed in my country's seas. It has a high oligosaccharide content, is non-toxic and has no significant side effects, providing a basis for the development of hypoglycemic active drugs or functional foods. Summary of the invention

[0005] The invention provides an enteromorpha oligosaccharide and a preparation method and application thereof. The enteromorpha oligosaccharide has good safety and obvious effect of alleviating type 2 diabetes.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention provides a Enteromorpha oligosaccharide, which has a polymerization degree of 2-10 and contains rhamnose, glucose, glucuronic acid and xylose.

[0008] Furthermore, the sulfate group in the Enteromorpha oligosaccharide is located at →3)-α-L-Fuc p -(1→C-4 and →4)-α-L-Fuc p -(1→C-3 position.

[0009] Further, the rhamnose is →3)-α-L-Rha p -(1→and→4)-α-L-Rha p -(1→; glucose is composed of →4)-β-D-Glc p- (1→ and →6)-β-D-Glc p- (1→composed of.

[0010] Furthermore, the Enteromorpha oligosaccharide is prepared by degrading Enteromorpha polysaccharide; the percentages of rhamnose, glucose, glucuronic acid and xylose in the Enteromorpha polysaccharide are 57.9%, 32.4%, 5.1% and 4.6% respectively.

[0011] Furthermore, the schematic diagram of the structure of the Enteromorpha polysaccharide is shown below:

[0012] ,

[0013] Among them, R1=H or HSO3-, R3=H, Rha p -(1→, Gal or Xylp-(1→.

[0014] The present invention also provides a method for preparing the Enteromorpha oligosaccharide, comprising the following steps:

[0015] (1) 95% ethanol was added to Enteromorpha powder at a solid-liquid ratio of 1:5 (W / V), and the mixture was heated at 80°C for 4 h for defatting. The Enteromorpha precipitate was collected by centrifugation and dried at 55°C.

[0016] (2) adding distilled water to the dried Enteromorpha precipitate in step (1) at a solid-liquid ratio of 1:10 (W / V), stirring continuously for 4 h at room temperature, and collecting the supernatant by centrifugation;

[0017] (3) Concentrating the supernatant in step (2) using a rotary evaporator, and then desalting the supernatant using a 3500 Da dialysis bag to obtain a concentrated solution;

[0018] (4) adding 95% ethanol to the concentrated solution of step (3) at a solid-liquid ratio of 1:4 (V / V) for alcohol precipitation, standing at 4°C overnight, and collecting the precipitate by centrifugation;

[0019] (5) decolorizing and dehydrating the precipitate in step (4) using acetone and ethanol, and drying at 55° C. to obtain a crude product of Enteromorpha polysaccharide;

[0020] (6) Using a Q-Sepharose Fast Flow anion exchange column to separate and purify Enteromorpha polysaccharide to obtain Enteromorpha polysaccharide;

[0021] (7) 0.1 mol / L H2SO4 was added to the Enteromorpha polysaccharide to a final concentration of 10 mg / mL, and the mixture was degraded by heating in a water bath at 80°C for 5 h. A saturated barium hydroxide solution was added to the degradation solution until no precipitation was produced. The supernatant was collected by centrifugation. The supernatant was dialyzed and desalted with a 500 Da dialysis bag, and then freeze-dried to obtain an oligosaccharide mixture. The oligosaccharide mixture was further purified by a Bio-Gel P4 gel column to obtain Enteromorpha oligosaccharides.

[0022] Furthermore, in step (6), the length of the Q-Sepharose Fast Flow anion exchange column is 40 cm, the diameter is 3.5 cm, and the elution conditions are 0 mol / L, 0.5 mol / L, 1 mol / L and 2 mol / L NaCl solutions.

[0023] Furthermore, in step (7), the length of the Bio Gel P4 gel column is 100 cm, the diameter is 1.5 cm, the elution condition is 0.2 mol / L NH4HCO3 solution, and the flow rate is 0.3 mL / min.

[0024] The present invention also provides the use of the Enteromorpha oligosaccharide in preparing a medicine for improving diabetes.

[0025] Furthermore, the diabetes is type 2 diabetes.

[0026] Furthermore, the effective dose of the Enteromorpha oligosaccharide for improving the function of type 2 diabetes is 50 mg / kg-200 mg / kg.

[0027] Optimally, the best effective dose of the Enteromorpha oligosaccharide for improving the function of type 2 diabetes is 150 mg / kg.

[0028] Furthermore, the Enteromorpha oligosaccharide can significantly reduce the fasting blood sugar and serum insulin levels of type 2 diabetic mice, improve insulin resistance, and effectively control the blood sugar levels of type 2 diabetic mice.

[0029] Furthermore, the Enteromorpha oligosaccharide can improve the lipid metabolism capacity of type 2 diabetic mice by reducing the serum glycoprotein levels, triglycerides, total cholesterol, and low-density lipoprotein of type 2 diabetic mice and increasing the content of high-density lipoprotein.

[0030] Furthermore, the Enteromorpha oligosaccharide can increase the accumulation of glycogen in the liver of type 2 diabetic mice and enhance the liver's glycogen synthesis capacity.

[0031] Furthermore, the Enteromorpha oligosaccharide can improve the antioxidant capacity of the body of type 2 diabetic mice and protect the function of pancreatic β cells.

[0032] Furthermore, the Enteromorpha oligosaccharide can protect the function of the pancreatic islets by alleviating the damage to the pancreatic islet tissue in type 2 diabetic mice.

[0033] Compared with the prior art, the beneficial effects and advantages of the present invention are:

[0034] The oligosaccharide raw material obtained by the present invention is the green alga Enteromorpha, which has abundant sources and is safe to use. The polysaccharide EP prepared by the present invention using Enteromorpha belongs to sulfated rhamnose, and the monosaccharide composition of EP is rhamnose, glucose, glucuronic acid and a small amount of xylose, with percentages of 57.9%, 32.4%, 5.1% and 4.6%. The rhamnose in EP is mainly composed of →3)-α-L-Rha p -(1→and→4)-α-L-Rha p -(1→, glucose is composed of →4)-β-D-Glc p-(1→ and →6)-β-D-Glc p- (1→composition, sulfate groups are mainly located at→3)-α-L-Rha p -(1→C-4 and →4)-α-L-Rha p -(1→C-3 position.

[0035] The present invention characterizes the structural characteristics of Enteromorpha oligosaccharide EPs, and focuses on exploring the active effects and mechanisms of EPs in improving type 2 diabetes in mice. The active effects of EPs in improving type 2 diabetes in mice are obvious, and can not only reduce fasting blood sugar and serum insulin levels, but also improve insulin resistance, enhance lipid metabolism capacity, and protect pancreatic islet function. The present invention verifies that Enteromorpha oligosaccharide EPs can be used as a natural product to alleviate type 2 diabetes, by improving the antioxidant capacity of the body of type 2 diabetic mice, reducing pancreatic islet tissue damage, and protecting pancreatic islet function. Therefore, EPs has great potential to develop into drugs and functional foods for treating and improving type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A This is the separation diagram of Enteromorpha polysaccharide EP Q Sepharose Fast Flow;

[0037] Figure 1B The monosaccharide composition analysis of Enteromorpha polysaccharide EP;

[0038] Figure 1C This is the infrared spectrum of Enteromorpha polysaccharide EP;

[0039] Figure 2A This is the methylation GC-MS total ion current diagram of Enteromorpha polysaccharide EP;

[0040] Figure 2B Enteromorpha polysaccharide EP 1 H spectrum;

[0041] Figure 2C Enteromorpha polysaccharide EP 13 C spectrum;

[0042] Figure 2D This is a schematic diagram of the structure of Enteromorpha polysaccharide EP;

[0043] Figure 3A The figure shows the degree of polymerization and composition of Enteromorpha oligosaccharide EPs;

[0044] Figure 3B It is the secondary mass spectrum of Enteromorpha oligosaccharide EPs with a mass-to-charge ratio (m / z) of 243;

[0045] Figure 3C The mass-to-charge ratio (m / z) of Enteromorpha oligosaccharide EPs is 389.

[0046] Figure 3D The mass-to-charge ratio (m / z) of Enteromorpha oligosaccharide EPs is 419.

[0047] Figure 4 The effects of EPs on blood glucose, serum insulin and insulin resistance in type 2 diabetic mice; A is the effect of EPs on fasting blood glucose in type 2 diabetic mice, B is the effect of EPs on insulin content in type 2 diabetic mice, and C is the effect of EPs on HOMA-IR in type 2 diabetic mice;

[0048] Figure 5 A is the effect of EPs on glucose and lipid metabolism in type 2 diabetic mice; A is the effect of EPs on GSP in type 2 diabetic mice; B is the effect of EPs on TC content in type 2 diabetic mice; C is the effect of EPs on TG content in the liver of type 2 diabetic mice; D is the effect of EPs on LDL-C in the liver of type 2 diabetic mice; E is the effect of EPs on HDL-C in the liver of type 2 diabetic mice; F is the effect of EPs on Gly in the liver of type 2 diabetic mice;

[0049] Figure 6 A is the effect of EPs on the antioxidant capacity of type 2 diabetic mice; A is the effect of EPs on the total antioxidant capacity of type 2 diabetic mice; B is the effect of EPs on SOD of type 2 diabetic mice; C is the effect of EPs on MDA of type 2 diabetic mice; D is the effect of EPs on GPX of type 2 diabetic mice;

[0050] Figure 7 The effect of EPs on the pathological morphology of pancreatic islets in type 2 diabetic mice. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.

[0052] Example 1: Extraction, separation and purification of Enteromorpha polysaccharide

[0053] The extraction, separation and purification of Enteromorpha polysaccharide in the present invention comprises the following steps:

[0054] (1) 95% ethanol was added to Enteromorpha powder (500 g) at a solid-liquid ratio of 1:5 (W / V), and the mixture was heated at 80°C for 4 h for defatting. The Enteromorpha precipitate was collected by centrifugation and dried at 55°C.

[0055] (2) adding distilled water to the Enteromorpha precipitate obtained in step (1) at a solid-liquid ratio of 1:10 (W / V), stirring continuously for 4 h at room temperature, and collecting the supernatant by centrifugation;

[0056] (3) Concentrating the supernatant in step (2) using a rotary evaporator, and then desalting using a 3500 Da dialysis bag;

[0057] (4) adding 95% ethanol to the desalted concentrate in step (3) at a material-liquid ratio of 1:4 (V / V) for alcohol precipitation, standing at 4°C overnight, and collecting the precipitate by centrifugation;

[0058] (5) decolorizing and dehydrating the precipitate in step (4) using acetone and anhydrous ethanol, and drying at 55° C. to obtain a crude product of Enteromorpha polysaccharide;

[0059] (6) Enteromorpha polysaccharide was separated and purified using Q-Sepharose Fast Flow anion exchange column and Bio-Gel P4 gel column to obtain Enteromorpha polysaccharide EP.

[0060] Example 2: Basic structural characteristics of Enteromorpha polysaccharide (EP)

[0061] 1. Separation and purification of Enteromorpha polysaccharides by ion exchange chromatography

[0062] The Enteromorpha polysaccharide prepared in Example 1 was graded and eluted using a Q-Sepharose Fast Flow strong anion exchange column with 0, 0.5, 1.0, 1.5, 2.0 and 4.0 mol / L NaCl, respectively. The eluted components were collected by an automatic collector, and the sugar content was detected by the sulfuric acid phenol method and the elution volume-absorbance curve was drawn. According to the elution curve, the appropriate NaCl elution concentration was determined, a large amount of preparation was performed, the eluate was collected and combined, the eluate was concentrated and dialyzed through a 3.5 kDa dialysis bag, and lyophilized to obtain a polysaccharide with uniform charge density.

[0063] like Figure 1A As shown, the fractions eluted with 1.0 mol / L NaCl were collected, concentrated, and freeze-dried and named EP.

[0064] 2. Determination of monosaccharide composition of EP by HPLC

[0065] (1) Accurately weigh 2 mg of EP prepared in Example 1, add 400 μL of 2 mol / L trifluoroacetic acid, and degrade under sealed conditions at 105°C for 6 h. After degradation, remove excess trifluoroacetic acid.

[0066] (2) Dissolve the EP degradation products and monosaccharide standards in 100 μL of distilled water, add 100 μL of 0.3 mol / L NaOH and 120 μL of 0.5 mol / L PMP methanol solution and place in a 70°C water bath for 60 min. After cooling, add HCl solution for neutralization, extract with dichloromethane three times, and filter through a 0.22 μm filter membrane for later use.

[0067] (3) The monosaccharide composition of EP was determined by HPLC. The chromatographic conditions were as follows: Eclipse XDB-C18 (5 μm, 4.6 μm×25.0 cm) column; mobile phase was acetonitrile: phosphate buffer (pH 6.7) = 17:83 (v / v); injection volume was 10 μL; column temperature was 35°C; UV detector (254 nm); flow rate was 1.0 mL / min.

[0068] The results are as follows Figure 1B As shown, the monosaccharide composition of EP is rhamnose, glucose, glucuronic acid and a small amount of xylose, with percentages of 57.9%, 32.4%, 5.1% and 4.6%, indicating that EP is a rhamnose containing part of glucuronic acid, xylose and glucose.

[0069] 3. Determination of infrared spectrum of EP by KBr tablet method

[0070] The polysaccharide sample EP was dried under reduced pressure with P2O5 at 50 °C for 48 h. About 1 mg of the sample was ground with an appropriate amount of dry potassium bromide powder and pressed into a transparent sheet for infrared spectroscopy. The infrared spectrometer measurement parameters were as follows: Nicolet Nexus 470 infrared spectrometer; background scan number 32 times; scanning range: 400~4000 cm -1 ; Resolution: 4.0 cm -1 ; Detector: DTGS.

[0071] The results are as follows Figure 1C As shown in the FT-IR spectrum of EP, at 3419 cm −1 The characteristic absorption band at 1608 cm −1 The absorption signal at 1345 cm is attributed to the bending vibration of -OH. −1 The peak at 1056 cm corresponds to the absorption peak of CH bond angular vibration. −1 The peak at 1258 cm-1 is from the stretching vibration of CO. Several peaks corresponding to sulfate groups were also observed. −1 and 814 cm -1 The stretching vibration at is due to S=O. The results of infrared spectroscopy showed that EP is a highly sulfated rhamnose.

[0072] 4. Analysis of the connection mode of polysaccharide EP by methylation reaction and temperament

[0073] The modified Hakomori method was used to carry out methylation reaction on polysaccharide EP. The methylation product was completely acid hydrolyzed, reduced and acetylated and then analyzed by GC-MS. The MS spectrum was compared with the spectrum of the standard to determine the connection mode of the sugar group. Figure 2A This is the methylation GC-MS total ion current diagram of Enteromorpha polysaccharide EP. The MS spectrum in the figure is compared with the spectrum of the standard to determine the connection mode of the sugar group.

[0074] The methylation GC-MS results before and after desulfurization are shown in Table 1. EP and its desulfurization product Ds-EP are mainly composed of four types of rhamnose and two types of glucose. p -(1→)-Glc has the highest content, followed by [→4)-Glc p -(1→],[→3)-Rha p -(1→],[Rha p -(1→] has the lowest content; the content of [→3)-Rha p -(1→] mainly, [→3,4)-Rha p -(1→] has the lowest content. By comparing the ratios of sugar groups with different connection modes before and after desulfurization, it can be seen that after desulfurization, [→3)-Rha p -(1→],[→4)-Rha p -(1→] content increased, [→3,4)-Rha p The -(1→] connection disappears, indicating that the sulfate group is located at [→3)-Rha p -(1→] at C-4 and a small amount at [→4)-Rha p -(1→] at C-3, and mainly at [→3)-Rha p -(1→] at C-4, the ratio of the two is about 3.0:1. Therefore, EP mainly contains [→3)-Rha p -(1→],[→4)-Rha p -(1→],[→3,4)-Rha p -(1→] and [→4)-Glc p -(1→] and [→6)-Glc p -(1→], the sulfate group is located at the C-3 position of 1,4-linked rhamnose and at the C-4 position of 1,3-linked rhamnose.

[0075] Table 1 Methylation analysis results of polysaccharide EP and its desulfurized component (Ds-EP)

[0076]

[0077] a Not Detected

[0078] 5. Polysaccharide NMR spectroscopy analysis

[0079] The polysaccharide samples were dissolved in D2O, freeze-dried, centrifuged and placed in NMR tubes. The one-dimensional ( 1 H-NMR, 13 C-NMR) nuclear magnetic resonance spectroscopy. Deuterated acetone was added as an internal standard. 1 The chemical shift of H was determined to be 2.225 ppm, 13 The chemical shift of C was determined to be 31.07 ppm and NMR spectroscopy was performed.

[0080] EP 1 H NMR Figure 2B As shown, the CH3 proton signals of rhamnose C-6 are around δ 1.35 ppm and δ 1.37 ppm; the signals of H-2-H-5 are in the region of δ 3.47-4.40 ppm, and the signals overlap; the signals of H-1 are in the region of δ 4.40-5.40 ppm. 13 C NMR Figure 2C As shown, δ 18.4 ppm is the signal of the methyl carbon at C-6 position of rhamnose, δ 176.2 ppm is the carbon signal of COOH at C-6 position of glucuronic acid, and the signal at δ 98.9-104.7 ppm is the terminal carbon signal. According to the number of anomeric hydrogen and terminal carbon, it can be preliminarily judged that there are 8 connection modes of monosaccharides in EP, which is consistent with the results of methylation analysis.

[0081] Example 3 Preparation of Enteromorpha Oligosaccharides (EPs) and Secondary Mass Spectrometry Analysis

[0082] 1. Preparation of Enteromorpha oligosaccharides (EPs)

[0083] Weigh an appropriate amount of Enteromorpha polysaccharide, add 0.1 M H2SO4 to a final concentration of 10 mg / mL, and degrade it by heating in a water bath at 80 °C for 5 hours; after the reaction is completed, add saturated barium hydroxide solution until no precipitation is produced, centrifuge, and take the supernatant; the supernatant is dialyzed and desalted with a 500 Da dialysis bag, and then freeze-dried to obtain an oligosaccharide mixture. After the oligosaccharide mixture is further purified by a Bio-Gel P4 gel column, Enteromorpha oligosaccharides are obtained, named EPs.

[0084] 2. Determination of polymerization degree and content of Enteromorpha oligosaccharide EPs

[0085] The oligosaccharide polymerization degree and content were determined by liquid chromatography-mass spectrometry (LC-MS).

[0086] (1) HPLC conditions: Agilent 1290 ultra-high pressure liquid chromatograph coupled with Thermo LTQ XL mass spectrometer; chromatographic column model: Phenomenex Luna 3 μm HILIC 200A (150×2.00 mm); spray voltage: 4.2 kV; ion source: ESI; detector: mass spectrometer detector (MSD); mass scan range: 300–2000.

[0087] (2) First, the mass spectrometry data was deconvoluted using DeconTools software, and then the oligosaccharide information was matched using Glycresoft software. Finally, the relative content was determined by the peak area of ​​the mass spectrometry ion flow, and the oligosaccharide connection mode was determined by the secondary mass spectrometry data.

[0088] The results are as follows Figure 3A As shown in the figure, the oligosaccharide fragments in EPs are relatively more tetrasaccharide, trisaccharide and disaccharide, and the proportions of these three oligosaccharide fragments are 43.1%, 10.4% and 22.8% respectively. Among them, there are two tetrasaccharides, namely [Xyl+2Rha+GlcA+2SO3 - ] accounted for 31.4%, [Xyl+2Rha+Glc+GlcA+SO3 - ] accounted for 11.7%, and disaccharides also included two types, namely [Rha+GlcA+SO3 - ] and [Xyl+Rha+SO3 - ], accounting for 10.0% and 7.8% respectively. Example 4

[0089] By analyzing the electrospray collision induced fragmentation secondary mass spectrometry (ESI-CID-MS / MS) in negative ion mode, the secondary mass spectrometry performs specific fragmentation on ions with a specific mass-to-charge ratio based on the primary mass spectrometry, thereby obtaining rich structural information and analyzing the structure of the oligosaccharide by attributing the broken fragments. In the secondary mass spectrometry of oligosaccharides, the breakage occurs not only at the glycosidic bond, but also inside the sugar ring, and may also break in the form of losing small molecules.

[0090] The mass-to-charge ratio (m / z) 243 is monosulfated rhamnose, and its ESI-CID-MS 2 Spectrum Figure 3B As shown, m / z97 is caused by the detachment of the sulfate group. According to the results of NMR and methylation analysis, it is speculated that the sulfate group is located at the C-3 position of rhamnose, and m / z183 is caused by the cleavage of the ring. 0,2 A is produced;

[0091] The mass-to-charge ratio (m / z) 389 is monosulfated rhamnobiose, such as Figure 3CAs shown in the figure, the fragment ions m / z 225 and m / z 243 were generated by the cleavage of the rings and were assigned to B1 and C1, respectively. The disaccharide structure was inferred to be [α-L-Rha p -(1→4)-α-L-Rha p 3S];

[0092] The mass-to-charge ratio (m / z) 419 is monosulfated glucuronyl rhamnobiose, such as Figure 3D As shown, the fragment ions m / z193, m / z 225 and m / z 243 are all generated by inter-ring cleavage and are labeled C1, Z1 and Y1, respectively. Since the peak of m / z 243 is the largest, that is, monosulfated rhamnose, it is determined that the sulfate substitution occurs at the position of rhamnose. m / z 193 is the peak of glucuronic acid, so it is determined that the disaccharide is composed of 1,2 or 1,4 linked glycosidic bonds, in which glucuronic acid is located at the reducing end. The structure is as follows: [β-D-GlcA p -(1→2)-α-L-Rha p 3S] or [β-D-GlcA p -(1→4)-α-L-Rha p 3S]. Example 5

[0093] After 1 week of adaptive feeding, SPF C57 male mice were randomly divided into a normal control group (n=15) and a modeling group (n=65) according to their body weight. The normal group and the modeling group were fed with a basic diet and a high-fat diet for 4 weeks, respectively. After four weeks of high-fat feeding, the mice were fasted for 12 hours. The modeling group was intraperitoneally injected with 95 mg / kg·BW streptozotocin (STZ) for three consecutive days, and the normal group was injected with the same volume of citric acid buffer (pH=4). Blood was collected from the tail vein on the 7th day after modeling (the mice in each group fasted for 8 hours before blood collection and drank normal water), and the fasting blood glucose level was tested. Mice with a blood glucose level exceeding 11.1 mmol / L and accompanied by the "three more and one less" characteristics were considered to be successfully modeled. For mice that did not form a model, STZ was continued to be supplemented. Next, the mice were randomly divided into different groups for oligosaccharide function verification experiments. The experimental mice were gavaged for 30 consecutive days, and the relevant indicators were detected.

[0094] 1. Serum biochemical index determination: serum samples frozen at -20℃ were taken, thawed, and determined according to the method in the instructions of the glycosylated serum protein (GSP) kit; insulin (INS) content was determined according to the method in the instructions of the ELISA kit and the insulin resistance index (HOMA-IR) of mice was calculated according to the following formula:

[0095] HOMA-IR=[FBG(mmol / L)xINS(mIU / L)] / 22.5

[0096] Lipid metabolism index determination The concentrations of lipid metabolism markers such as TG, TC, LDL-C, and HDL-C in mouse serum were determined according to the kit instructions.

[0097] The results are as follows Figure 4 As shown in Figures AC, after EPs intervention, the fasting blood glucose level of the MG group was significantly different from that of the EPs-L, EPs-M, and EPs-H intervention groups (P<0.05). The INS content and HOMA-IR in the serum of the mice in the MG group were significantly higher than those in the NG group (P<0.05), and the symptoms of insulin resistance appeared. After 30 days of intervention with Enteromorpha oligosaccharides, the INS content and HOMA-IR in the serum of the mice in the EPs-L, EPs-M, EPs-H, and PG groups were significantly reduced compared with the MG group (P<0.05). Therefore, Enteromorpha oligosaccharides can reduce the FBG and serum INS content of type 2 diabetic mice, thereby effectively improving the symptoms of islet resistance (reduced HOMA-IR) in type 2 diabetic mice.

[0098] Depend on Figure 5 As shown in A, the GSP content in the serum of mice in the MG group was significantly higher than that in the NG group (P<0.05); after EPs intervention, the GSP content in the EPs-L group and the EPs-M group was significantly lower than that in the MG group (P<0.05); the GSP content in the PG group was also significantly reduced (P<0.05). Therefore, Enteromorpha oligosaccharides can reduce the GSP content in the serum of type 2 diabetic mice, delay the rate of non-enzymatic glycosylation reaction, and improve glucose metabolism disorders in diabetic mice.

[0099] Depend on Figure 5 From the BE, compared with the NG group, the TC, TG, and LDL-C levels of mice in the MG group were significantly increased (P<0.05), and the HDL-C content was significantly decreased (P<0.05), indicating that type 2 diabetic mice had lipid metabolism disorders. After 30 days of EPs intervention, the TG, TC, and LDL-C levels in the serum of mice in the EPs-L, EPs-M, EPs-H, and PG groups were significantly decreased (P<0.05); compared with the MG group, the HDL-C content in the EPs-M and EPs-H groups was significantly increased (P<0.05). This shows that Enteromorpha oligosaccharides can improve the lipid metabolism ability of type 2 diabetic mice to a certain extent.

[0100] 2. Determination of liver glycogen content Take liver tissue samples frozen at -20°C, thaw, roughly cut out 10 mg, add alkali solution at a ratio of 1:3 (sample weight mg: alkali solution volume μL), react in a boiling water bath for 20 minutes, and obtain glycogen hydrolyzate after cooling, and determine the liver glycogen content of mice liver.

[0101] The results are as follows Figure 5 As shown in F, the liver glycogen content of mice in the MG group was significantly lower than that in the NG group (P<0.05). After 30 days of EPs intervention, the liver glycogen accumulation of mice in the EPs-M, EPs-H, and PG groups was significantly increased (P<0.05). Glycogen is a highly branched glucose polymer and also the energy storage form of glucose in the liver. It is the main control factor of blood sugar and plays a key role in maintaining blood sugar homeostasis. As an important organ of glucose metabolism in the body, the synthesis and decomposition of liver glycogen can reflect the body's absorption and utilization of glucose. If the liver glycogen content is low, it means that the ability to synthesize liver glycogen is weakened and gluconeogenesis is increased. Therefore, Enteromorpha oligosaccharide EPs can improve the ability to synthesize liver glycogen in mouse liver.

[0102] 3. Take serum samples frozen at -20℃, thaw them, and measure the T-AOC, MDA content and antioxidant enzyme (SOD, GSH-PX) activities in mouse serum.

[0103] The results are as follows Figure 6 As shown in the results, the MDA content in the serum of the mice in the MG group was significantly higher than that in the NG group (P<0.05), and the activities of T-AOC, SOD and GPX were significantly lower than those in the NG group (P<0.05). After 30 days of EPs intervention, compared with the MG group, the T-AOC in the serum of the mice in the EPs-L, EPs-M, EPs-H and PG groups was significantly increased (P<0.05); the MDA content in the serum of the mice in the EPs-L, EPs-M, EPs-H and PG groups was significantly lower than that in the MG group (P<0.05); the activities of the antioxidant enzyme SOD in the serum of the mice in the EPs-L, EPs-M, EPs-H and PG groups were significantly higher than those in the MG group (P<0.05). The reduction of the body's antioxidant level is closely related to diabetes. When the blood glucose concentration in the body is too high, it will stimulate tissue cells to produce a large amount of reactive oxygen species (ROS), which may damage the function of pancreatic B cells and cause insufficient insulin secretion. This shows that Enteromorpha oligosaccharides help improve the antioxidant capacity of type 2 diabetic mice and play a positive role in the prevention and treatment of type 2 diabetes.

[0104] 4. Immediately place the removed pancreatic islet tissue in formalin fixative (4% concentration, fixed at room temperature), dehydrate the fixed tissue with high ethanol for several hours, embed and keep warm in liquid paraffin after xylene treatment, and slice the embedded paraffin tissue. After HE staining, set the light microscope magnification to 400x, take pictures of the colored tissue sections and observe their morphological characteristics.

[0105] The results are as follows Figure 7 As shown in the figure, through the analysis of pancreatic pathological structure, H&E staining showed that the pancreatic acini and islets in the NG group were intact, the pancreatic islet tissue in the MG group was dispersed, the volume was significantly reduced, most of the islet cells were atrophied, the number of cells was reduced, the cytoplasm was significantly reduced, the nuclei were condensed on a large scale, and lymphocytes were widely infiltrated around the islets. The number of islets and β cells in the EPs-L, EPs-M, and EPs-H intervention groups increased significantly, the morphological structure was basically normal, the cytoplasm was uniform and large in number, the nucleoli were clear, and a small amount of lymphocyte infiltration was occasionally seen. In summary, Enteromorpha oligosaccharides have an alleviating effect on pancreatic damage in mice with type 2 diabetes.

[0106] 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, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein 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 claimed to be protected by the present invention.

Claims

1. A Enteromorpha oligosaccharide, characterized in that: The degree of polymerization of the Enteromorpha oligosaccharide is 2-10, which contains rhamnose, glucose, glucuronic acid and xylose, and is prepared by degrading Enteromorpha polysaccharide; the percentages of rhamnose, glucose, glucuronic acid and xylose in the Enteromorpha polysaccharide are 57.9%, 32.4%, 5.1% and 4.6% respectively; the structural schematic diagram of the Enteromorpha polysaccharide is as follows: ， Among them, R1=H or HSO3-, R3=H, Rhap-(1→, Gal or Xylp-(1→.

2. The Enteromorpha oligosaccharide according to claim 1, characterized in that: The sulfate groups in the Enteromorpha oligosaccharide are located at the C-4 position of →3)-α-L-Fucp-(1→) and the C-3 position of →4)-α-L-Fucp-(1→.

3. The Enteromorpha oligosaccharide according to claim 1, characterized in that: The rhamnose is composed of →3)-α-L-Rhap-(1→ and →4)-α-L-Rhap-(1→); and the glucose is composed of →4)-β-D-Glcp-(1→ and →6)-β-D-Glcp-(1→.

4. The method for preparing enteromorpha oligosaccharide according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) adding ethanol to Enteromorpha powder, heating to defat, collecting Enteromorpha precipitate by centrifugation and drying; (2) adding distilled water to the Enteromorpha precipitate dried in step (1), stirring evenly, and then centrifuging to collect the supernatant; (3) concentrating the supernatant of step (2), and then performing a desalting treatment to obtain a concentrated solution; (4) adding ethanol to the concentrated solution of step (3) for alcohol precipitation, standing overnight, and collecting the precipitate by centrifugation; (5) decolorizing and dehydrating the precipitate of step (4), and drying to obtain a crude product of Enteromorpha polysaccharide; (6) separating and purifying the crude enteromorpha polysaccharide obtained in step (5) to obtain enteromorpha polysaccharide; The Enteromorpha polysaccharide was graded and eluted with 0, 0.5, 1.0, 1.5, 2.0 and 4.0 mol / L NaCl respectively using Q-Sepharose Fast Flow strong anion exchange column, and the fraction eluted with 1.0 mol / L NaCl was collected, concentrated and freeze-dried to obtain Enteromorpha polysaccharide. (7) degrading, separating and purifying the Enteromorpha polysaccharide obtained in step (6); The specific degradation steps are as follows: adding 0.1 mol / L H2SO4 to the Enteromorpha polysaccharide, degrading it under water bath heating conditions, adding saturated barium hydroxide solution to the degradation solution until no precipitation is produced, centrifuging to obtain the supernatant, dialyzing the supernatant with a dialysis bag, desalting it, and then further purifying it using a gel column to obtain Enteromorpha oligosaccharides.

5. The method for preparing enteromorpha oligosaccharide according to claim 4, characterized in that: In the steps (1) and (4), the ethanol is 95% ethanol; the solid-liquid ratio of the Enteromorpha powder to ethanol is 1:5-10 W / V; and the solid-liquid ratio of the concentrated solution to ethanol is 1:4-8 V / V.

6. The method for preparing enteromorpha oligosaccharide according to claim 4, characterized in that: In the step (2), the solid-liquid ratio of enteromorpha precipitate to distilled water is 1:5-10 W / V.

7. Use of the Enteromorpha oligosaccharide according to any one of claims 1 to 3 in the preparation of a drug for improving diabetes, characterized in that: The diabetes is type 2 diabetes.

8. The use according to claim 7, characterized in that: The effective dosage of the Enteromorpha oligosaccharide for improving the function of type 2 diabetes is 50 mg / kg-200 mg / kg.

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