阅读说明：本技术 β-1,4型葡萄糖醛酸/甘露糖醛酸直链杂合寡糖及其制备方法和应用 (Beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide and preparation method and application thereof ) 是由 张真庆 吴芳霞 李笃信 易琳 欧阳艺兰 于 2021-08-31 设计创作，主要内容包括：本发明公开了β-1,4型葡萄糖醛酸/甘露糖醛酸直链杂合寡糖,其具有以下通式结构：该杂合寡糖具有分子量低、水溶性好、生物利用度高、易于质量控制等优点,具有显著的治疗缺血性脑疾病的活性。其制备工艺为：称取魔芋葡甘聚糖,依次加入4-乙酰氨基-2,2,6,6-四甲基哌啶-1-氧自由基、亚氯酸钠和酸性缓冲溶液搅拌均匀；再加入次氯酸钠溶液进行氧化,之后加入有机试剂终止反应即得β-1,4型葡萄糖醛酸/甘露糖醛酸直链杂合多糖；将上述杂合多糖水解、除盐、冷冻干燥即制备得到直链杂合寡糖。其反应条件温和,合成工艺简单、得率高,制备原料易得,成本较低,易于产业化。(The invention discloses a beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide, which has the following general structure: the hybrid oligosaccharide has the advantages of low molecular weight, good water solubility, high bioavailability, easy quality control and the like, and has remarkable activity of treating ischemic brain diseases. The preparation process comprises the following steps: weighing konjac glucomannan, sequentially adding 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxylUniformly stirring sodium chlorite, sodium chlorite and acid buffer solution; adding sodium hypochlorite solution for oxidation, and adding an organic reagent to terminate the reaction to obtain beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide; hydrolyzing the hybrid polysaccharide, desalting, and freeze drying to obtain straight-chain hybrid oligosaccharide. The method has the advantages of mild reaction conditions, simple synthesis process, high yield, easily obtained preparation raw materials, low cost and easy industrialization.)

1. The beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide is characterized by comprising the following general structure:

wherein the content of the first and second substances,

m is independently selected from any one of hydrogen and cation;

m₁and m₂One or more integers selected from 0 to 20;

n₁is one or more integers selected from 0 to 5.

2. The linear hybrid oligosaccharide of β -1,4 glucuronic acid/mannuronic acid according to claim 1, characterized in that: the cation is selected from any one or more of lithium ion, sodium ion, potassium ion, beryllium ion, magnesium ion, calcium ion, iron ion, ferrous ion, zinc ion, selenium ion, vanadium ion, tin ion or strontium ion.

3. The linear hybrid oligosaccharide of β -1,4 glucuronic acid/mannuronic acid according to claim 1, characterized in that: the molar ratio of glucuronic acid to mannuronic acid is 1: 2-1: 6.

4. the linear hybrid oligosaccharide of β -1,4 glucuronic acid/mannuronic acid according to claim 1, characterized in that: the percentage of glucuronic acid and mannuronic acid is greater than or equal to 90%.

5. The preparation method of the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide is characterized by comprising the following steps:

1) weighing konjac glucomannan, sequentially adding 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxygen radical, sodium chlorite and sodium acetate/acetic acid buffer solution, and stirring uniformly; adding sodium hypochlorite solution for oxidation reaction, adding organic reagent for terminating the reaction, desalting the obtained sample, removing insoluble substances, rotary steaming for concentration, and freeze drying to obtain beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid polysaccharide;

2) reacting the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide under any acidolysis condition for different time periods to obtain the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide.

6. The method for preparing beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide according to claim 5, characterized in that: in the step 1), the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide comprises the following general structure:

wherein the content of the first and second substances,

m is independently selected from any one of hydrogen and cation;

m₁and m₂One or more integers selected from 100-500;

n₁is one or more integers selected from 0 to 50.

7. The method for preparing beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide according to claim 6, characterized in that: the cation is selected from any one or more of lithium ion, sodium ion, potassium ion, beryllium ion, magnesium ion, calcium ion, iron ion, ferrous ion, zinc ion, selenium ion, vanadium ion, tin ion or strontium ion.

8. The method for preparing beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide according to claim 6, characterized in that: the molar ratio of glucuronic acid to mannuronic acid is 1: 2-1: 6.

9. the method for preparing beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide according to claim 6, characterized in that: the percentage of glucuronic acid and mannuronic acid is greater than or equal to 90%.

10. The use of the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide according to claim 1 in the preparation of a medicament for treating ischemic brain diseases.

Technical Field

The invention belongs to the technical field of medical compounds, and particularly relates to beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide and a preparation method and application thereof.

Background

In recent years, with the improvement of the living standard and the increase of aging degree of people, the morbidity and mortality of cardiovascular and cerebrovascular diseases are on the rise, ischemic stroke accounts for the main part of various brain diseases, and the medicines clinically used for preventing and treating the ischemic stroke at present comprise aspirin and tissue plasminogen activator tPA. Aspirin prevents further thrombus formation by preventing platelet aggregation, and although aspirin is currently used very widely throughout the world, the effect of aspirin in acute stroke is not ideal. The tissue plasminogen activator tPA can dissolve thrombus, dredge blood vessels and reduce brain injury, but the effective treatment time window is only short 4.5h, so that only 3-5% of patients are effectively treated. Therefore, there is a need to develop new drugs for treating ischemic stroke.

Polysaccharide substances have been hindered from developing the drug properties to a certain extent due to their complex structures, and gradually enter the field of scientific researchers with the advantages of definite therapeutic effects and low toxicity along with the improvement of various separation and analysis technical means. If a TEMPO system is used for oxidizing natural polysaccharides such as starch, microcrystalline cellulose and the like to obtain a series of glucuronic acid oligosaccharides, two kinds of uronic acid oligosaccharides have good protection effect on mouse hippocampal neuronal cells under the condition of sugar deficiency and oxygen deficiency, and if a TEMPO system is used for selectively oxidizing konjac glucomannan to obtain a glucuronic acid/mannuronic acid oligosaccharide, but the reducing ends of the three kinds of oligosaccharides are subjected to ring opening in different degrees, so that the quality control is difficult to a certain degree in process amplification.

Therefore, there is a need to develop a beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide, a preparation method and application thereof to solve the problems.

Disclosure of Invention

The invention aims to provide beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide and a preparation method and application thereof.

The invention has a technical scheme that:

the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide comprises the following general structure:

wherein the content of the first and second substances,

m is independently selected from any one of hydrogen and cation;

m₁and m₂One or more integers selected from 0 to 20;

n₁is one or more integers selected from 0 to 5.

Further, the cation is selected from any one or more of lithium ion, sodium ion, potassium ion, beryllium ion, magnesium ion, calcium ion, iron ion, ferrous ion, zinc ion, selenium ion, vanadium ion, tin ion or strontium ion.

Further, the molar ratio of glucuronic acid to mannuronic acid is 1: 2-1: 6.

further, the percentage content of glucuronic acid and mannuronic acid is greater than or equal to 90%.

The other technical scheme of the invention is as follows:

the preparation method of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide comprises the following steps:

1) weighing konjac glucomannan, sequentially adding 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxygen radical, sodium chlorite and sodium acetate/acetic acid buffer solution (pH is 4.7), and stirring; adding sodium hypochlorite solution for oxidation reaction, adding organic reagent for terminating the reaction, desalting the obtained sample, removing insoluble substances, rotary steaming for concentration, and freeze drying to obtain beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid polysaccharide;

2) reacting the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide under any acidolysis condition for different time periods to obtain the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide.

Further, in step 1), the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide comprises the following general structure:

wherein the content of the first and second substances,

m is independently selected from any one of hydrogen and cation;

m₁and m₂One or more integers selected from 100-500;

n₁is one or more integers selected from 0 to 50.

Further, the cation is selected from any one or more of lithium ion, sodium ion, potassium ion, beryllium ion, magnesium ion, calcium ion, iron ion, ferrous ion, zinc ion, selenium ion, vanadium ion, tin ion or strontium ion.

Further, the molar ratio of glucuronic acid to mannuronic acid is 1: 2-1: 6.

further, the percentage content of glucuronic acid and mannuronic acid is greater than or equal to 90%.

The beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide prepared by the method is applied to the preparation of medicines for treating ischemic brain diseases.

The invention provides beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide which is obtained by selecting a 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxygen radical/sodium chlorite/sodium hypochlorite system to selectively oxidize and hydrolyze glucomannan, wherein the reducing end of the oligosaccharide does not generate open loop, and the oligosaccharide is friendly to the subsequent quality control link and has the following specific advantages:

1) the method comprises the following steps of (1) synthesizing beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide shown in a formula (II) by taking konjac glucomannan as a raw material through a 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxygen radical/sodium chlorite/sodium hypochlorite system, and obtaining beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide shown in the formula (I) through acidolysis of the polysaccharide;

2) the preparation method is simple, mild in condition, low in cost, high in purity and yield and easy to industrialize;

3) the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide prepared by the method has a definite structure, and has the advantages of low molecular weight, high bioavailability and simple and easy quality control;

4) the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide obtained by the invention has a remarkable effect of treating ischemic brain diseases, and has a good application prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein the content of the first and second substances,

FIG. 1 is the nuclear magnetic spectrum of beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid polysaccharide, wherein A is beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid polysaccharide¹³C NMR carbon spectrum, B is beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid polysaccharide¹H-¹³C HSQC spectrogram;

FIG. 2 is an ultraviolet spectrum and a mass spectrum at 210nm of the beta-1, 4-type glucuronic acid/mannuronic acid linear chain hybrid polysaccharide, wherein A is the ultraviolet spectrum at 210nm of the beta-1, 4-type glucuronic acid/mannuronic acid linear chain hybrid polysaccharide, and B is the mass spectrum of the beta-1, 4-type glucuronic acid/mannuronic acid linear chain hybrid polysaccharide;

FIG. 3 is a chromatogram of monosaccharide composition ions of the beta-1, 4 glucuronic acid/mannuronic acid linear hybrid polysaccharide of the present invention;

FIG. 4 is a graph showing the molecular weight distribution of the beta-1, 4 type glucuronic acid/mannuronic acid linear chain hybrid polysaccharide of the present invention;

FIG. 5 is a UV chromatogram at 210nm of beta-glucuronic acid/mannuronic acid oligosaccharides obtained by different hydrolysis times;

FIG. 6 is an ultraviolet chromatogram and a mass spectrogram at 210nm of beta-1, 4 type glucuronic acid/mannuronic acid oligosaccharide after 6h hydrolysis, wherein A is the ultraviolet chromatogram of beta-1, 4 type glucuronic acid/mannuronic acid linear chain hybrid oligosaccharide at 210nm, and B is the ion mass spectrogram of beta-1, 4 type glucuronic acid/mannuronic acid linear chain hybrid oligosaccharide;

FIG. 7 is an enlarged view of mass spectra of beta-1, 4 type glucuronic acid/mannuronic acid oligosaccharides with different polymerization degrees, which are mass spectra of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide and octasaccharide respectively;

FIG. 8 is a chromatogram of monosaccharide composition ions of a beta-1, 4 glucuronic acid/mannuronic acid linear hybrid oligosaccharide of the invention;

FIG. 9 shows the effect of a beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide prepared under the conditions of example 1 of the invention on normal mouse hippocampal neuronal cells;

FIG. 10 shows the protection effect of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide prepared under the condition of example 1 on mouse hippocampal neuron cells under the condition of sugar deficiency and hypoxia.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.

First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The present invention is described in detail by using the schematic structural diagrams, etc., and for convenience of illustration, the schematic diagrams are not enlarged partially according to the general scale when describing the embodiments of the present invention, and the schematic diagrams are only examples, which should not limit the scope of the present invention. In addition, the actual fabrication process should include three-dimensional space of length, width and depth.

Example 1

The preparation process of the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide comprises the following steps:

step 1: weighing 1g konjac glucomannan, sequentially adding 192mg 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxyl free radical and 1360mg sodium chlorite, adding 200ml 0.2M sodium acetate/acetic acid buffer solution (pH 4.7), and stirring.

Step 2: 4mL of 5% sodium hypochlorite solution was added to the solution of step 1, oxidation was carried out at 25 ℃ and 5mL of absolute ethanol was added after 48 hours to terminate the reaction.

And step 3: and (3) putting the obtained sample into a 500Da dialysis bag for dialysis for 72h for desalting, filtering through a rapid filter membrane to remove insoluble substances, taking the lower clear liquid, carrying out rotary evaporation concentration, and freeze-drying to obtain the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide solid substance.

And 4, step 4: dissolving the polysaccharide substance in 100mM trifluoroacetic acid solution, and performing acidolysis at 100 deg.C for 6 hr to obtain beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide solution.

And 5: removing acid from the obtained oligosaccharide solution, removing insoluble substances, rotary evaporating for concentration, and freeze drying to obtain beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid oligosaccharide solid substance.

The structure confirmation and the protection effect verification of mouse hippocampal neuron cells HT-22 under the sugar-deficient and oxygen-deficient conditions are carried out on the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide/oligosaccharide prepared by the method, and the method and the results are as follows:

product analysis 1

Nuclear magnetic structure confirmation of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide:

¹³c NMR carbon spectrum and¹H-¹³referring to fig. 1, fig. 1 is a nuclear magnetic spectrum of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide of the present invention, wherein a is beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide¹³C NMR carbon spectrum, B is beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid polysaccharide¹H-¹³C HSQC spectrum. As shown in figure 1, the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide is prepared by respectively measuring the C1 bit anomeric carbon signal peak of glucuronic acid and mannuronic acid which are respectively connected with beta-1 and 4 at the chemical shifts of 103ppm and 101ppm, and measuring the carbonyl carbon signal peak of the carboxyl which is obtained by oxidizing the 6 bit primary hydroxyl of a sugar ring into carboxyl at 177ppm, wherein the carbon signal peak of the 6 bit primary hydroxyl of the sugar ring at the chemical shift of 60ppm is completely disappeared, which shows that the oxidation is basically complete, and the glucose/mannose unit in the konjac glucomannan sugar chain is basically oxidized into the glucuronic acid/mannuronic acid.

Product analysis 2

Mass spectrum structure confirmation of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide:

referring to fig. 2, fig. 2 is an ultraviolet spectrum and a mass spectrum at 210nm of the β -1, 4-glucuronic acid/mannuronic acid linear chain hybrid polysaccharide, wherein a is an ultraviolet spectrum at 210nm of the β -1, 4-glucuronic acid/mannuronic acid linear chain hybrid polysaccharide, and B is a mass spectrum of the β -1, 4-glucuronic acid/mannuronic acid linear chain hybrid polysaccharide. As shown in FIG. 2, the polysaccharide was broken into oligosaccharides of different polymerization degrees (dp 2-dp 9 in FIG. 2B) at 300v under ion source, since a small amount of glucose/mannose in the sugar chain was not coatedThe oxidation is glucuronic acid/mannuronic acid, the oligosaccharide with each polymerization degree has two forms of complete oxidation and partial oxidation in the ion source, and the phenomena of dehydration and decarboxylation and water addition to a certain degree occur simultaneously. Further performing primary mass spectrometry on a dp5 mass spectrum peak in FIG. 2B, wherein m/z 897 corresponds to a structure beta-1, 4 type glucuronic acid/mannuronic acid pentasaccharide, as shown in formula I (m is 5, n)₁0), denoted dp5-DO 5; m/z 879 corresponds to a structure of removing one molecule of water from beta-1, 4 type glucuronic acid/mannuronic acid pentasaccharide, which is marked as dp5-DO5-H₂O; the structure corresponding to m/z 865 is that one molecule of water is removed from beta-1, 4 type glucuronic acid/mannuronic acid pentasaccharide, 1 glucose exists in the oligosaccharide fragment or mannose units are not oxidized into glucuronic acid and mannuronic acid due to incomplete oxidation, and the oligosaccharide fragment is shown in the formula (I) (m is 4, n is₁1) as dp5-DO4-H₂O; the structure corresponding to m/z 851 is that a molecule of water is removed from beta-1, 4-type glucuronic acid/mannuronic acid pentasaccharide, 2 glucose units or mannose units are not oxidized into glucuronic acid and mannuronic acid in the oligosaccharide fragment due to incomplete oxidation, and the oligosaccharide fragment is shown as formula (I) (m is 3, n1 is 2) which is recorded as dp5-DO3-H₂O; m/z 835 corresponds to a structure of beta-1, 4 type glucuronic acid/mannuronic acid pentasaccharide without molecular water and carboxyl, and is marked as dp5-DO5-H₂O-CO₂(ii) a The structure corresponding to m/z 821 is that one molecule of water and carboxyl are removed from incompletely oxidized beta-1, 4 type glucuronic acid/mannuronic acid pentasaccharide, and the mark is dp5-DO4-H₂O-CO₂(ii) a m/z 807 corresponding to beta-1, 4 type glucuronic acid/mannuronic acid pentasaccharide with incompletely oxidized structure, one molecule of water and carboxyl are removed, and the structure is marked as dp5-DO3-H₂O-CO₂。

Product analysis 3

Monosaccharide composition analysis of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide:

referring to fig. 3, fig. 3 is a monosaccharide composition ion chromatogram of the β -1,4 glucuronic acid/mannuronic acid linear chain hybrid polysaccharide according to the present invention. As shown in fig. 3, a β -1,4 glucuronic acid/mannuronic acid linear chain hybrid polysaccharide sample prepared under the conditions of this example 1 contains a large amount of glucuronic acid and mannuronic acid, and a small amount of glucose and mannose units, the molar ratio of glucuronic acid and mannuronic acid measured by the external standard method is about 4, and the sum of the percentage content of glucuronic acid and mannuronic acid is greater than or equal to 90%.

Product analysis 4

Referring to FIG. 4, FIG. 4 is a graph showing the molecular weight distribution of beta-1, 4 type glucuronic acid/mannuronic acid linear chain hybrid polysaccharide according to the present invention. As shown in fig. 4, the molecular weight Mw of a β -1,4 type glucuronic acid/mannuronic acid linear hybrid polysaccharide sample obtained in this example is 114.9kDa, Mn is 69.8kDa, and polydispersity is 1.6.

Product analysis 5

Mass spectrum structure confirmation of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide:

referring to fig. 6, fig. 6 shows an ultraviolet spectrum and a mass spectrum at 210nm of β -1,4 type glucuronic acid/mannuronic acid oligosaccharide after 6h hydrolysis, wherein a is an ultraviolet spectrum at 210nm of β -1,4 type glucuronic acid/mannuronic acid linear chain hybrid oligosaccharide, and B is an ion mass spectrum of β -1,4 type glucuronic acid/mannuronic acid linear chain hybrid oligosaccharide. As shown in FIG. 6, since a size exclusion column is used, substances with high molecular weight flow out of the column first, and are arranged from high to low according to the degree of polymerization from left to right. The first-order mass spectrometry analysis of the substances corresponding to the mass spectrum peaks in FIG. 6 is performed to respectively correspond to disaccharide to undecanose (dp 2-dp 11, see FIG. 6B in FIG. 6), and the specific analysis results are as follows: wherein m/z 351, 545, 721, 897, 1073, 1249 and 1425 are mass spectrum ion peaks with a charge respectively, the corresponding structures are beta-1, 4 type glucuronic acid/mannuronic acid disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide and octasaccharide, taking pentasaccharide as an example, 897.1683 is pentasaccharide which is completely oxidized and is recorded as dp5-DO5, 883.1882 is pentasaccharide which is partially oxidized and is recorded as dp5-DO4, namely one neutral saccharide unit in the pentasaccharide unit is not oxidized into an acidic saccharide unit. Wherein m/z 448, 624, 800 and 976 are mass spectrum ion peaks with two charges respectively, and the corresponding structures are beta-1, 4 type glucuronic acid/mannuronic acid pentasaccharide, heptasaccharide, nonasaccharide and undecanose. Referring to fig. 7, fig. 7 is an enlarged view of mass spectra of β -1,4 type glucuronic acid/mannuronic acid oligosaccharides with different polymerization degrees, which are mass spectra of β -1,4 type glucuronic acid/mannuronic acid straight-chain hybrid trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide and octasaccharide, respectively.

Product analysis 6

Monosaccharide composition analysis of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide:

the monosaccharide composition results of the beta-1, 4 type glucuronic acid/mannuronic acid linear hybrid oligosaccharide sample are shown in figure 8. The beta-1, 4 glucuronic acid/mannuronic acid linear chain hybrid oligosaccharide sample prepared under the condition of the embodiment 1 contains a large amount of glucuronic acid and mannuronic acid, and a small amount of glucose and mannose units, the molar ratio of the glucuronic acid and the mannuronic acid is determined to be about 4 by an external standard method, and the sum of the percentage contents of the glucuronic acid and the mannuronic acid is greater than or equal to 90%.

Product analysis 7

Effect of a β -1,4 type glucuronic acid/mannuronic acid linear hybrid oligosaccharide on survival of HT-22 cells under conditions of sugar oxygen deprivation (OGD):

7.1 establishment and Experimental grouping of OGD model

Culturing HT-22 cells in prepared high-glucose DMEM medium at 37 deg.C with 5% CO continuously introduced₂Gas, when the cell density is 90% and the growth is good, can be used for experiments.

Inoculating HT-22 cells in normal culture on 96-well plate, setting 3 parallel samples in each group, culturing in incubator for 24 hr, absorbing original high-sugar DMEM in administration group of control group, adding 100 μ L fresh high-sugar DMEM culture medium, respectively adding 10 μ L samples (working concentration 10ug/mL, 100ug/mL, 500ug/mL) prepared with PBS at different concentrations, adding 10 μ L PBS in blank group of control group, placing at 37 deg.C and 5% CO₂Culturing in an incubator for 2 h.

The administration group of the model group was aspirated of the original high-sugar DMEM, 100. mu.L of fresh sugar-free DMEM medium was added, and 10. mu.L of samples (working concentration 10ug/mL, 100ug/mL, 500ug/mL) prepared with PBS at different concentrations were added, respectivelymL), 10 μ L of PBS was added to the model control group. Model group 96 well plates were placed in an anoxic tank (95% N)₂、5％CO₂) Then, the mixture was cultured in a 5% CO2 incubator at 37 ℃ for 2 hours.

After the normal group and the model group are co-cultured for 2h, the cell survival rate is measured to determine the influence of the obtained beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide on HT-22 cells under OGD conditions.

7.2 determination of cell viability by CCK-8 method

After the culture is finished, adding 10 mu LCCK-8 reagent into each hole of the control group and the model group in a super clean bench in a dark place, continuously incubating for 2h in an incubator, measuring absorbance at 450nm, and calculating the cell survival rate.

7.3 results

The results of the cell viability calculation for each group by the CCK-8 method are shown in tables 1 and 2 and FIGS. 9 and 10. Table 1 shows the effect of a β -1,4 type glucuronic acid/mannuronic acid linear hybrid oligosaccharide on the survival rate of HT-22 cells cultured normally (n ═ 3), table 2 shows the effect of a β -1,4 type glucuronic acid/mannuronic acid linear hybrid oligosaccharide on the survival rate of HT-22 cells cultured under OGD (n ═ 3), and fig. 9 shows the effect of a β -1,4 type glucuronic acid/mannuronic acid linear hybrid oligosaccharide prepared under the conditions of example 1 of the present invention on normal mouse hippocampal neurons; FIG. 10 shows the protection effect of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide prepared under the condition of example 1 on mouse hippocampal neuron cells under the condition of sugar deficiency and hypoxia.

TABLE 1

TABLE 2

From FIG. 9, it can be seen that different doses of the beta-1, 4 type glucuronic acid/mannuronic acid linear hybrid oligosaccharide had no effect on the survival rate of normally cultured HT-22 cells, indicating that this sample is not toxic to HT-22 cells.

From fig. 10, it can be seen that the cell survival rate of the model group is lower than that of the normal culture cell control group, and has significant difference, indicating that the sugar-deficient hypoxia condition can effectively inhibit the cell survival rate. And the administration group of the model group can effectively improve the cell survival rate along with the increase of the administration concentration, which shows that the prepared beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide can obviously improve the survival rate of HT-22 cells.

In tables 1 and 2, the data are expressed as mean + SEM; comparison with the control group: p <0.05 was significantly different.

Example 2

The preparation method of the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide comprises the following steps:

step 1: weighing 10g of konjac glucomannan, sequentially adding 3840mg of 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxyl free radical and 2720mg of sodium chlorite, adding 2000ml of 0.2M sodium acetate/acetic acid buffer solution (pH 4.7), and stirring uniformly.

Step 2: and (3) adding 50mL of 5% sodium hypochlorite solution into the solution in the step 1, carrying out oxidation reaction at 25 ℃, and adding 30mL of absolute ethyl alcohol after 72 hours to stop the reaction.

And step 3: and (3) putting the obtained sample into a 500Da dialysis bag for dialysis for 72h for desalting, filtering through a rapid filter membrane to remove insoluble substances, taking the lower clear liquid, carrying out rotary evaporation concentration, and freeze-drying to obtain the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide solid substance.

And 4, step 4: dissolving the polysaccharide substance in 1M trifluoroacetic acid solution, and performing acidolysis for 3h at 100 ℃ to obtain oligosaccharide solution.

And 5: removing acid from the obtained oligosaccharide solution, removing insoluble substances, rotary evaporating for concentration, and freeze drying to obtain beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid oligosaccharide solid substance.

Step 6: and (3) putting the obtained sample into a 500Da dialysis bag for dialysis for 48h, carrying out rotary evaporation and concentration, centrifuging for 30min by using a 3K ultrafiltration centrifugal tube under the condition of 5000r/min, taking the lower-layer clear liquid, and carrying out freeze drying to obtain the partially oxidized beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide.

Example 3

A method for preparing beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide comprises the following steps:

step 1: weighing 2g konjac glucomannan, sequentially adding 192mg 4-acetamido-2, 2,6, 6-tetramethylpiperidine-1-oxyl free radical and 1360mg sodium chlorite, adding 200ml 0.2M sodium acetate/acetic acid buffer solution (pH 4.7), and stirring.

Step 2: and adding 12mL of 5% sodium hypochlorite solution into the solution in the step 1, carrying out oxidation reaction at 25 ℃, and adding 10mL of absolute ethyl alcohol after 96 hours to stop the reaction.

And step 3: and (3) putting the obtained sample into a 500Da dialysis bag for dialysis for 72h for desalting, filtering through a rapid filter membrane to remove insoluble substances, taking the lower clear liquid, carrying out rotary evaporation concentration, and freeze-drying to obtain the beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid polysaccharide solid substance.

And 4, step 4: dissolving the polysaccharide substance in 100mM trifluoroacetic acid solution, and performing acidolysis for 1h, 3h, 6h, 9h and 12h at 100 ℃ to obtain acidolysis oligosaccharide solutions with different molecular weight distributions and different polymerization degree oligosaccharide monomers in relative composition ratios, specifically referring to fig. 5, wherein fig. 5 is an ultraviolet chromatogram of beta-1, 4 type glucuronic acid/mannuronic acid oligosaccharide obtained after different hydrolysis time at 210 nm.

And 5: removing acid from the obtained oligosaccharide solution, removing insoluble substances, rotary evaporating for concentration, and freeze drying to obtain several beta-1, 4 type glucuronic acid/mannuronic acid straight chain hybrid oligosaccharide solid substances.

Step 6: and putting the obtained sample in a 500Da dialysis bag for dialysis for 72h, carrying out rotary evaporation and concentration, centrifuging for 30min by using a 3K ultrafiltration centrifugal tube under the condition of 3000r/min, taking the lower-layer clear liquid, and carrying out freeze drying to obtain a plurality of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharides.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a preparation method of beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide, which has the advantages of mild reaction conditions, simple synthesis process, high product purity, easily obtained preparation raw materials, lower cost and easy industrialization; the prepared beta-1, 4 type glucuronic acid/mannuronic acid straight-chain hybrid oligosaccharide has the advantages of definite structure, low molecular weight and high bioavailability, and has excellent application prospect.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.