阅读说明：本技术 葡甘寡糖系列组合物、其制备方法及应用 (Glucomycooligosaccharide series composition, preparation method and application thereof ) 是由 王春明 殷小雨 张哲� 王一涛 于 2021-01-06 设计创作，主要内容包括：本发明公开了葡甘寡糖系列组合物、其制备方法及应用,涉及酶降解技术领域。葡甘多糖的降解方法包括：对葡甘多糖进行酶解,在酶解过程中采用的酶选自内切-1,4-β-甘露聚糖、β-甘露糖苷酶、β-葡糖苷酶和纤维素酶中的至少一种。酶解得到的葡甘寡糖组合物分子量均匀且纯度较高,具有显著抑炎生物活性,可以在制备抗炎药物中得到应用,也可以作为免疫细胞促进白细胞介素-10和转化生长因子-β1中至少一种物质表达的促进剂,还可以作为免疫细胞抑制肿瘤坏死因子TNF-α和白细胞介素-1β中至少一种物质表达的抑制剂。(The invention discloses a glucomannooligosaccharide series composition, a preparation method and application thereof, and relates to the technical field of enzymatic degradation. The degradation method of the glucomannan comprises the following steps: the glucomannan is subjected to enzymolysis, and the enzyme adopted in the enzymolysis process is at least one selected from endo-1, 4-beta-mannan, beta-mannosidase, beta-glucosidase and cellulase. The glucoglycerooligosaccharide composition obtained by enzymolysis has uniform molecular weight and higher purity, has obvious inflammation inhibiting bioactivity, can be applied to the preparation of anti-inflammatory drugs, can also be used as an accelerant for promoting the expression of at least one substance of interleukin-10 and transforming growth factor-beta 1 by immune cells, and can also be used as an inhibitor for inhibiting the expression of at least one substance of tumor necrosis factor TNF-alpha and interleukin-1 beta by immune cells.)

1. A glucomannooligosaccharide series composition, which comprises glucomannobiose, glucomannotriose, glucomannotetraose, glucomannopentaose and glucomannohexaose, wherein the structure of the glucomannobiose is beta-D-Manp- (1 → 4) -beta-D-Glcp, the structure of the glucomannotriose is beta-D-Manp- (1 → 4) -beta-D-Glcp, and the structure of the glucomannohexaose is beta-D-Manp- (1 → 4) -beta-D-Glcp or beta-D-Manp- (1 → 4) -beta-D-Glcp -D-Glcp, wherein said structure of said glucopentasaccharide is β -D-Manp- (1 → 4) - β -D-Glcp, and said structure of said glucohexasaccharide is β -D-Manp- (1 → 4) - β -D-Glcp;

preferably, the molecular weight of the glucomannan disaccharide, the glucomannan trisaccharide, the glucomannan tetrasaccharide, the glucomannan pentasaccharide and the glucomannan hexasaccharide is 342Da, 503Da, 666Da, 828Da and 991Da in sequence;

preferably, the glucomannooligosaccharide series composition is a freeze-dried powder or an aqueous solution.

2. A method for preparing the gluco-oligosaccharide series composition of claim 1, which comprises: the glucomannan is subjected to enzymolysis, and the enzyme adopted in the enzymolysis process is at least one selected from endo-1, 4-beta-mannan, beta-mannosidase, beta-glucosidase and cellulase.

3. The preparation method according to claim 2, wherein the enzymes used in the enzymatic hydrolysis process comprise, in mass fraction, endo-1, 4- β -mannan 0-10%, β -mannosidase 0-10%, β -glucosidase 0-10%, and cellulase 70-100%;

or, the enzyme adopted in the enzymolysis process comprises 0-10% of endo-1, 4-beta-mannan, 0-10% of beta-mannosidase, 70-100% of beta-glucosidase and 0-10% of cellulase by mass fraction;

or, the enzyme adopted in the enzymolysis process comprises 0-10% of endo-1, 4-beta-mannan, 70-100% of beta-mannosidase, 0-10% of beta-glucosidase and 0-10% of cellulase by mass fraction;

or, the enzyme adopted in the enzymolysis process comprises 70-100% of endo-1, 4-beta-mannan, 0-10% of beta-mannosidase, 0-10% of beta-glucosidase and 0-10% of cellulase by mass fraction;

or, the enzyme adopted in the enzymolysis process comprises 10-30% of endo-1, 4-beta-mannan, 10-30% of beta-mannosidase, 20-40% of beta-glucosidase and 20-40% of cellulase by mass fraction.

4. The method of claim 2 or 3, wherein the step of enzymatic hydrolysis comprises: mixing and dissolving the glucomannan lyophilized powder and enzyme, and reacting at 35-50 deg.C for 24-72 h.

5. The preparation method of claim 4, wherein the mass ratio of the lyophilized glucomannan powder to the enzyme is 5-20: 1; preferably 8-12: 1.

6. The preparation method of claim 4, wherein the preparation of the lyophilized glucomannan powder comprises: uniformly mixing glucomannan and water, and freeze-drying; preferably, the glucomannan and water are mixed for a time of 12-24 hours.

7. The process according to claim 4, wherein the oligosaccharide mixture is obtained by filtration after completion of the reaction, followed by separation and purification;

preferably, the separation and purification process is to separate by using a method of separating by using dextran chromatography, hydroxypropyl dextran gel chromatography, silica gel chromatography or liquid chromatography, and collecting the eluent;

more preferably, the separation and purification are carried out by a glucan chromatography method, and the eluent is deionized water;

more preferably, the separation and purification are carried out by adopting a liquid chromatography separation method, the eluent is water and acetonitrile, and the mass ratio of the water to the acetonitrile is controlled to be 1: 0.8-1.2;

more preferably, the method further comprises freeze-drying the collected eluate.

8. Use of the composition of the gluco-oligosaccharides series as defined in claim 1 or the composition of the gluco-oligosaccharides series produced by the production method as defined in any one of claims 2 to 7 for the production of anti-inflammatory drugs.

9. Use of the composition of the glucomannan series according to claim 1 or the composition of the glucomannan series prepared by the preparation method according to any one of claims 2 to 7 as an agent for promoting the expression of at least one of interleukin-10 and transforming growth factor-beta 1 by immune cells;

preferably, the immune cells include monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes, mast cells, accessory cells and the corresponding precursor cells of the above cells.

10. Use of the composition of the glucomannooligosaccharide series according to claim 1 or the composition of the glucomannooligosaccharide series prepared by the preparation method according to any one of claims 2 to 7 as an inhibitor for suppressing the expression of at least one of tumor necrosis factor TNF- α and interleukin-1 β by immune cells.

Technical Field

The invention relates to the technical field of enzymatic degradation, and in particular relates to a glucomannooligosaccharide series composition, a preparation method and application thereof.

Background

The immune system is divided into innate immunity and adaptive immunity, and the innate immunity is used as a first defense line and acts together with the adaptive immunity in a human body, so that the innate immunity has a great influence on resisting infection and inflammation. At present, the traditional synthetic immunosuppressants used clinically, such as azathioprine, methotrexate, hydroxychloroquine and the like, have certain adverse reactions, such as leukopenia, thrombocytopenia or anemia, liver function damage and the like, need to be checked regularly, and have the problems of high early development cost and the like.

In recent years, polysaccharides and derivatives thereof have great potential in the biopharmaceutical field. Glucomannan is a highly polymeric polysaccharide which is extracted from natural sources such as plant tubers, bulbs and roots. Glucomannan derived from natural konjac plant resources is a very promising bioactive polysaccharide, which consists of D-glucose and D-mannose units in a molar ratio of about 1: 1.6, the sugar units are linked by a β -1,4 linkage. Few acetyl groups are randomly distributed at the C-6 position, the percentage is about 5% -10%, the G-M main chain of the konjac glucomannan is provided with short side chains, and the structure is close to linear polysaccharide. The molecular weight of natural konjac glucomannan is usually between 500,000 and 2,000,000Da, which varies from plant source to plant source and from production method to plant source. Similarly, natural bletilla striata glucomannan is a macromolecular polysaccharide formed by combining glucose and mannan by beta-1, 4 glycosidic bonds, the ratio of mannose to glucose is 3:1, and each 12 hexose units contain 1.7 hexose branches and two acetyl groups.

The engineered glucomannan can be used as a drug delivery system or a biomaterial and applied in the field of treatment. However, the biological application of glucomannan is greatly limited due to the uncertain relationship between its structure and function. At present, the preparation of the glucomannooligosaccharides with definite structures is very necessary, and on the basis of the high-precision structures of the glucomannooligosaccharides, deeper biological activity mechanisms of the glucomannooligosaccharides are explored, so that the application fields of the glucomannooligosaccharides are expanded in the future.

At present, the methods for degrading natural glucomannan with high molecular weight mainly include ultrasonic physical methods and chemical hydrolysis methods (including acid hydrolysis, alkali hydrolysis and the like). The physical ultrasonic method is characterized in that glucomannan fragments are correspondingly changed under different ultrasonic treatment time and power, but typical physical properties such as viscosity, a storage module, a loss module and the like are also changed after ultrasonic degradation. Acid treatment hydrolyzes glucomannan, but the reaction conditions are severe, and the post-treatment process of an acid degradation means is more complicated, especially the process of removing acid. The method of alkali treatment and heating combined degradation of glucomannan can also obtain low viscosity glucomannan, however glucomannan has a disadvantageous tendency to form gel network under alkaline conditions, and the process is not stable.

Therefore, the existing degradation method of natural glucomannan can not obtain high-purity oligo-glucomannan with uniform molecular weight. In view of this, the present application is specifically made.

Disclosure of Invention

The invention aims to provide a gluco-glyco-oligosaccharide series composition which has good inflammation-inhibiting biological activity and is suitable for popularization and application.

The second purpose of the invention is to provide a preparation method of the glucomannooligosaccharide series composition, which adopts an enzyme degradation mode, and the prepared glucomannooligosaccharide composition has remarkable inflammation inhibition bioactivity, uniform molecular weight and high purity.

The third purpose of the invention is to provide the application of the gluco-glyco-oligosaccharide series composition.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

The invention provides a glucomannooligosaccharide series composition, which comprises glucomannobiose, glucomannotriose, glucomannotetraose, glucomannopentaose and glucomannohexaose, wherein the structure of the glucomannobiose is beta-D-Manp- (1 → 4) -beta-D-Glcp, the structure of the glucomannotriose is beta-D-Manp- (1 → 4) -beta-D-Glcp, and the structure of the glucomannohexaose is beta-D-Manp- (1 → 4) -beta-D-Glcp or beta-D-Manp- (1 → 4) -beta- D-Glcp, wherein the structure of the glucopentasaccharide is beta-D-Manp- (1 → 4) -beta-D-Glcp, and the structure of the glucohexasaccharide is beta-D-Manp- (1 → 4) -beta-D-Glcp.

The invention also provides a preparation method of the glucomannooligosaccharide series composition, which comprises the following steps: the glucomannan is subjected to enzymolysis, and the enzyme adopted in the enzymolysis process is at least one selected from endo-1, 4-beta-mannan, beta-mannosidase, beta-glucosidase and cellulase.

The invention also provides application of the glucomannooligosaccharide series composition in preparing anti-inflammatory drugs.

The invention also provides the application of the glucomannooligosaccharide series composition in serving as an accelerant for promoting the expression of at least one of interleukin-10 and transforming growth factor-beta 1 by immune cells.

The invention also provides the application of the gluco-glyco-oligosaccharide series composition in serving as an inhibitor for immune cells to inhibit the expression of at least one substance of tumor necrosis factors TNF-alpha and interleukin-1 beta.

The embodiment of the invention provides a degradation method of glucomannan and a series of glucomannan oligosaccharide compositions prepared by the degradation method, which have the following beneficial effects: the method adopts at least one enzyme of endo-1, 4-beta-mannan, beta-mannosidase, beta-glucosidase and cellulase to carry out enzymolysis on the glucomannan, the glucomannan oligosaccharide composition obtained by enzymolysis has uniform molecular weight and higher purity, has obvious inflammation inhibiting biological activity, can be applied to the preparation of anti-inflammatory drugs, can also be used as an accelerant for promoting the expression of at least one substance of interleukin-10 and transforming growth factor-beta 1 by immune cells, and can also be used as an inhibitor for inhibiting the expression of at least one substance of tumor necrosis factor TNF-alpha and interleukin-1 beta by the immune cells.

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 embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a structure of a glucomannan oligosaccharide composition deduced by NMR one-dimensional and two-dimensional spectrum analysis;

FIG. 2 is a High Performance Liquid Chromatography (HPLC) and Gel Permeation Chromatography (GPC) profile of different glucomannooligosaccharide compositions;

FIG. 3 shows the identification of the components of the glucomannan oligosaccharide composition by High Performance Liquid Chromatography (HPLC);

FIG. 4 is a graph showing the results of measurements of the expression levels of IL-10 in macrophages by the different glucomannan compositions obtained in examples 1-3, as measured by real-time fluorescent quantitative PCR experiments;

FIG. 5 is a graph showing the results of measurement of the expression level of TGF-. beta.1 of macrophages by the different glucomannan compositions obtained in examples 1-3 according to a real-time fluorescent quantitative PCR experiment;

FIG. 6 is a graph showing the results of measurements of the expression levels of TNF- α in macrophages from different glucomannan compositions obtained in examples 1-3 by real-time fluorescent quantitative PCR assay;

FIG. 7 is a graph showing the results of a test for determining the expression level of IL-1. beta. of macrophages by the different glucomannooligosaccharide compositions obtained in examples 1-3, using a real-time fluorescent quantitative PCR experiment;

FIG. 8 shows a comparison of the separation effect of different separation methods.

Detailed Description

In order to make the objects, 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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following provides specific descriptions of the gluco-oligosaccharides, their preparation methods and applications.

The embodiment of the invention provides a degradation method of glucomannan, which degrades the glucomannan by adopting an enzymolysis mode, and the inventor creatively adopts at least one enzyme of endo-1, 4-beta-mannan (EC:3.2.1.78), beta-mannosidase (EC:3.2.1.25), beta-glucosidase (EC:3.2.1.21) and cellulase (EC:232.734.4) for enzymolysis to prepare the glucomannan disaccharide, the glucomannan trisaccharide, the glucomannan tetrasaccharide, the glucomannan pentasaccharide and the glucomannan hexasaccharide, and the degradation method has higher purity, uniform molecular weight and certain biological activity of immunosuppression, and provides a new thought for developing natural immunosuppressants.

Specifically, the glucomannan can be glucomannan of natural origin, such as bletilla polysaccharide and konjac polysaccharide, for example, extracted from rhizome of bletilla striata or konjac polysaccharide extracted from tuber of konjac.

The enzyme adopted in the enzymolysis process can adopt the following five technical schemes: (1) the enzyme adopted in the enzymolysis process comprises 0-10% of endo-1, 4-beta-mannan, 0-10% of beta-mannosidase, 0-10% of beta-glucosidase and 70-100% of cellulase by mass fraction. (2) The enzyme adopted in the enzymolysis process comprises 0-10% of endo-1, 4-beta-mannan, 0-10% of beta-mannosidase, 70-100% of beta-glucosidase and 0-10% of cellulase by mass fraction. (3) The enzyme adopted in the enzymolysis process comprises 0-10% of endo-1, 4-beta-mannan, 70-100% of beta-mannosidase, 0-10% of beta-glucosidase and 0-10% of cellulase by mass fraction. (4) The enzyme adopted in the enzymolysis process comprises 70-100% of endo-1, 4-beta-mannan, 0-10% of beta-mannosidase, 0-10% of beta-glucosidase and 0-10% of cellulase by mass fraction. (5) The enzyme adopted in the enzymolysis process comprises 10-30% of endo-1, 4-beta-mannan, 10-30% of beta-mannosidase, 20-40% of beta-glucosidase and 20-40% of cellulase by mass fraction.

The five technical schemes are all suitable for the enzymolysis process in the embodiment of the application, and can be used for enzymolysis to obtain the glucomannose, the glucotriose, the glucotetraose, the glucopentaose and the glucohexaose, the purity can reach more than 95% after separation, and the purity identification of the glucohexaose can be carried out by adopting High Performance Liquid Chromatography (HPLC).

Further, the step of enzymatic hydrolysis comprises: mixing and dissolving the glucomannan freeze-dried powder and enzyme, and reacting for 24-72h at 35-50 ℃; the mass ratio of the glucomannan lyophilized powder to the enzyme is 5-20: 1. The yield and the purity of the product are further improved by further controlling the reaction temperature and time and the raw material dosage ratio in the enzymolysis process.

Further, the preparation of the lyophilized glucomannan powder comprises the following steps: uniformly mixing glucomannan and water, and freeze-drying, wherein the glucomannan and water are mixed and stirred for 12-24h to achieve a uniform mixing state. In other embodiments, the lyophilized powder of glucomannan can be prepared from commercially available raw materials without the above-mentioned lyophilization step.

In some embodiments, the oligosaccharide mixture is filtered after the reaction is completed, and then is separated and purified; the separation and purification process comprises separating by dextran chromatography, hydroxypropyl dextran gel chromatography, silica gel chromatography or liquid chromatography, collecting eluate, and separating to obtain glucosabiose, glucostriose, glucosetraose, glucospentaose and glucoshexaose with purity of above 95%.

In the preferred embodiment of the invention, the separation and purification are carried out by using a glucan chromatography method, and the eluent is deionized water. The separation degree can be further improved by adopting a glucan chromatography method, and the purity of the product is improved.

In the preferred embodiment of the invention, the separation and purification are carried out by adopting a liquid chromatography separation method, the eluent is water and acetonitrile, and the mass ratio of the water to the acetonitrile is controlled to be 1: 0.8-1.2; the product can be effectively separated by liquid chromatography, but the ratio of water to acetonitrile is controlled.

In some embodiments, the collected eluate may be further lyophilized to prepare a lyophilized powder; the collected eluate may be formed into an aqueous product.

The embodiment of the invention also provides a glucomannooligosaccharide series composition which is prepared by the degradation method; the prepared glucomannooligosaccharide series composition comprises glucomannobiose, glucomannotriose, glucotetraose, glucopentaose and glucohexaose, wherein the structure of the glucomannobiose is beta-D-Manp- (1 → 4) -beta-D-Glcp, the structure of the glucohexaose is beta-D-Manp- (1 → 4) -beta-D-Glcp, and the structure of the glucotetraose is beta-D-Manp- (1 → 4) -beta-D-Glcp or beta-D-Manp- (1 → 4) -beta-D-Glcp- Glcp, wherein the structure of the glucopentasaccharide is beta-D-Manp- (1 → 4) -beta-D-Glcp, the structure of the glucohexasaccharide is beta-D-Manp- (1 → 4) -beta-D-Glcp, and the structure is shown in figure 1 (arranged from top to bottom according to the sequence of the structures), the connection mode of the saccharide units and the units are 1, 4-beta-D-Man-D-Glc or 1, 4-alpha-D-Man-D-Glc.

The molecular weight (Mw) of the glucomannan disaccharide, the glucomannan trisaccharide, the glucomannan tetrasaccharide, the glucomannan pentasaccharide and the glucomannan hexasaccharide is 342Da, 503Da, 666Da, 828Da and 991Da in sequence; identification of molecular weight of glucoglycerooligosaccharide is preferably carried out by Gel Permeation Chromatography (GPC) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS).

The glucomannooligosaccharide series composition provided by the embodiment of the invention can be applied to the preparation of anti-inflammatory drugs, can also be used as an accelerant for promoting the expression of at least one substance of interleukin-10 and transforming growth factor-beta 1 by immune cells, and can also be used as an inhibitor for inhibiting the expression of at least one substance of tumor necrosis factor TNF-alpha and interleukin-1 beta by immune cells. The gluco-glyco-oligosaccharide series composition has the effect of obviously improving the expression of immunosuppressive genes in the aspect of natural immune stimulation, wherein immune cells comprise monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes, mast cells, accessory cells and precursor cells corresponding to the cells.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a degradation method of glucomannan, which comprises the following specific steps:

fully dissolving 5g of bletilla striata polysaccharide of natural source in 200ml of deionized water, heating to 60 ℃, and stirring for 24 hours. And (4) carrying out freeze-drying operation on the bletilla striata polysaccharide solution to obtain the bletilla striata polysaccharide freeze-dried powder. 500mg of bletilla striata polysaccharide freeze-dried powder is weighed, dissolved in 150ml of deionized water and placed in a round bottom flask. Then 50mg of the combined enzyme is weighed, and the combined enzyme comprises the following components in percentage by mass: 5% of endo-1, 4-beta-mannase, 5% of beta-mannosidase, 5% of beta-glucosidase and 85% of cellulase, fully dissolving the combined enzyme in a round-bottom flask at the same time, heating to 40 ℃, and carrying out the reaction for 24 hours to carry out the reaction of degrading bletilla striata polysaccharide by the combined enzyme. After the reaction is finished, heating to 100 ℃, heating for 10min, and inactivating the enzyme so as to terminate the enzyme degradation reaction. Then filtering and centrifuging the reaction solution, and collecting oligosaccharide supernatant; and (3) further separating and purifying the oligosaccharide mixture by Sephadex G-25 glucan chromatography, collecting separated eluent, and freeze-drying to obtain a bletilla striata oligosaccharide composition sample, namely DGM-1.

Example 2

The embodiment provides a degradation method of glucomannan, which comprises the following specific steps:

fully dissolving natural konjac polysaccharide with the weight of 5g in 200ml of deionized water, heating to 60 ℃, and stirring for 24 hours. And (4) carrying out freeze-drying operation on the konjac polysaccharide solution to obtain the konjac polysaccharide freeze-dried powder. 500mg of konjac glucomannan freeze-dried powder is weighed, dissolved in 150ml of deionized water and placed in a round-bottom flask. Then 50mg of the combined enzyme is weighed, and the combined enzyme comprises the following components in percentage by mass: fully dissolving the combined enzyme in a round-bottom flask simultaneously by 10 percent of endo-1, 4-beta-mannase, 10 percent of beta-mannosidase, 10 percent of beta-glucosidase and 70 percent of cellulase, heating to 40 ℃, and carrying out the reaction for 24 hours to carry out the reaction for degrading the konjac polysaccharide by the combined enzyme. After the reaction is finished, heating to 100 ℃, heating for 10min, and inactivating the enzyme so as to terminate the enzyme degradation reaction. Then filtering and centrifuging the reaction solution, and collecting oligosaccharide supernatant; and (3) further separating and purifying the oligosaccharide mixture by SephadexLH-20 hydroxypropyl dextran gel chromatography, collecting separated eluent, and freeze-drying to obtain a konjac oligosaccharide composition sample DGM-2.

Example 3

The embodiment provides a degradation method of glucomannan, which comprises the following specific steps:

fully dissolving natural konjac polysaccharide with the weight of 5g in 200ml of deionized water, heating to 60 ℃, and stirring for 24 hours. And (4) carrying out freeze-drying operation on the konjac polysaccharide solution to obtain the konjac polysaccharide freeze-dried powder. 500mg of konjac glucomannan freeze-dried powder is weighed, dissolved in 150ml of deionized water and placed in a round-bottom flask. Then 50mg of the combined enzyme is weighed, and the combined enzyme comprises the following components in percentage by mass: fully dissolving the combined enzyme in a round-bottom flask simultaneously by 10 percent of endo-1, 4-beta-mannase, 10 percent of beta-mannosidase, 70 percent of beta-glucosidase and 10 percent of cellulase, heating to 40 ℃, and carrying out the reaction for 24 hours to carry out the reaction for degrading the konjac polysaccharide by the combined enzyme. After the reaction is finished, heating to 100 ℃, heating for 10min, and inactivating the enzyme so as to terminate the enzyme degradation reaction. Then filtering and centrifuging the reaction solution, and collecting oligosaccharide supernatant; further separating and purifying the oligosaccharide mixture by preparative HPLC-C18 chromatography, collecting the eluate, and lyophilizing to obtain rhizoma Amorphophalli oligosaccharide composition sample, DGM-3.

Example 4

This example provides a method for degrading glucomannan, which differs from example 1 only in that: the enzymes used in the enzymatic degradation process include only endo-1, 4-beta-mannanase.

Example 5

This example provides a method for degrading glucomannan, which differs from example 1 only in that: the enzymes used in the enzymatic degradation process include only beta-mannosidase.

Example 6

This example provides a method for degrading glucomannan, which differs from example 1 only in that: the enzymes used in the enzymatic degradation process include only beta-glucosidase.

Example 7

This example provides a method for degrading glucomannan, which differs from example 1 only in that: the enzymes used in the enzymatic degradation process include only cellulases.

Example 8

This example provides a method for degrading glucomannan, which differs from example 1 only in that: different combined enzyme ratios comprise the following components in percentage by mass: 20% endo-1, 4-beta-mannanase, 20% beta-mannosidase, 30% beta-glucosidase and 30% cellulase.

Example 9

This example provides a method for degrading glucomannan, which differs from example 1 only in that: separating and purifying by liquid chromatography, wherein the eluent is water and acetonitrile, and the volume ratio of water to acetonitrile is controlled to be 1: 1.

Example 10

This example provides a method for degrading glucomannan, which differs from example 1 only in that: the method adopts hydroxypropyl sephadex chromatography, the eluent is deionized water and methanol, and the volume ratio of the deionized water to the methanol is controlled to be 2: 1.

Example 11

This example provides a method for degrading glucomannan, which differs from example 1 only in that: the method adopts a silica gel chromatography method, the eluent is deionized water and methanol, and the volume ratio of the deionized water to the methanol is controlled to be 5: 1.

Test example 1

The structure of the product obtained in examples 1-3 was tested and analyzed by NMR one-dimensional and NMR two-dimensional spectra to obtain the results of fig. 1, which in turn were: beta-D-Manp- (1 → 4) -beta-D-Glcp (glucomannoside), beta-D-Manp- (1 → 4) -beta-D-Glcp (glucotetraose), beta-D-Manp- (1 → 4) -beta-D-Glcp (glucotetraose), beta-D-Manp- (1 → 4) -beta- -Manp- (1 → 4) - β -D-Manp- (1 → 4) - β -D-Glcp (glucopentaose), β -D-Manp- (1 → 4) - β -D-Manp- (1 → 4) - β -D-Manp- (1 → 4) - β -D-Glcp (glucohexaose).

Test example 2

The purity and molecular weight of the glucomannooligosaccharide compositions obtained in preparation examples 1 to 3 were analyzed by a combination of High Performance Liquid Chromatography (HPLC) and Gel Permeation Chromatography (GPC), and the purity and molecular weight of the glucomannooligosaccharide composition samples were integrated, and the results are shown in fig. 2 and table 1.

Table 1 product purity and molecular weight test results

Sample (I)	Purity of	Molecular weight	Yield of
				Example 1(DGM-1)	99.99％	2109.16	87.4％
Example 2(DGM-2)	99.98％	1482.95	86.3％
				Practice ofExample 3(DGM-3)	97.67％	1220.71	77.9％
Example 4	78.42％	432.21	34.62％
				Example 5	52.13％	3238.12	65.7％
Example 6	36.43％	531.2	45.73％
				Example 7	87.69％	1118.32	88.7％
Example 8	90.23％	894.5	88.84％
				Example 9	87.83％	1221	87.83％
Example 10	58.04％	1221	58.04％
				Example 11	63.55％	1221	63.55％

Test example 3

The components of the glucooligosaccharide composition were identified by High Performance Liquid Chromatography (HPLC), and the results are shown in fig. 3.

As is apparent from FIG. 3, there are six oligosaccharide substances, including glucomannan, pentasaccharide and glucomannan, which constitute the glucomannan oligosaccharide composition.

Test example 4

The single glucomannooligosaccharide composition in the example 1 is further separated, and six samples obtained after purification and separation are analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) for molecular weight identification.

The results of the tests show that the molecular weights (Mw) of the six samples are 342Da, 503Da, 666Da, 828Da and 991Da respectively.

Test example 5

An induction stimulation experiment was performed on mouse-derived bone marrow transplant cells (mBMDM, primary cells extracted from C57BL/6J mice by a laboratory method) using the glucomannooligosaccharide composition prepared in preparation examples 1 to 3: macrophage derived from mouse bone marrow (5X 10)⁶Cells/10 cm petri dish each) was inoculated in 10cm petri dish, cultured with RPMI-1640 medium + 10% fetal bovine serum + 1% penicillin-streptomycin, placed at 37 ℃, 5% CO₂After the cells adhere to the wall in the incubator, 100ug/ml of DGM-1, DGM-2 and DGM-3 are respectively usedAnd 100ng/ml LPS for 24h, setting a blank group, and respectively setting three groups of parallel experiments. After incubation for 24h, RNA was extracted, and the expression levels of inflammatory-related cytokines before and after macrophages were measured by real-time quantitative PCR, as shown in FIGS. 4-7.

As can be seen from fig. 4 to 7: the glucomannan oligosaccharide compositions prepared in examples 1-3 all expressed strongly on interleukin-10 (IL-10, a typical inflammatory and immunosuppressive factor, IL-10 being able to inhibit the synthesis of pro-inflammatory cytokines such as IFN-. gamma., IL-2, IL-3, TNF. alpha., and GM-CSF produced by cells such as macrophages and Th1T cells) and transforming growth factor-. beta.1 (TGF-. beta.1 being able to inhibit the proliferation of immunocompetent cells), exhibited a certain degree of immunosuppressive activity; while the transcriptional levels of tumor necrosis factor (TNF-alpha) and interleukin-1 beta (IL-1 beta) were not much changed relative to the blank, indicating that the glucomannooligosaccharide composition has little activity in promoting inflammation.

Test example 6

The separation effect of the products of example 1 and examples 9-11 was tested and the results are shown in FIG. 8.

As can be seen from FIG. 8, the separation method using dextran chromatography and liquid chromatography can achieve a better separation effect, and the separation methods using hydroxypropyl dextran gel chromatography and silica gel chromatography are less effective.

In summary, the degradation method of glucomannan provided by the invention adopts at least one enzyme of endo-1, 4-beta-mannan, beta-mannosidase, beta-glucosidase and cellulase to carry out enzymolysis on the glucomannan, the glucomannan composition obtained by enzymolysis has uniform molecular weight and higher purity, has obvious inflammation-inhibiting biological activity, can be applied to the preparation of anti-inflammatory drugs, can be used as an accelerant for promoting the expression of at least one substance of interleukin-10 and transforming growth factor-beta 1 by immune cells, and can be used as an inhibitor for inhibiting the expression of at least one substance of tumor necrosis factor TNF-alpha and interleukin-1 beta by immune cells.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.