A kind of high-purity japonicus japonicus glycosaminoglycan and its preparation method and application

Abstract

The invention provides a high-purity apostichopus japonicas glycosaminoglycan as well as a preparation method and application thereof. The apostichopus japonicas glycosaminoglycan has the following structural formula as shown in the description and has a skeleton structure of ->4GlcUA beta 1->3GalNAc beta 1->(m is equal to 80-98), the O-4 / 6 position of acetamidogalactose is substituted by sulfate, each disaccharide repeating structure contains a fucose branched chain (R) which has three substitutable positions, wherein 47.4% of branched chains are linked at the O-3 position of glucuronic acid, 43.8% and 8.9% of branched chains are linked at the O-4 position and O-6 position of acetamidogalactose; 55.2% of fucose branched chains are substituted by O-2,4-bissulfuric acid groups, 27.1% of fucose branched chains are substituted by O-3,4-bis sulfuric acid groups, 17.7% of fucose branched chains are substituted by O-4-mono-sulfuric acid groups. The content of the sulfuric acid groups are 33.00%-34.00%, the weight-average molecular weight is 90.89 + / - 9.12KDa and the molecular weight distribution is 1.48 + / - 0.05 and the purity is greater than 99%. A medicine has the functions of significantly promoting mouse dendritic cells, enhancing the capability of peritoneal macrophages, promoting the proliferation of lymphocytes, enhancing the humoral immune function and significantly promoting the hematopoietic function.

Description

technical field

[0001] The invention relates to the field of immunostimulator drugs, in particular to a high-purity imitation sea cucumber glycosaminoglycan and its preparation method and application. Background technique

[0002] Sea cucumber glycosaminoglycans are a class of fucosylated glycosaminoglycans with novel structures extracted from echinoderm sea cucumber wall cells and connective tissues in recent decades. It has a backbone structure similar to chondroitin sulfate, and highly sulfated fucose branches that are not found in other glycosaminoglycan polymers. At present, there are two main types of sea cucumber glycosaminoglycans that have been studied in detail, which are derived from Japanese sea cucumber ( S. japonicus ) and Brazilian sea cucumber ( L. grisea ). They are all composed of glucuronic acid, acetylgalactosamine, fucose and sulfate groups. Its main chain structure is a disaccharide repeating unit composed of equimolar glucuronic acid and acetylgal...

Examples

Embodiment 1

[0040] Example 1: Preparation of high-purity japonicus japonicus glycosaminoglycan AJ-GAG.

[0041] 1. Fresh imitation sea cucumber ( Apostichopus japonicus ), remove the water from the stomach, and mash the body wall tissue to make a homogenate. Take 1kg of homogenate and make it into an aqueous solution with a concentration of 50% (M / V), add potassium hydroxide to the aqueous solution to a concentration of 1mol / L, stir and react at 50~60°C for 1 hour, and cool after the reaction is terminated .

[0042] 2. Adjust the pH value to 8.5 with glacial acetic acid, add 0.5% (M / V) trypsin (EC3.4.21.4), stir and react at 50°C for 3 hours, cool down after the reaction is terminated. The pH was adjusted to 7 with glacial acetic acid, and the precipitate was removed by centrifugation.

[0043] 3. Add an equal volume of 95% ethanol to the supernatant, let stand overnight at 4°C, collect the precipitate by centrifugation, wash with 80% ethanol, absolute ethanol and acetone, and dry to...

Embodiment 2

[0056] Example 2: The main immune activity experiment of the high-purity japonicus japonicus glycosaminoglycan.

[0057] 1. The effect of AJ-GAG on dendritic cells (DC).

[0058] 1) The effect of FA-A on the morphology of DC cells.

[0059] Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation, adhered to the wall to obtain mononuclear cells, GM-CSF and IL-4 were added to induce differentiation, LPS or AJ-GAG induced maturation, and observed under an inverted microscope. Set blank group, positive control group (LPS: 1μg / ml) and AJ-GAG group (1μg / ml, 10μg / ml, 50μg / ml), the observation results are shown in Figure 4 .

[0060] The experimental results showed that the cells in the positive control group and the AJ-GAG group were significantly larger in size, protruded on the surface, and had obvious DC cell characteristics, and were better than the LPS group in the concentration range of 1-10 μg / ml. It shows that AJ-GAG can promo...

technical field

[0001] The invention relates to the field of immunostimulator drugs, in particular to a high-purity imitation sea cucumber glycosaminoglycan and its preparation method and application. Background technique

[0002] Sea cucumber glycosaminoglycans are a class of fucosylated glycosaminoglycans with novel structures extracted from echinoderm sea cucumber wall cells and connective tissues in recent decades. It has a backbone structure similar to chondroitin sulfate, and highly sulfated fucose branches that are not found in other glycosaminoglycan polymers. At present, there are two main types of sea cucumber glycosaminoglycans that have been studied in detail, which are derived from Japanese sea cucumber ( S. japonicus ) and Brazilian sea cucumber ( L. grisea ). They are all composed of glucuronic acid, acetylgalactosamine, fucose and sulfate groups. Its main chain structure is a disaccharide repeating unit composed of equimolar glucuronic acid and acetylgal...

Examples

Embodiment 1

[0040] Example 1: Preparation of high-purity japonicus japonicus glycosaminoglycan AJ-GAG.

[0041] 1. Fresh imitation sea cucumber ( Apostichopus japonicus ), remove the water from the stomach, and mash the body wall tissue to make a homogenate. Take 1kg of homogenate and make it into an aqueous solution with a concentration of 50% (M / V), add potassium hydroxide to the aqueous solution to a concentration of 1mol / L, stir and react at 50~60°C for 1 hour, and cool after the reaction is terminated .

[0042] 2. Adjust the pH value to 8.5 with glacial acetic acid, add 0.5% (M / V) trypsin (EC3.4.21.4), stir and react at 50°C for 3 hours, cool down after the reaction is terminated. The pH was adjusted to 7 with glacial acetic acid, and the precipitate was removed by centrifugation.

[0043] 3. Add an equal volume of 95% ethanol to the supernatant, let stand overnight at 4°C, collect the precipitate by centrifugation, wash with 80% ethanol, absolute ethanol and acetone, and dry to...

Embodiment 2

[0056] Example 2: The main immune activity experiment of the high-purity japonicus japonicus glycosaminoglycan.

[0057] 1. The effect of AJ-GAG on dendritic cells (DC).

[0058] 1) The effect of FA-A on the morphology of DC cells.

[0059] Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation, adhered to the wall to obtain mononuclear cells, GM-CSF and IL-4 were added to induce differentiation, LPS or AJ-GAG induced maturation, and observed under an inverted microscope. Set blank group, positive control group (LPS: 1μg / ml) and AJ-GAG group (1μg / ml, 10μg / ml, 50μg / ml), the observation results are shown in Figure 4 .

[0060] The experimental results showed that the cells in the positive control group and the AJ-GAG group were significantly larger in size, protruded on the surface, and had obvious DC cell characteristics, and were better than the LPS group in the concentration range of 1-10 μg / ml. It shows that AJ-GAG can promo...

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