Radix achyranthis bidentatae crude polysaccharide, radix achyranthis bidentatae polysaccharide component, radix achyranthis bidentatae homogeneous polysaccharide, and preparation methods and uses of radix achyranthis bidentatae crude polysaccharide, radix achyranthis bidentatae polysaccharide component and radix achyranthis bidentatae homogeneous polysaccharide

Abstract

The present invention belongs to the technical field of medicine, and particularly to the field of vaccines and immunizations, more particularly to a radix achyranthis bidentatae crude polysaccharide, a radix achyranthis bidentatae polysaccharide component and a radix achyranthis bidentatae homogeneous polysaccharide extracted from radix achyranthis bidentatae, wherein the radix achyranthis bidentatae crude polysaccharide is selected from a radix achyranthis bidentatae crude polysaccharide 1 and a radix achyranthis bidentatae crude polysaccharide 2, wherein the radix achyranthis bidentatae crude polysaccharide 1 mainly comprises fructose, has a molecular weight of 1000-3000 Da, and preferably contains a small amount of glucose, and the radix achyranthis bidentatae crude polysaccharide 2 mainly comprises arabinose, glucose, Rhamnose, galactose and galacturonic acid, and has a molecular weight of 1.0*10<4>-2.0*10<5>Da. The present invention further relates to a pharmaceutical composition containing the polysaccharide, preparation methods of the polysaccharides, and vaccine uses of the polysaccharides. According to the present invention, the radix achyranthis bidentatae crude polysaccharide, the radix achyranthis bidentatae polysaccharide component and the radix achyranthis bidentatae homogeneous polysaccharide respectively have good immunoadjuvant activity and good immunomodulatory effects, and have the potential in preparation of vaccine adjuvants and immunomodulatory drugs.

Description

technical field

[0001] The present invention belongs to the field of medical technology, in particular, to the field of vaccines and immunization. Specifically, the present invention relates to crude polysaccharides, polysaccharide fractions and homogeneous polysaccharides extracted from Achyranthes. The present invention also relates to pharmaceutical compositions containing them, their preparation methods, and their use in vaccine adjuvants, immunomodulators, or in the preparation of vaccine adjuvants, vaccine preparations, vaccine compositions or antibodies. Background technique

[0002] Achranthis Bidentata (Achranthis Bidentata and Cyathula officinalis) is the dry root of Achyranthes officinalis, which has the functions of promoting blood circulation, clearing meridians, invigorating liver and kidney, and strengthening muscles and bones. Achyranthes contains a variety of secondary metabolites, such as oleanolic acid and its saponins, anthraquinone compounds, etc., and ...

Examples

Embodiment 1

[0162] Example 1: Preparation of Achyranthes crude polysaccharides ABP-50 and ABP-100

[0163] 1. Preparation of Achyranthes crude polysaccharide ABP-50

[0164] Take 1kg of Achyranthe huaihuai (purchased from Beijing Tongrentang Pharmacy), pulverize it, add 15L of distilled water to it, soak it for 5 hours at 50°C, and stir from time to time; then filter with two layers of gauze, and the filtrate is centrifuged (3000r / min×20min) , the residue after leaching is extracted for the second time under the same conditions to obtain the residue of Achyranthes (residue is used for the preparation of ABP-100 later) and water extract. The water extracts from the two extractions were combined, and concentrated under reduced pressure at 50-55°C to obtain a water extract concentrate. Then, 4 times the volume of 95% ethanol of the concentrated solution was added to carry out alcohol precipitation for 48 hours (the concentration of ethanol in the supernatant was 80%). Centrifuge, separate ...

Embodiment 2

[0167] Example 2: ABP-50-A, ABP-50-B, and ABP-100-A, ABP-50-B, ABP-100-A, Preparation of ABP-100-B and ABP-100-C

[0168] 1. Preparation of ABP-50-A and ABP-50-B components of Achyranthes polysaccharide

[0169] Weigh the ABP-501g ABP-501g obtained in Example 1, add 20 mL of water to dissolve it, and load it on a DEAE-cellulose chromatography column (Φ7.0cm×50cm), followed by H 2 O and 0.25mol / L NaHCO 3 Elution, the elution speed is 1mL / min, each test tube collects 10mL, and the anthrone-sulfuric acid method detects the effluent sugar absorption peak (OD). 620nm ).

[0170] ABP-50-A (H 2 O elution) and ABP-50-B (0.25mol / LNaHCO 3 elution) two polysaccharide components, see the elution curve figure 1 . The ABP-50-B fraction was dialyzed with distilled water to remove salt.

[0171] 2. Preparation of ABP-100-A, ABP-100-B and ABP-100-C of Achyranthes polysaccharide components

[0172] Weigh the ABP-1001g ABP-1001g obtained in Example 1, add 20 mL of water to dissolve ...

Embodiment 3

[0174] Example 3: Achyranthes homogeneous polysaccharide ABP-50-A-1, and ABP-100-C-1, Preparation of ABP-100-C-2 and ABP-100-C-3

[0175] 1. Preparation of Achyranthes homogeneous polysaccharide ABP-50-A-1

[0176] Weigh 200mg of the polysaccharide component ABP-50-A, add 2mL of water to dissolve, load the sample on a SephadexG50 gel chromatography column (Ф2.0cm×120cm), and elute with distilled water (see the elution curve in image 3 ), the eluted sugar absorption peak (OD) was detected by the sulfuric acid-anthrone method. 620nm ), concentrated under reduced pressure and freeze-dried to obtain homogeneous polysaccharide ABP-50-A-1.

[0177] 2. Preparation of Achyranthes homogeneous polysaccharide ABP-100-C-1, ABP-100-C-2, ABP-100-C-3

[0178] Weigh 200 mg of the polysaccharide component ABP-100-C, add 3 mL of water to dissolve, load on a Sephadex G100 gel chromatography column (Ф2.0cm×120cm), and elute with 0.1mol / L NaCl (see the elution curve in Figure 4 ), three s...

technical field

[0001] The present invention belongs to the field of medical technology, in particular, to the field of vaccines and immunization. Specifically, the present invention relates to crude polysaccharides, polysaccharide fractions and homogeneous polysaccharides extracted from Achyranthes. The present invention also relates to pharmaceutical compositions containing them, their preparation methods, and their use in vaccine adjuvants, immunomodulators, or in the preparation of vaccine adjuvants, vaccine preparations, vaccine compositions or antibodies. Background technique

[0002] Achranthis Bidentata (Achranthis Bidentata and Cyathula officinalis) is the dry root of Achyranthes officinalis, which has the functions of promoting blood circulation, clearing meridians, invigorating liver and kidney, and strengthening muscles and bones. Achyranthes contains a variety of secondary metabolites, such as oleanolic acid and its saponins, anthraquinone compounds, etc., and ...

Examples

Embodiment 1

[0162] Example 1: Preparation of Achyranthes crude polysaccharides ABP-50 and ABP-100

[0163] 1. Preparation of Achyranthes crude polysaccharide ABP-50

[0164] Take 1kg of Achyranthe huaihuai (purchased from Beijing Tongrentang Pharmacy), pulverize it, add 15L of distilled water to it, soak it for 5 hours at 50°C, and stir from time to time; then filter with two layers of gauze, and the filtrate is centrifuged (3000r / min×20min) , the residue after leaching is extracted for the second time under the same conditions to obtain the residue of Achyranthes (residue is used for the preparation of ABP-100 later) and water extract. The water extracts from the two extractions were combined, and concentrated under reduced pressure at 50-55°C to obtain a water extract concentrate. Then, 4 times the volume of 95% ethanol of the concentrated solution was added to carry out alcohol precipitation for 48 hours (the concentration of ethanol in the supernatant was 80%). Centrifuge, separate ...

Embodiment 2

[0167] Example 2: ABP-50-A, ABP-50-B, and ABP-100-A, ABP-50-B, ABP-100-A, Preparation of ABP-100-B and ABP-100-C

[0168] 1. Preparation of ABP-50-A and ABP-50-B components of Achyranthes polysaccharide

[0169] Weigh the ABP-501g ABP-501g obtained in Example 1, add 20 mL of water to dissolve it, and load it on a DEAE-cellulose chromatography column (Φ7.0cm×50cm), followed by H 2 O and 0.25mol / L NaHCO 3 Elution, the elution speed is 1mL / min, each test tube collects 10mL, and the anthrone-sulfuric acid method detects the effluent sugar absorption peak (OD). 620nm ).

[0170] ABP-50-A (H 2 O elution) and ABP-50-B (0.25mol / LNaHCO 3 elution) two polysaccharide components, see the elution curve figure 1 . The ABP-50-B fraction was dialyzed with distilled water to remove salt.

[0171] 2. Preparation of ABP-100-A, ABP-100-B and ABP-100-C of Achyranthes polysaccharide components

[0172] Weigh the ABP-1001g ABP-1001g obtained in Example 1, add 20 mL of water to dissolve ...

Embodiment 3

[0174] Example 3: Achyranthes homogeneous polysaccharide ABP-50-A-1, and ABP-100-C-1, Preparation of ABP-100-C-2 and ABP-100-C-3

[0175] 1. Preparation of Achyranthes homogeneous polysaccharide ABP-50-A-1

[0176] Weigh 200mg of the polysaccharide component ABP-50-A, add 2mL of water to dissolve, load the sample on a SephadexG50 gel chromatography column (Ф2.0cm×120cm), and elute with distilled water (see the elution curve in image 3 ), the eluted sugar absorption peak (OD) was detected by the sulfuric acid-anthrone method. 620nm ), concentrated under reduced pressure and freeze-dried to obtain homogeneous polysaccharide ABP-50-A-1.

[0177] 2. Preparation of Achyranthes homogeneous polysaccharide ABP-100-C-1, ABP-100-C-2, ABP-100-C-3

[0178] Weigh 200 mg of the polysaccharide component ABP-100-C, add 3 mL of water to dissolve, load on a Sephadex G100 gel chromatography column (Ф2.0cm×120cm), and elute with 0.1mol / L NaCl (see the elution curve in Figure 4 ), three s...

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