Astragalus oligosaccharide extract, its preparation method and application

Abstract

This application relates to an astragalus oligosaccharide extract, its preparation method, and its application. The preparation method includes the following steps: dissolving the crude astragalus polysaccharide precipitate obtained by water extraction in water and dialyzing it through a dialysis membrane to retain astragalus oligosaccharides with a molecular weight cutoff of <2 kDa; eluting the astragalus oligosaccharides using a polar macroporous resin column with a 5%–15% (v / v) ethanol aqueous solution to obtain a first astragalus oligosaccharide fraction; eluting the astragalus oligosaccharides using a polar macroporous resin column with a 20%–40% (v / v) ethanol aqueous solution to obtain a second astragalus oligosaccharide fraction; eluting the astragalus oligosaccharides using a non-polar macroporous resin column with a 20%–40% (v / v) ethanol aqueous solution to obtain a third astragalus oligosaccharide fraction; and mixing the first, second, and third astragalus oligosaccharide fractions at a mass ratio of 1:1–20:1–10 to obtain the astragalus oligosaccharide extract. This astragalus oligosaccharide extract exhibits high activity and good immune-enhancing effects.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to an astragalus oligosaccharide extract, its preparation method, and its application. Background Technology

[0002] Astragalus, a traditional Chinese medicine, has been shown to possess immunomodulatory activity in its polysaccharide components. Extraction methods for astragalus polysaccharides mainly include water extraction with alcohol precipitation, dialysis, and column chromatography. While these extraction methods currently yield astragalus polysaccharides with some activity in promoting lymphocyte proliferation, their immune-enhancing effects are limited. For example, commercially available astragalus polysaccharides show little effect in promoting lymphocyte proliferation and increasing immunoglobulin (IgA, IgM) levels in immunosuppressed animal models, failing to meet the need for highly effective immune enhancement. Summary of the Invention

[0003] Therefore, it is necessary to provide an Astragalus oligosaccharide extract, its preparation method, and its application to enhance its activity and immune-enhancing effects.

[0004] One aspect of this application provides a method for preparing an astragalus oligosaccharide extract, comprising the following steps:

[0005] Astragalus was subjected to water extraction to obtain an astragalus water extract;

[0006] An alcohol solvent was added to the aqueous extract of Astragalus membranaceus and allowed to stand for precipitation to obtain crude polysaccharide precipitate of Astragalus membranaceus.

[0007] The crude polysaccharide precipitate of Astragalus membranaceus was dissolved in water and dialyzed using a dialysis membrane to retain Astragalus oligosaccharides with a molecular weight of <2 kDa.

[0008] The astragalus oligosaccharide was eluted using a polar macroporous resin column with an ethanol aqueous solution of 5% to 15% by volume to obtain the first astragalus oligosaccharide fraction.

[0009] The astragalus oligosaccharide was subjected to a second elution using a polar macroporous resin column with an ethanol aqueous solution of 20%~40% by volume to obtain a second astragalus oligosaccharide fraction.

[0010] The astragalus oligosaccharide was subjected to a third elution using a non-polar macroporous resin column with an ethanol aqueous solution of 20%~40% by volume to obtain the third astragalus oligosaccharide fraction.

[0011] The first, second, and third astragalus oligosaccharide fractions are mixed in a mass ratio of 1:1 to 20:1 to 10 to obtain the astragalus oligosaccharide extract.

[0012] The above preparation method directionally enriches astragalus oligosaccharides with a molecular weight ≤2kDa from crude astragalus polysaccharides, and then performs a first elution treatment with a specific volume concentration of ethanol aqueous solution through polar macroporous resin, weakly polar macroporous resin and non-polar macroporous resin respectively to obtain a first astragalus oligosaccharide fraction, a second astragalus oligosaccharide fraction and a third astragalus oligosaccharide fraction. These three specific oligosaccharide fractions with smaller molecular weights are more easily absorbed by intestinal epithelial cells. The three oligosaccharide fractions in a specific mass ratio work together synergistically, enabling the prepared astragalus oligosaccharide extract to quickly penetrate the cell membrane of immune cells and enter the cell to activate the immune function of cells. It has a significant function in promoting the proliferation activity of mouse lymphocytes and can significantly increase the content of IgA and IgM immunoglobulins in the serum of immunosuppressed mice, thus having excellent immune-enhancing effects.

[0013] In some embodiments, the mass ratio of the first astragalus oligosaccharide fraction, the second astragalus oligosaccharide fraction, and the third astragalus oligosaccharide fraction is 1:2~10:1~5.

[0014] In some embodiments, during the first elution,

[0015] The polar macroporous resin column includes a NAK-9 type chromatographic column; and / or...

[0016] The volume fraction of the ethanol aqueous solution is 8% to 12%.

[0017] In some embodiments, during the second elution,

[0018] The polarity of the polar macroporous resin column in the second elution is weaker than that of the polar macroporous resin column in the first elution; and / or,

[0019] The polar macroporous resin column includes an AB-8 type chromatographic column; and / or,

[0020] The volume fraction of the ethanol aqueous solution is 25% to 35%.

[0021] In some embodiments, during the third elution,

[0022] The nonpolar macroporous resin column includes an XAD-4 type chromatographic column; and / or,

[0023] The volume fraction of the ethanol aqueous solution is 25% to 35%.

[0024] A second aspect of this application provides an astragalus oligosaccharide extract, which is prepared using the preparation method described in the first aspect.

[0025] A third aspect of this application provides a composition for enhancing immunity, the composition comprising the astragalus oligosaccharide extract and egg white extract described in the second aspect.

[0026] In some embodiments, the mass ratio of the astragalus oligosaccharide extract to the egg white extract is 1:1 to 20.

[0027] In some embodiments, the method for preparing the egg white extract includes the following steps:

[0028] The egg white was subjected to water extraction to obtain an egg white water extract;

[0029] The egg white aqueous extract was dried to obtain a crude egg white extract.

[0030] The crude egg white extract was dissolved in water and dialyzed using an ultrafiltration membrane to retain egg white oligopeptides with a molecular weight of <1 kDa.

[0031] The egg white oligopeptide was subjected to a fourth elution treatment using a polar macroporous resin column with an ethanol aqueous solution of 5%~15% by volume to obtain the egg white extract.

[0032] A fourth aspect of this application provides a pharmaceutical preparation for enhancing immunity, comprising the astragalus oligosaccharide extract described in the second aspect or the composition described in the third aspect. Detailed Implementation

[0033] To facilitate understanding of this application, a more complete description of the application will be provided below with reference to relevant embodiments. Preferred embodiments of the application are shown herein. However, the application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] Astragalus, a traditional Chinese medicine for tonifying qi, has polysaccharide components that have been proven to have immunomodulatory activity. Although the astragalus polysaccharides extracted so far have a certain activity in promoting lymphocyte proliferation, their effects on promoting lymphocyte proliferation and increasing the levels of immunoglobulins (IgA and IgM) in immunosuppressed animal models are not significant, making it difficult to meet the needs of efficiently enhancing immunity.

[0036] Egg white is rich in protein and polypeptides, and oligopeptides in its hydrolysate or extract have also been shown to have immunomodulatory functions. Currently, commercially available egg white peptides are mainly obtained through enzymatic hydrolysis or crude extraction, and the immune-enhancing effect of egg white extract is limited.

[0037] Based on this, the first aspect of this application provides a method for preparing an astragalus oligosaccharide extract, comprising the following steps:

[0038] Astragalus was subjected to water extraction to obtain an astragalus water extract;

[0039] An alcohol solvent was added to the aqueous extract of Astragalus membranaceus and allowed to stand for precipitation to obtain crude polysaccharide precipitate of Astragalus membranaceus.

[0040] The crude polysaccharide precipitate of Astragalus membranaceus was dissolved in water and dialyzed using a dialysis membrane to retain Astragalus oligosaccharides with a molecular weight <2kDa;

[0041] The astragalus oligosaccharide was eluted using a polar macroporous resin column with a 5%~15% (v / v) ethanol aqueous solution to obtain the first astragalus oligosaccharide fraction.

[0042] The astragalus oligosaccharide was eluted using a polar macroporous resin column with a 20%–40% (v / v) ethanol aqueous solution to obtain the second astragalus oligosaccharide fraction.

[0043] The astragalus oligosaccharide was eluted using a nonpolar macroporous resin column with a 20%–40% (v / v) ethanol aqueous solution to obtain the third astragalus oligosaccharide fraction.

[0044] The first, second, and third astragalus oligosaccharide fractions were mixed at a mass ratio of 1:1 to 20:1 to 10 to obtain the astragalus oligosaccharide extract.

[0045] The above preparation method directionally enriches astragalus oligosaccharides with a molecular weight <2kDa from crude astragalus polysaccharide, and then performs a first elution treatment with a specific volume concentration of ethanol aqueous solution through polar macroporous resin, weakly polar macroporous resin and non-polar macroporous resin respectively to obtain a first astragalus oligosaccharide fraction, a second astragalus oligosaccharide fraction and a third astragalus oligosaccharide fraction. These three specific oligosaccharide fractions with smaller molecular weights are more easily absorbed by intestinal epithelial cells. The three oligosaccharide fractions in a specific mass ratio work together synergistically, enabling the prepared astragalus oligosaccharide extract to quickly penetrate the cell membrane of immune cells and enter the cell to activate the immune function of cells. It has a significant function in promoting the proliferation activity of mouse lymphocytes and can significantly increase the content of IgA and IgM immunoglobulins in the serum of immunosuppressed mice, thus having excellent immune-enhancing effects.

[0046] Understandably, the differences between the first, second, and third astragalus oligosaccharide fractions stem from the dual separation caused by the resin polarity gradient and the concentration gradient of the eluting ethanol, resulting in differences in polarity, molecular weight, monosaccharide composition, and glycosidic bond type among the three oligosaccharide fractions. The first astragalus oligosaccharide fraction has a small molecular weight and high polarity, allowing it to rapidly enter cells and initiate a response; the second astragalus oligosaccharide fraction has a moderate molecular weight and polarity, providing broad-spectrum activation and amplification effects, which can enhance lymphocyte proliferation activity; the third astragalus oligosaccharide fraction has low polarity, playing a long-term regulatory role and consolidating memory. The three fractions work together synergistically to achieve comprehensive, efficient, and lasting immune enhancement.

[0047] As an example, the molecular weight of astragalus oligosaccharides can be 0.05 kDa, 0.10 kDa, 0.15 kDa, 0.20 kDa, 0.25 kDa, 0.30 kDa, 0.35 kDa, 0.40 kDa, 0.45 kDa, 0.50 kDa, 0.55 kDa, 0.60 kDa, 0.65kDa, 0.70 kDa, 0.75 kDa, 0.80 kDa, 0.85 kDa, 0.90 kDa, 0.95 kDa, 1.00 kDa, 1.05 kDa, 1.10 kDa, 1.15 kDa, 1.20 kDa, 1.25 kDa, 1.30 kDa, 1.35 kDa, 1.40 kDa, 1.45 kDa, 1.50kDa, 1.55 kDa, 1.60 kDa, 1.65 kDa, 1.70 kDa, 1.75 kDa, 1.80 kDa, 1.85 kDa, 1.90 kDa, or 1.95 kDa, or any two of the above point values ​​as endpoints within the range.

[0048] Furthermore, the molecular weight of astragalus oligosaccharides is 0.5kDa to 1kDa.

[0049] As an example, in the first elution, the volume fraction of the ethanol-water solution can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0050] Furthermore, in the first elution, the volume fraction of the ethanol-water solution is 8%–12%. The astragalus oligosaccharide extract prepared from the first astragalus oligosaccharide fraction obtained using an eluent within this volume fraction range can further enhance the proliferative activity and efficacy of mouse lymphocytes.

[0051] As an example, in the second elution, the volume fraction of the ethanol aqueous solution can be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0052] Furthermore, in the second elution, the volume fraction of the ethanol-water solution is 25%–35%. Using an eluent within this volume fraction range, the astragalus oligosaccharide extract prepared from the second astragalus oligosaccharide fraction can further enhance the proliferative activity of mouse lymphocytes and improve immune efficacy.

[0053] As an example, in the third elution, the volume fraction of the ethanol aqueous solution can be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0054] Furthermore, in the third elution, the volume fraction of the ethanol-water solution is 25%–35%. The astragalus oligosaccharide extract prepared from the third astragalus oligosaccharide fraction obtained using this volume fraction range can further enhance the proliferative activity and efficacy of mouse lymphocytes.

[0055] As an example, in the Astragalus oligosaccharide extract, the mass ratio of the first Astragalus oligosaccharide fraction, the second Astragalus oligosaccharide fraction, and the third Astragalus oligosaccharide fraction can be 1:1:1, 1:2:3, 1:2.5:2, 1:3:5, 1:4:7, 1:5:9, 1:6:2, 1:7:4, 1:8:6, 1:9:8, 1:10:10, 1:11:5, 1:12:6, 1:13:7, 1:14:8, 1:15:9, 1:16:10, 1:17:3, 1:18:6, 1:19:9, 1:20:7, or 1:20:10, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0056] Furthermore, the mass ratio of the first, second, and third astragalus oligosaccharide fractions in the astragalus oligosaccharide extract is 1:2~10:1~5. At this mass ratio, the astragalus oligosaccharide extract is more easily absorbed by intestinal epithelial cells and can rapidly penetrate the cell membrane of immune cells to enter the cell, further enhancing lymphocyte proliferation activity and immune efficacy.

[0057] Furthermore, in the Astragalus oligosaccharide extract, the mass ratio of the first Astragalus oligosaccharide fraction, the second Astragalus oligosaccharide fraction, and the third Astragalus oligosaccharide fraction is 1:2~4:1~3.

[0058] In some of these embodiments, the molecular weight of the first astragalus oligosaccharide fraction is <1 kDa.

[0059] In some embodiments, the molecular weight of the second astragalus oligosaccharide fraction is 1 kDa to 1.9 kDa.

[0060] In some embodiments, the molecular weight of the third astragalus oligosaccharide fraction is: 1.9kDa < molecular weight ≤ 1.95kDa.

[0061] In some of these embodiments, Astragalus includes the root of Astragalus membranaceus.

[0062] In some embodiments, during the first elution, the polar macroporous resin column includes a NAK-9 type chromatographic column.

[0063] In some embodiments, the weakly polar macroporous resin column used in the second elution includes an AB-8 type column.

[0064] In some embodiments, the polarity of the polar macroporous resin column in the second elution is weaker than that of the polar macroporous resin column in the first elution.

[0065] In some embodiments, the nonpolar macroporous resin column used in the third elution includes an XAD-4 type column.

[0066] A second aspect of this application provides an astragalus oligosaccharide extract, which is prepared using the method of the first aspect.

[0067] A third aspect of this application provides a composition for enhancing immunity, the composition comprising the astragalus oligosaccharide extract and egg white extract of the second aspect.

[0068] In the above composition, the astragalus oligosaccharide extract and egg white extract complement each other in terms of chemical structure, target of action and immunomodulatory mechanism, and have a synergistic effect. This results in a significant enhancement of the composition's ability to promote the proliferation of mouse lymphocytes and a significant increase in the levels of IgA and IgM immunoglobulins in the serum of immunosuppressed mice, thus exhibiting excellent immune-enhancing effects.

[0069] Egg white extract, also known as egg white extract, is extracted from egg whites of chicken eggs.

[0070] In some embodiments, the mass ratio of Astragalus oligosaccharide extract to egg white extract in the composition is 1:1 to 20.

[0071] As an example, the mass ratio of Astragalus oligosaccharide extract to egg white extract in the composition can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:5.5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0072] Furthermore, in the composition, the mass ratio of Astragalus oligosaccharide extract to egg white extract is 1:2~10.

[0073] In some embodiments, the preparation method of egg white extract includes the following steps:

[0074] The egg white was subjected to water extraction to obtain an egg white water extract;

[0075] The egg white aqueous extract was dried to obtain crude egg white extract.

[0076] The crude extract of egg white was dissolved in water and dialyzed using an ultrafiltration membrane to retain egg white oligopeptides with a molecular weight of <1kDa.

[0077] The oocyte oligopeptides were subjected to a fourth elution process using a polar macroporous resin column with an ethanol aqueous solution of 5%–15% by volume to obtain the oocyte extract.

[0078] The egg white extract prepared by the above method retains the complete spatial conformation of ovalbumin and specific active peptide sequences. The low molecular weight astragalus oligosaccharide extract is responsible for "rapid delivery," while the high molecular weight egg white extract is responsible for "long-lasting stimulation," and the two complement each other in terms of absorption kinetics. In addition, the astragalus oligosaccharide extract and the egg white extract work together to activate different branches of the immune system, significantly stimulating the proliferation of T and B lymphocytes and increasing the number of immune cells. Moreover, the low molecular weight astragalus oligosaccharide provides a highly bioavailable immune activation signal (TLR4 / NF-κB), while the high molecular weight egg white extract provides abundant antigenic epitopes and specific immune stimulation. Through a triple mechanism of receptor activation, antigen presentation, and complex stabilization, the two achieve comprehensive and high-intensity synergistic activation of innate and adaptive immunity.

[0079] In some embodiments, the polar macroporous resin column used in preparing the egg white extract includes a CG300 chromatographic column.

[0080] As an example, the molecular weight of oocyte oligopeptide can be 0.05 kDa, 0.1 kDa, 0.2 kDa, 0.3 kDa, 0.4 kDa, 0.5 kDa, 0.6 kDa, 0.7 kDa, 0.8 kDa, 0.9 kDa, or 0.95 kDa, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0081] Furthermore, the molecular weight of oocyte oligopeptide is 0.5 kDa ~ 0.9 kDa.

[0082] As an example, the volume fraction of the ethanol-water solution during the fourth elution process can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or it can be within the range formed by any two of the above point values ​​as endpoints.

[0083] A fourth aspect of this application provides a pharmaceutical preparation for enhancing immunity, comprising the astragalus oligosaccharide extract described in the second aspect or the composition described in the third aspect.

[0084] To make the objectives, technical solutions, and advantages of this application clearer and more concise, the following specific embodiments are used for illustration, but this application is by no means limited to these embodiments. The embodiments described below are merely preferred embodiments of this application and can be used to describe this application, but should not be construed as limiting the scope of this application. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

[0085] To better illustrate this application, the following description, in conjunction with specific embodiments, further explains its content. The following are specific embodiments.

[0086] Example 1

[0087] The preparation steps of Astragalus oligosaccharide extract are as follows:

[0088] 1) Mix 5kg of fresh Astragalus root with 8L of purified water, then crush and pulverize the mixture. After homogenization, heat and boil for 25 minutes. Filter the mixture while it is still hot using gauze, and collect the filtrate to obtain Astragalus water extract.

[0089] 2) Concentrate 1L of Astragalus aqueous extract to 200 mL using a rotary evaporator, then add ethanol to obtain a mixture with a volume concentration of 85% ethanol in the mixture. Place the mixture in a 2℃ refrigerator and let it stand for 12 hours to precipitate crude Astragalus polysaccharide. Filter to separate the crude Astragalus polysaccharide precipitate and freeze-dry it to obtain crude Astragalus polysaccharide.

[0090] 3) Dissolve 100 g of crude Astragalus polysaccharide in 100 mL of water and dialyze it with a dialysis membrane with a molecular weight cutoff of 2 kDa to obtain Astragalus oligosaccharide-a with a molecular weight of <2 kDa;

[0091] 4) Take three portions of astragalus oligosaccharide-a and perform first, second, and third elutions respectively. Collect the eluents and freeze-dry them to obtain the astragalus oligosaccharide fraction, wherein:

[0092] In the first elution, astragalus oligosaccharide-a was eluted with a polar macroporous resin column (NAK-9 type chromatographic column) using a 10% (v / v) ethanol aqueous solution. The elution fraction was collected for each column volume, and then the elution fraction was freeze-dried to obtain the first astragalus oligosaccharide fraction (astragalus oligosaccharide-a-NAK-9-1); the first astragalus oligosaccharide-a-NAK-9-1 is a pale yellow powder;

[0093] In the second elution, astragalus oligosaccharide-a was eluted with a weakly polar macroporous resin column (AB-8 type chromatographic column) using a 30% (v / v) ethanol aqueous solution. The elution fraction was collected for each column volume, and then the elution fraction was freeze-dried to obtain the second astragalus oligosaccharide fraction (astragalus oligosaccharide-a-AB-8-2); the second astragalus oligosaccharide-a-AB-8-2 was a pale yellow powder.

[0094] In the third elution, astragalus oligosaccharide-a was eluted with a nonpolar macroporous resin column (XAD-4 type column) using a 30% (v / v) ethanol aqueous solution. The elution fraction was collected for each column volume, and then the elution fraction was freeze-dried to obtain the third astragalus oligosaccharide fraction (astragalus oligosaccharide-a-XAD-4-2); the third astragalus oligosaccharide-a-XAD-4-2 was a white powder.

[0095] 5) The first, second, and third astragalus oligosaccharide fractions were mixed evenly in a mass ratio of 1:2.5:2 to obtain the astragalus oligosaccharide extract.

[0096] The preparation steps of egg white extract are as follows:

[0097] 1) Mix 1L of egg white with 4L of purified water to prepare a mixture. After ultrasonic extraction of the mixture for 20 minutes, centrifuge it. Freeze-dry the supernatant after centrifugation, which is the egg white water extract, to obtain crude egg white extract.

[0098] 2) Dissolve 100g of crude egg white extract in 100mL of purified water, and dialyze it with an ultrafiltration membrane with a cutoff of 1kDa to separate egg white oligopeptide-a with a molecular weight ≤1 kDa.

[0099] 3) The above-mentioned oocyte oligopeptide-a was subjected to a fourth elution treatment using a polar macroporous resin column (CG300) and a 10% (v / v) ethanol aqueous solution. The elution fraction was collected once for each column volume, and then the elution fraction was freeze-dried to obtain oocyte extract (oocyte extract-a-CG300).

[0100] The preparation steps of the composition are as follows:

[0101] The above-prepared astragalus oligosaccharide extract and egg white extract were mixed evenly at a mass ratio of 1:5.5 to obtain a composition with immune-enhancing activity.

[0102] In the preparation process of the active substance, the following exploratory experiments were also conducted in this application:

[0103] The first exploratory experiment is basically the same as Example 1, except that in step 3) of preparing the astragalus oligosaccharide extract, astragalus oligosaccharide-b with a molecular weight cutoff of 5 kDa and astragalus oligosaccharide-c with a molecular weight cutoff of 2 kDa to 5 kDa are obtained by dialysis with a molecular weight cutoff of 5 kDa.

[0104] The cell proliferation-promoting activities of Astragalus oligosaccharide-a, Astragalus polysaccharide-b, and Astragalus polysaccharide-c fractions were detected by mouse lymphocyte proliferation activity assay. Astragalus oligosaccharide-a showed the strongest cell proliferation activity.

[0105] Exploratory Experiment 2 is basically the same as Example 1, except that in step 4) of preparing the Astragalus oligosaccharide extract, in the first elution, Astragalus oligosaccharide-a is eluted with a polar macroporous resin column (NAK-9 type chromatographic column) with 30% and 45% ethanol aqueous solution respectively to obtain Astragalus oligosaccharide-a-NAK-9-2 and Astragalus oligosaccharide-a-NAK-9-3; in the second elution, Astragalus oligosaccharide-a is eluted with a weakly polar macroporous resin column (AB). The astragalus oligosaccharide-a was eluted with a nonpolar macroporous resin column (XAD-8 type column) using 10% and 45% ethanol aqueous solutions, respectively, to obtain astragalus oligosaccharide-a-AB-8-1 and astragalus oligosaccharide-a-AB-8-3. In the third elution, astragalus oligosaccharide-a was further eluted with a nonpolar macroporous resin column (XAD-4 type column) using 10% and 45% ethanol aqueous solutions, respectively, to obtain astragalus oligosaccharide-a-XAD-4-1 and astragalus oligosaccharide-a-XAD-4-3.

[0106] The cell proliferation-promoting activities of astragalus oligosaccharide-a-NAK-9-1, astragalus oligosaccharide-a-NAK-9-2, and astragalus oligosaccharide-a-NAK-9-3 were detected by mouse lymphocyte proliferation activity assay, and astragalus oligosaccharide-a-NAK-9-1 showed the strongest cell proliferation activity.

[0107] The cell proliferation-promoting activities of Astragalus oligosaccharide-a-AB-8-1, Astragalus oligosaccharide-a-AB-8-2, and Astragalus oligosaccharide-a-AB-8-3 were detected by mouse lymphocyte proliferation activity assay, and Astragalus oligosaccharide-a-AB-8-2 showed the strongest cell proliferation activity.

[0108] The cell proliferation-promoting activities of astragalus oligosaccharide-a-XAD-4-1, astragalus oligosaccharide-a-XAD-4-2, and astragalus oligosaccharide-a-XAD-4-3 were detected by mouse lymphocyte proliferation activity assay, and astragalus oligosaccharide-a-XAD-4-2 showed the strongest cell proliferation activity.

[0109] The third exploratory experiment is basically the same as Example 1, except that in step 2) of preparing the egg white extract, dialysis is also performed using ultrafiltration membranes with a cutoff of 5 kDa and 10 kDa to separate egg white oligopeptide-b (1 kDa < molecular weight ≤ 5 kDa), egg white oligopeptide-c (5 kDa < molecular weight ≤ 10 kDa) and egg white oligopeptide-d (> 10 kDa).

[0110] The cell proliferation-promoting activities of egg white extract-a, egg white extract-b, egg white extract-c and egg white extract-d were detected by mouse lymphocyte proliferation activity assay. Egg white extract-a showed the strongest cell proliferation activity.

[0111] Experiment 4 is basically the same as Example 1, except that in step 3) of preparing the egg white extract, egg white oligopeptide-a is further eluted with a polar macroporous resin column (XAD16N) with a 10% ethanol aqueous solution to obtain egg white extract-a-XAD16N; egg white oligopeptide-a is further eluted with a polar macroporous resin column (XAD7HP) with a 10% ethanol aqueous solution to obtain egg white extract-a-XAD7HP; and egg white oligopeptide-a is further eluted with a polar macroporous resin column (SP700 type) with a 10% ethanol aqueous solution to obtain egg white extract-a-SP700.

[0112] The cell proliferation activity of egg white extract-a-XAD16N, egg white extract-a-CG300, egg white extract-a-XAD7HP and egg white extract-a-SP700 was detected by mouse lymphocyte proliferation activity assay. Egg white extract-a-CG300 showed the strongest cell proliferation activity.

[0113] Comparative Example 1

[0114] The cell proliferation activity and immune function of commercially available Astragalus polysaccharides were evaluated. The commercially available Astragalus polysaccharides were purchased from Guangzhou Jiyu Technology Co., Ltd.

[0115] Comparative Example 2

[0116] The cell proliferation activity and immune function of commercially available ovalbumin were evaluated. The commercially available ovalbumin was purchased from Guangzhou Jiyu Technology Co., Ltd.

[0117] The following is a performance evaluation of the Astragalus oligosaccharide extract, egg white extract and composition prepared in Example 1, as well as commercially available Astragalus polysaccharides and commercially available egg white peptides.

[0118] (1) Evaluation of the activity of promoting the proliferation of mouse lymphocytes

[0119] ① Sample preparation: The Astragalus oligosaccharide extract, egg white extract and composition obtained in Example 1, commercially available egg white peptide and commercially available Astragalus polysaccharide were dissolved in distilled water to prepare samples with a concentration of 100 μg / mL for later use.

[0120] ② Preparation of lymphocyte suspension: BALB / c mice (SPF grade, male, weighing (22+2) g, 5 weeks old, purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were sacrificed to prepare lymphocyte suspension and adjust the cell concentration to 5×10⁻⁶. 4 Cells / mL were cultured in a cell culture incubator at 37°C for 12 h.

[0121] ③ Set up groups (8 holes per group):

[0122] Astragalus oligosaccharide extract group: 100 μL of the above lymphocyte suspension and 100 μL of the above astragalus oligosaccharide extract sample were added to each well of a 96-well plate;

[0123] Egg white extract group: 100 μL of the above lymphocyte suspension and 100 μL of the above egg white extract sample were added to each well of a 96-well plate.

[0124] Composition group: Add 100 μL of the above lymphocyte suspension and 100 μL of the above composition sample to each well of a 96-well plate;

[0125] Commercially available Astragalus polysaccharide group: 100 μL of the above lymphocyte suspension and 100 μL of the above commercially available Astragalus polysaccharide sample were added to each well of a 96-well plate;

[0126] Commercially available oocyte peptide group: Add 100 μL of the above lymphocyte suspension and 100 μL of the above commercially available oocyte peptide group sample to each well of a 96-well plate;

[0127] Blank control group: No active ingredient was added. The blank control group consisted of 100 μL of lymphocyte suspension and 100 μL of cell culture medium.

[0128] Cell culture conditions: The groups set above were placed in a cell culture incubator at 37°C and cultured for 12 hours. The effect of the samples on the proliferation activity of mouse lymphocytes was detected by the CCK 8 kit. The results are shown in Table 1.

[0129] In this process, 10 μL of CCK 8 solution was added to each well of each of the pre-set groups, and the cells were incubated in a cell culture incubator in the dark for 4 hours. After incubation, the absorbance (OD) of each well was measured using a microplate reader at a wavelength of 450 nm. 450 Cell proliferation activity = (experimental group OD) 450 / Blank control group OD 450 ) × 100%.

[0130] Table 1

[0131]

[0132] The lymphocyte proliferation activity of the blank control group was set at 100%, and the cell proliferation activity of other groups was a relative value.

[0133] As shown in Table 1 above, compared with the blank control group, the cell proliferation activity of the commercially available Astragalus polysaccharide group and the commercially available ovalbumin group increased by 10.3% (p<0.05) and 9.40% (p>0.05), respectively. This indicates that commercially available Astragalus polysaccharide and commercially available ovalbumin can promote the proliferation of mouse lymphocytes and have a certain cell proliferation-promoting activity, which also indicates that commercially available Astragalus polysaccharide and commercially available ovalbumin have a certain immune-enhancing function. Compared with commercially available Astragalus polysaccharide or commercially available ovalbumin at the same concentration, the Astragalus oligosaccharide extract and ovalbumin extract group of Example 1 showed stronger proliferation activity of mouse lymphocytes, indicating that under the same concentration conditions, the Astragalus oligosaccharide extract and ovalbumin extract of Example 1 enhanced the proliferation activity of mouse lymphocytes more strongly than commercially available Astragalus polysaccharide and commercially available ovalbumin. Furthermore, compared with commercially available Astragalus polysaccharide or commercially available ovalbumin or Astragalus oligosaccharide extract and ovalbumin extract of Example 1, under the same concentration conditions, the composition of this application has the best activity in enhancing the proliferation of mouse lymphocytes. Compared with the blank control group, the proliferation activity of mouse lymphocytes in the composition group of Example 1 increased by 48.2% (p<0.01).

[0134] (2) Evaluation of efficacy in enhancing the immunity of mice

[0135] The Astragalus oligosaccharide extract, egg white extract and composition obtained in Example 1, commercially available egg white peptide and commercially available Astragalus polysaccharide were dissolved in distilled water to prepare samples with a concentration of 1.0 g / mL for later use.

[0136] BALB / c mice (SPF grade, female, weighing (22+2) g, 5 weeks old, purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were randomly divided into a blank control group, a model group (an immunosuppressive model induced by cyclophosphamide), and an active ingredient group (the Astragalus oligosaccharide extract group of Example 1, the egg white extract group of Example 1, the combination group of Example 1, the commercially available egg white peptide group, and the commercially available Astragalus polysaccharide group), with 8 mice in each group. The blank group was injected intraperitoneally with physiological saline, while the model group and the active ingredient group were injected intraperitoneally with cyclophosphamide to establish an immunosuppressive model. The active ingredient group was administered the active ingredient by gavage at a dose of 0.2 mL / day, while the blank control group and the model group were administered an equal volume of purified water by gavage. The administration was continued for 14 days. After the experiment, the mice were sacrificed and serum was collected. The effect of the sample on the IgA and IgM immunoglobulin levels in the serum of immunosuppressed mice was detected by ELISA kit, as shown in Table 2 below.

[0137] Table 2

[0138]

[0139] As shown in Table 2 above, compared with the blank control group, the serum IgA and IgM immunoglobulin levels in the model group mice were significantly reduced, indicating a significant decrease in mouse immunity. Compared with the blank control group, the serum IgA and IgM immunoglobulin levels in the model group mice decreased by 55.1% (p<0.01) and 50.4% (p<0.01), respectively. Compared with the model group, the serum IgA immunoglobulin levels in the commercially available Astragalus polysaccharide group and the commercially available ovalbumin group increased by 10.5% (p<0.05) and 7.11% (p>0.05), respectively; compared with the model group, the serum IgM immunoglobulin levels in the commercially available Astragalus polysaccharide group and the commercially available ovalbumin group increased by 10.8% (p<0.05) and 7.87% (p<0.05), respectively, indicating that commercially available Astragalus polysaccharide and commercially available ovalbumin have a certain function of enhancing mouse immunity. Compared to commercially available Astragalus polysaccharides or commercially available ovalbumin at the same concentration, the Astragalus oligosaccharide extract and ovalbumin extract groups in Example 1 showed a more significant increase in the levels of IgA and IgM immunoglobulins in the mouse serum, indicating that under the same concentration conditions, the Astragalus oligosaccharide extract and ovalbumin extract in Example 1 have stronger immune-enhancing activity in mice than commercially available Astragalus polysaccharides and commercially available ovalbumin. Compared to the model group, the levels of IgA and IgM immunoglobulins in the serum of mice in the Example 1 composition group increased by 96.7% (p<0.01) and 95.3% (p<0.01), respectively. Under the same concentration conditions, compared to commercially available Astragalus polysaccharides or commercially available ovalbumin, or the Astragalus oligosaccharide extract and ovalbumin extract in Example 1, the composition in Example 1 has the strongest immune-enhancing activity in mice.

[0140] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0141] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for preparing an astragalus oligosaccharide extract, characterized in that, Includes the following steps: Astragalus was subjected to water extraction to obtain an astragalus water extract; An alcohol solvent was added to the aqueous extract of Astragalus membranaceus and allowed to stand for precipitation to obtain crude polysaccharide precipitate of Astragalus membranaceus. The crude polysaccharide precipitate of Astragalus membranaceus was dissolved in water and dialyzed using a dialysis membrane to retain Astragalus oligosaccharides with a molecular weight of <2 kDa. The astragalus oligosaccharide was eluted using a polar macroporous resin column with an ethanol aqueous solution of 5% to 15% by volume to obtain the first astragalus oligosaccharide fraction. The astragalus oligosaccharide was subjected to a second elution using a polar macroporous resin column with an ethanol aqueous solution of 20%~40% by volume to obtain a second astragalus oligosaccharide fraction. The astragalus oligosaccharide was subjected to a third elution using a non-polar macroporous resin column with an ethanol aqueous solution of 20%~40% by volume to obtain the third astragalus oligosaccharide fraction. The first, second, and third astragalus oligosaccharide fractions are mixed in a mass ratio of 1:1 to 20:1 to 10 to obtain the astragalus oligosaccharide extract.

2. The method for preparing the Astragalus oligosaccharide extract as described in claim 1, characterized in that, The mass ratio of the first, second, and third astragalus oligosaccharide fractions is 1:2 to 10:1 to 5.

3. The method for preparing the Astragalus oligosaccharide extract as described in claim 1 or 2, characterized in that, In the first elution, The polar macroporous resin column includes a NAK-9 type chromatographic column; and / or... The volume fraction of the ethanol aqueous solution is 8% to 12%.

4. The method for preparing the Astragalus oligosaccharide extract as described in claim 1 or 2, characterized in that, In the second elution, The polarity of the polar macroporous resin column in the second elution is weaker than that of the polar macroporous resin column in the first elution; and / or, The polar macroporous resin column includes an AB-8 type chromatographic column; and / or, The volume fraction of the ethanol aqueous solution is 25% to 35%.

5. The method for preparing the Astragalus oligosaccharide extract as described in claim 1 or 2, characterized in that, In the third elution... The nonpolar macroporous resin column includes an XAD-4 type chromatographic column; and / or, The volume fraction of the ethanol aqueous solution is 25% to 35%.

6. An astragalus oligosaccharide extract, characterized in that, The astragalus oligosaccharide extract was prepared by the preparation method described in any one of claims 1 to 5.

7. A composition for enhancing immunity, characterized in that, The composition comprises the astragalus oligosaccharide extract and egg white extract as described in claim 6.

8. The composition according to claim 7, characterized in that, The mass ratio of the Astragalus oligosaccharide extract to the egg white extract is 1:1~20.

9. The composition according to claim 7 or 8, characterized in that, The preparation method of the egg white extract includes the following steps: The egg white was subjected to water extraction to obtain an egg white water extract; The egg white aqueous extract was dried to obtain a crude egg white extract. The crude egg white extract was dissolved in water and dialyzed using an ultrafiltration membrane to retain egg white oligopeptides with a molecular weight of <1 kDa. The egg white oligopeptide was subjected to a fourth elution treatment using a polar macroporous resin column with an ethanol aqueous solution of 5%~15% by volume to obtain the egg white extract.

10. A pharmaceutical preparation for enhancing immunity, characterized in that, Includes the astragalus oligosaccharide extract according to claim 6 or the composition according to any one of claims 7 to 9.

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