Composition for improving immunity based on natural components of radix astragali and preparation method thereof

Abstract

The invention relates to the technical field of radix astragali, in particular to a composition for improving immunity based on natural ingredients of radix astragali and a preparation method. The method comprises the following steps: carrying out preliminary extraction, macroporous resin extraction, silica gel chromatography, gel chromatography and ODS column chromatography on astragalus membranaceus, so as to obtain the composition consisting of various active ingredients of astragalus membranaceus. The composition not only has the function of promoting the proliferation of CD4 + T cells and CD8 + T cells, but also can activate T cells, thereby enhancing humoral immunity, regulating immune response, maintaining immune tolerance, enhancing immune memory, providing long-term immune protection and reducing the risk of reinfection. Adenosine cyclophosphate and 2 '-methoxy adenosine both inhibit the activation of CD4 + T cells or CD8 + T cells, although the intensity of immune response can be reduced, humoral immunity is not favorably enhanced, and cytokines are not favorably secreted to remove pathogens.

Description

Technical Field

[0001] The present invention relates to the technical field of Astragalus membranaceus, and specifically relates to an immune-enhancing composition based on natural components of Astragalus membranaceus and a preparation method thereof. Background Art

[0002] The components of Astragalus membranaceus include Astragalus polysaccharide (APS), Astragalosides, alkaloids, flavonoids, folic acid, and various trace elements. Among them, Astragalus polysaccharide is the main component and has a wide range of pharmacological effects. Astragalus membranaceus is not only a commonly used traditional Chinese medicine for replenishing qi, but also a dietary therapy medicine with remarkable effects, having the efficacy of both medicine and food. In dietary therapy, Astragalus membranaceus is often used as the main ingredient in combination with other auxiliary materials as a dietary therapy formula for health care and disease prevention. Astragalus polysaccharide is the most abundant and the main active ingredient in the effective components of Astragalus membranaceus. The regulatory effect of Astragalus polysaccharide on immune function not only lies in enhancing the functions of immune organs and cells, but also in stimulating the release of cytokines, affecting the nerve-endocrine-immune system network, and promoting the synthesis and expression of related genes. Astragalus glycoprotein is an important natural active ingredient extracted from traditional Chinese medicine Astragalus membranaceus. Existing studies have shown that Astragalus glycoprotein has potential therapeutic effects in multiple disease fields such as pulmonary fibrosis, acute spinal cord injury, experimental autoimmune encephalomyelitis (EAE), and rheumatoid arthritis (RA). Summary of the Invention

[0003] In view of this, the present invention provides an immune-enhancing composition based on natural components of Astragalus membranaceus and a preparation method thereof.

[0004] The first aspect of the present invention provides a preparation method of an immune-enhancing composition based on natural components of Astragalus membranaceus, which includes: performing ethanol reflux extraction on dried Guangxi Astragalus membranaceus to obtain a first paste; extracting the first paste with macroporous adsorption resin to obtain a second paste; subjecting the second paste to silica gel column chromatography to obtain a third paste; subjecting the third paste to gel chromatography to obtain a fourth paste; subjecting the fourth paste to ODS column chromatography to obtain a fifth paste; and freeze-drying the fifth paste to obtain an Astragalus membranaceus extract composition.

[0005] In a specific embodiment, the dried Guangxi Astragalus membranaceus is added to a 58% ethanol solution at a solid-liquid ratio of 1:20, fully soaked for 48 h, then heated under reflux at 75 °C for 3 times, 3 h each time, and the extraction solutions are combined and concentrated to obtain a first paste.

[0006] In a specific embodiment, the first paste is suspended in water and statically loaded onto a pretreated D101 macroporous adsorption resin column (the column height-to-diameter ratio is 10:1). After sequentially eluting with water and 30% ethanol at a flow rate of 0.15% of the column bed volume per minute for 3 column bed volumes, the 30% ethanol eluate is collected, concentrated, and the second paste is obtained.

[0007] In a specific embodiment, the second paste is formulated into a 1 g / mL solution with 30% ethanol aqueous solution and statically loaded onto a pretreated silica gel chromatography column (the column bed height-to-diameter ratio is 30:1). The gradient elution is carried out with a mixed solvent of dichloromethane and methanol at a flow rate of 0.5% of the column bed volume per minute. The elution gradient is that the mixed volume ratio of dichloromethane to methanol is from 9:1 to 8:2, and a total of 2 column volumes are eluted. The eluate is collected, combined, and concentrated to obtain the third paste.

[0008] In a specific embodiment, the third paste is formulated into a 350 mg / mL sample solution with a mixed solution of dichloromethane and methanol with a volume ratio of 8.5:1.5 and statically loaded onto a pre-equilibrated Sephadex LH-20 gel column (the column bed height-to-diameter ratio is 30:1). Methanol is used for elution at a flow rate of 0.5% of the column bed volume per minute. The eluate with 0.25 - 1.16 elution column volumes is collected, combined, and concentrated to obtain the fourth paste.

[0009] In a specific embodiment, the fourth paste is formulated into a 200 mg / mL sample solution with methanol and statically loaded onto an ODS column. The gradient elution is carried out with a 22% - 35% ethanol aqueous solution at a flow rate of 0.5% of the column bed volume per minute for 50 column volumes. The eluate with 30 - 40 elution column volumes is collected, combined, and concentrated to obtain the fifth paste.

[0010] In the second aspect of the present invention, there is provided a composition obtained by the preparation method of the first aspect, which comprises adenosine cyclic phosphate with a mass percentage of 8.32%, 2'-methoxyadenosine with a mass percentage of 8.78%, 1-ribosyl-2,3-diketone-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline with a mass percentage of 10.28%, (S)-N-(1-hydroxy-3-phenylpropan-2-yl)acetamide with a mass percentage of 11.09%, cyclo(glycyl-L-leucyl) with a mass percentage of 12.12%, cyclo(isoleucine-alanine) with a mass percentage of 7.69%, cyclo(L-leucine-L-proline) with a mass percentage of 6.72%, cyclo(L-phenylalanyl-L-serine) with a mass percentage of 5.99%, cyclo(L-phenylalanine-L-proline) with a mass percentage of 5.67%, and cyclo-(L-proline-L-proline) with a mass percentage of 6.31%.

[0011] The third aspect of the present invention provides the use of the composition obtained by the preparation method of the first aspect in the preparation of a reagent for promoting T cell proliferation.

[0012] The fourth aspect of the present invention provides the use of the composition obtained by the preparation method of the first aspect in the preparation of a reagent for promoting T cell activation.

[0013] The fifth aspect of the present invention provides the use of the composition obtained by the preparation method of the first aspect in the preparation of a reagent for preventing or treating nephritis proteinuria.

[0014] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present invention.

[0015] Advantageous technical effects of the present invention:

[0016] In the present invention, astragalus membranaceus is preliminarily extracted, extracted by macroporous resin, silica gel chromatography, gel chromatography and ODS column chromatography to obtain a composition composed of various astragalus active ingredients. This astragalus extraction composition not only has the function of promoting the proliferation of CD4+ T cells and CD8+ T cells, can activate T cells, thereby enhancing humoral immunity, regulating immune responses, maintaining immune tolerance, enhancing immune memory, providing long-term immune protection and reducing the risk of reinfection. While both cyclic adenosine monophosphate and 2'-methoxyadenosine inhibit the activation of CD4+ T cells or CD8+ T cells, although they can reduce the intensity of immune responses, it is not conducive to enhancing humoral immunity and is not conducive to secreting cytokines to achieve pathogen clearance.

[0017] Moreover, compared with the inhibitory effects of pure cyclic adenosine monophosphate or 2'-methoxyadenosine on T cell proliferation and activation, the astragalus extraction composition provided by the present invention, through the mutual synergy of multiple components, can instead promote T cell proliferation and activation, enhance humoral immunity, regulate immune responses, maintain immune tolerance, enhance immune memory, provide long-term immune protection and reduce the risk of reinfection. While both cyclic adenosine monophosphate and 2'-methoxyadenosine inhibit the activation of CD4+ T cells or CD8+ T cells, although they can reduce the intensity of immune responses, it is not conducive to enhancing humoral immunity and is not conducive to secreting cytokines to achieve pathogen clearance.

[0018] Moreover, the astragalus extract composition provided by the present invention can greatly reduce the urinary protein excretion of rats with nephritis proteinuria induced by bovine serum albumin and lower the levels of serum creatinine and serum urea nitrogen, compared with the slight improvement effect of simple adenosine cyclic phosphate or 2'-methoxyadenosine on nephritis proteinuria. The pathological results show that compared with the normal group, there are no obvious changes in the glomeruli of the model group, but the scores of protein casts and bleeding foci grading are significantly increased. The astragalus extract composition provided by the present invention can reduce the protein casts and the generation of bleeding foci in rats with nephritis induced by bovine serum albumin. That is to say, the astragalus extract composition provided by the present invention has an obvious improvement effect on rats with nephritis induced by bovine serum albumin and has the application prospect for the treatment of nephritis proteinuria. Description of the Drawings

[0019] Figure 1 It is the HPLC chart of the astragalus extract composition.

[0020] Figure 2 It is the effects of the astragalus extract composition (experimental group), adenosine cyclic phosphate (control group 1), or 2'-methoxyadenosine (control group 2) on the proliferation of CD4+ T cells, respectively.

[0021] Figure 3 It is the effects of the astragalus extract composition (experimental group), adenosine cyclic phosphate (control group 1), or 2'-methoxyadenosine (control group 2) on the proliferation of CD8+ T cells, respectively.

[0022] Figure 4 It is the effects of the astragalus extract composition (experimental group 1), adenosine cyclic phosphate (control group 1), or 2'-methoxyadenosine (control group 2) on the activation of CD4+ T cells, respectively.

[0023] Figure 5 It is the effects of the astragalus extract composition (experimental group 1), adenosine cyclic phosphate (control group 1), or 2'-methoxyadenosine (control group 2) on the activation of CD8+ T cells, respectively.

[0024] Figure 6 It is the results of the urinary protein, serum creatinine, and serum urea nitrogen contents after the intervention of the astragalus extract composition, astragaloside IV, adenosine cyclic phosphate, or 2'-methoxyadenosine on rats with nephritis proteinuria model, respectively.

[0025] Figure 7 It is the results of the protein cast score and bleeding focus grading score after the intervention of the astragalus extract composition, astragaloside IV, adenosine cyclic phosphate, or 2'-methoxyadenosine on rats with nephritis proteinuria model, respectively. Detailed Embodiments

[0026] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention. Reagents not specifically described in detail in the present invention are all conventional reagents and can be obtained from commercial sources; methods not specifically described in detail are all conventional test methods and can be learned from the prior art.

[0027] 1. Astragalus extraction

[0028] (1) Ethanol extraction

[0029] Dry material of Nogra guangxiensis C.F.Wei is added to a 58% ethanol solution at a solid-liquid ratio of 1:20. After fully soaking for 48 h, it is heated under reflux at 75 °C for extraction three times, each time for 3 h. The extraction solutions are combined and concentrated to obtain a first paste.

[0030] (2) D101 macroporous resin extraction

[0031] The first paste is suspended in water and statically loaded onto a pretreated D101 macroporous adsorption resin column (column height-to-diameter ratio is 10:1). Water and 30% ethanol are respectively eluted at a flow rate of 0.15% of the column bed volume per minute for three column bed volumes. The 30% ethanol eluate is collected and concentrated to obtain a second paste.

[0032] (3) Silica gel column chromatography

[0033] The second paste is prepared into a 1 g / mL solution with 30% ethanol aqueous solution and statically loaded onto a pretreated silica gel chromatography column (column bed height-to-diameter ratio is 30:1). A mixed solvent of dichloromethane and methanol is used for gradient elution at a flow rate of 0.5% of the column bed volume per minute. The elution gradient is that the mixed volume ratio of dichloromethane and methanol is from 9:1 to 8:2, and a total of two column volumes are eluted. The eluate is collected, combined, and concentrated to obtain a third paste.

[0034] (4) Gel chromatography

[0035] The third paste is prepared into a 350 mg / mL sample solution with a mixed solution of dichloromethane and methanol with a volume ratio of 8.5:1.5 and statically loaded onto a pre-equilibrated Sephadex LH-20 gel column (column bed height-to-diameter ratio is 30:1). Methanol is used for elution at a flow rate of 0.5% of the column bed volume per minute. The eluate with 0.25 - 1.16 elution column volumes is collected, combined, and concentrated to obtain a fourth paste.

[0036] (5) ODS column chromatography

[0037] The fourth paste was formulated into a sample solution of 200 mg / mL with methanol and statically loaded onto an ODS column (SinoChrom ODS-AP 5 μm ID 20 mm * 250 mm, Elite). It was gradient eluted with an ethanol aqueous solution of 22% to 35% at a flow rate of 0.5% of the column bed volume per minute for 50 column volumes, and the eluate of 30 - 40 elution column volumes was collected, combined and concentrated to obtain the fifth paste. The fifth paste was freeze-dried to obtain the astragalus extraction composition of the present invention.

[0038] 2. Component detection in the astragalus extraction composition

[0039] (1) Reagents

[0040] Cyclo(L-phenylalanyl-L-seryl), CAS: 35591-00-5, purity: 98% HPLC, Shenzhen Qimeike Biotechnology Co., Ltd. Cyclo(L-phenylalanine-L-proline), CAS: 3705-26-8, purity: 97%, Shanghai Ruihui Chemical Technology Co., Ltd. Cyclo(L-leucyl-L-proline), CAS: 2873-36-1, purity: >=97%, Shenzhen Innosaint Biotechnology Co., Ltd. (S)-N-(1-hydroxy-3-phenylpropan-2-yl)acetamide, CAS: 52485-51-5, purity: 98%, Shanghai Yunlian Medical Technology Co., Ltd. Cyclo(glycyl-L-leucyl), CAS: 5845-67-0, purity: 95% or 98%, Hangzhou Sinodata Medical Technology Co., Ltd. Cyclo(isoleucine-alanine) dipeptide, CAS: 90821-99-1, purity: >=98%, Wuhan Tianzhi Biotechnology Co., Ltd. Cyclo-(L-prolyl-L-proline), CAS: 19943-27-2, purity 98% HPLC, Hangzhou Sinodata Medical Technology Co., Ltd. 1-ribosyl-2,3-diketone-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline, CAS No. 18650-70-9, ETCM. 2'-methoxyadenosine, CAS: 2140-79-6, purity: >99.0%, Hefei Xinxin Medical Technology Co., Ltd. Cyclic adenosine monophosphate, CAS: 60-92-4, purity: 99% HPLC, Yantai Xianhua Technology Group Co., Ltd.

[0041] (2) Chromatographic conditions

[0042] Chromatographic column: ACQUITY UPLC BEH C18 Column, 1.7 μm, 2.1 mm × 100 mm, 1 / pk, SKU: 186002352, Waters. Mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile, flow rate 0.5 mL / min, column temperature 40 °C. Sample loading volume is 5 μL. Gradient elution conditions: 0 - 5 min: 5% B (initial equilibrium); 5 - 40 min: 5% → 40% B (slow gradient, improving early resolution); 40 - 55 min: 40% → 80% B (eluting hydrophobic components); 55 - 60 min: 80% → 95% B (strong elution of highly retained substances); 60 - 65 min: 95% B (column cleaning); 65 - 70 min: 5% B (re - equilibration). Detection wavelength: 210 nm; injection volume: 20 μL.

[0043] (3) Detection

[0044] Prepare solutions of cyclo(L - phenylalanyl - L - seryl) containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of cyclo(L - phenylalanine - L - proline) containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of cyclo(L - leucyl - L - proline) containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of (S)-N-(1 - hydroxy - 3 - phenylpropan - 2 - yl)acetamide containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of cyclo(glycyl - L - leucyl) containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of cyclo(isoleucine - alanine) dipeptide containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of cyclo-(L - prolyl - L - proline) containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of 1 - ribosyl - 2,3 - dione - 1,2,3,4 - tetrahydro - 6,7 - dimethylquinoxaline containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, solutions of 2'-methoxyadenosine containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, and solutions of cyclic adenosine monophosphate containing 50 mg / mL, 10 mg / mL, 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.05 mg / mL respectively, as standard solutions.

[0045] As Figure 1As shown, the retention time of adenosine cyclic phosphate is 6.2 min, the retention time of 2'-methoxyadenosine is 9.5 min, the retention time of 1-ribosyl-2,3-diketone-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline is 15.8 min, the retention time of (S)-N-(1-hydroxy-3-phenylpropan-2-yl)acetamide is 20.3 min, the retention time of cyclo(glycyl-L-leucyl) is 28.6 min, the retention time of cyclo(isoleucyl-alanine) is 32.4 min, the retention time of cyclo(L-leucyl-L-prolyl) is 38.9 min, the retention time of cyclo(L-phenylalanyl-L-seryl) is 45.2 min, the retention time of cyclo(L-phenylalanyl-L-prolyl) is 52.7 min, and the retention time of cyclo-(L-prolyl-L-prolyl) is 58.1 min, indicating that the detection method provided by the present invention can effectively detect these active ingredients in the astragalus extract composition.

[0046] Using the above standard solutions, standard curves of each active ingredient were obtained, and the standard equations of the active ingredient concentrations and chromatographic peak areas in the standard solutions were fitted. According to the standard equations and Figure 1 the chromatographic peak areas corresponding to each active ingredient in [[]], the mass percentages of adenosine cyclic phosphate, 2'-methoxyadenosine, 1-ribosyl-2,3-diketone-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline, (S)-N-(1-hydroxy-3-phenylpropan-2-yl)acetamide, cyclo(glycyl-L-leucyl), cyclo(isoleucyl-alanine), cyclo(L-leucyl-L-prolyl), cyclo(L-phenylalanyl-L-seryl), cyclo(L-phenylalanyl-L-prolyl), and cyclo-(L-prolyl-L-prolyl) in the astragalus extract composition were calculated to be 8.32%, 8.78%, 10.28%, 11.09%, 12.12%, 7.69%, 6.72%, 5.99%, 5.67%, and 6.31% respectively.

[0047] 3. Effects of astragalus extract composition on T cells

[0048] (1) Preparation of T cell and DC cell suspensions

[0049] Male BALB / c mice were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences, 6 - 8 weeks old, with a body weight of 20 ± 1 g. All animals were housed separately in a temperature-controlled room with a 12-hour light and 12-hour dark cycle.

[0050] The spleen was transferred to a 50 mL sterile centrifuge tube and a small amount of PBS was added. The spleen was cut into 1 - 2 mm pieces with ophthalmic scissors 3Small pieces. Transfer the minced spleen to a cell sieve, gently grind it with the plunger end of a syringe to allow the cells to pass through the sieve into the collection tube until there are no obvious red clumps. Add an appropriate amount of collagenase IV (100 U / mL) and DNAse I (20 μg / mL) digestion solution to the centrifuge tube, incubate at 37 °C for 20 - 30 minutes, and gently shake the centrifuge tube during this period. Filter the ground or digested cell suspension through a 70 μm cell sieve and collect the filtrate. Rinse the sieve with 5 - 10 mL of PBS to ensure that all cells pass through the sieve. Add an appropriate amount of red blood cell lysis buffer (such as ACK lysis buffer) to the collected cell suspension, gently resuspend the cells, and incubate at room temperature for 3 - 5 minutes to lyse the red blood cells. Add 2 - 3 volumes of cold PBS to terminate the lysis reaction. Centrifuge at 400 - 600 × g at 4 °C for 5 minutes and discard the supernatant. Repeat the washing once to ensure the removal of all red blood cells. Centrifuge the cell suspension at 400 - 600 × g at 4 °C for 5 minutes and discard the supernatant. Resuspend the cells with an appropriate amount of PBS or RPMI 1640 medium, adjust the cell concentration to 5×10 cells / mL, and use a hemocytometer or flow cytometer to count the cells to ensure the cell concentration and viability, which is the murine spleen cell suspension.

[0051] Use a CD4+ T cell isolation kit (Huizhi Heyuan Biotechnology) and a CD8+ T cell isolation kit (Huizhi Heyuan Biotechnology) to isolate unlabeled CD4+ T cells and CD8+ T cells from the murine spleen cell suspension. Use a mouse Dendritic Cells Enrichment Set - DM to negatively select spleen dendritic cells (DCs) from spleen cells. Count the cells using a hemocytometer and trypan blue staining method to ensure that the cell viability is greater than 90%.

[0052] (2) Cell proliferation assay

[0053] Adjust the CD4+ T cells and CD8+ T cells to 5×10 6 cells / mL respectively. In a 96 - well flat - bottom cell culture plate, add 200 μL of CD4+ T cell suspension or 200 μL of CD8+ T cell suspension to each well, along with RPMI 1640 medium containing 10% FCS with Astragalus membranaceus extraction composition at different mass concentrations, which are 0.05, 0.1, 0.5, 1.0, 5.0, 10.0 μg / mL respectively. Another control group is corresponding mass concentrations of cyclic adenosine monophosphate or 2'-methoxyadenosine, and an additional blank group with the same volume of RPMI 1640 medium containing 10% FCS. Culture at 37 °C, 5% CO2 for 4 days, detect the absorbance at 570 nM by the MTT method, and calculate the proliferation inhibition rate for the two types of T cells. Inhibition rate = 100%×(A570 value of the blank well - A570 value of the test well or control well) / A570 value of the blank well.

[0054] As Figure 2 and Figure 3 shown, whether CD4+ T cells or CD8+ T cells are co-cultured with cyclic adenosine monophosphate or 2'-methoxyadenosine respectively, their proliferation is inhibited. However, the astragalus extract composition provided by the present invention can promote T cell proliferation when co-cultured with CD4+ T cells or CD8+ T cells.

[0055] (3) Mixed lymphocyte reaction test

[0056] Antibodies: Magnetic beads coated with CD3 / CD28 antibodies (for stimulating T cells). CD4-FITC, CD8-PE, CD69-APC, CD25-PE-Cy7 fluorescently labeled antibodies (for labeling T cell subsets and activation markers).

[0057] Cell culture medium: RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin. Other reagents: PBS, trypan blue, 7-AAD (for excluding dead cells).

[0058] Treatment of DCs: DCs were treated with interferon-γ (IFN-γ) for 24 hours to enhance their antigen presentation ability. DCs were treated with mitomycin C (concentration 20 - 50 μg / mL) to make them lose their proliferative ability but retain their antigen presentation function. After treatment for 20 - 30 minutes, they were washed 3 times with PBS to remove mitomycin C.

[0059] Blank group 1: Only the treated DCs and CD4+ T cells were added to the medium and incubated in a cell culture incubator at 37°C, 5% CO 2 for 7 days.

[0060] Blank group 2: Only the treated DCs and CD8+ T cells were added to the medium and incubated in a cell culture incubator at 37°C, 5% CO 2 for 7 days.

[0061] Test group 1: In the system containing DCs and CD4+ T cells, 0.05, 0.1, 0.5, 1.0, 5.0 or 10.0 μg / mL of astragalus extract composition was added to the medium and incubated in a cell culture incubator at 37°C, 5% CO 2 for 7 days.

[0062] Control group 1: In the system containing DCs and CD4+ T cells, 0.05, 0.1, 0.5, 1.0, 5.0 or 10.0 μg / mL of cyclic adenosine monophosphate was added to the medium and incubated in a cell culture incubator at 37°C, 5% CO2 Incubate in a cell incubator for 7 days.

[0063] Control Group 2: Add 2'-methoxyadenosine at 0.05, 0.1, 0.5, 1.0, 5.0 or 10.0 μg / mL to the medium containing DCs and CD4+ T cells, and incubate at 37°C and 5% CO 2 in a cell incubator for 7 days.

[0064] Experimental Group 2: Add the astragalus extract composition at 0.05, 0.1, 0.5, 1.0, 5.0 or 10.0 μg / mL to the medium containing DCs and CD8+ T cells, and incubate at 37°C and 5% CO 2 in a cell incubator for 7 days.

[0065] Control Group 3: Add cyclic adenosine monophosphate at 0.05, 0.1, 0.5, 1.0, 5.0 or 10.0 μg / mL to the medium containing DCs and CD8+ T cells, and incubate at 37°C and 5% CO 2 in a cell incubator for 7 days.

[0066] Control Group 4: Add 2'-methoxyadenosine at 0.05, 0.1, 0.5, 1.0, 5.0 or 10.0 μg / mL to the medium containing DCs and CD8+ T cells, and incubate at 37°C and 5% CO 2 in a cell incubator for 7 days.

[0067] Set up more than three replicate wells for each experimental condition to ensure data reliability.

[0068] Detection of activation markers: After the culture is completed, take out the culture plate from the incubator, gently pipette the cells to fully mix the cells with the medium. Wash the cells 2 - 3 times with PBS, and centrifuge to remove the supernatant. Resuspend the cells with an appropriate amount of PBS, add CD4-FITC, CD8-PE, CD69-APC, CD25-PE-Cy7 fluorescently labeled antibodies, and incubate at room temperature in the dark for 30 minutes to label CD4+ T cells, CD8+ T cells and their activation markers CD69 and CD25. Wash the cells 2 - 3 times with PBS, and centrifuge to remove the unbound antibodies. Add 7-AAD staining solution, and incubate at room temperature in the dark for 10 min to exclude dead cells.

[0069] The positive rates of CD69 and CD25 were detected by flow cytometry, and the inhibition rates of the astragalus extract composition, adenosine cyclic phosphate, and 2'-methoxyadenosine provided by the present invention on T cell activation were calculated. The cells were resuspended with an appropriate amount of PBS for flow cytometry detection. The parameters of the flow cytometer were set, and the parameters were adjusted according to the fluorescence intensity and scatter plot of the cells to ensure the accuracy of the data. By the flow cytometry analysis software, the positive rates of CD69 and CD25 in CD4+ T cells and CD8+ T cells in each group were statistically analyzed respectively. The inhibition rate was calculated as follows: Inhibition rate = (Positive rate of the blank group - Positive rate of the treatment group) / Positive rate of the control group × 100%. The positive rates of CD69 and CD25 were detected by flow cytometry, and the inhibition rates of the astragalus extract composition, adenosine cyclic phosphate, and 2'-methoxyadenosine provided by the present invention on T cell activation were calculated. The experimental data were statistically analyzed to compare the differences between different concentrations of the astragalus extract composition, adenosine cyclic phosphate, and 2'-methoxyadenosine and the blank group respectively.

[0070] As Figure 4 and Figure 5 shown, whether adenosine cyclic phosphate or 2'-methoxyadenosine was added to the culture medium of CD4+ T cells or CD8+ T cells, and co-cultured with DCs cells for the one-way MLR test, the activation of both types of T cells was inhibited. However, when the astragalus extract composition provided by the present invention was used to treat CD4+ T cells or CD8+ T cells, and after the MLR test, it could promote T cell activation.

[0071] The beneficial immunobiological functions after CD4+ T cell activation. For example, activated CD4+ T cells can assist B cells in producing antibodies and enhance humoral immunity. Activated CD4+ T cells can also activate macrophages and enhance their phagocytosis and bactericidal abilities. Activated CD4+ T cells can enhance their regulation of immune responses, maintain immune tolerance, prevent the occurrence of autoimmune diseases, and at the same time help restore immune homeostasis in the late stage of the immune response. After activation, CD4+ T cells can secrete cytokines such as IL-2, which can promote the proliferation and differentiation of CD8+ T cells and enhance their cytotoxicity. After activation, CD4+ T cells are more likely to differentiate into memory T cells. These memory cells can be rapidly activated when encountering the same antigen again, generating a faster and more effective immune response, providing long-term immune protection for the body and reducing the risk of reinfection.

[0072] Beneficial immunobiological functions after CD8+ T cell activation. For example, activated CD8+ T cells can directly kill target cells, such as by recognizing the antigen-MHC class I molecule complex on the surface of target cells, releasing perforin and granzyme, inducing apoptosis of target cells, and being able to eliminate virus-infected cells and tumor cells, preventing the spread of pathogens and the growth of tumors. Activated CD8+ T cells are more likely to secrete a variety of cytokines, such as IFN-γ, TNF-α, etc., which helps to eliminate pathogens and play a regulatory role in the immune response. After activation, CD8+ T cells are more likely to differentiate into memory T cells. These memory cells can be rapidly activated when encountering the same antigen again, generating a faster and more effective immune response, providing long-term immune protection for the body and reducing the risk of reinfection.

[0073] Thus, it can be seen that the astragalus extract composition provided by the present invention can activate T cells, thereby enhancing humoral immunity, regulating the immune response, maintaining immune tolerance, enhancing immune memory, providing long-term immune protection and reducing the risk of reinfection. However, both cyclic adenosine monophosphate and 2'-methoxyadenosine inhibit the activation of CD4+ T cells or CD8+ T cells. Although it can reduce the intensity of the immune response, it is not conducive to enhancing humoral immunity and secreting cytokines to achieve the elimination of pathogens.

[0074] 4. Effects of astragalus extract composition on T cells

[0075] (1) Experimental animals, reagents and instruments

[0076] SD rats, Cyagen Biosciences. Bovine serum albumin, imported and sub-packed by Shanghai Biochemical Company. Urinary protein kit, creatinine kit, and urea nitrogen kit were produced by Nanjing Jiancheng Bioengineering Company. Beckman DU-640 ultraviolet spectrophotometer; HH-60 type rapid constant temperature digital display water tank, produced by Changzhou Guohua Electric Appliance Co., Ltd.; Rat metabolic cage: produced by Suzhou Experimental Animal Cage Factory; Axioskop 2plus advanced biological microscope and Axiovision image analysis processing system: produced by Carl Zeiss AG, Germany.

[0077] (2) Methods

[0078] Rats (180 - 220 g in weight, with equal numbers of males and females) were all injected with bovine serum albumin via the tail vein once (dissolved in 1 mL of phosphate buffer (PBS) with pH 7.4. The doses from the 1st day to the 7th day were 1.0, 1.0, 1.0, 1.5, 1.5, 2.0, 2.0 mg in sequence. One week later, the urinary protein content of the mice was 42.08 ± 5.39 urinary protein / mg (the normal rats were 5.39 ± 0.87 urinary protein / mg), indicating that the model of proteinuria nephritis rats was successfully established.

[0079] From the second week, 1.5 mg of bovine serum albumin was injected into the caudal vein daily under aseptic conditions for 2 consecutive weeks. The model rats were divided into a model group, experimental group 1, experimental group 2, experimental group 3, and experimental group 4. In experimental group 1, the rats were intragastrically administered with the astragalus extract composition at a dose of 20 mg / Kg body weight daily in the second week. In experimental group 2, the rats were intragastrically administered with astragaloside at a dose of 20 mg / Kg body weight daily in the second week. In experimental group 3, the rats were intragastrically administered with adenosine cyclic phosphate at a dose of 20 mg / Kg body weight daily in the second week. In experimental group 4, the rats were intragastrically administered with 2'-methoxyadenosine at a dose of 20 mg / Kg body weight daily in the second week. After 2 consecutive weeks of administration, the rats were placed in a metabolic cage, 24-hour urine was collected, and the urine protein content was measured. At the same time, blood was collected to separate serum, and the serum creatinine and urea nitrogen concentrations were measured.

[0080] (3) Results

[0081] As Figure 6 shown, the urine protein, serum creatinine, and serum urea nitrogen contents of the rats in the model group were significantly higher than those in the normal group (**, p < 0.01, significantly higher than the normal group), indicating that the nephritis proteinuria model rats were successfully modeled. After intragastric administration of astragaloside to rats, the nephritis proteinuria index of the rats did not decrease (still significantly higher than the normal group, **, p < 0.01). After intragastric administration of adenosine cyclic phosphate or 2'-methoxyadenosine to rats (still significantly higher than the normal group, *, p < 0.05), the nephritis proteinuria index of the rats decreased, but neither was as obvious as the astragalus extract composition provided by the present invention (no significant difference from the normal group, ns, p < 0.05).

[0082] In this experiment, the rats in each group were also sacrificed, and both kidneys were fixed in 10% formaldehyde for pathological histological examination. The protein cast score and blood focus grading score in the pathological histological pictures were analyzed per 10*10 fields of view by Axiovision image analysis. The experimental results were statistically analyzed by the t-test between groups to analyze the differences between groups.

[0083] As Figure 7 shown, the protein cast score and blood focus grading score of the model group were significantly higher than those of the normal group (**, p < 0.01, significantly higher than the normal group), indicating that the nephritis proteinuria model rats were successfully modeled. After intragastric administration of astragaloside to rats, the protein cast score and blood focus grading score did not decrease (still significantly higher than the normal group, **, p < 0.01). After intragastric administration of adenosine cyclic phosphate or 2'-methoxyadenosine to rats, the protein cast score and blood focus grading score of the rats decreased, but were still significantly higher than the normal group (*, p < 0.05). However, the astragalus extract composition provided by the present invention had no significant difference from the normal group (ns, p < 0.05).

[0084] This shows that the astragalus extract composition provided by the present invention can significantly reduce the urinary protein excretion of rats with nephritis induced by bovine serum albumin, and lower the levels of serum creatinine and serum urea nitrogen. The pathological results show that compared with the normal group, there are no obvious changes in the glomeruli of the model group, but the scores of protein casts and bleeding foci grading are significantly increased. The astragalus extract composition provided by the present invention can reduce the protein casts and the generation of bleeding foci in rats with nephritis induced by bovine serum albumin. That is to say, the astragalus extract composition provided by the present invention has an obvious improvement effect on rats with nephritis induced by bovine serum albumin and has the application prospect for treating nephritis proteinuria.

[0085] The above is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a composition for improving immunity based on natural ingredients of Astragalus, characterized in that: include: Performing ethanol reflux extraction on the dried material of Guangxi native astragalus to obtain a first paste; Extracting the first paste with a macroporous adsorption resin to obtain a second paste; The second paste is subjected to silica gel column chromatography to obtain a third paste; The third paste is subjected to gel chromatography to obtain a fourth paste; The fourth paste is subjected to ODS column chromatography to obtain a fifth paste; The fifth paste is freeze-dried to obtain the astragalus extract composition.

2. The preparation method according to claim 1, characterized in that: The dried material of Guangxi native astragalus was added to 58% ethanol solution at a solid-liquid ratio of 1:20, and after being fully soaked for 48 hours, it was heated to reflux and extracted for 3 times at 75°C, each time for 3 hours, and the extracts were combined and concentrated to obtain the first paste.

3. The preparation method according to claim 1, characterized in that: The first paste was suspended in water and then statically loaded onto a pretreated D101 macroporous adsorption resin column (column height-to-diameter ratio of 10:1). Three column bed volumes were eluted with water and 30% ethanol at a flow rate of 0.15% bed volume / min, and the 30% ethanol eluate was collected and concentrated to obtain a second paste.

4. The preparation method according to claim 1, characterized in that: The second paste was prepared into a 1 g / mL solution with 30% ethanol aqueous solution and statically loaded onto a pretreated silica gel chromatography column (column bed height-to-diameter ratio of 30:1), and gradient eluted with a mixed solvent of dichloromethane and methanol at a flow rate of 0.5% column volume / min, with an elution gradient of a mixed volume ratio of dichloromethane to methanol of 9:1 to 8:2, for a total of 2 column volumes, the eluate was collected, combined and concentrated to obtain a third paste.

5. The preparation method according to claim 1, characterized in that: The third paste was prepared into a 350 mg / mL sample solution with a mixed solution of dichloromethane and methanol in a volume ratio of 8.5:1.5, and statically loaded onto a pre-equilibrated Sephadex LH-20 gel column (column bed height-to-diameter ratio of 30:1), eluted with methanol at a flow rate of 0.5% column volume / min, and 0.25 to 1.16 column volumes of the eluate were collected, combined and concentrated to obtain a fourth paste.

6. The preparation method according to claim 1, characterized in that: The fourth paste was prepared into a 200 mg / mL sample solution with methanol and statically loaded onto the ODS column. It was gradient eluted with 22% to 35% ethanol aqueous solution for 50 column volumes at a flow rate of 0.5% column volume / min. 30 to 40 column volumes of the eluate were collected, combined and concentrated to obtain the fifth paste.

7. The composition obtained by the method according to any one of claims 1 to 6, comprising 8.32% by mass of cyclic adenosine monophosphate, 8.78% by mass of 2'-methoxyadenosine, 10.28% by mass of 1-ribosyl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline, 11.09% by mass of (S)-N-(1-hydroxy-3-phenylpropan-2-yl)acetamide, and 11.28% by mass of 1-ribosyl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline. The mass percentage of the cyclic (glycyl-L-leucyl) is 12.12%, the mass percentage of the cyclic (isoleucine-alanine) is 7.69%, the mass percentage of the cyclic (L-leucine-L-proline) is 6.72%, the mass percentage of the cyclic (L-phenylalanyl-L-seryl) is 5.99%, the mass percentage of the cyclic (L-phenylalanine-L-proline) is 5.67%, and the mass percentage of the cyclic (L-proline-L-proline) is 6.31%.

8. Use of the composition obtained by the method of any one of claims 1 to 6 in the preparation of an agent for promoting T cell proliferation.

9. Use of the composition obtained by the method of any one of claims 1 to 6 in the preparation of an agent for promoting T cell activation.

10. Use of the composition obtained by any one of claims 1 to 6 in the preparation of an agent for preventing or treating nephritic proteinuria.

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