An immune enhancement composition derived from BP and its preparation method and application

Abstract

The present invention provides an immunopotentiation composition derived from BP, and a preparation method and application thereof, and relates to the technical field of effective component separation. The preparation method of the immunopotentiation composition is as follows: (1) the BP raw material is dissolved in water and ultrafiltration is performed to obtain an ultrafiltrate and a concentrated solution; an ultrafiltration membrane with a molecular weight of 2kDa-5kDa is used for ultrafiltration; (2) the ultrafiltrate is passed through a D101 macroporous adsorption resin for adsorption; (3) at least one of water and an ethanol solution is used for elution, and each eluate is collected to obtain an immunopotentiation composition; the volume concentration of the ethanol solution is 40%-95%; when the ethanol solution is used for elution, the elution method is gradient elution. The separated immunopotentiation composition is used for the preparation of immunomodulatory drugs, has a high cell proliferation effect, improves the killing rate of natural killer cells, and can also enhance the phagocytic ability of phagocytes.

Description

Technical Field

[0001] The invention belongs to the technical field of effective component separation, and specifically relates to an immune enhancement composition derived from BP factor, and a preparation method and application thereof. Background Art

[0002] BP element (ribonucleic acid II for injection) is a biochemical drug made from healthy bovine pancreas using modern bioengineering technology, membrane separation, low-temperature centrifugation and other biological extraction and purification methods. The main components of BP element are ribonucleotide substances, which contain active ingredients such as ribonucleic acid, phospholipids, total sugar, protein, amino sugar, and the main component is ribonucleic acid.

[0003] Chinese invention patent CN102818867A discloses a quality control method for RNAi II for injection, wherein the nuclease hydrolysis solution of the test substance is subjected to high performance liquid chromatography analysis, the chromatographic column used is an Agilent ZORBAX SB-AQC18 chromatographic column, and the mobile phase is a mixture of formic acid solution and acetonitrile solution; after component analysis by the method of the invention, it is determined that the test substance contains five substances, namely, cytosine nucleotide, uracil nucleotide, guanine nucleotide, guanosine nucleoside and adenine nucleoside, and the test substance is or can be RNAi II for injection.

[0004] As an immunomodulatory drug, BP is suitable for the adjuvant treatment of pancreatic cancer, liver cancer, gastric cancer, lung cancer, breast cancer, soft tissue sarcoma and other cancers. The drug also has a good effect on the adjuvant treatment of hepatitis B, and can also be used for various diseases caused by other low immune function. Chinese invention patent CN118477034A discloses the use of RNA II for injection in the preparation of drugs for the treatment of hematopoietic dysfunction. The study found that RNA II for injection can improve the pathological condition of bone marrow with hematopoietic dysfunction, increase the number of hematopoietic stem cells, long-term hematopoietic stem cells, B lymphocytes and macrophages in the bone marrow, and has the function of promoting hematopoiesis. In addition, RNA II for injection can promote the polarization of macrophages to M1 type, and increase the level of hematopoietic promoting factors released by M1 macrophages, so as to play a hematopoietic function. Therefore, RNA II for injection can improve hematopoietic dysfunction and has a good hematopoietic function.

[0005] Although BP has an immunoenhancing effect, the immunomodulatory effects of the interactions between different components in BP are different. At present, there is no research on the separation and extraction of effective components in BP to improve immune efficacy. Based on this, it is necessary to study the separation method of the effective components of BP in order to obtain a BP-derived isolated and extracted substance with significant immunoenhancing effect. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides an immunoenhancing composition derived from BP, and a preparation method and application thereof. The BP raw material is first dissolved in water, ultrafiltered, and the obtained ultrafiltrate is then adsorbed by D101 macroporous adsorption resin, and finally eluted in sequence with water and ethanol solutions of different volume concentrations to obtain active parts with different immunoenhancing activities, i.e., an immunoenhancing composition. The separated immunoenhancing composition is applied to the preparation of immunomodulatory drugs, which has a high cell proliferation effect, improves the killing rate of natural killer cells, and can also enhance the phagocytic ability of phagocytes.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] First, the present invention provides a method for preparing an immunoenhancing composition, comprising the steps of:

[0009] (1) The BP raw material is dissolved in water and subjected to ultrafiltration treatment to obtain an ultrafiltrate (BPU) and a concentrate (BPC); the ultrafiltration treatment specifically uses an ultrafiltration membrane with a molecular weight of 2 kDa-5 kDa for ultrafiltration;

[0010] (2) Passing the ultrafiltrate through D101 macroporous adsorption resin for adsorption;

[0011] (3) Eluting with at least one of water and ethanol solution, collecting each eluate to obtain an immunopotentiating composition; the volume concentration of the ethanol solution is 40%-95%; when using the ethanol solution for elution, the elution method is gradient elution.

[0012] Preferably, in step (1), during the process of adding water to dissolve, the solid-liquid ratio of BP raw material to water is 400-600:800-1200 g / mL.

[0013] Further preferably, in step (1), during the process of adding water to dissolve, the solid-liquid ratio of BP raw material to water is 500:1000 g / mL.

[0014] Preferably, in step (1), the ultrafiltration treatment uses an ultrafiltration membrane with a molecular weight of 3 kDa.

[0015] Preferably, in step (1), if the BP raw material contains ethanol, after dissolving in water, it is placed under heating conditions of 40-50°C for volatilization of the ethanol, and then ultrafiltration is performed after the alcohol taste is gone; more preferably, the heating condition is 45°C.

[0016] Preferably, in step (2), during the adsorption process, the column diameter-to-height ratio is 1:4-12, and the sample volume ratio is 1:20-30.

[0017] Further preferably, in step (2), during the adsorption process, the column diameter-to-height ratio is 1:8 and the sample volume ratio is 1:25.

[0018] Preferably, in step (3), the volume concentration of the ethanol solution used for elution is an endpoint value, an arbitrary point value in the middle, or an arbitrary range value within the range, and is not limited to 40%-70%, 40%-50%, 60%-80%, 90%-95%, 70%-95%, 40%, 70% or 95%.

[0019] Further preferably, in step (3), if an ethanol solution is used for elution, the obtained eluate needs to be subjected to ethanol recovery until there is no alcohol taste in the eluate.

[0020] Further preferably, in step (3), the elution is specifically as follows: first adding water for elution, then using an ethanol solution with a volume concentration of 40%-50% for elution, then using an ethanol solution with a volume concentration of 60%-80% for elution, and finally using an ethanol solution with a volume concentration of 90%-95% for elution.

[0021] More preferably, in step (3), the elution is specifically as follows: first, water is added for elution, then, an ethanol solution with a volume concentration of 40% is used for elution, then, an ethanol solution with a volume concentration of 70% is used for elution, and finally, an ethanol solution with a volume concentration of 95% is used for elution.

[0022] Preferably, in step (3), the elution solution is water.

[0023] Preferably, in step (3), the elution flow rate is 1-3 BV / h, and the amount of each eluent used is 3-6 BV.

[0024] Further preferably, in step (3), the elution flow rate is 2BV / h, and the amount of each eluent used is 4BV.

[0025] Preferably, step (3) further comprises the steps of concentrating and freeze-drying each eluate after elution.

[0026] In addition, the present invention provides an immune enhancement composition derived from BP, which is prepared by the above preparation method.

[0027] Preferably, the immune enhancing composition is a water eluate substance.

[0028] Preferably, when the immunoenhancing composition is an ethanol solution eluent substance, it is 40%-50% ethanol solution eluent substance, 60%-80% ethanol solution eluent substance, 90%-95% ethanol solution eluent substance respectively.

[0029] More preferably, the immune enhancing composition is a 40%-50% ethanol solution eluent substance.

[0030] Further preferably, the immunoenhancing composition is a 40% ethanol solution eluate substance, a 70% ethanol solution eluate substance, or a 95% ethanol solution eluate substance.

[0031] Further preferably, the immune enhancing composition is a water eluent substance or a 40% ethanol solution eluent substance.

[0032] Preferably, the ingredients of the immune enhancing composition include uracil, hypoxanthine and xanthine.

[0033] Then, the present invention provides an immune enhancement composition, which comprises the following components by weight: 68-92 parts of uracil, 5-22 parts of hypoxanthine and 3.5-8 parts of xanthine.

[0034] Preferably, the immune enhancing composition comprises the following components, measured by weight: 74-86 parts of uracil, 10-20 parts of hypoxanthine and 4-6 parts of xanthine.

[0035] Further preferably, in the immune enhancing composition, the mass ratio of uracil, hypoxanthine and xanthine is 78:12:5.

[0036] Finally, the present invention provides the use of the above-mentioned immune enhancement composition in the preparation of immune regulation drugs.

[0037] Preferably, the immunomodulatory drug is a drug that has the effects of proliferating lymphocytes, increasing the killing rate of natural killer cells and / or increasing the phagocytic ability of phagocytes.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. In the immune enhancement composition derived from BP of the present invention, the components uracil, hypoxanthine and xanthine interact with each other and have the effect of significantly enhancing immunity.

[0040] 2. The immunopotentiating composition of the present invention is a product obtained by ultrafiltration of BP, adsorption by D101 macroporous adsorption resin, and elution with water and / or ethanol solution. The eluted immunopotentiating composition has different immune effects. The present invention adopts specific adsorption materials and eluents to significantly improve the killing rate of the composition on natural killer cells and the phagocytic ability of phagocytes, and improves the ability of cell proliferation.

[0041] 3. The immunopotentiating composition of the present invention is used in the preparation of immunomodulatory drugs, and has the effects of significantly proliferating lymphocytes, increasing the killing rate of natural killer cells, and improving the phagocytic ability of phagocytes. DETAILED DESCRIPTION

[0042] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0043] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0044] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0045] In the present invention, the manufacturer of BP raw material alcohol ointment is Jilin Aodong Pharmaceutical Group Yanji Co., Ltd.; uracil control preparation is purchased from China Food and Drug Inspection Institute; hypoxanthine control preparation is purchased from China Food and Drug Inspection Institute; xanthine control preparation is purchased from China Food and Drug Inspection Institute. Products from different manufacturers have no significant effect on the effect.

[0046] Basic Example

[0047] The immunoenhancing composition comprises the following components by weight: 74-86 parts of uracil, 10-20 parts of hypoxanthine and 4-6 parts of xanthine. Within the scope of this basic embodiment, the technical effects of the present invention can be achieved and the technical problems of the present invention can be solved.

[0048] Example 1

[0049] A method for preparing an immune enhancement composition derived from BP:

[0050] (1) Dissolve 500 g of BP raw material alcohol paste in 1000 mL of water, place in a 45°C water bath for heating, evaporate the ethanol until there is no alcohol smell, and then use a 3 kDa ultrafiltration membrane for ultrafiltration for 24 h to obtain ultrafiltrate (BPU) and concentrate (BPC);

[0051] (2) The ultrafiltrate was passed through D101 macroporous adsorption resin (column diameter-to-height ratio was 1:8, sample volume ratio was 1:25) for adsorption;

[0052] (3) After adsorption, the mixture was eluted with water, 40% ethanol solution, 70% ethanol solution and 95% ethanol solution in sequence, and the eluates were collected to obtain: water eluate, 40% ethanol solution eluate, 70% ethanol solution eluate and 95% ethanol solution eluate; ethanol was recovered from the eluate until there was no alcohol taste, and the mixture was freeze-dried to obtain four kinds of immune enhancing compositions: BPU-1 (water eluate substance), BPU-2 (40% ethanol solution eluate substance), BPU-3 (70% ethanol solution eluate substance) and BPU-4 (95% ethanol solution eluate substance).

[0053] After component analysis, the components of BPU-1, BPU-2, BPU-3 and BPU-4 are uracil, hypoxanthine and xanthine.

[0054] In the immune enhancing composition BPU-1, the mass ratio of uracil (compound 1), hypoxanthine (compound 2), and xanthine (compound 3) is 78:12:5.

[0055] In the immunopotentiating composition BPU-2, the mass ratio of uracil, hypoxanthine and xanthine is 75:15:4.5.

[0056] In the immune enhancing composition BPU-3, the mass ratio of uracil, hypoxanthine and xanthine is 68:5:8.

[0057] In the immune enhancing composition BPU-4, the mass ratio of uracil, hypoxanthine and xanthine is 92:22:3.5.

[0058] Example 2

[0059] The difference from Example 1 is that the molecular weight of the ultrafiltration membrane in step (1) is 2 kDa. The rest is the same as Example 1.

[0060] According to component analysis, the mass ratio of uracil: hypoxanthine: xanthine in the immune enhancement composition BPU-1 is 76:11.5:4.8.

[0061] In the immunopotentiating composition BPU-2, the mass ratio of uracil, hypoxanthine and xanthine is 74.5:17:4.2.

[0062] In the immune enhancing composition BPU-3, the mass ratio of uracil, hypoxanthine and xanthine is 68:6:7.5.

[0063] In the immune enhancing composition BPU-4, the mass ratio of uracil, hypoxanthine and xanthine is 90:21:3.6.

[0064] Example 3

[0065] Different from Example 1, the elution in step (3) is specifically as follows: elution is carried out in sequence with water, 50% by volume ethanol solution, 80% by volume ethanol solution, and 90% by volume ethanol solution to obtain BPU-1 (water eluent substance), BPU-2 (50% ethanol solution eluent substance), BPU-3 (80% ethanol solution eluent substance), and BPU-4 (90% ethanol solution eluent substance), respectively.

[0066] The rest are the same as in Example 1.

[0067] According to component analysis, the mass ratio of uracil, hypoxanthine and xanthine in the immune enhancement composition BPU-1 is 78:12:5.

[0068] In the immune enhancing composition BPU-2, the mass ratio of uracil, hypoxanthine and xanthine is 73:10:6.

[0069] In the immunopotentiating composition BPU-3, the mass ratio of uracil, hypoxanthine and xanthine is 70:8:7.

[0070] In the immune enhancing composition BPU-4, the mass ratio of uracil, hypoxanthine and xanthine is 89:17:5.5.

[0071] Example 4

[0072] An immune enhancement composition comprises the following components by weight: 74 parts of uracil, 20 parts of hypoxanthine and 6 parts of xanthine.

[0073] Example 5

[0074] An immune enhancement composition comprises the following components by weight: 86 parts of uracil, 10 parts of hypoxanthine and 4 parts of xanthine.

[0075] Comparative Example 1

[0076] The difference from Example 1 is that in the method for preparing the immunopotentiating composition, the D101 macroporous adsorption resin in step (2) is replaced by HPD826 macroporous adsorption resin. The rest is the same as Example 1.

[0077] (1) Dissolve 500 g of BP raw material alcohol paste in 1000 mL of water, place in a 45°C water bath for heating, evaporate the ethanol until there is no alcohol smell, and then use a 3 kDa ultrafiltration membrane for ultrafiltration for 24 h to obtain ultrafiltrate (BPU) and concentrate (BPC);

[0078] (2) The ultrafiltrate was passed through D101 macroporous adsorption resin (column diameter-to-height ratio was 1:8, sample volume ratio was 1:25) for adsorption;

[0079] (3) After adsorption, the mixture was eluted with water, 40% ethanol solution, 70% ethanol solution and 95% ethanol solution in sequence, and the eluates were collected to obtain: water eluate, 40% ethanol solution eluate, 70% ethanol solution eluate and 95% ethanol solution eluate; ethanol was recovered from the eluate until there was no alcohol taste, and the mixture was freeze-dried to obtain four kinds of immune enhancing compositions: BPU-1 (water eluate substance), BPU-2 (40% ethanol solution eluate substance), BPU-3 (70% ethanol solution eluate substance) and BPU-4 (95% ethanol solution eluate substance).

[0080] Comparative Example 2

[0081] The difference from Example 1 is that in the ultrafiltration treatment in step (1), the ultrafiltration membrane used has a molecular weight of 8 kDa.

[0082] The rest is the same as implementation 1.

[0083] Comparative Example 3

[0084] The difference from Example 1 is that the elution order in step (3) is: after adsorption, firstly perform elution with 95% ethanol solution to obtain an immune enhancing composition (Comparative Example 4 BPU-1); then perform elution with 70% ethanol solution to obtain an immune enhancing composition (Comparative Example 4 BPU-2); then perform elution with 40% ethanol solution to obtain an immune enhancing composition (Comparative Example 4 BPU-3); finally perform elution with water to obtain an immune enhancing composition (Comparative Example 4 BPU-4).

[0085] The rest is the same as implementation 1.

[0086] Comparative Example 4

[0087] An immune enhancement composition comprises the following components by weight: 10 parts of uracil, 4 parts of hypoxanthine and 86 parts of xanthine.

[0088] Comparative Example 5

[0089] An immunoenhancing composition comprises the following components by weight: 78 parts of cytosine, 12 parts of hypoxanthine and 5 parts of guanine.

[0090] Test for immune enhancement

[0091] 1. Materials and reagents

[0092] Test samples: BP factor (BP factor raw material alcohol paste); ultrafiltrate (BPU, the ultrafiltrate obtained in step (1) is concentrated under reduced pressure, freeze-dried, and then tested for immunopotentiating effect); concentrated solution (BPC, the concentrated solution in step (1) is concentrated under reduced pressure, freeze-dried, and then tested for immunopotentiating effect); BPU-1, BPU-2, BPU-3 and BPU-4 of each embodiment and comparative example after freeze-drying.

[0093] Experimental animals: ICR male mice (20.0±2.0 g) ICR mice, clean grade, provided by the Experimental Animal Center of Jilin University, Animal Certificate No.: (Jilin) ​​2008-0005.

[0094] Experimental reagents: RPMI 1640 medium, D-Hank's, penicillin / streptomycin solution, all purchased from Gibco; fetal calf serum FCS, purchased from Lanzhou Minhai Bioengineering Co., Ltd., a Sino-US joint venture; concanavalin A (ConA), bacterial lipopolysaccharide (LPS), HEPES, MTT, all purchased from Sigma; dimethyl sulfoxide and neutral red, all purchased from Amersco; EDTA, purchased from Shanghai Shenggong Bioengineering Co., Ltd.

[0095] Experimental instruments: Bio-rad 680 microplate reader, produced by Bio-rad, USA; CO2 incubator, produced by Sanyo, Japan; TGL-16G-A low-temperature centrifuge, produced by Shanghai Anting Scientific Instrument Factory.

[0096] Cell line: YAC-1 cell line was provided by Jilin Cancer Hospital.

[0097] 2. Immunity enhancement

[0098] 2.1 Effect on proliferation of spleen lymphocytes in normal mice

[0099] (1) Effect on the proliferation rate of spleen lymphocytes in normal mice induced by ConA

[0100] ICR mice (18-22 g) were randomly divided into groups of 7 and housed in a standard environment to adapt to the new environment.

[0101] Mouse groups: BP high-dose group, Example 1 BPU high-dose group, Example 1 BPC high-dose group, Example 1 BPU-1 high-dose group, Example 1 BPU-1 low-dose group, Example 1 BPU-2 high-dose group, Example 1 BPU-3 high-dose group, Example 1 BPU-4 high-dose group, Example 2 BPU-1 high-dose group, Example 2 BPU-2 high-dose group, Example 2 BPU-3 high-dose group, Example 2 BPU-4 high-dose group, Example 3 BPU-2 high-dose group, Example 3 BPU-3 high-dose group, Example 3 BPU-4 high-dose group, Example 4 high-dose group, Example 5 high-dose group, Comparative Example 1 BPU-1 high-dose group, Comparative Example 1 BPU-2 high-dose group, Comparative Example 1 BPU-3 high-dose group, Comparative Example 1 BPU-4 high-dose group, Comparative Example 2 BPU-1 high-dose group, Comparative Example 3 BPU-1 high-dose group, Comparative Example 3 BPU-2 high-dose group, Comparative Example 3 BPU-3 high-dose group, comparative example 3 BPU-4 high-dose group, comparative example 4 high-dose group, comparative example 5 high-dose group; each group was intraperitoneally injected with the corresponding composition. The normal control group was intraperitoneally injected with normal saline. The injection amount of the high-dose group was 35 mg / kg, and the injection amount of the low-dose group was 17.5 mg / kg.

[0102] The mice were given once a day for 10 consecutive days. One hour after the last administration, the animals were killed. The mice were opened aseptically on a clean bench, and the spleen was taken out and placed in 5 mL Hank ' s plate to wash, remove blood, and absorb Hank ' s, ground with a glass slide with a grinding surface, and ' s gently rinse the sieve to prepare spleen cell suspension, and the 2% trypan blue test confirmed that the cell survival rate was >95%. The prepared cell suspension was centrifuged at 1500r / min, the supernatant was discarded, 2mL of red blood cell lysis buffer was added, and it was placed in a 37℃ water bath for 5min, centrifuged at 1500r / min, the supernatant was discarded, and the suspension was purified by Hank's ' The cells were washed twice and the cell number was adjusted to 5 × 10 6 / mL, inoculated into 96-well cell culture plates, 180μL per well, placed in a 37℃ constant temperature incubator with 5% CO2 for 4 hours, added 10μL of ConA (final concentration 5μg / mL) to each well, continued to culture for 48h, added 10μl of MTT solution with a concentration of 5mg / mL to each well, set up 3 replicates and continued to culture for 4h, aspirated the culture medium, added 100μL of DMSO solution, oscillated for 10min, measured the optical density (OD) value at a wavelength of 570nm on a microplate reader, and calculated the proliferation rate.

[0103] Splenic lymphocyte proliferation rate (%) = OD value of experimental group / OD value of control group × 100%.

[0104] The results of the effects of the tested samples on the proliferation rate of normal mouse spleen lymphocytes induced by ConA are shown in Table 1.

[0105] Table 1

[0106]

[0107] In Table 1, compared with the normal group, △ P<0.05, △△ P<0.01, with significant difference; compared with the high-dose group of BPU-1 in Example 1, # P<0.05, ## P<0.01, there is a significant difference.

[0108] The results in Table 1 show that, compared with the normal control group, the immunoenhancing compositions BP, BPU, BPU-1, BPU-2 and BPU-3 of the present invention have significantly higher mouse lymphocyte proliferation rates under concanavalin stimulation than the normal group, and there is a significant difference. The immunoenhancing compositions of BPC and the comparative example group have lower effects than those of the embodiments.

[0109] (2) Effect on the proliferation rate of normal mouse spleen lymphocytes induced by LPS

[0110] ICR mice (18-22 g) were randomly divided into groups of 7 and housed in a standard environment to adapt to the new environment.

[0111] The mice were divided into groups: high-dose BP group, high-dose BPU group in Example 1, high-dose BPC group in Example 1, high-dose BPU-1 group in Example 1, low-dose BPU-1 group in Example 1, high-dose BPU-2 group in Example 1, high-dose BPU-3 group in Example 1, high-dose BPU-4 group in Example 1, high-dose group in Comparative Example 4, and high-dose group in Comparative Example 5; each group was intraperitoneally injected with the corresponding composition. The normal control group was intraperitoneally injected with physiological saline. The injection amount of the high-dose group was 35 mg / kg, and the injection amount of the low-dose group was 17.5 mg / kg.

[0112] The mice were given once a day for 10 consecutive days. One hour after the last administration, the animals were killed. The mice were opened aseptically on a clean bench, and the spleen was taken out and placed in 5 mL Hank ' s plate to wash, remove blood, and absorb Hank ' s, ground with a glass slide with a grinding surface, and 's gently rinse the sieve to prepare spleen cell suspension, and the 2% trypan blue test confirmed that the cell survival rate was >95%. The prepared cell suspension was centrifuged at 1500r / min, the supernatant was discarded, 2mL of red blood cell lysis buffer was added, and it was placed in a 37℃ water bath for 5min, centrifuged at 1500r / min, the supernatant was discarded, and the suspension was purified by Hank's ' The cells were washed twice and the cell number was adjusted to 5 × 10 6 / mL, inoculated into 96-well cell culture plates, 180μL per well, placed in a 37℃ constant temperature incubator with 5% CO2 for 4 hours, added 10μL of LPS (final concentration 10μg / mL) to each well, continued to culture for 48h, added 10μl of MTT solution with a concentration of 5mg / mL to each well, set up 3 replicates and continued to culture for 4h, aspirated the culture medium, added 100μL of DMSO solution, oscillated for 10min, measured the optical density (OD) value at a wavelength of 570nm on a microplate reader, and calculated the proliferation rate.

[0113] Splenic lymphocyte proliferation rate (%) = OD value of experimental group / OD value of control group × 100%.

[0114] The results of the effects of the tested samples on the proliferation rate of normal mouse spleen lymphocytes induced by LPS are shown in Table 2.

[0115] Table 2

[0116]

[0117] In Table 2, compared with the normal group, △△△ P < 0.001, △△ P<0.01, △ P<0.05, with significant difference. Compared with the high-dose group of BPU-1 in Example 1, # P<0.05, ## P<0.01, # P<0.05, there is a significant difference.

[0118] 2.2 Effect on the killing rate of NK cells in normal mice

[0119] ICR mice (18-22 g) were randomly divided into groups, with 7 mice in each group.

[0120] The mice were divided into groups: high-dose BP group, high-dose BPU group in Example 1, high-dose BPC group in Example 1, high-dose BPU-1 group in Example 1, low-dose BPU-1 group in Example 1, high-dose BPU-2 group in Example 1, high-dose BPU-3 group in Example 1, high-dose BPU-4 group in Example 1, high-dose group in Comparative Example 4, and high-dose group in Comparative Example 5; each group was intraperitoneally injected with the corresponding composition. The normal control group was intraperitoneally injected with physiological saline. The injection amount of the high-dose group was 35 mg / kg, and the injection amount of the low-dose group was 17.5 mg / kg.

[0121] The animals were sacrificed 1 hour after the last administration, once a day, for 10 consecutive days. The animals were immersed in 75% ethanol for about 2 minutes, and the abdomen was opened aseptically in a clean bench. The spleen was removed and placed in a plate containing 5 mL of Hank's to clean and remove the blood. The Hank's was aspirated and ground with a sand glass slide to prepare a spleen cell suspension. The prepared spleen cell suspension was centrifuged at 1500 r / min twice, 2 mL of red blood cell lysis buffer was added, and the suspension was allowed to stand for 5 minutes in a constant temperature water bath at 37°C, and then centrifuged at 1500 r / min for 10 minutes, and the supernatant was discarded. ' s were washed twice, and the number of spleen cells was adjusted to a concentration of 1×10 7 / mL cell suspension, these cells are used as effector cells, and are reserved. YAC-1 cells are used as target cells to detect NK cell cytotoxicity. Add 96-well plates according to the effector-target ratio of 1:100, 1:50, and 1:20, i.e., 1×10 spleen cells 6 cells / well, while YAC-1 cells were 1.0×10 4 , 2×10 4 , 5.0×10 4 / well. After 6 hours of co-culture in a CO2 incubator, measure OD 540nm The absorbance value of the cells was measured and the killing rate was calculated.

[0122] Killing rate (%) = (OD value of target cells - OD value of experimental group) / OD value of target cells × 100%.

[0123] The results of the test sample activity against normal mouse natural killer cells are shown in Table 3.

[0124] Table 3

[0125]

[0126] In Table 3, compared with the normal group, △ P<0.05, △△ P<0.01, △△△ P<0.001 has significant difference. Compared with the high-dose group of BPU-1 in Example 1,# P<0.05, ## P<0.01, ### P<0.001, there is a significant difference.

[0127] 2.3 Effects on the function of peritoneal macrophages in normal mice

[0128] Experimental methods: ICR mice (18-22 g) were randomly divided into groups, with 7 mice in each group.

[0129] The mice were grouped as follows: high-dose BP group, high-dose BPU group in Example 1, high-dose BPC group in Example 1, high-dose BPU-1 group in Example 1, high-dose BPU-2 group in Example 1, high-dose BPU-3 group in Example 1, high-dose BPU-4 group in Example 1, high-dose BPU-1 group in Comparative Example 1, high-dose BPU-1 group in Comparative Example 2, high-dose BPU-1 group in Comparative Example 3, high-dose BPU-2 group in Comparative Example 3, high-dose BPU-3 group in Comparative Example 3, high-dose BPU-4 group in Comparative Example 3; each group was intraperitoneally injected with the corresponding composition. The normal control group was intraperitoneally injected with physiological saline. The injection amount of the high-dose group was 35 mg / kg, and the injection amount of the low-dose group was 17.5 mg / kg.

[0130] For 10 consecutive days, 1 hour after administration on the 10th day, 0.4 mL of 5% chicken red blood cells was injected into the peritoneum of each mouse. After 8 hours, the animals were killed and fixed on the mouse board in the supine position. The abdomen was disinfected, the skin was cut open, and 2 mL of normal saline was injected into the peritoneum. The fixed plate was rotated for 1 minute, and then 1 mL of peritoneal fluid was drawn and dropped on a clean slide, 0.2 mL per slide, a total of 2 slides, placed in an enamel box with wet gauze, placed in a 37°C incubator for 30 minutes, and the slides were removed and rinsed with normal saline to remove the unattached cells. Dry, fix with acetone-methanol solution (1:1) for 5 minutes, then stain with 4% Giemsa-Wright stain for 3 minutes, rinse with distilled water, and dry. The phagocytosis of mouse peritoneal macrophages was observed under a microscope, and the phagocytosis rate was calculated.

[0131] Phagocytic rate (%) = number of phagocytes phagocytosing chicken red blood cells / 200 macrophages × 100%.

[0132] The results of the phagocytic rates of the test samples on normal mouse macrophages are shown in Table 4.

[0133] Table 4

[0134]

[0135] In Table 4, compared with the normal group, △ P<0.05, △△ P<0.01, with significant difference. Compared with the high-dose group of BPU-1 in Example 1,# P<0.05, ## P<0.01, there is a significant difference.

[0136] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an immunopotentiating composition, characterized in that: Includes steps: (1) Dissolving the BP raw material in water and performing ultrafiltration to obtain an ultrafiltrate and a concentrate; the ultrafiltration treatment specifically uses an ultrafiltration membrane with a molecular weight of 2 kDa-5 kDa for ultrafiltration; (2) Passing the ultrafiltrate through D101 macroporous adsorption resin for adsorption; (3) first adding water for elution, then using an ethanol solution with a volume concentration of 40%-50% for elution, then using an ethanol solution with a volume concentration of 60%-80% for elution, and finally using an ethanol solution with a volume concentration of 90%-95% for elution to obtain an immune enhancing composition; The immunoenhancing composition is a water eluent substance, a 40%-50% ethanol solution eluent substance, or a 60%-80% ethanol solution eluent substance.

2. The preparation method according to claim 1, characterized in that: In step (1), during the process of adding water to dissolve, the solid-liquid ratio of BP raw material to water is 400-600:800-1200 g / mL; in step (1), the ultrafiltration treatment uses an ultrafiltration membrane with a molecular weight of 3 kDa.

3. The preparation method according to claim 1, characterized in that: In step (2), during the adsorption process, the column diameter-to-height ratio is 1:4-12, and the sample volume ratio is 1:20-30.

4. The preparation method according to claim 1, characterized in that: In step (3), the elution flow rate is 1-3 BV / h, and the amount of each eluent used is 3-6 BV.

5. The preparation method according to claim 1, characterized in that: In step (3), the elution is specifically as follows: first, water is added for elution, then an ethanol solution with a volume concentration of 40% is used for elution, then an ethanol solution with a volume concentration of 70% is used for elution, and finally an ethanol solution with a volume concentration of 95% is used for elution.

6. The immunopotentiating composition prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the immunopotentiating composition obtained by the preparation method according to any one of claims 1 to 5 or the immunopotentiating composition according to claim 6 in the preparation of immunomodulatory drugs.

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