Use of Ejiao Qianggu Oral Liquid in Regulating Immunity

Abstract

The application discloses a use of Ejiao Qianggu oral liquid in regulating immunity, and particularly relates to an improving effect of the Ejiao Qianggu oral liquid on an immune system, which is realized by the following aspects. On one hand, the Ejiao Qianggu oral liquid promotes proliferation and transformation (mainly early T lymphocytes and part of B lymphocytes) and active functions of spleen lymphocytes; on the other hand, the Ejiao Qianggu oral liquid improves phagocytosis of macrophages. The Ejiao Qianggu oral liquid has the following characteristics: firstly, the Ejiao Qianggu oral liquid acts on early lymphocyte maturation, and has no significant effect on proliferation of peripheral blood lymphocytes; secondly, the Ejiao Qianggu oral liquid can significantly act on immune target organs, thymus and spleen tissues, and does not excessively stimulate the spleen, unlike positive drugs such as colony stimulating factor which can cause significant edema of the spleen; and thirdly, the Ejiao Qianggu oral liquid has a significant recovery effect on body weight, white blood cells, lymphocytes, red blood cells and hemoglobin in blood of patients with low immunity.

Description

Technical Field

[0001] This invention relates to the application of donkey-hide gelatin oral liquid in the preparation of drugs that enhance immunity; it belongs to the field of pharmaceutical technology. Background Technology

[0002] The Ejiao Qianggu Oral Liquid (National Medicine Approval Number Z20000039) produced by Xinjiang Huashidan Pharmaceutical Co., Ltd. contains the following ingredients: Rehmannia glutinosa, donkey-hide gelatin, wolfberry, oyster shell, Astragalus membranaceus, and Codonopsis pilosula. Excipients include honey, aspartame, and sorbic acid. Its main functions and indications are: tonifying the liver and kidneys, replenishing essence and strengthening bones; used for primary osteoporosis, liver and kidney deficiency syndrome, lower back pain or weakness in the lower back and knees, numbness and convulsions, inability to bear weight, dizziness and tinnitus, restlessness and insomnia, as well as rickets in children with liver and kidney deficiency syndrome; sparse hair, sallow complexion, excessive sweating, night terrors or night crying. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a novel immune-enhancing drug, that is, to provide...

[0004] Application of donkey-hide gelatin oral liquid in the preparation of drugs that enhance immunity.

[0005] Application of donkey-hide gelatin oral liquid in the preparation of drugs that increase white blood cell count.

[0006] Application of donkey-hide gelatin oral liquid in the preparation of drugs that enhance lymphocytes.

[0007] Application of donkey-hide gelatin oral liquid in the preparation of drugs that enhance red blood cell count.

[0008] Application of donkey-hide gelatin oral liquid in the preparation of drugs that increase hemoglobin.

[0009] Application of donkey-hide gelatin oral liquid in the preparation of drugs that promote macrophage phagocytic function.

[0010] Application of donkey-hide gelatin oral liquid in the preparation of drugs that promote the proliferation and transformation of splenic lymphocytes.

[0011] Application of donkey-hide gelatin oral liquid in the preparation of drugs that promote spleen lymphocyte function.

[0012] Application of Ejiao Strong Bone Oral Liquid in the preparation of drugs to restore drug-induced weight loss.

[0013] Application of donkey-hide gelatin oral liquid in the preparation of drugs that improve thymus index.

[0014] The preparation of Ejiao Strong Bone Oral Liquid enhances immunity through cellular immunity and humoral immunity components, as further confirmed by the following experiments.

[0015] (1) Effect of donkey-hide gelatin oral liquid on delayed-type hypersensitivity (DTH) induced by DNCB;

[0016] (2) Effects of donkey-hide gelatin oral liquid on thymus and spleen weight index and peripheral blood lymphocyte transformation in mice;

[0017] (3) Effect of donkey-hide gelatin oral liquid on the hemolytic ability of spleen plaque-forming cells (PFC);

[0018] (4) Effect on restoring serum IgG and IgM levels in animal models with low immunoglobulin levels;

[0019] (5) The regulatory effects of donkey-hide gelatin oral liquid on serum IgA, IgG and IgM in an immunosuppressed animal model, its effect on intracellular cAMP concentration in spleen cells and its effect on the proliferation of spleen lymphocytes.

[0020] (6) Macrophage phagocytosis of neutral red and Staphylococcus aureus experiment.

[0021] Experimental results show that the effect of donkey-hide gelatin oral liquid on the immune system of mice is mainly to promote the proliferation and transformation of spleen lymphocytes (mainly early T lymphocytes and some B lymphocytes) and their activity function; and to enhance the phagocytic function of macrophages.

[0022] Its characteristics are as follows: First, it acts on lymphocytes in the early stages of maturation, and its effect on the proliferation of peripheral blood lymphocytes is not significant; second, it acts very significantly on immune target organs, namely the thymus tissue, but without overstimulation, unlike positive drug colony-stimulating factors that can cause severe splenic edema; third, it has a significant restorative effect on body weight, white blood cells, lymphocytes, red blood cells and hemoglobin in immunocompromised animals.

[0023] This experiment preliminarily clarifies the main immune organs and immune cell types involved in the immune enhancement effect of donkey-hide gelatin oral liquid. However, further research is needed to explore its effects on lymphocyte subtypes, cytokine networks, etc.

[0024] The immune system-enhancing effect of donkey-hide gelatin oral liquid is mainly achieved through the following aspects: Firstly, it promotes the proliferation and transformation of splenic lymphocytes (mainly early T lymphocytes and some B lymphocytes) and their activity; secondly, it enhances the phagocytic function of macrophages. Its characteristics include: firstly, it acts on lymphocytes in the early stages of maturation, with little effect on the proliferation of peripheral blood lymphocytes; secondly, it significantly affects the immune target organ, particularly the thymus tissue, without overstimulating it or causing edema; and thirdly, it has a significant restorative effect on body weight, white blood cells, lymphocytes, red blood cells, and hemoglobin in immunocompromised patients.

[0025] Specifically, this invention verifies the regulatory effect of donkey-hide gelatin oral liquid on the immune systems of normal mice and immune-abnormal mice from different perspectives through five embodiments; wherein immune-abnormal mice include immunocompromised mice and hypersensitive mice.

[0026] In verifying the phagocytic activity of macrophages, Ejiao Qianggu Oral Liquid significantly promoted the phagocytic function of macrophages. This invention examined the effects of Ejiao Qianggu Oral Liquid on macrophage activity in three aspects: phagocytosis of neutral red, phagocytosis of Staphylococcus aureus, and carbon clearance. The results showed that macrophage phagocytosis of Staphylococcus aureus was significantly enhanced compared to the normal group (p < 0.01), exceeding the normal group by more than 5 times (Example 5); the phagocytic capacity for neutral red was significantly enhanced compared to the normal group (p < 0.01), exceeding the normal group by 2 times (Examples 2, Example 5); and the effect of Ejiao Qianggu Oral Liquid on the carbon clearance index was not significant in vivo.

[0027] This invention demonstrates through experiments that, in terms of the proliferation and transformation of splenic lymphocytes and peripheral blood lymphocytes, donkey-hide gelatin oral liquid has a significant effect on the proliferation and transformation of splenic lymphocytes, but has no promoting effect on the proliferation of peripheral blood lymphocytes.

[0028] This invention experimentally demonstrates that in the assay of splenic lymphocyte immune function (PFC plaque assay), a significant increase in the number of hemolytic plaques can be observed in the medium-dose group of Ejiao Qianggu oral liquid. Its promoting effect on the AOI index (plaque area ratio) is 6 times that of the normal group and 3 times that of the positive control group. Furthermore, experimental observation of the cAMP / cGMP ratio in splenic cells and serum reveals that Ejiao Qianggu oral liquid also enhances the cAMP / cGMP ratio in splenic cells. This indicates that Ejiao Qianggu oral liquid has a very significant effect in promoting splenic lymphocyte function.

[0029] In comparing the body weight of normal and immunosuppressed mouse models, this invention experimentally demonstrated that Ejiao Qianggu Oral Liquid can restore drug-induced weight loss in mice. On day 14 of administration, the body weight of mice in the model group was still significantly lower than that in the normal group (p < 0.01). After 14 days of use of Ejiao Qianggu Oral Liquid, the body weight of mice was significantly restored relative to the body weight after modeling, as shown in Table 17.

[0030] Immunosuppressed animal studies showed that, after 14 days of administration, the donkey-hide gelatin oral liquid group significantly increased the number of white blood cells, lymphocytes, and monocytes in mice compared to the model group (p < 0.05); while the positive control group's colony-stimulating factor (SCGF) increased white blood cells by 28 times. Observation of organ coefficients in mice revealed that each dose group of the donkey-hide gelatin oral liquid significantly increased the thymus coefficient compared to the model group (p < 0.01), suggesting that the donkey-hide gelatin oral liquid may act on T cell maturation and proliferation. Furthermore, the positive control group increased the spleen coefficient by 6 times, and spleen edema also occurred.

[0031] In this experiment, ELISA kits were used to detect serum IgA, IgM, and IgG in mice from different groups. Serum samples from 5 mice in each group were used for ELISA detection in 2 replicate wells. The results showed that the three immunoglobulins were significantly lower in the model group compared with the normal group. After 14 days of administration, IgM in the low-dose group of the donkey-hide gelatin oral liquid was significantly higher than that in the model group (p < 0.05), and IgG in the medium- and high-dose groups of the donkey-hide gelatin oral liquid was significantly higher than that in the model group (p < 0.05).

[0032] Comprehensive experimental results show that Ejiao Qianggu Oral Liquid can act on the thymus of an immunocompromised mouse model, significantly increasing the total number of white blood cells, lymphocytes, and monocytes in the model mice. Its effect is relatively mild compared to colony-stimulating factor. In terms of immunoglobulins, the kit results show that Ejiao Qianggu Oral Liquid has a significant effect on increasing IgM and IgG.

[0033] This invention experimentally demonstrates that, regarding the organ coefficients of the spleen and thymus, donkey-hide gelatin oral liquid significantly increases the thymus index in an immunosuppressed mouse model (p < 0.01). However, in normal animals, high-dose donkey-hide gelatin oral liquid significantly increases the spleen index (p < 0.05) but has no effect on the thymus index. Comparison with positive drug colony-stimulating factors reveals that its increasing effect is very mild and does not induce excessive splenomegaly. Attached Figure Description

[0034] Figure 1 Changes in the toes of mice after Ejiao Qianggu Oral Liquid was applied to a DTH mouse model.

[0035] Figure 2 The oral liquid for strengthening bones and spleen with donkey-hide gelatin has an effect on hemolytic plaques in spleen cells.

[0036] a: Newly added rabbit red blood cells 0h; b: Blank group; c: Levamisole group; d: Low-dose group; e: Medium-dose group; f: High-dose group

[0037] Figure 3 The results of splenic lymphocyte transformation were shown (100×).

[0038] Figure 4 IL-6 standard curve.

[0039] Figure 5 The oral liquid for strengthening bones and spleen with donkey-hide gelatin has an effect on hemolytic plaques in spleen cells.

[0040] a: Newly added rabbit red blood cells 0h; b: Blank group; c: Levamisole group; d: Low-dose group; e: Medium-dose group; f: High-dose group

[0041] Figure 6 IgA standard curve.

[0042] Figure 7 IgA expression level.

[0043] Figure 8 IgM standard curve.

[0044] Figure 9 IgM expression level.

[0045] Figure 10 IgG standard curve.

[0046] Figure 11 The expression levels of IgG.

[0047] Figure 12 , is the standard curve for cyclic guanosine monophosphate (cGMP).

[0048] Figure 13 It is the standard curve for cyclic adenosine monophosphate (cAMP).

[0049] Figure 14 This is a PI fluorescent staining image of macrophages phagocytizing Staphylococcus aureus. Detailed Implementation

[0050] Example 1. Effect of donkey-hide gelatin oral liquid on delayed-type hypersensitivity (DTH) induced by rabbit erythrocytes in mouse paws.

[0051] Type IV delayed-type hypersensitivity (DTH) is a type of cellular immune response mediated by specifically sensitized effector T cells. In guinea pigs, rats, and mice, DTH responses to most protein antigens are passively transferred via CD4+ T cells. Recently, it has been shown that CD8+ T cells can also passively transfer DTH-like responses. For example, antiviral DTH responses are primarily mediated by CD8+ T cells, while responses to injected proteins or extracellular antigens are mainly mediated by CD4+ T cells. The final effector cells in DTH responses are activated mononuclear phagocytes. This type of reaction always occurs 24 hours after exposure to the antigen, hence the name delayed-type hypersensitivity. DTH responses are observed in many allergic reactions to intracellular parasites such as mycobacteria and Listeria monocytogenes, viruses, and fungal infections; in contact dermatitis caused by certain simple chemicals (such as DNCB); and in rejection reactions to transplanted tissues and organs (such as SRBC).

[0052] I. Experimental Methods

[0053] 1.1 Laboratory Animals

[0054] BALB / c mice, weighing approximately 18-20g, half male and half female, 60 mice in total. Rabbit: 1 mouse.

[0055] 1.2 Reagents

[0056] Rabbit red blood cells (RRBCs): Collect 5-10 mL of anticoagulated blood from the heart of a rabbit. Centrifuge at 2000 rpm for 5 min, and aspirate the plasma layer. Add 2-3 times the volume of physiological saline to the tube and gently mix using a capillary dropper. Centrifuge again at 2000 rpm for 5 min, discarding the supernatant. Repeat this process three times. For the final centrifugation, the centrifugation time can be appropriately extended to 10 min. Prepare a 10% cell suspension.

[0057] Donkey-hide gelatin oral liquid for strengthening bones: 19021201; Xinjiang Huashidan Pharmaceutical Co., Ltd.;

[0058] Dexamethasone acetate tablets (immunosuppressant): 1906211; Suicheng Pharmaceutical Co., Ltd. 1.3 Test Methods

[0059] Modeling and drug administration method: Sixty BALB / c mice (half male and half female) were randomly divided into six groups according to sex and weight: blank group, model control group, positive drug group (dexamethasone 5 mg / kg), and three dosage groups of donkey-hide gelatin oral liquid (4 ml, 8 ml, and 16 ml / kg). All groups were administered the drug via gavage for 13 consecutive days. Except for the normal control group, all other groups of mice received an injection of 0.05 ml / mouse of 10% sheep red blood cell saline suspension (S RBC·NS), while the normal control group received an equal volume of saline. Five days later, except for the normal control group, all other groups of mice received an injection of 0.05 ml / mouse of 10% RRBC / NS on their right paw pad, while the normal control group received an equal volume of saline. Twenty-four hours later, the swelling of the right paw pad was measured, and blood and spleen samples were collected for relevant tests.

[0060] 1.4 Detection Indicators:

[0061] Weight monitoring: Record the changes in mouse weight after drug administration;

[0062] Detection of paw pad swelling: 24 hours after injecting RRBC·NS into the right paw pad of mice, the left and right paw pads of mice were measured with an outside micrometer, and the difference was taken as the degree of paw pad swelling.

[0063] Complete blood count (CBC): Focus on observing granulocytes, white blood cells, and red blood cells.

[0064] Spleen and thymus coefficient detection: Determining organ coefficients.

[0065] II. Experimental Results

[0066] 1. Effects of donkey-hide gelatin oral liquid on delayed-type hypersensitivity (DTH) induced by rabbit erythrocytes in mouse paws.

[0067] 1.1 Changes in body weight in the DTH mouse model

[0068] Table 1. Changes in body weight of mice on day 20

[0069]

[0070]

[0071] 1.2 Changes in immune organ coefficients in the DTH mouse model

[0072] Table 2 shows that the spleen coefficient of mice changed significantly after 5 days of drug treatment, with a significant increase in the model group compared to the normal group. However, compared to the model group, the spleen coefficient of the donkey-hide gelatin oral liquid significantly recovered. This indicates that the donkey-hide gelatin oral liquid has a significant restorative effect on the splenomegaly caused by hypersensitivity reactions, although its effect is slightly lower than that of the anti-allergic glucocorticoid drug dexamethasone.

[0073] Table 2 Immune Organ Coefficient

[0074] Group number Spleen coefficient thymus coefficient normal group 0.24±0.09 0.06±0.03 Model group 0.65±0.36* 0.19±0.17 Dexamethasone group 0.17±0.08## 0.14±0.19 Low-dose group of donkey-hide gelatin for strengthening bones 0.33±0.12# 0.09±0.09 Medium dose group of donkey-hide gelatin for strengthening bones 0.27±0.09# 0.07±0.01 High-dose group of donkey-hide gelatin for strengthening bones 0.26±0.07# 0.07±0.02

[0075] Note: Compared with the normal group "*": p < 0.05, significant difference; compared with the model group "#": p < 0.05, significant difference; compared with the model group "##": p < 0.01, extremely significant difference;

[0076] Changes in blood routine indices in a 1.3DTH mouse model

[0077] Table 3 shows that the number of neutrophils in the model group was significantly higher than that in the normal group, and that donkey-hide gelatin oral liquid had a significant restorative effect on neutrophils. Neutrophils have chemotactic, phagocytic, and bactericidal functions. Neutrophils originate from bone marrow and have lobed or rod-shaped nuclei. Their cytoplasm contains a large number of neutral fine granules that are neither basophilic nor eosinophilic. These granules are mostly lysosomes, containing abundant enzymes such as myeloperoxidase, lysozyme, alkaline phosphatase, and acid hydrolases, which are related to the phagocytic and digestive functions of cells.

[0078] Table 3 Blood Routine Tests

[0079]

[0080]

[0081] Changes in blood routine indices in a 1.4DTH mouse model

[0082] From Table 4 and Figure 1 It can be seen that the toe thickness of the DTH model group was significantly increased compared with that of normal animals, and obvious edema was observed in the morphology. In contrast, the toe thickness of the donkey-hide gelatin oral liquid group was significantly increased compared with that of the model group.

[0083] Table 4. Changes in toe thickness

[0084] Group Changes in toe thickness (mm) normal group 0.014±0.017 Model group 0.023±0.026* Dexamethasone group 0.012±0.005# Low-dose group of donkey-hide gelatin for strengthening bones 0.041±0.036# Donkey-hide gelatin for strengthening bones (medium dose group) 0.041±0.012# High-dose group of donkey-hide gelatin for strengthening bones 0.049±0.023#

[0085] III. Summary

[0086] This experiment observed the degree of delayed anaphylaxis in mice after 14 days of treatment with Ejiao Qianggu oral liquid. Type IV delayed anaphylaxis is a type of cellular immune response mediated by specific sensitized effector T cells. In guinea pigs, rats, and mice, the DTH response to most protein antigens can be passively transferred via CD4+ T cells. Observations of different drugs acting on allergic animals showed that the spleen coefficient, the largest immune organ, was significantly upregulated in the model group, while the positive drug and Ejiao Qianggu oral liquid showed a significant decrease compared to the model group, with the dexamethasone tablet group showing the strongest decrease. Regarding toe thickness, compared to the normal control group, the model group and the high, medium, and low dose drug groups showed significantly increased paw pad swelling (P<0.05). Compared to the model group, the positive dexamethasone group showed a significant decrease in paw pad swelling (P<0.05).

[0087] Example 2. Effects of donkey-hide gelatin oral liquid on thymus and spleen weight index, and peripheral blood lymphocyte transformation in mice.

[0088] When lymphocytes are cultured in vitro, they will transform into lymphoblasts after being stimulated by non-specific mitogens or specific antigens (such as ConA, LPS, PHA). These lymphocytes are characterized by increased size, ability to divide, and increased metabolic level. The transformation rate can reflect the level of cellular immunity in the body and is often used as an indicator to detect the body's immunity.

[0089] Experimental methods

[0090] 2.1 Laboratory Animals

[0091] BALB / c mice, weighing approximately 18-20g, were divided into equal numbers of males and females, totaling 50 mice.

[0092] 2.2 Reagents and medicinal materials

[0093] Levamisole hydrochloride: Manufacturer: Shanxi Yunpeng Pharmaceutical Co., Ltd., Batch No.: 20151101;

[0094] Plant lectin PHA: Manufacturer: Solabio, USA; Product No.: 403N032;

[0095] Canavon protein A: Manufacturer: Sigma, USA; Product number: C2010-2016;

[0096] LPS: Manufacturer: Solabio, USA; Product No.: 403N032;

[0097] Lymphocyte separation solution, manufacturer: Tianjin Haoyang Biotechnology, batch number 11015;

[0098] Donkey-hide gelatin oral liquid: 19021201; Xinjiang Huashidan Pharmaceutical Co., Ltd.

[0099] 2.3 Model Methods

[0100] Fifty mice were randomly divided into five groups: a blank control group, a positive control group (levamisole hydrochloride group (LM): 50 mg / mL levamisole hydrochloride was injected subcutaneously, 0.2 mL / mouse), and three doses of donkey-hide gelatin oral liquid (4 ml, 8 ml, 16 ml / kg). Each group was administered the drug by gavage for 14 consecutive days. The model control group was given an equal amount of distilled water (DW).

[0101] 2.4 Detection methods and indicators

[0102] (1) Weight index of thymus and spleen in mice

[0103] Fourteen days after drug administration, the test mice were weighed, euthanized by cervical dislocation, and their spleens and thymus were harvested. The thymus was rinsed with pre-cooled physiological saline, blotted dry with filter paper, and weighed (accurate to 0.001). The immune organ index was calculated using the following formula:

[0104] Immune organ index = Immune organ mass (g) / Body mass (g) × 100%

[0105] (2) Effects on the proliferation and transformation of mouse spleen lymphocytes:

[0106] Spleen cell isolation and culture: Mouse spleens were aseptically harvested, and the outer membrane of the spleen was carefully removed. The spleen was homogenized until it reached a liquid state using a homogenizer. The mixture was then filtered through a 120-mesh cell sieve, and the filtrate was collected and added to an equal volume of lymphocyte separation medium. The mixture was incubated at 2500 rpm. -1 After 20 minutes, lymphocytes and monocytes were collected and placed in centrifuge tubes. Five volumes of erythrocyte lysis buffer were added, and the mixture was incubated at 37°C for 10 minutes. The cells were washed twice with D-Hank's solution and diluted to 1×10⁻⁶. 7 / mL.

[0107] Splenic lymphocyte proliferation: 100 μL of mixed lymphocyte suspension was added to each well of a 96-well plate and cultured for 5 h. PHA (final concentration 50 μg / mL) was then added as an inducer. -1 After incubating the cell plates at 37°C and 5% CO2 for 48 hours, the proliferation rate of lymphocytes in different groups was measured at 570 nm using the MTT assay.

[0108] Transformation: The T / B lymphocyte transformation rate was detected using Giemsa staining. Three random fields of view were photographed under a microscope to calculate the T / B lymphocyte transformation rate.

[0109] Transformation rate = (Number of transformed cells / Total number of cells) × 100%;

[0110] (3) Detection of peripheral blood mixed lymphocyte transformation:

[0111] Aseptically collect 1 mL of anticoagulated blood from mice, dilute with an equal volume of Hank's solution, add an equal volume of lymphocyte separation medium, and incubate at 2500 rpm. -1 After 20 minutes, lymphocytes and monocytes were collected and placed in centrifuge tubes. Five volumes of erythrocyte lysis buffer were added, and the mixture was incubated at 37°C for 10 minutes. The cells were washed twice with Hank's solution and diluted to 1×10⁻⁶. 7 / mL.

[0112] Add 100 μL of mixed lymphocyte suspension to each well of a 96-well plate and incubate for 5 h. Add inducing agent (LPS, final concentration 25 μg / mL). -1 Or a final ConA concentration of 5 μg / mL -1 Cell plates were incubated at 37°C and 5% CO2 for 48 hours. Lymphocyte proliferation rates in different groups were measured at 570 nm using the MTT assay. The stimulation index of T lymphocytes was measured using concanavalin A, and the proliferation index of B lymphocytes was measured using lipopolysaccharide (LPS). Giemsa staining was performed (in the following order: wash 3 times with PBS; fix in methanol: glacial acetic acid = 3:1 fixative for 30 minutes → stain with Giemsa solution for 10 minutes → rinse with tap water).

[0113] T / B lymphocyte transformation rate was detected using Giemsa staining. T / B lymphocyte transformation rate was calculated by randomly photographing three fields of view under a microscope.

[0114] Transformation rate = (Number of transformed cells / Total number of cells) × 100%;

[0115] II. Experimental Results

[0116] 2. Effects of donkey-hide gelatin oral liquid on thymus and spleen weight index, and peripheral blood lymphocyte transformation in mice. 2.1 Changes in mouse body weight.

[0117] Table 5. Changes in mouse body weight (n=10)

[0118]

[0119]

[0120] 2.2 Changes in spleen and thymus coefficients in mice

[0121] Table 6. Organ coefficients in mice (n=10)

[0122] Group number Spleen coefficient thymus coefficient Model group 0.38±0.08 0.13±0.05 Levamisole group 0.40±0.08 0.12±0.04 Low-dose group of donkey-hide gelatin for strengthening bones 0.38±0.04 0.12±0.04 Medium dose group of donkey-hide gelatin for strengthening bones 0.31±0.04 0.16±0.06 High-dose group of donkey-hide gelatin for strengthening bones 0.34±0.05 0.13±0.03

[0123] 2.3 Changes in immune-related cells in mouse blood

[0124] As shown in Table 7, after normal animals were given Ejiao Qianggu Oral Liquid for 14 days, the number of white blood cells in the low-dose and high-dose groups of Ejiao Qianggu Oral Liquid was significantly increased compared with the blank group, while the number of lymphocytes in the high-dose group was also significantly increased (p<0.05).

[0125] Table 7. Mouse blood immune cells (n=10)

[0126]

[0127] Note: Compared with the normal group, "**" indicates p < 0.01, indicating a highly significant difference; compared with the normal group, "*" indicates p < 0.05, indicating a significant difference; 2.4 Effect on spleen cell proliferation rate

[0128] Table 8 shows that the spleen cell proliferation rate of mice in the low-to-medium dose group of Ejiao Qianggu Oral Liquid was significantly higher than that in the normal group.

[0129] Table 8. Splenocyte proliferation rate in mice (n=3)

[0130]

[0131]

[0132] Note: Compared with the normal group, "**" indicates p < 0.01, meaning the difference is extremely significant; compared with the normal group, "*" indicates p < 0.05, meaning the difference is significant.

[0133] 2.5 Effects on splenic lymphocyte transformation

[0134] Table 9. Splenic lymphocyte transformation rate (n=3)

[0135] Group number Conversion rate (%) normal group 22.83 Levamisole group 23.44 Low-dose group of donkey-hide gelatin for strengthening bones 35.54* Donkey-hide gelatin for strengthening bones (medium dose group) 28.85 High-dose group of donkey-hide gelatin for strengthening bones 32.39*

[0136] Compared with the normal group "*": p < 0.05, the difference is statistically significant.

[0137] 2.6 Effects on the transformation of peripheral blood lymphocytes into T / B lymphocytes

[0138] Table 10. Influence of donkey-hide gelatin bone-strengthening oral liquid on the proliferation rate of peripheral blood lymphocytes in mice (n=3)

[0139]

[0140] 2.7 Phagocytic capacity of macrophages

[0141] Table 11 Effects of donkey-hide gelatin oral liquid on the phagocytic capacity of peritoneal macrophages in mice (n=3)

[0142] Group number Neutral Red OD Value Absorption capacity (%) normal group 0.65±0.22 100 Levamisole group 0.67±0.23 102.31 Low-dose group of donkey-hide gelatin for strengthening bones 0.99±0.39 149.95* Medium dose group of donkey-hide gelatin for strengthening bones 0.72±0.09 109.9 High-dose group of donkey-hide gelatin for strengthening bones 0.82±0.23 124.10

[0143] Note: Compared with the normal group, "*" indicates p < 0.05, indicating a significant increase;

[0144] Table 12 Effect of donkey-hide gelatin oral liquid on IL-6 content in mouse macrophages (n=5)

[0145] Group number OD value IL-6 level (pg / mL) normal group 0.35±0.03 18.58 Levamisole group 0.38±0.02 20.61 Low-dose group of donkey-hide gelatin for strengthening bones 0.36±0.01 19.29 Donkey-hide gelatin for strengthening bones (medium dose group) 0.39±0.03 20.93 High-dose group of donkey-hide gelatin for strengthening bones 0.34±0.02 17.64

[0146] III. Summary

[0147] The purpose of this experiment is twofold: firstly, to detect the effect of donkey-hide gelatin oral liquid on the immune organs (thymus and spleen) and the degree of transformation of their lymphocytes in mice; and secondly, to observe the changes in the phagocytic capacity of peritoneal macrophages in mice after treatment with donkey-hide gelatin oral liquid.

[0148] Experimental results showed that after two weeks of treatment with Ejiao Qianggu oral liquid, mice exhibited significantly higher white blood cell counts (low and high dose groups) and significantly higher total lymphocyte counts (high dose), erythrocyte counts, and hemoglobin levels (high dose group) compared to normal control mice. Further observation of the effects of Ejiao Qianggu oral liquid on the proliferation and transformation of mouse splenic lymphocytes revealed that while it did not significantly increase the spleen coefficient, it significantly promoted the proliferation and transformation of splenic lymphocytes. This effect was not dose-dependent. Comparison of the effects of Ejiao Qianggu oral liquid on the proliferation of mouse peripheral blood lymphocytes showed that its proliferative effect was not significant, indicating that the drug selectively targets lymphocytes, requiring further classification and verification.

[0149] In verifying the phagocytosis of chemical drugs by macrophages (neutral red), the experiment found that the effect of donkey-hide gelatin oral liquid was also significant (low-dose group), with a 50% increase in phagocytic capacity, but dose differences still existed. The secretion capacity of macrophages of IL-6 was improved, but not significantly.

[0150] In summary, donkey-hide gelatin oral liquid promotes the proliferation and transformation of splenic lymphocytes in the mouse immune system, but has no significant effect on the proliferation of mature peripheral blood lymphocytes. It can also increase the ability of peritoneal macrophages to phagocytose chemically toxic substances.

[0151] Example 3. Effect of donkey-hide gelatin oral liquid on the hemolytic ability of spleen plaque-forming cells (PFC);

[0152] The plaque-induced hemolytic assay is a method for detecting the formation of B lymphocytes stimulated by antibodies. The principle is to stimulate mice with SRBCs, extract the spleen lymphocytes of the immunized mice and prepare a suspension, and incubate them with the antigen SRBCs again in vitro. Immune cells will release hemolysin against SRBCs, and with the participation of complement, the surrounding SRBCs will be dissolved to form plaques. The strength of the mouse's immunity can be observed by observing the size of the well plate.

[0153] I. Experimental Methods

[0154] 3.1 Laboratory Animals

[0155] BALB / c mice, weighing approximately 18-20g, were divided into equal numbers of males and females, totaling 50 mice.

[0156] 3.2 Reagents and medicinal materials

[0157] Agarose gel, manufacturer: Thermo Fisher Scientific, batch number: 191101; RPMI-1640 medium, rabbit red blood cells (RRBC), homemade, batch number 20201112, fetal bovine serum, serum from 2 guinea pigs.

[0158] Donkey-hide gelatin oral liquid: 19021201; Xinjiang Huashidan Pharmaceutical Co., Ltd.

[0159] 3.3 Model Methods

[0160] Fifty mice, half male and half female, weighing 18-20g, were randomly divided into four groups according to sex and weight: a blank control group, a positive control group (levamisole hydrochloride group (LM): subcutaneous injection of 50mg / mL levamisole hydrochloride, 0.2mL / mouse), and three doses of donkey-hide gelatin and bone-strengthening oral liquid (4ml, 8ml, 16ml / kg). Each group was administered the drug by gavage for 14 consecutive days. The model control group was given an equal volume of distilled water (DW).

[0161] The mice in the above groups were injected intraperitoneally with RRBC suspension (4×10⁻⁶) on day 14. 8 Mice were sacrificed on day 4 post-immunization (1 ml / mouse).

[0162] 3.4 Detection Methods

[0163] (1) Spleen cell isolation and culture: Mouse spleens were aseptically harvested, the outer membrane of the spleen was carefully removed, and the spleen was homogenized to a liquid state using a homogenizer. The mixture was then filtered through a cell sieve, and the filtrate was collected and added to an equal volume of lymphocyte separation medium. The mixture was incubated at 2500 rpm. -1 After 20 minutes, lymphocytes and monocytes were collected and placed in centrifuge tubes. Five volumes of erythrocyte lysis buffer were added, and the mixture was incubated at 37°C for 10 minutes. The cells were washed twice with Hank's solution and diluted to 1×10⁻⁶. 7 / mL.

[0164] (2) Preparation of the bottom gel: Add 0.5g agarose to 100ml sterile physiological saline and heat to dissolve. Add 0.5g agarose to 100ml Hank's solution with pH 7.2 and heat to dissolve. Add 0.5ml / test tube to a test tube at a constant temperature of 46-50℃.

[0165] (3) Observe the number of plaques in each group under a microscope. Calculate the number of plaques generated per million spleen cells and take pictures.

[0166] II. Experimental Results

[0167] 3. Effect of donkey-hide gelatin oral liquid on the hemolytic ability of spleen plaque-forming cells (PFC);

[0168] 3.1 Changes in mouse body weight

[0169] Table 13 Changes in mouse body weight (n=10)

[0170] Group weight normal group 21.05±0.96 Levamisole group 21.71±1.29 Low-dose group of donkey-hide gelatin for strengthening bones 21.37±2.12 Donkey-hide gelatin for strengthening bones (medium dose group) 22.12±1.35 High-dose group of donkey-hide gelatin for strengthening bones 21.0±1.37

[0171] 3.2 Mouse blood routine index

[0172] Table 14 shows that the high-dose group of donkey-hide gelatin for strengthening bones has a significant effect on increasing the number of white blood cells and lymphocytes.

[0173] Table 14 Blood Routine Test Table (n=10)

[0174]

[0175]

[0176] 3.3 Hemolytic plaque effect

[0177] Using ImageJ software to detect the area of ​​hemolytic plaques, it was found that Ejiao Qianggu Oral Liquid significantly (p<0.01) increased the area and number of hemolytic plaques formed in spleen cells.

[0178] Table 15 Area of ​​hemolytic plaques (AOI) (n=3)

[0179] Group Hemolytic empty spot AOI normal group 115.21±18.20 Levamisole group 289.73±22.46** Low-dose group of donkey-hide gelatin for strengthening bones 305.84±32.11** Donkey-hide gelatin for strengthening bones (medium dose group) 608.22±37.80** High-dose group of donkey-hide gelatin for strengthening bones 404.69±27.25**

[0180] III. Summary

[0181] The plaque-induced hemolytic assay is a method for detecting the formation of B lymphocytes stimulated by antibodies. The principle is to stimulate mice with heterologous red blood cells, extract the spleen lymphocytes of the immunized mice and prepare a suspension, and incubate them with the antigen red blood cells in vitro. The immune cells will release hemolysin against the red blood cells, and with the participation of complement, the surrounding red blood cells will be dissolved to form plaques. The strength of the mouse's immunity can be observed by observing the size of the well plate.

[0182] The blood routine results and plaque effect diagrams in this experiment confirm that Ejiao Qianggu Oral Liquid can increase the number of B lymphocytes in splenic lymphocytes, thereby enhancing the body's immunity. In other words, Ejiao Qianggu Oral Liquid has a significant regulatory effect on humoral immunity.

[0183] Example 4: The regulatory effect of donkey-hide gelatin bone-strengthening oral liquid on serum IgA, IgG, and IgM in an immunosuppressed animal model

[0184] The immunodeficiency mouse model established in this experiment was based on the existing method for creating a bone marrow suppression mouse model. Cyclophosphamide was used to enhance the stimulation of the model on the 7th day of modeling. The modeling results showed that the body weight, white blood cell count, and monocyte count of the mice were significantly lower than those of the normal group after modeling. The modeling effect was still significantly different between the modeling group and the normal group 14 days after drug administration, indicating that the modeling method is still feasible.

[0185] I. Experimental Methods

[0186] 1. Test reagents and methods

[0187] 4.1 Laboratory Animals

[0188] BALB / c mice, weighing approximately 18-20g, were divided into males and females, totaling 84 mice.

[0189] 4.2 Experimental Reagents

[0190] Chloramphenicol tablets: Chongqing Dikang Changjiang Pharmaceutical Co., Ltd., batch number 200202;

[0191] Cyclophosphamide: Baxter International Ltd., lot number OK426A;

[0192] Recombinant human colony-stimulating factor injection: Beijing Shuanglu Pharmaceutical Co., Ltd., batch number 20191010;

[0193] Donkey-hide gelatin oral liquid for strengthening bones: Xinjiang Huashidan Pharmaceutical Co., Ltd., batch number 20020401

[0194] Mouse IgA Reagent Kit: LOT: A27410841; Hangzhou Lianke Biotechnology Co., Ltd.

[0195] Mouse IgM Reagent Kit: LOT: A27610841; Hangzhou Lianke Biotechnology Co., Ltd.

[0196] Mouse IgG Reagent Kit: LOT: A27110735; Hangzhou Lianke Biotechnology Co., Ltd.

[0197] 4.3 Modeling Method

[0198] (1) Solvent preparation: Chloramphenicol injection (chloramphenicol tablets are diluted with propylene glycol to a final concentration of 8 mg / ml, filtered through a 0.22 μm filter membrane for sterilization and then stored); Cyclophosphamide injection (cyclophosphamide is dissolved in physiological saline for injection to a final concentration of 10 mg / ml).

[0199] (2) Model preparation: After acclimatizing to the environment for 3 days, 84 normal mice (half male and half female) were randomly divided into a normal group (n=12) and a model group (n=72) according to body weight. The model group mice were given intraperitoneal injections of chloramphenicol 50 mg / kg / day for 3-5 consecutive days. Two mice from the normal group and three mice from the model group were used to detect the levels of IgG and IgM in their blood and complete blood counts (lymphocytes and white blood cells). The mortality rate and model success rate of the mice were recorded. Seven days after the initial administration, cyclophosphamide 40 mg / kg / day was administered again.

[0200] 4.4 Experimental Methods

[0201] (1) Animal grouping: Approximately 55 mice that successfully developed the model were randomly divided into 5 drug groups (n=10 per group) for 20 consecutive days of drug administration. The mice were then divided into the following groups: blank control group, model control group, recombinant human colony-stimulating factor GM-GFM group, and donkey-hide gelatin oral liquid (4ml, 8ml, 16ml / kg). The mice were administered the drug for 14 consecutive days. Body weight was measured and recorded daily, and general physical signs were observed.

[0202] (2) Animal handling and sample collection: After 14 days of gavage administration to each group of animals, half of the animals were collected, fasted, weighed, and their weight was recorded. Blood was collected from the inner canthus, with 0.5 ml of blood placed in a tube containing anticoagulant. The anticoagulated blood was used for routine blood analysis. Approximately 1.5 ml of blood was placed in a clean EP tube, incubated overnight at 4 degrees Celsius, and centrifuged at 3500 rpm for 15 minutes to collect the serum.

[0203] (3) Indicator Testing

[0204] Organ coefficients were determined by collecting animal thymus and spleen.

[0205] Animal serum was collected for IgA, IgG, and IgM content detection;

[0206] Collect anticoagulated blood for routine blood tests.

[0207] II. Experimental Results

[0208] 4.1 Results of Immunohistolytic Mouse Model

[0209] The body weight of mice in the model group was significantly lower than that in the normal group (P≤0.01). Two mice from the normal group and three mice from the model group were randomly selected for blood routine tests. The results showed that the white blood cell and lymphocyte counts in the model group were significantly lower than those in the normal group (P≤0.01) (Table 16). Therefore, the immunodeficient mouse model was established. A total of 72 mice were used for this modeling experiment. 14 mice died during the modeling process, and blood routine tests were performed on 3 of them. 55 animals were enrolled in the modeling group. The modeling success rate in this experiment was 80.56%, and the mortality rate was 19.44%.

[0210] Table 16 Blood routine indicators of mice 8 days after modeling (n=3)

[0211] Group leukocyte Total number of lymphocytes Total number of monocytes Total number of neutrophils Total number of red blood cells hemoglobin Total platelet count normal group 3.65±0.36 2.61±0.001 0.40±0.21 0.52±0.09 7.22±0.39 134±2.83 784±66.47 Modeling group 0.73±0.20** 0.21±0.26** 0.02±0.03** 0.44±0.35 6.53±0.70 118.5±11.79 572.25±148.76

[0212] Note: Compared with the normal group, "**" indicates a significant decrease (p < 0.01).

[0213] 4.2 Changes in mouse body weight

[0214] The experimental results showed that on day 14 of administration, the body weight of mice in the model group was still significantly lower than that in the normal group (p < 0.01). After 14 days of administration of Ejiao Qianggu Oral Liquid, the body weight of mice recovered significantly compared with that of mice after modeling, as shown in Table 17.

[0215] Table 17 Changes in mouse body weight (n=10)

[0216]

[0217] Note: Compared with the normal group, "**" indicates a significant increase (p < 0.01).

[0218] 4.3 Mouse immune organ coefficient

[0219] Table 18 shows that the different dosage groups of Ejiao Qianggu Oral Liquid significantly increased the thymus coefficient in mice compared to the model group (p < 0.01), suggesting that Ejiao Qianggu Oral Liquid may act on T cell maturation and proliferation. Furthermore, the positive control drug increased the spleen coefficient by 6 times, and spleen edema also occurred, consistent with the results of the previous experiment.

[0220] Table 18 Weight coefficients of mouse immune organs (n=10)

[0221] Group Spleen coefficient thymus coefficient normal group 0.18±0.03 0.13±0.05 Model group 0.20±0.04 0.10±0.03 G-CSF Group 1.28±0.33## 0.07±0.04 Low-dose group of donkey-hide gelatin for strengthening bones 0.32±0.07## 0.08±0.03 Medium dose group of donkey-hide gelatin for strengthening bones 0.31±0.10## 0.10±0.04 High-dose group of donkey-hide gelatin for strengthening bones 0.27±0.06## 0.08±0.04

[0222] Note: Compared with the model group: "##": p < 0.01 significantly increased;

[0223] 4.4 Blood routine test results

[0224] Table 19 shows that after 14 days of administration, the total number of white blood cells, lymphocytes, and monocytes in the model group and the normal group mice were significantly reduced (p<0.05); while compared with the model group, Ejiao Qianggu Oral Liquid had a significant effect on increasing white blood cells, lymphocytes, and monocytes (p<0.05); the positive drug colony-stimulating factor had a 28-fold effect on increasing white blood cells.

[0225] Table 19 Blood routine tests of mice after 14 days of administration of donkey-hide gelatin and bone-strengthening oral liquid (n=5)

[0226]

[0227]

[0228] Note: Compared with the normal group, "**" indicates p < 0.01, "*" indicates p < 0.05, indicating a significant increase in the difference; compared with the model group, "##" indicates p < 0.01, "#" indicates p < 0.05, indicating a significant increase in the difference.

[0229] 4.5 Immunoglobulin Detection Results

[0230] Serum IgA, IgM, and IgG levels in mice from different groups were detected using an ELISA kit. Serum samples from five mice in each group were used, with two replicate wells for ELISA testing. The results showed that the model group mice had significantly lower levels of the three immunoglobulins compared to the normal group mice. After 14 days of administration, the low-dose group of the donkey-hide gelatin oral liquid showed a significantly higher IgM level compared to the model group (p < 0.05), while the high-dose group showed a significantly higher IgG level compared to the model group (p < 0.05), as shown in Table 20.

[0231] Table 20 Three Immunoglobulins in the Immunosuppression Model (n = 5 * 2)

[0232] Group <![CDATA[IgA(×10 3 ng / mL)]]> <![CDATA[IgM(×10 4 ng / mL)]]> <![CDATA[IgG(×10 4 ng / mL)]]> normal group 29.51±6.43 30.22±4.91 363.79±20.50 Model group 22.97±6.11* 23.76±1.79* 143.59±36.00* GM-GSF Group 21.68±3.07 17.38±6.90 118.03±43.36 Low-dose group of donkey-hide gelatin for strengthening bones 17.02±3.48 28.01±9.00# 159.69±26.50 Medium dose group of donkey-hide gelatin for strengthening bones 19.72±3.43 22.99±5.52 194.19±17.37# High-dose group of donkey-hide gelatin for strengthening bones 18.67±3.74 26.09±7.53 207.02±21.50#

[0233] Note: Compared with the normal group, "*" indicates a significant decrease (p < 0.05); compared with the model group, "#" indicates a significant increase (p < 0.05).

[0234] III. Summary

[0235] The immunodeficiency mouse model established in this experiment was based on the existing method for creating a bone marrow suppression mouse model. Cyclophosphamide was used to enhance the stimulation of the model on the 7th day of modeling. The modeling results showed that the body weight, white blood cell count, and monocyte count of the mice were significantly lower than those of the normal group after modeling. The modeling effect was still significantly different between the modeling group and the normal group 14 days after drug administration, indicating that the modeling method is still feasible.

[0236] The animal model established showed that, after 14 days of administration, the donkey-hide gelatin oral liquid group significantly increased the number of white blood cells, lymphocytes, and monocytes in mice compared to the model group (p < 0.05); while the positive control group's colony-stimulating factor (SCGF) increased the number of white blood cells by 28 times. Observation of organ coefficients in mice revealed that each dose group of the donkey-hide gelatin oral liquid significantly increased the thymus coefficient compared to the model group (p < 0.01), suggesting that the donkey-hide gelatin oral liquid may act on T cell maturation and proliferation. Furthermore, the positive control group increased the spleen coefficient by 6 times, and spleen edema also occurred, consistent with the results of the previous experiment.

[0237] In this experiment, an Elisa kit was used to detect serum IgA, IgM, and IgG in mice of different groups. Serum samples from 5 mice were taken in each group, and ELISA was performed with 2 replicate wells. The experimental results showed that the levels of the three immunoglobulins in the model group of mice were significantly lower than those in the normal group; after 14 days of administration, the low-dose group of Ejiao Qianggu Oral Liquid had a significantly increased IgM compared with the model group (p < 0.05), and the high- and medium-dose groups of Ejiao Qianggu Oral Liquid had a significantly increased IgG compared with the model group (p < 0.05).

[0238] Based on the comprehensive experimental results, Ejiao Qianggu Oral Liquid can act on the thymus of the immune-deficient mouse model, significantly increase the total number of white blood cells, lymphocytes, and monocytes in the model mice, and its effect is milder compared with colony-stimulating factor. In terms of immunoglobulins, the kit results showed that Ejiao Qianggu Oral Liquid has a significant enhancing effect on IgM and IgG.

[0239] Example 5. Effects of Ejiao Qianggu Oral Liquid on the contents of serum IgG, IgM, IgA in mice, the concentration of cAMP in splenocytes, the proliferation of splenic lymphocytes, and the experiments of macrophages phagocytosing neutral red and Staphylococcus aureus

[0240] Immunoglobulins are a group of proteins with antibody activity, mainly present in the blood, tissue fluid, and external secretions of organisms, and are an important indicator for examining the humoral immune function of the body. Human immunoglobulins are divided into five categories, namely IgG, IgA, IgM, IgD, and IgE. Among them, the contents of IgD and IgE are very low, so the Ig commonly measured by us is mainly IgG, IgA, and IgM.

[0241] I. Experimental methods

[0242] 5.1 Experimental animals

[0243] BALB / c mice, weighing about 18 - 20 g, with half males and half females, 80 in total. Provided by the Animal Experiment Center of Xinjiang Medical University, license number: SCXK(Xin)2019 - 0001

[0244] 5.2 Reagents and medicinal materials

[0245] Mouse cAMP enzyme-linked immunosorbent assay kit: product number JL13362, Shanghai Jianglai Industrial Co., Ltd.;

[0246] Mouse cGMP enzyme-linked immunosorbent assay kit: product number JL12644, Shanghai Jianglai Industrial Co., Ltd.;

[0247] Phytohemagglutinin PHA: manufacturer: Solabio, USA, product number: 403N032;

[0248] Canavon protein A: Manufacturer: Sigma, USA; Product number: C2010-2016;

[0249] LPS: Manufacturer: Solabio, USA; Product No.: 403N032;

[0250] Lymphocyte separation solution, manufacturer: Tianjin Haoyang Biotechnology, batch number 11015;

[0251] Donkey-hide gelatin oral liquid for strengthening bones: 19021201; Xinjiang Huashidan Pharmaceutical Co., Ltd.;

[0252] Levamisole hydrochloride: Manufacturer: Shanxi Yunpeng Pharmaceutical Co., Ltd., Batch No.: 20151101;

[0253] D-Hanks balanced salt solution (i.e., a Hanks solution free of calcium and magnesium ions): 8g NaCl, 0.4g KCl, 0.06g Na2HPO4·2H2O, 0.06g KH2PO4·2H2O, and 0.35g NaHCO3 are dissolved in 1000ml ultrapure water without adding phenol red. The solution is then sterilized by high-temperature steam and stored at 4℃.

[0254] PBS solution: Dissolve 8g NaCl, 0.2g KCl, 1.56g Na2HPO4·2H2O, and 0.2g KH2PO4·2H2O in 1000ml of ultrapure water, sterilize by high-temperature steam, and store at 4℃.

[0255] PI solution: Weigh 6 mg of PI powder and dissolve it in 1 ml of PBS solution to prepare a 100× stock solution. Store at 4°C protected from light. Use 1× working solution to achieve a final concentration of 1-5 μg / ml.

[0256] Staphylococcus aureus: ATCC25923, American Center for Type Culture Collection;

[0257] 5.3 Model Methods

[0258] Eighty Balb / c mice were randomly divided into six groups: a blank control group, a positive control group (levamisole hydrochloride group (LM): intraperitoneal injection of 50 mg / mL levamisole hydrochloride, 0.2 mL / mouse; Zhenqi granules: gavage), and three dosage groups of donkey-hide gelatin and bone-strengthening oral liquid (4 ml, 8 ml, 16 ml / kg). Each group was administered the drug by gavage for 14 consecutive days. The model control group was given an equal volume of distilled water (DW).

[0259] 5.4 Detection methods and indicators

[0260] (1) Effects on the proliferation and transformation of mouse spleen lymphocytes:

[0261] Spleen cell isolation and culture: Mouse spleens were aseptically harvested, and the outer membrane of the spleen was carefully removed. The spleen was homogenized until it reached a liquid state using a homogenizer. The mixture was then filtered through a 120-mesh cell sieve, and the filtrate was collected and added to an equal volume of lymphocyte separation medium. The mixture was incubated at 2500 rpm. -1 After 20 minutes, lymphocytes and monocytes were collected and placed in centrifuge tubes. Five volumes of erythrocyte lysis buffer were added, and the mixture was incubated at 37°C for 10 minutes. The cells were washed twice with Hank's solution and diluted to 1×10⁻⁶. 7 / mL. Splenic lymphocyte proliferation: 100 μL of mixed lymphocyte suspension was added to each well of a 96-well plate and cultured for 5 h. PHA (final concentration 50 μg·mL⁻¹) was added, and the cell plate was incubated at 37℃ in a 5% CO₂ incubator for 48 h. The lymphocyte proliferation rate of different groups was then measured at 570 nm using the MTT assay.

[0262] (2) Effects on the levels of serum immunoglobulins IgG, IgM, and IgA in mice;

[0263] Preparation of mouse blood: 1 ml of blood was collected from the eyeballs of each group of mice, placed in an anticoagulant tube, and sent to the Laboratory Department of Urumqi County Hospital for testing of serum IgG, IgM, and IgA levels. The reagent kit was used to detect serum cAMP and cGMP levels.

[0264] (3) Effect on intracellular cAMP concentration in spleen cells

[0265] Take an equal amount of spleen cells (1×10⁻⁶) from each group of mice. 7 1 ml of suspension ( / mL) was washed three times with pre-cooled PBS, centrifuged to collect cells, sonicated on ice, centrifuged at 12000 r / min for 10 min at 4℃, and the supernatant was collected. The contents of cAMP and cGMP were detected by enzyme-linked immunosorbent assay kit.

[0266] (4) Carbon clearance of mouse macrophages

[0267] One hour after the last administration, three mice in each group were injected intravenously with an Indian ink saline solution (1:4), at a rate of 0.1 ml per 10 g body weight. Two minutes (T1) and ten minutes (T2) after injection, 20 μl of blood was collected from the orbital cavity of each mouse and added to 2 ml of 0.1% Na2CO3 solution. After mixing, the absorbance values ​​A1 and A2 were measured at 600 nm. The animals were then sacrificed, and the liver and spleen were weighed. The carbon clearance index K and phagocytic index α were calculated.

[0268] K=(logAl-logA2) / (T2-T1).

[0269]

[0270] (5) Effects of mouse macrophages on the phagocytosis of neutral red blood cells

[0271] Mice were euthanized by cervical dislocation, and 5 ml of pre-cooled physiological saline was injected intraperitoneally. 5 ml of peritoneal exudate was collected in a centrifuge tube (ice bath). The tube was centrifuged at 1600 rpm for 10 minutes, the supernatant was discarded, and the volume was adjusted to 5 ml with 4°C physiological saline. Macrophages were counted under a hemocytometer. The macrophage concentration was adjusted to 1 × 10⁻⁶ cells / mL with physiological saline. 6 / ml, take 4ml, centrifuge at 1600rpm for 10 minutes, discard the supernatant, add 5ml, 10% FCS, RPM 1640 medium to resuspend. Add macrophages to culture flasks for purification and culture for 2h, discard the culture supernatant, digest with 0.25% trypsin or scrape cells, and resuspend in culture medium.

[0272] Inoculate 1×10 per well 6 Cells per ml, 200 μl of cells adhered and cultured for 2-3 h, then the culture medium was discarded and different concentrations of drugs were added and cultured for 24 h, then the supernatant was discarded;

[0273] Add 200 μL of 0.1% Neutral Red, incubate for 1 h, then wash twice with PBS, add 200 μL of cell lysis buffer (acetic acid: anhydrous ethanol = 1:1) per well, incubate overnight at 4 °C or for 30 min at 37 °C, and measure OD value at 570 nm wavelength.

[0274] (6) PI fluorescence staining of macrophages phagocytizing Staphylococcus aureus

[0275] Bacterial culture preparation: Staphylococcus aureus stored at -80℃ was inoculated at a ratio of 1% into centrifuge tubes containing tryptic soy broth, and incubated overnight at 37℃ and 220 rpm. An appropriate amount of the bacterial culture was centrifuged at 3000 rpm for 5 min; the supernatant was discarded, and 1×PBS was added and mixed well. The mixture was then centrifuged twice at 3000 rpm for 3 min each time. After incubation, the bacteria were mixed with culture medium and detected at 600 nm using a UV spectrophotometer. The bacterial concentration was calculated by comparing with a standard curve, and the bacterial culture was diluted to 1×10⁻⁶. 6 CFU / ml.

[0276] Peritoneal macrophage preparation: Following the macrophage preparation method described above, 1 × 10⁶ macrophages were seeded per well for each group. 6 / ml, 200ul cells were cultured adherently (the culture medium did not contain antibiotics);

[0277] Infection experiment: Macrophages were incubated at a ratio of 1:1.5 per well for 2 h, cooled on ice, and washed 3 times with PBS. 100 μL of fixative methanol:glacial acetic acid (3:1) was added, and the mixture was incubated for 30 min. After washing 3 times with PBS, 200 μL of PI staining solution was added, mixed well, and counterstained at 4 °C in the dark for 10 min. The mixture was washed twice with PBS and then observed and analyzed under a fluorescence microscope.

[0278] II. Experimental Results

[0279] 5. Effects of donkey-hide gelatin oral liquid on serum IgG, IgM, IgA, splenic lymphocyte proliferation, and splenic cell cAMP content in normal mice.

[0280] 5.1 Changes in body weight in mice 14 days after drug administration

[0281] As shown in Table 22, the body weight of mice in the drug group decreased significantly after treatment, while no significant change was observed in the high-dose donkey-hide gelatin bone-strengthening group. This indicates that donkey-hide gelatin bone-strengthening oral liquid has a lower effect on body weight compared with the positive control drugs levamisole and Zhenqi granules.

[0282] Table 22 Body weight of mice 14 days after drug administration (n=14)

[0283] Group number Body weight (g) normal group 26.21±1.94 Levamisole group 22.89±1.67** Zhenqi Granules Group 22.13±3.17** Low-dose group of donkey-hide gelatin for strengthening bones 21.82±1.51** Medium dose group of donkey-hide gelatin for strengthening bones 23.96±1.88* High-dose collagen-strengthening group 25.45±3.38

[0284] Note: Compared with the normal group, "**" indicates p < 0.01, which is highly significant; compared with the normal group, "*" indicates p < 0.05, which is significant.

[0285] 5.2 Coefficients of immune organs: thymus and spleen

[0286] The thymus is primarily the site of T lymphocyte development and maturation, while the spleen is mainly an organ for the proliferation and transformation of B cells and some T cells. The results of this experiment show that the positive control drugs (levamisole and Zhenqi granules) and the high-dose group of Ejiao Qianggu oral liquid significantly increased the spleen coefficient in mice. Anatomical observation revealed that the spleen in the positive control drug group was significantly enlarged and edematous. In contrast, compared to the positive control drugs, Ejiao Qianggu oral liquid increased the spleen coefficient while restoring the degree of splenic swelling in mice without causing pathological changes.

[0287] Table 23 Immune organ coefficients in mice after drug administration

[0288]

[0289]

[0290] Note: Compared with the normal group, "*" indicates p < 0.05, which is a significant difference; compared with the Zhenqi granules group, "#" indicates p < 0.05, which is a significant difference.

[0291] 5.3 Serum IgA, IgM and IgG levels in mice

[0292] IgM has the ability to lyse bacteria and blood cells, and can neutralize viruses; its efficacy is more than 100 times higher than that of IgG. Many natural antimicrobial antibodies, hemagglutinins (anti-A and anti-B blood types), rheumatoid factor in rheumatoid arthritis, and complement-fixing antibodies in syphilis all belong to IgM. Results showed that the high-dose group of donkey-hide gelatin oral liquid significantly promoted and increased IgM levels in normal mice (p < 0.05).

[0293] Table 24 Serum IgA, IgM, and IgG levels in mice (n=7)

[0294] Group number IgG IgA IgM normal group 0.48±0.05 0.17±0.01 0.41±0.03 Levamisole group 0.43±0.13 0.17±0.01 0.60±0.08 Zhenqi Granules Group 0.41±0.12 0.17±0.02 0.54±0.08 Low-dose group of donkey-hide gelatin for strengthening bones 0.47±0.05 0.17±0.03 0.59±0.02 Donkey-hide gelatin for strengthening bones (medium dose group) 0.41±0.08 0.17±0.04 0.58±0.07 High-dose group of donkey-hide gelatin for strengthening bones 0.43±0.05 0.17±0.05 0.65±0.05*

[0295] Compared with the normal group (*): p < 0.05, the difference is significant.

[0296] 5.4 Complete blood count in mice

[0297] Blood routine tests showed that, compared with the normal group, the medium-dose group of donkey-hide gelatin oral liquid significantly increased the total number of white blood cells and total number of lymphocytes in mice (p<0.05).

[0298] Table 25. Routine blood tests in mice (n=7)

[0299]

[0300] Note: Compared with the normal group "*": p < 0.05, significant difference; compared with the model group "#": p < 0.05, significant difference.

[0301] 5.5 Changes in cAMP and cGMP levels in mouse spleen and serum

[0302] An increase in the cAMP to cGMP ratio can reflect an increase in the body's immune level, while a decrease can indicate a decrease in the body's immune capacity. Figure 12 It is the standard curve of cyclic guanosine monophosphate (cGMP). Figure 13 It is the standard curve for cyclic adenosine monophosphate (cAMP).

[0303] The results of this experiment showed that Ejiao Qianggu Oral Liquid had an increasing effect on cGMP in the spleen compared with the normal group, but the effect was not significant; while the cAMP / cGMP ratio results showed that the ratio in serum was increased in both groups compared with the normal group, but the effect was not significant.

[0304] Table 26. cGMP and cAMP levels in mice (n=7)

[0305]

[0306] 5.6 Proliferation of mouse spleen cells

[0307] The results of the spleen cell proliferation experiment showed that the proliferation of the positive drug levamisole and donkey-hide gelatin oral liquid group was significantly increased compared with the normal group.

[0308] Table 27 Changes in spleen cell proliferation rate in mice (n=7)

[0309]

[0310]

[0311] Note: Compared with the normal group, "*" indicates p < 0.05, which is statistically significant;

[0312] 5.7 Mouse carbon clearance

[0313] Table 28. Carbon clearance index in mice (n=7)

[0314] Group number k a normal group 0.03 6.44 Levamisole group 0.05 7.71 Zhenqi Granules Group 0.01 5.67 Low-dose group of donkey-hide gelatin for strengthening bones 0.02 6.01 Medium dose group of donkey-hide gelatin for strengthening bones 0.01 5.32 High-dose group of donkey-hide gelatin for strengthening bones 0.01 4.74

[0315] 5.8 Macrophage phagocytosis of neutral red experiment

[0316] Table 29. Phagocytosis of neutral red by mouse macrophages (n=7)

[0317] Group number Neutral Red OD value (570) normal group 0.65±0.05 Levamisole group 0.80±0.17 Zhenqi Granules Group 0.91±0.07** Low-dose group of donkey-hide gelatin for strengthening bones 1.11±0.18** Medium dose group of donkey-hide gelatin for strengthening bones 1.11±0.46** High-dose group of donkey-hide gelatin for strengthening bones 0.65±0.22

[0318] Note: Compared with the normal group "*": p < 0.05, significant difference; compared with the model group "#": p < 0.05, significant difference.

[0319] 5.8 Macrophage phagocytosis of Staphylococcus aureus with fluorescent staining experiment

[0320] Propidium iodide (PI) is a nucleic acid intercalating dye that can penetrate the macrophage cell membrane, staining the DNA and RNA of macrophages and their internal bacteria. The average fluorescence intensity of the cell is directly proportional to the number of bacteria phagocytosed in each cell, thus reflecting the degree of cell staining; the greater the number of bacteria phagocytosed, the stronger the fluorescence. Figure 14 This is a PI fluorescent staining image of macrophages phagocytizing Staphylococcus aureus.

[0321] Table 9 Fluorescence Intensity Analysis (n=3)

[0322]

[0323]

[0324] Note: Compared with the normal group, "**" indicates p < 0.0, meaning the difference is highly significant;

[0325] III. Summary

[0326] The purpose of this experiment was to detect changes in serum levels of three immunoglobulins (IgA, IgG, and IgM), spleen and blood levels of cAMP and cGMP, and macrophage activity in normal mice after treatment with donkey-hide gelatin oral liquid.

[0327] The results showed that in promoting immune organs, the positive control drugs (levamisole and Zhenqi granules) and the high-dose group of Ejiao Qianggu oral liquid could significantly increase the spleen coefficient in mice. However, anatomical observation revealed that the spleen in the positive control drug group was significantly enlarged and edematous. In contrast, compared with the positive control drugs, Ejiao Qianggu oral liquid could increase the spleen coefficient in restoring the degree of spleen swelling in mice without causing pathological changes.

[0328] Regarding the three immunoglobulins, the results of this experiment showed that the high-dose group of Ejiao Qianggu Oral Liquid significantly promoted and increased IgM levels. IgM has the ability to dissolve bacteria and blood cells, and can neutralize viruses; its efficacy is more than 100 times higher than that of IgG. Many natural antimicrobial antibodies, hemagglutinins (anti-A and anti-B blood types), rheumatoid factor in rheumatoid arthritis, and complement-fixing antibodies in syphilis all belong to IgM.

[0329] An increased cAMP / cGMP ratio reflects a rise in the body's immune level, while a decreased ratio indicates a decline in immunity. The results of this experiment showed that Ejiao Qianggu Oral Liquid had a slight effect on increasing cGMP levels in the spleen compared to the normal group; however, the increase was not significant. The cAMP / cGMP ratio in serum was also slightly higher in the Ejiao Qianggu Oral Liquid group compared to the normal group. Spleen cell proliferation experiments showed that the proliferation of cells in the positive control group (levamisole) and the Ejiao Qianggu Oral Liquid group was significantly increased compared to the normal group.

[0330] Experiments on macrophage phagocytosis of neutral red and Staphylococcus aureus showed that the donkey-hide gelatin and bone-strengthening oral liquid group could significantly enhance the phagocytic capacity of macrophages.

Claims

1. Application of Ejiao Qianggu Oral Liquid in the preparation of a medicament for treating or improving delayed-type hypersensitivity.

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