Composition for enhancing immunity as well as preparation method, product and application thereof

Abstract

The invention belongs to the technical field of food, and particularly relates to a composition for enhancing immunity as well as a preparation method, a product and application thereof. The composition comprises the following components in parts by weight: 40-60 parts of shiitake mushroom powder, 20-40 parts of lucid ganoderma powder, 5-15 parts of paecilomyces hepialid powder and 2-5 parts of an astragalus extract. Through specific compatibility, the pharmaceutical composition provided by the invention can significantly improve immune organ indexes, immune cell proliferation ability and activity, and can regulate intestinal flora and increase the number of beneficial bacteria. The components of the composition provided by the invention are all medicinal and edible substances, and have small toxicity or side effects on human bodies, so that the composition has high safety and is suitable for popularization and long-term eating.

Description

Technical Field

[0001] This invention belongs to the field of food technology, specifically relating to an immune-enhancing composition, its preparation method, product, and application. Background Technology

[0002] Strengthening immunity refers to enhancing one's own immune system through certain means. Immunity is the body's own defense mechanism, its ability to recognize and eliminate any invading foreign substances (viruses, bacteria, etc.), process aging, damaged, dead, and degenerated cells, and recognize and deal with mutated and virus-infected cells. The immune system is a crucial line of defense against invading pathogens. However, under certain circumstances, the immune system can be damaged, leading to decreased immunity. For example, chronic diseases, long-term stress, malnutrition, and drug side effects can all adversely affect the immune system.

[0003] Immunostimulant drugs can be used as adjunctive therapy to improve the effectiveness of disease treatment. In cancer treatment, immunotherapy has become an important approach, working by regulating the function of the immune system to kill tumor cells. Furthermore, in the treatment of infectious diseases, immunostimulant drugs can help patients clear pathogens more quickly and shorten the course of the disease. For healthy individuals, immunostimulant drugs can also be a choice for daily health maintenance. Long-term use of appropriate amounts of immunostimulant drugs can strengthen the body, improve immunity, and prevent the occurrence of diseases.

[0004] Food-medicine homology substances play a crucial role in enhancing immunity, and this importance is reflected in several aspects. First, these substances are typically composed of various natural plant, animal, or mineral components, which are widely found in nature and have extremely low toxicity and side effects. Second, the multiple components in these substances can synergistically work together to enhance immunity, resulting in a more comprehensive and profound effect on boosting immunity.

[0005] Food and medicine homologous substances play an irreplaceable role in enhancing immunity. Through natural components, synergistic effects, personalized customization, and multiple immune-enhancing mechanisms, they provide effective solutions for improving human immunity. Therefore, it remains necessary to continue developing drugs to enhance immunity to meet the needs of different populations, especially those with compromised or weakened immune systems. Summary of the Invention

[0006] To address the aforementioned problems, the present invention provides a composition that exhibits superior synergistic effects in enhancing immunity. The components of the composition are both edible as food and possess certain medicinal properties, exhibiting characteristics such as high efficiency, low toxicity, and broad spectrum.

[0007] On the one hand, the present invention provides a composition for enhancing immunity, comprising, by weight, 40-60 parts of shiitake mushroom powder, 20-40 parts of Ganoderma lucidum powder, 5-15 parts of Paecilomyces hepiali powder and 2-5 parts of Astragalus membranaceus extract.

[0008] Specifically, the combination comprises, by weight, 40 parts of shiitake mushroom powder, 40 parts of Ganoderma lucidum powder, 5 parts of Paecilomyces hepiali powder, and 5 parts of Astragalus membranaceus extract.

[0009] Specifically, the combination comprises, by weight, 60 parts of shiitake mushroom powder, 20 parts of Ganoderma lucidum powder, 15 parts of Paecilomyces hepiali powder, and 2 parts of Astragalus membranaceus extract.

[0010] Specifically, the combination comprises, by weight, 50 parts of shiitake mushroom powder, 30 parts of Ganoderma lucidum powder, 10 parts of Paecilomyces hepiali powder, and 3 parts of Astragalus membranaceus extract.

[0011] In another aspect, the present invention provides a method for preparing the aforementioned composition, comprising the following steps: S1. Take the prescribed amounts of shiitake mushroom powder, Ganoderma lucidum powder, Paecilomyces hepiali powder and Astragalus membranaceus extract and mix them to obtain mixture A; S2. Add the mixture A obtained in step S1 to the solvent, and after dissolution, the composition is obtained.

[0012] Specifically, the solvent in step S2 includes, but is not limited to, water, and the dissolution is complete.

[0013] Specifically, the content of mixture A in the composition described in step S2 is not less than 30% m / m.

[0014] Preferably, the content of mixture A in the composition described in step S2 can be 30% m / m.

[0015] In another aspect, the present invention provides the use of the aforementioned composition in the preparation of products that enhance immunity.

[0016] Specifically, the products include, but are not limited to, food or medicine.

[0017] In another aspect, the present invention provides a food product comprising the aforementioned composition.

[0018] Specifically, the food also includes food additives that can be used on food.

[0019] Preferably, the excipients available for use on the food are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0020] Preferably, the excipients available for use on the food are selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0021] Specifically, the food products include, but are not limited to: gel particles, crystal balls, solid beverages, liquid beverages, ice cream, hard candies, shortbread candies, caramel candies, gel candies, milk candies, chocolate, gum-based candies, aerated candies, compressed candies, and / or jellies.

[0022] Specifically, the food may be a health food.

[0023] In another aspect, the present invention provides a medicament comprising the aforementioned composition.

[0024] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0025] More specifically, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0026] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0027] The technical effects achieved by this invention are as follows: (1) The composition provided by the present invention has no significant effect on the thymus index and spleen index of mice.

[0028] (2) The composition provided by the present invention can significantly improve the proliferation capacity of mouse lymphocytes.

[0029] (3) The composition provided by the present invention can significantly improve the activity of antibody-producing cells and the level of serum hemolysin.

[0030] (4) The composition provided by the present invention can significantly improve the ability of macrophages to engulf chicken red blood cells.

[0031] (5) The composition provided by the present invention can significantly improve NK cell activity.

[0032] (6) The composition provided by the present invention can significantly increase the total number of intestinal flora and increase the number of beneficial bacteria Bifidobacterium and Lactobacillus. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0034] Example 1 Shiitake mushroom powder: Pingquan Tiance Edible Fungus Professional Cooperative, conforming to GB 7096.

[0035] Ganoderma lucidum powder: Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd., polysaccharide ≥0.9% (m / m).

[0036] Hepialus fasciatus powder: Jiangsu Shenhua Pharmaceutical Co., Ltd., adenosine ≥0.28 (m / m).

[0037] Astragalus extract: Zhejiang Yangzhikang Biotechnology Co., Ltd., polysaccharide ≥20% (m / m).

[0038] Codonopsis pilosula extract: Shaanxi Jiahe Biotechnology Co., Ltd., polysaccharide ≥10% (m / m).

[0039] Atractylodes macrocephala extract: Shaanxi Jiahe Biotechnology Co., Ltd., crude polysaccharide ≥15% (m / m).

[0040] 1.1 Composition of the composition By weight: 40 parts shiitake mushroom powder, 40 parts Ganoderma lucidum powder, 5 parts Paecilomyces hepiali powder, and 5 parts Astragalus membranaceus extract.

[0041] 1.2 Preparation process (1) Take the formula amount of shiitake mushroom powder, Ganoderma lucidum powder, Paecilomyces cerevisiae powder and Astragalus membranaceus extract, and put them into a high-speed mixer in sequence for mixing (the mixer is kept running at low speed when feeding the materials). After feeding the materials, run the mixer at high speed for 5 minutes to obtain the mixture.

[0042] (2) Add the above mixture to a certain amount of distilled water in batches, and stir at a low speed of 200 rpm until the mixture is completely dissolved. The mixture ratio is 30% (m / m), and the composition is obtained.

[0043] Example 2 The difference between Example 2 and Example 1 is that the proportions of each component are different, but otherwise they are the same as in Example 1.

[0044] The composition of Example 2 (by weight) consists of: 60 parts of shiitake mushroom powder, 20 parts of Ganoderma lucidum powder, 15 parts of Paecilomyces hepiali powder, and 2 parts of Astragalus membranaceus extract.

[0045] Example 3 The difference between Example 3 and Example 1 is that the proportion of the composition is different, but otherwise the same as Example 1.

[0046] The composition of Example 3 (by weight): 50 parts of shiitake mushroom powder, 30 parts of Ganoderma lucidum powder, 10 parts of Paecilomyces hepiali powder, and 3 parts of Astragalus membranaceus extract.

[0047] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Astragalus extract was replaced with an equal amount of Codonopsis extract, otherwise the same as in Example 1.

[0048] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that Astragalus extract was replaced with an equal amount of Atractylodes macrocephala extract, otherwise the same as in Example 1.

[0049] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that Astragalus extract was not added; otherwise, they are the same as in Example 1.

[0050] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that no shiitake mushroom powder, Ganoderma lucidum and Paecilomyces hepiali powder were added, that is, only Astragalus membranaceus extract was added, and the rest was the same as in Example 1.

[0051] Comparative Example 5 The difference between Comparative Example 5 and Comparative Example 1 is that no shiitake mushroom powder, Ganoderma lucidum powder and Paecilomyces hepiali powder were added, that is, only Codonopsis pilosula extract was added, and the rest was the same as in Example 1.

[0052] Comparative Example 6 The difference between Comparative Example 6 and Comparative Example 2 is that no shiitake mushroom powder, Ganoderma lucidum powder and Paecilomyces hepiali powder were added, that is, only Atractylodes macrocephala extract was added, and the rest was the same as in Example 1.

[0053] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that no shiitake mushroom powder was added; otherwise, they are the same as Example 1.

[0054] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that shiitake mushroom powder and astragalus extract were not added, but otherwise the same as in Example 1.

[0055] Example 1: Effects of the composition on mouse body weight and organ / body weight ratio (1) Effects of the examples and comparative examples on the relative weight of organs in mice When mice are healthy, their thymus-to-body weight and spleen-to-body weight ratios are usually relatively stable. If mice are exposed to toxins or develop lesions during the experiment, the weight of the damaged organs will be affected and altered, and their organ indices may change.

[0056] Mice: Healthy SPF-grade BALB / c mice, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0057] Experimental grouping and intervention methods: The experiment was divided into a blank control group, Examples 1-3, and Comparative Examples 1-8, with 10 mice in each group. Each mouse was administered a gavage dose of 3100 mg / kg BW at a volume of 20 mL / kg BW. The blank control group was administered an equal volume of distilled water by gavage. All treatments were given orally once daily for 30 consecutive days. During this period, mice in all groups were housed in a barrier-level animal room at a temperature range of (22±2)℃ and a relative humidity of 50%-60%, with a day / night cycle of 12h:12h. They were fed an SPF-grade basal diet and provided with free access to food and water.

[0058] The results are shown in Table 1. There was no significant difference in body weight among the mice in all groups at the end of the experiment. P The results showed that the mouse body weight was not affected by the composition, and there were no significant differences in the spleen / body weight ratio and thymus / body weight ratio (P>0.05), indicating that the composition had no significant effect on the mouse organs (see Table 1).

[0059] Table 1. Effects of intervention on mouse body weight and organ / body weight ratio in the examples and control groups.

[0060] Example 2: Effect of the composition on delayed-type hypersensitivity in mice Delayed-type hypersensitivity (DTH) testing is a method for assessing cellular immune function. It involves subcutaneously injecting an antigen and observing whether mice develop a skin allergic reaction to evaluate their cellular immune function. Sheep erythrocytes (SRBCs) can stimulate and attack mice, causing swelling of the paw pads. The degree of swelling directly reflects the effect on delayed-type hypersensitivity (DTH) in mice, and thus reflects the effect on the cellular immune function of mice. (Mouse lymphocyte activity assay.)

[0061] (1) Experimental methods Mice: Healthy SPF-grade BALB / c mice, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0062] Experimental grouping and intervention methods: The experiment was divided into a blank control group, Examples 1-3, and Comparative Examples 1-8, with 10 mice in each group. Each mouse was administered a gavage dose of 3100 mg / kg BW at a volume of 20 mL / kg BW. The blank control group was administered an equal volume of distilled water by gavage. All treatments were given orally once daily for 30 consecutive days. During this period, mice in all groups were housed in a barrier-level animal room at a temperature range of (22±2)℃ and a relative humidity of 50%-60%, with a day / night cycle of 12h:12h. They were fed an SPF-grade basal diet and provided with free access to food and water.

[0063] Delayed-type hypersensitivity was used to detect cellular immunity in vivo. First, sheep red blood cells (SRBCs) were prepared: sheep blood was collected via the jugular vein and transferred under sterile conditions to a sterile Erlenmeyer flask containing glass beads. The flask was agitated in one direction for 30 minutes to defibrinate the blood. The defibrinated blood was washed three times with physiological saline, centrifuged at 2500 rpm for 15 minutes each time, to prepare packed SRBCs. The packed SRBCs were diluted with physiological saline to a 2% (v / v) cell suspension (approximately 1 × 10⁻⁶ cells / v). 8 Mice in each group were immunized by intraperitoneal injection of 0.2 mL of 2% (v / v) SRBC on day 26 of the intervention with the compositions of the examples and comparative examples. On day 30, the thickness of the left hind paw metatarsal region of the mice was measured using calipers, and 20 μL of 20% (v / v) SRBC was injected subcutaneously at each measurement site. The thickness of the left hind paw metatarsal region of each mouse was measured 24 h after injection, and the average of three measurements was taken as the thickness of the metatarsal region. The same caliper was used for both measurements, and the measurements were performed by a specialist. The caliper was placed firmly against the left hind paw metatarsal region of the mouse, but no pressure was applied. The difference in the thickness of the left hind paw metatarsal region of the mice on day 30 and day 31 was used to represent the degree of DTH.

[0064] (2) Experimental results The results of the delayed-type hypersensitivity test in mice are shown in Table 2: Compared with Comparative Examples 1-8, the difference in paw swelling in mice after intervention in Examples 1-3 was significantly increased (p < 0.05), indicating that Examples 1-3 could significantly improve the delayed-type hypersensitivity ability of mice; there was no significant difference in the difference in paw swelling in Comparative Example 3 (without Astragalus extract) compared with Comparative Example 7 (without shiitake mushroom powder) and Comparative Example 8 (without shiitake mushroom powder and Astragalus extract) (p > 0.05), and there was also no significant difference between Comparative Example 7 and Comparative Example 8 (p > 0.05). This indicates that shiitake mushroom powder and Astragalus extract can improve the delayed-type hypersensitivity of mice. In terms of ability, it has a synergistic effect; in addition, compared with Comparative Example 5 (Codonopsis pilosula extract) and Comparative Example 6 (Atractylodes macrocephala extract), the difference in paw swelling of mice after intervention with Comparative Example 4 (Astragalus membranaceus extract) was lower, and the difference was significant (p<0.05). However, compared with Comparative Example 1 containing Codonopsis pilosula extract and Comparative Example 2 containing Atractylodes macrocephala extract, the difference in paw swelling of mice after intervention with Astragalus membranaceus extract was higher, and the difference was significant (p<0.05). This indicates that there is a synergistic effect between Astragalus membranaceus extract and other components in Examples 1-3 in improving the delayed-type hypersensitivity ability of mice.

[0065] Table 2. Measurement of paw edema in mice (mean ± standard error)

[0066] Note: Different lowercase letters indicate significant differences. P <0.05), the same lowercase letter indicates no significant difference ( P >0.05).

[0067] Effect of the composition on humoral immune function in mice (Example 3) The mice used in the antibody-producing cell detection and serum hemolysin assay in the humoral immune function experiment were from the same batch.

[0068] Laboratory animals: Healthy SPF-grade BALB / c mice, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0069] Experimental grouping and intervention methods: The experiment was divided into a blank control group, Examples 1-3, and Comparative Examples 1-8, with 10 mice in each group. Each mouse was administered a gavage dose of 3100 mg / kg BW at a volume of 20 mL / kg BW. The blank control group was administered an equal volume of distilled water by gavage. All treatments were given orally once daily for 30 consecutive days. During this period, mice in all groups were housed in a barrier-level animal room at a temperature range of (22±2)℃ and a relative humidity of 50%-60%, with a day / night cycle of 12h:12h. They were fed an SPF-grade basal diet and provided with free access to food and water.

[0070] (1) Detection of antibody-producing cells Antibody-producing cell assay is a method for measuring the immune response in mice. It assesses the immune response by observing whether the spleen cells or bone marrow cells of mice produce antibody-producing cells. The number of antibody-producing cells in mice is determined using a modified Jerne slide assay. The resulting hemolytic plaque data represents the number of antibodies produced; a higher number of hemolytic plaques indicates better antibody-producing cell activity.

[0071] Guinea pig: Hartley guinea pig, weighing 300-400g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0072] Complement preparation: Blood from guinea pigs untreated by the composition was collected via the femoral artery, allowed to stand for 60 min, and then centrifuged at 3000 rpm for 15 min to separate the serum. Serum from 10 guinea pigs was mixed and then combined with compressed SRBC (prepared using the same method as for delayed-type hypersensitivity reactions in Example 2) at a ratio of 5:1 (v / v). The mixture was incubated at 4 °C for 30 min (with occasional shaking), centrifuged at 2500 rpm for 15 min to separate the serum, and stored at -80 °C.

[0073] Slide coating: Dissolve 0.5 g of agarose in double-distilled water to 100 mL by heating to prepare an agarose solution. Brush a thin layer of agarose solution onto a clean slide and let it air dry. The mouse immunization method is the same as the delayed-type hypersensitivity reaction in Example 2. Five days after SRBC immunization, mice were euthanized by cervical dislocation, and their spleens were harvested. The spleens were gently ground in a petri dish containing Hank's solution using a grinding rod, filtered through a 200-mesh sieve, and a single-cell suspension was prepared. The suspension was washed twice with Hank's solution, centrifuged at 1000 rpm for 10 min each time, and then cultured in RPMI 1640 complete medium containing 10% fetal bovine serum to prepare 5×10⁶ cells / mL of agarose solution. 6 Spleen cell suspension was prepared at 1 g agarose / mL. 1 g agarose was dissolved in double-distilled water to 100 mL by heating to prepare the surface culture medium, which was then incubated at 50 °C. The surface culture medium was mixed with an equal volume of 2×Hank's solution (pH 7.2), and 0.5 mL was dispensed into each small test tube. 50 μL of 10% (v / v) SRBC (prepared with SA buffer) and 20 μL of spleen cell suspension were added to the tube, and the mixture was quickly mixed and poured onto agarose-coated slides. Three replicates were prepared for each spleen cell sample. After the agarose solidified, the slides were placed horizontally on a slide holder and incubated at 37 °C for 1.5 h in a 5% CO2 incubator. Complement was diluted 1:8 with SA buffer and added to the grooves of the slide holder. The slides were returned to the incubator and incubated for another 1.5 h. The number of hemolytic plaques was then counted. The average number of plaques from the three replicates was taken as the hemolytic plaque count for that sample, and the result was calculated as hemolytic plaque count / 10. 6 Spleen cells are represented.

[0074] (2) Serum hemolysin assay The preparation method of SRBC and the mouse immunization method were the same as in Example 2. Five days after immunization, the mice were enucleated and blood was collected. After standing in a centrifuge tube for 1 hour, the serum was separated by centrifugation at 3000 rpm for 10 minutes. The serum was diluted 200 times with SA buffer. 1 mL of the diluted serum was transferred to a 5 mL centrifuge tube, and 0.5 mL of 10% (v / v) SRBC and 1 mL of complement (SA buffer diluted 1:8) were added sequentially. Another 1 mL of SA buffer was used as a control tube instead of serum. All tubes were incubated at 37 ℃ for 30 minutes and then the reaction was stopped by ice bath. After centrifugation at 2000 rpm for 10 minutes, 1 mL of the supernatant was collected and 3 mL of Duchenne's reagent was added. At the same time, 0.25 mL of 10% (v / v) SRBC was collected and Duchenne's reagent was added to a final volume of 4 mL. After thorough mixing, the tubes were incubated for 10 minutes. The optical density of each tube was measured at 540 nm using an ELISA reader, with the control tube as a blank. Serum hemolysin levels are expressed as the half hemolysis value (HC50): HC50 = (Optical density value of the composition / Optical density value of SRBC half-hemolysis) × Dilution factor.

[0075] (3) Experimental results ① In spleen cells, antibody-producing cells release anti-SRBC antibodies, sensitizing surrounding SRBCs. With the participation of complement, SRBCs are dissolved, forming visible hemolytic plaques. Each plaque contains one antibody-producing cell in its center; the number of plaques represents the number of antibody-producing cells. A visible plaque forms around each plaque, with each plaque representing one antibody-producing cell. The number of hemolytic plaques reflects the body's humoral immune function. A higher number of hemolytic plaques indicates better antibody-producing cell activity and better immune function in the mouse.

[0076] The results are shown in Table 3. The number of plaques formed after the intervention of Examples 1-3 was significantly higher than that of Comparative Examples 1 and 2 (p < 0.05), and extremely significantly higher than that of Comparative Examples 3-8. However, there was no significant difference among Comparative Examples 3, 7, and 8 (p > 0.05). This indicates that Examples 1-3 have a good effect on improving the immune function of mice, and that shiitake mushroom powder and astragalus extract have a synergistic effect.

[0077] The number of hemolytic plaques produced by the spleen cells of mice after intervention with Comparative Example 4 (Astragalus extract) was significantly less than that of Comparative Example 5 (Codonopsis extract) and Comparative Example 6 (Atractylodes macrocephala extract), while the number of hemolytic plaques produced by the spleen cells of mice after intervention with Examples 1-3 (containing Astragalus extract) was significantly greater than that of Comparative Example 1 (containing Codonopsis extract) and Comparative Example 2 (containing Atractylodes macrocephala extract). This indicates that Astragalus extract has a synergistic effect with the components other than Astragalus extract in Examples 1-3 in increasing the number of antibody cells and improving the immune function of mice.

[0078] ② Mouse serum hemolysin is calculated using the half-hemolysin value (CH50). A higher CH50 value indicates a higher serum hemolysin level. In humoral immunity, a higher serum hemolysin content indicates a better immune-enhancing effect of the composition.

[0079] The results are shown in Table 3: The serum hemolysin levels generated after the intervention in Examples 1-3 were significantly higher than those in Comparative Examples 1 and 2 (p < 0.05), and also significantly higher than those in Comparative Examples 3-8 (p < 0.05). p <0.05), indicating that Examples 1-3 significantly increased serum hemolysin levels in mice; compared with Comparative Example 7 (without shiitake mushroom powder) and Comparative Example 8 (without shiitake mushroom powder and Astragalus extract), there was no significant difference in serum hemolysin levels in mice after intervention in Comparative Example 3 (without Astragalus extract) (p>0.05), and there was no significant difference between Comparative Example 7 and Comparative Example 8 (p>0.05), indicating that shiitake mushroom powder and Astragalus extract have a synergistic effect on serum hemolysin levels; in addition, compared with Comparative Example 5 (Codonopsis pilosula extract) and Compared with Comparative Example 6 (Atractylodes macrocephala extract), the serum hemolysin level of mice after intervention with Comparative Example 4 (Astragalus membranaceus extract) was lower, with a significant difference (p < 0.05). However, compared with Comparative Example 1 (containing Codonopsis pilosula extract) and Comparative Example 2 (containing Atractylodes macrocephala extract), the serum hemolysin level of mice after intervention with intervention with Astragalus membranaceus extract was higher, with a significant difference (p < 0.05). This indicates that the Astragalus membranaceus extract in Examples 1-3 has a synergistic effect with other components in increasing serum hemolysin levels.

[0080] Table 3 Number of hemolytic plaques and CH50 (mean ± standard error) in mice

[0081] Note: In the same column, different lowercase letters indicate significant differences. p <0.05), the same lowercase letter indicates no significant difference ( p >0.05).

[0082] Example 4: Effect of the composition on mouse monocyte-macrophage function (1) Mouse carbon clearance test Laboratory animals: Healthy SPF-grade BALB / c mice, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0083] Experimental grouping and intervention methods: The experiment was divided into a blank control group, Examples 1-3, and Comparative Examples 1-8, with 10 mice in each group. Each mouse was given a gavage dose of 3100 mg / kg BW at a volume of 20 mL / kg BW. The blank control group was given an equal volume of distilled water by gavage. All treatments were administered orally once daily for 30 consecutive days. During this period, mice in all groups were housed in a barrier-grade animal room at a temperature range of (22±2)℃ and a relative humidity of 50%-60%, with a day:night ratio of 12h:12h. They were fed an SPF-grade basal diet and provided with free access to food and water.

[0084] After gavage, diluted Indian ink (10 mL / kg) was injected into the tail vein of the mice according to their body weight, and the timing was started immediately after injection. Two and ten minutes after ink injection, 20 μL of blood was collected from the internal canthal venous plexus and immediately added to 2 mL of 0.1% Na₂CO₃ solution. The optical density (OD) value was measured at 600 nm. The mice were then sacrificed, and the liver and spleen were harvested. Blood stains on the surface of the organs were blotted dry with filter paper, and the organs were weighed separately. The phagocytic index α was calculated to represent the carbon clearance capacity of the mice.

[0085]

[0086]

[0087] In the above formula, k — rate of absorption, ɑ —Eat-out Index OD1 —OD measured by blood sample taken 2 minutes after ink injection. OD2 —OD measured by blood sample taken 10 minutes after ink injection. t1 —2 min, t2 —10 min.

[0088] (2) Experimental results The results are shown in Table 4: The carbon clearance capacity of mice after intervention in Examples 1-3 was significantly higher than that in Comparative Examples 1-8 (P < 0.05), indicating that Examples 1-3 could significantly improve the carbon clearance capacity of mice. However, there was no significant difference in carbon clearance capacity among Comparative Examples 3, 7, and 8, and also no significant difference between Comparative Examples 7 and 8 (p > 0.05). This indicates that shiitake mushroom powder and Astragalus extract have a synergistic effect in improving the carbon clearance capacity of mice. Furthermore, compared with Comparative Example 5 (Codonopsis pilosula extract...) Compared with Comparative Example 6 (Atractylodes macrocephala extract), mice treated with Comparative Example 4 (Astragalus membranaceus extract) showed significantly lower carbon clearance ability (p < 0.05). However, compared with Comparative Example 1 (containing Codonopsis pilosula extract) and Comparative Example 2 (containing Atractylodes macrocephala extract), mice treated with Astragalus membranaceus extract showed significantly stronger carbon clearance ability (p < 0.05). This indicates that the Astragalus membranaceus extract in Examples 1-3 has a synergistic effect with other components in improving the delayed-type hypersensitivity ability of mice.

[0089] Table 4. Results of carbon clearance rate determination in mice (mean ± standard error)

[0090] Note: In the same column, different lowercase letters indicate significant differences. P <0.05), the same lowercase letter indicates no significant difference ( P >0.05).

[0091] Example 5: Effect of the composition on the killing activity of mouse NK cells NK cell immune function is assessed by observing the ability of mouse NK cells to kill target cells.

[0092] (1) Experimental methods Laboratory animals: Healthy SPF-grade BALB / c mice, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0093] Experimental grouping and intervention methods: The experiment was divided into a blank control group, Examples 1-3, and Comparative Examples 1-8, with 10 mice in each group. Each mouse was given a gavage dose of 3100 mg / kg BW at a volume of 20 mL / kg BW. The blank control group was given an equal volume of distilled water by gavage. All treatments were administered orally once daily for 30 consecutive days. During this period, mice in all groups were housed in a barrier-grade animal room at a temperature range of (22±2)℃ and a relative humidity of 50%-60%, with a day:night ratio of 12h:12h. They were fed an SPF-grade basal diet and provided with free access to food and water.

[0094] Twenty-four hours prior to the experiment, mouse T-lymphoma cells (YAC-1) were passaged as target cells. Mice were continuously treated with the composition for 30 days. After euthanasia by cervical dislocation, the spleen was aseptically harvested and gently ground in a petri dish containing Hank's solution using a grinding rod. The mixture was then filtered through a 200-mesh sieve to prepare a single-cell suspension. The cells were washed twice with Hank's solution, centrifuged at 1000 rpm for 10 min each time. The supernatant was discarded, and the cells were resuspended. 0.5 mL of sterile double-distilled water was added for 20 seconds to lyse the red blood cells. Then, 0.5 mL of 2× Hank's solution and 8 mL of Hank's solution were added, mixed well, and centrifuged at 1000 rpm for 10 min. After discarding the supernatant, add 1 mL of RPMI 1640 complete culture medium containing 10% (v / v) fetal bovine serum and resuspend the cells. Dilute with 1% (v / v) glacial acetic acid and perform trypan blue staining for cell counting (viable cell count should be above 95%). Finally, adjust the cell concentration to 2 × 10⁶ cells / mL using RPMI 1640 complete culture medium. 7 Cells / mL were prepared as effector cells. Before use, target cells were washed three times with Hank's solution and adjusted to a concentration of 4 × 10⁻⁶ cells / mL using RPMI 1640 complete culture medium. 5 Cells / mL. At an effector-to-target ratio of 50:1, 100 μL each of target cells and effector cells were added to each of the following 96-well U-shaped culture plates as reaction wells: 100 μL each of target cells and culture medium were added to each well for target cell spontaneous release; and 100 μL each of target cells and 2.5% (v / v) Triton were added to each well for maximum target cell release. Three replicates were prepared for each well. The plates were incubated at 37 °C for 4 h in a 5% CO2 incubator, followed by centrifugation at 1500 rpm for 5 min. 100 μL of the supernatant from each well was transferred to a flat-bottomed 96-well culture plate, and 100 μL of lactate dehydrogenase substrate solution was added to each well. The reaction was carried out for 7 min, followed by the addition of 30 μL of 1 mol / L HCl. The OD value of each well was measured at 490 nm using a microplate reader.

[0095]

[0096] (2) Experimental results The strength of immunity is closely related to the body's immune cells, and NK cell activity is positively correlated with immunity. As shown in Table 5, the NK cell activity after intervention in Examples 1-3 was significantly higher than that in Comparative Examples 1-8 (p < 0.05), indicating that Examples 1-3 can significantly improve the NK cell activity of mice. However, there was no significant difference in NK cell activity between Comparative Example 3 (without Astragalus extract) and Comparative Examples 7 (without Lentinus edodes powder) and 8 (without Lentinus edodes powder and Astragalus extract) (p > 0.05), and there was also no significant difference between Comparative Examples 7 and 8 (p > 0.05). This indicates that Lentinus edodes powder and Astragalus extract have limited effect on improving the NK cell activity of mice. In addition, compared with Comparative Example 5 (Codonopsis pilosula extract) and Comparative Example 6 (Atractylodes macrocephala extract), the NK cell activity of mice after intervention with Comparative Example 4 (Astragalus membranaceus extract) was lower, with a significant difference (p < 0.05). However, compared with Comparative Example 1 containing Codonopsis pilosula extract and Comparative Example 2 containing Atractylodes macrocephala extract, the NK cell activity of mice after intervention with Astragalus membranaceus extract was higher, with a significant difference (p < 0.05). This indicates that there is a synergistic effect between Astragalus membranaceus extract and other components in Examples 1-3 in improving NK cell activity.

[0097] The NK cell activity level of mice after intervention with Comparative Example 4 (Astragalus extract) was significantly lower than that of Comparative Example 5 (Codonopsis extract) and Comparative Example 6 (Atractylodes macrocephala extract), while the NK cell activity level of mice after intervention with Examples 1-3 (containing Astragalus extract) was significantly higher than that of Comparative Example 1 (Codonopsis extract) and Comparative Example 2 (Atractylodes macrocephala extract). This indicates that Astragalus extract and the components in Examples 1-3 other than Astragalus extract have a synergistic effect in improving the NK cell activity level of mice.

[0098] Table 5. Mouse NK cell activity assay (mean ± standard error)

[0099] Note: In the same column, different lowercase letters indicate significant differences. P <0.05), the same letter indicates no significant difference ( P＞ 0.05).

[0100] Effect 6: The effect of the composition on the gut microbiota (1) In vitro fermentation test ① Prepare the fermentation medium 1-2 days in advance and store it at 4℃ for later use; Fermentation medium (1 L): NaCl 0.1 g, K2HPO4 0.04 g, KH2PO4 0.04 g, MgSO4 0.01 g, CaCl2 0.01 g, NaHCO3 2 g, peptone 2.5 g, yeast extract 4 g, glucose 0.4 g, mucin 2 g, bile salts 0.5 g, cysteine ​​hydrochloride 0.46 g, Tween-80 2 mL. After mixing, adjust the pH to 7.0 (0.5M HCl), autoclave at 121℃ for 15 min, and cool before use.

[0101] ② Preparation of fecal fermentation broth: Fresh feces were collected from a healthy volunteer without gastrointestinal diseases. The feces were mixed and diluted with sterile PBS buffer at pH 7.0 at a ratio of 1:9 (w / v), homogenized for 10 min, and centrifuged at 1000 rpm for 10 min to remove impurities. The supernatant was kept as the fecal fermentation broth for later use.

[0102] ③ In vitro simulated colon fermentation experiment: a. Fermentation: Substrate culture medium and fecal fermentation broth were added at a ratio of 2:1 according to the total fermentation system volume, and then the composition of Examples 1-3 and Comparative Examples 1-8 was added at a ratio of 5% (m / m). The mixture was cultured in an anaerobic environment at 37°C for 48 h; b. Sampling: Samples (5 mL) were collected at 0, 6, 12, 24 and 48 h, centrifuged at 12000 rpm for 10 min, and the precipitate was used for bacterial count determination.

[0103] ④ Bacterial count determination: a. The collected fermentation broth precipitate can be used to extract fermentation microbial gDNA. Follow the instructions of the fecal genomic DNA extraction kit (centrifuge column type, catalog number: D213, Beijing Jinbaite Biotechnology Co., Ltd.) for relevant operations; b. Dilute the extracted in vitro fermentation broth gDNA to 1 ng / μL with TB. Construct a standard curve using the corresponding bacterial plasmid standard that was successfully sequenced, and perform absolute quantitative qPCR detection. Refer to the "Real-Time quantitative PCR kit instructions" and use 2x SYBR Green qPCR Mix (NO ROX) (catalog number: P313, Beijing Jinbaite Biotechnology Co., Ltd.) in a 20 μL system. Perform 45 cycles of amplification at 60℃ for annealing / extension for 30 seconds.

[0104] ⑤ A standard curve was generated with the Logcopies (log value of DNA replication copy number) of the standard plasmid as the x-axis and the cq value (PCR cycle number when the fluorescence signal is greater than the fluorescence threshold) as the y-axis.

[0105] Standard curve for total microbiota (Universal-3) = -2.5084x + 45.985, R² = 0.9703; Bifidobacterium ( Bifidobacteriumy The standard curve is y = -3.0018x + 37.5, with R² = 0.9844. Lactobacillus ( Lactobacillus johnsonii The standard curve is y = -2.9041x + 35.796, with R² = 0.9794.

[0106] The average value of three sets of cq values ​​for different compositions at different fermentation times of 0h, 6h, 12h, 24h, and 48h was calculated, and the Logcopies for different fermentation times were obtained according to the standard curve.

[0107] (2) Experimental results ①The primer sequences are shown in Table 6.

[0108] Table 6. Primer sequences for gut microbiota expansion

[0109] ② Expression analysis of three gut microbiota The average value of three sets of cq values ​​for different compositions at different fermentation times of 0h, 6h, 12h, 24h, and 48h was calculated, and the Logcopies for different fermentation times were obtained according to the standard curve.

[0110] As shown in Table 7, at 6h, 12h, 24h, and 48h after fermentation, the total bacterial count after intervention in Examples 1-3 was significantly higher than that in Comparative Examples 1-8, indicating that the in vitro colonic fermentation products of Examples 1-3 can increase the total number of intestinal flora. Meanwhile, there was no significant difference in the total number of intestinal flora among Comparative Examples 3, 7, and 8 (p > 0.05), indicating that shiitake mushroom powder and astragalus extract have a synergistic effect in increasing the number of intestinal flora. Furthermore, at 6h after fermentation, the total bacterial count after intervention in Comparative Examples 1-3, 7, and 8 was significantly higher than that in Comparative Examples 4-6.

[0111] Table 7. Measurement of total gut microbiota expression (Logcopies / μL)

[0112] Note: In the same column, different lowercase letters indicate significant differences. P <0.05).

[0113] As shown in Table 8, at 6 h after fermentation, the number of Bifidobacteria after intervention in Examples 1-3, Comparative Examples 1, and Comparative Examples 2 was significantly higher than that in Comparative Examples 3-8 (p < 0.05), while there was no significant difference between Examples 1-3 and Comparative Examples 1 and 2 (p > 0.05). At 12 h, 24 h, and 48 h after fermentation, the number of Bifidobacteria after intervention in Examples 1, 2, and 3 was significantly higher than that in Comparative Examples 1-8 (p < 0.05), while there was no significant difference in the number of Bifidobacteria after intervention in Comparative Examples 3, 7, and 8 (p > 0.05). This indicates that the in vitro colonic fermentation products of Examples 1-3 can significantly increase the number of Bifidobacteria, and that shiitake mushroom powder and astragalus extract have a synergistic effect in increasing the number of intestinal Bifidobacteria.

[0114] Bifidobacteria not only help regulate the balance of gut microbiota, but also have multiple functions such as enhancing the immune system, promoting nutrient absorption, and regulating intestinal inflammation, resulting in a comprehensive effect. Currently, scientific research has accumulated substantial evidence demonstrating the high reliability and effectiveness of Bifidobacteria in improving gut microecological balance and enhancing immunity.

[0115] Table 8. Effects of the examples and comparative examples on Bifidobacterium expression (Logcopies / μL)

[0116] Note: In the same column, different lowercase letters indicate significant differences. P <0.05).

[0117] As shown in Table 9, at 6 h after fermentation, the number of *Lactobacillus johnsonii* after intervention in Examples 1-3 was significantly higher than that in Comparative Examples 3-6 (p < 0.05), while there was no significant difference between Examples 1-3 and Comparative Examples 1 and 2 (p > 0.05). At 12 h, 24 h, and 48 h after fermentation, the number of *Lactobacillus johnsonii* after intervention in Examples 1-3 was significantly higher than that in Comparative Examples 1-8 (p < 0.05), indicating that the in vitro colonic fermentation products of Examples 1-3 can significantly increase the number of *Lactobacillus johnsonii*. Meanwhile, there was no significant difference in the number of *Lactobacillus johnsonii* after intervention in Comparative Examples 3, 7, and 8 (p > 0.05), indicating that shiitake mushroom powder and astragalus extract have a synergistic effect in increasing the number of *Lactobacillus johnsonii* in the intestine.

[0118] Lactobacillus, as a probiotic, has a wide range of effects on human health. Its main functions and effects include enhancing immunity, promoting digestion and absorption, preventing and treating diarrhea, inhibiting the growth of harmful bacteria, and improving skin health. Lactobacillus colonizes the intestines, forming a biological barrier to prevent the invasion of pathogens. At the same time, Lactobacillus can stimulate the activity of immune cells and promote antibody production, thereby improving the body's resistance to disease.

[0119] Table 9. Effects of the Examples and Comparative Examples on Lactobacillus Johnsoni Expression (Logcopies / μL)

[0120] Note: In the same column, different lowercase letters indicate significant differences. P <0.05).

[0121] In summary, compared with Comparative Examples 1-8, Examples 1-3 significantly increased the total number of gut microbiota, with a significant increase in the number of Bifidobacterium and Lactobacillus johnsonii.

Claims

1. A composition for enhancing immunity, characterized by comprising: The composition comprises by weight 40-60 parts of Lentinula edodes powder, 20-40 parts of Ganoderma lucidum powder, 5-15 parts of Paecilomyces hepiali powder and 2-5 parts of Astragalus extract.

2. The composition of claim 1, wherein, The composition comprises by weight 40 parts of Lentinula edodes powder, 40 parts of Ganoderma lucidum powder, 5 parts of Paecilomyces hepiali powder and 5 parts of Astragalus extract.

3. The composition of claim 1, wherein, The composition comprises by weight 60 parts of Lentinula edodes powder, 20 parts of Ganoderma lucidum powder, 15 parts of Paecilomyces hepiali powder and 2 parts of Astragalus extract.

4. The composition of claim 1, wherein, The composition comprises by weight 50 parts of Lentinula edodes powder, 30 parts of Ganoderma lucidum powder, 10 parts of Paecilomyces hepiali powder and 3 parts of Astragalus extract.

5. Process for the preparation of a composition according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: S1, mixing the formula amount of Lentinula edodes powder, Ganoderma lucidum powder, Paecilomyces hepiali powder and Astragalus extract to obtain a mixture A; S2, adding the mixture A obtained in step S1 into a solvent to obtain the composition.

6. The production method according to claim 5, wherein The solvent in step S2 is water.

7. The preparation method according to claim 5, characterized in that, The weight content of the mixture A in the composition in step S2 is not less than 30%.

8. Use of the composition of any one of claims 1-4 or the preparation method of any one of claims 5-7 in the preparation of an immune-enhancing product.

9. Use according to claim 8, characterized in that, The product comprises a food or a medicine.

10. A food product, characterized by, The food comprises the composition of any one of claims 1-4.

11. The food product of claim 10, wherein, The food further comprises a food-acceptable excipient.

12. The food according to claim 10 or 11, characterized in that, The food is a gel particle, a crystal ball, a solid beverage, a liquid beverage, an ice cream, a hard candy, a crisp candy, a caramel candy, a gel candy, a toffee candy, a chocolate, a gum candy, an aerated candy, a pressed candy and / or a jelly.

13. The food according to claim 10 or 11, characterized in that, The food is a health food.

14. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The medicine comprises the composition of any one of claims 1-4.

15. The medicament according to claim 14, characterized in that, The medicine further comprises a pharmaceutically acceptable excipient.

16. The medicament according to claim 14 or 15, characterized in that, The pharmaceutically acceptable excipient is selected from the group consisting of a wetting agent, an emulsifying agent, a preservative, an antioxidant, a buffering agent, an excipient, a diluent, a lubricant, a bacteriostatic agent, a suspending agent, a suspending aid, a solubilizing agent, a thickening agent, a stabilizing agent, a sweetener and a flavoring agent, or a combination of one or more thereof.

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