Immunoregulation solid beverage and preparation method thereof

Abstract

The invention belongs to the field of functional beverages, and particularly discloses an immunoregulation solid beverage and a preparation method thereof. The Pu'er tea polysaccharide and the catechin are combined to form the composite solid beverage, spleen and thymus tissue damage and immune imbalance induced by Cy can be improved, and a basis is provided for developing the Pu'er tea polysaccharide and the catechin as potential functional food for improving human health.

Description

Technical Field

[0001] The invention relates to the field of functional beverages, and in particular to an immunomodulatory solid beverage and a preparation method thereof. Background Art

[0002] In recent years, tea polysaccharides have attracted much attention for their safety and immunomodulatory activity. Studies have found that Fuzhuan tea polysaccharides can affect the body's immunomodulatory function by affecting the abundance of intestinal microorganisms; Liubao brick tea polysaccharides can activate nuclear factor kappa-B (NF-κB) and AMP-dependent protein kinase (Adenosine 5'-monophosphate (AMP)-activated protein kinase, AMPK) signaling pathways by enhancing the phagocytic ability of cells and the release of nitric oxide (NO), thereby exerting its immunomodulatory function; Pu'er ripe tea is rich in tea polysaccharides, which also show significant biological efficacy in multiple fields such as enhancing immunity, regulating metabolism and anti-tumor.

[0003] Catechins, as the main component of polyphenol compounds in tea, account for about 80% of the total tea polyphenols. On December 1, 2023, the National Health Commission officially listed catechins as new food ingredients, triggering a wave of development in the industry. Studies have shown that catechins play a significant role in immune regulation. Cheng J et al. found that catechins can restore Salmonella infection caused by a high-fat diet by changing the composition and diversity of intestinal flora (Cheng J, Yang M, Wu T, et al. Protective effects of catechin (C) and epigallocatechin gallate (EGCG) against high-fat diet (HFD)-exacerbated Salmonella infection in mice[J]. Journal of Functional Foods, 2024, 120106389-106389.); Aditya Ganeshpurkar et al. found that catechins have the potential to regulate immune activity through cellular and humoral mechanisms (Ganeshpurkar A, Saluja K A. Protective effect of catechin on humoral and cell mediated immunity in rat model[J]. International Immunopharmacology, 2018, 54261-266.).

[0004] The relevant mechanisms of Pu-erh tea polysaccharide and catechin regulating immunity alone have been relatively clear. However, the combination of Pu-erh tea polysaccharide and catechin to form a compound beverage has not been publicly reported. Therefore, the present invention explores the combination of Pu-erh tea polysaccharide and catechin to obtain a functional beverage, providing an important reference for the research and development of Pu-erh tea polysaccharide and catechin in food and functional products. Summary of the Invention

[0005] In order to develop a beverage with immunomodulatory function, the present invention combines Pu-erh tea polysaccharide and catechin to form a compound solid beverage, which is of great significance for improving human health.

[0006] The specific technical solution of the present invention is as follows: An immunomodulatory solid beverage comprising Pu-erh tea polysaccharide and catechin.

[0007] Pu-erh tea polysaccharide is an acidic glycoprotein composed of carbohydrates, pectin, proteins, etc. in tea leaves, combined with a large amount of mineral elements, called tea polysaccharide complex, abbreviated as tea polysaccharide (Tea Polysaccharide). Pu-erh tea polysaccharide is mainly composed of glucose, arabinose, xylose, fucose, ribose, galactose, etc., and also contains mineral elements such as calcium, magnesium, iron, manganese and a small amount of trace elements. Pu-erh tea polysaccharide has a variety of biological activity functions, including hypoglycemic, hypolipidemic, immunomodulatory, anticoagulant, antithrombotic, antioxidant, etc. It can also slow down the heart rate, increase coronary blood flow and hypoxia tolerance, etc. In recent years, it has been found that Pu-erh tea polysaccharide also has the effect of treating diabetes. In addition, Pu-erh tea polysaccharide exerts a variety of physiological functions by regulating the composition of intestinal flora, promoting the growth of probiotics, and increasing the content of short-chain fatty acids (SCFAs), the metabolites of intestinal flora.

[0008] Catechin, also known as catechuic acid and catechin, is a class of phenolic active substances extracted from natural plants such as tea leaves, with the molecular formula C 15 H 14 O6. Catechin has a variety of pharmacological effects such as anti-tumor, antioxidant, anti-bacterial and protecting heart and brain organs.

[0009] Preferably, the addition dosage range of Pu-erh tea polysaccharide is 100 - 700 mg / kg, and the addition dosage range of catechin is 40 - 80 mg / kg. Through the screening experiment of the optimal immunomodulatory dosage, it is found that within this preferred range, the effect is the best.

[0010] Furthermore, based on the results of the dosage screening experiment, the preferred addition dosage of Pu-erh tea polysaccharide is 400 mg / kg, and the preferred addition dosage of catechin is 60 mg / kg.

[0011] Moreover, the results of this study indicate that the addition of catechins enhances the immunomodulatory effect of Pu-erh tea polysaccharides. Therefore, in this patent, Pu-erh tea polysaccharides and catechins are simultaneously added to prepare an immunomodulatory solid beverage without adding other additives.

[0012] The Pu-erh tea polysaccharides described above are prepared by the following method: Using ripe Pu-erh tea as the raw material, it is soaked in hot water at 80 - 100 °C for 2 - 2.5 h and then filtered by suction. The solid-liquid ratio is 1:5 - 15, and it is passed through a 400-mesh sieve. Then, it is concentrated under reduced pressure at 50 - 60 °C and subjected to ethyl acetate extraction. The aqueous phase is taken, ethanol is added to make the alcohol concentration of the solution ≥ 80 degrees for alcohol precipitation and centrifugal separation. The precipitate is washed twice with absolute ethanol and then vacuum dried to obtain Pu-erh tea polysaccharides.

[0013] Furthermore, using the ripe Pu-erh tea in 2022 as the raw material, it is soaked in hot water at 90 °C for 2.25 h and then filtered by suction. The solid-liquid ratio is 1:10, and it is passed through a 400-mesh sieve. Then, it is concentrated under reduced pressure at 50 - 60 °C and subjected to ethyl acetate extraction. The aqueous phase is taken, ethanol is added to make the alcohol concentration of the solution ≥ 80 degrees for alcohol precipitation and centrifugal separation. The precipitate is washed twice with absolute ethanol and then vacuum dried to obtain Pu-erh tea polysaccharides.

[0014] This invention mainly uses the alcohol precipitation method for the extraction of Pu-erh tea polysaccharides. This method does not require complex equipment and mainly relies on the precipitation of polysaccharides in high-concentration ethanol and centrifugal separation. The operation steps are simple, suitable for laboratory and industrial production. Among them, ethanol can be recycled and reused, reducing production costs and having high economic efficiency. In addition, the alcohol precipitation method is usually carried out at room temperature or low temperature, avoiding the destruction of the structure of tea polysaccharides by high temperature (such as degradation or denaturation), which helps to retain their biological activity.

[0015] The catechins described above can be prepared by the methods of the existing technology. For example, it can be prepared by the following method: Using ripe Pu-erh tea as the raw material, it is soaked in hot water at 80 - 100 °C for 2 - 2.5 h and then filtered by suction. The solid-liquid ratio is 1:5 - 15, and it is passed through a 400-mesh sieve. Then, it is concentrated under reduced pressure at 50 - 60 °C and subjected to ethyl acetate extraction. The organic phase is taken, passed through macroporous adsorption resin, and gradient eluted with ethanol aqueous solutions of different concentrations, and then spray dried to obtain catechins.

[0016] Preferably, using ripe Pu-erh tea as the raw material, it is soaked in hot water at 90 °C for 2.25 h and then filtered by suction. The solid-liquid ratio is 1:10, and it is passed through a 400-mesh sieve. Then, it is concentrated under reduced pressure at 50 - 60 °C and subjected to ethyl acetate extraction. The organic phase is taken, passed through macroporous adsorption resin, and gradient eluted with ethanol aqueous solutions of different concentrations, and then spray dried to obtain catechins.

[0017] This application also provides a preparation method of an immunomodulatory solid beverage as follows: The polysaccharide of Pu'er tea and catechin are fully and evenly mixed by a high-speed mixing device, wherein the added amount of polysaccharide of Pu'er tea is 100-700 mg / kg, the added amount of catechin is 40-80 mg / kg, and no other additives are added.

[0018] Preferably, the Pu'er tea polysaccharide and catechins are fully and evenly mixed by a high-speed mixing device, wherein the added amount of Pu'er tea polysaccharide is 400 mg / kg, and the added amount of catechins is 60 mg / kg.

[0019] The present invention mainly adopts a mixing method to prepare solid beverages. The advantage is that the high-speed mixing equipment can complete the uniform mixing of materials in a short time, and the materials are fully broken and circulated during the mixing process, avoiding the problem of particle stratification or agglomeration. In addition, the mixing method is combined with a low-temperature drying process (spray drying) to reduce the damage of high temperature to heat-sensitive components (such as volatile flavor substances), ensuring the retention of nutrition and flavor.

[0020] Beneficial effects of the present invention: This invention combines Pu'er tea polysaccharides and catechins to form a composite beverage for the first time. The results of this study show that the addition of catechins enhances the immunomodulatory effect of Pu'er tea polysaccharides. Using cyclophosphamide-induced immunosuppressive mice as the research object, the immunomodulatory effect of TPC was studied from three aspects: immune organ damage, immune disorder and serum metabolism. It was found that TPC can significantly improve the spleen and thymus index of mice, regulate the levels of interleukin-6 (IL-6) and interferon-γ (IFN-γ) factors in mouse serum, and increase the levels of immunoglobulin G (IgG) and immunoglobulin M (IgM). Serum metabolomics analysis showed that TPC significantly up-regulated and down-regulated 43 and 87 metabolites, respectively, among which many metabolites are related to choline metabolism and vitamin digestion and absorption in cancer. That is, TPC can improve Cy-induced spleen and thymus tissue damage and immune imbalance. The present invention provides a basis for developing Pu'er tea polysaccharides and catechins as potential functional foods for improving human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The animal experiment design is the same as that of Example 1. CK: normal control group; MD: model control group; LH: positive control group; LP: low-dose polysaccharide group; MP: medium-dose polysaccharide group; HP: high-dose polysaccharide group; TPC: Pu'er tea polysaccharide solid beverage group.

[0022] Figure 2Results of the optimal dose screening experiment for the immune regulation of Pu-erh tea polysaccharide in Example 1. Among them, A: spleen index; B: thymus index; C: IL-6 level; D: IFN-γ level; E: IgG level; F: spleen HE; CK: normal control group; MD: model control group; LH: positive control group; LP: low-dose polysaccharide group; MP: medium-dose polysaccharide group; HP: high-dose polysaccharide group. Compared with the CK group, "*" indicates significant difference (P < 0.05), and "**" indicates extremely significant difference (P < 0.01); compared with the MD group, "#" indicates significant difference (P < 0.05), and "##" indicates extremely significant difference (P < 0.01). The same applies hereinafter. Figure 3 Results of the immune regulation experiment of the compound solid beverage of Pu-erh tea polysaccharide and catechins in Example 1. Among them, A: spleen index; B: thymus index; C: IL-6 level; D: IFN-γ level; E: IgG level; F: IgM level; G: spleen HE; Figure 4 Metabolome results of the immune regulation experiment of the compound solid beverage of Pu-erh tea polysaccharide and catechins in Example 1. Among them, A: PLS-DA analysis of CK and MD groups; B: PLS-DA analysis of MD and TPC groups; C: volcano plot of differential metabolites between MD and TPC groups; D: clustering heat map of differential metabolites between MD and TPC groups; Figure 5 Bubble chart of significant pathways of differential metabolites between MD and TPC groups in Example 1. Detailed implementation manners

[0023] To more clearly elaborate the purpose, technical solution and advantages of the present invention, the following will be described in detail with reference to the accompanying drawings and specific embodiments. The exemplary embodiments shown in the drawings are only for the present invention, not a limitation on its implementation manner. The present invention can be implemented in various forms, and its design concept and core technology are not limited by the embodiments shown in the drawings. The purpose of providing these embodiments is to more conveniently enable those skilled in the art to understand the principle and function of the present invention, so as to better master and apply its technical solution. The terms used in this specification are only for describing specific embodiments and do not constitute a limitation on the present invention.

[0024] Example 1: The materials used in this example are as follows: (1) Animals, materials and reagents 120 specific pathogen free (SPF)-level BALB / c female mice (5 weeks old) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and the product license number is SCXK (Xiang) 2019-0004. The breeding environmental conditions are a temperature of 24 ± 2 °C, a relative humidity of 45% - 55%, and a 12 h light-dark cycle.

[0025] Cyclophosphamide (Cy, no. PHR1404), Sigma-Aldrich (Shanghai, China). Levamisole hydrochloride (LH), Shandong Renhetang Pharmaceutical Co., Ltd. Interleukin-6 (IL-6), interferon-γ (IFN-γ), immunoglobulin G (IgG), immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) kits, Wuhan Huamei Bioengineering Co., Ltd.

[0026] Pu-er tea polysaccharides (TP) were prepared by the following process: 2022 Pu-er ripe tea was used as raw material, soaked in 90°C hot water for 2.25 h and then filtered, with a solid-liquid ratio of 1:10, passed through 400 mesh, and then concentrated under reduced pressure at 50-60°C and low temperature, and then extracted with ethyl acetate. The aqueous phase was taken, ethanol was added to make the solution alcohol content ≥80 degrees, and then precipitated and centrifuged. The precipitate was washed twice with anhydrous ethanol and vacuum dried to obtain Pu-er tea polysaccharides.

[0027] Catechins (CA) were prepared by the following process: 2022 Pu'er ripe tea was used as raw material, soaked in 90°C hot water for 2.25 h and then filtered, with a solid-liquid ratio of 1:10, passed through 400 mesh, and then concentrated under reduced pressure at 50-60°C and then extracted with ethyl acetate. The organic phase was taken, passed through a macroporous adsorption resin, and gradient eluted with ethanol aqueous solution of different concentrations, and spray-dried to obtain catechins.

[0028] (2) Instruments and equipment BSA323S electronic balance, Startorius, Switzerland; D3024R desktop high-speed refrigerated centrifuge, Beijing Dalong Xingchuang Experimental Instrument Co., Ltd.; MDF-86V6408 ultra-low temperature refrigerator, Anhui Zhongke Duling Commercial Appliance Co., Ltd.; MX-F vortex mixer, Wuhan Sevier Biotechnology Co., Ltd.; Epoch enzyme label detector, BioTeK, USA; high performance liquid chromatography-quadrupole time-of-flight tandem high-resolution mass spectrometry, Agilent Technologies, USA.

[0029] The experimental method used in this embodiment is as follows: (1) Experimental design for screening the optimal dose of Pu'er tea polysaccharides for immune regulation likeFigure 1 As shown in Figure A in , after one week of adaptive feeding, the mice were randomly divided into 6 groups: normal control group (CK), model control group (MD), positive control group (LH), low-dose group (LP), medium-dose group (MP), and high-dose group (HP), with 11 mice in each group. The mice in the CK group were intraperitoneally injected with normal saline (80 mg / kg / d), and the mice in the remaining groups were intraperitoneally injected with cyclophosphamide (Cy, 80 mg / kg / d) for 3 days to establish the model. Subsequently, for 10 days, the CK group and the MD group were gavaged with pure water, the LP group, MP group, and HP group were respectively gavaged with TP at 100 mg / kg / d, 200 mg / kg / d, and 400 mg / kg / d, and at the same time, the LH group was gavaged with levamisole hydrochloride (30 mg / kg / d), and the addition amount of LH was converted from the recommended daily dose for adults (2.5 mg / kg) according to the instructions for humans and mice. On the 14th day, the mice were sacrificed after being anesthetized with sodium pentobarbital aqueous solution. Blood, thymus, and spleen tissues were taken for further analysis to select the dose of Pu-erh tea polysaccharide with the best immune regulation effect.

[0030] (2) Experimental design for the regulation of immunity by Pu-erh tea polysaccharide solid beverage As Figure 1 shown in Figure B in , after one week of adaptive feeding, the mice were randomly divided into 5 groups: normal control group (CK), model control group (MD), Pu-erh tea polysaccharide group (TP), compound solid beverage group of Pu-erh tea polysaccharide and catechin (TPC), and positive control group (LH), with 12 mice in each group. Similarly, the mice in the CK group were intraperitoneally injected with normal saline (80 mg / kg / d), and the mice in the remaining groups were intraperitoneally injected with cyclophosphamide (Cy, 80 mg / kg / d) for 3 days to establish the model. Subsequently, for 10 days, the CK group and the MD group were gavaged with pure water, the TP group was gavaged with TP (400 mg / kg / d), the TPC group was gavaged with TP (400 mg / kg / d) and CA (60 mg / kg / d), and at the same time, the LH group was gavaged with levamisole hydrochloride (30 mg / kg / d). On the 14th day, the mice were sacrificed after being anesthetized with sodium pentobarbital aqueous solution. Blood, thymus, and spleen were taken for further analysis.

[0031] (3) Analysis of spleen and thymus indices The collected thymus and spleen were accurately weighed, and the organ index was calculated as: Organ index (mg / g) = Organ weight (mg) / Body weight (g).

[0032] (4) Serum biochemical analysis The sera of the mice in each treatment group were taken, and according to the instructions of the ELISA kit, the concentrations of IL-6, IFN-γ, IgG, and IgM in the sera of the mice were detected.

[0033] (5) Spleen section analysis The spleen tissues of mice in each treatment group were fixed with 4% paraformaldehyde fixative, made into paraffin sections, stained with hematoxylin-eosin (HE) staining method, the pathological changes of spleen tissues were observed under a light microscope, and images were collected by scanning method.

[0034] (6) Serum metabolome analysis Untargeted metabolomics analysis was performed on the sera of mice in each group, and detected by an ultra-high performance liquid chromatography-quadrupole time-of-flight tandem high-resolution mass spectrometer. Chromatographic column: ACQUIY UPLC BEH C 18 (2.1 mm×100 mm, 1.7 µm); Chromatographic conditions: column temperature 25 °C; flow rate 0.5 mL / min; injection volume 2 μL; mobile phases A and B were water + 25 mM ammonium acetate + 25 mM ammonia water and acetonitrile respectively. Mass spectrometry conditions: spray gas pressure: 60 psi; auxiliary gas pressure: 60 psi; curtain gas pressure: 30 psi; ion source temperature: 600 °C; ion source voltage: ±5500 V (positive and negative dual modes); collision energy: 35±15 V.

[0035] The QI software was used to process the metabolomics data obtained from mass spectrometry detection, the METLIN and HumanDatabase (HMDB) databases were selected for compound matching and identification, and the data were subjected to pathway analysis, univariate and multivariate statistical analysis through the website of Metaboanalyst 5.0 (http: / / www.metaboanalyst.ca). Adobe Illustrator CS5 software was used for figure grouping.

[0036] The data statistics and analysis methods of this example are as follows: Excel worksheet was used for data processing, GraphPad Prism 9.0 software was used for statistical analysis and graphing, and the experimental data were all expressed as "mean ± standard deviation". One-way ANOVA was used for data comparison between groups, P<0.05 indicated significant difference, and P<0.01 indicated extremely significant difference.

[0037] The results and analysis of this example are as follows: (1) Optimal dose screening experiment for Pu-erh tea polysaccharide to regulate immunity Effects of different doses of Pu-erh tea polysaccharide on the organ indexes of Cy-induced immunosuppressed mice: as Figure 2 A in Figure 2As shown in B of [Figure 0], compared with the CK group, Cy treatment significantly increased the spleen index of mice and significantly decreased the thymus index, indicating successful modeling. LP, MP, and HP could all significantly restore the abnormal spleen index and thymus index of mice caused by Cy to the level of the CK group, and the HP group (high-dose Pu-erh tea polysaccharide) had the best effect.

[0038] Effects of different doses of Pu-erh tea polysaccharide on serum indexes of Cy-induced immunosuppressed mice: By detecting the changes of cytokines in the serum of mice in each group, the immunomodulatory effect of the optimal dose of TP was screened. As Figure 2 shown in C, D, and E of [Figure 0], after Cy treatment, the cytokines IL-6 and IFN-γ in the serum increased significantly, and the immunoglobulin IgG decreased significantly, indicating successful modeling. LP, MP, and HP groups could all restore the abnormalities of IL-6, IFN-γ, and IgG in mice caused by Cy to the level of the CK group, and the HP group (high-dose Pu-erh tea polysaccharide) had the best effect.

[0039] Effects of different doses of Pu-erh tea polysaccharide on the histology of the spleen of Cy-induced immunosuppressed mice: The spleen is an important immune organ in the body, and the immune status of mice can be evaluated by observing the histological structure of the spleen. As Figure 2 shown in F of [Figure 0], in the normal control group, the white pulp of the spleen was large in volume and numerous in quantity, and the boundary between the red and white pulp was obvious. In the model control group, the white pulp was small in volume and few in quantity, and the boundary between the red and white pulp was not obvious. After LP, MP, and HP intervention, the white pulp increased, and the boundary between the red and white pulp was more obvious than that in the model control group. Among them, the white pulp in the MP and HP groups was significantly more.

[0040] (2)Immunomodulatory experiment of the compound solid beverage of Pu-erh tea polysaccharide and catechins Effects of TPC on the organ indexes of Cy-induced immunosuppressed mice: As Figure 3 shown in A and B of [Figure 1], compared with the CK group, Cy treatment significantly increased the spleen index of mice and significantly decreased the thymus index, indicating successful modeling. Both TP and TPC could significantly restore the abnormal spleen index and thymus index of mice caused by Cy to the level of the CK group, and the effect of TPC was slightly better than that of TP.

[0041] Effects of TPC on the serum indexes of Cy-induced immunosuppressed mice: As Figure 3 shown in C, D, E, and F of [Figure 1], after Cy treatment, the cytokines IL-6 and IFN-γ in the serum increased significantly, and the immunoglobulin IgG and IgM decreased significantly, indicating successful modeling. Both TP and TPC groups could restore the abnormalities of IL-6, IFN-γ, IgG, and IgM in mice caused by Cy to the level of the CK group. Except for the level of INF-γ, the effects of the other immune factors showed that TPC was slightly better than TP.

[0042] Effect of TPC on the histology of the spleen in Cy-induced immunosuppressed mice: As shown in G of Figure 3 , in the normal control group, the white pulp of the spleen was large in volume and numerous in quantity, and the boundary between the red and white pulp was obvious. In the model control group, the white pulp was small in volume and few in quantity, and the boundary between the red and white pulp was not obvious. After intervention with TP and TPC, the white pulp increased, the boundary between the red and white pulp was clearer than that in the model control group, and the volume and quantity of the white pulp in the TPC group were slightly better than those in the TP group.

[0043] Effect of TPC on the metabolite composition of Cy-induced immunosuppressed mice: Partial least squares discriminant analysis (PLS-DA) was performed on the detected metabolites, and the results are shown in Figure 4 . As can be seen from A and B in Figure 4 , the samples of each group could be clearly distinguished. Subsequently, VIP>1.0 and P<0.05 were used as the criteria for screening differential metabolites. Among them, compared with the MD group, 43 differential metabolites were significantly up-regulated in the TPC group, and 87 differential metabolites were significantly down-regulated in the TPC group. The volcano plot of differential metabolites is shown in C of Figure 4 . In addition, a cluster heat map analysis of differential metabolites was performed between the MD group and the TPC group. As shown in D of Figure 4 , the differential metabolites in the two groups were mainly various alkaloids, phosphocholine, pyridoxamine and other compounds. Most of the alkaloids were up-regulated in the MD group and down-regulated in the TPC group, while compounds such as phosphocholine and pyridoxamine were down-regulated in the MD group and up-regulated in the TPC group.

[0044] In order to systematically and comprehensively understand the biological changes in the metabolic effects of Pu-erh tea polysaccharide and catechins on mice, further pathway enrichment analysis was performed on the differential metabolites. The pathways involved in the differential metabolites between the MD group and the TPC group mainly included: Choline metabolism in cancer, Vitumindgestion and absorption, etc. The bubble plot of the significantly enriched pathways is shown in Figure 5 .

[0045] The results of the examples are specifically discussed as follows: Cyclophosphamide (Cy) is an immunosuppressant widely used in the field of anticancer drugs. However, Cy has strong side effects. It can damage the DNA structure, interfere with the differentiation of B cells and T cells, thereby reducing the body's immunity. In addition, it can also cause intestinal mucosal inflammation and lead to intestinal microbial imbalance. Therefore, Cy is often used to establish a mouse immune deficiency model.

[0046] In this example, immunosuppressed mice induced by Cy were used as the research objects. First, they were gavaged with Pu-erh tea polysaccharides at low, medium, and high doses. Through the study of spleen and thymus organ indices, HE staining sections of the spleen, and the levels of immune-related factors in the serum, the optimal dose for regulating immunity was screened out. Then, gavage with a compound solid beverage of Pu-erh tea polysaccharides and catechins (TPC) was carried out. By comparing the normal control group, the model control group, and the positive control group, the immunomodulatory effect of TPC on mice was explored from the changes in three aspects: immune factors and metabolites in mouse tissues and serum.

[0047] The spleen and thymus are common immune organs of the body. As a peripheral immune organ, the spleen provides a place for mature immune cells to respond to immune stimuli. The thymus is the place where immune cells divide, differentiate, and mature. Therefore, the spleen and thymus indices are original indicators reflecting immune ability. In this example, after the mice were treated with Cy by intraperitoneal injection, the spleen index increased significantly, the thymus index decreased significantly, and the immune organs were significantly damaged. After treatment with each dose of TP, the immune organ indices of the LP, MP, and HP groups could enhance those of the mice, and the effect of the HP group was slightly better. In addition, through further study of the HE sections of the spleen, it was found that each dose of TP could restore the white pulp damage caused by Cy to a certain extent, and the phenomenon that the boundary between the red and white pulp was not obvious. It shows that TP can restore the immune organ damage caused by Cy to a certain extent, and the effect of the high dose is the best.

[0048] Cytokines have a variety of biological functions, including regulating cell growth, differentiation, and immune responses. IL-6 is a cell inflammatory factor that can regulate immunity by stimulating the maturation of B lymphocytes and the production of immunoglobulins. IFN-γ is a class of glycoproteins that participate in cell-mediated immune responses, by processing and presenting antigens, upregulating pathogen recognition, participating in leukocyte transport, and inhibiting cell proliferation and apoptosis. The experimental results of this example showed that after treatment with Cy by intraperitoneal injection, the levels of IL-6 and IFN-γ in the serum of the mice were significantly abnormal. Compared with the MD group, each dose of TP could improve the levels of IL-6 and IFN-γ in Cy-induced immunosuppressed mice to a certain extent, thereby regulating the body's immunity, and the effect of the high dose of TP was the best.

[0049] IgG and IgM are relatively common immunoglobulins in serum. Among them, IgG is the main antibody component in serum, accounting for about 75% of immunoglobulins, and can be used to treat B-cell lymphoproliferative syndrome; IgM is the first antibody synthesized and secreted during individual development, accounting for about 5%-10% of the total serum immunoglobulin, and is the earliest antibody to appear in the primary humoral immune response. During the experimental process of this example, it was found that after Cy intraperitoneal injection in mice, the IgG level decreased significantly. After treatment with various doses of TP, the IgG concentration increased significantly. Through the above experiments, it was found that TP at various doses had a restorative effect on the immune damage caused by Cy in mice, and the high-dose TP had a more prominent effect. Therefore, combined with the analysis results of immune tissues, high-dose TP was selected and combined with CA to form a compound solid beverage of pu-erh tea polysaccharide and catechin, and its immunomodulatory effect and mechanism were studied.

[0050] The research results showed that after TPC intervention, compared with the MD group, the spleen index decreased significantly, the thymus index increased significantly, the levels of IL-6 and IFN-γ decreased significantly, the levels of IgG and IgM increased significantly, and the spleen HE section showed that the TPC group could restore the white pulp damage caused by Cy, the phenomenon of unclear boundary between red and white pulp, and except for the INF-γ level, the other immune indexes of the TPC group were slightly better than those of the TP group. It was speculated that the addition of CA could enhance the immunomodulatory effect of TP.

[0051] As the activity site of various nutrients and immune cells in the body, the serum is where material exchange, signal recognition, and various immune phenomena occur all the time, making it an important battlefield for immune responses. Therefore, performing serum metabolome analysis in research helps to understand the phenomena that have occurred, are ongoing, or may occur in the future in mice. The research results show that the differences in metabolites among groups are relatively significant. In the screening of differential metabolites, compared with the MD group, 130 metabolites in the TPC group showed significant changes, and these metabolites are mainly compounds such as alkaloids, phosphorylcholine, and pyridoxamine. Through KEGG enrichment analysis, it was found that these compounds are also involved in multiple metabolic pathways, including choline metabolism in cancer, vitamin digestion and absorption, etc. Among them, phosphorylcholine, as a key metabolite in choline metabolism, is an essential component for cancer cell growth and is closely related to immune regulation; pyridoxamine, as a form of vitamin B6, is related to vitamin digestion and absorption. Previous studies have shown that pyridoxamine has anti-inflammatory effects, and inflammation is interrelated with immunity and is a defense response of the immune system to pathogens or injuries. Thus, it is speculated that TPC may participate in the immune regulation of the body through metabolic pathways such as choline metabolism in cancer and vitamin digestion and absorption.

[0052] In summary, TPC has an obvious restorative effect on the immune function of Cy-induced immunosuppressed mice, mainly manifested as improving the immune organ index, immune organ phenotype, and regulating the cytokine levels of IL-6, IFN-γ, IgG, and IgM in the serum. In addition, TPC can also exert its immune regulatory function through metabolic pathways such as choline metabolism in cancer and vitamin digestion and absorption.

[0053] Finally, we need to emphasize that the examples provided here are only a part of the embodiments of the present invention, not all of them. Based on the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. An immune-regulating solid beverage, characterized in that: It includes Pu-erh tea polysaccharide and catechin, without adding other additives.

2. The immunomodulatory solid beverage according to claim 1, wherein: The added dosage of Pu-erh tea polysaccharide is 100 - 700 mg / kg, and the added dosage of catechin is 40 - 80 mg / kg.

3. The immunomodulatory solid beverage according to claim 1, characterized in that: The added dosage of Pu-erh tea polysaccharide is 350 - 450 mg / kg, and the added dosage of catechin is 55 - 65 mg / kg.

4. The immunomodulatory solid beverage according to claim 1, characterized in that: The added dosage of Pu-erh tea polysaccharide is 400 mg / kg, and the added dosage of catechin is 60 mg / kg.

5. The immunomodulatory solid beverage according to claim 1, characterized in that: The Pu-erh tea polysaccharide is prepared by the following method: Using ripe Pu-erh tea as raw material, soaking it in hot water at 80 - 100 °C for 2 - 2.5 h and then performing suction filtration, with the solid-liquid ratio of 1:5 - 15, passing through 400 meshes, then performing vacuum concentration at 50 - 60 °C under reduced pressure and then carrying out ethyl acetate extraction, taking the aqueous phase, adding ethanol to make the alcohol concentration of the solution ≥ 80 degrees for alcohol precipitation and centrifugal separation, washing the precipitate with absolute ethanol twice, and then drying it under vacuum to obtain Pu-erh tea polysaccharide.

6. The immunomodulatory solid beverage according to claim 5, wherein: The Pu-erh tea polysaccharide is prepared by the following method: Using ripe Pu-erh tea as raw material, soaking it in hot water at 90 °C for 2.25 h and then performing suction filtration, with the solid-liquid ratio of 1:10, passing through 400 meshes, then performing vacuum concentration at 50 - 60 °C under reduced pressure and then carrying out ethyl acetate extraction, taking the aqueous phase, adding ethanol to make the alcohol concentration of the solution ≥ 80 degrees for alcohol precipitation and centrifugal separation, washing the precipitate with absolute ethanol twice, and then drying it under vacuum to obtain Pu-erh tea polysaccharide.

7. The immunomodulatory solid beverage according to claim 1, characterized in that: The catechin is prepared by the following method: Using ripe Pu-erh tea as raw material, soaking it in hot water at 80 - 100 °C for 2 - 2.5 h and then performing suction filtration, with the solid-liquid ratio of 1:5 - 15, passing through 300 - 500 meshes, then performing vacuum concentration at 50 - 60 °C under reduced pressure and then carrying out ethyl acetate extraction, taking the organic phase, passing it through macroporous adsorption resin, and performing gradient elution with ethanol aqueous solutions of different concentrations, and then obtaining catechin after spray drying.

8. The immunomodulatory solid beverage according to claim 7, wherein: The catechin is prepared by the following method: Using ripe Pu-erh tea as raw material, soaking it in hot water at 85 - 95 °C for 2 - 2.5 h and then performing suction filtration, with the solid-liquid ratio of 1:8 - 12, passing through 350 - 450 meshes, then performing vacuum concentration at 50 - 60 °C under reduced pressure and then carrying out ethyl acetate extraction, taking the organic phase, passing it through macroporous adsorption resin, and performing gradient elution with ethanol aqueous solutions of different concentrations, and then obtaining catechin after spray drying.

9. The immunomodulatory solid beverage according to claim 8, characterized in that: The catechin is prepared by the following method: Using ripe Pu-erh tea as raw material, soaking it in hot water at 90 °C for 2.25 h and then performing suction filtration, with the solid-liquid ratio of 1:10, passing through 400 meshes, then performing vacuum concentration at 50 - 60 °C under reduced pressure and then carrying out ethyl acetate extraction, taking the organic phase, passing it through macroporous adsorption resin, and performing gradient elution with ethanol aqueous solutions of different concentrations, and then obtaining catechin after spray drying.

10. The preparation method of the immunomodulatory solid beverage according to any one of claims 1-9, characterized in that: Mix the Pu-erh tea polysaccharide and catechin thoroughly and evenly through a high-speed mixing device to obtain it, where the added dosage of Pu-erh tea polysaccharide is 100 - 700 mg / kg, and the added dosage of catechin is 40 - 80 mg / kg; Preferably, the added dosage of Pu-erh tea polysaccharide is 350 - 450 mg / kg, and the added dosage of catechin is 55 - 65 mg / kg; More preferably, the added dosage of Pu-erh tea polysaccharide is 400 mg / kg, and the added dosage of catechin is 60 mg / kg.

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