Black tea and walnut peptide compound and preparation and application thereof

Abstract

The invention belongs to the field of health food, and discloses a black tea and walnut peptide compound, a preparation method thereof and application of the black tea and walnut peptide compound in relieving immunosuppression induced by cyclophosphamide. Researches find that FT-WP treatment significantly reverses weight, thymus and spleen indexes of mice induced by cyclophosphamide, improves balance of jejunum CD4 + T cell subsets, and balances levels of cytokines and immune globulins. In addition, the research shows that FT-WP affects metabolic pathways such as metabolism of glycine, serine and threonine, metabolism of pyrimidine, digestion and absorption metabolism of protein and the like and regulation of metabolites such as choline, trimethylamine-N-oxide, prothioconazole, stearoyl carnitine, prostaglandin F2. Beta and the like. The black tea and walnut peptide composite food can relieve immunosuppression induced by cyclophosphamide and regulate abnormal serum metabolism.

Description

Technical Field

[0001] This invention belongs to the field of health food, and discloses the black tea and walnut peptide complex, its preparation, and its application in alleviating cyclophosphamide-induced immunosuppression. Background Technology

[0002] In recent years, with increasing emphasis on healthy eating, the development and research of functional foods has become a hot topic. Tea, a traditional Chinese beverage, has been beloved since ancient times. With the advancement of science and technology, the health benefits of tea have been gradually confirmed by scientific research. Tea mainly contains chemical components such as tea polyphenols, alkaloids, amino acids, and polysaccharides, all of which play important roles in human health. Numerous studies have shown that tea has a positive effect on the immune system. Furthermore, tea polyphenols possess antioxidant, anti-inflammatory, and antibacterial biological activities, activating immune cells and enhancing the body's immunity. Black tea, a traditional Chinese health food, boasts rich nutritional value and various biological activities. It is rich in various beneficial antioxidant polyphenols, such as catechins, theaflavins, and thearubigins. Research has found that black tea polyphenols not only exhibit a strong ability to scavenge reactive oxygen free radicals in vitro but also inhibit free radical generation by maintaining the activity of antioxidant enzymes, thereby achieving anti-aging and weight-loss effects. Other studies have found that black tea polyphenols can significantly inhibit tumor growth in the body, enhance immunomodulatory activity, and improve antioxidant capacity (Liu S, Huang H. Assessments of antioxidant effect of black tea extract and its rationals by erythrocyte haemolysis assay, plasma oxidation assay and cellular antioxidant activity (CAA) assay[J]. Journal of Functional Foods, 2015, 181095-1105.).

[0003] Bioactive peptides (BAPs) are a class of peptide compounds composed of 20 natural amino acids in different compositions and arrangements. They are multifunctional fragments of proteins. Bioactive peptides can be derived from proteins in vivo or in vitro through enzymatic hydrolysis, including those from animals, plants, and microorganisms. These peptides possess various physiological functions, such as hormonal effects, immune regulation, antibacterial and antiviral activity, blood pressure reduction, cholesterol reduction, and anticancer effects, playing an important role in maintaining and restoring bodily health. Walnut peptides are small molecule polypeptides extracted from walnuts. As a novel bioactive component, walnut peptides are not only easily absorbed by the human body but are also considered a highly promising natural health-promoting substance due to their antioxidant, blood pressure-lowering, and anti-hyperuric acid activities (Huang R, Yu H. Extraction methods, chemical compositions, molecular structure, health functions, and potential applications of teapolysaccharides as a promising biomaterial: A review. J. International journal of biological macromolecules, 2024, 277(P3): 134150.).

[0004] The immune system, as a robust barrier protecting the body, constantly monitors and defends against external threats; while the metabolic system, like a sophisticated energy coordinator, ensures that every aspect of life receives an adequate energy supply. The relationship between metabolism and immunity is interdependent and mutually coordinated, representing a complex and crucial area of ​​research in maintaining homeostasis. Studies have shown that metabolic reprogramming of tumor cells not only affects their growth and survival but also influences immune molecules through the release of metabolites, thereby affecting the activity and function of immune cells. This competition for energy and nutrients creates metabolic competition within the tumor ecosystem, limiting the effective supply of nutrients and leading to microenvironment acidosis, thus hindering the function of immune cells.

[0005] Cyclophosphamide is commonly used in cancer treatment and as an immunosuppressant. It suppresses the body's immune system by reducing spleen and thymus indices and disrupting the intestinal mucosal barrier. Therefore, cyclophosphamide can be used to establish immunosuppressive models. As a chemotherapeutic drug and immunosuppressant, cyclophosphamide is widely used in tumor treatment and for autoimmune diseases (such as rheumatoid arthritis and systemic lupus erythematosus). However, the non-specific cytotoxicity of cyclophosphamide can damage immune cells (such as T cells, B cells, and NK cells), leading to thymus and spleen atrophy, leukopenia, and cytokine imbalance, significantly increasing the risk of infection and tumor recurrence. Cyclophosphamide interferes with metabolic pathways such as glycine and serine, affecting the energy supply and proliferation capacity of immune cells. For example, cyclophosphamide-induced serum metabolic abnormalities (such as changes in choline and prostaglandin F2β levels) may exacerbate immune microenvironment dysregulation. Targeted regulation of these metabolic pathways can restore immune cell function. Therefore, mitigating its immunosuppressive side effects is crucial for improving treatment safety and patient quality of life. The immunosuppressive effect of cyclophosphamide may weaken its anti-tumor efficacy. For example, in studies of BCMA / CD3 dual antibody combined with cyclophosphamide for the treatment of multiple myeloma, while cyclophosphamide can inhibit excessive T cell activation, long-term use may lead to T cell depletion, affecting immune memory function and thus limiting durable efficacy. By modulating immunosuppression, the cytotoxic and immune-activating effects of cyclophosphamide can be balanced, enhancing the synergistic effect of combination therapy. Summary of the Invention

[0006] The purpose of this invention is to address the problems in the application of immunosuppressants by providing a composition and its preparation method for alleviating cyclophosphamide-induced immunosuppression.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention provides a black tea and walnut peptide complex, which is prepared by mixing black tea water extract and walnut protein peptide in a certain proportion.

[0009] The walnut protein peptides are prepared as follows: walnut meal is used as raw material. After crushing and homogenization, the protein is extracted under acidic conditions. Then, a compound enzyme is used to convert the large molecule protein into small molecule peptides through compound enzymatic hydrolysis technology. Subsequently, impurities are removed and the mixture is concentrated using purification processes such as ultrafiltration and nanofiltration. Finally, the walnut protein peptides are obtained by spray drying.

[0010] Preferably, the compound enzyme is an alkaline protease; preferably, the mixing ratio of black tea water extract to walnut peptide is 1-15:8, more preferably 3-12:8, and in preferred embodiments, it is 19:40, 19:20, or 57:40.

[0011] The black tea and walnut peptide complex is a food product.

[0012] The black tea and walnut peptide complex is a beverage, but it can also be a health food and nutritional supplement, a functional baked food, a tea bag or instant tea powder, or a pet nutritional additive.

[0013] This invention provides the application of the aforementioned black tea and walnut peptide complex in alleviating cyclophosphamide-induced immunosuppression.

[0014] The relief of cyclophosphamide-induced immunosuppression is achieved by reversing cyclophosphamide-induced weight, thymus, and spleen indices in mice, improving the balance of jejunal CD4+ T cell subsets, or restoring the levels of cytokines and immunoglobulins.

[0015] The relief of cyclophosphamide-induced immunosuppression is achieved by influencing metabolic pathways such as the metabolism of serum metabolites glycine, serine, and threonine, pyrimidine metabolism, and protein digestion and absorption, as well as the regulation of choline, trimethylamine-N-oxide, prothioconazole, stearoylcarnitine, and prostaglandin F2.β.

[0016] The present invention also provides the use of the aforementioned black tea and walnut peptide complex in the preparation of formulations that alleviate cyclophosphamide-induced immunosuppression.

[0017] The relief of cyclophosphamide-induced immunosuppression is achieved by reversing cyclophosphamide-induced weight, thymus, and spleen indices in mice, improving the balance of jejunal CD4+ T cell subsets, or restoring the levels of cytokines and immunoglobulins.

[0018] The relief of cyclophosphamide-induced immunosuppression is achieved by influencing metabolic pathways such as the metabolism of serum metabolites glycine, serine, and threonine, pyrimidine metabolism, and protein digestion and absorption, as well as the regulation of choline, trimethylamine-N-oxide, prothioconazole, stearoylcarnitine, and prostaglandin F2.β.

[0019] This invention also provides a method for preparing a black tea and walnut peptide complex, the method comprising:

[0020] ① Black tea water extract is made from black tea raw materials;

[0021] ② Using walnut meal as raw material, after crushing and homogenization, protein is extracted under acidic conditions. Then, a compound enzyme is used to convert the large molecule protein into small molecule peptides through compound enzymatic hydrolysis technology. Subsequently, impurities are removed and concentrated using purification processes such as ultrafiltration and nanofiltration. Finally, walnut protein peptides are obtained by spray drying.

[0022] ③ Mix black tea water extract and walnut protein peptides in a certain proportion to make a black tea and walnut peptide complex;

[0023] Preferably, the compound enzyme is an alkaline protease. Preferably, the mixing ratio of black tea water extract to walnut peptide is 1-15:8, more preferably 3-12:8, and in preferred embodiments, it is 19:40, 19:20 or 57:40. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of mouse grouping in an experiment.

[0025] Among them, CK group, i.e. normal control group; MD group, i.e. immunosuppression model group; LH group, i.e. positive drug group; WP group, i.e. walnut peptide group; LTD group, i.e. low-dose FT-WP group; MTD group, i.e. medium-dose FT-WP group; HTD group, i.e. high-dose FT-WP group; n=12.

[0026] Figure 2 The effects of FT-WP on body weight, organ index and serum biochemical parameters in immunosuppressed mice.

[0027] Among them, (A) mouse body weight change; (B) thymus index; (C) spleen index; (D) IL-6 level; (E) IFN-γ level; (F) IgM level; (G) IgG level. Different letters a, b, c, d indicate significant differences between different groups for the same indicator, while the same letter indicates no significant difference. For example, bcd means that this group has no significant difference from groups labeled b, c, or d, but a significant difference from the group containing only a.

[0028] Figure 3 The effects of FT-WP on spleen and intestinal immunity.

[0029] (A) H&E staining of spleen tissue, magnified 100 times; (B) Immunohistochemical detection of the effect of FT-WP on CD4+ T cell expression, magnified 200 times.

[0030] Figure 4 This study investigates metabolites in mouse serum.

[0031] (A) OPLS-DA plot of CK and MD groups under cationic mode; (B) OPLS-DA plot of MD and HTD groups under cationic mode; (C) OPLS-DA plot of CK and MD groups under anionic mode; (D) OPLS-DA plot of MD and HTD groups under anionic mode; (E) Displacement test analysis plot of CK and MD groups under cationic mode; (F) Displacement test analysis plot of MD and HTD groups under cationic mode; (G) Displacement test analysis plot of CK and MD groups under anionic mode; (H) Displacement test analysis plot of MD and HTD groups under anionic mode; (I) Volcano plot of differential metabolites between CK and MD groups under mixed mode; (J) Volcano plot of differential metabolites between MD and HTD groups under mixed mode; (K) Bar chart and Venn diagram of differential metabolites between CK, MD and HTD groups under mixed mode.

[0032] Figure 5 Differences in serum metabolites between groups.

[0033] (A) Significantly different metabolites in the CK, MD and HTD groups under cationic mode; (B) Significantly different metabolites in the CK, MD and HTD groups under anionic mode; (C) Differential metabolite classification between the CK and MD groups based on the HMDB database; (D) Differential metabolite classification between the MD and HTD groups based on the HMDB database; (E) KEGG enrichment metabolic pathways of differential metabolites between the CK and MD groups; (M) KEGG enrichment metabolic pathways of differential metabolites between the MD and HTD groups under mixed mode.

[0034] Figure 6 For differential metabolite heatmaps.

[0035] (A) The top 20 significantly different metabolites between the CK group and the MD group; (B) The top 20 significantly different metabolites between the MD group and the HTD group.

[0036] Figure 7 The mechanism by which FT-WP improves cyclophosphamide-induced immunosuppression in mice. Detailed Implementation

[0037] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0038] Example 1

[0039] 1. Materials and Methods

[0040] 1.1 Materials and Reagents

[0041] The aqueous extract of black tea (the raw material of Fengqing black tea, provided by Yunnan Shuangjiang Mengku Tea Co., Ltd., hereinafter referred to as "Yunnan black tea") (extracted according to the operation steps of tea sample aqueous extraction in GB / T 8305—2013, with an extraction rate of 23%, purity: GA 1.17%, CAF 9.26%, EGC 0.53%, DL-C 0.19%, EC 0.45%, EGCG 0.77%, GCG 0.09%, ECG 1.32%) was extracted and produced by Hunan Aijia Biotechnology Co., Ltd.

[0042] Walnut peptide (WP) was provided by Guangdong Huatai Biotechnology Co., Ltd. Using walnut meal as raw material, after crushing and homogenization, proteins were extracted under acidic conditions, and then macromolecular proteins were converted into small peptides through alkaline protease hydrolysis technology. Subsequently, purification processes such as ultrafiltration and nanofiltration were used to remove impurities and concentrate, and finally the finished product was obtained by spray drying; moisture ≤ 7.0%, ash ≤ 8.0%, peptide content ≥ 40.0%, protein ≥ 60.0% (calculated on dry basis, N×5.30).

[0043] Cyclophosphamide (cyclophosphamide, no. PHR1404) was purchased from Sigma-Aldrich (Shanghai, China).

[0044] Levamisole hydrochloride (LH) was purchased from Shandong Renhetang Pharmaceutical Co., Ltd.

[0045] ELISA kits for interferon-γ (IFN-γ), interleukin 6 (IL-6), immunoglobulin M (IgM) and immunoglobulin G (IgG) were purchased from Wuhan Huamei Biotechnology Co., Ltd.

[0046] 1.2 Animal experiments

[0047] 84 SPF-grade female BALB / c mice at 5 weeks of age were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (production license number: SCXK (Xiang) 2019-0004). The mice were housed in the animal house of the Tea Research Institute of Hunan Agricultural University. Before the experiment, the mice underwent a one-week adaptation period under controlled conditions (temperature 24 ± 2°C, relative humidity 45% - 65%, regular lighting from 9:00 to 21:00), and had unrestricted access to food and water.

[0048] After domestication, the mice were randomly divided into 7 groups: normal control group (CK), immunosuppressive model group (MD), positive drug group (LH), walnut peptide group (WP), low-dose group (LTD), medium-dose group (MTD) and high-dose group (HTD), with 12 mice in each group.

[0049] Mice in the CK group were injected intraperitoneally with saline (90 mg / kg / d), while mice in the other groups were injected intraperitoneally with cyclophosphamide (80 mg / kg / d) for 3 days.

[0050] For the next 10 days, the CK and MD groups were administered purified water by gavage, the LH group was administered levamisole hydrochloride (30 mg / kg / d) by gavage, the WP group was administered walnut peptide (800 mg / kg / d) by gavage, and the LTD, MTD, and HTD groups were administered a mixture of walnut peptide and Yunnan black tea extract by gavage. The LTD group (Yunnan black tea extract 380 mg / kg / d + walnut peptide 800 mg / kg / d), the MTD group (Yunnan black tea extract 760 mg / kg / d + walnut peptide 800 mg / kg / d), and the HTD group (Yunnan black tea extract 1140 mg / kg / d + walnut peptide 800 mg / kg / d) were administered the mixture.

[0051] During the experiment, the body weight of mice in each group was measured daily before gavage; the growth and condition of the mice were also observed. The mice in our study were handled in accordance with the National Research Council's guidelines on the ethical care and use of laboratory animals. The Laboratory Animal Ethics Committee of Hunan Agricultural University carefully reviewed and approved the experiment to ensure it met ethical standards. The intragastric administration procedure for laboratory animals is detailed below. Figure 1 .

[0052] 1.3 Blood Collection

[0053] Blood was collected from the mouse orbital cavity into centrifuge tubes and centrifuged (4℃, 3500 r·min⁻¹, 10 min) to obtain serum samples.

[0054] 1.4 Thymus and Spleen Indices

[0055] The collected thymus and spleen were accurately weighed, and the organ index was calculated as follows:

[0056] Organ index (mg / g) = organ weight (mg) / mouse body weight (g).

[0057] 1.5 Organizational Morphological Analysis

[0058] After dissecting the mice, the spleen tissue was fixed in 4% paraformaldehyde solution for 24 h, gradually dehydrated with anhydrous ethanol to achieve transparency, wrapped in paraffin blocks and sectioned (3-5 μm thick) to make paraffin sections, stained with hematoxylin-eosin (H&E), dehydrated with anhydrous ethanol and mounted, and then the pathological changes of the spleen tissue were observed under an optical microscope and images were acquired using scanning methods.

[0059] 1.6 Immunohistochemical test

[0060] Jejunum sections were fixed in 4% paraformaldehyde and then destained in PBS (pH 7.4). Tissue sections were then placed in EDTA antigen retrieval buffer (pH 9.0) for antigen retrieval. Sections were then immersed in 3% hydrogen peroxide solution to inhibit endogenous peroxidase activity, rinsed with PBS, and blocked with 10% normal rabbit serum. Additionally, mouse primary antibody was added to the sections, and they were incubated overnight at 4°C. After rinsing, sections were treated with secondary antibody at room temperature. After destaining, slides were stained with diaminobenzidine (DAB) and counterstained with hematoxylin (Harris). After dehydration with anhydrous ethanol, slides were mounted with neutral resin. The area of ​​CD4+ T cells was counted using ImageJ-64 software 6.0.

[0061] 1.7 Biochemical Indicators

[0062] The levels of cytokines (IFN-γ, IL-6) and immunoglobulins (IgM, IgG) in mouse serum were detected using an ELISA kit. Cytokine concentrations were then calculated using a standard curve. The detection technique was performed according to the ELISA kit instructions.

[0063] 1.8 Serum Metabolome

[0064] After slow thawing of serum samples at 4°C, 100 μL of each sample was added to 400 μL of pre-cooled methanol / acetonitrile solution (1:1, v / v), vortexed, incubated at -20°C for 30 min, centrifuged at 14000 g at 4°C for 20 min, and the supernatant was collected, vacuum dried, and reconstituted with 100 μL of acetonitrile aqueous solution (acetonitrile:water = 1:1, v / v) for mass spectrometry analysis. The mixture was then vortexed, centrifuged at 14000 g at 4°C for 15 min, and the supernatant was injected for analysis. Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with a HILIC column. Throughout the analysis, samples were placed in an autosampler at 4°C. To avoid the influence of instrument signal fluctuations, continuous analysis of samples was performed in a randomized order. The raw data was converted to .MzML format using ProteoWizard (v3.0.6428), and then peak alignment, retention time correction, and peak area extraction were performed using XCMS (online 3.7.1). The data extracted by XCMS underwent metabolite structure identification and data preprocessing (null filtering: removing ion peaks with missing values ​​> 50%; null filling: KNN filling; data filtering: filtering features with RSD > 50%), and finally, data analysis was performed.

[0065] 1.9 Statistical Analysis

[0066] Images were processed using ImageJ software. All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using IBM SPSS Statistics 27. For data analysis among multiple groups, ANOVA one-way ANOVA was performed first, followed by Fisher's LSD test. A p-value < 0.05 was considered statistically significant. Figures in this study were created using GraphPadPrism 9, Adobe Illustrator 2024, and Origin 2024 software.

[0067] 2 Results and Analysis

[0068] 2.1 Effects of FT-WP on body weight and organ indices in immunosuppressed mice

[0069] Changes in mouse body weight as follows Figure 2 As shown in (A). The results showed that the body weight of mice in the blank group (CK) increased normally during the experiment, while the body weight of mice in the model group (MD) (P<0.01), levamisole hydrochloride group (LH), walnut peptide group (WP), and low, medium, and high dose groups (LTD, MTD, HTD) (P<0.001) decreased significantly 3 days after injection of cyclophosphamide, indicating that the model was successfully established and that cyclophosphamide caused serious damage to the mice.

[0070] After 10 consecutive days of oral administration of levamisole hydrochloride, walnut peptide, and different doses of FT-WP, the weight of mice recovered well, suggesting that the positive control group (LH), the walnut peptide group (WP), and the low, medium, and high dose groups (FT-WP) can alleviate the weight loss in mice with cyclophosphamide-induced liver injury. Thymus index and spleen index were also observed. Figure 2 As shown in (B) and (C), after a 14-day feeding period, compared with the control group (CK), cyclophosphamide induced a significant decrease in the thymus index in mice (p<0.05), indicating that cyclophosphamide can damage the function of immune organs. Compared with the model group (MD), the FT-WP group showed significantly increased thymus and spleen indices (p<0.05), suggesting that FT-WP can improve cyclophosphamide-induced immune organ damage.

[0071] 2.2 Effects of FT-WP on pathological changes in spleen and jejunum tissues of immunosuppressed mice

[0072] The spleen is an important immune organ, and the immune status of mice treated with cyclophosphamide can be assessed by examining the histological structure of the spleen. Figure 3As shown in (A) and (B), the control group (CK) exhibits neatly and tightly arranged cells with a high number of white blood cells; the splenic nodules are well-developed with a clear structure, a distinct boundary between the white and red pulp, no proliferation in the marginal zone, and very few germinal centers. Compared to the control group (CK), the model group (MD) shows thickened capsules extending into the splenic parenchyma to form trabeculae, significant proliferation in the marginal zone, increased and thickened marginal zone, severe damage to the splenic nodules, and lymphoid follicle proliferation leading to blurred boundaries between the white and red pulp. Abnormal atrophy of the white pulp indicates damage to lymphatic tissue, a reduced number of lymphocytes, a higher number of germinal centers, severe damage to the splenic nodules, and severe congestion in the red pulp area. Compared with the model group (MD), the high-dose group (HTD) showed no obvious germinal centers, a significantly reduced thickness of the marginal zone compared with the model group (MD), slight congestion of the red pulp, an increased area of ​​white pulp with denser internal lymphocytes, and a distribution ratio of white pulp and red pulp that was close to that of the normal group. The walnut peptide group (WP), low-dose group (LTD), medium-dose group (MTD) and the number of marginal zones and white pulp in the spleen showed significant improvement, but not as significant as the recovery effect of the high-dose group (HTD).

[0073] CD4+ T cells are important immune cells that, upon antigen stimulation, can differentiate into different subtypes of Th cells and perform various functions. Cyclophosphamide can affect the intestinal immune status of mice by inhibiting T cell differentiation. Therefore, the area of ​​CD4+ T cells in each group was studied using immunohistochemistry. Figure 3 As shown in (B), CD4+ T cells were stained brown. It is clearly visible that the blank group and all drug-treated groups had a higher number of brown cells, while the model group had almost no brown cells. CD4+ T cell expression in the MD group was significantly lower than that in the CK group (P<0.05). After FT-WP administration, CD4+ T cell levels significantly increased (P<0.05), with the highest increase observed in the HTD group. Therefore, FT-WP can effectively improve the balance of CD4+ T cell levels in cyclophosphamide-induced jejunal immunity.

[0074] 2.3 Effects of FT-WP on serum biochemical parameters in immunosuppressed mice

[0075] Cytokines are primarily small, soluble proteins induced by immune cells or other stimuli, participating in various immune functions. Based on these findings, we further investigated the levels of inflammatory cytokines and immune factors IL-6, IFN-γ, IgM, and IgG in serum. Figure 2As shown in (DG), compared with the CK group, the serum levels of IL-6, IFN-γ, IgM, and IgG in the MD group were significantly decreased (p<0.05); compared with the MD group, the serum levels of IL-6, IFN-γ, IgM, and IgG in the LH, WP, and FT-WP groups were significantly increased (p<0.05), and the effects were better at higher doses. This indicates that Cy can significantly inhibit the secretion of immune factors in mice, and LH, WP, and FT-WP treatments reversed the reduction of immune factors to some extent.

[0076] 2.4 Regulatory effect of FT-WP on serum metabolism in immunosuppressed mice

[0077] Based on the analysis of the previous experimental results, it was found that the high-dose (HTD) FT-WP study showed the best results. Further analysis using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) was conducted to analyze non-targeted metabolomics, comparing the metabolites in the serum of the CK, MD, and HTD groups. In the cation ( Figure 4 In the A and B) and anion mode ( Figure 4 C and D), orthogonal partial least squares discriminant analysis (OPLS-DA) showed that the MD group was clearly separated from the CK and HTD groups, indicating that the serum metabolites of the MD group were significantly different from those of the CK and HTD groups. And in cations ( Figure 4 E and F) and anion mode ( Figure 4 In the G and H permutation test analysis, all the Q2 values ​​after permutation were lower than the Q2 values ​​of the original model, indicating that the model's prediction results are reliable, have good explanatory and predictive abilities, and there is no overfitting. This experiment can continue with further analysis. In the mixed mode, the selection criteria of VIP>2 and P<0.05 were used; |log2 fold change|>1. The volcano plot showed that compared with the CK group, the MD group induced changes in 134 metabolites, of which 82 metabolites were significantly upregulated and 52 metabolites were significantly downregulated. Figure 4 (I). Furthermore, compared to the MD group, the HTD group induced changes in 139 metabolites, of which 20 metabolites were upregulated and 119 were downregulated ( ). Figure 4 The number of differentially expressed metabolites between the CK and MD groups, the MD and HTD groups, and the CK and HTD groups were 134, 139, and 152, respectively. Figure 4 (Middle K).

[0078] Hierarchical clustering heatmap analysis showed significant differences in serum metabolite profiles among the three groups. Figure 5 (A and B). These differentially metabolized substances were classified in the HMDB and KEGG databases, respectively. Based on HMDB, the top four categories compared to MD and HTD, CK, MD, and HTD, were carboxylic acids and their derivatives, fatty acyl groups, benzene and its substituted derivatives, and organic oxygen compounds, each accounting for more than 9%. Figure 5 (C and D). KEGG enrichment analysis showed that, based on the number of compounds, the top three enriched pathways were amino acid metabolism, lipid metabolism, and carbohydrate metabolism. The three most significantly enriched pathways in the KEGG pathways for differentially regulated metabolites between the MD and CK groups were arginine and proline metabolism, D-amino acid metabolism, and glycine, serine, and threonine metabolism. Differentially regulated metabolites between the MD and HTD groups were significantly enriched in glycine, serine and threonine metabolism, pyrimidine metabolism, and protein digestion and absorption metabolism. Figure 5 (E and F in the middle).

[0079] The top 20 differentially expressed metabolites based on the OPLS-DA model were selected as potential biomarkers (VIP > 2). For example, heat... Figure 6 As shown in (A), all 20 compounds showed significant differences between the CK and MD groups (p<0.05). Compared with the CK group, the MD group had an increase in 12 compounds (Xipamide, Thymidine 5'-monophosphate, Trimethylamine n-oxide, Pyridoxal phosphate, Prothioconazole, Terfenadine, Sarcosine, Resazurin, Xanthine, Taurine, Pyruvaldehyde, Pseudouridine), and a decrease in the remaining 8 compounds (Tetrabenazine, Succinic semialdehyde, Stachydrine, Quinolinate, Ricinoleic acid). (ricinoleic acid, solanidine, quinoline, pyruvate); such as heat Figure 6As shown in (B), there was a significant difference between the HTD group and the MD group (p<0.05). Compared with the MD group, the HTD group had a decrease in 19 compounds (Thymidine 5'-monophosphate, Taurine, Pi 36:4, Quinolinate, Trimethylamine n-oxide, Prothioconazole, Uracil, Terfenadine, Pyridoxalphosphate, Xipamide, Profenofos, Piperidine, Sarcosine, Pyruvaldehyde, Pseudouridine, Pi 38:4, Stearoylcarnitine, Prostaglandin F2.β, Pi 36:5) and an increase in one compound (Retinene). Among them, 10 metabolites, namely Xipamide, Thymidine 5'-monophosphate, Trimethylamine n-oxide, Pyridoxal phosphate, Prothioconazole, Terfenadine, Sarcosine, Taurine, Pyruvaldehyde, and Pseudouridine, showed an increase in the MD group, while HTD effectively improved the changes in metabolite levels induced by cyclophosphamide.

[0080] Since many diseases are related to immune system dysfunction, immune regulation plays a crucial role in maintaining good health. Components in black tea, such as tea polyphenols and theanine, possess various bioactivities, including antioxidant, anti-inflammatory, and blood pressure-lowering effects. Walnut peptides, as a plant-based bioactive peptide, also exhibit similar physiological functions. Both have significant impacts on immune regulation. Cyclophosphamide, commonly used in cancer treatment and as an immunosuppressant, exerts its inhibitory effect on the body's immune system by reducing spleen and thymus indices and disrupting the intestinal mucosal barrier. Therefore, cyclophosphamide can be used to establish immunosuppression models.

[0081] In this study, cyclophosphamide-induced symptoms in mice included weight loss, decreased spleen and thymus indices, and reduced secretion of related cytokines IFN-γ, IL-6, IgM, and IgG. Furthermore, H&E staining of the spleen showed indistinct boundaries between the red and white medulla. This indicates the successful establishment of the immune injury model. Immunohistochemical sections of the jejunum showed thinning of the intestinal wall and a decrease in lymphocytes in the MD group. The percentage of T lymphocyte subsets accurately reflects immune function. CD4+ and CD8+ T cells play important roles in the immune system. Studies have shown that increasing the proportion of CD8+ T cells in tumor-bearing mice can inhibit tumor growth; a decreased proportion of CD4+ T cells leads to intestinal immune dysfunction. Immunohistochemical results showed a significant decrease in the proportion of CD4+ T cells in the MD group (P<0.05). FT-WP administration significantly increased CD4+ T cell levels (P<0.05), with the most significant increase observed in the HTD group. Therefore, FT-WP can effectively improve the balance of CD4+ T cell levels in Cy-induced jejunal immunity. Cytokines, proteins secreted by activated T cells, are crucial for the immune response. Initially, CD4+ T cells differentiate into four distinct Th subsets: Th1 secretes IFN-γ and TNF-α, primarily for cellular immunity; Th2 secretes IL-6 and IL-10, participating in humoral immunity. T-bet and GATA 3 (also known as GATA-binding protein 3) play key roles in the differentiation of type 1 and type 2 helper T lymphocytes. This study further demonstrates that FD-WP can alleviate immunosuppression by increasing the expression levels of IFN-γ and IL-6, effectively reducing Cyclic acid (Cy) damage to the intestinal barrier and maintaining intestinal homeostasis.

[0082] To better elucidate the effects of FT-WP on serum metabolites in mice, we used non-targeted metabolomics to further investigate the effects of high-dose FT-WP treatment (HTD) on metabolites. This study found that, compared to the CK group, the MD group showed significant enrichment of differentially expressed metabolites in the KEGG pathway. The three most significant enrichment pathways were arginine and proline metabolism, D-amino acid metabolism, and glycine, serine, and threonine metabolism. Studies have shown that increased catabolism of tryptophan (Trp) and arginine (Arg) exacerbates inflammatory processes, thereby promoting tumorigenesis, hindering immune responses, and potentially causing cancer-related neurological syndromes. FT-WP primarily improves cyclophosphamide-induced metabolic disorders by regulating metabolic pathways such as glycine, serine, and threonine metabolism.

[0083] In this study, cyclophosphamide induced elevated levels of trimethylamine n-oxide (TMAO), and HTD administration decreased choline levels. Previous research has shown that choline is a major precursor to TMAO, which is metabolized by gut microbes to trimethylamine (TMA), subsequently oxidized in the liver to form TMAO. TMAO is associated with a variety of health outcomes, including all-cause mortality, cardiovascular disease, hypertension, diabetes, cancer, and kidney dysfunction; TMAO can activate inflammatory pathways, induce reactive oxygen species (ROS) production, reduce antioxidant activity, and disrupt intracellular redox balance; TMAO leads to a range of biological dysfunctions, including oxidative stress, blood-brain barrier disruption, decreased synaptic plasticity, inflammation, mitochondrial dysfunction, and abnormal protein aggregation; TMAO has been shown to accelerate brain aging and cognitive decline by inducing neuronal senescence and exacerbating neuroinflammation and oxidative stress. Previous studies have shown that prothioconazole induces mitochondrial dysfunction, leading to oxidative stress and apoptosis, causing adverse cardiovascular effects, and exhibiting developmental toxicity in early-life zebrafish. However, after gavage administration of FD-WP, choline levels decreased, and among the metabolites regulated by FD-WP, compared with the MD group, FD-WP reduced the levels of trimethylamine-N-oxide and prothioconazole, suggesting that FD-WP can improve cyclophosphamide-induced immune damage.

[0084] Compared to the MD group, the FD-WP (HTD group) showed reduced levels of prostaglandin F2.β and stearoylcarnitine. Prostaglandins (PGs) play a crucial role in the pathogenesis of skin cancer and melanoma by influencing complex signaling pathways involving inflammation, angiogenesis, and immunosuppression. Prostaglandin F2.β is an isomer of the prostaglandin F2 series and belongs to the class of isoprostaglandin compounds. Besides serving as a biomarker of oxidative stress, it also acts as a mediator for maintaining physiological homeostasis and participates in the biological activities of inflammatory and immune responses associated with disease pathology. Stearoylcarnitine is a fatty ester lipid molecule belonging to the endogenous metabolites in the human body. It plays an important role in the body, for example, serving as a metabolomics biomarker for early-onset and late-onset preeclampsia. Furthermore, stearoylcarnitine can be synthesized from perfused palmitate during fatty acid chain elongation in perfused rat hearts. Multi-omics analyses have shown that stearoylcarnitine is a novel biomarker for the development of cardiometabolic diseases in the offspring of gestational diabetes rats. Studies in HIV-infected individuals have found that the accumulation of acylcarnitine in plasma is associated with poor immune recovery after antiretroviral therapy (ART). Interestingly, the MD group showed decreased stachydrine levels. Studies have shown that stachydrine plays an important role in cardiovascular and cerebrovascular diseases, neuroprotection, anticancer activity, uterine regulation, anti-inflammatory responses, obesity treatment, and respiratory diseases. Unfortunately, administration of FD-WP did not significantly alter stachydrine levels, but it mitigated immune damage by regulating metabolites such as choline, trimethylamine-N-oxide, prothioconazole, stearoylcarnitine, and prostaglandin F2.β.

[0085] The above results indicate that FD-WP can improve cyclophosphamide-induced immunosuppression mechanisms (such as... Figure 7 ).

[0086] Our results indicate that FT-WP intervention significantly increased Cy-induced body weight, thymus and spleen indices in mice, alleviated splenic atrophy, improved the balance of CD4+ T cell levels, and simultaneously increased cytokine and immunoglobulin levels. Furthermore, the study showed that FT-WP regulates metabolic pathways such as glycine, serine, and threonine metabolism, affecting the levels of metabolites such as choline, trimethylamine-N-oxide, and prostaglandin F2.β. In conclusion, the black tea / walnut peptide complex can alleviate Cy-induced immunosuppression and regulate abnormal serum metabolism, providing a theoretical basis for the utilization of black tea / walnut peptides.

Claims

1. A black tea and walnut peptide complex, characterized in that, Black tea water extract and walnut protein peptides are mixed in a certain proportion to make a black tea and walnut peptide complex. The walnut protein peptides are prepared as follows: walnut meal is used as raw material. After crushing and homogenization, the protein is extracted under acidic conditions. Then, a compound enzyme is used to convert the large molecule protein into small molecule peptides through compound enzymatic hydrolysis technology. Subsequently, impurities are removed and the mixture is concentrated using purification processes such as ultrafiltration and nanofiltration. Finally, the walnut protein peptides are obtained by spray drying. Preferably, the complex enzyme is an alkaline protease; Preferably, the mixing ratio of black tea water extract to walnut peptide is 1-15:8, more preferably 3-12:8, and in preferred embodiments, it is 19:40, 19:20 or 57:

40.

2. The black tea and walnut peptide complex as described in claim 1, characterized in that, The complex is a food complex.

3. The black tea and walnut peptide complex as described in claim 1, characterized in that, The complex is a beverage, health food and nutritional supplement, functional baked goods, tea bags or instant tea powder, or pet nutritional additive.

4. The use of the black tea and walnut peptide complex as described in any one of claims 1-3 in alleviating cyclophosphamide-induced immunosuppression.

5. The application as described in claim 4, characterized in that, The relief of cyclophosphamide-induced immunosuppression is achieved by reversing cyclophosphamide-induced weight, thymus, or spleen indices in mice, improving the balance of jejunal CD4+ T cell subsets, or balancing the levels of cytokines and immunoglobulins.

6. The application as described in claim 4, characterized in that, The relief of cyclophosphamide-induced immunosuppression is achieved by influencing the metabolism of serum metabolites glycine, serine, and threonine, pyrimidine metabolism, protein digestion and absorption metabolism, or the regulation of choline, trimethylamine-N-oxide, prothioconazole, stearoylcarnitine, and prostaglandin F2.β.

7. Use of the black tea and walnut peptide complex as described in any one of claims 1-3 in the preparation of an agent to alleviate cyclophosphamide-induced immunosuppression.

8. The use as described in claim 7, characterized in that, The relief of cyclophosphamide-induced immunosuppression is achieved by reversing cyclophosphamide-induced weight, thymus, or spleen indices in mice, improving the balance of jejunal CD4+ T cell subsets, or balancing the levels of cytokines and immunoglobulins.

9. The use as described in claim 7, characterized in that, The relief of cyclophosphamide-induced immunosuppression is achieved by influencing the metabolism of serum metabolites glycine, serine, and threonine, pyrimidine metabolism, protein digestion and absorption metabolism, or the regulation of choline, trimethylamine-N-oxide, prothioconazole, stearoylcarnitine, and prostaglandin F2.β.

10. The method for preparing the black tea and walnut peptide complex according to claim 1, characterized in that, ① Black tea water extract is made from black tea raw materials; ② Using walnut meal as raw material, after crushing and homogenization, protein is extracted under acidic conditions. Then, the large molecule protein is converted into small molecule peptides through compound enzymatic hydrolysis technology. Subsequently, impurities are removed and concentrated by purification processes such as ultrafiltration and nanofiltration. Finally, walnut protein peptides are obtained by spray drying. ③ Mix black tea water extract and walnut protein peptides in a certain proportion to make a black tea and walnut peptide complex; Preferably, the complex enzyme is an alkaline protease; Preferably, the mixing ratio of black tea extract to walnut peptide is 1-15:8, more preferably 3-12:8, and in preferred embodiments, it is 19:40, 19:20 or 57:40.

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