A terminalia active polypeptide and a preparation method and application thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a Terminalia chebula active polypeptide, its preparation method, and its application. The preparation method uses Terminalia chebula slices as raw material, which are pulverized, homogenized with water, and then subjected to a series of enzymatic hydrolysis steps: a first hydrolysis with neutral protease, a second hydrolysis with pepsin, and a third hydrolysis with trypsin. Enzyme inactivation is performed after each hydrolysis. Subsequent steps include filtration, removal of alkaline proteins, ultrafiltration, and fine filtration to obtain the Terminalia chebula active polypeptide. The preparation method of this invention, through a three-step progressive enzymatic hydrolysis method, can efficiently prepare highly active Terminalia chebula polypeptides. The obtained Terminalia chebula active polypeptides have small molecular weight, are easily absorbed, and are non-toxic. These active polypeptides exhibit significant anti-tumor and liver fibrosis-improving effects, effectively reshaping the tumor microenvironment, enhancing anti-tumor immune responses, and repairing liver tissue damage. They have broad application prospects in the preparation of anti-tumor and liver fibrosis-improving drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Terminalia chebula active polypeptide, its preparation method, and its application. Background Technology

[0002] The dried, ripe fruit of Terminalia chebula Retz. and its variety, T. chebula Retz. Var. tomentella Kurz, is a core ingredient in Mongolian and Tibetan medicine, hailed as the "King of Herbs." It is also a traditional Chinese medicine listed in the Pharmacopoeia of the People's Republic of my country, and is abundant in Yunnan, Tibet, Guangdong, Guangxi, and Fujian provinces. With a long history of medicinal use, it has traditionally been used for its astringent, antidiarrheal, lung-clearing, cough-suppressing, heat-clearing, and throat-soothing effects. Modern pharmacological studies have further confirmed its various biological activities, including antioxidant, anti-inflammatory, anticancer, antibacterial, cardiotonic, immunomodulatory, and hepatoprotective properties, and it has been widely used in the clinical treatment of tumors and liver diseases.

[0003] Currently, the development and application of Terminalia chebula mainly focuses on Terminalia chebula extract, which is made by water extraction from Terminalia chebula or Terminalia velutipes fruit. It is a brownish-yellow powder containing Terminalia chebulic acid, Terminalia chebulic acid, gallic acid and other components. It has been applied in the fields of medicine, food and cosmetics, and can be used as a natural preservative, a raw material for functional health products and an antioxidant component for skin care products.

[0004] Bioactive peptides, a class of bioactive substances composed of 2-20 amino acids with specific regulatory functions, have become a research hotspot in nutritional science and the fields of food, health products, and pharmaceuticals due to their unique advantages such as small molecular weight, easy absorption, no residue in the body, broad bioactivity, and strong molecular specificity. Numerous studies have shown that bioactive peptides have beneficial functions in the prevention and treatment of tumors, metabolic diseases, and geriatric diseases. Therapies based on bioactive peptides are considered innovative and promising alternatives for treating dangerous diseases such as cancer. Novel bioactive peptides derived from plants can serve as adjuvant or preventative and therapeutic agents for cancer treatment, providing an important foundation for the development of drugs and functional foods.

[0005] Although the medicinal value of Terminalia chebula is widely recognized, current technology still has many shortcomings that urgently need to be addressed: First, Terminalia chebula is mostly used as the principal ingredient in Mongolian medicine in compound formulations. The complex composition of these compound formulations makes it difficult to accurately explain the specific efficacy of individual components, thus hindering in-depth research and precise application of its medicinal mechanisms. Second, the large molecular weight of existing Terminalia chebula extracts leads to low cellular absorption efficiency, severely affecting the full realization of its medicinal effects. Third, Terminalia chebula itself contains some potentially toxic components, and improper or excessive use can easily cause adverse reactions or even poisoning. Current extraction technologies cannot effectively remove or reduce this toxicity. Fourth, current research on Terminalia chebula is mostly focused on the extract level. A mature solution for the preparation process of Terminalia chebula bioactive peptides has not yet been developed. Research on its specific peptide composition, structural characteristics, and precise anti-tumor and hepatoprotective mechanisms is still insufficient, failing to fully explore the medicinal potential of Terminalia chebula and limiting the modernization of Mongolian medicine and its promotion in the international market.

[0006] Against this backdrop, developing a preparation process for Terminalia chebula bioactive peptides that can overcome the shortcomings of existing Terminalia chebula extracts and possess low toxicity, high activity, and easy absorption, and clarifying its anti-tumor and hepatoprotective mechanisms, has significant theoretical value and application prospects for promoting the modernization of Mongolian medicine and expanding the sources of anti-tumor and hepatoprotective drugs and functional health products. Summary of the Invention

[0007] Based on the above technical background, the main objective of this invention is to provide a Terminalia chebula active polypeptide, its preparation method and application, so as to overcome the shortcomings of the prior art.

[0008] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0009] The first aspect of this invention is to provide a method for preparing Terminalia chebula active polypeptide, the method comprising the following steps: Step 1: Crush the Terminalia chebula slices to obtain Terminalia chebula powder, add water to the Terminalia chebula powder and stir well; Step 2: Heat the mixture after adding water in Step 1, add neutral protease for one enzymatic hydrolysis, then heat to inactivate the enzyme, cool, and obtain the enzymatically inactivated Terminalia chebula one-time enzymatic hydrolysate. Step 3: Add hydrochloric acid to the primary hydrolysate of Terminalia chebula after enzyme inactivation to adjust the pH, then add pepsin for secondary hydrolysis, then heat to inactivate the enzyme, cool to obtain the secondary hydrolysate of Terminalia chebula after enzyme inactivation. Step 4: Add sodium hydroxide to the inactivated Terminalia chebula secondary enzymatic hydrolysate, adjust the pH, add trypsin for tertiary enzymatic hydrolysis, then heat to inactivate the enzyme, cool to obtain the inactivated Terminalia chebula tertiary enzymatic hydrolysate. Step 5: Filter the three enzymatic hydrolysates of Terminalia chebula after enzyme inactivation, add sodium hydroxide to adjust the pH, heat and stir to obtain a polypeptide solution with basic proteins removed. Filter, cool, ultrafilter and fine filter the polypeptide solution to obtain active Terminalia chebula polypeptide.

[0010] The steps described above are described in detail below.

[0011] In step 1, the Terminalia chebula slices are pulverized to 60-100 mesh.

[0012] Preferably, the Terminalia chebula slices are pulverized to 80 mesh.

[0013] The amount of water added is 2 to 5 times the weight of the Terminalia chebula slices.

[0014] Preferably, the amount of water added is three times the mass of the Terminalia chebula slices.

[0015] In step 2, the mixture after adding water in step 1 is heated to 40-60°C, preferably to 50°C, to homogenize the Terminalia chebula.

[0016] The conditions for the first enzymatic hydrolysis are as follows: the amount of neutral protease added is 2-4‰ of the weight of Terminalia chebula, the enzymatic hydrolysis is carried out at 40-60℃ for 2-4 hours, and the hydrolysis is carried out intermittently during the enzymatic hydrolysis process, with each stirring time being 2-6 minutes.

[0017] Preferably, the conditions for the first enzymatic hydrolysis are as follows: the amount of neutral protease added is 3‰ of the mass of Terminalia chebula, the enzymatic hydrolysis is carried out at 50°C for 3 h, and the hydrolysis is carried out intermittently, with each stirring time being 3 to 5 min.

[0018] The enzyme inactivation conditions are as follows: under stirring conditions, the temperature is raised to 70-90℃ and stirred and kept at that temperature for 5-15 minutes.

[0019] Preferably, the enzyme inactivation conditions are: heating to 80°C under stirring conditions, and stirring and holding at that temperature for 10 min.

[0020] After enzyme inactivation, the solution is cooled to 40–60°C, preferably to 50°C, to obtain the enzyme-inactivated Terminalia chebula primary hydrolysate.

[0021] In step 3, hydrochloric acid is added to the enzyme-inactivated Terminalia chebula primary hydrolysate to adjust the pH to 2-4, preferably to 3.

[0022] The conditions for the secondary enzymatic hydrolysis are as follows: the amount of pepsin added is 2-4‰ of the weight of Terminalia chebula, the enzymatic hydrolysis is carried out at 35-40℃ for 3-5 hours, and the hydrolysis is carried out intermittently during the process, with each stirring time being 2-6 minutes.

[0023] Preferably, the conditions for the secondary enzymatic hydrolysis are as follows: the amount of pepsin added is 3‰ of the mass of Terminalia chebula, the enzymatic hydrolysis is carried out at 38°C for 4 hours, and the hydrolysis is carried out intermittently, with each stirring time being 3 to 5 minutes.

[0024] The enzyme inactivation conditions are as follows: under stirring conditions, the temperature is raised to 70-90℃ and stirred and kept at that temperature for 5-15 minutes.

[0025] Preferably, the enzyme inactivation conditions are: heating to 80°C under stirring conditions, and stirring and holding at that temperature for 10 min.

[0026] After enzyme inactivation, the solution is cooled to 30–50°C, preferably to 40°C, to obtain the secondary enzymatic hydrolysate of Terminalia chebula after enzyme inactivation.

[0027] In step 4, sodium hydroxide is added to the secondary enzymatic hydrolysate of Terminalia chebula after enzyme inactivation. The pH is adjusted to 7.5–9, preferably to 8.

[0028] The conditions for the three enzymatic hydrolysis steps are as follows: the amount of trypsin added is 2-4‰ of the weight of Terminalia chebula, the enzymatic hydrolysis is carried out at 35-45℃ for 1-3 hours, and the hydrolysis is carried out intermittently, with each stirring time being 2-6 minutes.

[0029] Preferably, the conditions for the three enzymatic hydrolysis are as follows: the amount of trypsin added is 3‰ of the mass of Terminalia chebula, the enzymatic hydrolysis is carried out at 40℃ for 2 h, and the mixture is stirred intermittently during the enzymatic hydrolysis, with each stirring time being 3 to 5 min.

[0030] The enzyme inactivation conditions are as follows: under stirring conditions, the temperature is raised to 60-80℃ and stirred and kept at that temperature for 5-15 minutes.

[0031] Preferably, the enzyme inactivation conditions are: heating to 70°C under stirring conditions, and stirring and holding at that temperature for 10 min.

[0032] In step 5, sodium hydroxide is added to adjust the pH to 7.5-9, the temperature is raised to 80-95℃, and the mixture is stirred and kept warm for 5-15 minutes to remove the basic protein, thus obtaining a polypeptide solution with the basic protein removed.

[0033] Preferably, sodium hydroxide is added to adjust the pH to 8, the temperature is raised to 90°C, and the mixture is stirred and kept warm for 10 min to remove the basic protein, thereby obtaining a polypeptide solution with the basic protein removed.

[0034] The filtered polypeptide solution was cooled to 35–45°C and subjected to ultrafiltration using a membrane with a molecular weight cutoff of 6 kDa. Subsequently, it was subjected to fine filtration using sterile filters with pore sizes of 0.40–0.50 and 0.05–0.2 μm.

[0035] Preferably, the filtered polypeptide solution is cooled to 40°C and ultrafiltration is performed using a membrane with a molecular weight cutoff of 6 kDa, followed by fine filtration using sterile filters with pore sizes of 0.45 and 0.1 μm.

[0036] A second aspect of the present invention is to provide a Terminalia chebula active polypeptide prepared by the preparation method described in the first aspect of the present invention.

[0037] A third aspect of the present invention is to provide the application of the Terminalia chebula active polypeptide described in the second aspect of the present invention in the preparation of anti-tumor drugs or functional foods that improve liver damage and liver function.

[0038] The beneficial effects of this invention are as follows: (1) The preparation method of the present invention adopts a three-step progressive enzymatic hydrolysis process of neutral protease, pepsin and trypsin, combined with precise control of enzymatic hydrolysis parameters and enzyme inactivation conditions, which can specifically degrade macromolecular substances in Terminalia chebula. This preparation method does not require complex and expensive equipment, is easy to scale up for industrial production, and solves the problems of crude preparation process and low conversion rate of target components in existing Terminalia chebula extract preparation processes.

[0039] (2) The active polypeptide of Terminalia chebula prepared by this invention has a small molecular weight (screened by ultrafiltration with a molecular weight cutoff of 6 kDa), which is more easily absorbed by cells than traditional Terminalia chebula extract, thus maximizing its efficacy. At the same time, this invention can effectively decompose the potential toxic components in Terminalia chebula through three-step enzymatic hydrolysis. The safety experiment in rats has verified that no adverse toxic reactions were produced in different dosage groups, and there were no abnormalities in blood biochemical indicators, inflammatory factor levels and organ tissue morphology. It has naturalness and high safety, and solves the defects of potential toxicity risk and low absorption efficiency in the use of traditional Terminalia chebula.

[0040] (3) The active polypeptide of Terminalia chebula obtained in this invention can significantly inhibit the growth of tumors derived from MKN45 and MFC. It enhances the body's anti-tumor immune response by destroying the tumor tissue structure, increasing immune cell infiltration, reshaping the tumor microenvironment and activating immune-related pathways. Moreover, the gene characteristics it regulates are related to the improvement of the survival rate of gastric cancer patients, providing a new candidate drug for tumor immunotherapy and making up for the shortcomings of the existing Terminalia chebula-related products in terms of unclear anti-tumor mechanism and poor targeting.

[0041] (4) The active polypeptide of Terminalia chebula prepared in this invention can effectively improve early liver cirrhosis induced by CCl4 in rats, significantly reduce abnormally elevated biochemical indicators such as ALT, AST, TG, TC and LDL in the serum of rats with liver injury, increase HDL content, regulate serum inflammatory factors (IL-1β, IL-10, TNF-α) levels, improve the antioxidant capacity of liver tissue (increase SOD, TAOC, GSH-px content, and reduce MDA level), inhibit collagen fiber deposition in liver tissue, repair liver tissue damage, and clearly exert liver protection through the Hh-EMT signaling pathway, providing a new effective substance for the prevention and treatment of chronic liver disease.

[0042] (5) This invention breaks through the limitation that Terminalia chebula is mostly used in compound prescriptions and the efficacy of single components is difficult to explain, and realizes the modern and precise development of traditional Mongolian medicine. The active polypeptide of Terminalia chebula obtained by this invention can be used in the preparation of anti-tumor and liver-protecting drugs, expanding the application field of Terminalia chebula. Attached Figure Description

[0043] Figure 1 This shows the tumor growth in mice after the application of Terminalia chebula active peptides; Figure 2 This study demonstrates the impact of single-cell sequencing analysis on the active peptides of Terminalia chebula on the tumor microenvironment. Figure 3 The results of ALT, AST, TC, TG, LDL and HDL levels in different groups of rats in Experiment Example 2 are shown. Figure 4 The results of serum inflammatory factor level detection in different groups of rats in Experiment Example 2 are shown. Figure 5 The results of antioxidant index detection in different groups of rats in Experiment Example 2 are shown; Figure 6 The results of Masson's trichrome staining of rat livers from different groups in Experiment Example 2 are shown. Figure 7 The results of PAS staining of the livers of rats in different groups in Experiment Example 2 are shown. Figure 8 The results of Victoria blue staining of rat livers in different groups in Experiment Example 2 are shown. Figure 9 The results of reticular fiber staining of the livers of different groups of rats in Experiment Example 2 are shown. Figure 10 The spectrum of the Terminalia chebula active polypeptide prepared in Example 1 is shown; Figure 11 The diagram shows the peptide length distribution of the Terminalia chebula active polypeptide prepared in Example 1. Detailed Implementation

[0044] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0045] Example The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention are commercially available.

[0046] Example 1 The Terminalia chebula slices were pulverized to 80 mesh using a traditional Chinese medicine pulverizer to obtain Terminalia chebula powder, which was then set aside. The Terminalia chebula powder was poured into a reaction vessel, and pure water was added at 3 times the net weight of the Terminalia chebula. The mixture was homogenized using a colloid mill and extracted using a flash extractor for 5 minutes, stirring thoroughly.

[0047] The mixture with added water was heated to 50°C with stirring. After heating, the Terminalia chebula was homogenized. Neutral protease was weighed out at 3‰ of the net weight of the Terminalia chebula. The neutral protease was first dissolved in a small amount of water and then slowly poured into the reaction vessel containing the Terminalia chebula powder with stirring. The mixture was stirred evenly and kept at 50°C for 3 hours. During this time, the mixture was stirred intermittently for 3-5 minutes each time to perform enzymatic hydrolysis. The hydrolysate was heated to 80°C with continuous stirring and kept at this temperature for 10 minutes to inactivate the enzyme. Then it was cooled to 50°C.

[0048] Add HCl to the primary hydrolysate of Terminalia chebula after enzyme inactivation and cooling to adjust the pH to 3.0. Weigh pepsin at 3‰ of the net weight of Terminalia chebula. Dissolve the pepsin in a small amount of pure water and slowly pour it into the reaction vessel while stirring. Stir well and keep warm at 38℃ for 4 hours. During this period, stir intermittently for 3-5 minutes each time to carry out secondary hydrolysis. Heat the secondary hydrolysate to 80℃ while stirring continuously, stir and keep warm for 10 minutes to inactivate the enzyme, and then cool to 40℃.

[0049] Add NaOH to the second enzymatic hydrolysate of Terminalia chebula after enzyme inactivation and cooling to adjust the pH to 8.0. Weigh out 3‰ of the net weight of Terminalia chebula as trypsin. Dissolve the trypsin in a small amount of pure water first, and slowly pour it into the reaction vessel while stirring. Stir well and keep warm at 40℃ for 2 hours. During this period, stir intermittently for 3-5 minutes each time to perform three enzymatic hydrolysates. While stirring continuously, raise the temperature of the three enzymatic hydrolysates to 70℃ and stir and keep warm for 10 minutes to inactivate the enzyme.

[0050] The enzyme-inactivated products from the three enzymatic hydrolysates were filtered hot using a stainless steel plate and frame filter to obtain the filtrate. NaOH was added to the filtrate to adjust the pH to 8.0, and the temperature was raised to 90°C and stirred for 10 min to remove alkaline proteins. The peptide solution after removing alkaline proteins was filtered hot using a stainless steel plate and frame filter to obtain the filtrate, and then cooled to 40°C. Ultrafiltration was performed using a membrane with a molecular weight cutoff of 6 kDa. The ultrafiltrate was collected and then finely filtered through 0.45 and 0.1 μm sterile filters to obtain the Terminalia chebula active peptides, which were then divided into two portions for subsequent processing. The finely filtered solution was aliquoted into 3 ml vials, sterilized, and stored for later use.

[0051] Example 2 The preparation of Terminalia chebula active polypeptides was carried out in a manner similar to that in Example 1, except that: the mixture after adding water was heated to 40°C with stirring; after heating, the Terminalia chebula was homogenized; neutral protease was weighed at 2‰ of the net weight of Terminalia chebula; the neutral protease was first dissolved in a small amount of water; and then slowly poured into a reaction vessel containing Terminalia chebula powder with stirring; the mixture was stirred evenly; and enzymatic hydrolysis was carried out at 40°C for 4 h, with intermittent stirring for 3-5 min each time. The hydrolysate was heated to 80°C with continuous stirring and kept at this temperature for 10 min to inactivate the enzyme, and then cooled to 50°C.

[0052] Example 3 The preparation of Terminalia chebula active polypeptide was carried out in a manner similar to that in Example 1, except that: HCl was added to the primary enzymatic hydrolysate of Terminalia chebula after enzyme inactivation and cooling to adjust the pH to 3.0; pepsin was weighed at 4‰ of the net weight of Terminalia chebula; the pepsin was first dissolved in a small amount of pure water and then slowly poured into the reaction vessel under stirring; the mixture was stirred evenly and enzymatically hydrolyzed at 40°C for 3 h, with intermittent stirring for 3-5 min each time for secondary enzymatic hydrolysis; the secondary enzymatic hydrolysate was heated to 80°C under continuous stirring and kept at this temperature for 10 min for enzyme inactivation, and then cooled to 40°C.

[0053] Example 4 The preparation of Terminalia chebula active polypeptide was carried out in a manner similar to that in Example 1, except that: NaOH was added to the secondary enzymatic hydrolysate of Terminalia chebula after enzyme inactivation and cooling to adjust the pH to 8.0; trypsin was weighed at 3‰ of the net weight of Terminalia chebula; the trypsin was first dissolved in a small amount of pure water and then slowly poured into the reaction vessel with stirring. The mixture was stirred evenly and enzymatically hydrolyzed at 45°C for 1 h, with intermittent stirring for 3-5 min each time, for three enzymatic hydrolyses. The three enzymatic hydrolysates were heated to 60°C with continuous stirring and kept at this temperature for 15 min to inactivate the enzyme.

[0054] Experimental Example Experimental Example 1: Regulatory Role of the Tumor Immune Microenvironment The study investigated the regulatory role of Terminalia chebula peptides on the tumor microenvironment through single-cell sequencing, with a focus on the regulatory effect of peptides on immune cells.

[0055] Research Methods: Mice bearing MKN45 and MFC-derived tumors were administered Terminalia chebula active peptides (prepared in Example 1), and their effect on tumor growth was evaluated. Figure 1 As shown in the figure. Tumor tissue pathology and immune cell infiltration were analyzed by staining, while single-cell RNA sequencing (scRNA-seq) elucidated the changes in tumor microenvironment composition and transcriptional profile induced by Terminalia chebula active peptides. The test results are as follows: Figure 2 As shown. Genetic signatures regulated by Terminalia chebula active peptides were correlated with clinical outcomes using Cancer Genome Atlas (TCGA) data.

[0056] from Figure 1 It can be seen that the active polypeptide of Terminalia chebula significantly inhibited tumor growth in mice. Histopathological analysis showed that in tissues treated with the active polypeptide of Terminalia chebula, tumor structure was disrupted and immune cell infiltration increased. Figure 2 The results show that single-cell RNA sequencing further demonstrates the alteration of tumor microenvironment cellular composition and activation of immune-related pathways by the active peptides of Terminalia chebula. Differential gene expression analysis identified key immune factors regulated by the active peptides of Terminalia chebula. These results indicate that the active peptides of Terminalia chebula play an important role in enhancing anti-tumor immunity. Kaplan-Meier analysis confirmed that gene signatures associated with the active peptides of Terminalia chebula are associated with improved survival rates in gastric cancer patients.

[0057] In summary, the described Terminalia chebula active peptides exert potent antitumor effects in gastric cancer models by remodeling the tumor microenvironment and enhancing the immune response. These results make Terminalia chebula active peptides a promising candidate for gastric cancer immunotherapy.

[0058] Experimental Example 2: The regulatory effect of Terminalia chebula active polypeptide on liver function (1) Safety test of Terminalia chebula active polypeptide: 32 SD rats were randomly divided into: blank control group, low-dose group, medium-dose group, and high-dose treatment group. There were 8 rats in each group, half male and half female. 1 mL of Terminalia chebula active polypeptide of different concentrations was administered by gavage each time. The low-dose group was administered 6.25 mg / 100 g each time, the medium-dose group was administered 12.5 mg / 100 g each time, and the high-dose group was administered 25 mg / 100 g each time, twice a week. The blank group was given 1 mL of physiological saline by gavage each time. The experiment lasted for 90 days. The weight of the rats was recorded weekly.

[0059] During the 90-day experimental period, no abnormalities were observed in any of the rat groups. There were no significant differences in food intake and body weight among the rat groups (P>0.05). No poisoning or discomfort symptoms were observed in any of the rat groups. This indicates that the dosage of the Terminalia chebula active polypeptide, which is 6.25–25 mg / 100 g, is a safe dose for rats. Within the above dosage range, the Terminalia chebula active polypeptide has no toxic side effects on rats.

[0060] (2) Liver function regulation test: Thirty SD rats were randomly divided into three groups: a blank control group, a Terminalia chebula bioactive peptide intervention group, and a liver fibrosis model group, with 10 rats in each group (half male and half female). The blank control group received no treatment. For the Terminalia chebula bioactive peptide intervention group and the liver fibrosis model group, CCl4 induction was used to induce liver fibrosis in the rats. After successful model establishment, the rats in the Terminalia chebula bioactive peptide intervention group were administered Terminalia chebula bioactive peptide by gavage at a dose of 12.5 mg / 100 g twice a week for 8 consecutive weeks. Samples were collected from each group for testing.

[0061] Blood samples were taken for biochemistry testing: ALT, AST, TC, TG, LDL, and HDL levels. Test results are as follows: Figure 3 As shown; the serum inflammatory factors IL-10, IL-1β, and TNF-α were detected, and the results are as follows. Figure 4 As shown; the results of the antioxidant index test are as follows: Figure 5 As shown; histopathological, Masson, PAS, and immunohistochemical staining examinations were performed, such as... Figure 6 As shown.

[0062] Figure 3 In this context, ALT represents alanine aminotransferase (ALT), AST represents aspartate aminotransferase (AST), TC represents total cholesterol, TG represents triglycerides, HDL represents high-density lipoprotein (HDL), and LDL represents low-density lipoprotein (LDL). In the bar chart, NC represents the blank control group, Model represents the CCl4-induced liver fibrosis model group, and BAPT represents the Terminalia chebula bioactive peptide intervention group. Different lowercase letters in the bar chart indicate significant differences in experimental data (P < 0.05).

[0063] Figure 4 In the figure, NC was the blank control group, Model was the CCl4-induced liver fibrosis model group, and BAPT was the Terminalia chebula bioactive peptide intervention group. Different lowercase letters in the bar chart indicate significant differences in experimental data (P<0.05).

[0064] Figure 5In the above, SOD represents superoxide dismutase, MDA represents malondialdehyde, TAOC represents total antioxidant capacity, and GSH-px represents glutathione peroxidase. In the bar chart, NC represents the blank control group, Model represents the CCl4-induced liver fibrosis model group, and BAPT represents the Terminalia chebula bioactive peptide intervention group.

[0065] from Figures 3-5 As can be seen, the levels of blood biochemical indicators (ALT, AST, TG, TC, HDL and LDL) and inflammatory factors (IL-10, IL-1β and TNF-α) in the Terminalia chebula bioactive peptide intervention group did not show significant changes compared with the normal control group (P>0.05). The expression levels of inflammatory factors in the liver and kidneys also showed no abnormalities, with no significant differences (P>0.05).

[0066] Before the experiment, the rats in all groups were in good condition, but significant changes occurred after modeling. Treatment with Terminalia chebula active peptides improved the rats' condition. Figure 3 As can be seen, in the CCl4-induced liver fibrosis model, the levels of ALT, AST, TG, TC, and LDL in rat serum were significantly increased, while the level of HDL was decreased. After intervention with Terminalia chebula active polypeptide, the levels of ALT, AST, TG, TC, and LDL in rat serum were significantly decreased, while the level of HDL increased, and the differences were statistically significant (P<0.05).

[0067] Figure 4 The results of the detection of inflammatory factors in the serum of rats in each group showed that the serum levels of IL-1β, IL-10 and TNF-α in the CCl4-induced liver fibrosis model rats were significantly higher than those in the blank control group, and the differences were statistically significant (P<0.05). After intervention with Terminalia chebula active polypeptide, the serum levels of IL-1β, IL-10 and TNF-α in rats were significantly reduced compared with the fibrosis model group (P<0.05).

[0068] from Figure 5 As can be seen, the levels of SOD, TAOC, and GSH-px in the CCl4-induced liver fibrosis model rats were all lower than those in the blank control group, and the data analysis results showed significant differences (P<0.05). Simultaneously, the MDA level in the liver tissue of the liver fibrosis model rats was significantly increased, showing a significant difference compared to the blank control group (P<0.05). Compared with the liver fibrosis model group, after treatment with Terminalia chebula bioactive peptides, the levels of SOD, TAOC, and GSH-px in the rat liver tissue significantly increased, while the MDA level significantly decreased, with statistically significant differences (P<0.05).

[0069] Figures 6-10 The pathology is shown in HE and Masson ( Figure 6 ), PAS ( Figure 7 Victoria Blue Figure 8 ) and reticular fiber staining ( Figure 9 The results of the inspection.

[0070] Figure 6 In the figure, NC was the blank control group, Model was the CCl4-induced liver fibrosis model group, and BAPT was the Terminalia chebula bioactive peptide intervention group. Scale bar = 50 μm. Figure 6 Masson's trichrome staining results showed that a small amount of collagen fibers were distributed around the blood vessel walls in normal liver tissue, while a large amount of collagen fibers were deposited in the liver tissue of the model group, appearing blue and extending outward from the portal area. The fiber strands were thicker and stained darker, indicating that the liver tissue of the model group had more collagen fibers, which were wrapped and had formed pseudolobules. The liver tissue of the Terminalia chebula bioactive peptide intervention group was between the two, indicating that the Terminalia chebula bioactive peptide described in this invention can improve CCl4-induced liver fibrosis and is beneficial to improving liver damage.

[0071] Figure 7 In the study, NC represented the blank control group, Model represented the CCl4-induced liver fibrosis model group, and BAPT represented the Terminalia chebula bioactive peptide intervention group (scale bar = 50 μm). PAS staining is a routine pathological staining method that can display glycogen, neutral mucous substances, and certain acidic substances. To further clarify the effect of bioactive peptides on rat liver fibrosis, glycogen staining was performed. As shown in the NC group, negative PAS staining results showed that glycogen granules were red or purplish-red, cytoplasm was colorless, and cell nuclei and basophilic substances were blue. In the Model group, cell nuclei and basophilic substances were purple, while the Terminalia chebula bioactive peptide intervention group (BAPT group) fell between the two.

[0072] Figure 8 In this study, NC represented the blank control group, Model represented the CCl4-induced liver fibrosis model group, and BAPT represented the Terminalia chebula bioactive peptide intervention group. The scale bar was 50 μm. To further clarify the effect of Terminalia chebula bioactive peptides on rat liver fibrosis, this invention used Victoria blue staining to detect and analyze the collagen arrangement in rat liver tissue. As a specific collagen staining method, Victoria blue staining resulted in a pale red color in the collagen tissue, which can visually display the collagen accumulation level in the liver tissue. Figure 8As can be seen, the collagen fibers in the normal control group (blank control group) were neatly arranged and lightly colored, while the extracellular collagen fibers in the fibrosis model group showed obvious deposition, with disordered arrangement and darker staining. This further indicates that CCl4 treatment induced the accumulation of collagen fibers in liver tissue. After treatment with Terminalia chebula bioactive peptides, the extracellular collagen accumulation in liver cells was effectively improved. At the same time, the arrangement of collagen fibers in liver tissue after intervention with Terminalia chebula bioactive peptides was significantly improved and more neat than that in the fibrosis model group. This shows that the Terminalia chebula bioactive peptides described in this invention can improve the accumulation of collagen fibers in liver tissue induced by CCl4 and increase the arrangement of collagen fibers in liver tissue.

[0073] Figure 9 In the diagram, NC represents the blank control group, Model represents the CCl4-induced liver fibrosis model group, and BAPT represents the Terminalia chebula bioactive peptide intervention group. Scale bar = 50 μm. Reticular fiber staining is shown below. Figure 9 As shown, the reticular fibers are black, and the collagen fibers are dark yellow. From Figure 9 As can be seen, in the normal group, the reticular fibers were evenly distributed in the liver tissue in a flocculent pattern, without thickening, fusion, or collapse of the reticular fibers. In the model group, the reticular fibers in the liver tissue lost their normal distribution, and from the portal area, the reticular fibers collapsed, fused, thickened, and encapsulated, forming pseudolobules. The Terminalia chebula bioactive peptide group was intermediate between the two. These results indicate that the Terminalia chebula bioactive peptide described in this invention can improve CCl4-induced thickening, fusion, collapse, and encapsulation of reticular fibers in liver tissue.

[0074] from Figures 6-9 It can be seen that the liver cells in the blank control group have normal morphology and the hepatocyte cords are arranged radially; the liver cells in the fibrosis model rats are arranged disorderedly, with a large number of pseudolobules, enlarged portal areas, and inflammatory cell infiltration; after intervention with Terminalia chebula bioactive peptides, the liver damage in rats was significantly improved.

[0075] In summary, no adverse toxic effects were observed in rats during the experiment, and no unexpected adverse effects were found. Early-stage cirrhosis in rats was successfully established by intraperitoneal injection of a CCl4 / olive oil (2:3) mixture, 1 ml / kg, twice a week for 8 weeks. The Terminalia chebula bioactive peptide improved liver function in CCl4-induced early-stage cirrhosis. This peptide effectively inhibited the infiltration of inflammatory cells, deposition of extracellular matrix such as collagen and elastic fibers in the liver of rats with early-stage cirrhosis, thus playing a role in intervening in early-stage cirrhosis. During the experiment, the lack of difference in cell survival rate between different concentrations of Terminalia chebula bioactive peptide confirmed the conclusion of Experiment 1, namely, that the Terminalia chebula bioactive peptide has no toxic side effects. Treatment with this peptide improved the proliferation capacity, mitochondrial activity level, and oxidative stress homeostasis of liver cells in fibrotic rats. The gene and protein expression levels further verified the ameliorative effect of the Terminalia chebula bioactive peptide based on the Hh-EMT signaling pathway on rat liver fibrosis.

[0076] Experimental Example 3: Liquid Chromatography-Mass Spectrometry Analysis The active polypeptide of Terminalia chebula prepared in Example 1 was analyzed by liquid chromatography-mass spectrometry (LC-MS). The specific analytical procedure is as follows: The active polypeptide of Terminalia chebula was dissolved in mobile phase A of the LC system and then separated using an EASY-nLC 1200 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The LC gradient settings were: 0-62 min, 4%–21% B; 62-82 min, 21%–32% B; 82-86 min, 32%–80% B; 86-90 min, 80% B, with the flow rate maintained at 500 nL / min. After separation by the ultra-high performance liquid chromatography system, the active polypeptide of Terminalia chebula was injected into an NSI ion source for ionization and then analyzed by Q Exactive™ HF-X mass spectrometry. The ion source voltage was set to 2.1 kV. High-resolution Orbitrap was used to detect and analyze the precursor ions and secondary fragments of the active polypeptide from Terminalia chebula. The primary mass spectrometry scan range was set to 350–1800 m / z with a scan resolution of 120,000; the secondary scan resolution was set to 15,000. Data acquisition was performed using a data-dependent scanning (DDA) procedure, where the top 10 peptide precursor ions with the highest signal intensity were sequentially introduced into the HCD collision cell after the primary scan and fragmented using 28% of the fragmentation energy, followed by secondary mass spectrometry analysis. To improve the efficiency of mass spectrometry, automatic gain control (AGC) was set to 5E4, the signal threshold to 2.5E5 ions / s, the maximum injection time to 40 ms, and the dynamic exclusion time for tandem mass spectrometry scans to 30 s to avoid repeated scanning of precursor ions. Detection results are as follows: Figure 10 As shown, the peptide length distribution is as follows: Figure 11 As shown.

[0077] Figure 10 In this context, Total spectrums represents the total number of spectra, the number of secondary spectra generated by mass spectrometry, Matchedspectrums represents the number of valid spectra, the number of spectra that match the theoretical secondary spectra, and Identified peptides represents the number of identified peptides, the number of peptide sequences resolved from the matching results. Figure 10 The results show that the number of peptides in the Terminalia chebula active polypeptide prepared in Example 1 is 3.

[0078] from Figure 11 As can be seen, the peptides of the Terminalia chebula active polypeptide obtained by this invention are mostly distributed in the range of 7–20 amino acids, which conforms to the general rules based on enzymatic digestion and mass spectrometry fragmentation. The distribution of peptide lengths identified by mass spectrometry meets the quality control requirements.

[0079] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing an active polypeptide from Terminalia chebula, characterized in that, The preparation method includes the following steps: Step 1: Crush the Terminalia chebula slices to obtain Terminalia chebula powder, add water to the Terminalia chebula powder and stir well; Step 2: Heat the mixture after adding water in Step 1, add neutral protease for one enzymatic hydrolysis, then heat to inactivate the enzyme, cool, and obtain the enzymatically inactivated Terminalia chebula one-time enzymatic hydrolysate. Step 3: Add hydrochloric acid to the primary hydrolysate of Terminalia chebula after enzyme inactivation to adjust the pH, then add pepsin for secondary hydrolysis, then heat to inactivate the enzyme, cool to obtain the secondary hydrolysate of Terminalia chebula after enzyme inactivation. Step 4: Add sodium hydroxide to the inactivated Terminalia chebula secondary enzymatic hydrolysate, adjust the pH, add trypsin for tertiary enzymatic hydrolysis, then heat to inactivate the enzyme, cool to obtain the inactivated Terminalia chebula tertiary enzymatic hydrolysate. Step 5: Filter the three enzymatic hydrolysates of Terminalia chebula after enzyme inactivation, add sodium hydroxide to adjust the pH, heat and stir to obtain a polypeptide solution with basic proteins removed. Filter, cool, ultrafilter and fine filter the polypeptide solution to obtain active Terminalia chebula polypeptide.

2. The preparation method according to claim 1, characterized in that, In step 1, The Terminalia chebula slices were pulverized to 60-100 mesh.

3. The preparation method according to claim 1, characterized in that, In step 2, The conditions for the first enzymatic hydrolysis are as follows: the amount of neutral protease added is 2-4‰ of the weight of Terminalia chebula, the enzymatic hydrolysis is carried out at 40-60℃ for 2-4 hours, and the hydrolysis is carried out intermittently during the enzymatic hydrolysis process, with each stirring time being 2-6 minutes.

4. The preparation method according to claim 1, characterized in that, In step 2, The enzyme inactivation conditions are as follows: under stirring conditions, the temperature is raised to 70-90℃ and stirred and kept at that temperature for 5-15 minutes; after enzyme inactivation, the temperature is cooled to 40-60℃.

5. The preparation method according to claim 1, characterized in that, In step 3, Add hydrochloric acid to the enzyme-inactivated primary hydrolysate of Terminalia chebula to adjust the pH to 2-4. The conditions for the secondary enzymatic hydrolysis are as follows: the amount of pepsin added is 2-4‰ of the weight of Terminalia chebula, the enzymatic hydrolysis is carried out at 35-40℃ for 3-5 hours, and the hydrolysis is carried out intermittently during the process, with each stirring time being 2-6 minutes.

6. The preparation method according to claim 1, characterized in that, In step 3, The enzyme inactivation conditions are as follows: under stirring conditions, the temperature is raised to 70-90℃ and stirred and kept at that temperature for 5-15 minutes.

7. The preparation method according to claim 1, characterized in that, In step 4, Add sodium hydroxide to the secondary enzymatic hydrolysate of Terminalia chebula after enzyme inactivation to adjust the pH to 7.5-9; The conditions for the three enzymatic hydrolysis steps are as follows: the amount of trypsin added is 2-4‰ of the weight of Terminalia chebula, the enzymatic hydrolysis is carried out at 35-45℃ for 1-3 hours, and the mixture is stirred intermittently during the enzymatic hydrolysis process, with each stirring time being 2-6 minutes.

8. The preparation method according to claim 1, characterized in that, In step 5, Add sodium hydroxide to adjust the pH to 7.5–9, heat to 80–95°C, stir and keep warm for 5–15 minutes to obtain a polypeptide solution with basic proteins removed.

9. A Terminalia chebula active polypeptide prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the Terminalia chebula active polypeptide according to claim 9 in the preparation of anti-tumor drugs or functional foods that improve liver fibrosis, improve liver damage, and improve liver function.

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