Application of an amaranthus hybridus polypeptide in the preparation of a drug for improving ulcerative colitis

Abstract

The present invention relates to the use of Amaranthus hybridus L. polypeptides or proteins in the preparation of drugs or functional foods for improving ulcerative colitis. The Amaranthus hybridus L. polypeptides in the present invention are a complex system. Experimental results have proven that the whole medicine is effective, but the amino acid sequences of the Amaranthus hybridus L. polypeptides cannot be determined yet. Specifically, the experimental results of the present invention show that the analysis of colon pathological tissue sections after HE staining in the administration group prepared in the implementation shows that, compared with the model group, the degree of colon lesions in the Amaranthus hybridus L. polypeptide administration group is significantly improved; the expressions of ZO-1 and Occludin proteins are significantly increased, suggesting that Amaranthus hybridus L. polypeptides have a certain protective effect on the process of ulcerative colitis. Since polypeptides are hydrolysis products of proteins, it is suggested that Amaranthus hybridus L. polypeptides or proteins have the effect of protecting colon tissues.

Description

Technical Field

[0001] The present invention relates to the use of amaranthus hybridus polypeptide as a drug or functional food for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a non-specific ulcerative colitis disease, which is a common disease in the department of gastroenterology. The lesions mostly invade the mucosa and submucosa of the colon and rectum, and there is currently no effective treatment. Its clinical symptoms mainly include abdominal pain, diarrhea, hematochezia, etc. [1] , and the commonly used clinical drug is mesalazine [1,2] , which can only partially reduce the intestinal inflammatory response of patients. In recent years, the incidence of UC has been showing an increasing trend, and the carcinogenic risk has increased. Therefore, it is particularly urgent to find a safer and more effective drug for UC.

[0003] The dextran sulfate sodium (DSS)-induced colitis model is widely used because of its simplicity and many similarities to human ulcerative diseases [3] , and it is generally considered to be related to mechanisms such as macrophage dysfunction, intestinal flora dysregulation, the toxic effect of DSS on the colonic epithelium, and the important role of cytokines in the pathogenesis of the DSS colitis model (tumor necrosis factor, interleukin, interferon, IL-10, and IL-12). At present, it is generally believed that there is a disorder of intestinal mucosal inflammatory immune tolerance in the UC body, the intestinal mucosal barrier is damaged, and the weakening of the intestinal mucosal barrier function is one of the key factors causing ulcerative colitis [4] . Ulcerative colitis will cause an imbalance between the intestinal flora and mucosal immunity [5] . The colonic epithelium promotes host-microbial interactions to control mucosal immunity, coordinate nutrient circulation, and form a mucus barrier. The disruption of the epithelial barrier is the basis of inflammatory bowel disease (IBD) [6] . The immune dysfunction of UC can be manifested as an imbalance of inflammatory cytokines, which ultimately leads to the destruction of intestinal epithelial tissue. Common cytokines such as endotoxin (LPS), IL-17, IL-23, and TNF-α change. In the late stage of the disease, a large section of the intestinal lumen will be affected, leading to toxic colitis and colon cancer, and the clinical cure rate is low [7,8,9] .

[0004] Due to the characteristics of traditional Chinese medicine such as overall treatment, safety and reliability, and small toxic and side effects, traditional Chinese medicine has obvious natural advantages in the treatment or prevention of inflammatory bowel disease. Traditional Chinese medicine can regulate the intestinal flora, prevent the translocation of intestinal bacteria and endotoxin, balance beneficial bacteria and bacteria and viruses [7] , significantly improve the regeneration ability of mucosal cells, reduce the excessive mucosal permeability, and thus improve the barrier function of the mucosa. A large number of studies have shown that traditional Chinese medicine can protect the intestinal mucosal barrier through various different mechanisms of action[10,11] 。

[0005] In patients with UC, activated nuclear factor κB (NF-κB) was observed in inflammatory mucosal biopsy specimens and was confined to macrophages and epithelial cells. Transcription factors of the NF-κB family are essential in the expression of genes involved in inflammation. Generally, this family contains 5 different members, including p65 (RelA), RelB, c-Rel, p50, and p52

[12] 。Increasing evidence indicates that polypeptides in the diet can effectively relieve IBD by reducing pro-inflammatory cytokine levels, alleviating oxidative stress, and manipulating the gut microbiota

[13] 。However, the effects of peptide intervention before and after inducing colitis are different. Severe colitis reduces the absorption and digestion of nutrients, leaving more peptides and amino acids to be fermented by the colonic microbiota.

[0006] During the immune response process in UC, T cells play an important role in regulating, suppressing, and maintaining the inflammatory state. The innate immune system is considered to be possibly the cause of the occurrence and development of IBD. Toll-like receptors are crucial in the host's defense against pathogens. Studies have shown that Toll-like receptor 4 (TLR4) is expressed at a low level in normal colonic tissue, but significantly increased in UC, indicating that the increased expression of TLR4 may initiate and maintain intestinal inflammation

[14] 。

[0007] Amaranthus hybridus L. is often used as a coarse grain food and a forage crop. It is rich in nutritional value, has an ideal amino acid composition, various vitamins, mineral elements, and bioactive secondary metabolites [15,16] 。It has excellent protein quality, a balanced AA ratio, and a high proportion of essential amino acids, meeting the human protein consumption standards recommended by FAO / WHO. It is one of the ideal food ingredients for humans. Amaranthus hybridus L. has the effects of replenishing qi and removing heat, killing insect poisons, being able to expel pathogenic factors, promote urination and defecation, eliminate worms, relieve cold and heat, dredge blood vessels, and expel stasis. The polyphenolic substances in Amaranthus hybridus L. have been proven to have antioxidant, hypoglycemic, lipid-lowering, and cholesterol-lowering effects

[17] 。Currently, the research on Amaranthus hybridus L. polypeptides mainly focuses on hypoglycemic and antihypertensive aspects. There has been no report on the role of Amaranthus hybridus L. polypeptides in improving ulcerative colitis and their uses in drugs or functional foods for improving this disease.

[0008] References

[0009] [1] Jing Jie. Efficacy of mesalazine combined with Bifidobacterium triple viable in the treatment of ulcerative colitis [J]. Medical Information, 2021, 34; No. 540(13): 163 - 165.

[0010] [2]Turner D, Yerushalmi B, Kori M, et al. Once-Versus Twice-daily Mesalazine to Induce Remission in Paediatric Ulcerative Colitis: A Randomised Controlled Trial[J]. J Crohns Colitis, 2017, 11(5): 527-33.

[0011] [3] Li Qinmei, Wang Yuhan, Lü Feifei, et al. Comparative Study on Rat Models of Ulcerative Colitis and Damp-Heat Syndrome Ulcerative Colitis[J]. Acta Laboratorium Animalis Scientia Sinica, 2021, 29(03): 354-363.

[0012] [4] Yuan Liuyi, Li Xiaojin, Yin Qingsheng, et al. Research Progress on Improvement of Intestinal Mucosal Barrier Function by Traditional Chinese Medicine Intervention in Intestinal Flora[J]. Chinese Traditional and Herbal Drugs, 2018, 49(08): 1932-1938.

[0013] [5] Zhao-hua Shen, Chang-xin Zhu, Yong-Sheng Quan, et al. Relationship between intestinal microbiota and ulcerative colitis: Mechanisms and clinical application of probiotics and fecal microbiota transplantation[J]. World Journal of Gastroenterology. 2018, 24(01): 5-14.

[0014] [6] Parikh K, Antanaviciute A, Fawkner-Corbett D, et al. Colonic Epithelial Cell Diversity in Health and Inflammatory Bowel Disease[J]. Nature, 2019, 567(7746): 49-55.

[0015] [7] Xiao Xiuling. Relationship between Intestinal Flora and Blood TNF-α and Endotoxin Levels in Patients with Ulcerative Colitis[J]. Journal of Clinical and Experimental Medicine, 2012, 11(17): 1364-1365+1368.

[0016] [8] Quan Shuai, Lü Kaiyuan, Li Xinyu, et al. Effects and mechanism of action of oligomeric proanthocyanidins from grape seeds on dextran sulfate sodium-induced ulcerative colitis in mice [J]. Chinese Traditional and Herbal Drugs, 2020, 51(01): 149-156.

[0017] [9] Cristina-Sorina Cǎanǎ, Ioana Berindan Neagoe, Vasile Cozma, Cristian Magdas, et al. Contribution of the IL-17 / IL-23 axis to the pathogenesis of inflammatory bowel disease [J]. World Journal of Gastroenterology, 2015, 21(19): 5823-5830.

[0018]

[10] Ma Lingling, Ji Jianbin. Improvement effect and mechanism of Qingchi San enema on intestinal mucosal permeability in ulcerative colitis mice [J]. Shandong Medical Journal, 2021, 61(33): 24-29.

[0019]

[11] Yuan Liuyi, Li Xiaojin, Yin Qingsheng, et al., Zhuang Pengwei. Research progress on improving intestinal mucosal barrier function by traditional Chinese medicine intervention in intestinal flora [J]. Chinese Traditional and Herbal Drugs, 2018, 49(08): 1932-1938.

[0020]

[12] Li X X, Chen S G, Yue G L, et al. Natural flavone tricin exerted anti-inflammatory activity in macrophage via NF-κB pathway and ameliorated acute colitis in mice [J]. Phytomedicine, 2021, 90: 153625.

[0021]

[13] Sun Chongzhen, Tang Xin, Shao Xin, et al. Mulberry (Morus atropurpurea Roxb.) leaf protein hydrolysates ameliorate dextran sodium sulfate-induced colitis via integrated modulation of gut microbiota and immunity[J]. Journal of Functional Foods, 2021, 84(84).

[0022]

[14] Tartey S, Takeuchi O. Pathogen recognition and Toll-like receptor targeted therapeutics in innate immune cells[J]. Int Rev Immunol, 2017, 36(2):57-73.

[0023]

[15] Tovar-Pérez, Erik G, Lugo-Radillo A, Aguilera-Aguirre S. Amaranth grain as a potential source of biologically active peptides: a review of their identification, production, bioactivity, and characterization[J]. Food Reviews International, 2018, 35:1-25.

[0024]

[16] Development of a nutrient-dense complementary food using amaranth-sorghum grains[J]. Food Science & Nutrition, 2016, 5(1):86-93.

[0025]

[17] Sarker U,Oba S.Phenolic profiles and antioxidant activities in selected drought-tolerant leafy vegetable amaranth[J].Scientific Reports,2020,10(1):18287.

[0026] Object of the Invention

[0027] The object of the invention of this project is to discover a new medical use of amaranth viridis polypeptide for the treatment of ulcerative colitis.

[0028] Technical Solution

[0029] Application of an amaranth viridis polypeptide or protein in the preparation of a drug for improving ulcerative colitis.

[0030] The application as described above is characterized in that the amaranth viridis polypeptide is prepared by the pepsin enzymolysis method.

[0031] The application as described above is characterized in that the amaranth viridis polypeptide and protein are prepared by the following steps: powder the amaranth viridis seeds, pass through a 60-mesh sieve, defat with n-hexane three times the volume of the crude drug for 24 h, and dry in a fume hood. Ultrasonically extract the defatted polypeptide three times with 0.06 mol / L NaOH solution 15 times the volume of the crude drug for 1.5 h each time. After combining the filtrates, adjust the pH to 4, centrifuge at 4000 r / min for 15 min, and collect the precipitate to obtain amaranth viridis seed protein; dissolve the protein in ultrapure water, and add pepsin for enzymatic hydrolysis to obtain amaranth viridis polypeptide.

[0032] Specifically:

[0033] The described Amaranthus hybridus L. polypeptide is prepared by the following steps: The seeds of Amaranthus hybridus L. are ground into powder, passed through a 60-mesh sieve, defatted with n-hexane in an amount three times the volume of the crude drug for 24 h, and dried in a fume hood. The defatted polypeptide is ultrasonically extracted three times with 0.06 mol / L NaOH solution at 15 times the volume of the crude drug, each time for 1.5 h. After combining the filtrates, the pH is adjusted to 4, and centrifuged at 4000 r / min for 15 min to collect the precipitate to obtain the protein of Amaranthus hybridus L. seeds. The protein is dissolved in ultrapure water, the pH of enzymatic hydrolysis is adjusted to 3.25, the temperature is 35.90 °C, the substrate concentration is 24 mg / mL, the enzymatic hydrolysis time is 1 h, and the enzyme addition amount is 245.21 U / g. After adding protease and shaking well, it is placed in a water bath for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, it is inactivated in a boiling water bath for 15 min, and the enzymatic hydrolysate is centrifuged (4000 r / min) for 15 min to collect the supernatant. Trichloroacetic acid (TCA) is used to precipitate the unhydrolyzed protein in the supernatant, and centrifuged (4000 r / min) for 15 min. Under these conditions, the yield of Amaranthus hybridus L. polypeptide is 27.67%-37.84%.

[0034] Specifically:

[0035] This invention is proved through research that the Amaranthus hybridus L. polypeptide can improve the ulcerative colitis model of mice induced by DSS. By observing the pathological sections of colon tissues after HE staining, the protein expression levels of ZO-1 and Occludin in the colon tissues of each group of mice are detected. The experimental results show that: The Amaranthus hybridus L. polypeptide in this invention can improve the inflammatory degree of the colon tissues of model mice, showing an intervention effect on the ulcerative colitis model of mice, and can be used for the new medical use of treating ulcerative colitis diseases.

[0036] Beneficial effects

[0037] 1. Factors such as intestinal barrier dysfunction, innate immunity, and adaptive immunity play a key role in the pathogenesis of ulcerative colitis. At present, there is no research report that the Amaranthus hybridus L. polypeptide or a certain monomer component thereof can be used for the treatment of ulcerative colitis. The inventor of this invention proves through in-vivo experiments that the Amaranthus hybridus L. polypeptide significantly improves ulcerative colitis in mice induced by dextran sulfate sodium. The Amaranthus hybridus L. polypeptide and protein of this invention have good stability and can be used for preparing drugs or functional foods for treating ulcerative colitis.

[0038] 2. The amaranthus hybridus polypeptide in the present invention is a complex system. The experimental results prove that the whole medicine is effective, but the specific amino acid sequence of the polypeptide cannot be determined yet. Specifically, the experimental results of the present invention show that in the administration group prepared in the implementation, the HE staining pathological sections show that the degree of colon lesions in the amaranthus hybridus polypeptide administration group is significantly improved, and the expressions of ZO-1 and Occludin are significantly higher than those in the model group, indicating that the amaranthus hybridus polypeptide or protein has a certain protective effect on the process of ulcerative colitis. Since the polypeptide is the hydrolysis product of the protein, it is suggested that the amaranthus hybridus polypeptide or protein has the effect of protecting the colon tissue.

[0039] 3. The experimental materials involved in the present invention are from the original plant, which has a wide range and low cost, and has extensive practical value. Brief Description of the Drawings

[0040] Figure 1 Response surface test results of obtaining polypeptide by enzymolysis of amaranthus hybridus protein; Response surface analysis diagrams of the effects of enzymolysis pH (A) and enzymolysis temperature (B) on the extraction rate of amaranthus hybridus polypeptide; (B) Response surface analysis diagrams of the effects of enzymolysis pH (C) and enzyme dosage (D) on the extraction rate of amaranthus hybridus polypeptide; Response surface analysis diagrams of the effects of enzymolysis temperature (E) and enzyme dosage (F) on the extraction rate of amaranthus hybridus polypeptide.

[0041] Figure 2 The amaranthus hybridus polypeptide in the example reduces the changes in the morphological structure and colon length of the colon tissue of mice induced by dextran sulfate sodium. (A) Morphological changes of the colon tissue in each group. (B) Statistical results of the degree of change in the colon length of mice induced by dextran sulfate sodium reduced by amaranthus hybridus polypeptide. *p<0.05, **p<0.01, ***p<0.001.

[0042] Figure 3 The amaranthus hybridus polypeptide in the example reduces the histopathological structure changes and histopathological scores of the colon tissue of mice induced by dextran sulfate sodium. (A) Degree of inflammation of the colon tissue in each group (HE staining) (×200, scale bar = 50μm). Control blank; Modle model; AM-L amaranthus hybridus polypeptide low-dose group; AM-H amaranthus hybridus polypeptide high-dose group; Mes mesalazine. *p<0.05, **p<0.01, ***p<0.001. (B) Histopathological inflammation scores of the colon tissue in each group. Control blank; Modle model; AM-L amaranthus hybridus polypeptide low-dose group; AM-H amaranthus hybridus polypeptide high-dose group; Mes mesalazine. *p<0.05, **p<0.01, ***p<0.001.

[0043] Figure 4Effect of Amaranthus hybridus polypeptides in the examples on the expression of ZO-1 and Occludin proteins in mice with dextran sulfate sodium-induced ulcerative colitis model. (A) Representative western blot pictures of ZO-1 protein expression in colon tissues of mice in each group. Control: blank; Modle: model; AM-L: low-dose group of Amaranthus hybridus polypeptides; AM-H: high-dose group of Amaranthus hybridus polypeptides; Mes: mesalazine. *p<0.05, **p<0.01, ***p<0.001. (B) Quantitative analysis results of Occludin protein expression in colon tissues of each group. Control: blank; Modle: model; AM-L: low-dose group of Amaranthus hybridus polypeptides; AM-H: high-dose group of Amaranthus hybridus polypeptides; Mes: mesalazine. *p<0.05, **p<0.01, ***p<0.001. Detailed implementation manners

[0044] Example 1

[0045] I. Preparation of Amaranthus hybridus polypeptides

[0046] Amaranthus hybridus, a plant of the genus Amaranthus, was purchased from Zhenping, Shaanxi. Reagents such as sodium hydroxide are all of analytical purity.

[0047] The Amaranthus hybridus polypeptides were prepared by the following method: The seeds of Amaranthus hybridus were ground into powder, passed through a 60-mesh sieve, defatted with n-hexane at three times the volume of crude drug for 24 h, and dried in a fume hood. The defatted polypeptides were ultrasonically extracted 3 times with 0.06 mol / L NaOH solution at 15 times the volume of crude drug, 1.5 h each time. After combining the filtrates, the pH was adjusted to 4, and centrifuged at 4000 r / min for 15 min to collect the precipitate to obtain Amaranthus hybridus seed proteins. The proteins were dissolved in ultrapure water, protease was added and shaken well, and then placed in a water bath for enzymatic hydrolysis. The pH of enzymatic hydrolysis was adjusted to 3.25, the temperature was 35.90 °C, the substrate concentration was 24 mg / mL, the enzymatic hydrolysis time was 1 h, and the enzyme addition amount was 245.21 U / g.

[0048] After the enzymatic hydrolysis was completed, it was inactivated in a boiling water bath for 15 min, and the enzymatic hydrolysate was centrifuged (4000 r / min) for 15 min. The supernatant was collected, and trichloroacetic acid (TCA) was used to precipitate the unhydrolyzed proteins in the supernatant. Centrifuged (4000 r / min) for 15 min, and the Amaranthus hybridus polypeptides were diluted with distilled water to prepare a medicinal solution for activity research.

[0049] II. Optimization of the extraction process of Amaranthus hybridus polypeptides

[0050] 1. The method for measuring the protein content in the Amaranthus hybridus extract is as follows:

[0051] ① Preparation of standard curve: Prepare a standard solution of BSA (bovine serum albumin) with a concentration of 10 mg / mL. Take 6 clean test tubes and add 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of the BSA standard solution respectively, then add distilled water to 1.0 mL respectively. Finally, add 4 mL of biuret reagent to each test tube, mix well and let stand for 30 min. Then, perform colorimetry at a wavelength of 540 nm to measure the absorbance and record the data. Using the concentration of the BSA standard solution as the abscissa and its absorbance as the ordinate, draw the standard curve, and obtain the linear regression equation as C = 0.2325A, R 2 = 0.9995.

[0052] ② Determination of protein content in the sample: Pipette 1 mL of the sample solution, add 4 mL of biuret reagent, mix well and let stand for 30 min. Then, perform colorimetry at 540 nm to measure its absorbance value, record the data, and calculate the protein content in the sample using the standard curve.

[0053] 2. Single-factor experiment design for protein extraction from Amaranthus hybridus L.

[0054] ① Weigh 0.5 g of dry powder of Amaranthus hybridus L. into a 50 mL conical flask, add 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L NaOH solution to the conical flask according to a solid-liquid ratio of 1:10, place it in an ultrasonic cleaner, and ultrasonically extract for 1 h. Centrifuge the extract (3000 r / min) for 10 min, collect the supernatant, and determine the protein content according to the previous determination method to study the effect of sodium hydroxide concentration on the protein extraction effect.

[0055] ② Weigh 0.5 g of dry powder of Amaranthus hybridus L. into a 50 mL conical flask, add 0.05 mol / L NaOH solution to the conical flask according to a solid-liquid ratio of 1:10, place it in an ultrasonic cleaner, and ultrasonically extract for 30 min, 60 min, 90 min, 120 min, 150 min. Centrifuge the extract (3000 r / min) for 10 min, collect the supernatant, and determine the protein content according to the previous determination method to study the effect of extraction time on the protein extraction effect.

[0056] ③ Weigh 0.5 g of dry powder of Amaranthus hybridus L. into a 50 mL conical flask, add 0.05 mol / L NaOH solution to the conical flask according to solid-liquid ratios of 1:10, 1:15, 1:20, 1:25, 1:30, place it in an ultrasonic cleaner, and ultrasonically extract for 1 h. Centrifuge the extract (3000 r / min) for 10 min, collect the supernatant, and determine the protein content according to the previous determination method to study the effect of the solid-liquid ratio on the protein extraction effect.

[0057] ④ Weigh 0.5 g of dry green amaranth powder into a 50 mL conical flask, add 0.05 mol / L NaOH solution to the conical flask according to the solid-liquid ratio of 1:10, place it in an ultrasonic cleaner, extract ultrasonically for 1 h, centrifuge the extract (3000 r / min) for 10 min, collect the supernatant, extract the residue twice again under the above conditions, and measure the protein content according to the previous measurement method to study the effect of extraction times on the protein extraction effect.

[0058] 3. Orthogonal experiment design for green amaranth polypeptide:

[0059] Through single-factor experiments, study the effects of four factors on the extraction rate of ultrasonic extraction of green amaranth protein. Based on the results of single-factor experiments, design a four-factor and three-level orthogonal experiment (L93 4 ) with four factors: sodium hydroxide solution concentration, extraction time, solid-liquid ratio, and extraction times, as shown in Table 1.

[0060] Table 1 Factor-level table of orthogonal experiment for extraction rate of green amaranth protein

[0061]

[0062]

[0063] From the experimental data, it can be obtained that the optimal process parameters in terms of the protein content in the extract should be A3B2C1D3, that is, the sodium hydroxide solution concentration is 0.06 mol / L, the solid-liquid ratio is 1:15, the extraction time is 90 min, and the extraction is carried out 3 times. This combination is determined as the optimal condition combination for ultrasonic extraction of green amaranth protein, and the protein yield is 10.61%-13.03%.

[0064] III. Process optimization for preparing anti-inflammatory peptides by enzymatic hydrolysis of green amaranth protein

[0065] 1. Determination of polypeptide content in green amaranth enzymatic hydrolysate

[0066] ① Drawing of standard curve: Draw the standard curve for polypeptide content determination according to the method of drawing the standard curve for protein content determination described above.

[0067] 2. Single-factor experiment design for enzymatic hydrolysis of green amaranth protein

[0068] ① Select four proteases: alkaline protease, neutral protease, papain, and trypsin, with the enzyme addition amount of 3000 U / g for each, and the enzyme addition amount of pepsin is 300 U / g. According to the characteristics of different enzymes, set the most suitable hydrolysis conditions for each enzyme, adjust to the optimal temperature and pH value, carry out enzymatic hydrolysis of green amaranth protein, and finally screen out that the best protease for enzymatic hydrolysis of green amaranth is pepsin.

[0069] ② The amount of protease added was 100 U / g. Under the conditions of substrate protein concentrations of 16 mg / ml, 20 mg / ml, 24 mg / ml, 28 mg / ml, and 32 mg / ml, an enzymatic hydrolysis solution pH of 3.0, an enzymatic hydrolysis temperature of 37 °C, and an enzymatic hydrolysis time of 1 h, it was found that the polypeptide yield was the highest when the substrate concentration was 24 mg / mL.

[0070] ③ The enzymatic hydrolysis temperatures were set to 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C in sequence. Under the conditions of a substrate protein concentration of 16 mg / ml, an enzyme addition amount of 100 U / g, an enzymatic hydrolysis solution pH of 3.0, and an enzymatic hydrolysis time of 1 h, enzymatic hydrolysis was carried out according to the above enzymatic hydrolysis process. It was obtained that the polypeptide yield was the highest when the enzymatic hydrolysis temperature was 35 °C.

[0071] ④ The pH values of the protein solution were adjusted to 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 in sequence. Under the conditions of a substrate protein concentration of 16 mg / ml, an enzyme addition amount of 100 U / g, an enzymatic hydrolysis temperature of 37 °C, and an enzymatic hydrolysis time of 1 h, enzymatic hydrolysis was carried out according to the above enzymatic hydrolysis process. It was obtained that the polypeptide yield was the highest when the enzymatic hydrolysis pH was 3.

[0072] ⑤ The amounts of protease added were 50 U / g, 100 U / g, 150 U / g, 200 U / g, 250 U / g, and 300 U / g in sequence. Under the conditions of a substrate protein concentration of 16 mg / ml, an enzymatic hydrolysis temperature of 37 °C, an enzymatic hydrolysis solution pH of 3.0, and an enzymatic hydrolysis time of 1 h, enzymatic hydrolysis was carried out according to the above enzymatic hydrolysis process. It was obtained that the polypeptide yield was the highest when the enzyme addition amount was 250 U / g.

[0073] ⑥ The enzymatic hydrolysis times were set to 30 min, 60 min, 90 min, 120 min, and 150 min respectively. Under the conditions of a substrate protein concentration of 16 mg / ml, an enzyme addition amount of 100 U / g, an enzymatic hydrolysis pH of 3.0, and an enzymatic hydrolysis temperature of 37 °C, enzymatic hydrolysis was carried out according to the above enzymatic hydrolysis process. The research results showed that the polypeptide yield was the highest at 90 min.

[0074] 3. Orthogonal test design for the enzymatic hydrolysis of Amaranthus hybridus L. protein:

[0075] Based on the results of the single-factor experiment, with the substrate mass concentration fixed at 24 mg / mL and the enzymatic hydrolysis time at 1 h, using Design Expert 8.0.6 software, according to the Box-Behnken central composite design principle, with the enzymatic hydrolysis pH (A), enzymatic hydrolysis temperature (B), and enzyme addition amount (C) as the investigation factors and the polypeptide yield (Y) as the response value, a response surface experiment with three factors and three levels was designed for quadratic multiple regression equation fitting and its optimization analysis. The factor levels of the response surface experiment are shown in Table 1, and the response surface experiment design and results are shown in Table 2.

[0076] Table 2 Factor table of the response surface experiment

[0077]

[0078] From the results of the response surface experiment, it can be obtained that the enzymatic hydrolysis pH is 3.25, the temperature is 35.90 °C, the substrate concentration is 24 mg / mL, the enzymatic hydrolysis time is 1 h, and the enzyme dosage is 245.21 U / g. Under these conditions, the yield of amaranth green spike polypeptide is 27.67%-37.84%.

[0079] IV. Improvement of dextran sulfate sodium-induced ulcerative colitis model in mice by amaranth green spike polypeptide

[0080] The amaranth green spike polypeptide prepared in the example was selected for the following in vivo pharmacodynamic study. A mouse ulcerative colitis model was replicated by drinking an aqueous solution containing 4% dextran sulfate sodium for 7 days.

[0081] 1 Materials and Instruments Adult female C57BL / 6 mice, 6-8 weeks old, 18-20 g. Provided by Nanjing Bikai Animal Breeding Farm. Wahaha purified water. Dextran sulfate sodium (MP Biomedicals).

[0082] 2 Experimental Methods Female C57BL / 6 mice were divided into a blank group, a model group, a positive drug group, a low-dose and a high-dose amaranth green spike polypeptide group, with 20 mice in each group. Except for the blank group, which drank an aqueous solution containing 4% dextran sulfate sodium for 7 days to replicate the mouse ulcerative colitis model and then drank purified water for 1 day, the blank group drank purified water throughout the experiment.

[0083] The blank group and the model group were intragastrically administered with normal saline every day, and the positive drug group was intragastrically administered with mesalazine at a dose of 200 mg / kg / d. The amaranth green spike polypeptide prepared in the example was taken for the administration group. After prophylactic administration at a dose of 1 g / kg for one week, continuous intragastric administration was carried out for 8 days during the modeling period. After 8 days, the mice were sacrificed by orbital blood collection. After standing, centrifugation was carried out at 4 °C, 3000 r / min for 15 min, and stored at -80 °C for later use. The colon tissue was taken out, fixed in 4% formaldehyde, dehydrated step by step with alcohol, cleared with xylene, infiltrated with wax, paraffin-embedded, sectioned routinely, stained with HE, and observed for changes in colon tissue injury.

[0084] All data were expressed as mean ± SD (x±s). Processed with GraphPad Prism 8 statistical software, and one-way ANOVA was used for statistics. P<0.05 indicated that the difference was statistically significant.

[0085] The pathological tissue sections were stained with HE. The results showed that the colonic barrier structure of the mice in the model group was damaged, the intervals were widened, and a large number of inflammatory cells infiltrated, and ulcerative colitis was formed, which basically conformed to the characteristics of ulcerative colitis, indicating that the ulcerative colitis model of experimental mice was successfully prepared. After treatment with the prepared amaranth paniculatus polypeptide, the colonic tissue structure of the mice was found to be intact and clear, and the degree of inflammatory cell infiltration was relatively light. After treatment with the positive drug mesalazine, the colonic barrier structure of the mice in the positive control group was relatively clear, with more inflammatory cell infiltration, and the degree of lesion was reduced compared with the model group. Compared with the model group, the degree of inflammation in each administration group and the positive drug group was reduced, indicating that amaranth paniculatus polypeptide can improve the lesions of ulcerative colitis mice.

[0086] 3. Effects of amaranth paniculatus polypeptide on ZO-1 and Occludin in the colonic tissue of model mice.

[0087] Using Western Blot technology, the protein expressions of ZO-1 and Occludin in the colonic tissue of mice in each group were detected at the end of the experiment. Compared with the control group, the protein expressions of ZO-1 and Occludin in the model group were significantly decreased. Compared with the model group, after intragastric administration of the prepared amaranth paniculatus polypeptide, the protein expression levels of ZO-1 and Occludin in the colonic tissue of model mice were significantly increased (P<0.05). The research results suggest that the prepared amaranth paniculatus polypeptide can increase the protein expression levels of ZO-1 and Occludin to varying degrees, and can better intervene in the degree of ulcerative colitis induced by dextran sulfate sodium in mice.

[0088] V. Discussion

[0089] The results of this experiment showed that in the polypeptide administration group prepared in this study, the colonic length of the mice in the administration group increased significantly. The pathological sections and Western Blot results showed that the degree of ulcerative colitis in the administration group was significantly improved, indicating that amaranth paniculatus polypeptide has a certain protective effect on the process of ulcerative colitis, suggesting that amaranth paniculatus polypeptide has the effect of protecting colonic tissue, and confirming the drug use of amaranth paniculatus polypeptide in improving ulcerative colitis.

Claims

1. Use of amaranth viridis polypeptide in the preparation of a drug for improving ulcerative colitis, characterized in that, The described Amaranthus hybridus L. polypeptide is prepared by the following steps: The Amaranthus hybridus L. seeds are ground into powder, passed through a 60-mesh sieve, defatted with n-hexane three times the volume of the crude drug for 24 h, and dried in a fume hood; The defatted polypeptide is ultrasonically extracted three times with 0.06 mol / L NaOH solution 15 times the volume of the crude drug, each time for 1.5 h. After combining the filtrates, the pH is adjusted to 4, and centrifuged at 4000 r / min for 15 min. The precipitate is collected to obtain Amaranthus hybridus L. seed protein. The protein is dissolved in ultrapure water, and pepsin is added for enzymatic hydrolysis to obtain Amaranthus hybridus L. polypeptide.

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