Carthamus tinctorius seed meal protein and peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to safflower seed meal protein and peptide as well as a preparation method and application thereof. The safflower seed meal protein and peptide provided by the invention are prepared through the steps of degreasing, alkali extraction, ultrasonic-assisted extraction, acid precipitation, ammonium sulfate precipitation, enzymolysis and the like, and have good antioxidant activity. Animal experiments show that the protein and the peptide can effectively relieve DSS-induced mouse colitis, remarkably improve hematochezia, diarrhea, colon shortening and other symptoms, regulate immune organ indexes and T lymphocyte subgroups and enhance the oxidation resistance of intestinal tracts, and do not show toxicity under high dosage. The traditional Chinese medicine composition can be effectively used for preparing a medicine for treating colitis, and a natural and safe new candidate medicine is provided for colitis treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to safflower seed meal protein, peptides, their preparation methods and applications. Background Technology

[0002] With increasing health awareness, the concept of "prevention is better than cure" is becoming more widespread. Natural health products with both immune-regulating and anti-inflammatory / soothing functions have become a core focus in the consumer market and research and development field. Modern populations are increasingly experiencing gut microbiota imbalance and chronic low-grade inflammation due to factors such as irregular eating habits, excessive stress, and environmental pollution. Long-term gut inflammation can easily lead to digestive disorders, while decreased immune function weakens the body's resistance, increasing the risk of infection and disease. However, current immune-regulating health products on the market mostly rely on traditional ingredients such as probiotics and vitamins, while anti-inflammatory products are often limited to single soothing effects. Furthermore, some products have issues such as unclear ingredient sources and unstable activity, failing to meet consumers' demands for "natural sources, multi-effect synergy, and safety and gentleness." Developing highly active, multifunctional health products based on plant by-products has become a breakthrough direction for the industry.

[0003] Safflower seed meal, a major byproduct of safflower oil processing, has long been used as animal feed or disposed of as waste, resulting in resource waste and missing its core value—the high-quality plant protein it contains. Existing research has confirmed that plant-derived proteins can enhance immunity by activating immune cells and regulating immune organ function, and can also alleviate local chronic inflammation by inhibiting the release of inflammatory factors. However, research on safflower seed meal protein has significant shortcomings: First, its application potential in gut microbiota-friendly health products has not been explored, especially regarding whether it can improve the intestinal immune barrier function by regulating intestinal immunity (such as enhancing macrophage phagocytic activity and balancing T lymphocyte subsets), which lacks targeted research. Second, whether its anti-inflammatory activity can precisely target chronic low-grade intestinal inflammation and alleviate discomfort symptoms such as bloating and intestinal sensitivity lacks clear data support. More importantly, whether safflower seed meal protein can achieve a synergistic effect of "immunomodulation-anti-inflammatory relief," meeting the complex needs of modern people for gut health, remains a research gap in the industry. Therefore, providing a protein with immunomodulatory and anti-inflammatory functions has become a pressing technical problem to be solved in this field.

[0004] There are some key shortcomings in the development and utilization of safflower seed meal protein, mainly concerning its core components. Currently produced safflower seed meal protein is rich in phenolic substances, which can form irreversible complexes with proteins, severely reducing their activity and function. This is a core obstacle to its application. The presence of phenolic substances also interferes with its functional performance. Summary of the Invention

[0005] The purpose of this invention is to provide safflower seed meal protein, peptides, their preparation methods, and applications.

[0006] This invention provides a method for preparing safflower seed meal protein, comprising the following steps: S1. Safflower seed meal powder and petroleum ether are mixed at a mass-volume ratio of 0.5~1.5g:2.5~3.5mL, and stirred for 4~6min to degrease until the petroleum ether becomes transparent, to obtain degreased safflower seed meal powder. S2. Mix the defatted safflower seed meal powder with water at a mass-volume ratio of 0.5~1.5g:45~55mL, adjust the pH to 12~14, and ultrasonically extract at 45~55℃ and 750~850W for 0.5~1.5h. After filtering the extract, adjust the pH of the filtrate to 2~4, centrifuge to collect the first precipitate, add ammonium sulfate to the supernatant obtained by centrifugation until the ammonium sulfate saturation is 85%~95%, and centrifuge to obtain the second precipitate. S3. Combine the first and second precipitates and resuspend them to obtain a resuspended solution. Adjust the pH of the resuspended solution to 6-8, desalt it by dialysis, concentrate and freeze dry it after dialysis to obtain safflower seed meal protein. The molecular weight cutoff for the dialysis is 900-1100 Da.

[0007] This invention extracts protein from safflower seed meal with low phenol content using an ultrasound-assisted alkaline extraction and acid precipitation process, and then prepares protein peptides through alkaline protease hydrolysis. The invention systematically verifies the immunomodulatory activity (enhancing the thymus / spleen immune organ index and regulating the function of intestinal immune cells) and anti-inflammatory and soothing effects (reducing the level of intestinal inflammatory factors and relieving mild inflammation of the intestinal mucosa). At the same time, safety experiments (high-dose gavage toxicity test) ensure that it meets safety standards.

[0008] The present invention also provides safflower seed meal protein prepared by the aforementioned method.

[0009] This invention also provides safflower seed meal protein peptide SPH-A, which is prepared by the following method: safflower seed meal protein is resuspended to form a safflower seed meal protein solution with a mass fraction of 1% to 3%, the pH value is adjusted to 10 to 11 to denature the protein in the protein solution, then 11 to 13 KU / g alkaline protease is added, and enzymatic hydrolysis is performed under ultrasonic assistance for 5 to 7 hours. After enzyme inactivation, safflower seed meal protein peptide SPH-A is obtained.

[0010] Furthermore, the ultrasonic conditions are 250~350W and 40~50℃.

[0011] The present invention also provides the application of the safflower seed meal protein in the preparation of a drug for treating colitis.

[0012] The present invention also provides the application of the safflower seed meal protein peptide SPH-A in the preparation of a drug for treating colitis.

[0013] The present invention also provides the application of the safflower seed meal protein in the preparation of health products that enhance immunity.

[0014] The present invention also provides the application of the safflower seed meal protein peptide SPH-A in the preparation of health products that enhance immunity.

[0015] The present invention has the following beneficial effects: This protein peptide exhibits excellent immunomodulatory and anti-inflammatory soothing activities: it can increase the thymus / spleen index in immunosuppressed mice, regulate the ratio of CD3⁺CD4⁺ T cells and the CD4⁺ / CD8⁺ ratio, and enhance macrophage activity; it can also reduce pro-inflammatory factors such as intestinal TNF-α and IL-6, and alleviate mucosal inflammatory infiltration. At a high dose of 400 mg / kg, it has no effect on liver and kidney function in mice, demonstrating good safety.

[0016] This invention enables the high-value utilization of safflower seed meal, and the phenol content of the safflower seed meal protein provided is significantly reduced, resulting in better protein functional activity. The protein peptides obtained from the enzymatic hydrolysis can be used as core functional ingredients in health products. They can be combined with water or physiological saline and other diluents to make oral liquid preparations, providing new ingredients for natural and safe immune-regulating and anti-inflammatory synergistic health products, and also providing a pathway for the industrialization of plant by-products. Attached Figure Description

[0017] Figure 1 Degree of hydrolysis of protein in enzymatically hydrolyzed safflower seed meal.

[0018] Figure 2 Fourier transform infrared spectrum of safflower seed meal protein hydrolysate.

[0019] Figure 3 The graph shows the antioxidant capacity of safflower seed meal protein hydrolysate. In the graph, A is the ABTS free radical scavenging capacity, B is the DPPH free radical scavenging capacity, and C is the reducing capacity.

[0020] Figure 4 The images show the in vitro immunomodulatory activity of safflower seed meal protein hydrolysate. In the images, A represents cell viability, B represents nitric oxide content, C represents phagocytic capacity, D represents IL-1β level, E represents TNF-α level, and F represents IL-6 level.

[0021] Figure 5 The graphs show the effects of different conditions on the degree of hydrolysis of SPH-A. In the graphs, A represents the effect of different ultrasonic powers on the degree of hydrolysis of SPH-A, B represents the effect of different enzyme amounts on the degree of hydrolysis of SPH-A, and C represents the effect of different treatment times on the degree of hydrolysis of SPH-A.

[0022] Figure 6 The response surface and contour plots are shown, where A is the contour plot, B is the response surface plot of ultrasonic time and enzyme addition amount, C is the response surface plot of ultrasonic power and enzyme addition amount, and D is the response surface plot of ultrasonic power and ultrasonic time.

[0023] Figure 7 The graphs show the effects of SP and SPH-A on renal and hepatic function indicators in mice. In the graphs, A represents creatinine level, B represents blood urea nitrogen level, C represents alanine aminotransferase level, and D represents aspartate aminotransferase level.

[0024] Figure 8 This is the experimental design diagram for DSS-induced ulcerative colitis in mice.

[0025] Figure 9 The graphs show the effects of SP and SPH-A on the rate of change in body weight, DAI, organ index, and colon length in mice with ulcerative colitis. In the graphs, A is the statistical graph of the rate of change in body weight, B is the statistical graph of DAI, C is the graph of the thymus organ index, D is the graph of the spleen organ index, and E is the graph of colon length.

[0026] Figure 10 Figure showing the effects of SP and SPH-A on the histopathology of mouse colon tissue.

[0027] Figure 11 The graph shows the effects of SP and SPH-A on antioxidant intestinal tissue in mice. In the graph, A is the statistical graph of CAT activity and B is the statistical graph of MDA level.

[0028] Figure 12 This is a design diagram for an experiment on CTX-induced immunosuppressed mice.

[0029] Figure 13 The graphs show the effects of SP and SPH-A on the rate of change in body weight and organ indices in immunosuppressed mice. In the graphs, A is the statistical graph of the rate of change in body weight, B is the graph of the thymus organ index, and C is the graph of the spleen organ index.

[0030] Figure 14 The graph shows the effects of SP and SPH-A on T cell subsets in mouse spleen. In the graph, A is a flow cytometry sorting statistical graph of CD3⁺CD4⁺ T cells, B is a flow cytometry sorting statistical graph of CD3⁺CD8⁺ T cells, and C is a flow cytometry sorting statistical graph of CD4⁺CD8⁺ T cells.

[0031] Figure 15 Figure showing the effects of SP and SPH-A on the histopathology of mouse colon tissue.

[0032] Figure 16 Figure showing the effects of SP and SPH-A on the histopathology of mouse spleen tissue.

[0033] Figure 17The graph shows the effects of SP and SPH-A on antioxidant intestinal tissue in mice. In the graph, A is the statistical graph of CAT activity in small intestinal tissue, B is the statistical graph of SOD activity in small intestinal tissue, and D is the statistical graph of MDA content in small intestinal tissue.

[0034] Figure 18 The graph shows the effects of SP and SPH-A on cytokines in mouse intestinal tissue. In the graph, A is the level of TNF-α cytokine in small intestinal tissue, B is the level of IL-6 cytokine in small intestinal tissue, and C is the level of IL-10 cytokine in small intestinal tissue. Detailed Implementation

[0035] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0036] Example 1: Extraction and determination of protein (SP) from safflower seed meal.

[0037] I. Protein extraction.

[0038] The fat in safflower seed meal was removed using petroleum ether. Safflower seed meal powder was treated with petroleum ether at a ratio of 1g:3mL for three rounds, each round lasting 5 minutes, with continuous stirring until the petroleum ether became transparent. The solid phase obtained after solid-liquid separation was the degreased safflower seed meal powder.

[0039] Protein was extracted from safflower seed meal using an ultrasound-assisted alkaline extraction and acid precipitation method. The steps were as follows: Degreased safflower seed meal powder was mixed with ultrapure water at a ratio of 1g:50mL. The pH of the solution was adjusted to 13.0 with 0.1M NaOH, and ultrasonic extraction was performed at 50℃ and 800W for 1 hour. After filtration, the pH of the solution was adjusted to 3.0 with 0.1M HCl, and the mixture was allowed to stand overnight before centrifugation to collect the precipitate. Ammonium sulfate powder was added to the supernatant to achieve an ammonium sulfate saturation of 90%, and the mixture was allowed to stand overnight before centrifugation to collect the precipitate. The two precipitates were mixed to obtain safflower seed meal protein, abbreviated as SP. The safflower seed meal protein was resuspended in ultrapure water, and the pH of the resuspended solution was adjusted to 7.0. Then, it was transferred to a dialysis bag with a molecular weight cutoff of 1000Da for dialysis and desalting. After dialysis, the solution was concentrated and finally freeze-dried to obtain lyophilized safflower seed meal protein powder.

[0040] II. Determination of the physicochemical properties of safflower seed meal protein.

[0041] The physicochemical properties of safflower seed meal protein were determined. The contents of protein, phenols, polysaccharides, and flavonoids were determined using the Coomassie brilliant blue method, gallic acid method, phenol-sulfuric acid method, and quercetin-AlCl3 method, respectively. The results are shown in Table 1.

[0042] Table 1: Chemical composition of safflower seed meal protein As shown in Table 1, the protein content of SP is as high as 62.97±0.74%.

[0043] III. Preparation of safflower seed meal protein peptides.

[0044] First, a 2% (w / w) safflower seed meal protein solution was prepared. After adjusting the pH to 7.0, the solution was incubated in a 95°C water bath for 20 minutes to ensure complete protein denaturation. Following this, the following experiments were conducted:

[0045] (1) Add 4000 U / g of alkaline protease to the completely denatured safflower seed meal protein solution, and enzymatically hydrolyze for 4 h at 45℃, pH 10.5 and 100W ultrasound-assisted conditions. After enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 20 min to obtain safflower seed meal hydrolysate SPH-A.

[0046] (2) Add 4000 U / g neutral protease to the completely denatured safflower seed meal protein solution, and enzymatically hydrolyze for 4 h at 40℃, pH 7.0 and 100W ultrasound-assisted conditions. After enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 20 min to obtain safflower seed meal hydrolysate SPH-N.

[0047] (3) Add 4000 U / g flavor enzyme to the completely denatured safflower seed meal protein solution, and enzymatically hydrolyze for 4 h at 55℃, pH 7.0 and 100W ultrasound-assisted conditions. After enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 20 min to obtain safflower seed meal hydrolysate SPH-F.

[0048] (4) Add 4000 U / g bromelain to the completely denatured safflower seed meal protein solution, and enzymatically hydrolyze for 4 h at 55℃, pH 7.0 and 100W ultrasound-assisted conditions. After enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 20 min to obtain safflower seed meal hydrolysate SPH-B.

[0049] IV. Determination of degree of hydrolysis.

[0050] The safflower seed meal enzymatic hydrolysate was centrifuged at 8000 rpm / s for 10 min at 4℃. The protein content of the supernatant was determined using the BCA method and recorded as C. The degree of hydrolysis of the enzymatic hydrolysate was determined using the o-phthalaldehyde method. 20 μL of the supernatant was mixed with 150 μL of OPA reagent and reacted precisely for 2 min. The absorbance at 340 nm was measured and recorded as ODs. Simultaneously, 0.9516 meqv / L standard serine and ultrapure water were used as standard and blank controls, respectively, and recorded as ODs. st and OD bThe degree of hydrolysis DH is calculated using the following formula:

[0051] ; Serine NH2 The calculation formula is: ; In the formula, DH represents the degree of hydrolysis, expressed as % . Equivalent to the number of peptide bonds produced by hydrolysis, in meqv / L; α and β are 0.970 and 0.342, respectively; OD s The absorbance at 340 nm is the result of reacting 20 μL of supernatant with 150 μL of OPA reagent; OD b The absorbance value of the blank control at 340 nm; OD st C represents the absorbance of standard serine at 340 nm; C is the protein concentration of the enzymatic digest supernatant, in mg / mL; h hot The total number of peptide bonds in the protein, h is the protein content of safflower seed meal. hot It is 8.

[0052] The results are as follows Figure 1 As shown, the degrees of hydrolysis of SPH-A, SPH-N, SPH-F, and SPH-B were determined to be 39.48±0.12%, 37.93±0.13%, 32.44±0.08%, and 51.72±1.31%, respectively. The degree of hydrolysis of SPH-B was significantly higher than that of the other three proteases, followed by SPH-A.

[0053] V. Infrared spectroscopy detection of safflower seed meal protein peptides.

[0054] SP and potassium bromide powders were compressed into tablets. Then, the tablets were analyzed using a Nexus 470 FT-IR spectrometer at 4000 cm⁻¹. -1 Up to 400cm -1 FT-IR spectra were tested within the specified range. Results are as follows: Figure 2 As shown, SP contains abundant NH and amide bonds. The peaks of SPH-A, SPH-N, and SPH-F in the Amine A and Amide I regions are significantly weakened, indicating that the number of NH and C=O groups decreases after enzymatic cleavage of peptide bonds. The peak of SPH-B almost disappears, indicating that its enzymatic hydrolysis is the most profound, the protein is hydrolyzed more thoroughly, and the number of NH and C=O groups is greatly reduced, which corresponds to the highest degree of hydrolysis in SPH-B.

[0055] VI. Determination of antioxidant activity.

[0056] The ABTS radical scavenging capacity, DPPH radical scavenging capacity, and reducing capacity of safflower seed meal protein peptides were determined. Ascorbic acid (Vc) was used as a positive control. At a concentration of 5 mg / mL, SPH-A exhibited the highest ABTS radical scavenging rate, reaching 91.44 ± 0.70%, significantly higher than other SPHs (P < 0.05). Figure 3 As shown in A. Regarding reducing power, as... Figure 3 As shown in Figure C, at a concentration of 5 mg / mL, the reducing abilities of SPH-A, SPH-N, SPH-F, and SPH-B were 0.63±0.006, 0.43±0.02, 0.36±0.01, and 0.60±0.04, respectively. With increasing concentration, the DPPH scavenging activity gradually increased, as shown in Figure C. Figure 3 As shown in Figure B, at a concentration of 5 mg / mL, the DPPH scavenging abilities of SPH-A, SPH-N, SPH-F, and SPH-B were 67.40±0.57%, 50.77±3.48%, 51.53±1.72%, and 61.57±1.20%, respectively. SPH-A showed significantly higher efficacy than the other SPHs (P<0.05). Combining the three antioxidant abilities, SPH-A exhibited the best relative antioxidant capacity. In this study, the SPHs obtained by the ultrasound-assisted alkaline extraction and acid precipitation method all demonstrated multiple antioxidant properties, including free radical scavenging activity and reducing capacity.

[0057] VII. Determination of immunomodulatory activity.

[0058] The effect of SPHs on phagocytic activity was assessed using neutral red staining. Furthermore, the levels of NO, IL-6, IL-1β, and TNF-α in the cell supernatant were measured strictly according to the manufacturer's instructions. The proliferative effect of SPHs on RAW264.7 cells was evaluated using the CCK-8 assay. Figure 4 As shown in Figure A. The results indicated that SPSs significantly enhanced the proliferation of RAW264.7 cells, with no cytotoxicity observed at a concentration of 100 μg / mL. Nitric oxide (NO) is an important effector molecule of macrophages and a key indicator of macrophage activation. (As shown in Figure A.) Figure 4 As shown in Figure B, NO production in the SPHs group was significantly higher than that in the control group, but its activity was still significantly lower than that in the LPS group (P<0.05). Macrophages are important immune cells involved in phagocytosis, antigen presentation, and the synthesis and secretion of signaling molecules; their phagocytic activity assay is a fundamental method for evaluating the body's immune function. Figure 4As shown in Figure C, the phagocytic proliferative capacity of SPHs was significantly higher than that of the control group (P<0.05). Furthermore, the phagocytic proliferative activity of SPH-A (138.62±0.20%) was significantly higher than that of the other three SPHs and the LPS group (P<0.05). Therefore, these results indicate that SPHs significantly promote macrophage proliferation, NO release, and phagocytosis, with SPH-A potentially serving as a novel immunomodulator with significant potential in regulating the immune process.

[0059] Cytokine levels were measured using an ELISA kit, such as... Figure 4 As shown, the cytokines included IL-1β, TNF-α, and IL-6. Compared with the control group, SPHs significantly affected the release of IL-6, IL-1β, and TNF-α (P<0.05). These experimental results indicate that SPHs can promote the release of TNF-α, IL-6, and IL-1β cytokines, activating macrophages to exert their immune activity.

[0060] 8. Optimization of SPH-A enzymatic hydrolysis process.

[0061] SPH-A exhibits superior antioxidant and in vitro immunomodulatory activity compared to the other three SPHs, therefore, the enzymatic hydrolysis process of SPH-A was optimized. Single-factor experiments and response surface methodology (RSM) were used to optimize the enzymatic hydrolysis conditions of SPH-A. In the single-factor experiments, three factors were selected: ultrasonic power (100W, 200W, 300W, and 400W), enzyme dosage (4KU / g, 8KU / g, 12KU / g, and 16KU / g), and ultrasonic time (2h, 4h, 6h, and 8h). Figure 5 As shown. Optimization employed a Box-Behnken design, with each variable coded into three levels (-1, 0, +1). RSM design and regression analysis were performed using Design-Expert 8.0.6. Optimal extraction conditions were obtained using modeling calculations, such as... Figure 6 As shown, the results were verified through three repeated experiments under the same conditions. The actual conditions for RSM verification were: ultrasonic power 300W, enzyme dosage 12KU / g, ultrasonic time 6h, and the optimal DH was 62.61±0.91%, which was close to the predicted value (63.722%) (P<0.05).

[0062] Example 2: Safety evaluation of SP and SPH-A.

[0063] Mice were randomly divided into a blank group, an SP group (400 mg / kg), and an SPH-A group (400 mg / kg). They were administered the mice by gavage for 14 consecutive days, and their general condition, growth, and organ toxicity were observed.

[0064] Mice were randomly divided into three groups (n=8): a control group (NC), an SP group, and an SPH-A group. The SP group received SP via gavage at a dose of 400 mg / kg, the SPH-A group received SPH-A via gavage at a dose of 400 mg / kg, and the control group received an equal volume of 0.9% saline. Gavage was administered for 14 consecutive days. Mice were fasted for 12 hours after gavage on the last day and then sacrificed by weighing. Results are as follows: Figure 7 As shown, there were no significant differences in liver and kidney function between the SP and SPH-A groups and the NC group, indicating that SP and SPH-A have no obvious toxicity to mice at a concentration of 400 mg / kg and have good safety.

[0065] Example 3: Preliminary evaluation of SP and SPH-A in alleviating DSS-induced colitis in mice.

[0066] I. Immune regulation detection.

[0067] The immunomodulatory effects of SP were investigated using a DSS-induced immunosuppressive mouse model. Animal experimental design: as follows... Figure 8 As shown, mice were randomly divided into 7 groups (n=8). These groups included a blank control group (NC), a model group (MC), a positive control group (5-ASA), a low-dose SP group (SPL), a high-dose SP group (SPH), a low-dose SPH-A group (SPHAL), and a high-dose SPH-A group (SPHAH). Except for the blank control group, all other groups received free access to DSS solution (3% v / v) in drinking water for 7 consecutive days. After modeling, the 5-ASA group was administered 400 mg / kg of methalazine, the SPL group was administered 200 mg / kg of SP, the SPH group was administered 400 mg / kg of SP, the SPHAL group was administered 200 mg / kg of SPH-A, and the SPHAH group was administered 400 mg / kg of SPH-A, all administered by gavage for 7 consecutive days.

[0068] Mice were fasted for 12 hours after gavage on the last day, then weighed and sacrificed, and the rate of weight change was calculated. The spleen and thymus of the mice were collected and weighed, and the spleen and thymus indices were calculated. Results are as follows: Figure 9 As shown in Figure A, compared with the NC group, the rate of weight change in the MC group was significantly lower (P<0.05). Compared with the MC group, the 5-ASA group, different doses of SP, and different doses of SPH-A intervention all significantly reversed the decrease in weight change rate caused by DSS (P<0.05), indicating that SP and SPH-A can improve the weight loss caused by DSS. The change of DAI score over time is shown in Figure A. Figure 9 On day 7, the DAI scores of the SP group and the SPH-A group with different doses were lower than those of the MC group, effectively alleviating the symptoms of bloody stools and diarrhea in mice with colitis.

[0069] As a major immune organ, the thymus undergoes changes in size and quality when the immune system is compromised. For example... Figure 9 As shown in Figure C, compared with the NC group, the thymus index in the MC group was significantly decreased (P<0.05), indicating that DSS causes severe damage to the immune organs of mice. After intervention with different doses of SP and different doses of SPH-A, the thymus index significantly increased (P<0.05). Among them, the high-dose SP group was more effective in regulating immune organ function, while the low-dose SPH-A group showed better results.

[0070] Ulcerative colitis increases spleen weight in mice. The main reason is likely the activation of the immune system and a systemic inflammatory response, a compensatory response of the body to inflammation and the immune system. Therefore, changes in spleen weight can serve as an important indicator for assessing the degree of inflammation. Figure 9 As shown in Figure D, the spleen index was significantly higher in the MC group compared with the NC group (P<0.05). After SP and SPH-A intervention, the spleen index decreased, and the difference from the MC group was statistically significant (P<0.05).

[0071] The pathogenesis of colitis in mice involves colonic wall thickening, fibrotic deposition, and tissue destruction, ultimately resulting in colonic shortening. These structural changes provide a direct correlation with the intensity of inflammation. Figure 9 As shown in Figure E, the colon length in the NC group was 10.07 ± 0.75 cm. After DSS induction, the colon length in the MC group was significantly shorter than that in the NC group (P < 0.05), with a mean length of 6.05 ± 0.17 cm. After positive drug intervention, the colon length significantly increased (P < 0.05), with a mean length of 7.42 ± 0.33 cm. Colon length also significantly increased after SP and SPH-A intake (P < 0.05), indicating that both SP and SPH-A can inhibit colonic shortening symptoms in ulcerative colitis mice.

[0072] To further confirm the protective effects of SP and SPH-A on the mouse intestine, pathological studies were conducted on the colon tissue of mice treated with SP and SPH-A. Figure 10 As shown, the colonic lamina propria glands in the NC group were neatly distributed with no inflammatory cell infiltration. The colonic crypt depth and goblet cell count were increased in the SP and SPH-A groups. These results indicate that both SP and SPH-A can alleviate colitis induced by DSS.

[0073] II. Effects of SP and SPH-A on antioxidant indices in mouse colon tissue.

[0074] Colonic tissue oxidative stress markers CAT and MDA levels. For example... Figure 11As shown, compared with the NC group, the MC group had significantly lower CAT levels and significantly higher MDA levels (P<0.05), indicating that DSS-induced colitis leads to increased oxidative stress levels. Compared with the MC group, SP and SPH-A interventions reduced MDA levels and increased CAT levels (P<0.05), indicating that both SP and SPH-A can alleviate DSS-induced oxidative damage to colonic tissue, and SPH-A has a stronger ability to alleviate colonic tissue oxidative damage than SP. In particular, the low-dose SPH-A group can more effectively reduce MDA levels and increase CAT levels, thereby better alleviating damage.

[0075] In summary, SP and its SPH-A exerted a good therapeutic effect on DSS-induced colitis in mice through multiple pathways, including protecting immune organs, alleviating colonic inflammation and structural damage, and inhibiting oxidative stress. Among them, SPH-A, especially at low doses, exhibited superior antioxidant effects and thymprotective activity compared to SP, demonstrating potential application and development value.

[0076] Example 4: Immunomodulatory effects of SP and SPH-A on cyclophosphamide-mediated immunosuppressed mice.

[0077] I. Experimental setup.

[0078] The immunomodulatory effects of SP were investigated using a cyclophosphamide (CTX)-induced immunosuppressed mouse model. Animal experimental design: as follows... Figure 12 As shown, mice were randomly divided into 5 groups (n=8): blank control group (NC), model group (MC), positive control group (LH), low-dose SP group (SPL), and high-dose SP group (SPH). Except for the blank control group, all other groups were injected with 80 mg / kg CTX for 3 consecutive days. The blank control group was given the same amount of 0.9% saline. After modeling, the LH group was given levamisole 40 mg / kg, the SPL group was given SP 200 mg / kg, the SPH group was given SP 400 mg / kg, the SPHAL group was given SPH-A 200 mg / kg, and the SPHAH group was given SPH-A 400 mg / kg. The blank control group and the model group were given the same amount of 0.9% saline. All groups were administered by gavage for 14 consecutive days.

[0079] Mice were fasted for 12 hours after gavage on the last day, then weighed and sacrificed, and the rate of weight change was calculated. The thymus glands of the mice were collected and weighed, and the thymus index was calculated. Results are as follows: Figure 13As shown, compared with the NC group, the weight change rate of mice in the MC group was significantly reduced (P<0.05). Compared with the MC group, the LH group and different doses of SP and different doses of SPH-A interventions all significantly reversed the decrease in weight change rate induced by CTX (P<0.05), indicating that SP and SPH-A can improve the weight loss induced by CTX. The spleen and thymus, as major immune organs, change in size and mass when the immune system is impaired. Figure 8 As shown, compared with the NC group, the spleen and thymus indices in the MC group decreased significantly after CTX injection, indicating that CTX causes severe damage to the immune organs of mice. The LH group and different doses of SP and SPH-A showed a significant increasing trend in the spleen index of mice (P<0.05). These results indicate that both SP and SPH-A can improve the immune organ damage induced by CTX, and that low doses of SPH-A are more effective in regulating immune organ function.

[0080] II. The proportion of T lymphocyte subsets in the spleen of mice.

[0081] Flow cytometry was used to analyze T lymphocyte subsets in mouse spleen. Antibodies used for spleen cell staining were PE Anti-Mouse CD8a (53-6.7), APC Anti-Mouse CD3e (145-2C11), and PerCP-Cyanine 5.5 Anti-Mouse CD4. Results are as follows: Figure 14 As shown, under conditions of immune system damage, CTX-induced reduction in the proportion of CD3⁺CD4⁺ cells and the CD4⁺ / CD8⁺ T cell ratio in the spleen of mice significantly decreased (P<0.05), while the proportion of CD3⁺CD8⁺ cells significantly increased (P<0.05). However, compared with the MC group, different doses of SP and SPH-A significantly upregulated the proportion of CD3⁺CD4⁺ T cells and the CD4⁺ / CD8⁺ T cell ratio (P<0.05), while significantly decreasing the proportion of CD3⁺CD8⁺ cells (P<0.05). These results indicate that SPH-A intervention is more effective than SP, especially the low-dose SPH-A group, which has a more significant effect on reversing the reduction of T lymphocytes induced by CTX in mice.

[0082] III. Effects of SP on the tissue structure of the colon and spleen in mice.

[0083] Figure 15It can be seen that the MC group showed damaged epithelial cells, a significant reduction in goblet cells, and marked crypt loss in the colonic tissue, accompanied by inflammatory cell infiltration of the lamina propria. In contrast, the NC group showed intact colonic mucosal structure, with no epithelial cell shedding or inflammatory cell infiltration. After intervention with different doses of SP and SPH-A, crypt depth increased, epithelial cell recovery was significant, but some inflammatory cell infiltration still existed. Figure 16 It can be seen that the splenic tissue in the NC group has a clearly regular border, and the white pulp is deeply stained, indicating a higher number of lymphocytes. In the MC group, there is no clear boundary between the white pulp and red pulp, the border is almost invisible, and the tissue is severely deformed. In the SP group, the boundary between the red pulp and white pulp gradually becomes clearer, the white pulp area increases, and the number of lymphocytes increases. In the SPH-A group, the boundary between the red pulp and white pulp gradually becomes clearer, and the white pulp area increases. These results indicate that both SP and SPH-A have a protective effect against CTX-induced intestinal and spleen damage.

[0084] IV. Effects of SP and SPH-A on antioxidant indices in mouse intestinal tissue.

[0085] Oxidative damage is caused by the excessive production of free radicals and an imbalance in antioxidant mechanisms, and is related to immune system dysfunction. CTX reduces the antioxidant capacity of the gut and increases the abundance of free radicals, thereby leading to immune dysfunction. Figure 17 As shown, compared with the NC group, the MDA content in the small intestine of the MC group was significantly increased (P<0.05), and the CAT and SOD activities in the LH group were significantly higher than those in the MC group (P>0.05). After SP intervention, both CAT and SOD activities increased in a dose-dependent manner. After SPH-A intervention, both CAT and SOD activities increased, with the low-dose group showing better activity, even exceeding that of the LH group. SP and SPH-A intervention reduced MDA content to a level not significantly different from the NC group (P>0.05). In conclusion, SP and SPH-A can enhance the activity of CAT and SOD and reduce MDA content in immunosuppressed mice. Compared with SP, SPH-A has a more significant effect on increasing CAT and SOD activity and reducing MDA content. This indicates that SP and SPH-A can alleviate CTX-induced oxidative damage in the small intestine and exert immunomodulatory effects.

[0086] V. Effects of SP and SPH-A on cytokines in mouse intestinal tissue.

[0087] Cytokines, as intercellular signaling molecules, regulate cell differentiation, maturation, and activation, directly or indirectly affecting the body's immune function. Compared to the NC group, the levels of cytokines (TNF-α, IL-6, and IL-10) in the small intestine tissue of the MC group were significantly reduced. Compared to MC, the secretion of TNF-α, IL-6, and IL-10 increased in a dose-dependent manner after SP treatment. Figure 18As shown, there was no significant difference between the SP and LH groups (P<0.05). After SPH-A treatment, the secretion of TNF-α, IL-6, and IL-10 all increased, with the low-dose group showing better results, even exceeding those of the LH group. In conclusion, SP and SPH-A effectively alleviated CTX-induced intestinal immune dysfunction by increasing the secretion of intestinal cytokines, and the regulatory effect of SPH-A was superior to that of SP.

[0088] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0089] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0090] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing safflower meal protein, characterized by, Comprising the following steps: S1, defat the safflower meal powder by mixing with petroleum ether at a mass-volume ratio of 0.5-1.5 g:2.5-3.5 mL, stirring for 4-6 min, until the petroleum ether becomes transparent, to obtain defatted safflower meal powder; S2, mix the defatted safflower meal powder with water at a mass-volume ratio of 0.5-1.5 g:45-55 mL, adjust the pH to 12-14, and ultrasonically extract at 45-55℃, 750-850 W for 0.5-1.5 h, filter the extract, adjust the pH of the filtrate to 2-4, centrifuge to collect the first precipitate, add ammonium sulfate to the supernatant obtained by centrifugation to a saturation degree of 85%-95%, and centrifuge to obtain the second precipitate; S3, combine the first and second precipitates and resuspend to obtain a resuspension, adjust the pH of the resuspension to 6-8, dialyze to remove salt, concentrate and freeze-dry after dialysis to obtain safflower meal protein; The dialysis has a molecular weight cutoff of 900-1100 Da.

2. The safflower meal protein prepared by the preparation method of claim 1.

3. Safflower meal protein peptide SPH-A, characterized in that, The preparation method of the safflower meal protein peptide SPH-A is as follows: resuspend the safflower meal protein of claim 1 to obtain a safflower meal protein solution with a mass fraction of 1%-3%, adjust the pH to 10-11 to denature the protein in the protein solution, then add 11-13 KU / g of alkaline protease, and perform ultrasonic-assisted enzymolysis for 5-7 h, and obtain the safflower meal protein peptide SPH-A after enzyme inactivation. 4.The safflower meal protein peptide SPH-A according to claim 2, characterized in that, The ultrasonic conditions are 250-350 W and 40-50℃.

5. The safflower meal protein of claim 1 for use in the preparation of a drug for treating colitis.

6. The safflower meal protein peptide SPH-A of claim 3 for use in the preparation of a drug for treating colitis.

7. The safflower meal protein of claim 1 for use in the preparation of a health product for enhancing immunity.

8. The safflower meal protein peptide SPH-A of claim 3 for use in the preparation of a health product for enhancing immunity.

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