Preparation method and application of carboxymethylated mulberry leaf polysaccharide with improved antioxidant activity

Abstract

The application discloses a preparation method of carboxymethyl mulberry leaf polysaccharide with improved antioxidant activity, which comprises the following steps: (1) crude mulberry leaf polysaccharide is dissolved in an alkali solution, acid is added under stirring, and then the reaction is continuously stirred; (2) the reaction is terminated by adjusting to neutral with acid, and the reaction liquid is dialyzed in a dialysis bag; (3) after centrifugation, alcohol precipitation is carried out in an ethanol system, and freeze-drying is carried out, so that the carboxymethyl mulberry leaf polysaccharide with improved antioxidant activity is prepared. The carboxymethyl mulberry leaf polysaccharide product prepared by the application has excellent solubility and increased antioxidant activity. After the mouse liver injury symptoms are obviously improved, the decrease of the liver index is relieved, and the liver lesion is reduced. Therefore, the carboxymethyl mulberry leaf polysaccharide can effectively prevent liver injury and oxidative stress caused by aflatoxin B1, has a wide application prospect in the antioxidant preparation research of the mulberry leaf polysaccharide, and provides a train of thought for subsequent research and development and optimization and innovation of the polysaccharide.

Description

Technical Field

[0001] This invention belongs to the field of mulberry leaf polysaccharide preparation technology, specifically relating to a method for preparing and applying carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity. Technical Background

[0002] Molecular modification refers to the process of altering the structure of compound molecules through chemical or other means to obtain derivatives with different structural types. Polysaccharides are natural macromolecules that are widely available and possess biological activity, biocompatibility, and low or no cytotoxicity. The biological activity of polysaccharides is closely related to their structure. Carboxymethylation modification refers to the reaction of introducing carboxymethyl groups onto the polysaccharide macromolecular chain. After modification, the water solubility and biological activity of polysaccharides can be improved.

[0003] Mulberry leaves belong to the Moraceae family and are mainly distributed in Asia, Europe, and North America. my country is the world's largest mulberry-growing country, with a cultivation history of over 4,000 years. Mulberry leaves are the dried leaves of the mulberry tree, also known as iron fan leaves or silkworm leaves. Modern research shows that mulberry leaves contain various natural active ingredients such as flavonoids, alkaloids, polysaccharides, polyphenols, and organic acids, playing important roles in anti-inflammation, obesity treatment, blood sugar reduction, arteriosclerosis control, and antioxidant effects. They have been widely used in food additives and the biomedical industry. In 2002, they were listed as a food with medicinal properties by the National Health Commission.

[0004] Aflatoxin B1 (AFB1) is a fungal toxin with strong toxicity and carcinogenicity, posing adverse effects on human and animal health. According to the Food and Agriculture Organization (FAO), approximately 25% of the world's crops are contaminated with fungal toxins. AFB1 is widely found in livestock feed and human food, causing significant economic losses to agriculture and animal husbandry. Long-term exposure to low doses of AFB1 can lead to chronic toxicity and even liver cancer; AFB1 has been found to trigger liver tissue damage and impaired liver function. According to incomplete statistics, the high temperature and humidity in the Yangtze River basin and South my country cause 31 million tons of grain loss annually due to fungal contamination (mainly aflatoxin, zearalenone, and vomitoxin), resulting in economic losses of 68-85 billion yuan.

[0005] Studies on the antioxidant activity of mulberry leaf polysaccharides (MLP) have been reported, but their low antioxidant activity has limited their clinical application and promotion. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing carboxymethylated mulberry leaf polysaccharide with improved antioxidant activity and its application. The carboxymethylated mulberry leaf polysaccharide product obtained by the modification method has excellent solubility, can enhance the body's antioxidant capacity, and can effectively prevent AFB1-induced liver damage.

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

[0008] A method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity includes the following steps:

[0009] (1) Dissolve the crude polysaccharide of mulberry leaves in an alkaline solution, add acid while stirring, and then continue stirring to react;

[0010] (2) Adjust to neutral with acid to terminate the reaction. Place the reaction solution in a dialysis bag for dialysis.

[0011] (3) After centrifugation, the polysaccharide was precipitated with ethanol and freeze-dried to obtain carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity.

[0012] Preferably, the purity of the crude polysaccharide from mulberry leaves in step (1) is 68%.

[0013] Preferably, the alkaline solution in step (1) is a NaOH solution with a concentration of 3 mol / L.

[0014] Preferably, the acid in step (1) is monochloroacetic acid with a concentration of 1.5 mol / L.

[0015] Preferably, the temperature of the stirring reaction in step (1) is 60°C and the reaction time is 2.5h.

[0016] Preferably, the acid in step (2) is HCl with a concentration of 2.0 mol / L.

[0017] Preferably, the ethanol system in step (3) is 80%.

[0018] Preferably, the degree of carboxymethyl substitution of the carboxymethylated mulberry leaf polysaccharide in step (3) is 0.69.

[0019] This invention also provides a study on the application of carboxymethylated mulberry leaf polysaccharide in in vitro antioxidant capacity, and the application of the carboxymethylated mulberry leaf polysaccharide in a drug for alleviating AFB1-induced liver injury in mice.

[0020] The present invention has the following beneficial effects:

[0021] (1) To address the issue of poor antioxidant activity of mulberry leaf polysaccharides (MLP), this invention attempts to modify them by carboxymethylation to improve their physicochemical properties and antioxidant capacity, making them suitable for clinical production. To this end, this invention establishes a method for preparing carboxymethylated mulberry leaf polysaccharides with enhanced antioxidant activity. Crude mulberry leaf polysaccharides are dissolved in NaOH solution, and monochloroacetic acid is added with stirring. The reaction is then continued with stirring. The solution is adjusted to neutral with HCl to terminate the reaction. The reaction solution is dialyzed in a dialysis bag, centrifuged, precipitated with ethanol, and freeze-dried to obtain carboxymethylated mulberry leaf polysaccharides (CM-MLP). This invention uses response surface methodology to optimize the preparation method of carboxymethylated mulberry leaf polysaccharides. A three-factor, three-level response surface experimental scheme is established with chloroacetic acid concentration, temperature, and time as independent variables. Experimental results showed that when the concentration of chloroacetic acid was 1.5 mol / L, the preparation temperature was 60℃, and the reaction time was 2.5 h, the carboxymethylated mulberry leaf polysaccharide had the highest degree of substitution of 0.69.

[0022] (2) This invention studies the in vitro antioxidant activity of CM-MLP by measuring its hydroxyl radical scavenging ability, DPPH radical scavenging ability, and superoxide anion radical scavenging ability. The results show that CM-MLP is superior to MLP in terms of hydroxyl radical scavenging ability and DPPH radical scavenging ability.

[0023] (3) Using KM mice as experimental subjects, a liver injury model was established through AFB1. The protective effect of CM-MLP on AFB1-induced liver injury was further studied by measuring mouse liver index, serum indicators (AST, ALT, ALP, GST, GSH, SOD, CYP2E1), and liver tissue homogenate indicators (CYP2E1, Nrf2, and SOD1). The experimental results showed that the liver injury symptoms of mice treated with CM-MLP were significantly improved, the decrease in liver index was alleviated, and the liver lesions were reduced. Analysis showed that CM-MLP improves the body's antioxidant level and slows down liver injury through the CYP2E1 / Nrf2 signaling pathway. Therefore, the carboxymethylated mulberry leaf polysaccharide of this invention can effectively prevent liver injury caused by AFB1 and has broad application prospects in the research of antioxidant preparations of mulberry leaf polysaccharide, providing ideas for the subsequent research and optimization of this polysaccharide. Attached Figure Description

[0024] Figure 1 This is a standard curve diagram of the preparation method of carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity according to the present invention: standard curve of chromium acid-sulfuric acid method.

[0025] Figure 2This is a 3D visualization of the response surface methodology for optimizing the preparation method of carboxymethylated mulberry leaf polysaccharide. In the figure: A represents time and chloroacetic acid concentration, B represents chloroacetic acid and temperature, and C represents time and temperature.

[0026] Figure 3 This is a graph showing the solubility of carboxymethylated mulberry leaf polysaccharide.

[0027] Figure 4 This is a scanning electron microscope (SEM) characterization image of carboxymethylated mulberry leaf polysaccharide. In the image, D represents mulberry leaf polysaccharide, and E represents carboxymethylated mulberry leaf polysaccharide.

[0028] Figure 5 For the in vitro antioxidant activity study of CM-MLP, F in the figure represents the scavenging capacity of hydroxyl radicals, G represents the scavenging capacity of DPPH radicals, and H represents the scavenging capacity of superoxide anion radicals.

[0029] Figure 6 This is a graph of the liver index in mice.

[0030] Figure 7 This is a graph showing the measurement of ALT, AST, and ALP in mouse serum. In the graph, I represents AST, J represents ALT, and K represents ALP.

[0031] Figure 8 This is a graph showing the detection of biochemical indicators in mouse liver. In the graph, L represents GST, M represents GSH, and N represents SOD.

[0032] Figure 9 The images show the HE staining results of mouse livers. In the images, a is the blank group, b is the model group, c is the positive control group, d is the low-dose MLP group, e is the high-dose MLP group, f is the low-dose CM-MLP group, and g is the high-dose CM-MLP group.

[0033] Figure 10 This is a graph showing the measurement of CYP2E1 in mouse serum.

[0034] Figure 11 This is a detection diagram of genes related to the CYP2E1 / Nrf2 pathway in mouse liver tissue. In the diagram, O represents CYP2E1, P represents Nrf2, and Q represents SOD1. Detailed Implementation

[0035] 1. Materials and Reagents

[0036] 1.1 Drug: Mulberry leaf polysaccharide was extracted by the Traditional Chinese Veterinary Laboratory of Guangxi University, with a purity of 68%.

[0037] 1.2 Main reagents: Chloroacetic acid (MCA), glycolic acid (GA), chromotropic acid (CP), and ammonium acetate (ACS) were all purchased from Shanghai Macklin Biochemical Co., Ltd. (product numbers were C832199, G810373, C804988, A800996 respectively); sodium hydroxide (NaOH, Chengdu Jinshan Chemical Reagent Co., Ltd., 20200308); aflatoxin B1 (AFB1, Chengdu Lingliu Biotechnology Co., Ltd., 1162 - 65 - 8); alanine aminotransferase ALT kit, aspartate aminotransferase AST kit, and malondialdehyde kit were all purchased from Nanjing Jiancheng Bioengineering Institute (product numbers were C009 - 2 - 1, C010 - 2 - 1, A003 - 1 - 2 respectively); mouse glutathione S - transferase (GST) ELISA kit, mouse superoxide dismutase (SOD) ELISA kit, mouse cytochrome cyp2e1 (cyp2e1) ELISA kit, and mouse alkaline phosphatase (ALP) ELISA kit were all purchased from Nanjing Boyan Biotechnology Co., Ltd. (product numbers were BY - EM230078, BY - EM230143, BY - EM228259, BY - EM230140 respectively); TRNGeneReagent, StarScript II RT Mix with gDNA Remover, and 2×RealStar Fast SYBR qPCR Mix were all purchased from GenStar Company. Other reagents were all of analytical grade.

[0038] The experimental animals were specific pathogen - free (SPF) grade

[0039] The experimental KM male mice, 70 in number, 4 - week - old mice with a body weight of 20 ± 2 g, were purchased from Changsha Tianqin Biotechnology Co., Ltd., with the license number SCXK(Xiang)2019 - 0014.

[0040] 2. Experimental methods

[0041] 2.1 Optimization of the preparation method of carboxymethylated mulberry leaf polysaccharide by response surface methodology

[0042] Drawing of the standard curve of glycolic acid. Accurately weigh 250 mg of glycolic acid and place it in a beaker, add an appropriate amount of distilled water, fully dissolve it and transfer it to a 100 - mL volumetric flask for volume fixation to obtain a glycolic acid standard solution with a concentration of 2.5 mg / mL. Accurately pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1 mL of the glycolic acid standard solution into stoppered test tubes numbered 0 to 6, and respectively supplement distilled water to make the solution volume of each test tube reach 1 mL. Measure the absorbance by chromotropic acid - sulfuric acid colorimetry, and fit the linear regression equation as Figure 1 ;

[0043] Step 1: Preparation of carboxymethylated mulberry leaf polysaccharide. Accurately weigh 0.1 g of crude mulberry leaf polysaccharide sample into a 250 mL beaker, dissolve it in 20 mL of 3 mol / L NaOH solution, and slowly add 20 mL of monochloroacetic acid of a certain concentration while stirring vigorously. After stirring and reacting for a period of time at the set temperature, adjust the pH to neutral with 2.0 mol / L HCl to terminate the reaction. Dialyze the reaction solution in a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h. After centrifugation, precipitate with 80% ethanol and freeze-dry to obtain carboxymethylated mulberry leaf polysaccharide.

[0044] Step 2: Determination of carboxymethyl substitution degree. Following the relevant procedures of the chromium-sulfuric acid method, calculate the carboxymethyl substitution degree of carboxymethylated mulberry leaf polysaccharide based on the standard curve equation. The relevant calculations refer to the following formula:

[0045] Degree of carboxymethyl substitution (DS) = 162B / [76-(59-1)]B (1)

[0046] In formula (1): B-the mass of each gram of carboxymethylated mulberry leaf polysaccharide sample is equivalent to the mass of glycolic acid; 76-the molar mass of glycolic acid, g / mol; 59-the molar mass of CH2COOH, g / mol.

[0047] A three-factor, three-level response surface methodology using a box-behnken design was employed, with the degree of carboxymethyl substitution (DS) as the response value, to explore the optimal preparation process of carboxymethylated mulberry leaf polysaccharide based on chloroacetic acid concentration, temperature, and time.

[0048]

[0049] 2.2 Preparation of carboxymethylated mulberry leaf polysaccharide

[0050] Carboxymethylated mulberry leaf polysaccharide was prepared by dissolving 5g of mulberry leaf polysaccharide (68% purity) in 1000mL of 3mol / L sodium hydroxide solution according to the optimal preparation method. A certain amount of carboxymethylated mulberry leaf polysaccharide was then prepared into an oral liquid of the corresponding dosage.

[0051] 2.3 Determination of the solubility of carboxymethylated mulberry leaf polysaccharide

[0052] The time for complete dissolution of polysaccharides under different temperature conditions was determined. 0.02 g of MLP and CM-MLP were weighed and added to 20 mL of distilled water. The polysaccharides were heated and stirred simultaneously using a magnetic stirrer, and the time for complete dissolution at different temperatures (20, 40, 60, 80, 100°C) was recorded.

[0053] 2.4 Scanning Electron Microscopy (SEM) Characterization

[0054] Approximately 5 mg of dried sample was adhered to a conductive carbon film containing double-sided adhesive and placed in the sample chamber of an ion sputtering instrument for approximately 40 seconds of gold sputtering. After removal, the sample was placed in the observation chamber of a scanning electron microscope at an accelerating voltage of 5 kV for observation and analysis, and photographed at 20,000x magnification.

[0055] 2.5 Determination of the in vitro antioxidant capacity of carboxymethylated mulberry leaf polysaccharides

[0056] 2.5.1 Hydroxyl radical scavenging ability

[0057] Using ascorbic acid as a positive control, 50 µL of ferrous sulfate solution (0.009 mol / L), 50 µL of salicylic acid-ethanol solution (0.009 mol / L), and 50 µL of vitamin C (VC) solution, MLP solution, and CM-MLP solution (concentrations of 0.125, 0.250, 0.500, 1.000, and 2.000 g / L, respectively) were added to each well of a 96-well plate. Distilled water was used as a blank control. All solutions were mixed thoroughly, and 50 µL of hydrogen peroxide solution (0.009 mol / L) was added. The mixture was incubated at 37 °C for 30 min, and the photoluminescence was measured at 510 nm.

[0058] Hydroxyl radical scavenging rate = [A0 - (A - AX)] / A0 × 100% (2)

[0059] In formula (2): A0 represents the absorbance of distilled water instead of the sample; A represents the absorbance of the test well with the sample solution added; AX represents the absorbance of the polysaccharide or the modified polysaccharide itself.

[0060] 2.5.2 DPPH free radical scavenging ability

[0061] DPPH was dissolved in anhydrous ethanol to prepare a 0.2 mmol / L solution. Using VC as a positive control, 1 mL of MLP and CM-MLP (concentrations of 2.000, 1.000, 0.5000, 0.250, and 0.125 g / L, respectively) were added to test tubes. 4 mL of the pre-prepared DPPH ethanol solution was added, mixed well, and reacted at room temperature for 30 min in the dark. Distilled water was used as a blank control instead of the polysaccharide solution. The absorbance was measured at 517 nm. The DPPH scavenging rate was calculated.

[0062] DPPH free radical scavenging rate = [1-(E2-E1) / E0]×100% (3)

[0063] In formula (3): E2 represents the absorbance of each concentration of polysaccharide itself; E1 represents the absorbance of distilled water instead of DPPH ethanol solution; E0 represents the absorbance of distilled water instead of polysaccharide solution.

[0064] Superoxide anion free radical scavenging ability

[0065] Gallic acid was dissolved in distilled water to prepare a 25 mmol / L solution; 25 mL of 0.1 mol / L tris(hydroxymethyl)aminomethane (Tris) solution was added to hydrochloric acid to adjust the pH to 8, and the volume was brought to 50 mL; distilled water was used as a blank control instead of the polysaccharide solution, and vitamin C was used as a positive control. 180 µL of buffer solution, 40 µL of MLP solution, and AC-MLP solution were added sequentially to a 96-well plate and mixed thoroughly. The mixture was incubated at 37 °C for 10 min; then 20 µL of gallic acid solution was added and mixed thoroughly, and the reaction was incubated for 4 min. The absorbance was measured at 325 nm. The superoxide anion radical scavenging rate was calculated.

[0066] Superoxide anion radical scavenging rate = [A0 - (A - AX)] / A0 × 100% (4)

[0067] In formula (4): A0 represents the absorbance of distilled water instead of the sample; A represents the absorbance of the sample solution; AX represents the absorbance of the polysaccharide solution itself.

[0068] 2.6 Animal Experiment Design

[0069] After the acclimatization period, the mice were randomly divided into 7 groups (n=10): blank group (C), model group (A), positive control group (B), low-dose MLP group (G), high-dose MLP group (L), low-dose CM-MLP group (Y), and high-dose CM-MLP group (Z). The experimental groups were: blank control group (0.1 ml / 10 g bw saline administered by gavage at 9:00 AM and 11:00 AM daily), model group (0.75 mg / kg bw AFB1 administered by gavage at 9:00 AM and 0.1 ml / 10 g bw saline administered by gavage at 11:00 AM daily), positive control group (0.75 mg / kg bw AFB1 administered by gavage at 9:00 AM and 100 mg / kg bw silymarin administered by gavage at 11:00 AM daily), low-dose MLP group and low-dose CM-MLP group (0.75 mg / kg bw AFB1 administered by gavage at 9:00 AM and 200 mg / kg bw polysaccharide administered by gavage at 11:00 AM daily), high-dose MLP group and high-dose CM-MLP group (0.75 mg / kg bw AFB1 administered by gavage at 9:00 AM and 400 mg / kg bw polysaccharide administered by gavage at 11:00 AM daily). The experimental period was 14 days, and the amount of food consumed and the mental state were recorded and observed daily. Mice were fasted for 12 hours after their last exposure to the drug, but had normal water intake. The following day, after fasting and weighing, blood and liver tissue were collected. Part of the mouse liver was stored at -80 °C, while another part was fixed in 10% neutral formalin solution and prepared into sections.

[0070] 2.7 Measurement of in vivo indicators in mice

[0071] 2.7.1 Determination of mouse liver coefficient

[0072] Record the mouse body weight and liver tissue weight, and calculate the liver organ coefficient using the formula b:

[0073] b = Liver tissue weight (g) / Mouse body weight (g) × 100%

[0074] 2.7.2 Determination of serum ALT, AST and ALP.

[0075] Perform ALT, AST, and ALP measurements according to the kit instructions.

[0076] 2.7.3 Liver biochemical index detection

[0077] Perform GST, GSH, and SOD testing according to the kit instructions.

[0078] 2.7.4 Observation of liver tissue morphology

[0079] Liver tissue fixed in formaldehyde solution was obtained and routinely sectioned in paraffin.

[0080] 2.7.5 Determination of CYP2E1 in serum

[0081] Perform the assay according to the ELISA kit instructions.

[0082] 2.7.6 Detection of Nrf2 pathway-related genes

[0083] Total RNA was extracted according to the TRNGene Reagent kit instructions, and the concentration of extracted total RNA was determined. Reverse transcription was performed according to the StarScript II RT Mix with gDNA Remover kit instructions, and the synthesized cDNA was stored at -20℃ for later use. The target gene sequence was obtained from Genebank, and comparative analysis was performed using BLAST. Highly specific primers were selected and synthesized by Beijing Qingke Biotechnology Co., Ltd. qPCR reactions were performed strictly according to the kit instructions.

[0084] 3. Experimental Results and Analysis

[0085] 3.1 The experimental design scheme and experimental results of response surface methodology are shown in Tables 2 and 3.

[0086]

[0087] Analyzing the model, the regression equation for the degree of carboxymethylation substitution (DS) of mulberry leaf polysaccharides and the coded values ​​of the three selected factors is: Y = +0.6631 -0.0149A -0.0038B +0.0035C +0.0176AB +0.0225AC -0.0401BC -0.2360A² -0.0969B² -0.1043C²

[0088]

[0089] Analysis of variance was performed on the regression model, and the model coefficients were tested for significance. As shown in Table 3, in this experimental model, P < 0.0001 indicates extreme significance. The model's lack-of-fit term P = 0.4087 > 0.05, indicating that the lack of fit relative to pure error is not significant. This suggests that the model equation is suitable for determining the degree of substitution of carboxymethylated mulberry leaf polysaccharides. The model's coefficient of determination (R = 0.9905) indicates that the established model is applicable. The coefficient of variation (CV = 4.86%) is low, indicating high accuracy and reliability of the experimental values. The significance of each coefficient was tested based on the P-value; the smaller the P-value, the more significant the corresponding coefficient. The first-order terms A, B, and C of the model have an extremely significant effect on the degree of substitution of carboxymethylated mulberry leaf polysaccharides (P < 0.01), while the interaction terms AB, AC, and BC have no significant effect on the degree of substitution of carboxymethylated mulberry leaf polysaccharides. The quadratic term A... 2 B 2 and C 2 The p-values ​​were all less than 0.01, indicating they were all highly significant. In conclusion, the model fit conforms to the principles of mathematical statistics, and the carboxymethylation conditions of mulberry leaf polysaccharides can be predicted using this equation.

[0090] Response surface methodology for carboxymethyl substitution degree: The influence of various factors on DS can be seen from... Figure 2 The three sets of interactive influence diagrams visually illustrate this: the steeper the slope of the response surface, the greater the impact of changes in the corresponding conditions on the response value, and vice versa. As shown in the diagrams, MCA concentration and temperature have the most significant impact on DS, corresponding to steep response surface slopes and noticeable changes in contour density. Time has a relatively smaller impact on the degree of substitution, and its corresponding curve is relatively flat. Through response surface analysis, the factor levels at which DS reaches its maximum value are: MCA concentration 1.468 mol / L, reaction temperature 60.368℃, and reaction time 2.473 h, predicting a DS of 0.663. Considering the feasibility of the experimental operation, the optimal preparation process for carboxymethylated mulberry leaf polysaccharide is determined to be: MCA concentration 1.5 mol / L, reaction temperature 60℃, and reaction time 2.5 h.

[0091] 3.2 Polysaccharide solubility determination

[0092] like Figure 3As shown, within the temperature range of 20-100℃, the dissolution time of CM-MLP and MLP decreased with increasing temperature. At the same temperature, the dissolution time of CM-MLP was shorter than that of MLP. The results indicate that carboxymethyl modification of mulberry leaf polysaccharides improves polysaccharide solubility and reduces the time required for complete dissolution.

[0093] 3.3 Scanning electron microscope image

[0094] like Figure 4 As shown, the surface of the MLP consists of tightly adhered granules, while the interior forms a network of interconnected clumps. The granular mosaics of CM-MLP are spherical or elliptical, adhered to each other, and of varying sizes. The results indicate that carboxymethyl modification of mulberry leaves alters the surface structure, possibly related to increased solubility.

[0095] 3.4 In vitro antioxidant activity study

[0096] 3.4.1 Scavenging ability of hydroxyl radicals

[0097] like Figure 5 As shown in F, the scavenging ability of hydroxyl radicals gradually increases with increasing sample concentration. The scavenging ability of CM-MLP is higher than that of MLP. At a concentration of 2 mg / mL, CM-MLP has a scavenging ability of 65.9% and MLP has a scavenging ability of 35.6%.

[0098] 3.4.2 Scavenging ability of DPPH free radicals

[0099] like Figure 5 As shown in G, the scavenging ability of DPPH free radicals gradually increases with the increase of sample concentration. The scavenging ability of CM-MLP for DPPH free radicals is higher than that of MLP. At a concentration of 2 mg / mL, CM-MLP has a scavenging ability of 83.4% and MLP has a scavenging ability of 80.1%.

[0100] 3.4.3 Scavenging ability of superoxide anion free radicals

[0101] like Figure 5 As shown in H, the scavenging ability of superoxide anion radicals gradually increases with increasing sample concentration. The scavenging abilities of CM-MLP and MLP for superoxide anion radicals are comparable. At a concentration of 2 mg / mL, CM-MLP has a scavenging ability of 73.3% and MLP has a scavenging ability of 76.1%.

[0102] 3.5 Prophylactic use of CM-MLP can alleviate symptoms of AFB1-induced liver injury.

[0103] To investigate the preventive effect of CM-MLP on AFB1-induced liver injury, different polysaccharide groups were administered different doses of polysaccharide via gavage from day 1 to day 14. During the experiment, mice gradually developed loss of appetite, lethargy, and ruffled fur in the later stages of AFB1 administration. The liver is an important detoxification organ in the body, participating in various processes such as digestion, metabolism, detoxification, and immunity. Liver indices can indirectly reflect potential liver lesions such as edema, hyperplasia, and atrophy. Mouse liver indices are shown below. Figure 6 Compared with the control group, the liver index in the model group was significantly decreased (P<0.01), possibly due to hepatocyte damage and atrophy. Compared with the model group, the liver index at each dose of CM-MLP and MLP was significantly increased (P<0.01), and the high-dose CM-MLP group could recover to normal levels. This indicates that CM-MLP and MLP can effectively improve the decrease in liver index caused by AFB1 and prevent liver atrophy.

[0104] This indicates a significant difference compared to the control group (P<0.05). # indicates a highly significant difference compared to the blank group (P<0.01); # indicates a significant difference compared to the model group (P<0.05); ## indicates a highly significant difference compared to the model group (P<0.01).

[0105] When hepatocytes are damaged, ALT and AST are rapidly released into the bloodstream. Serum ALT and AST levels are important biochemical markers for assessing liver function. Elevated ALP levels can also evaluate liver damage, but they are not liver-specific. Figure 7 As shown, compared with the blank group, the activities of the three enzymes in the model group were significantly increased (P<0.01), indicating that hepatocytes were damaged and the model was feasible. Compared with the model group, the activities of the three enzymes in the positive drug group, each dose group of CM-MLP and each dose group of MLP were significantly decreased (P<0.05), indicating that CM-MLP and MLP have a protective effect against AFB1-induced liver injury.

[0106] GST is one of the important metabolic enzymes for biotransformation in the body and a major detoxification system for cellular damage resistance. GSH is an anti-lipid peroxidation substance, mainly found in hepatocytes, which not only regulates intracellular redox reactions but also has detoxification functions. SOD can catalyze the generation of superoxide free radicals and effectively scavenge free radicals; therefore, detecting SOD activity can reflect the ability to scavenge free radicals and antioxidant capacity. The measurement results are obtained from... Figure 8Compared with the control group, the model group showed significantly lower levels of GST and GSH (P<0.01), indicating liver damage and decreased detoxification function. Simultaneously, the level of the reductase SOD was significantly lower (P<0.01), indicating increased liver oxidative stress. Compared with the model group, the high-dose MLP and CM-MLP groups showed significantly higher levels of GST (P<0.01), while the low-dose CM-MLP group showed significantly higher levels of GSH (P<0.01). GSH expression was not significant in the low-dose MLP group, while SOD expression was significantly higher in the low-dose CM-MLP group (P<0.05). SOD expression was not significant in the low-dose MLP group, while SOD expression was significantly higher in both the high-dose MLP and CM-MLP groups (P<0.01). At the same dosage, CM-MLP showed significantly higher levels of GSH and SOD than MLP. Both CM-MLP and MLP can enhance the antioxidant level in AFB1-induced liver injury, achieving a hepatoprotective effect. CM-MLP's antioxidant capacity was slightly better than that of MLP.

[0107] Results of HE staining of mouse liver tissue are shown in the figure. Figure 9 In the blank group, the hepatocyte cords were intact and the hepatocyte morphology was normal; in the model group, the hepatocytes were arranged in a disordered manner, the nuclei were severely broken, and severe vacuolar degeneration occurred; after the administration of polysaccharides, the vacuolar degeneration of hepatocytes in each MLP dose group and each CM-MLP dose group was reduced, and the cells were arranged more neatly; in the positive control group, the vacuolar degeneration of hepatocytes was reduced, but some hepatocyte nuclei were still dissolved.

[0108] Cytochrome P450 2E1 (CYP2E1) is a member of the CYP450 family, responsible for the metabolism of endogenous and exogenous compounds in the body. CYP2E1 is closely related to the progression of liver damage. Figure 10 It can be seen that compared with the blank group, the CYP2E1 level in the model group was significantly increased (P<0.01); compared with the model group, the CYP2E1 level in each MLP dose group and each CM-MLP dose group was significantly decreased (P<0.01).

[0109] Nrf2 is a key regulator of oxidative stress, playing an antioxidant role in maintaining cellular, protein, and metabolic homeostasis, and also serving a crucial function in cellular detoxification pathways. Normally, Nrf2 is bound to Keap1 in the cytoplasm. When oxidative homeostasis is disrupted, Nrf2 dissociates from Keap1, and subsequently translocates to the nucleus, binding to antioxidant response element (ARE) sites. This promotes the transcription of downstream antioxidant genes such as SOD1 and HO1 to maintain oxidative homeostasis in the body. Therefore, activating Nrf2 for antioxidant activity can achieve a liver-protective effect. Figure 11Compared with the control group, CYP2E1 mRNA expression in the model group was significantly increased (P<0.01); compared with the model group, CYP2E1 mRNA expression in the low-dose CM-MLP group and the low-dose MLP group was significantly decreased (P<0.01), while CYP2E1 mRNA expression in the high-dose CM-MLP group and the high-dose MLP group was not significant. Compared with the control group, Nrf2 mRNA expression in the model group was significantly decreased (P<0.01); compared with the model group, Nrf2 mRNA expression in each dose group of CM-MLP and each dose group of MLP was significantly increased (P<0.01). Furthermore, compared with the control group, SOD1 mRNA expression in the model group was significantly decreased (P<0.05); compared with the model group, SOD1 mRNA expression in each dose group of CM-MLP was significantly increased (P<0.01), and SOD1 mRNA expression in the high-dose MLP group was significantly increased (P<0.05). At the same dosage, CM-MLP regulates CYP2E1 and SOD1 expression more strongly than MLP.

[0110] The above results indicate that MLP and CM-MLP can play a protective role against AFB1-induced liver injury. Their mechanism of action may be related to improving the antioxidant capacity of the liver and regulating the CYP2E1 / Nrf2 signaling pathway. CM-MLP is slightly better than MLP in improving the body's antioxidant capacity and regulating the Nrf2 pathway.

[0111] The above content should not be construed as limiting the specific implementation of this invention to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of this invention, and all such deductions or substitutions should be considered as belonging to this invention as determined by the submitted claims.

Claims

1. A method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity, characterized in that, Includes the following steps: (1) Dissolve the crude polysaccharide of mulberry leaves in an alkaline solution, add monochloroacetic acid with a concentration of 1.5 mol / L while stirring, and then continue stirring to react; (2) Adjust to neutral with acid to terminate the reaction. Place the reaction solution in a dialysis bag for dialysis. (3) After centrifugation, the polysaccharide was precipitated with ethanol and freeze-dried to obtain carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity.

2. The method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity according to claim 1, characterized in that: The purity of the crude polysaccharide from mulberry leaves mentioned in step (1) is 68%.

3. The method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity according to claim 1, characterized in that: The alkaline solution mentioned in step (1) is a NaOH solution with a concentration of 3 mol / L.

4. The method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity according to claim 1, characterized in that: The temperature of the stirring reaction in step (1) is 60℃ and the reaction time is 2.5h.

5. The method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity according to claim 1, characterized in that: The acid mentioned in step (2) is HCl with a concentration of 2.0 mol / L.

6. The method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity according to claim 1, characterized in that: In step (3), the ethanol system is 80%.

7. The method for preparing carboxymethylated mulberry leaf polysaccharide with enhanced antioxidant activity according to claim 1, characterized in that: The degree of carboxymethyl substitution of the carboxymethylated mulberry leaf polysaccharide in step (3) is 0.

69.

8. The use of a carboxymethylated mulberry leaf polysaccharide prepared according to any one of claims 1-7 in the preparation of an antioxidant drug.

9. The application of the carboxymethylated mulberry leaf polysaccharide according to claim 8, characterized in that: The application of the carboxymethylated mulberry leaf polysaccharide in the preparation of a drug to alleviate AFB1-induced liver injury in mice.

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