Zizania latifolia waste leaf polysaccharide component, and preparation method and use thereof

Abstract

The present application relates to the technical field of comprehensive utilization of Zizania latifolia, and particularly relates to a preparation method, components and uses of polysaccharide from Zizania latifolia discarded leaves. The cellulase synergistic microwave extraction method provided by the present application can efficiently prepare polysaccharide from Zizania latifolia discarded leaves. The polysaccharide component with high efficient antioxidant and anticoagulant activity can be separated from the polysaccharide from Zizania latifolia discarded leaves prepared by the method. When the concentration is 2.5 mg / mL, the DPPH free radical scavenging rate is 43.7±0.56%, and the hydroxyl free radical scavenging rate is 52.1±2.83%. When the concentration is 5.0 mg / mL, the TT value is 16.1±0.36 s, and the PT value is 17.1±1.05 s. The polysaccharide component can be used for preparing oral liquid, tablets, capsules and other related products.

Description

Technical Field

[0001] The present invention relates to the technical field of comprehensive utilization of wild rice stem, and specifically relates to a preparation method, component and use of polysaccharide from abandoned wild rice stem leaves. Background Art

[0002] Wild rice stem is a perennial Gramineae aquatic plant widely cultivated in China and Southeast Asia. China is one of the first countries to cultivate wild rice stem as a vegetable. China is extremely rich in wild rice stem resources, which is the second largest aquatic vegetable after lotus root, mainly planted in the provinces along the middle and lower reaches of the Yangtze River. Wild rice stem is a kind of plant with homologous medicine and food, which is rich in nutrients and has unique medicinal value, enjoying the reputation of "ginseng in water". The wild rice stem plant is tall, and the mass of its bract leaves (inedible part) accounts for 50%-70% of the total mass of the wild rice stem plant. According to statistics, the annual output of abandoned wild rice stem leaves in Jiangsu Province, China alone is 350,000 to 490,000 tons (fresh mass). During the annual wild rice stem harvest season, a large number of abandoned wild rice stem leaves are discarded in rivers, village roads and even highways, becoming a breeding ground for diseases and insect pests, and some are dried in the sun and then burned in a centralized manner, causing serious environmental pollution.

[0003] Therefore, the comprehensive development and utilization of abandoned wild rice stem leaf resources is an urgent problem and challenge to be solved in the wild rice stem industry at present. Researchers have tried to use abandoned wild rice stem leaves as silage to feed ruminants such as cattle and sheep. However, the energy utilization rate is low only after the livestock pass through the digestive tract and return to the field. In fact, agricultural wastes are also rich in functional components with development value.

[0004] Cellulose is the main structural component of plant cell walls, usually combined with hemicellulose, pectin and lignin. There are hydrogen bonds between cellulose molecules, which are very stable at room temperature. The crude fiber content in abandoned wild rice stem leaves is as high as about 50%. These fibers enclose the active components and nutrients in the bract leaves within the cell wall, resulting in the disadvantages of high extraction difficulty, long extraction time and low yield in the preparation of polysaccharides from abandoned wild rice stem leaves. Modern extraction technologies emerging in recent years, such as hot water extraction, enzyme-assisted extraction, ultrasonic-assisted extraction, microwave-assisted extraction, etc., have shown great advantages in breaking walls and promoting release. However, there has been no report on the extraction of polysaccharides from abandoned wild rice stem leaves so far.

[0005] Cardiovascular diseases (CVD) are among the leading causes of death worldwide and a major health problem in my country. In my country, there are approximately 290 million CVD patients, with a mortality rate exceeding that of malignant tumors, accounting for over 40% of all disease-related deaths. Thrombosis plays a crucial role in the development of CVD; an imbalance between the coagulation and anticoagulation systems leads to increased blood coagulability, thereby increasing the risk of thrombosis. Therefore, maintaining a balance between blood coagulation and anticoagulation is essential. Heparin and its derivatives are the most widely used anticoagulants in clinical practice. Although they play an important role in the prevention and treatment of CVD, heparin use can cause side effects such as bleeding, thrombocytopenia, osteoporosis, allergic reactions, and hyperkalemia. Furthermore, heparin is an animal-derived polysaccharide, which also increases the risk of infection by animal pathogens. In recent years, the anticoagulant effects of plant polysaccharides have gradually attracted attention. It has been found that polysaccharides from wolfberry, green tea, and pumpkin all possess anticoagulant functions. Plant-derived polysaccharides are widely available and can avoid pathogen infection caused by animal-derived polysaccharides, making them an important source of novel anticoagulant functional factors. In the process of studying the anticoagulant effects of plant polysaccharides, researchers have also discovered that polysaccharides with antioxidant activity often exhibit strong anticoagulant effects. Therefore, it is necessary to further explore the antioxidant and anticoagulant activities of polysaccharides from waste leaves of water chestnut. This will help reveal the antioxidant and anticoagulant mechanisms of polysaccharides from waste leaves of water chestnut, providing a basis for the development and application of new antioxidant and anticoagulant products.

[0006] In summary, no reports have been found on the microwave-enzymatic extraction of polysaccharides from waste leaves of water bamboo shoots and their antioxidant and anticoagulant activities. This invention provides a method for microwave extraction of polysaccharide components from waste leaves of water bamboo shoots using cellulase synergistically and its application in antioxidant and anticoagulant properties. Summary of the Invention

[0007] The purpose of this invention is to provide a polysaccharide component from waste leaves of water bamboo, its preparation method, and its uses. Compared with traditional extraction methods, the cellulase-assisted microwave extraction method provided by this invention can efficiently prepare polysaccharides from waste leaves of water bamboo. Polysaccharide components with highly efficient antioxidant and anticoagulant activities can be isolated from the polysaccharides from waste leaves of water bamboo prepared by this method.

[0008] According to a first aspect of the present invention, the present invention provides a method for preparing polysaccharide components from waste leaves of water chestnut, wherein waste leaf powder of water chestnut is soaked in distilled water, then a certain amount of cellulase is added for enzymatic hydrolysis, the enzyme is inactivated and then extracted in a microwave extractor for a certain period of time; the crude polysaccharide components of waste leaves of water chestnut are obtained by centrifugation, concentration of supernatant, ethanol precipitation, freeze drying and deproteinization.

[0009] Preferably, the preparation method of the polysaccharide component from waste leaves of water chestnut specifically includes the following steps:

[0010] Fresh water bamboo waste leaves were washed, dried at 60℃ to constant weight, pulverized, and passed through a 40-mesh sieve to obtain water bamboo waste leaf powder for later use. The water bamboo waste leaf powder was soaked in warm water at a certain material-to-liquid ratio for 8 hours, then the pH was adjusted to 5.0±0.2, a certain amount of cellulase was added, and the mixture was incubated in a shaker at 50℃ for 2 hours. After incubation, the enzyme hydrolysate was placed in a boiling water bath for 15 minutes to inactivate the enzyme. After cooling, the pH was adjusted to 7.0±0.2, and the enzyme hydrolysate was placed in a microwave extractor and heated to a certain power. After a period of treatment, the supernatant was collected by centrifugation. The supernatant was concentrated, and three times the volume of anhydrous ethanol was added for alcohol precipitation at 4°C for 12 hours. The precipitate was obtained by centrifugation, and then water was added to dissolve it. The precipitate was then mixed with Sevag reagent (n-butanol: chloroform = 1:4 (v / v)) at a ratio of 4:1 and shaken thoroughly for 15 minutes before centrifugation. The supernatant was aspirated after centrifugation, and the Sevag deproteinization was repeated until no more protein was produced. The supernatant was freeze-dried to obtain crude polysaccharide from waste leaves of water chestnut.

[0011] Preferably, the ratio of waste water bamboo leaf powder to water during the distilled water soaking process is 1:20 g / mL to 1:60 g / mL, and more preferably 1:40 g / mL.

[0012] Preferably, the amount of cellulase added is 0.3% to 0.7% of the weight of the waste water chestnut leaf powder, more preferably 0.5%;

[0013] Preferably, the enzymatic hydrolysis temperature is 45℃~65℃, more preferably 55℃;

[0014] Preferably, the enzymatic hydrolysis time is 40 min to 80 min, more preferably 60 min;

[0015] Preferably, the microwave duration is 1 min to 15 min, more preferably 7 min;

[0016] Preferably, the microwave power is 200W to 600W, and more preferably 425W;

[0017] Preferably, the microwave temperature is 60℃ to 100℃, and more preferably 80℃.

[0018] Preferably, the ratio of waste water chestnut leaf powder to water is 1:40 g / mL, the amount of cellulase added is 0.5% of the weight of the waste water chestnut leaf powder, the enzymatic hydrolysis temperature is 55℃, the enzymatic hydrolysis time is 60 min, the microwave time is 7 min, the microwave power is 425 W, and the microwave temperature is 80℃. Under these conditions, the polysaccharide yield of waste water chestnut leaves is 0.82%, which is significantly higher than that of hot water extraction (0.38±0.08%), enzymatic extraction (0.46±0.05%), and hot water / microwave extraction (0.42±0.06%).

[0019] The method for preparing polysaccharide components from waste leaves of water bamboo according to the present invention further includes separating and purifying the prepared crude polysaccharide from waste leaves of water bamboo to obtain purified polysaccharide from waste leaves of water bamboo. The separation and purification step is to purify the crude polysaccharide from waste leaves of water bamboo sequentially by passing it through a diethylaminoethyl (DEAE) cellulose-52 column and a Sephadex G-100 dextran gel column.

[0020] The specific method is as follows: 100 mg of waste crude water chestnut leaf polysaccharide was weighed and dissolved in 20 mL of deionized water, and loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm); eluted with deionized water at a flow rate of 1 mL / min, 5 mL per tube, and plotted an elution curve; the polysaccharide fraction eluted by deionized water was collected, concentrated, dialyzed three times, and freeze-dried; 50 mg of freeze-dried waste water chestnut leaf polysaccharide was weighed and dissolved in 10 mL of deionized water, and loaded onto a Sephadex G-100 dextran gel column for further separation and purification, using deionized water as the eluent at a flow rate of 0.25 mL / min, 4 mL per tube, and plotted an elution curve; the polysaccharide fraction eluted by deionized water was collected, concentrated, dialyzed three times, and freeze-dried to obtain the purified polysaccharide fraction from the waste water chestnut leaves.

[0021] According to a second aspect of the present invention, a polysaccharide component from waste leaves of water chestnut is provided, wherein the total sugar content of the waste leaves of water chestnut is 79.13±2.5%, the uronic acid content is 5.16±0.24%, and the sulfate group content is 3.98±0.12; the waste leaves of water chestnut are composed of mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose in a molar ratio of 12.25:4.75:2.25:1.00:2.50:7.00:24.25:2.00:17.25, and the waste leaves of water chestnut are homogeneous polysaccharides with a molecular weight of 8.06×10⁻⁶. 3 kDa.

[0022] According to a third aspect of the present invention, the present invention provides the use of the polysaccharide component of the waste leaves of wild rice in products with antioxidant and anticoagulant functions; it can be used to prepare oral liquids, capsules, tablets and other formulations.

[0023] The present invention has the following beneficial effects:

[0024] 1) The cellulase-synergistic microwave extraction method provided by this invention has significant advantages in cell wall disruption and release, and has a synergistic effect on the extraction yield of crude polysaccharides from waste leaves of water chestnut. It is especially suitable for bract-leaved plants rich in cellulose and is expected to be applied to the efficient extraction of polysaccharides from agricultural wastes such as waste leaves of water chestnut.

[0025] 2) This invention optimizes the purification method of crude polysaccharides from waste leaves of water chestnut. The resulting polysaccharide fraction exhibits a DPPH radical scavenging rate of 43.7±0.56% and a hydroxyl radical scavenging rate of 52.1±2.83% at a concentration of 2.5 mg / mL. At a concentration of 5.00 mg / mL, the TT value reaches 16.1±0.36 s and the PT value reaches 17.1±1.05 s. This polysaccharide can be used to prepare related antioxidant and anticoagulant products. Attached Figure Description

[0026] Figure 1 The effect of the material-to-liquid ratio on the yield of polysaccharides from waste leaves of water chestnut;

[0027] Figure 2 The effect of enzyme addition on the yield of polysaccharides from waste leaves of water chestnut;

[0028] Figure 3 The effect of enzymatic hydrolysis temperature on the yield of polysaccharides from waste leaves of water chestnut;

[0029] Figure 4 Effect of enzymatic hydrolysis time on the yield of polysaccharides from waste leaves of water chestnut;

[0030] Figure 5 Effect of microwave time on the yield of polysaccharides from waste leaves of wild rice;

[0031] Figure 6 The effect of microwave power on the yield of polysaccharides from waste leaves of wild rice;

[0032] Figure 7 Effect of microwave temperature on the yield of polysaccharides from waste leaves of wild rice;

[0033] Figure 8 DEAE elution curve of polysaccharides from waste leaves of wild rice;

[0034] Figure 9 Elution curve of polysaccharide-glucan gel from waste leaves of wild rice;

[0035] Figure 10 The DPPH free radical scavenging ability of polysaccharide components from waste leaves of water bamboo;

[0036] Figure 11 The scavenging ability of polysaccharide components from waste leaves of water bamboo shoots against hydroxyl radicals;

[0037] Figure 12 Anticoagulant activity of polysaccharide components from waste leaves of wild rice;

[0038] Figure 13 UV-Vis scanning spectrum of polysaccharide components from waste leaves of water bamboo shoots;

[0039] Figure 14 Infrared scanning spectra of polysaccharide components from waste leaves of wild rice (Zizania latifolia);

[0040] Figure 15 Monosaccharide composition of polysaccharide components from waste leaves of water bamboo shoots;

[0041] Figure 16 Atomic weight distribution of polysaccharide components from waste leaves of wild rice. Detailed Implementation

[0042] Waste leaves of water bamboo were collected in June 2023 from riverbanks surrounding the Southeast Development Zone of Changshu City, Jiangsu Province; cellulase (10,000 U / g) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; monosaccharide standards and T-series dextran standards were purchased from Sinopharm Chemical Reagent Co., Ltd.; microwave extraction apparatus (model XH-300B) was provided by Beijing Xianghu Technology Development Co., Ltd.; all experiments were performed in triplicate, and data are expressed as mean ± SD. Statistical analysis of the data was performed using t-test or ANOVA, and P < 0.05 was considered statistically significant.

[0043] Example 1: Preparation of crude polysaccharide from waste leaves of water chestnut

[0044] Fresh water chestnut waste leaves were washed, dried at 60℃ to constant weight, pulverized, and passed through a 40-mesh sieve to obtain water chestnut waste leaf powder for later use. The water chestnut waste leaf powder was soaked in warm water at a certain material-to-liquid ratio for 8 hours, then the pH was adjusted to 5.0±0.2 with phosphate buffer, a certain amount of cellulase was added, and incubated in a shaker at 50℃ for 2 hours. After incubation, the enzyme hydrolysate was placed in a boiling water bath for 15 minutes to inactivate the enzyme. After cooling, the pH was adjusted to 7.0±0.2 with phosphate buffer, and the enzyme hydrolysate was placed in a microwave extractor for further processing. After processing at high power for a period of time, the supernatant was collected by centrifugation. The supernatant was concentrated to obtain a concentrate. Three times the volume of anhydrous ethanol was added to the concentrate and precipitated at 4°C for 12 hours. The precipitate was obtained by centrifugation, and then water was added to dissolve it. The concentrate was then mixed with Sevag reagent (n-butanol: chloroform = 1:4 (v / v)) at a ratio of 4:1 and shaken thoroughly for 15 minutes before centrifugation. After centrifugation, the supernatant was aspirated, and the Sevag deproteinization was repeated until no more protein was produced. The supernatant was freeze-dried to obtain crude polysaccharide from waste leaves of water chestnut.

[0045] 1.1 Single-factor experiment

[0046] Process parameters: Following the method in Example 1, experiments were conducted by changing individual factors. The parameters were set as follows: material-to-liquid ratio 1:40 g / mL, enzyme addition amount 0.7%, enzymatic hydrolysis temperature 50℃, enzymatic hydrolysis time 60 min, microwave time 10 min, microwave power 500 W, and microwave temperature 70℃.

[0047] 1.1.1 Effect of material-to-liquid ratio on the yield of polysaccharides from waste leaves of water chestnut

[0048] When investigating the effect of the material-to-liquid ratio on the crude polysaccharide yield, the material-to-liquid ratio was varied while other factors remained constant. Crude polysaccharide yield (%) = m / M × 100, where: m is the mass (g) of crude polysaccharide from freeze-dried water chestnut waste leaves; M is the mass (g) of water chestnut waste leaf powder (the same applies below). The results are as follows... Figure 1 As shown.

[0049] Depend on Figure 1 It was found that the highest yield of bract polysaccharides (0.37%) was achieved when the material-to-liquid ratio was 1:40. When the solvent volume was small, the mixing effect deteriorated, hindering sufficient contact between the plant material, the extraction solvent, and the enzymes, and also impeding the diffusion and dissolution of the target compound. Conversely, when the solvent volume was too large, the temperature rise after absorbing microwave energy decreased, leading to a lower yield. Therefore, the optimal material-to-liquid ratio was determined to be 1:40 g / mL.

[0050] 1.1.2 Effect of enzyme addition amount on the yield of polysaccharides from waste leaves of water chestnut

[0051] When investigating the effect of enzyme dosage on crude polysaccharide yield, the enzyme dosage was varied while other factors remained constant (the material-to-liquid ratio was set to 1.1.1; the following operations were similar, meaning each single factor was studied based on the previous single factor). The results are as follows... Figure 2 As shown.

[0052] Depend on Figure 2 It can be seen that as the amount of enzyme added increases, the yield of polysaccharides from waste water chestnut leaves initially rises and then falls, reaching its maximum at 0.44% when the enzyme addition is 0.5%. This is likely because, with other factors remaining constant, the continuous increase in cellulase addition disrupts the cellulose structure, promoting polysaccharide dissolution. Once the enzyme concentration in the solution reaches saturation, further increasing the addition will block the polysaccharide dissolution channels, and the increased enzyme concentration can also cause competition or even inhibition between enzymes. Therefore, the optimal cellulase addition amount is 0.5%.

[0053] 1.1.3 Effect of enzymatic hydrolysis temperature on the yield of polysaccharides from waste leaves of water chestnut

[0054] In investigating the effect of enzymatic hydrolysis temperature on the yield of crude polysaccharides, the enzymatic hydrolysis temperature was varied while other factors remained constant. The results are as follows: Figure 3 As shown.

[0055] Depend on Figure 3 It can be seen that as the enzymatic hydrolysis temperature increases, the polysaccharide yield first increases and then decreases, reaching its highest at 55℃. This is because the enzyme's catalytic effect is maximized at 55℃; excessively high or low temperatures will lead to a decrease in enzyme activity and a weakening of the enzymatic reaction, thus reducing the polysaccharide yield. Therefore, the optimal enzymatic hydrolysis temperature is 55℃.

[0056] 1.1.4 Effect of enzymatic hydrolysis time on the yield of polysaccharides from waste leaves of water chestnut

[0057] In investigating the effect of enzymatic hydrolysis time on the yield of crude polysaccharides, the hydrolysis time was varied while other factors remained constant. The results are as follows: Figure 4 As shown.

[0058] Depend on Figure 4 It can be seen that the polysaccharide yield is highest when the enzymatic hydrolysis time is 60 minutes. This is because if the hydrolysis time is too short, the reaction between the enzyme and the substrate is insufficient, resulting in a lower yield. Furthermore, as the hydrolysis time increases, the polysaccharide yield no longer increases. When the enzyme is sufficient, extending the hydrolysis time can lead to polysaccharide degradation, actually decreasing the yield. Therefore, the optimal hydrolysis time is 60 minutes.

[0059] 1.1.5 Effect of microwave time on the yield of polysaccharides from waste leaves of water chestnut

[0060] In investigating the effect of microwave time on the yield of crude polysaccharides, the microwave time was varied while other factors remained constant. The results are as follows: Figure 5 As shown.

[0061] Depend on Figure 5 It was found that the highest yield of polysaccharides from waste water chestnut leaves (0.59%) was achieved when the microwave treatment time was 7 minutes. The yield decreased significantly after 7 minutes. This is likely because most of the polysaccharides in the sample had already dissolved after 7 minutes of microwave-assisted extraction; further extending the treatment time would exacerbate the degradation of polysaccharides by microwaves and the generation of impurities such as proteins. Therefore, the optimal microwave treatment time is 7 minutes.

[0062] 1.1.6 Effect of microwave power on the yield of polysaccharides from waste leaves of water chestnut

[0063] In investigating the effect of microwave power on the yield of crude polysaccharides, the microwave power was varied while other factors remained constant. The results are as follows: Figure 6 As shown.

[0064] Depend on Figure 6 It can be seen that within the microwave power range of 200-600W, the yield of polysaccharides from waste water chestnut leaves initially increases and then decreases with increasing microwave power, reaching a maximum of 0.73% at a power of 400W. Microwaves help raise the internal temperature of the material, accelerating the dissolution of polysaccharides and thus significantly increasing the yield of polysaccharides from waste water chestnut leaves. However, excessive microwave power hinders the penetration of the medium, and excessive heat can also damage the polysaccharide structure, leading to a decrease in polysaccharide yield. Therefore, the optimal microwave power is 400W.

[0065] 1.1.7 Effect of microwave temperature on the yield of polysaccharides from waste leaves of water chestnut

[0066] In investigating the effect of microwave temperature on the yield of crude polysaccharides, the microwave temperature was varied while other factors remained constant. The results are as follows: Figure 7 As shown.

[0067] Depend on Figure 7 It is observed that the yield of polysaccharides initially increases slowly with increasing microwave temperature, then gradually decreases, reaching its optimal level at 80℃. Increased temperature intensifies molecular motion, expanding cell diffusion and leading to an increase in polysaccharides in the solvent. When the temperature exceeds 80℃, the polysaccharide structure is disrupted, and impurities increase accordingly. Therefore, the optimal microwave temperature is 80℃.

[0068] 1.2 Plackett-Burman Test

[0069] Based on the single-factor experiments, Plackett-Burman experiments were conducted using Design-Expert V8.0.6.1 software to design experiments for seven factors: enzyme addition amount A (%), material-liquid ratio B (g / mL), enzymatic hydrolysis time C (min), microwave time D (min), microwave power E (w), enzymatic hydrolysis temperature F (°C), and microwave temperature G (°C). Each factor was set to two levels: high (1) and low (-1) (Table 1). Four additional virtual terms were also set up to design 12 PB experiments. The design scheme and results are shown in Table 2, and the results of the analysis of variance are shown in Table 3.

[0070] Table 1 Plackett-Burman Design Factor Levels

[0071]

[0072] Table 2 Results of the Plackett-Burman Experimental Design

[0073]

[0074] Table 3. Analysis of Variance for Plackett-Burman Experimental Design

[0075]

[0076] Note: R 2 =0.9869, Adj R 2 =0.9639, Pred R 2 =0.9013; Significance: * indicates a significant difference, P<0.05; ** indicates an extremely significant difference, P<0.01; ns indicates no significant difference, P>0.05.

[0077] As shown in Table 3 above, the enzyme addition amount, microwave time, and microwave power have a highly significant impact on the yield of polysaccharides from waste leaves of water chestnut. Therefore, in the subsequent response surface methodology analysis, only the effects of these three factors on the response values ​​will be considered.

[0078] 1.3 Response Surface Experiment

[0079] Based on the experiment in 1.2, the Box-Behnken method in Design-Expert V8.0.6.1 software was used to design the experiment. A three-factor, three-level response surface optimization experiment was designed with enzyme addition (%), microwave time (min), and microwave power (W) as independent variables and polysaccharide yield (Y) as the response value. The levels of each factor in the response surface experiment are shown in Table 4, and the experimental scheme and results of the response surface analysis are shown in Table 5.

[0080] Table 4 Box-Behnken Design Factor Levels

[0081]

[0082] Table 5. Experimental scheme and results of response surface methodology.

[0083]

[0084]

[0085] Through multiple regression fitting, the quadratic regression model equation of the predicted value of Y on the encoded values ​​of X1, X2, and X3 is obtained:

[0086] Y=0.80+0.037×X1-0.016×X2+0.088×X3-0.070×X1 X2-0.065×X1 X3+0.041×X2 X3-0.21×X1 2 -0.25×X2 2 -0.17×X3 2

[0087] The results of the analysis of variance for the regression model are shown in Table 6.

[0088] Table 6. Results of ANOVA for the Regression Model

[0089]

[0090] Note: R 2 =0.9881, Adj R 2 =0.9728, Pred R 2 =0.9052; Significance: * indicates a significant difference, P<0.05; ** indicates an extremely significant difference, P<0.01; ns indicates no significant difference, P>0.05.

[0091] Table 6 shows that the p-value of this model is <0.0001, indicating that the selected model is highly significant; the p-value of the lack-of-fit term is >0.05 and not significant, indicating that the model fits well and the unknown factors have little interference with the experimental results; the coefficient of determination R is... 2 =0.9881, indicating that 98.81% of the change in the response value originates from the selected variable; R 2 adj =0.9728, meaning the model can explain 97.28% of the variability in the experimental data. Analysis of variance of the lack-of-fit term shows that the lack-of-fit term is not significant (P>0.05), indicating that the model is stable and can predict the actual variation in polysaccharide yield from discarded water chestnut leaves well.

[0092] Analysis using Design-Expert software revealed the following conditions for the synergistic microwave preparation of crude polysaccharides from waste water chestnut leaves using cellulase: cellulase dosage 0.505%, microwave treatment time 6.938 min, and microwave power 425.39 W. Under these conditions, the model predicted a yield of 0.808% for crude polysaccharides from waste water chestnut leaves. Based on the software predictions and the feasibility of the actual process settings, the extraction conditions were verified using 0.5% cellulase dosage, 7.0 min microwave treatment time, and 425 W microwave power. The experiment verified that the actual yield of polysaccharides from waste water chestnut leaves was 0.82 ± 0.08%, with a relative error of 1.49% (< 5.0%) compared to the theoretical prediction. This indicates that the optimized extraction process for polysaccharides from waste water chestnut leaves based on this response surface methodology is effective and feasible.

[0093] Comparative Example

[0094] Based on the optimal process obtained in Example 1 (i.e., the ratio of waste water bamboo leaf powder to water is 1:40 g / mL, the amount of cellulase added is 0.5% of the weight of waste water bamboo leaf powder, the enzymatic hydrolysis temperature is 55℃, the enzymatic hydrolysis time is 60 min, the microwave time is 7 min, the microwave power is 425 W, and the microwave temperature is 80℃), in order to prove that the method of cellulase-assisted microwave extraction of polysaccharides from waste water bamboo leaves provided by the present invention is superior to the methods of hot water extraction, without enzymes, and without microwave treatment, the present invention conducted the following comparative experiment:

[0095] Option A: Hot Water Extraction (HWE)

[0096] Waste water chestnut leaf powder was soaked in water at a material-to-liquid ratio of 1:40 g / mL for 8 hours, and then extracted at 80℃ for 2.5 hours. After cooling, the supernatant was removed by centrifugation. The supernatant was concentrated, and three times the volume of anhydrous ethanol was added for alcohol precipitation at 4℃ for 12 hours. The precipitate was obtained by centrifugation, and then dissolved in water. It was then mixed with Sevag reagent (n-butanol: chloroform = 1:4 (v / v)) at a ratio of 4:1 and shaken thoroughly for 15 minutes before centrifugation. After centrifugation, the supernatant was aspirated, and the Sevag deproteinization was repeated until no more protein was produced. The supernatant was freeze-dried to obtain crude polysaccharide from waste water chestnut leaves. The yield of crude polysaccharide from waste water chestnut leaves prepared by the HWE method was calculated.

[0097] The results showed that the yield of crude polysaccharide from waste leaves of water chestnut was only 0.38±0.08% without the addition of enzymes or microwave treatment.

[0098] Option B: Cellulase Extraction Method (CCE)

[0099] Waste water chestnut leaf powder was soaked in water at a ratio of 1:40 g / mL for 8 hours. The pH was then adjusted to approximately 5.0, and 0.5% cellulase (based on the weight of the water chestnut powder) was added. The mixture was incubated in a shaker at 50°C for 2 hours. After incubation, the enzyme was inactivated in a boiling water bath for 15 minutes. After cooling, the pH was adjusted to 7.0, and the supernatant was collected by centrifugation. The supernatant was concentrated, and three times its volume of anhydrous ethanol was added for alcohol precipitation at 4°C for 12 hours. The precipitate was obtained by centrifugation, dissolved in water, and then mixed with Sevag reagent (n-butanol: chloroform = 1:4 (v / v)) at a ratio of 4:1. The mixture was shaken thoroughly for 15 minutes and then centrifuged. The supernatant was aspirated after centrifugation, and the Sevag deproteinization process was repeated until no more protein was produced. The supernatant was freeze-dried to obtain crude polysaccharide from waste water chestnut leaves. The yield of crude polysaccharide from waste water chestnut leaves prepared by the CCE method was calculated.

[0100] The results showed that the yield of crude polysaccharide from waste leaves of water chestnut was only 0.46±0.05% when only enzyme was added and no microwave treatment was added.

[0101] Option C: Hot water / microwave extraction (HME)

[0102] Waste water chestnut leaf powder was soaked in water at a ratio of 1:40 g / mL for 8 hours, then extracted at 80℃ for 2.5 hours. After cooling, the extract was placed in a microwave extractor and treated for 7.0 minutes at a microwave power of 425W. After treatment, the supernatant was collected by centrifugation, concentrated, and precipitated with 3 times the volume of anhydrous ethanol at 4℃ for 12 hours. The precipitate was obtained by centrifugation, dissolved in water, and then mixed with Sevag reagent (n-butanol: chloroform = 1:4 (v / v)) at a ratio of 4:1 and shaken thoroughly for 15 minutes before centrifugation. The supernatant was aspirated after centrifugation, and the Sevag deproteinization was repeated until no more protein was produced. The supernatant was freeze-dried to obtain crude polysaccharide from waste water chestnut leaves. The yield of crude polysaccharide from waste water chestnut leaves prepared by the HME method was calculated.

[0103] The results showed that the yield of crude polysaccharide from waste leaves of water chestnut was only 0.42±0.06% under the condition of hot water combined with microwave treatment without enzyme addition.

[0104] Therefore, the yield of crude polysaccharide from waste leaves of water bamboo shoots obtained by the present invention using cellulase-assisted microwave extraction is higher than that obtained by hot water extraction, cellulase extraction, and hot water / microwave extraction. This indicates that the addition of cellulase and synergistic microwave treatment can effectively promote the release of polysaccharides from waste leaves of water bamboo shoots.

[0105] Example 2: Isolation, purification, and determination of antioxidant and anticoagulant activities of crude polysaccharides from waste leaves of water chestnut.

[0106] Weigh 100 mg of crude polysaccharide from waste leaves of water chestnut (prepared using the optimal process parameters in Example 1) and dissolve it in 20 mL of deionized water. Load the solution into a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm). Elute sequentially with deionized water and 0.1 mol / L to 0.4 mol / L NaCl solution at a flow rate of 1 mL / min, 5 mL per tube. Plot the elution curve. Figure 8 Collect the polysaccharide fraction eluted with deionized water, concentrate it, dialyze it three times, and freeze-dry it. Weigh 50 mg of freeze-dried water chestnut waste leaf polysaccharide, dissolve it in 10 mL of deionized water, and load it onto a Sephadex G-100 dextran gel column for further separation and purification. Use deionized water as the eluent, with a flow rate of 0.25 mL / min. Collect 4 mL from each tube and plot the elution curve. Figure 9 Collect the polysaccharide components washed with deionized water, concentrate them, dialyze them three times, and freeze-dry them to obtain the purified polysaccharide components from the waste leaves of water chestnut.

[0107] Using DPPH radical scavenging capacity and hydroxyl radical scavenging capacity as evaluation indicators, the purified polysaccharide fraction from waste leaves of water chestnut was prepared into 0.5 mg / mL groups. -1 ~2.5mg / mL -1Vitamin C was used as a positive control; the experimental methods and procedures were as per the kit instructions.

[0108] Thrombin time (TT) and prothrombin time (PT) were used as indicators to evaluate anticoagulant activity; the purified polysaccharide from waste leaves of water chestnut was prepared into a fraction of 0.31 mg / mL. -1 ~5.00mg.mL -1 Use physiological saline as a blank control; refer to the kit instructions for experimental methods and procedures.

[0109] Depend on Figure 10 , 11 It can be seen that the DPPH radical scavenging ability and hydroxyl radical scavenging ability of the polysaccharide components of waste leaves of water chestnut increased with the increase of polysaccharide concentration (0.5-2.5 mg / mL). When the concentration was 2.5 mg / mL, the DPPH radical scavenging rate was 43.7% and the hydroxyl radical scavenging rate was 52.1%.

[0110] Depend on Figure 12 It can be seen that when the concentration of polysaccharide component from waste leaves of water bamboo is 5.00 mg / mL, the TT value reaches 16.1±0.36s, which is significantly higher than that of physiological saline (10.8±0.85s); at the same concentration, the polysaccharide component from waste leaves of water bamboo can also significantly prolong the PT value, which is 17.1±1.05s, which is significantly higher than that of physiological saline (12.1±0.6s).

[0111] Comparative Example

[0112] Based on Example 2, in order to demonstrate that the polysaccharide component of waste water chestnut leaves provided by this invention has superior antioxidant and anticoagulant effects compared to the polysaccharide component eluted with NaCl solution, the following comparative experiment was conducted:

[0113] Scheme E: Determination of the antioxidant and anticoagulant activities of the polysaccharide fraction obtained by elution with 0.1 mol / L NaCl

[0114] 100 mg of crude polysaccharide from waste water chestnut leaves (prepared using the optimal process parameters in Example 1) was weighed and dissolved in 20 mL of deionized water. The solution was loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm). Elution was performed with 0.1 mol / L NaCl solution at a flow rate of 1 mL / min, 5 mL per tube. An elution curve was plotted. The eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and then freeze-dried. 50 mg of freeze-dried waste water chestnut leaf polysaccharide was weighed and dissolved in 10 mL of deionized water. The solution was loaded onto a Sephadex G-100 dextran gel column for further separation and purification. Deionized water was used as the eluent at a flow rate of 0.25 mL / min, 4 mL per tube. An elution curve was plotted. The eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and then freeze-dried to obtain the purified waste water chestnut leaf polysaccharide fraction eluted with 0.1 mol / L NaCl solution.

[0115] At a concentration of 2.5 mg / mL, the DPPH radical scavenging rate and hydroxyl radical scavenging rate reached their highest values, at (8.59±1.23)% and (39.29±3.12)%, respectively. At a concentration of 5.00 mg / mL, the TT and PT values ​​also reached their highest values, at 11.2±0.15 s and 12.9±0.85 s, respectively. The antioxidant and anticoagulant activities were lower than those of the purified polysaccharide fraction from waste leaves of *Zizania latifolia* prepared at the same concentration in Example 2.

[0116] Scheme F: Determination of the antioxidant and anticoagulant activities of polysaccharide fractions eluted with 0.2 mol / L NaCl

[0117] 100 mg of crude polysaccharide from waste water chestnut leaves (prepared using the optimal process parameters in Example 1) was weighed and dissolved in 20 mL of deionized water. The solution was loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm). Elution was performed with 0.2 mol / L NaCl solution at a flow rate of 1 mL / min, 5 mL per tube. An elution curve was plotted. The eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and then freeze-dried. 50 mg of freeze-dried waste water chestnut leaf polysaccharide was weighed and dissolved in 10 mL of deionized water. The solution was loaded onto a Sephadex G-100 dextran gel column for further separation and purification. Deionized water was used as the eluent at a flow rate of 0.25 mL / min, 4 mL per tube. An elution curve was plotted. The eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and then freeze-dried to obtain the waste water chestnut leaf polysaccharide fraction eluted with 0.2 mol / L NaCl solution. At a concentration of 2.5 mg / mL, the DPPH radical scavenging rate and hydroxyl radical scavenging rate reached their highest values, at (8.59±1.23)% and (39.29±3.12)%, respectively. At a concentration of 5.00 mg / mL, the TT and PT values ​​also reached their highest values, at 12.1±0.25 s and 12.6±1.05 s, respectively. The antioxidant and anticoagulant activities were lower than those of the purified polysaccharide fraction from waste leaves of *Zizania latifolia* prepared at the same concentration in Example 2.

[0118] Scheme G: Determination of the antioxidant and anticoagulant activities of the polysaccharide fraction obtained by elution with 0.4 mol / L NaCl

[0119] 100 mg of crude polysaccharide from waste water chestnut leaves (prepared using the optimal process parameters in Example 1) was weighed and dissolved in 20 mL of deionized water. The solution was loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm). Elution was performed with 0.4 mol / L NaCl solution at a flow rate of 1 mL / min, 5 mL per tube. An elution curve was plotted. The eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and then freeze-dried. 50 mg of freeze-dried waste water chestnut leaf polysaccharide was weighed and dissolved in 10 mL of deionized water. The solution was loaded onto a Sephadex G-100 dextran gel column for further separation and purification. Deionized water was used as the eluent at a flow rate of 0.25 mL / min, 4 mL per tube. An elution curve was plotted. The eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and then freeze-dried to obtain the waste water chestnut leaf polysaccharide fraction eluted with 0.4 mol / L NaCl solution. At a concentration of 2.5 mg / mL, the DPPH and hydroxyl radical scavenging rates reached their highest levels, at (26.66±1.57)% and (11.45±0.51)%, respectively. At a concentration of 5.00 mg / mL, the TT and PT values ​​also reached their highest levels, at 11.7±0.34 s and 11.9±1.23 s, respectively. The antioxidant and anticoagulant activities were lower than those of the waste leaf polysaccharide fraction from *Zizania latifolia* at the same concentration.

[0120] Therefore, using antioxidant activity (DPPH free radical and hydroxyl free radical scavenging ability) and anticoagulant activity (TT, PT) as evaluation indicators, the optimal separation and purification conditions for crude polysaccharides from waste leaves of water chestnut were determined as follows: The crude polysaccharide solution from waste leaves of water chestnut was loaded into a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm); eluted with deionized water at a flow rate of 1 mL / min, 5 mL per tube; the eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and freeze-dried; this polysaccharide solution was then loaded onto a Sephadex G-100 dextran gel column for further separation and purification, using deionized water as the eluent at a flow rate of 0.25 mL / min, 4 mL per tube; the eluted polysaccharide fraction was collected, concentrated, dialyzed three times, and freeze-dried to obtain the purified polysaccharide fraction from waste leaves of water chestnut.

[0121] Example 3: Component analysis and structural characterization of polysaccharide components purified from waste leaves of water chestnut (obtained under optimal separation and purification conditions).

[0122] The total sugar content in the polysaccharide fraction of waste leaves of water chestnut was determined to be 79.13±2.5% by the phenol-sulfuric acid method; the uronic acid content was determined to be 5.16±0.24% by the m-hydroxybiphenyl method; and the sulfate group content was determined to be 3.98±0.12% by the barium chloride gelatin turbidimetric method.

[0123] Ultraviolet-visible spectroscopy determination: A 1 mg / mL solution of polysaccharide from waste leaves of water chestnut was prepared, and distilled water was used as a blank for zeroing. The polysaccharide solution was scanned using an ultraviolet-visible spectrophotometer with wavelength parameters set to 200–400 nm and a scan interval of 0.5 nm.

[0124] Infrared spectroscopy determination: 1 mg of dried water chestnut waste leaf polysaccharide fraction was mixed with 100 mg of dried potassium bromide, compressed into a tablet, and then placed in an FTIR-650 infrared spectrometer for measurement at 4000 cm⁻¹. -1 ~400cm -1 Spectral scanning was performed within the wavelength range (International Journal of Biological Macromolecules, 2019, 123:280-290).

[0125] Monosaccharide composition determination: Accurately weigh 2 mg of polysaccharide fraction from waste leaves of water chestnut, place it in a hydrothermal reactor, add 2 mL of 2 mol / L trifluoroacetic acid (TFA), seal, and hydrolyze at 110℃~120℃ for 6 h; remove residual TFA, then add 200 μL of 0.5 mol / L methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) and 200 μL of 0.3 mol / L NaOH solution, mix, and react at 70℃ for 1.5 h; after cooling, add 0.3 mol / L HCl solution to stop the reaction; extract three times with chloroform, collect the aqueous layer, which is the PMP-derived monosaccharide; filter the sample with a 0.45 μm Millipore microporous membrane before injection, and the sample volume is 20 μL. Instruments and chromatographic conditions: Therm Ultimate 3000 high performance liquid chromatograph, equipped with Thermo U3000 diode array detector; Supersil ODS2 column (5μm, 4.6mm×250mm); mobile phase: PBS (pH=6.8): acetonitrile = 82:18 (v / v); flow rate: 0.8mL / min; column temperature: 30℃; detector wavelength: 245nm; run time: 50min (International Journal of Biological Macromolecules, 2019, 155:1105-1113).

[0126] Molecular weight determination: Polysaccharide components from waste leaves of water chestnut were prepared into a 2 mg / mL solution; the sample was filtered through a 0.45 μm Millipore microporous membrane before injection, and the injection volume was 20 μL; the instrument was an Elite P230II high performance liquid chromatograph equipped with a differential refractive index detector, and the chromatographic column was a TSK-GELG4000PWXL (7.8 mm × 300 mm) saccharide liquid chromatographic column; a series of standard sugar solutions with different molecular weights were prepared using dextran as standard sugars, and a third-order calibration curve for polysaccharide molecular weight determination was established based on retention time and molecular weight values; the chromatographic conditions were as follows: mobile phase: ultrapure water; flow rate: 1.0 mL / min; column temperature: 50 ℃; detector temperature: 30 ℃; run time: 30 min (International Journal of Biological Macromolecules, 2019, 123:280-290).

[0127] Depend on Figure 13 It was found that the polysaccharide component from waste water chestnut leaves showed no characteristic absorption peaks at 260 nm and 280 nm. This indicates that the polysaccharide component contained no impurities such as proteins and nucleic acids, and its polysaccharide purity was high. Furthermore, the UV-Vis spectrum of the polysaccharide component from waste water chestnut leaves showed a gradually decreasing trend, which is consistent with the general characteristics of polysaccharides.

[0128] Depend on Figure 14 It can be seen that at 3460cm -1 ~3430cm -1 Within the wavenumber range, a broad absorption peak appeared in the infrared spectrum of the polysaccharide component from waste leaves of *Zizania latifolia*. This peak was caused by the OH stretching vibration of the hydroxyl groups, indicating the presence of intramolecular or intermolecular hydrogen bonds in the polysaccharide structure. Based on this, the polysaccharide component from waste leaves of *Zizania latifolia* can be identified as a saccharide compound. The absorption peak was observed in the range of 1665–1635 cm⁻¹. -1 Within the wavenumber range, a relatively strong absorption peak appeared in the spectrum, which was caused by the hydroxyl vibration of the hydrate and is a characteristic absorption peak of sugar hydrates, indicating the presence of water of crystallization in the polysaccharide component of waste leaves of water chestnut. Additionally, the infrared spectrum showed a peak at 1616 cm⁻¹. -1 There were no -NH2 and -NH3 at the wavenumber. + The characteristic absorption peaks indicate that there are no proteoglycans in the polysaccharide components of waste leaves of water chestnut, which is consistent with the results of ultraviolet-visible spectroscopy analysis.

[0129] Depend on Figure 15 It can be seen that the polysaccharide component of waste leaves of water bamboo is composed of mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 12.3:4.75:2.25:1.00:2.50:7.00:24.3:2.00:17.3.

[0130] Depend on Figure 16 It can be seen that the polysaccharide component of waste leaves of water bamboo is a homogeneous polysaccharide with a molecular weight of 8.06 × 10⁻⁶. 3 kDa.

[0131] Example 4: Preparation of Oral Liquid Containing Purified Polysaccharide Components from Waste Water Bamboo Leaves

[0132] Add 1.0g of waste water chestnut leaf polysaccharide component to 100mL of purified water, stir at room temperature until dissolved, add 0.1g of sorbitol and 0.1g of sodium benzoate respectively, stir evenly, bottle and sterilize instantly, bottle (10mL / bottle), seal to obtain oral solution of waste water chestnut leaf polysaccharide component.

[0133] Example 5: Preparation of tablets containing purified polysaccharide components from waste leaves of water chestnut

[0134] Take 1.0g of waste water chestnut leaf polysaccharide component, mix it with 150mg of sodium carboxymethyl cellulose and 1.0mg of calcium hydrogen phosphate, grind it into a fine powder, and pass it through a 100-mesh sieve. While stirring, add 5% of 95% ethanol solution of povidone K30 to make a soft mass, and wet granulate it. After drying at 60℃, sieving and granulating, add 1% magnesium stearate and 2% micronized silica gel, mix evenly, and compress into tablets to obtain water chestnut waste leaf polysaccharide component tablets.

[0135] Example 6: Preparation of purified polysaccharide components from waste water chestnut leaves into capsules

[0136] Take 1.0g of polysaccharide component from waste leaves of water chestnut that has passed through a 40-mesh sieve, spray it with 95% ethanol at a 1:1 ratio, mix it evenly, add 10% starch to make a soft material, granulate it through a 20-mesh sieve, dry it in an oven at 60℃, then granulate it through a 20-mesh sieve again, and fill it into No. 3 empty capsules in an environment with a relative humidity of less than 65% to obtain capsules containing polysaccharide component from waste leaves of water chestnut.

[0137] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing polysaccharide components from waste leaves of water bamboo, comprising soaking waste leaf powder of water bamboo in distilled water, then adding a certain amount of cellulase for enzymatic hydrolysis, inactivating the enzyme, and then extracting it in a microwave extractor for a certain period of time; obtaining crude polysaccharide components from waste leaves of water bamboo by centrifugation, concentrating the supernatant, precipitating with ethanol, freeze-drying, and deproteinizing. The specific steps are as follows: Wash fresh water chestnut waste leaves, dry them to constant weight, crush them, and sieve them to obtain water chestnut waste leaf powder for later use; soak the water chestnut waste leaf powder in warm water at a certain material-to-liquid ratio, then adjust the pH value to 5.0±0.2, add a certain amount of cellulase, incubate in a shaker, and inactivate the enzyme in the enzymatic hydrolysate after the incubation is complete; after cooling, adjust the pH value to 7.0±0.2, place the enzymatic hydrolysate in a microwave extractor, process it at a certain power for a period of time, centrifuge to collect the supernatant, concentrate the supernatant, add anhydrous ethanol for alcohol precipitation, centrifuge to obtain the precipitate, then add water to dissolve it, then shake it thoroughly with Sevag reagent and centrifuge, aspirate the upper solution after centrifugation, repeat Sevag deproteinization until no more protein is produced, and freeze-dry the upper solution to obtain crude polysaccharide from water chestnut waste leaves; During the distilled water soaking process, the ratio of the waste water bamboo leaf powder to water is 1:20 g / mL to 1:60 g / mL; The amount of cellulase added is 0.3% to 0.7% of the weight of the waste water chestnut leaf powder; The enzymatic hydrolysis temperature is 45℃ ~ 65℃; The enzymatic hydrolysis time is 40 min ~ 80 min; The microwave duration is 1 min to 15 min; The microwave power is 200 W ~ 600 W; The microwave temperature is 60℃ ~ 100℃; The ratio of waste water bamboo leaf powder to water is 1:40 g / mL. The amount of cellulase added is 0.5% of the weight of waste water bamboo leaf powder. The enzymatic hydrolysis temperature is 55℃, the enzymatic hydrolysis time is 60 min, the microwave time is 7 min, the microwave power is 425 W, and the microwave temperature is 80℃. The crude polysaccharide from the waste leaves of water bamboo was separated and purified to obtain purified polysaccharide from the waste leaves of water bamboo. The separation and purification steps included purifying the crude polysaccharide from the waste leaves of water bamboo sequentially through a diethylaminoethyl (DEAE) cellulose-52 column and a Sephadex G-100 dextran gel column. The specific steps for separating and purifying the crude polysaccharide from the waste leaves of wild rice are as follows: The crude polysaccharide from waste water chestnut leaves was weighed and dissolved in deionized water, then loaded onto a diethylaminoethyl (DEAE) cellulose-52 column. Elution was performed with deionized water at a flow rate of 1 mL / min, 5 mL per tube, and an elution curve was plotted. The polysaccharide fraction eluted by deionized water was collected, concentrated, dialyzed three times, and then freeze-dried. The freeze-dried polysaccharide from waste water chestnut leaves was weighed and dissolved in deionized water, then loaded onto a Sephadex G-100 dextran gel column for further separation and purification. Deionized water was used as the eluent at a flow rate of 0.25 mL / min, 4 mL per tube, and an elution curve was plotted. The polysaccharide fraction eluted by deionized water was collected, concentrated, dialyzed three times, and then freeze-dried to obtain the purified polysaccharide fraction from the waste water chestnut leaves.

2. A purified polysaccharide component from waste leaves of water chestnut prepared by the method of claim 1, wherein the total sugar content of the component is 79.13 ± 2.5%, the uronic acid content is 5.16 ± 0.24%, and the sulfate group content is 3.98 ± 0.12%; the purified polysaccharide component from waste leaves of water chestnut is composed of mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose, in a molar ratio of 12.25:4.75:2.25:1.00:2.50:7.00:24.25:2.00:17.25, and the polysaccharide component from waste leaves of water chestnut is a homogeneous polysaccharide.

3. The use of the purified polysaccharide component from waste leaves of water chestnut prepared by the method of claim 1, for use in the preparation of antioxidant functional products or anticoagulant drugs.

4. The use according to claim 3, wherein the product or drug is an oral liquid, capsule or tablet.

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