Moringa oleifera leaf polysaccharide with immune enhancement and antioxidant activity and preparation and application thereof

Abstract

This invention discloses Moringa leaf polysaccharides with both immune-enhancing and antioxidant activities, as well as their preparation and applications. The method involves liquid nitrogen pulverization, ethanol purification, drying, cellulase hydrolysis, high-pressure homogenization, high-temperature water extraction, centrifugation, alcohol precipitation, ultrafiltration, and freeze-drying to prepare Moringa leaf polysaccharides. The obtained Moringa leaf polysaccharides exhibit good antioxidant activity, showing excellent performance in DPPH, ABTS free radical scavenging, and FRAP total antioxidant capacity assays. Simultaneously, they can promote NO secretion from RAW264.7 macrophages and enhance their phagocytic activity, demonstrating good immune-enhancing activity. The process of this invention is relatively simple, has good applicability in the food industry, and can be applied to various types of food processing.

Description

Technical Field

[0001] This invention belongs to the field of moringa leaf refining and high-value utilization, specifically involving moringa leaf polysaccharides with both immune-enhancing and antioxidant activities, as well as their preparation and application. Background Technology

[0002] With the continued aging of the population, the development of foods for the elderly and functional foods has received widespread attention. As people age, their physiological functions gradually decline, often accompanied by decreased antioxidant defense capabilities and weakened immune function. Therefore, developing foods for the elderly with antioxidant and immune-enhancing effects is of great significance. Moringa leaves contain various nutrients and functional components such as polysaccharides, proteins, vitamins, minerals, and polyphenols. Moringa leaf polysaccharides, in particular, are one of its important active ingredients and show promising application prospects in antioxidant and immune-enhancing applications. Therefore, developing a food product for the elderly containing moringa leaf polysaccharides has high application value and market potential.

[0003] In recent years, the extraction methods and physiological activities of Moringa leaf polysaccharides have gradually attracted attention. Currently, there are several research schemes for the preparation and application of Moringa leaf polysaccharides. For example, one team used microwave and ultrasound-assisted processes combined with deproteinization, decolorization, dialysis, resin separation, and alcohol precipitation to prepare Moringa leaf polysaccharides (application number: CN201510269590.2). This method can achieve the extraction and purification of Moringa leaf polysaccharides and has been applied to research related to hyperuricemia. However, it involves many extraction and purification steps, a long process flow, and the purity of the obtained polysaccharides is relatively low, leaving room for further improvement. Another technique uses petroleum ether degreasing combined with the Sevag method for protein removal and resin purification to prepare Moringa leaf polysaccharides (application number: CN201611071660.4). This method can improve the product purification level to a certain extent and can be used in research related to alcoholic liver disease. However, its preparation process uses multiple organic reagents such as petroleum ether, chloroform, and n-butanol, which presents problems such as cumbersome operation, insufficient safety, and environmental impact. Other studies have used acetic anhydride to acetylate Moringa leaf polysaccharides to obtain acetylated Moringa leaf polysaccharides (application number: CN202011597256.7). This method can modify the polysaccharide structure through chemical modification and is mainly used for hypoglycemic activity studies. However, acetic anhydride has certain irritant and corrosive properties, and subsequent steps such as neutralization, dialysis, and alcohol precipitation are required, making the process relatively cumbersome. Another technique uses ion exchange column chromatography to separate and purify crude Moringa leaf polysaccharides to obtain relatively pure polysaccharide components (application number: CN201810415879.4) and conduct anti-inflammatory activity studies. However, ion exchange column chromatography has a slow elution process, a long purification cycle, and requires subsequent dialysis desalting and lyophilization, which is not conducive to improving preparation efficiency. Another approach extracts polysaccharides from Moringa leaves, roots, and seeds separately (application number: CN202211195493.X), expanding the development scope of polysaccharide resources from different parts of Moringa. However, this approach mainly focuses on optimizing the extraction process and does not further verify the bioactivity of the obtained polysaccharides, especially lacking systematic research on immune-enhancing and antioxidant activities. In addition, another technique uses enzymatic hydrolysis, resin adsorption, ultrafiltration, and dialysis to purify Moringa leaf polysaccharides (application number: CN202310145439.2), which can significantly improve polysaccharide purity and enhance its blood sugar and lipid-lowering activities. However, this method still involves a long purification process and is time-consuming overall. Furthermore, its research focus is mainly on blood sugar and lipid-lowering, with relatively little research on immune-enhancing and antioxidant activities.

[0004] In summary, the existing technologies for the preparation and application of Moringa leaf polysaccharides still have the following problems: (1) Some technical solutions involve multiple steps such as defatting, deproteinization, decolorization, dialysis, resin separation and column chromatography in the extraction and purification of Moringa leaf polysaccharides, resulting in a long process flow and complicated operation, which is not conducive to improving the preparation efficiency; (2) Some methods use organic reagents such as petroleum ether, chloroform, n-butanol and acetic anhydride in the preparation process, which have certain safety, environmental protection and food field application limitations; (3) Existing research focuses on activities such as hyperuricemia, alcoholic liver disease, blood sugar reduction, blood sugar control and lipid reduction and anti-inflammation, while there are relatively few studies on the immune-enhancing activity and antioxidant activity of Moringa leaf polysaccharides, which is difficult to meet the development needs of related functional foods; (4) Some technical solutions focus on chemical modification, fine separation or preparation of specific components, which is helpful for carrying out structural or activity research, but is not conducive to the direct preparation of natural Moringa leaf polysaccharides and their promotion and application in the food field.

[0005] Therefore, this invention employs liquid nitrogen to thoroughly pulverize Moringa leaves, reducing the particle size. Then, ethanol is used to remove some fat-soluble components and small molecule impurities, followed by drying to minimize the impact of impurities and solvent residues on subsequent extraction. Subsequently, cellulase is used to enzymatically break down cell walls and release intracellular polysaccharides. High-pressure homogenization and high-temperature water extraction further dissolve the Moringa leaf polysaccharides. Finally, centrifugation, alcohol precipitation, ultrafiltration, and freeze-drying are used to obtain the Moringa leaf polysaccharides. The obtained Moringa leaf polysaccharides are of high purity and possess excellent immunomodulatory and antioxidant activities. This technology combines the advantages of high polysaccharide extraction efficiency, good functional activity, and suitability for food applications, demonstrating its potential for industrial-scale application. Summary of the Invention

[0006] The present invention aims to provide a Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities, its preparation method, and its applications. The preparation method, guided by immune-enhancing and antioxidant activities, involves liquid nitrogen pulverization, ethanol purification, drying, cellulase hydrolysis, high-pressure homogenization, and high-temperature water extraction, combined with centrifugation, alcohol precipitation, ultrafiltration, and freeze-drying to obtain a Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities. The obtained Moringa leaf polysaccharide exhibits good antioxidant activity as evaluated by DPPH free radical scavenging, ABTS free radical scavenging, and FRAP total antioxidant capacity. Simultaneously, experiments using RAW264.7 macrophages to measure NO secretion and phagocytic activity confirm its good immune-enhancing activity, demonstrating potential applications in elderly foods, general foods, and health foods. The preparation process of this invention is relatively simple, and the entire process is suitable for food applications, showing good development and application prospects.

[0007] The technical solution of the present invention is as follows: This invention provides a method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities. Using Moringa leaves as raw material, the method employs a refined and optimized process including liquid nitrogen pulverization, ethanol purification, drying, cellulase hydrolysis, high-pressure homogenization, high-temperature water extraction, centrifugation, alcohol precipitation, ultrafiltration, and freeze-drying to obtain a Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities. The Moringa leaf polysaccharide contains 82.125%–83.526% total sugar and 1.278%–1.296% protein. It exhibits good antioxidant activity, with DPPH free radical scavenging capacity of 49.83%–51.21%, ABTS free radical scavenging capacity of 45.66%–46.95%, and FRAP total antioxidant capacity of 26.75–27.23 μg / mL. It also possesses good immune-enhancing activity, with RAW264.7 macrophage NO secretion of 15.56–16.45 μmol / L and phagocytic activity of 163.35%–165.25%. The extraction process of this invention is relatively simple, and the entire process is suitable for application in the food industry, applicable to ordinary foods, health products, and foods for the elderly. The specific preparation method is as follows: (1) Liquid nitrogen pulverization: The dried moringa leaves are pulverized into powder using a liquid nitrogen pulverization device to obtain moringa leaf powder; (2) Ethanol purification: The Moringa leaf powder obtained in step (1) is subjected to ethanol purification treatment to remove some fat-soluble components and small molecule impurities, and material 1 is obtained after separation; (3) Drying: The material 1 obtained in step (2) is dried to obtain material 2; (4) Cellulase hydrolysis: Mix the material 2 obtained in step (3) with water evenly, adjust the pH and add cellulase, and carry out enzymatic hydrolysis under constant temperature and stirring conditions to obtain suspension 1. (5) High-pressure homogenization: The suspension 1 obtained in step (4) is subjected to high-pressure homogenization to obtain suspension 2; (6) High-temperature water extraction: The suspension 2 obtained in step (5) is heated and extracted to obtain suspension 3; (7) Centrifugation: Centrifuge the suspension 3 obtained in step (6), take the supernatant, and obtain extract 1; (8) Alcohol precipitation: Add ethanol to the extract 1 obtained in step (7) for alcohol precipitation, and separate the precipitate after standing to obtain polysaccharide precipitate; (9) Ultrafiltration: The polysaccharide precipitate obtained in step (8) is redissolved and then subjected to ultrafiltration to obtain a purified polysaccharide solution; (10) Freeze-drying: Freeze-dry the polysaccharide purified solution obtained in step (9) to obtain Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities.

[0008] Furthermore, in step (1), the liquid nitrogen pulverizer is pulverized at -150~-120℃.

[0009] Furthermore, in step (2), the concentration of ethanol used for impurity removal is 40%~60%, the impurity removal temperature is 25~35℃, and the impurity removal time is 30~90min.

[0010] Furthermore, in step (3), the drying temperature is 30~50℃.

[0011] Further, in step (4), the ratio of material 2 to water is 1:15~1:25 (g:mL), the enzymatic hydrolysis pH is 4~5, the amount of cellulase added is 0.8%~1.2% of the mass of material 2, the enzymatic hydrolysis temperature is 45~55℃, and the enzymatic hydrolysis time is 1~3h.

[0012] Furthermore, in step (5), the homogenization pressure of the high-pressure homogenization process is 10~30MPa.

[0013] Furthermore, in step (6), the extraction temperature of high-temperature water extraction is 80~100℃, and the extraction time is 30~90min.

[0014] Furthermore, in step (7), the centrifugation speed is 6000~10000 r / min and the centrifugation time is 10~20 min.

[0015] The present invention also provides a Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities prepared by the above preparation method. The total sugar content of the Moringa leaf polysaccharide is 82.125%~83.526%, and the protein content is 1.278%~1.296%.

[0016] This invention also provides the application of the above-mentioned Moringa leaf polysaccharide, which has both immune-enhancing and antioxidant activities, in the preparation of ordinary foods, health products, and foods for the elderly.

[0017] The present invention has the following beneficial effects: (1) The present invention uses liquid nitrogen pulverization, ethanol purification, cellulase hydrolysis, high pressure homogenization and high temperature water extraction, which can effectively promote the cell breakage of Moringa leaf and the dissolution of polysaccharide components, thereby preparing Moringa leaf polysaccharide with good quality and clear activity.

[0018] (2) The entire preparation process of this invention is relatively simple, suitable for application in the food industry, and has certain potential for industrial application.

[0019] (3) The Moringa leaf polysaccharide obtained in this invention has both immune-enhancing and antioxidant activities. It has good antioxidant activity and performs well in the determination of DPPH, ABTS free radical scavenging and FRAP total antioxidant capacity.

[0020] (4) The Moringa leaf polysaccharide obtained in this invention, which has both immune-enhancing and antioxidant activities, can promote NO secretion from RAW264.7 macrophages and enhance their phagocytic activity, thus exhibiting good immune-enhancing activity.

[0021] (5) The Moringa leaf polysaccharide obtained by the present invention has both immune-enhancing and antioxidant activities, and can be applied in the fields of ordinary food, health products and elderly food. Attached Figure Description

[0022] Figure 1 Bar chart showing the total sugar content of MHWA and MHWB, the hot water extract polysaccharides from Moringa leaves in Comparative Examples 1-2, MCA and MCB, the enzymatic hydrolysis extract polysaccharides from Moringa leaves in Comparative Examples 3-4, and MEA, MEB, and MEC, the Moringa leaf polysaccharides in Examples 1-3 that have both immune-enhancing and antioxidant activities.

[0023] Figure 2 Bar charts showing the protein content of MHWA and MHWB, hot water extract polysaccharides from Moringa leaves in Comparative Examples 1-2, MCA and MCB, enzymatic hydrolysis extract polysaccharides from Moringa leaves in Comparative Examples 3-4, and MEA, MEB, and MEC, polysaccharides from Moringa leaves in Examples 1-3 that have both immune-enhancing and antioxidant activities.

[0024] Figure 3 Bar charts showing the DPPH free radical scavenging rates of MHWA and MHWB, hot water extract polysaccharides from Moringa leaves in Comparative Examples 1-2, MCA and MCB, enzymatic hydrolysis extract polysaccharides from Moringa leaves in Comparative Examples 3-4, and MEA, MEB, and MEC, polysaccharides from Moringa leaves in Examples 1-3 that possess both immune-enhancing and antioxidant activities.

[0025] Figure 4 Bar charts showing the ABTS free radical scavenging rates of MHWA and MHWB, hot water extract polysaccharides from Moringa leaves in Comparative Examples 1-2, MCA and MCB, enzymatic hydrolysis extract polysaccharides from Moringa leaves in Comparative Examples 3-4, and MEA, MEB, and MEC, polysaccharides from Moringa leaves in Examples 1-3 that possess both immune-enhancing and antioxidant activities.

[0026] Figure 5 Bar charts showing the total FRAP antioxidant capacity of Moringa leaf polysaccharides MHWA and MHWB (comparative Examples 1-2), enzymatic hydrolysis and water extraction polysaccharides MCA and MCB (comparative Examples 3-4), and Moringa leaf polysaccharides MEA, MEB, and MEC (comparative Examples 1-3) which have both immune-enhancing and antioxidant activities.

[0027] Figure 6The bar chart shows the effects of hot water extract polysaccharides MHWA and MHWB from Moringa leaves (Comparative Examples 1-2), enzymatic hydrolysis extract polysaccharides MCA and MCB from Moringa leaves (Comparative Examples 3-4), and Moringa leaf polysaccharides MEA, MEB, and MEC (Examples 1-3) which have both immune-enhancing and antioxidant activities on NO secretion from RAW264.7 macrophages.

[0028] Figure 7 Bar chart showing the effects of hot water extract polysaccharides MHWA and MHWB from Moringa leaves (Comparative Examples 1-2), enzymatic hydrolysis extract polysaccharides MCA and MCB from Moringa leaves (Comparative Examples 3-4), and Moringa leaf polysaccharides MEA, MEB, and MEC (Examples 1-3) with both immune-enhancing and antioxidant activities on the phagocytic activity of RAW264.7 macrophages. Detailed Implementation Plan To better understand the present invention, the present invention will be further described below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0029] 1. DPPH free radical scavenging ability The polysaccharide sample was prepared to a concentration of 1 mg / mL, and then mixed with 0.2 mmol / L DPPH free radical solution at a ratio of 1:2 (V / V) to make a total volume of 3 mL. After mixing, the mixture was reacted in the dark for 30 min, and the absorbance at 517 nm was measured. The scavenging rate was calculated using the following formula.

[0030] DPPH free radical scavenging rate =

[0031] In the formula: A i Indicates the absorbance of the sample and the DPPH radical solution; A j A represents the absorbance of the sample solution; A0 represents the absorbance of the DPPH free radical solution.

[0032] 2. ABTS cationic free radical scavenging ability ABTS was prepared by mixing 2.5 mmol / L K2S2O8 solution and 7 mmol / L ABTS solution in a 1:1 (V / V) ratio and incubating in the dark for 12 hours. ＋ The mother liquor was diluted to an absorbance of 0.7 ± 0.02 at a wavelength of 734 nm, thus obtaining ABTS. + Working solution. Take 100 μL of the 1 mg / mL sample and mix it with 1 mL of ABTS. + The working solution was thoroughly mixed in a 1.5 mL centrifuge tube and reacted in the dark for 10 min. The absorbance was then measured immediately at a wavelength of 734 nm. The clearance rate was calculated using the following formula.

[0033] ABTS cationic radical scavenging rate = % In the formula: Ai Indicates sample and ABTS + Absorbance of the working fluid; A j Indicates the absorbance of the sample; A0 indicates ABTS. + Absorbance of the working fluid.

[0034] 3. FRAP method for determining total antioxidant capacity Prepare a FRAP working solution by mixing 0.3 mol / L sodium acetate buffer (pH 3.6), 0.01 mol / L TPTZ solution (prepared with 40 mmol / L HCl), and 0.02 mol / L FeCl3 aqueous solution in a 10:1:1 (V / V / V) ratio. In a 1.5 mL centrifuge tube, add 200 μL of a 1 mg / mL sample to 1000 μL of the FRAP working solution and react in the dark for 10 min. Measure the absorbance at 593 nm. Plot a standard curve using ascorbic acid (0-0.1 mg / mL) as the standard (y = 27.077x - 0.0496, R0). 2 The total antioxidant capacity of FRAP was calculated using the formula (=0.9963).

[0035] 4. Griess method for determining NO content RA264.7 cells were fed at a dose of 1×10⁻⁶. 6 Cells were seeded at a density of 100 cells / mL in 96-well plates and cultured for 24 h to allow them to adhere. Then, 100 μL of fresh culture medium and 100 μL of polysaccharide solution (400 μg / mL) were added to each well, and the cells were cultured for another 24 h. The cell culture supernatant was collected, and Griess' reagent 1 (sulfonamide) was added. The reaction was carried out in the dark for 10 min, followed by the addition of Griess' reagent 2 (naphthylethylenediamine dihydrochloride) and a further 10 min reaction in the dark. The absorbance at 540 nm was measured, and a standard curve was plotted using NaNO2 (0-50 μmol / L) as the standard. The NO content in the supernatant was calculated.

[0036] 5. Neutral red phagocytosis experiment RAW264.7 cells were fed at a concentration of 1×10⁻⁶. 6 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured for 24 h to allow cell adhesion. Then, 100 μL of fresh culture medium and 100 μL of polysaccharide solution (400 μg / mL) were added to each well, while the normal control group received 200 μL of fresh culture medium. After culturing for another 24 h, the supernatant was discarded, and the cells were washed twice with PBS. The original culture medium was replaced with 100 μL of 1 mg / mL neutral red, and the cells were cultured for another 1 h. After washing three times with PBS, 100 μL of cell lysis buffer (ethanol:acetic acid = 1:1 (V / V)) was added to each well, and the absorbance was measured at 540 nm.

[0037] Devouring ability (%) =

[0038] A1, A2, and A0 represent the absorbance of the sample group, the normal control group (without sample), and the blank control group (without cell seeding and without neutral red addition), respectively.

[0039] The above methods were used to determine the DPPH free radical scavenging capacity, ABTS cationic free radical scavenging capacity, NO secretion amount, and neutral red phagocytic capacity of the Moringa leaf polysaccharides obtained in the examples and comparative examples.

[0040] Example 1 A method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities includes the following steps: (1) Liquid nitrogen pulverization: 1000g of dried moringa leaves are placed in a liquid nitrogen pulverization device at -150℃ and pulverized into powder to obtain moringa leaf powder. Take 30g of moringa leaf powder as M1A.

[0041] (2) Ethanol removal: Moringa leaf powder M1A obtained in step (1) is added to a 40% (V / V) ethanol solution and removed for 30 min at 25℃. After separation, material M2A is obtained.

[0042] (3) Drying: The material M2A obtained in step (2) is dried at 30°C to obtain material M3A.

[0043] (4) Cellulase hydrolysis: Mix the material M3A obtained in step (3) with deionized water at a ratio of 1:15 (g:mL), adjust the pH to 4.0, mix thoroughly, add cellulase, the amount of cellulase added is 0.8% of the mass of material M3A obtained in step (3), and hydrolyze at a constant temperature of 45℃ for 1 h to obtain suspension S1A.

[0044] (5) High pressure homogenization: The suspension S1A obtained in step (4) is subjected to high pressure homogenization at a pressure of 10 MPa to obtain suspension S2A.

[0045] (6) High-temperature water extraction: The suspension S2A obtained in step (5) is heated and extracted at 80°C for 30 min to obtain suspension S3A.

[0046] (7) Centrifugation: Centrifuge the suspension S3A obtained in step (6) at 6000 r / min for 10 min, take the supernatant, and obtain the extract E1A.

[0047] (8) Alcohol precipitation: Add four times the volume of anhydrous ethanol to the extract E1A obtained in step (7) for alcohol precipitation. After standing for 12 hours, separate the precipitate to obtain polysaccharide precipitate M4A.

[0048] (9) Ultrafiltration: The polysaccharide precipitate M4A obtained in step (8) is reconstituted and then subjected to ultrafiltration to obtain polysaccharide purified solution P1A.

[0049] (10) Freeze-drying: Freeze-dry the polysaccharide purified solution P1A obtained in step (9) to obtain Moringa leaf polysaccharide sample MEA, which is Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities.

[0050] Example 2 A method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities includes the following steps: (1) Liquid nitrogen pulverization: 1000g of dried moringa leaves are placed in a liquid nitrogen pulverization device at -135℃ and pulverized into powder to obtain moringa leaf powder. Take 30g of moringa leaf powder as M1B.

[0051] (2) Ethanol removal: The Moringa leaf powder M1B obtained in step (1) is added to a 50% (V / V) ethanol solution and removed for 60 min at 30℃. After separation, material M2B is obtained.

[0052] (3) Drying: The material M2B obtained in step (2) is dried at 40°C to obtain material M3B.

[0053] (4) Cellulase hydrolysis: Mix the material M3B obtained in step (3) with deionized water at a ratio of 1:20 (g:mL), adjust the pH to 4.5, mix thoroughly, add cellulase, the amount of cellulase added is 1.0% of the mass of material M3B obtained in step (3), and hydrolyze at 50℃ for 2 hours to obtain suspension S1B.

[0054] (5) High pressure homogenization: The suspension S1B obtained in step (4) is subjected to high pressure homogenization at a pressure of 20 MPa to obtain suspension S2B.

[0055] (6) High-temperature water extraction: The suspension S2B obtained in step (5) is heated and extracted at 90°C for 60 min to obtain suspension S3B.

[0056] (7) Centrifugation: Centrifuge the suspension S3B obtained in step (6) at 8000 r / min for 15 min, take the supernatant, and obtain the extract E1B.

[0057] (8) Alcohol precipitation: Add four times the volume of anhydrous ethanol to the extract E1B obtained in step (7) for alcohol precipitation. After standing for 12 hours, separate the precipitate to obtain polysaccharide precipitate M4B.

[0058] (9) Ultrafiltration: The polysaccharide precipitate M4B obtained in step (8) is redissolved and then subjected to ultrafiltration to obtain polysaccharide purified solution P1B.

[0059] (10) Freeze-drying: Freeze-dry the polysaccharide purified solution P1B obtained in step (9) to obtain Moringa leaf polysaccharide sample MEB, which is Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities.

[0060] Example 3 A method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities includes the following steps: (1) Liquid nitrogen pulverization: 1000g of dried moringa leaves are placed in a liquid nitrogen pulverization device at -120℃ and pulverized into powder to obtain moringa leaf powder. Take 30g of moringa leaf powder as M1C.

[0061] (2) Ethanol removal: The Moringa leaf powder M1C obtained in step (1) is added to a 60% (V / V) ethanol solution and removed for 90 min at 35℃. After separation, material M2C is obtained.

[0062] (3) Drying: The material M2C obtained in step (2) is dried at 50°C to obtain material M3C.

[0063] (4) Cellulase hydrolysis: Mix the material M3C obtained in step (3) with deionized water at a ratio of 1:25 (g:mL), adjust the pH to 5.0, mix thoroughly, add cellulase, the amount of cellulase added is 1.2% of the mass of material M3C obtained in step (3), and hydrolyze at 55℃ for 3 hours to obtain suspension S1C.

[0064] (5) High pressure homogenization: The suspension S1C obtained in step (4) is subjected to high pressure homogenization at a pressure of 30 MPa to obtain suspension S2C.

[0065] (6) High-temperature water extraction: The suspension S2C obtained in step (5) is heated and extracted at 100℃ for 90 min to obtain suspension S3C.

[0066] (7) Centrifugation: Centrifuge the suspension S3C obtained in step (6) at 10000 r / min for 20 min, take the supernatant, and obtain the extract E1C.

[0067] (8) Alcohol precipitation: Add four times the volume of anhydrous ethanol to the extract E1C obtained in step (7) for alcohol precipitation. After standing for 12 hours, separate the precipitate to obtain polysaccharide precipitate M4C.

[0068] (9) Ultrafiltration: The polysaccharide precipitate M4C obtained in step (8) is redissolved and then subjected to ultrafiltration to obtain polysaccharide purified solution P1C.

[0069] (10) Freeze-drying: Freeze-dry the polysaccharide purified solution P1C obtained in step (9) to obtain Moringa leaf polysaccharide sample MEC, which is Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities.

[0070] Comparative Example 1 Hot water extraction method: 1000g of dried Moringa leaves were placed in a liquid nitrogen pulverizer at -135℃ and pulverized into powder to obtain Moringa leaf powder. 30g of Moringa leaf powder was taken as M1B-2. The obtained material M1B-2 was mixed with deionized water at a material-to-liquid ratio of 1:20 (g:mL) and extracted at 90℃ for 60min. After centrifugation at 8000r / min for 15min, the supernatant was collected. Four times the volume of anhydrous ethanol was added for alcohol precipitation. After standing for 12h, the precipitate was separated, reconstituted, and dialyzed at 3000Da. After freeze-drying, Moringa leaf polysaccharide sample MHWA was obtained, which is the hot water extract polysaccharide of Moringa leaves.

[0071] Comparative Example 2 Hot water extraction method: 1000g of dried Moringa leaves were placed in a liquid nitrogen pulverizer at -135℃ and pulverized into powder to obtain Moringa leaf powder. 30g of Moringa leaf powder was taken as M1B-3. The obtained Moringa leaf powder M1B-3 was added to a 50% (V / V) ethanol solution and purified at 30℃ for 60min. The purified material was dried at 40℃ and mixed evenly with deionized water at a material-to-liquid ratio of 1:20 (g:mL). The mixture was heated and extracted at 90℃ for 60min and centrifuged at 8000r / min for 15min. The supernatant was collected. Four times the volume of anhydrous ethanol was added for alcohol precipitation. After standing for 12h, the precipitate was separated, reconstituted, and dialyzed at 3000Da. The precipitate was then freeze-dried to obtain Moringa leaf polysaccharide sample MHWB, which is the hot water extracted polysaccharide of Moringa leaves.

[0072] Comparative Example 3 Enzymatic hydrolysis and water extraction: 1000g of dried Moringa leaves were placed in a liquid nitrogen pulverizer at -135℃ and pulverized into powder to obtain Moringa leaf powder. 30g of Moringa leaf powder was designated as M1B-4. The obtained Moringa leaf powder M1B-4 was added to a 50% (V / V) ethanol solution and purified at 30℃ for 60 min. The resulting material was then dried at 40℃. The dried material was mixed with deionized water at a material-to-liquid ratio of 1:20 (g:mL), the pH was adjusted to 4.5, and after thorough mixing, fiber was added. The amount of vitaminase and cellulase added was 1.0% of the material mass. The mixture was enzymatically hydrolyzed at 50℃ for 2 hours to obtain a suspension. The obtained suspension was heated and extracted at 90℃ for 60 minutes, centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. Four times the volume of anhydrous ethanol was added to the obtained supernatant for alcohol precipitation. After standing for 12 hours, the precipitate was separated. The obtained precipitate was redissolved and dialyzed. The precipitate was then freeze-dried to obtain the Moringa leaf polysaccharide sample MCA, which is the Moringa leaf enzymatic hydrolysis water-extracted polysaccharide.

[0073] Comparative Example 4 Enzymatic hydrolysis and water extraction: 1000g of dried Moringa leaves were placed in a liquid nitrogen pulverizer at -135℃ and pulverized into powder to obtain Moringa leaf powder. 30g of this powder was designated as M1B-5. The obtained Moringa leaf powder M1B-5 was added to a 50% (v / v) ethanol solution and purified at 30℃ for 60 minutes. The resulting material was then dried at 40℃. The dried material was mixed with deionized water at a ratio of 1:20 (g:mL), the pH was adjusted to 4.5, and after thorough mixing, cellulase was added. The amount added was 1.0% of the material mass, and the mixture was enzymatically hydrolyzed at 50℃ for 2 hours to obtain a suspension. The obtained suspension was homogenized under high pressure at 20MPa, then extracted at 90℃ for 60 minutes, centrifuged at 8000r / min for 15 minutes, and the supernatant was collected. Four times the volume of anhydrous ethanol was added to the obtained supernatant for alcohol precipitation. After standing for 12 hours, the precipitate was separated, the precipitate was redissolved and dialyzed, and then freeze-dried to obtain Moringa leaf polysaccharide sample MCB, which is Moringa leaf enzymatic hydrolysis water-extracted polysaccharide.

[0074] Results Analysis 1. Analysis of total sugar content of Moringa leaf polysaccharides Figure 1 The bar chart shows the total sugar content of the hot water extract polysaccharides MHWA and MHWB from Moringa leaves in Comparative Examples 1-2, the enzymatic hydrolysis extract polysaccharides MCA and MCB from Moringa leaves in Comparative Examples 3-4, and the polysaccharides MEA, MEB, and MEC from Moringa leaves in Examples 1-3, which have both immune-enhancing and antioxidant activities.

[0075] Figure 1The results show that as the process is gradually improved, the total sugar content of Moringa leaf polysaccharides shows a continuous upward trend, and the complete process group (Examples 1-3) always maintains the highest level. The total sugar content of MHWA obtained by hot water extraction is only 59.665±0.606%, indicating that under the condition of lack of pretreatment purification and enhanced extraction, the sample still retains a lot of non-sugar components. After adding ethanol to remove impurities, the total sugar content of MHWB increases to 69.546±0.859%, an increase of about 16.6%, and the difference is significant, indicating that ethanol pretreatment has a significant effect on removing some non-sugar impurities and increasing the relative proportion of polysaccharides. After further introducing cellulase enzymatic hydrolysis, the total sugar content of MCA continues to increase to 71.263±0.420%, indicating that enzymatic hydrolysis can destroy the cell wall structure and promote polysaccharide release. After adding high-pressure homogenization, the total sugar content of MCB further increases to 76.414±1.153%, indicating that homogenization treatment can further enhance tissue disruption and mass transfer. The total sugar content of the complete process group reached 82.125%~83.526%, significantly higher than that of the comparative groups, with no significant differences within the complete process group itself. Compared with MHWA, the complete process group showed an increase of approximately 37.6%~40.0%; compared with MCB, it still showed an increase of approximately 7.5%~9.3%. These results indicate that the complete process of this invention not only increases the polysaccharide release but also further enhances the polysaccharide enrichment in the final purification stage, with ultrafiltration being more effective than dialysis in removing small molecule impurities and improving the final purity.

[0076] 2. Analysis of the polysaccharide and protein content of Moringa leaves Figure 2 The bar chart shows the protein content of MHWA and MHWB, which are extracted from hot water from Moringa leaves in Comparative Examples 1-2; MCA and MCB, which are extracted from enzymatic hydrolysis of Moringa leaves in Comparative Examples 3-4; and MEA, MEB, and MEC, which are extracted from Moringa leaves in Examples 1-3 and have both immune-enhancing and antioxidant activities.

[0077] Figure 2 This more directly demonstrates the effectiveness of removing non-target components. The protein content of MHWA obtained by hot water extraction was as high as 36.682±1.088%, indicating that a large amount of protein and other accompanying components remained in the crude sample, resulting in low purity. After adding ethanol for impurity removal, the protein content of MHWB significantly decreased to 10.137±0.707%, a reduction of approximately 72.4%, demonstrating the significant effect of ethanol treatment in reducing impurity residue. Further enzymatic hydrolysis with cellulase further reduced the protein content of MCA to 1.422±0.152%, indicating that while polysaccharide release increased, the proportion of the target component in the sample significantly increased, and protein impurities were further "diluted" and separated.

[0078] It is noteworthy that there was no significant difference in protein content between the MCB after high-pressure homogenization and the complete process group, with the protein content of the complete process group remaining between 1.278% and 1.296%. This indicates that the key stages of protein removal mainly occur during ethanol purification and cellulase hydrolysis, while high-pressure homogenization and ultrafiltration are primarily used to improve polysaccharide quality and activity, rather than to further significantly reduce protein residue. In other words, the advantage of this invention is not simply reducing protein content, but rather, on the basis of already significantly reduced protein content, continuing to enhance the functional performance of the polysaccharide components.

[0079] 3. Analysis of the DPPH free radical scavenging ability of Moringa leaf polysaccharides Figure 3 The bar chart shows the DPPH free radical scavenging rate of MHWA and MHWB, which are extracted from hot water of Moringa leaves in Comparative Examples 1-2; MCA and MCB, which are extracted from enzymatic hydrolysis of Moringa leaves in Comparative Examples 3-4; and MEA, MEB, and MEC, which are extracted from Moringa leaves in Examples 1-3 and have both immune-enhancing and antioxidant activities.

[0080] Figure 3 The results showed that DPPH results were not synchronized with total sugar content. The most prominent finding was that MHWA, obtained through hot water extraction, exhibited the highest DPPH scavenging rate, at 58.65±0.57%. This indicates that the DPPH index is significantly affected by residual polyphenols and other small-molecule antioxidants in the sample, and the "high value" in the crude extract cannot be simply attributed to the polysaccharides themselves. After adding ethanol for impurity removal, the DPPH scavenging rate of MHWB significantly decreased to 38.05±0.88%, a reduction of approximately 35.1%, further demonstrating that the removed polyphenols and other components made a significant contribution to DPPH scavenging.

[0081] Subsequently, after enzymatic hydrolysis with cellulase, the DPPH scavenging ability of MCA significantly rebounded, indicating that after removing some small-molecule antioxidant components, the antioxidant effect of the polysaccharides themselves began to manifest as polysaccharide release increased. However, there was no significant difference between MCB and MCA after high-pressure homogenization, suggesting that high-pressure homogenization has a limited direct effect on increasing DPPH. The DPPH scavenging rate of the complete process group was 49.83%–51.21%, significantly higher than MCA and MCB, but still lower than MHWA. These results indicate that although the complete process group no longer relies on a large amount of residual polyphenols to provide apparent antioxidant value, it can still maintain a strong DPPH scavenging ability under high-purity conditions, thus better reflecting the antioxidant potential of the Moringa leaf polysaccharide prepared in this invention, which possesses both immune-enhancing and antioxidant activities.

[0082] 4. Analysis of the ABTS free radical scavenging ability of Moringa leaf polysaccharides Figure 4The bar chart shows the ABTS free radical scavenging rate of MHWA and MHWB, which are extracted from hot water of Moringa leaves in Comparative Examples 1-2; MCA and MCB, which are extracted from enzymatic hydrolysis of Moringa leaves in Comparative Examples 3-4; and MEA, MEB, and MEC, which are extracted from Moringa leaves in Examples 1-3 and have both immune-enhancing and antioxidant activities.

[0083] Compared with DPPH results, Figure 4 The ABTS results better demonstrate the advantages of the complete process in "preserving activity after purification". The ABTS scavenging rate of MHWA obtained by hot water extraction was 52.25±0.72%, still the highest among all groups, indicating that the polyphenols and small molecule reducing components retained in the crude extract without impurity removal significantly contribute to the ABTS system. After adding ethanol for impurity removal, the ABTS scavenging rate of MHWB decreased to 29.09±0.46%, a decrease of approximately 44.3%, indicating that ethanol treatment significantly weakened the antioxidant effect of this non-polysaccharide source.

[0084] Unlike DPPH, the recovery of ABTS in subsequent processes was more pronounced. After cellulase hydrolysis, the ABTS scavenging rate of MCA significantly increased, while there was no significant difference between MCB and MCA after high-pressure homogenization. The complete process group showed significantly higher ABTS scavenging rates than both MCA and MCB, reaching 45.66%–46.95%, an increase of approximately 23.5%–27.0% compared to MCB. This indicates that the complete process, especially end-stage ultrafiltration compared to dialysis, is more effective in retaining or enriching polysaccharide components with antioxidant properties, allowing the sample to maintain strong ABTS scavenging ability even under high-purity conditions. The ABTS results further demonstrate that this invention not only improves sample purity but also effectively preserves its activity.

[0085] 5. Analysis of total antioxidant capacity of FRAP Figure 5 The bar charts show the total FRAP antioxidant capacity of the moringa leaf hot water extract polysaccharides MHWA and MHWB of Comparative Examples 1-2, the moringa leaf enzymatic hydrolysis extract polysaccharides MCA and MCB of Comparative Examples 3-4, and the moringa leaf polysaccharides MEA, MEB, and MEC of Examples 1-3, which have both immune-enhancing and antioxidant activities.

[0086] Figure 5 The FRAP results shown reflect the changes in the overall reducing power of the samples. MHWA, obtained by hot water extraction, had the highest FRAP value of 30.31 ± 1.22 μg / mL, indicating that residual polyphenols, pigments, and other reducing components in the crude extract significantly increased its total FRAP antioxidant capacity. After adding ethanol to remove impurities, the FRAP value of MHWB decreased to 15.16 ± 1.13 μg / mL, a decrease of approximately 50.0%, indicating that these small molecule reducing components were significantly removed.

[0087] The subsequent trend was similar to the first two antioxidant indicators: after adding cellulase for enzymatic hydrolysis, the FRAP value increased again, while after high-pressure homogenization, there was no significant difference compared to the previous step. The FRAP value of the complete process group was 26.75~27.23 μg / mL, significantly higher than MCA and MCB, but lower than MHWA. Compared with MCB, the complete process group showed an increase of approximately 32.6%~34.9%. This result indicates that the complete process can improve the purity of polysaccharides while better preserving the total antioxidant capacity of the sample's FRAP. Combined with the DPPH and ABTS results, the high antioxidant performance of MHWA mainly comes from impurities, while the complete process group maintained a high FRAP level even after significantly reducing the impurity background. Therefore, it further demonstrates that the Moringa leaf polysaccharide prepared in this invention, which has both immune-enhancing and antioxidant activities, itself has good antioxidant activity.

[0088] 6. Analysis of NO secretion by RAW264.7 macrophages Figure 6 The bar chart shows the effects of hot water extract polysaccharides MHWA and MHWB from Moringa leaves (Comparative Examples 1-2), enzymatic hydrolysis extract polysaccharides MCA and MCB from Moringa leaves (Comparative Examples 3-4), and Moringa leaf polysaccharides MEA, MEB, and MEC (Examples 1-3) which have both immune-enhancing and antioxidant activities on NO secretion from RAW264.7 macrophages.

[0089] Figure 6 The NO secretion results shown differed significantly from the trends of the three antioxidant indicators, with the increasing trend more clearly reflecting the process's promoting effect on immune-enhancing activity. The lowest NO secretion was observed in MHWA obtained through hot water extraction, at only 1.05 ± 0.57 μmol / L, indicating that the high impurities and low purity of the crude extract were detrimental to macrophage stimulation. While the NO secretion of MHWB increased slightly after ethanol removal, the increase was limited. Further enzymatic hydrolysis with cellulase showed no significant difference between MCA and MHWB, indicating that simply increasing polysaccharide release was insufficient to significantly enhance NO secretion.

[0090] The most significant turning point for this indicator occurred during the high-pressure homogenization step. After adding high-pressure homogenization, the NO secretion of MCB significantly increased to 12.05 ± 0.37 μmol / L, an increase of approximately 246% compared to MCA, indicating that high-pressure homogenization is a key step in enhancing immunomodulatory activity. This is likely because homogenization not only improves extraction efficiency but also enhances the polysaccharide particle size distribution and dispersion, making it easier for them to interact with macrophage surface receptors. The complete process group further achieved 15.56–16.45 μmol / L, significantly higher than MCB, an increase of approximately 29.1%–36.5%. This demonstrates that ultrafiltration is more effective than dialysis in enriching polysaccharide components with genuine immunostimulatory effects, thereby maximizing the NO-promoting capacity of the sample.

[0091] 7. Analysis of macrophage phagocytic activity in RAW264.7 macrophages Figure 7 The bar chart shows the effects of hot water extract polysaccharides MHWA and MHWB from Moringa leaves (Comparative Examples 1-2), enzymatic hydrolysis extract polysaccharides MCA and MCB from Moringa leaves (Comparative Examples 3-4), and Moringa leaf polysaccharides MEA, MEB, and MEC (Examples 1-3) which have both immune-enhancing and antioxidant activities on the phagocytic activity of RAW264.7 macrophages.

[0092] Figure 7 The changes in phagocytic activity were highly consistent with the NO secretion results, indicating that the samples obtained from the complete process have good stability and reliability in terms of immune enhancement. The phagocytic activity of MHWA obtained by hot water extraction was 100.68±0.97%, close to the baseline level, indicating that the high proportion of impurities in the crude extract sample cannot effectively promote macrophage phagocytic function. After adding ethanol for impurity removal, the phagocytic activity of MHWB increased by approximately 19.1%, indicating that front-end purification helps reduce inhibitory interfering substances. Further enzymatic hydrolysis with cellulase showed no significant difference between MCA and MHWB, indicating that simply increasing polysaccharide release is insufficient to significantly enhance phagocytic activity.

[0093] Similar to the NO secretion results, after high-pressure homogenization, the phagocytic activity of MCB significantly increased to 149.82±1.19%, an increase of approximately 26.3% compared to MCA, indicating that high-pressure homogenization makes a crucial contribution to enhancing immune function. The complete process group further increased to 163.35%~165.25%, significantly higher than MCB, an increase of approximately 9.0%~10.3%. This suggests that after front-end extraction and homogenization enhancement, using ultrafiltration instead of dialysis at the end can further optimize the distribution of active components in the sample, thereby achieving the best effect of Moringa leaf polysaccharide in promoting macrophage phagocytic function.

[0094] Based on the results of total sugar content, protein content, antioxidant activity, and immune-enhancing activity, it can be seen that each process step in this invention has different but complementary roles in the preparation of Moringa leaf polysaccharides. Ethanol purification is mainly used for front-end purification, which can significantly increase the relative content of total sugar and significantly reduce impurities such as protein, while also removing some small molecule antioxidant activity derived from polyphenols; cellulase hydrolysis mainly promotes polysaccharide release by disrupting the cell wall, further increasing the total sugar content and enabling the antioxidant effect of the polysaccharides themselves to begin to manifest; high-pressure homogenization further increases the total sugar content, but its more prominent contribution is reflected in the immune activity, being a key step in significantly enhancing NO secretion and phagocytic activity; ultrafiltration is superior to dialysis in the final purification, not only further improving the purity of total sugar but also more effectively enriching polysaccharide components with antioxidant and immune-enhancing effects.

[0095] Overall, the hot water extraction groups (Comparative Examples 1 and 2) showed higher performance in some antioxidant indicators, mainly reflecting the combined contribution of impurities such as polyphenols in the crude extract samples. In contrast, the complete process group maintained strong antioxidant capacity while significantly improving polysaccharide purity and reducing protein impurities, and achieved optimal levels in immune-enhancing indicators. These results indicate that the complete process of this invention is more conducive to preparing Moringa leaf polysaccharides with both immune-enhancing and antioxidant activities.

[0096] This invention provides a method for preparing and applying Moringa leaf polysaccharides with both immune-enhancing and antioxidant activities. The method uses Moringa leaves as raw material, improves the dispersibility of the raw material through liquid nitrogen pulverization, reduces impurity interference by using ethanol for purification, and combines cellulase enzymatic hydrolysis, high-pressure homogenization, and high-temperature water extraction to promote the full release of polysaccharide components. The final product is obtained through alcohol precipitation, ultrafiltration, and freeze-drying. The obtained Moringa leaf polysaccharides have a high total sugar content and a low protein content, and exhibit good DPPH free radical scavenging ability, ABTS free radical scavenging ability, and FRAP total antioxidant capacity. Simultaneously, it can promote NO secretion from RAW264.7 macrophages and enhance their phagocytic activity, indicating that it possesses both good antioxidant and immune-enhancing activities. The process flow of this invention is reasonable, the equipment used is conventional and easy to operate, suitable for development and utilization in the food industry, and has good prospects for widespread application.

[0097] The above embodiments are merely preferred embodiments of the present invention and are used only to illustrate the present invention, not to limit the present invention. Any changes, substitutions, or modifications made to the present invention by those skilled in the art without departing from the spirit and substance of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities, characterized in that, Includes the following steps: (1) Liquid nitrogen pulverization: The dried moringa leaves are pulverized into powder using a liquid nitrogen pulverization device to obtain moringa leaf powder; (2) Ethanol purification: The Moringa leaf powder obtained in step (1) is subjected to ethanol purification treatment, and material 1 is obtained after separation; (3) Drying: The material 1 obtained in step (2) is dried to obtain material 2; (4) Cellulase hydrolysis: Mix the material 2 obtained in step (3) with water evenly, adjust the pH, add cellulase, and carry out enzymatic hydrolysis under constant temperature and stirring conditions to obtain suspension 1; (5) High-pressure homogenization: The suspension 1 obtained in step (4) is subjected to high-pressure homogenization to obtain suspension 2; (6) High-temperature water extraction: The suspension 2 obtained in step (5) is heated and extracted to obtain suspension 3; (7) Centrifugation: Centrifuge the suspension 3 obtained in step (6), take the supernatant, and obtain extract 1; (8) Alcohol precipitation: Add ethanol to the extract 1 obtained in step (7) for alcohol precipitation, and separate the precipitate after standing to obtain polysaccharide precipitate; (9) Ultrafiltration: The polysaccharide precipitate obtained in step (8) is redissolved and then subjected to ultrafiltration to obtain a purified polysaccharide solution; (10) Freeze-drying: Freeze-dry the polysaccharide purified solution obtained in step (9) to obtain Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities.

2. The method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities according to claim 1, characterized in that, In step (1), the liquid nitrogen pulverization is carried out at -150~-120℃.

3. The method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities according to claim 1, characterized in that, In step (2), the volume fraction of ethanol used for ethanol purification is 40%~60%, the purification temperature is 25~35℃, and the purification time is 30~90min.

4. The method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities according to claim 1, characterized in that, In step (3), the drying temperature is 30~50℃.

5. The method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities according to claim 1, characterized in that, In step (4), the ratio of material 2 to water is 1:15 to 1:25 in g / mL, the enzymatic hydrolysis pH is 4 to 5, the amount of cellulase added is 0.8% to 1.2% of the mass of material 2, the enzymatic hydrolysis temperature is 45 to 55℃, and the enzymatic hydrolysis time is 1 to 3 hours.

6. The method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities according to claim 1, characterized in that, In step (5), the homogenization pressure of the high-pressure homogenization process is 10~30MPa.

7. The method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities according to claim 1, characterized in that, In step (6), the extraction temperature of the high-temperature water extraction is 80~100℃ and the extraction time is 30~90min.

8. The method for preparing Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities according to claim 1, characterized in that, In step (7), the centrifugation speed is 6000~10000 r / min and the centrifugation time is 10~20 min.

9. A Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities, prepared by the method described in any one of claims 1 to 8, characterized in that, The total sugar content of the Moringa leaf polysaccharide, which has both immune-enhancing and antioxidant activities, is 82.125%~83.526%.

10. The application of the Moringa leaf polysaccharide with both immune-enhancing and antioxidant activities as described in claim 9 in the preparation of ordinary food, health products or food for the elderly.

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