A prickly pear polysaccharide extracted by an enzymatic method, its preparation method and application

Abstract

This invention discloses an enzymatically extracted prickly pear polysaccharide, its preparation method, and its applications. The preparation method mainly includes prickly pear powder pretreatment, enzymatic extraction, deproteinization, alcohol precipitation, and freeze-drying. Compared to the traditional hot water extraction method, the yield of prickly pear polysaccharide obtained by this invention is increased by more than three times, with a maximum yield of up to 14%. The prickly pear polysaccharide of this invention has a low molecular weight and is mainly composed of glucose, arabinose, galactose, galacturonic acid, and other small amounts of monosaccharides, exhibiting long side chains and numerous branched structures. The prickly pear polysaccharide of this invention possesses significant antioxidant, immunostimulatory, hepatocyte autophagy activation, and liver function improvement effects. It can be used as a functional ingredient in the preparation of health foods, dietary supplements, cosmetics, and other products for the food, daily chemical, biological, and pharmaceutical industries, and is of great significance for the deep processing and high-value utilization of prickly pear.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of prickly pear and preparation of functional foods, specifically relating to an enzymatically extracted prickly pear polysaccharide, its preparation method and application. Background Technology

[0002] Prickly pear is a regionally distinctive plant used for both food and medicine, mainly distributed in mountainous areas of South and Southwest my country at altitudes of 500-2500 meters, with Guizhou Province being the primary production area. The fruit of the prickly pear is a unique, highly nutritious, medicinally valuable, and versatile third-generation fruit, recorded in works such as the *Compendium of Materia Medica* and the *Chinese Pharmacopoeia*. Prickly pear contains various active nutritional components, including polysaccharides, polyphenols, vitamin C, superoxide dismutase, and trace elements, earning it the title of "Three Kings Sacred Fruit." With in-depth research, its nutritional and bioactive value has been gradually confirmed. Currently, it has been found to possess multiple functions such as anti-oxidation, anti-atherosclerosis, hypoglycemia, anti-radiation, anti-cancer, and anti-aging, attracting widespread attention.

[0003] Natural polysaccharides are an important class of bioactive macromolecules with few side effects, are easily digested and absorbed, have multiple targets, and offer multi-layered effects, making them of significant development and utilization value. As one of the important functional components of the prickly pear fruit, prickly pear polysaccharide has been extensively studied in recent years. For example, Chinese invention patent 201010550315.5 discloses a novel application of prickly pear polysaccharide for ionizing radiation protection. This patent describes a method of water extraction and alcohol precipitation, which demonstrates protective effects against V79 cells irradiated with ionizing radiation and Kunming mice irradiated with 60Co γ-rays. Chinese invention patent 200510110737.X discloses a novel function of prickly pear polysaccharide in treating neural stem cell damage. Chinese invention patent 201811209724.1 discloses a hot water extraction method for prickly pear polysaccharide, with a yield of 3.57%. The polysaccharide has an average molecular weight of 67.2 kDa and is composed of glucose, galactose, arabinose, xylose, and fucose. It has the ability to regulate intestinal flora and inhibit α-glucosidase, and can be used as an adjunct treatment for intestinal diseases and diabetes. Chinese invention patent 20221046880.7 discloses a prickly pear polysaccharide with significant lipid-lowering and cholesterol-lowering effects, its preparation method and application. This patent extracts prickly pear polysaccharide by alkaline extraction, with a yield of 6.26% and an average molecular weight of 37-42 kDa. It is composed of glucose, galactose, arabinose, xylose, mannose and uronic acid. It has a strong ability to adsorb bile salts and cholesterol, inhibits the enzymatic hydrolysis rate and fatty acid release during the digestion of fats, and reduces the triglyceride and cholesterol content in high-fat induced HepG2 cells.

[0004] Furthermore, existing plant polysaccharide extraction technologies primarily use water as the extraction solvent, supplemented by other methods to improve polysaccharide utilization, such as ultrasonic, microwave, and enzyme-assisted extraction. Among these, enzyme-assisted extraction utilizes the properties of enzymes to break down plant tissues and accelerate polysaccharide release. This technology is a mild, specific, rapid, highly efficient, environmentally friendly, and structurally sound extraction method. Compared to traditional hot water extraction, this method offers advantages such as high extraction efficiency, short extraction time, good reproducibility, and minimal impact on polysaccharide structure.

[0005] However, polysaccharides extracted by enzymatic methods differ significantly from those extracted by traditional hydrothermal methods in terms of composition and molecular weight. Therefore, it is of great significance and promise to find ways to obtain polysaccharides with higher value (such as high antioxidant activity, new drug activity, etc.) based on enzymatic extraction. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an enzymatically extracted prickly pear polysaccharide, its preparation method, and its applications. The enzymatically extracted prickly pear polysaccharide of the present invention possesses high antioxidant, immunomodulatory, autophagy-activating, and liver-damage-improving properties.

[0007] The objective of this invention is achieved through one of the following technical solutions:

[0008] A method for preparing prickly pear polysaccharide by enzymatic extraction includes the following steps:

[0009] 1) Raw material pretreatment: Wash and dry the dried prickly pear fruit, then remove the seeds, crush thoroughly, and sieve to obtain prickly pear powder; mix the prickly pear powder with ethanol at a material-to-liquid ratio of 1:5 to 1:10 g / mL, shake at room temperature for 1 to 3 hours, centrifuge to collect the residue, repeat the treatment 5 to 10 times, centrifuge to separate, collect the residue and dry to obtain pretreated prickly pear powder.

[0010] 2) Enzymatic extraction: Mix the pretreated prickly pear powder from step 1) with water at a mass-to-volume ratio of 1:35 to 1:50 g / mL, add 2 to 3 wt% of cellulase based on the dry weight of the prickly pear powder, adjust the pH of the solution to 5.5 to 6.5, and then heat and stir continuously at 40 to 50°C for 80 to 100 min. Then heat the extract at 90 to 100°C to inactivate the enzyme activity, centrifuge, discard the precipitate, and concentrate the supernatant to 1:4 to 1:10 of the original volume (volume before concentration).

[0011] 3) Deproteinization: Mix Sevag reagent with the concentrated extract from step 2) at a volume ratio of 1:3 to 1:5, shake for 10 to 20 minutes, centrifuge to obtain the upper layer of prickly pear polysaccharide solution, repeat 5 to 10 times, and evaporate under reduced pressure to remove residual Sevag reagent.

[0012] 4) Alcohol precipitation: Add ethanol to the prickly pear polysaccharide solution deproteinized in step 3) until the ethanol volume concentration is 80-90%, let it stand at 0-5℃ for 12-24h, centrifuge to collect the precipitate, wash it 2-3 times with anhydrous ethanol, then redissolve it with water, and freeze dry it at -45--60℃ to obtain the enzymatically extracted prickly pear polysaccharide.

[0013] Preferably, the sieving in step 1) is sieve 40 to 120 mesh.

[0014] Preferably, the centrifugal force in steps 1), 2), 3) and 4) is 4000-5000g, and the centrifugation time is 5-15min.

[0015] Preferably, the drying method in step 1) is blower drying, and the drying temperature is 40-50°C;

[0016] Preferably, the ethanol mentioned in steps 1) and 4) is ethanol with a volume fraction of 95-100%; the ethanol is food-grade ethanol.

[0017] Preferably, the water in steps 2) and 4) is deionized water.

[0018] Preferably, the reagent used to adjust the pH of the solution in step 2) is a hydrochloric acid solution with a concentration of 2-6M;

[0019] Preferably, the cellulase activity in step 2) is 50–100 U / mg;

[0020] Preferably, the heating time for inactivating enzyme activity in step 2) is 5 to 10 minutes.

[0021] Preferably, the Sevag reagent in step 3) is a mixed solvent of chloroform: n-butanol in a volume ratio of 4:1 to 6:1.

[0022] Preferably, the mass-to-volume ratio of the precipitate and water in step 4) is 1:5 to 1:10 g / mL.

[0023] A prickly pear polysaccharide extracted by an enzymatic method was prepared using the method described above. The prickly pear polysaccharide possesses antioxidant, immunostimulatory, hepatocyte autophagy activation, and liver damage-improving activities, and can be used as an ingredient in functional foods and pharmaceuticals.

[0024] Preferably, the prickly pear polysaccharide extracted by the enzymatic method comprises, by molar percentage, glucose (30-45%), arabinose (15-25%), galactose (15-25%), galacturonic acid (5-15%), and other monosaccharides (5-15%).

[0025] Preferably, the main components of the prickly pear polysaccharide extracted by the enzymatic method have a molecular weight in the range of 4 to 5 kDa and have long side chains and many branched structures.

[0026] Preferably, the prickly pear polysaccharides extracted by the enzymatic method include polysaccharides with a weight average molecular weight of 150-200 kDa and polysaccharides with a weight average molecular weight of 4-5 kDa, with weight percentages of 20-30% and 70-80%, respectively.

[0027] The above-mentioned enzymatically extracted prickly pear polysaccharides are used in the preparation of food or cosmetics with antioxidant, immunomodulatory (immune enhancement), hepatocyte autophagy activation, and liver damage improvement effects.

[0028] Preferably, the food is a beauty and skin care drink or nutritional product, an anti-aging drink or nutritional product, a liver-protecting and hangover-relieving drink or nutritional product, an immunity-boosting nutritional product, or a liver-function-enhancing nutritional product; the cosmetic is an anti-aging face cream or serum, a repairing face mask or lotion, or an anti-ultraviolet radiation face mask or lotion.

[0029] Preferably, the food product is in the form of liquid, solid, powder, granules, or capsules.

[0030] Compared with the prior art, the present invention has the following effects and advantages:

[0031] (1) Compared with the traditional hot water extraction method, the method of this invention increases the yield of prickly pear polysaccharides by more than 3 times, with a maximum yield of 14%. In the prior art, He Jingyu et al. disclosed an enzymatic extraction technology for seedless prickly pear polysaccharides, with a yield of 16.73%, exhibiting DPPH· scavenging activity and a half-maximal inhibitory concentration (IC50) of 16.73%. 50 The concentration of DPPH· was 1.75 mg / mL, while the prickly pear polysaccharide prepared by the method of this invention has significant DPPH· scavenging ability, with an IC50 value of 1.75 mg / mL. 50 The value is 11 μg / mL, which means that the DPPH scavenging ability of the prickly pear polysaccharide of the present invention is 159 times that of the seedless prickly pear polysaccharide [He Jingyu, et al. Optimization of enzymatic extraction process and antioxidant activity of seedless prickly pear polysaccharide [J]. Fragrance and Cosmetics, 2021]. However, the composition and structure information of the seedless prickly pear polysaccharide were not disclosed in this study. The inventors previously reported that the weight-average molecular weight of the seedless prickly pear polysaccharide was 124.144 and 228.298 kDa, respectively, and the monosaccharide composition included arabinose, galactose, glucose, xylose, fructose and galacturonic acid [Chen Qing, et al. Physicochemical properties, in vitro antioxidant and α-glucosidase inhibitory activity of prickly pear polysaccharide [J]. Modern Food Science and Technology, 2019]. Its composition and structure are significantly different from those of the seeded prickly pear polysaccharide of the present invention.

[0032] (2) Compared with the traditional hot water extraction method, the prickly pear polysaccharide prepared by the method of this invention has a lower molecular weight, with the main components having a molecular weight in the range of 4-5 kDa. The monosaccharide composition by molar percentage mainly includes glucose (35-40%), arabinose (18-23%), galactose (18-25%), and galacturonic acid (8-15%), as well as other monosaccharides. Compared with known reports on prickly pear polysaccharides, its molecular weight, monosaccharide composition, and biological activity are significantly different, belonging to a novel type of prickly pear polysaccharide.

[0033] (3) The prickly pear polysaccharide of the present invention can significantly enhance the ability of mouse macrophages RAW246.7 to secrete immune cytokines, thus improving immunity. Through the construction of a mouse model of liver injury, it was demonstrated that the prickly pear polysaccharide can effectively reduce the body weight and blood lipid levels of the model mice, alleviate lipid droplet deposition in the liver, and repair hepatocyte damage. Furthermore, the prickly pear polysaccharide of the present invention is the first to be found to have hepatocyte autophagy activation activity, including activation of lipophage, mitophagy, and autophagic degradation of inflammasomes, which can maintain healthy liver function. In summary, the present invention provides a high-yield, low-cost, energy-saving, environmentally friendly, and highly active prickly pear polysaccharide extraction method, suitable for large-scale industrial production. Attached Figure Description

[0034] Figure 1 The chromatogram shows the molecular weight distribution of prickly pear polysaccharides in the examples.

[0035] Figure 2 The image shows an ion chromatogram of the monosaccharide composition of prickly pear polysaccharide in the example.

[0036] Figure 3 The image shows the UV-Vis spectrum of the prickly pear polysaccharide and I2-KI reaction complex in the example.

[0037] Figure 4 The ABTS free radical scavenging activity of prickly pear polysaccharide in the examples is shown.

[0038] Figure 5 The DPPH free radical scavenging activity of prickly pear polysaccharide in the examples is shown.

[0039] Figure 6 The effect of prickly pear polysaccharide on NO production in mouse macrophage RAW264.7 cells is illustrated in the example.

[0040] Figure 7 The effect of prickly pear polysaccharide on TNF-α production in mouse macrophage RAW264.7 cells is illustrated in the example.

[0041] Figure 8 The change in body weight of mice in different experimental groups.

[0042] Figure 9 Serum triglyceride levels in mice from different experimental groups.

[0043] Figure 10 The total cholesterol levels of mice in different experimental groups.

[0044] Figure 11 Oil Red O staining images of liver tissue from mice in different experimental groups.

[0045] Figure 12 The expression of autophagy genes in the liver of mice in different experimental groups. Detailed Implementation

[0046] To better understand the present invention, the following description is provided in conjunction with embodiments, but the implementation of the present invention is not limited thereto.

[0047] Example 1 Enzymatic Extraction

[0048] 1) Raw material pretreatment: Wash the dried prickly pear fruit, dry it in an oven at 45℃ for 48 hours, then remove the seeds, crush it, and pass it through a 40-mesh sieve to obtain prickly pear powder. Then mix the prickly pear powder with 95% ethanol (food grade) at a material-to-liquid ratio of 1:6 g / mL, shake it at room temperature for 2 hours to remove fat-soluble substances, pigments, small molecule sugars, etc., collect the residue by centrifugation, repeat the treatment 5 times, centrifuge at 5000g for 10 minutes, separate, collect the residue and dry it in an oven at 45℃ for 24 hours to obtain pretreated prickly pear powder;

[0049] 2) Enzymatic extraction: The prickly pear powder pretreated in step 1) was mixed with deionized water at a mass-to-volume ratio of 1:40 g / mL. 2% of the dry weight of the prickly pear powder of cellulase was added, the pH of the solution was adjusted to 5.75, and then the mixture was heated under reflux at 46℃ for 90 min. The extract was then placed in a 90℃ water bath for 5 min to inactivate the enzyme. After centrifugation at 5000g for 10 min, the precipitate was discarded, and the supernatant was concentrated to approximately 1:4 of the original volume.

[0050] 3) Deproteinization: Mix Sevag reagent with the concentrated extract from step 2) at a volume ratio of 1:4, shake for 10 min, centrifuge to obtain the upper layer of prickly pear polysaccharide solution, repeat 5 times, and remove residual Sevag reagent by vacuum evaporation.

[0051] 4) Alcohol precipitation: Add anhydrous ethanol (food grade) to the prickly pear polysaccharide solution from step 3) and stir continuously until the ethanol volume concentration reaches 80%. Let it stand at 4℃ for 12 hours, centrifuge to collect the precipitate, wash twice with anhydrous ethanol, and redissolve in deionized water at a solid-liquid ratio of 1:10 g / mL. Freeze-dry at -50℃ to obtain prickly pear polysaccharide, named ERFP. The yield of prickly pear polysaccharide was calculated to be 14% (based on the weight of dried prickly pear raw material).

[0052] Example 2: Traditional hot water extraction method

[0053] 1) Raw material pretreatment: Wash the dried prickly pear fruit, dry it in an oven at 45℃ for 48 hours, then remove the seeds, crush it, and pass it through a 40-mesh sieve to obtain prickly pear powder. Then mix the prickly pear powder with 95% ethanol (food grade) at a material-to-liquid ratio of 1:6 g / mL, shake it at room temperature for 2 hours to remove fat-soluble substances, pigments, small molecule sugars, etc., collect the residue by centrifugation, repeat the treatment 5 times, centrifuge at 5000g for 10 minutes, separate, collect the residue and dry it in an oven at 45℃ for 24 hours to obtain pretreated prickly pear powder;

[0054] 2) Hydrothermal extraction: The prickly pear powder pretreated in step 1) was mixed with deionized water at a mass-to-volume ratio of 1:40 g / mL, heated and refluxed at 90℃ for 90 min, then centrifuged at 5000g for 10 min, the precipitate was discarded, and the supernatant was concentrated to approximately 1:4 of the original volume.

[0055] 3) Deproteinization: Mix Sevag reagent with the concentrated extract from step 2) at a volume ratio of 1:4, shake for 10 min, centrifuge to obtain the upper layer of prickly pear polysaccharide solution, repeat 5 times, and remove residual Sevag reagent by vacuum evaporation.

[0056] 4) Alcohol precipitation: Add anhydrous ethanol (food grade) to the prickly pear polysaccharide solution from step 3) and stir continuously until the ethanol volume concentration reaches 80%. Let it stand at 4℃ for 12 hours, collect the precipitate by centrifugation, wash twice with anhydrous ethanol, redissolve in deionized water at a solid-liquid ratio of 1:10 g / mL, and freeze-dry at -50℃ to obtain prickly pear polysaccharide, named RFP. The yield of prickly pear polysaccharide was calculated to be 3.98%.

[0057] Example 3: Determination of molecular weight

[0058] 5 mg of polysaccharide was dissolved in 1 mL of phosphate buffer (0.02 M), filtered through a 0.22 μm aqueous membrane, and then analyzed under the following conditions: Gel chromatography columns of G-5000PWXL (7.8 × 300 mm) and G-3000PWXL (7.8 × 300 mm) were used in series; an Agilent 1260 differential detector was used; column temperature was 35 °C; flow rate was 0.6 mL / min; and injection volume was 20 μL. Calibration curves were constructed using the logarithm of the molecular weight of pullulan standards (5.9, 9.6, 21.1, 47.1, 107, 200, 344, 708 kDa) versus elution volume, and the molecular weight of the sample was calculated.

[0059] The molecular weight distributions of ERFP and RFP were analyzed using high-performance liquid chromatography (HPLC), and the results are shown in the attached figure. Figure 1As shown in the figure. HPLC results show that both ERFP and RFP exhibit two symmetrical peaks, indicating the presence of two polysaccharide components with different molecular weights. According to the pullulan standard calibration curve equation (LogMw = -0.0008x...),... 3 +0.0698x 2 The weight-average molecular weight (Mw) of ERFP was calculated to be 187496.77 Da (24.6%) and 4749.9 Da (75.4%), while that of RFP was 165039.88 Da (55.7%) and 4329.18 Da (44.3%). The results indicate that different extraction methods affected the molecular weight distribution of prickly pear polysaccharides. Specifically, the proportion of lower molecular weight polysaccharide components in ERFP was 75.4%, while that in RFP was 44.3%, indicating that the polysaccharide obtained by enzymatic extraction was a low molecular weight prickly pear polysaccharide compared to the traditional hot water extraction method.

[0060] Example 4 Monosaccharide Composition Analysis

[0061] 5 mg of sample was weighed and dissolved in 4 mL of trifluoroacetic acid (2 M). Hydrolysis was carried out at 105 °C in a sealed ampoule for 6 h. After cooling, excess trifluoroacetic acid was removed under reduced pressure at 45 °C. The residue was redissolved in 4 mL of methanol and dried under reduced pressure. This process was repeated 6 times. Subsequently, the residue was dissolved in 5 mL of deionized water and brought to a final volume of 50 mL. The solution was filtered through a 0.22 μm aqueous phase filter and temporarily stored at -4 °C for analysis using a Dionex-600 ion chromatograph.

[0062] Test conditions: The chromatographic column was a Carbopac PA20 (2×250mm, 5μm); the detector was an electrochemical detector; the column temperature was 30℃; the flow rate was 0.5mL / min; the injection volume was 20μL; and the mobile phase gradient elution program was: 0-16.05min, 10% NaOH (20mM) and 90% ultrapure water; 16.05-30.05min, 10% NaOH (20mM), 20% CH3COONa (500mM) and 70% ultrapure water. Under the same experimental conditions, monosaccharide standards were analyzed, calibration curves were plotted, and the content of monosaccharides in the test samples was calculated based on the linear regression equation.

[0063] The monosaccharide composition of the two polysaccharide samples was analyzed using ion chromatography. The ion chromatograms of their monosaccharide compositions are shown in the attached figure. Figure 2As shown in the figure. The results showed that, based on the comparison with the chromatograms of monosaccharide standards, both ERFP and RFP were identified as being composed of arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid. The molar percentage of each monosaccharide component was calculated based on the calibration curve equations of each monosaccharide standard. ERFP mainly contained arabinose (20.66%), galactose (20.58%), glucose (38.93%), and galacturonic acid (10.94%), with small amounts of xylose (6.62%) and glucuronic acid (2.27%). RFP mainly contained arabinose (28.92%), galactose (25.41%), glucose (19.30%), and galacturonic acid (22.65%), with small amounts of xylose (1.34%) and glucuronic acid (2.38%). These results indicate that different extraction methods do not affect the types of monosaccharides in prickly pear polysaccharides, but they do affect their content, and these changes may affect their function and bioactivity.

[0064] Example 5 I2-KI reaction

[0065] 2.0 mL of polysaccharide sample solution (2.0 mg / mL) was mixed with 8 mL of I2-KI solution (containing 0.02% I2 and 0.2% KI), and the absorbance was scanned and recorded in the range of 300-700 nm using a UV-Vis spectrometer (Nano Ready F-1100 Shanghai Meite Instruments Co., Ltd., Shanghai, China).

[0066] The I2-KI assay can be used to determine whether a polysaccharide contains a relatively long, multi-branched structure. When I2 reacts with a polysaccharide with fewer branches and shorter side chains to form a complex, the UV absorption peak of I2 shifts from 350 nm to 565 nm. (See attached image) Figure 3 As shown, the complexes formed by RFP and ERFP with I2-KI have no absorption peak at 565 nm, indicating that RFP and ERFP contain long side chains and more branched structures.

[0067] Example 6: Determination of ABTS free radical scavenging ability

[0068] First, prepare 5 mL of ABTS solution (7 mM) and 5 mL of potassium persulfate solution (2.45 mM). Then, mix the two solutions in a centrifuge tube and incubate in the dark for 12–16 h to obtain the ABTS stock solution. Before use, dilute it to obtain the ABTS stock solution. The standard concentration is 0.70 ± 0.20 μg / mL, measured at 734 nm. Add 200 μL of polysaccharide samples of different concentrations (50, 100, 200, 400, and 800 μg / mL) to 1 mL of ABTS solution. Vortex the mixture and incubate in the dark at room temperature for 6–10 min. After incubation, plate the samples on a 96-well plate and measure the absorbance at 734 nm using a microplate reader. Vitamin C (VC) is used as a control. The formula for calculating the ABTS free radical scavenging rate is as follows:

[0069]

[0070] In the formula: A0 represents the absorbance of the mixture of ABTS working solution and distilled water; A1 represents the absorbance of the mixture of ABTS working solution and sample solution.

[0071] As attached Figure 4 As shown, within the range of 12.5-400 μg / mL, the scavenging ability of RFP and ERFP against ABTS free radicals gradually increased. Among them, the ABTS free radical scavenging rate of ERFP at a concentration of 400 μg / mL was 94.31%, which was basically the same as that of VC at the same concentration, indicating that ERFP has a good antioxidant capacity. The order of ABTS free radical scavenging ability of VC and prickly pear polysaccharide was: VC > ERFP > RFP.

[0072] Example 7: Determination of DPPH free radical scavenging ability

[0073] Polysaccharide samples of different concentrations (12.5, 25, 50, 100, 200, 400 μg / mL) were mixed with an equal volume of DPPH solution (0.2 mM, 70% methanol). The mixture was vortexed and incubated in the dark at room temperature for 30 min. After incubation, the samples were plated on 96-well plates, and the absorbance was measured at 517 nm using a microplate reader. Vitamin C was used as a control. The reaction system of 70% methanol solution plus DPPH solution served as the control group, and the reaction system of 70% methanol solution plus polysaccharide sample solution served as the blank. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0074]

[0075] In the formula, As is the absorbance of the polysaccharide sample reaction system, and A b and A c The absorbance values ​​are for the blank group and the control group reaction system, respectively.

[0076] As attached Figure 5 As shown, the scavenging ability of RFP and ERFP against DPPH· free radicals gradually increased with increasing concentration of prickly pear polysaccharides. When the polysaccharide concentration was only 50 μg / mL, the scavenging rate of RFP and ERFP against DPPH· free radicals reached over 80%, comparable to the scavenging rate of VC at the same concentration, indicating that RFP and ERFP possess strong antioxidant capabilities. The half-maximal inhibitory concentration (IC50) of RFP and ERFP for scavenging DPPH· free radicals was also shown. 50 The DPPH· free radical scavenging values ​​were 15 μg / mL and 11 μg / mL, respectively, indicating that ERFP had faster antioxidant activity than RFP. The order of DPPH· free radical scavenging ability of VC and prickly pear polysaccharides was: VC > ERFP > RFP. He Jingyu et al. extracted seedless prickly pear polysaccharides using an enzymatic method, and their DPPH· free radical scavenging capacity was [not specified]. 50 The concentration was 1.75 mg / mL [He Jingyu, et al. Optimization of enzymatic extraction process and its antioxidant activity of seedless prickly pear polysaccharide, Fragrance and Cosmetics, 2021], while the DPPH free radical scavenging activity of ERFP in this invention is 159 times that of seedless prickly pear polysaccharide. Studies have shown that the antioxidant activity of natural polysaccharides is closely related to their composition and chemical structure. Among them, low molecular weight or medium molecular weight polysaccharides have strong antioxidant activity, which may be the reason why ERFP has strong antioxidant activity.

[0077] Example 8: In vitro immunomodulation assay

[0078] Logarithmic growth phase mouse macrophages RAW246.7 were taken, added to fresh culture medium, and prepared into a single-cell suspension by pipetting. The cell concentration was adjusted to approximately 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 μL / well in 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. The experiment included a zeroing group, experimental group, blank control group, and positive control group. The zeroing group consisted of wells containing no 100 μL of cells; only 100 μL of cells were added during the experiment. The experimental group, blank control group, and positive control group each contained 100 μL of cell suspension. 200 μL of sterile PBS solution was added to the outermost edge of each well. After 24 hours in the incubator, once the cells had adhered, the culture medium was discarded. 100 μL of fresh culture medium was added to the zeroing group and blank control group. 100 μL of 100, 200, and 400 μg / mL ERFP and RFP solutions (the polysaccharide concentrations were non-toxic to cells) were added to the experimental group, respectively. 100 μL of lipopolysaccharide (LPS, 1 μg / mL) was added to the positive control group. Each group was divided into three replicates, and the cells were cultured for another 24 hours. The content of nitric oxide (NO) in cell culture medium was detected using Griess reagent, and the content of tumor necrosis factor-α was detected using an ELISA kit.

[0079] The experimental results are attached. Figure 6 and 7 Compared with the control group, different concentrations of ERFP and RFP significantly stimulated the secretion of NO and TNF-α by mouse macrophages RAW246.7. With increasing polysaccharide concentration, the levels of NO and TNF-α in the cell supernatant also gradually increased, indicating that both ERFP and RFP possess immunomodulatory activity. Furthermore, compared with RFP, the ERFP of this invention maintained better activity, indicating that enzymatic extraction did not destroy its immunomodulatory activity.

[0080] Example 9: In vivo animal experiments

[0081] This study used 50 six-week-old male C57BL / 6J mice weighing 19.0±1.0g as experimental subjects. All mice were housed in the SPF Experimental Animal Center of Guangdong Pharmaceutical University (Animal Experiment Batch No.: SPF2022344) under standardized conditions and in strict accordance with experimental animal ethics regulations. The C57BL / 6 mice were housed in an SPF-grade animal facility under the following conditions: 12-hour light cycle, 26±1℃, 50–60% relative humidity, and free access to food and water. After one week of acclimatization, they were randomly divided into four groups: a normal control group (NC), a high-fat and high-fructose diet-induced NAFLD model group (M), the prickly pear polysaccharide intervention model group of this invention (ERFP), and a hot water extraction prickly pear polysaccharide intervention model group (RFP). The normal control group was fed a regular diet and administered saline via gavage daily. The model group was fed a high-fat diet (HFD), high-sugar water, and administered saline via gavage daily. The two polysaccharide intervention model groups, in addition to the saline administration, received the corresponding polysaccharide solution (500 mg / kg body weight) via gavage. The experiment lasted for 8 weeks. Physiological indicators of the mice in each group were then measured and analyzed, including changes in body weight, blood lipid levels, and Oil Red O staining of liver tissue. Additionally, a new experimental group—following the model group—received daily rapamycin gavage (Rap) as a positive control for autophagy activation. The liver autophagy induction effect of the two polysaccharides was evaluated by analyzing gene expression. The liver gene expression analysis method is as follows:

[0082] Total RNA was extracted from frozen liver tissue under aseptic conditions using an RNA kit. The purity of 2.5 μL of total RNA was measured using a NanoDrop 1000 spectrometer system. 1 μg of RNA sample was reverse transcribed into cDNA using the Revert Aid first-strand cDNA synthesis kit with single nucleotide (dT)18 primers. The levels of cDNA encoding AMPK, mTOR, ATG5, ATG12, ULK1, and Parkin were quantified using a CFX96RT-PCR detection system. Primer sequence information is listed in Table 1. ΔΔThe CT formula calculates the fold increase in target gene expression relative to the GAPDH internal reference:

[0083] A = CT(target gene, sample to be tested) – CT(internal standard gene, sample to be tested)

[0084] B = CT(target gene, control sample) – CT(internal standard gene, control sample)

[0085] K = A – B; Multiple of expression = 2 -K

[0086] Table 1. Gene Primer Sequence Information

[0087]

[0088] As attached Figure 8 As shown, after 8 weeks of induction with a high-fat, high-sugar diet, the weight gain of mice in the model group was significantly faster than that of the normal control group. However, the weight gain of mice in the model group treated with ERFP and RFP was significantly lower than that of the model group. Furthermore, the weight gain inhibition effect of ERFP was better than that of RFP, which indicates that ERFP has great potential for anti-obesity effects.

[0089] Figure 9 and Appendix Figure 10 This indicates that a high-fat, high-sugar diet leads to elevated blood lipid levels (triglycerides and total cholesterol), which is an important indicator of non-alcoholic fatty liver disease. After ERFP treatment, the blood lipid levels in mice decreased significantly, with serum triglycerides returning to the same level as the normal control group, while the reduction in blood lipid levels in the RFP-treated model group mice was not significant.

[0090] Tissue staining is one of the common methods for analyzing liver tissue pathology. (See attached image.) Figure 11 Oil Red O staining of liver tissue showed that the distribution of lipid droplets in the model group was significantly higher than that in the normal control group, indicating a higher lipid metabolic burden in the livers of the model group mice. After treatment with ERFP or RFP, lipid droplet aggregation was reduced, with the ERFP group showing a particularly significant effect, superior to the RFP group, almost restoring the levels of the normal control group. This indicates that the prickly pear polysaccharide of this invention has a significant anti-non-alcoholic fatty liver effect, and this effect is significantly better than that of traditional hot water extraction of prickly pear polysaccharide.

[0091] Activating autophagy in liver cells can alleviate liver damage caused by lipid metabolism disorders by reducing lipid accumulation, mitigating excessive inflammation, and enhancing antioxidant defense capabilities. AMPK and mTOR are important intracellular protein "switches" regulating autophagy; their activation plays a positive and negative regulatory role, respectively, in the initiation and progression of autophagy. (See attached image) Figure 12As shown in Figure AB, compared with the normal group, the expression of AMPK and mTOR genes in the liver of mice treated with a high-fat, high-sugar diet was significantly reduced and increased, respectively, indicating that a high-fat diet led to impaired autophagy in liver cells. This condition was significantly improved after ERFP treatment. Meanwhile, RFP treatment was less effective than ERFP treatment. ATG5, ATG12, and ULK1 are all important proteins involved in the autophagy process and play a crucial role in the completion of autophagy. Figure 12 As shown in the CE diagram, compared to the normal control group, a high-fat diet significantly reduced the expression of the three autophagy-related genes in the mouse liver, while ERFP significantly or insignificantly increased their gene expression, further suggesting the hepatic autophagy activation activity of ERFP. In contrast, the autophagy activation effect of RFP was not as significant as that of ERFP. A high-fat diet leads to fatty acid oxidation overload, which damages healthy mitochondria. Cells clear damaged mitochondria through the mitophagy pathway to prevent apoptosis and maintain cellular homeostasis. Parkin protein is a key protein mediating mitophagy, and its gene expression level reveals the level of mitophagy to some extent. Compared with the model group, a high-fat diet significantly reduced Parkin gene expression, while ERFP treatment significantly improved this trend. In contrast, the Parkin gene expression level induced by RFP treatment was not significantly different from that in the model group. In summary, ERFP exhibits effective hepatic autophagy activation activity and may improve high-fat diet-induced liver injury by activating lipophage, mitophagy, and autophagic degradation of inflammasomes.

[0092] 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 prickly pear polysaccharide by enzymatic extraction, characterized in that, Includes the following steps: 1) Raw material pretreatment: Wash the dried prickly pear fruit, dry it in a 45 °C oven for 48 h, then remove the seeds, crush it, and pass it through a 40-mesh sieve to obtain prickly pear powder. Then mix the prickly pear powder with 95% ethanol at a material-to-liquid ratio of 1:6 g / mL, shake it at room temperature for 2 h to remove fat-soluble substances, pigments, and small molecule sugars. Centrifuge to collect the residue, repeat the treatment 5 times, centrifuge at 5000 g for 10 min, separate, collect the residue and dry it in a 45 °C oven for 24 h to obtain pretreated prickly pear powder; 2) Enzymatic extraction: The prickly pear powder pretreated in step 1) was mixed with deionized water at a mass-to-volume ratio of 1:40 g / mL. 2% of the dry weight of the prickly pear powder of cellulase was added, the pH of the solution was adjusted to 5.75, and then the mixture was heated under reflux at 46 °C for 90 min. The extract was then placed in a 90 °C water bath for 5 min to inactivate the enzyme. After centrifugation at 5000 g for 10 min, the precipitate was discarded, and the supernatant was concentrated to 1:4 of the original volume. 3) Deproteinization: Mix Sevag reagent with the concentrated extract from step 2) at a volume ratio of 1:4, shake for 10 min, centrifuge to obtain the upper layer of prickly pear polysaccharide solution, repeat 5 times, and evaporate under reduced pressure to remove residual Sevag reagent; the Sevag reagent is a mixed solvent of chloroform: n-butanol = 4:1~6:1 by volume. 4) Alcohol precipitation: Add anhydrous ethanol to the prickly pear polysaccharide solution from step 3) and stir continuously until the ethanol volume concentration reaches 80%. Let it stand at 4 °C for 12 h, centrifuge to collect the precipitate, wash twice with anhydrous ethanol, and redissolve in deionized water at a solid-liquid ratio of 1:10 g / mL. Prickly pear polysaccharide was obtained by freeze-drying at 50 °C.

2. A prickly pear polysaccharide extracted by enzymatic method, characterized in that, The prickly pear polysaccharide was prepared by the preparation method described in claim 1; the polysaccharide extracted by the enzymatic method comprises, by molar percentage, 30-45% glucose, 15-25% arabinose, 15-25% galactose, 5-15% galacturonic acid and other monosaccharides.

3. The application of the prickly pear polysaccharide extracted by enzymatic method according to claim 2 in the preparation of food or cosmetics.

4. The application according to claim 3, characterized in that, The food is a beverage or nutritional product; the cosmetic is an anti-aging face cream or serum, a repairing face mask or lotion, or an anti-ultraviolet radiation face mask or lotion.

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