Low molecular weight spirea ulmaria polysaccharide microspheres for relieving fatigue, and preparation method and application thereof

Abstract

This invention belongs to the field of food processing technology, specifically relating to a low molecular weight *Hydrangea hygroscopica* polysaccharide microsphere for relieving fatigue, its preparation method, and its application. The preparation method includes the following steps: (1) extraction of crude polysaccharide from *Hydrangea hygroscopica*; (2) degradation; (3) encapsulation: the low molecular weight *Hydrangea hygroscopica* polysaccharide obtained in step (2) is dripped into CaCl2 solution through electrospray treatment to form a gel core, and the gel core is encapsulated to obtain the low molecular weight *Hydrangea hygroscopica* polysaccharide microspheres. The beneficial effects of this invention are: this invention efficiently extracts *Hydrangea hygroscopica* polysaccharide through steam explosion-pulsed electric field synergy, accurately prepares 10-20 kDa high-activity low molecular weight fragments through compound enzymatic hydrolysis-ultrasound-ultrafiltration, and finally constructs colon-targeting microspheres using electrospray-layer-by-layer self-assembly. These microspheres can protect the active ingredients and allow them to reach the colon for release, significantly improving the bioavailability and anti-fatigue effect of the polysaccharide.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a low molecular weight Hydnocarpus fusiforme polysaccharide microsphere for relieving fatigue, its preparation method, and its application. Background Technology

[0002] Fatigue is a physiological response that occurs after sustained mental or physical exertion, manifesting as memory loss, difficulty concentrating, and decreased exercise endurance. When fatigue is not relieved promptly and effectively, it can lead to decreased immune function, metabolic disorders, and other physiological dysfunctions. Studies have confirmed that fatigue is associated with the risk of various chronic diseases, including cardiovascular and cerebrovascular dysfunction, digestive system disorders, and neuroendocrine imbalances, posing a serious threat to health. The causes of fatigue are complex and diverse, potentially involving multiple factors such as energy metabolism imbalance, accumulated oxidative stress, and inflammatory responses. Currently, dietary supplements for relieving fatigue mainly contain ingredients such as caffeine, taurine, and rhodiola rosea. However, long-term intake of these substances may damage the nervous and cardiovascular systems, causing serious adverse reactions and complications. Therefore, it is essential to explore and develop more safe, reliable, and effective methods for relieving fatigue.

[0003] Supplementing with edible fungi polysaccharides has been proven to be an effective measure for relieving fatigue. Studies have shown that edible fungi polysaccharides can be fermented and utilized by intestinal flora in the colon, generating metabolites such as short-chain fatty acids. These metabolites can mediate the "gut-muscle axis" to regulate the body's metabolism, thereby relieving fatigue. Research has found that *Hydrangea macrophylla* polysaccharides have excellent immunomodulatory and antioxidant activities, and have important development value in relieving fatigue. However, edible fungi polysaccharides suffer from low extraction rates and high molecular weights, resulting in low bioavailability. While low-molecular-weight polysaccharides obtained through degradation have stronger biological activity, their stability is poor, and they are easily degraded in the upper digestive tract or absorbed prematurely, making it difficult for them to reach the colonic target area to exert their effects.

[0004] For example, Chinese invention patent application CN111040046A discloses a highly efficient method for preparing *Hydrangea hygroscopica* polysaccharides, which mainly focuses on the extraction efficiency of polysaccharides but fails to solve the problems of targeted delivery and stability of low molecular weight active polysaccharides. Therefore, developing a *Hydrangea hygroscopica* polysaccharide preparation that can be efficiently extracted, activity-enhanced, and targetedly delivered to the colon has significant practical and industrial value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a low molecular weight Hydnocarpus fusiforme polysaccharide microsphere for relieving fatigue, its preparation method and application, wherein the microsphere can protect the low molecular weight Hydnocarpus fusiforme polysaccharide to reach the colon smoothly for release, thereby exerting an efficient anti-fatigue effect.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a method for preparing low molecular weight *Hydrangea hygroscopica* polysaccharide microspheres to relieve fatigue, comprising the following steps: (1) Extraction of crude polysaccharides from *Hydrangea macrophylla*; (2) Degradation: The crude polysaccharide of *Hydrangea macrophylla* obtained in step (1) was dissolved in water to prepare a crude polysaccharide solution of *Hydrangea macrophylla*. Enzymatic hydrolysis was carried out using a complex enzyme system containing β-glucanase, papain and pectinase, and ultrasonic treatment was added. After enzyme inactivation, the solution was separated by an ultrafiltration membrane with a molecular weight cutoff of 10-20 kDa to obtain low molecular weight *Hydrangea macrophylla* polysaccharide. (3) Encapsulation: The low molecular weight Hydnocarpus polysaccharide obtained in step (2) is mixed with a low ester pectin solution, and CaCl2 solution is dripped into it through electrospray treatment to form a gel core. Then, the gel core is coated with chitosan solution and hyaluronic acid solution in sequence using layer-by-layer self-assembly technology. Finally, after washing and freeze-drying, low molecular weight Hydnocarpus polysaccharide microspheres are obtained.

[0007] Preferably, in the above-mentioned method for preparing low molecular weight Hydrangea polysaccharide microspheres for relieving fatigue, step (1) specifically involves: using Hydrangea as raw material, pre-treating it with steam explosion, and then performing water bath extraction in combination with high voltage pulse electric field technology. The resulting extract is then subjected to dialysis, alcohol precipitation, centrifugation, and freeze-drying to obtain crude Hydrangea polysaccharide.

[0008] Preferably, in step (1) of the above-mentioned method for preparing low molecular weight *Hydrangea hygroscopica* polysaccharide microspheres for relieving fatigue, the pressure of the steam explosion pretreatment is 0.5-1.5 MPa, and the pressure holding time is 60-80 s; the electric field strength of the high-voltage pulse electric field treatment is 10-20 kV / cm, and the number of pulses is 5-15; the material-to-liquid mass ratio of the water bath extraction is 1:30, the temperature is 60-80℃, and the time is 1-3 h.

[0009] As described above, the specific parameter combination for extracting crude polysaccharides from *Hydrangea spp.* (such as medium-pressure short-time steam explosion and medium-low intensity multiple pulse electric fields) has been proven to produce the best synergistic cell disruption effect. Under the premise of ensuring the activity of polysaccharide structure, the extraction rate is significantly increased to more than 15% (see Experimental Example 1), which solves the problem that the extraction rate of traditional hot water extraction method is less than 7%.

[0010] Preferably, in step (2) of the above-mentioned method for preparing low molecular weight Hydrangea polysaccharide microspheres for relieving fatigue, the concentration of the Hydrangea crude polysaccharide solution is 8-10 mg / mL, the mass ratio of β-glucanase, papain and pectinase in the complex enzyme is (2-4):1:1, and the amount of complex enzyme added is 0.2%-0.5% of the mass of the Hydrangea crude polysaccharide solution.

[0011] As described above, the composition and ratio of the specific complex enzyme are defined. This enzyme system with a specific ratio can synergistically attack the complex structure of *Hydrangea hydrangea* polysaccharide from different bond sites (β-1,3 / 1,6 glycosidic bonds, peptide bonds, and pectin ester bonds), achieving efficient and targeted degradation, thus laying the foundation for obtaining active fragments with the target molecular weight (10-20 kDa).

[0012] Preferably, in step (2) of the above-mentioned method for preparing low molecular weight Hydnocarpus sylvestris polysaccharide microspheres for relieving fatigue, the ultrasonic treatment power is 200-400 W, the temperature is 40-60℃, and the time is 1-3 h.

[0013] As described above, the conditions for ultrasound-assisted degradation are defined. These mild ultrasound conditions (medium-low power, medium temperature) can continuously expose new cleavage sites and enhance mass transfer to accelerate degradation through cavitation effects, while avoiding random breakage and loss of activity of polysaccharide chains caused by excessive ultrasound, thus achieving controllability of the degradation process.

[0014] Preferably, in step (3) of the above-mentioned method for preparing low molecular weight Hydnocarpus fusiforme polysaccharide microspheres for relieving fatigue, the amount of low molecular weight Hydnocarpus fusiforme polysaccharide added is 50%-80% of the mass of low ester pectin, and the mass fraction of the low ester pectin solution is 2%-5%.

[0015] As described above, the ratio of active ingredient to wall material in the gel core is defined. This ratio ensures the formation of a dense gel core with high drug loading capacity, and low-ester pectin in Ca²⁺ + Cross-linking forms a network that can effectively encapsulate low molecular weight polysaccharides, which is the core of constructing stable microsphere carriers.

[0016] Preferably, in step (3) of the above-mentioned method for preparing low molecular weight Hydnocarpus sylvestris polysaccharide microspheres to relieve fatigue, the voltage of the electrospray treatment is 10-30 kV and the flow rate is 20-30 mL / h; the mass fraction of the CaCl2 solution is 3-5%.

[0017] As described above, the key parameters for electrospray molding are defined. This combination of voltage and flow rate ensures that the mixed solution forms stable and uniform microdroplets in a high-voltage electrostatic field, falling into a Ca²⁺ solution of a specific concentration. + Instant cross-linking in the bath produces gel micronuclei with uniform particle size and good sphericity, which forms the basis for subsequent layer-by-layer self-assembly.

[0018] Preferably, in step (3) of the above-mentioned method for preparing low molecular weight Hydrangea polysaccharide microspheres for relieving fatigue, the mass fraction of the chitosan solution is 1-2%, the mass fraction of the hyaluronic acid solution is 2-3%, the stirring speed during the layer-by-layer self-assembly process is 200-300 r / min, and the coating time for each layer is 30-90 min.

[0019] The materials and processing conditions of the coating layer were limited. Chitosan (positively charged) and hyaluronic acid (negatively charged) solutions of specific concentrations, with opposite charges, can be alternately and firmly adsorbed onto the surface of the negatively charged gel core through electrostatic interaction at a suitable stirring speed, constructing a multi-layered protective shell that can resist gastric acid and digestive enzymes, thus achieving colon-targeting.

[0020] Another technical solution of the present invention is to provide fatigue-relieving low molecular weight *Hydrangea hydrangea* polysaccharide microspheres prepared by the preparation method of fatigue-relieving low molecular weight *Hydrangea hydrangea* polysaccharide microspheres described in any one of the above-mentioned methods.

[0021] Another technical solution of the present invention is to provide the application of the above-mentioned low molecular weight Hydnocarpus sylvestris polysaccharide microspheres in the preparation of drugs, health foods or functional foods for relieving or resisting fatigue. The beneficial effects of this invention are as follows: The preparation method of the fatigue-relieving low molecular weight *Hydrangea hygroscopica* polysaccharide microspheres of this invention has the following advantages: (1) High extraction efficiency: The combined effect of steam explosion and high-voltage pulse electric field is used to physically break the cell wall, which significantly improves the extraction rate of crude polysaccharide of Hydrangea spp. (up to 18% or more). Moreover, the treatment conditions are relatively mild, which is conducive to maintaining the activity of polysaccharide.

[0022] (2) Controllable active ingredients: Through the three-stage technology of “compound enzymatic hydrolysis-ultrasound-ultrafiltration membrane separation”, the molecular weight of *Hydrangea hydrangea* polysaccharide was precisely controlled (10-20kDa), and low molecular weight polysaccharides with higher biological activity and concentrated molecular weight distribution were obtained.

[0023] (3) Targeted delivery and good stability: The innovative electrospray combined with layer-by-layer self-assembly technology was used to construct core-shell structured microspheres with colon-targeting function. This structure can effectively resist the damage of gastric acid and small intestinal environment, ensuring the targeted release of low molecular weight Hydnocarpus erinaceus polysaccharide in the colon (see Experiment 3, the effect of the microsphere group is significantly better than that of the unencapsulated low molecular weight polysaccharide group).

[0024] (4) Synergistic effect: The preparation method of this invention is interconnected and synergistic. Efficient extraction provides sufficient substrate for subsequent degradation; controllable degradation produces an active core with a suitable molecular weight; precise encapsulation solves the problem of delivering the active core, and finally achieves a "1+1+1>3" improvement in anti-fatigue effect. Attached Figure Description

[0025] Figure 1 The exhaustive swimming time of mice in each experimental group of Experiment Example 3, which is a specific embodiment of the present invention; Figure 2 The content of blood lactate in the serum of mice in each experimental group of Experimental Example 3 of the specific embodiment of the present invention; Figure 3 The blood urea nitrogen content in the serum of mice in each experimental group of Experimental Example 3, which is a specific embodiment of the present invention. Detailed Implementation

[0026] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0027] The key concept of this invention lies in: (1) This invention employs a synergistic cell wall disruption system combining steam explosion and high-voltage pulsed electric field treatment to achieve efficient extraction of crude polysaccharides from *Hydrangea hydrangea*. Steam explosion utilizes the mechanical force generated by the instantaneous release of high-temperature, high-pressure steam to disrupt the dense cell wall structure of *Hydrangea hydrangea*, fully exposing the polysaccharides. Meanwhile, the high-voltage pulsed electric field further induces electroporation of the cells, increasing cell membrane permeability and accelerating the exudation of internal polysaccharides. The two processes create a significant synergistic effect, greatly improving the polysaccharide mass transfer rate and leaching efficiency, solving the problem of low extraction rates in traditional hot water extraction, and achieving efficient preparation of crude polysaccharides from *Hydrangea hydrangea*.

[0028] (2) This invention employs a three-stage technology of "compound enzymatic hydrolysis - ultrasonic treatment - ultrafiltration membrane separation" to achieve precise and controllable preparation of low molecular weight Hydnocarpus fusiforme polysaccharides. The compound enzyme system consists of β-glucanase, papain and pectinase, which can specifically degrade polysaccharides from different bond positions on the main chain and side chain; ultrasonic treatment not only enhances the mass transfer efficiency between the enzyme and the substrate, but also exposes more enzyme cleavage sites through cavitation effect, significantly improving degradation efficiency; finally, the ultrafiltration membrane is used for directional retention, strictly controlling the molecular weight within the suitable 10-20 kDa range to obtain low molecular weight Hydnocarpus fusiforme polysaccharides with concentrated molecular weight distribution and higher activity.

[0029] (3) This invention employs electrospray combined with layer-by-layer self-assembly technology to construct low molecular weight *Hydrangea hydrangea* polysaccharide microspheres, thereby improving the stability of low molecular weight *Hydrangea hydrangea* polysaccharide in the gastrointestinal tract and achieving colon-targeted release. After forming a mixed solution of polysaccharide and low-ester pectin, it is dispersed into Ca²⁺ using electrospray treatment. + In the cross-linking bath, a gel core with uniform particle size and dense structure can be formed. Then, by utilizing the alternating electrostatic adsorption of chitosan (positively charged) and hyaluronic acid (negatively charged), a multi-layer coating structure is constructed on the surface of the gel core. This structure can effectively resist the destruction of gastric acid and small intestinal digestive juices, achieving precise delivery to the colonic target.

[0030] (4) The preparation method of this invention is tightly linked, forming a continuous synergistic system of "efficient extraction - precise degradation - targeted delivery". First, the steam explosion combined with the high-voltage pulsed electric field significantly improves the release efficiency of *Hydrangea hygroscopica* polysaccharide through the synergistic effect of mechanical cell disruption and electroporation, providing sufficient substrate for subsequent degradation. Second, the three-stage precise degradation of "compound enzymatic hydrolysis - ultrasonic assistance - ultrafiltration membrane separation" is the key central link in the preparation of the active core. The compound enzyme system can utilize multiple structural sites to achieve targeted degradation of polysaccharides, and ultrasonic cavitation can expose more enzyme cleavage sites. The two work together to achieve high efficiency and controllability of the degradation process. Then, combined with ultrafiltration membrane separation, the degradation products are graded and screened, so that the target polysaccharide is enriched in the low molecular weight range with better biological activity, thereby achieving precise preparation of active polysaccharides. Finally, the electrospray technology is used in Ca 2+ Cross-linking rapidly constructs a uniform gel core, which is then combined with electrostatically driven layer-by-layer self-assembly technology to form a multi-charge layer coating structure, ensuring that the low molecular weight polysaccharide can effectively resist digestion in the gastrointestinal environment and achieve colon-targeted release.

[0031] This invention provides a method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres to relieve fatigue, comprising the following steps: (1) Extraction of crude polysaccharides from *Hydrangea macrophylla* by steam explosion combined with high-voltage pulsed electric field: Fresh *Hydrangea macrophylla* was pretreated by steam explosion at a pressure of 0.5-1.5 MPa for 60-80 s, then dried and pulverized to obtain *Hydrangea macrophylla* powder. The powder was mixed with water at a mass ratio of 1:30 and extracted in a water bath at 60-80℃ for 1-3 h under a high-voltage pulsed electric field with an electric field strength of 10-20 kV / cm and a pulse number of 5-15. The resulting extract was dialyzed (3000 Da), precipitated with alcohol, centrifuged, and freeze-dried to obtain crude polysaccharide from *Hydrangea macrophylla*. (2) Preparation of low molecular weight Hydatids polysaccharide by a three-stage process of “compound enzymatic hydrolysis-ultrasonic treatment-ultrafiltration membrane separation”: The crude polysaccharide of *Hylocereus undatus* obtained in step (1) was dissolved in water to prepare a crude polysaccharide solution of *Hylocereus undatus* with a concentration of 8-10 mg / mL. A complex enzyme containing β-glucanase, papain and pectinase (mass ratio of (2-4):1:1) was added to the crude polysaccharide solution at an addition rate of 0.2%-0.5%. The solution was then ultrasonically treated for 1-3 h at a power of 200-400 W and a temperature of 40-60℃ to obtain a polysaccharide degradation solution. The polysaccharide degradation solution was heated in a boiling water bath at 100℃ to inactivate the enzyme. The supernatant was collected by centrifugation. The supernatant was then separated by ultrafiltration membrane separation technology (molecular weight cutoff of 10-20 kDa). The resulting solution was dialyzed, precipitated with alcohol, centrifuged and freeze-dried to obtain low molecular weight *Hylocereus undatus* polysaccharide. (3) Electrospray treatment combined with layer-by-layer self-assembly to construct low molecular weight Hydatids polysaccharide microspheres: Low molecular weight *Hydrangea hygroscopica* polysaccharide (50%-80% by weight of low-ester pectin) was added to a 2%-5% low-ester pectin solution and mixed thoroughly to obtain a mixed solution. The mixed solution was then added dropwise to a 3%-5% CaCl2 solution using an electrospray irradiation system with a voltage of 10-30 kV, a flow rate of 20-30 mL / h, and a needle size of 20-30 G to form a gel core. The gel core was collected and washed, and then sequentially placed in a 1-2% chitosan solution and a 2-3% hyaluronic acid solution, stirred at 200-300 r / min for 30-90 min. The gel core was repeatedly encapsulated using electrostatically driven layer-by-layer self-assembly technology. Finally, after washing and freeze-drying, low molecular weight *Hydrangea hygroscopica* polysaccharide microspheres were obtained.

[0032] Example 1 A method for preparing low molecular weight Hydatids urticaria polysaccharide microspheres to relieve fatigue includes the following steps: (1) Extraction of crude polysaccharides from *Hydrangea macrophylla* by steam explosion combined with high-voltage pulsed electric field: Fresh *Hydrangea macrophylla* was pretreated by steam explosion at a pressure of 0.5 MPa for 60 s, then dried and pulverized to obtain *Hydrangea macrophylla* powder. The powder was mixed with water at a mass ratio of 1:30 and extracted in a water bath at 70℃ for 2 h under a high-voltage pulsed electric field of 10 kV / cm and 5 pulses. The extract was then dialyzed (3000 Da), precipitated with alcohol, centrifuged, and freeze-dried to obtain crude polysaccharide from *Hydrangea macrophylla*. (2) Preparation of low molecular weight Hydatids polysaccharide by a three-stage process of “compound enzymatic hydrolysis-ultrasonic treatment-ultrafiltration membrane separation”: The crude polysaccharide of *Hylocereus undatus* obtained in step (1) was dissolved in water to prepare a crude polysaccharide solution with a concentration of 8 mg / mL. A complex enzyme containing β-glucanase, papain and pectinase (mass ratio of 2:1:1) was added to the crude polysaccharide solution at an addition rate of 0.2%. The solution was then sonicated for 1 h at a power of 200 W and a temperature of 40 °C to obtain a polysaccharide degradation solution. The polysaccharide degradation solution was heated in a boiling water bath at 100 °C to inactivate the enzyme. The supernatant was collected by centrifugation. The supernatant was then separated by ultrafiltration membrane separation technology (molecular weight cutoff of 20 kDa). The resulting solution was dialyzed, precipitated with alcohol, centrifuged and freeze-dried to obtain low molecular weight *Hylocereus undatus* polysaccharide. (3) Electrospray treatment combined with layer-by-layer self-assembly to construct low molecular weight Hydatids polysaccharide microspheres: Low molecular weight *Hydrangea hygroscopica* polysaccharide (50% by weight of low-ester pectin) was added to a 2% low-ester pectin solution and mixed thoroughly to obtain a mixed solution. The mixed solution was then added dropwise to a 3% CaCl2 solution using an electrospray irradiation system (10 kV, 20 mL / h, 20 G needle) to form a gel core. The gel cores were collected and washed, then sequentially placed in a 1% chitosan solution and a 2% hyaluronic acid solution and stirred at 200 r / min for 30 min. The gel cores were repeatedly encapsulated using electrostatically driven layer-by-layer self-assembly technology. Finally, after washing and freeze-drying, low molecular weight *Hydrangea hygroscopica* polysaccharide microspheres were obtained.

[0033] Example 2 A method for preparing low molecular weight Hydatids urticaria polysaccharide microspheres to relieve fatigue includes the following steps: (1) Extraction of crude polysaccharides from *Hydrangea macrophylla* by steam explosion combined with high-voltage pulsed electric field: Fresh *Hydrangea macrophylla* was pretreated by steam explosion at a pressure of 1.0 MPa for 70 s, then dried and pulverized to obtain *Hydrangea macrophylla* powder. The powder was mixed with water at a mass ratio of 1:30 and extracted in a water bath at 70℃ for 2 h under a high-voltage pulsed electric field with an electric field strength of 15 kV / cm and 10 pulses. The resulting extract was dialyzed (3000 Da), precipitated with alcohol, centrifuged, and freeze-dried to obtain crude polysaccharide from *Hydrangea macrophylla*. (2) Preparation of low molecular weight Hydatids polysaccharide by a three-stage process of “compound enzymatic hydrolysis-ultrasonic treatment-ultrafiltration membrane separation”: The crude polysaccharide of *Hylocereus undatus* obtained in step (1) was dissolved in water to prepare a crude polysaccharide solution of *Hylocereus undatus* with a concentration of 9 mg / mL. A complex enzyme containing β-glucanase, papain and pectinase (mass ratio of 3:1:1) was added to the crude polysaccharide solution at an addition rate of 0.4%. The solution was then sonicated for 2 h at a power of 300 W and a temperature of 50 °C to obtain a polysaccharide degradation solution. The polysaccharide degradation solution was heated in a boiling water bath at 100 °C to inactivate the enzyme. The supernatant was collected by centrifugation. The supernatant was then separated by ultrafiltration membrane separation technology (molecular weight cutoff of 20 kDa). The resulting solution was dialyzed, precipitated with alcohol, centrifuged and freeze-dried to obtain low molecular weight *Hylocereus undatus* polysaccharide. (3) Electrospray treatment combined with layer-by-layer self-assembly to construct low molecular weight *Hydatida* polysaccharide colon microspheres: Low molecular weight *Hydrangea hygroscopica* polysaccharide (70% by weight of low-ester pectin) was added to a 3.5% low-ester pectin solution and mixed thoroughly to obtain a mixed solution. The mixed solution was then added dropwise to a 4% CaCl2 solution using an electrospray irradiation system (20 kV, 25 mL / h, 20 G needle) to form a gel core. The gel cores were collected and washed, then sequentially placed in a 1.5% chitosan solution and a 2.5% hyaluronic acid solution and stirred at 250 r / min for 60 min. The gel cores were repeatedly encapsulated using electrostatically driven layer-by-layer self-assembly technology. Finally, after washing and freeze-drying, low molecular weight *Hydrangea hygroscopica* polysaccharide microspheres were obtained.

[0034] Example 3 A method for preparing low molecular weight Hydatids urticaria polysaccharide microspheres to relieve fatigue includes the following steps: (1) Extraction of crude polysaccharides from *Hydrangea macrophylla* by steam explosion combined with high-voltage pulsed electric field: Fresh *Hydrangea macrophylla* was pretreated by steam explosion at a pressure of 1.5 MPa for 80 s, then dried and pulverized to obtain *Hydrangea macrophylla* powder. The powder was mixed with water at a mass ratio of 1:30 and extracted in a water bath at 70℃ for 2 h under a high-voltage pulsed electric field with an electric field strength of 20 kV / cm and 15 pulses. The resulting extract was dialyzed (3000 Da), precipitated with alcohol, centrifuged, and freeze-dried to obtain crude polysaccharide from *Hydrangea macrophylla*. (2) Preparation of low molecular weight Hydatids polysaccharide by a three-stage process of “compound enzymatic hydrolysis-ultrasonic treatment-ultrafiltration membrane separation”: The crude polysaccharide of *Hylocereus undatus* obtained in step (1) was dissolved in water to prepare a crude polysaccharide solution of *Hylocereus undatus* with a concentration of 10 mg / mL. A complex enzyme containing β-glucanase, papain and pectinase (mass ratio of 4:1:1) was added to the crude polysaccharide solution at an addition rate of 0.5%. The solution was then sonicated for 3 h at a power of 400 W and a temperature of 60 °C to obtain a polysaccharide degradation solution. The polysaccharide degradation solution was heated in a boiling water bath at 100 °C to inactivate the enzyme. The supernatant was collected by centrifugation. The supernatant was then separated by ultrafiltration membrane separation technology (molecular weight cutoff of 20 kDa). The resulting solution was dialyzed, precipitated with alcohol, centrifuged and freeze-dried to obtain low molecular weight *Hylocereus undatus* polysaccharide. (3) Electrospray treatment combined with layer-by-layer self-assembly to construct low molecular weight Hydatids polysaccharide microspheres: Low molecular weight *Hydrangea hydrangea* polysaccharide (80% by weight of low-ester pectin) was added to a 5% low-ester pectin solution and mixed thoroughly to obtain a mixed solution. The mixed solution was then added dropwise to a 5% CaCl2 solution using an electrospray irradiation system (30 kV, 30 mL / h, 20 G needle) to crosslink and form gel cores. The gel cores were collected and washed, then sequentially placed in a 2% chitosan solution and a 3% hyaluronic acid solution and stirred at 300 r / min for 90 min. The gel cores were repeatedly encapsulated using electrostatically driven layer-by-layer self-assembly technology. Finally, after washing and freeze-drying, low molecular weight *Hydrangea hydrangea* polysaccharide microspheres were obtained.

[0035] Comparative Example The crude polysaccharide of *Hydrangea macrophylla* was prepared by conventional water extraction and alcohol precipitation. Fresh *Hydrangea macrophylla* was dried and pulverized to obtain *Hydrangea macrophylla* powder. The *Hydrangea macrophylla* powder was mixed with water at a mass ratio of 1:30 and extracted at 70℃ for 2 h. The resulting extract was dialyzed (3000 Da), precipitated with alcohol, centrifuged, and freeze-dried to obtain crude polysaccharide of *Hydrangea macrophylla*.

[0036] Experimental example: Experimental Example 1: Determination of Crude Polysaccharide Extraction Rate from *Hydrangea macrophylla* The extraction rates of *Hydrangea macrophylla* crude polysaccharide extracted using the conventional method in the comparative example and *Hydrangea macrophylla* crude polysaccharide obtained by steam explosion combined with high-voltage pulsed electric field extraction in Examples 1-3 were determined. The calculation formula was: Extraction rate = ((weight of *Hydrangea macrophylla* crude polysaccharide / weight of *Hydrangea macrophylla* powder) × 100%). The results are shown in Table 1.

[0037] Table 1 As shown in Table 1, steam explosion combined with high-pressure pulsed electric field treatment greatly improved the extraction rate of crude polysaccharide from *Hydrangea hydrangea*.

[0038] Experimental Example 2: Determination of the molecular weight of low molecular weight *Hydrangea rubra* polysaccharides The molecular weights of the crude polysaccharide of *Hydrangea hydrangea* extracted using the conventional method in the comparative example and the low molecular weight polysaccharides obtained from the three-stage treatment process of "compound enzymatic hydrolysis-ultrasonic treatment-ultrafiltration membrane separation" in Examples 1-3 were determined. The molecular weights of the samples were determined using gel permeation chromatography with 0.1 M NaNO3 as the mobile phase, a flow rate of 1.0 mL / min, a column temperature of 40℃, and an injection volume of 100 μL. The results are shown in Table 2.

[0039] Table 2 As shown in Table 2, compared with the conventional water extraction and alcohol precipitation method, the three-stage treatment of "compound enzymatic hydrolysis-ultrasonic treatment-ultrafiltration membrane separation" effectively reduced the weight-average molecular weight of Hydrangea macrophylla polysaccharide.

[0040] Experiment Example 3: Determination of Fatigue Relief Activity Experimental grouping: The crude polysaccharide of *Hydrangea macrophylla* obtained in Example 2 (sample 1), low molecular weight *Hydrangea macrophylla* polysaccharide (sample 2), and low molecular weight *Hydrangea macrophylla* polysaccharide microspheres (sample 3) were used for animal experiments. Thirty-two male Balb / c mice were randomly divided into four groups: control group (physiological saline), sample 1 group (100 mg / kg), sample 2 group (100 mg / kg), and sample 3 group, and were administered by gavage for 28 days.

[0041] Exhaustion swimming time: 30 minutes after gavage, mice were placed in a swimming tank with a tail weight of 5% of their body weight in water at a depth of 30 cm and a water temperature of about 25°C. They were forced to exercise until exhaustion. Exhaustion was defined as when a mouse sank into the water and could not surface to breathe within 5 seconds. The time from the start of swimming to exhaustion was recorded with a stopwatch.

[0042] Blood lactate and blood urea nitrogen: 30 min after the last gavage, mice in each group swam without load in a constant-temperature water bath for 30 min and rested for 30 min. Blood was then collected from the orbital fossa and centrifuged to prepare serum. Serum lactate and blood urea nitrogen levels were determined using a Solarbio reagent kit.

[0043] The results of the exhaustive swimming time of mice in each group are shown below. Figure 1 .Depend on Figure 1 It can be seen that, compared with the control group, mice treated with low molecular weight Hydnocarpus sylvestris polysaccharide microspheres can significantly increase the exhaustive swimming time of mice.

[0044] The results of blood lactate levels in each group of mice are shown below. Figure 2 .Depend on Figure 2 It was found that, compared with the control group, all intervention groups significantly reduced the serum lactate content in mice. Among them, the low molecular weight *Hydrangea hydrangea* polysaccharide microspheres showed the best effect.

[0045] The results of blood urea nitrogen levels in each group of mice are shown below. Figure 3 .Depend on Figure 3 It was found that, compared with the control group, all groups of mice showed a significant reduction in serum urea nitrogen levels. The low molecular weight *Hydrangea hydrangea* polysaccharide microspheres showed the best effect.

[0046] The experimental results above show that the low molecular weight Hydnocarpus fusiforme polysaccharide microspheres prepared in this invention can significantly improve the exercise endurance of mice and effectively reduce the levels of blood lactate and blood urea nitrogen in mouse serum, making it a potential high-quality anti-fatigue ingredient.

[0047] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres to relieve fatigue, characterized in that, Includes the following steps: (1) Extraction of crude polysaccharides from *Hydrangea macrophylla*; (2) Degradation: The crude polysaccharide of *Hydrangea macrophylla* obtained in step (1) was dissolved in water to prepare a crude polysaccharide solution of *Hydrangea macrophylla*. Enzymatic hydrolysis was carried out using a complex enzyme system containing β-glucanase, papain and pectinase, and ultrasonic treatment was added. After enzyme inactivation, the solution was separated by an ultrafiltration membrane with a molecular weight cutoff of 10-20 kDa to obtain low molecular weight *Hydrangea macrophylla* polysaccharide. (3) Encapsulation: The low molecular weight Hydnocarpus polysaccharide obtained in step (2) is mixed with a low ester pectin solution, and CaCl2 solution is dripped into it through electrospray treatment to form a gel core. Then, the gel core is coated with chitosan solution and hyaluronic acid solution in sequence using layer-by-layer self-assembly technology. Finally, after washing and freeze-drying, low molecular weight Hydnocarpus polysaccharide microspheres are obtained.

2. The method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres for relieving fatigue according to claim 1, characterized in that, The specific steps (1) are as follows: using *Hydrangea macrophylla* as raw material, after steam explosion pretreatment, water bath extraction is carried out in combination with high voltage pulse electric field technology, and the obtained extract is obtained by dialysis, alcohol precipitation centrifugation and freeze drying to obtain crude polysaccharide of *Hydrangea macrophylla*.

3. The method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres for relieving fatigue according to claim 2, characterized in that, In step (1), the pressure of the steam explosion pretreatment is 0.5-1.5 MPa, and the pressure holding time is 60-80 s; the electric field strength of the high-voltage pulse electric field treatment is 10-20 kV / cm, and the number of pulses is 5-15; the mass ratio of the material to the liquid in the water bath extraction is 1:30, the temperature is 60-80℃, and the time is 1-3 h.

4. The method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres for relieving fatigue according to claim 1, characterized in that, In step (2), the concentration of the crude polysaccharide solution of *Hydrangea hydrangea* is 8-10 mg / mL, the mass ratio of β-glucanase, papain and pectinase in the complex enzyme is (2-4):1:1, and the amount of complex enzyme added is 0.2%-0.5% of the mass of the crude polysaccharide solution of *Hydrangea hydrangea*.

5. The method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres for relieving fatigue according to claim 1, characterized in that, In step (2), the ultrasonic treatment power is 200-400 W, the temperature is 40-60℃, and the time is 1-3 h.

6. The method for preparing low molecular weight *Hydrangea zebrina* polysaccharide microspheres for relieving fatigue according to claim 1, characterized in that, In step (3), the amount of low molecular weight Hydrangea polysaccharide added is 50%-80% of the mass of low ester pectin, and the mass fraction of the low ester pectin solution is 2%-5%.

7. The method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres for relieving fatigue according to claim 1, characterized in that, In step (3), the voltage of the electrospray treatment is 10-30 kV and the flow rate is 20-30 mL / h; the mass fraction of the CaCl2 solution is 3-5%.

8. The method for preparing low molecular weight *Hypericum hygroscopicum* polysaccharide microspheres for relieving fatigue according to claim 1, characterized in that, In step (3), the mass fraction of the chitosan solution is 1-2%, and the mass fraction of the hyaluronic acid solution is 2-3%; the stirring speed during the layer-by-layer self-assembly process is 200-300 r / min, and the coating time for each layer is 30-90 min.

9. A low molecular weight Hericium erinaceus polysaccharide microsphere for relieving fatigue, characterized in that, The low molecular weight Hydnocarpus polysaccharide microspheres for relieving fatigue are prepared by any one of claims 1 to 8.

10. The use of the low molecular weight Hydnocarpus sylvestris polysaccharide microspheres according to claim 9 in the preparation of drugs, health foods or functional foods for relieving or combating fatigue.

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