Method for preparing Janus particles of polysaccharide of mycena acicula by acoustic fusion and application thereof

Abstract

This invention provides a method for preparing Janus polysaccharide particles from *Amanita argentea* using sonofusion and its application. The preparation method is as follows: (1) Prepare aqueous solutions of *Amanita argentea* polysaccharide and sodium alginate, respectively; (2) Preliminarily emulsify the aqueous solution of *Amanita argentea* polysaccharide with an oil phase without cross-linking ions to obtain a primary emulsion A; (3) Preliminarily emulsify the aqueous solution of sodium alginate with an oil phase without cross-linking ions to obtain a primary emulsion B; (4) Mix primary emulsion A and primary emulsion B with an oil phase containing cross-linking ions to obtain a mixed emulsion; (5) Treat the mixed emulsion using sonofusion to obtain a mixture; (6) Centrifuge the mixture, collect the Janus gel particles, wash with a washing solvent to remove the oil phase, and freeze-dry to obtain the final product. The *Amanita argentea* polysaccharide particles of this invention exhibit significant efficacy in protecting against alcoholic and chemical liver injury.

Description

Technical Field

[0001] This invention belongs to the field of natural active polysaccharide materials technology, specifically relating to a method for preparing Janus polysaccharide particles by sonofusion and its application. Background Technology

[0002] Natural active polysaccharides, due to their excellent biocompatibility, biodegradability, and low toxicity, show broad application prospects in the biomedical field. Studies have shown that polysaccharides can effectively reduce liver damage caused by harmful factors such as alcohol and drugs by regulating key signaling pathways such as Nrf2 / Keap1 and TLR4 / NF-κB, thus exerting a significant hepatoprotective effect. Edible and medicinal fungal polysaccharides, such as Ganoderma lucidum polysaccharide, Grifola frondosa polysaccharide, and Lentinus edodes polysaccharide, have been proven to possess multiple hepatoprotective activities, including antioxidant, anti-inflammatory, and immunomodulatory activities. They can protect hepatocytes through pathways such as scavenging free radicals, inhibiting lipid peroxidation, and regulating the activity of alcohol-metabolizing enzymes. Pholiota squarrosoides polysaccharide, as a natural polysaccharide with significant immunomodulatory, antitumor, and hypoglycemic activities, has hepatoprotective potential worthy of further exploration.

[0003] However, the direct application of natural active polysaccharides as active ingredients has the following drawbacks: rapid dissolution in vivo, poor stability, low bioavailability, and a lack of effective delivery carriers to achieve targeted release and sustained-release effects. Preparing natural active polysaccharides into particles with specific structures to improve their delivery and efficacy is an effective way to solve these problems.

[0004] Janus particles are asymmetric structural particles with two different chemical compositions, physical properties, or functions. Their unique bifacial heterogeneity makes them a promising candidate for drug delivery. In drug delivery systems, the Janus structure enables a "active-functional" separation design: one side loads the active ingredient, while the other side provides structural support and functionalization sites, thereby achieving controlled release and targeted delivery.

[0005] Therefore, developing an efficient and controllable method to prepare natural active polysaccharide particles with a Janus structure is of great significance for improving the delivery performance of Janus argentis polysaccharide, enhancing its hepatoprotective effects, and expanding its applications in functional foods and pharmaceuticals. Summary of the Invention

[0006] The technical problem to be solved: Given that Janus polysaccharide, as an active ingredient, dissolves rapidly in vivo, has poor stability and low bioavailability, and lacks an effective delivery carrier to achieve targeted release and sustained release effects, the present invention aims to provide a method for preparing Janus polysaccharide particles using sonofusion and its application, demonstrating significant efficacy in protecting against alcoholic and chemical liver injury.

[0007] Technical solution: A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion, comprising the following steps: (1) Prepare aqueous solutions of polysaccharide from *Amanita argentea* and sodium alginate, respectively; (2) The aqueous solution of *Amanita argentea* polysaccharide was preliminarily emulsified with an oil phase that did not contain cross-linking ions to obtain primary emulsion A; (3) The sodium alginate aqueous solution was preliminarily emulsified with the oil phase without crosslinking ions to obtain the primary emulsion B; (4) Mix primary emulsion A and primary emulsion B with an oil phase containing cross-linking ions to obtain a mixed emulsion; (5) The mixed emulsion is treated by sonofusion to obtain a mixture; during the ultrasonic process, the ultrasonic energy drives the primary emulsion A droplet and the primary emulsion B droplet to collide and fuse to form a fused droplet; when the fused droplet comes into contact with the oil phase continuous phase containing cross-linked ions, the sodium alginate at the interface undergoes a "egg box" structure gelation reaction with the cross-linked ions, instantly cross-linking and solidifying, locking the Janus structure, and obtaining Janus gel particles; (6) The mixture was centrifuged to collect Janus gel particles, washed with washing solvent to remove the oil phase, and freeze-dried to obtain solid Janus polysaccharide particles.

[0008] Furthermore, the concentration of the polysaccharide aqueous solution of *Amanita argentea* in step (1) is 0.5% to 5%; and the concentration of the sodium alginate aqueous solution is 2% to 8%.

[0009] Furthermore, the oil phase is at least one of corn oil, soybean oil, and sunflower oil.

[0010] Furthermore, in step (2), the volume ratio of the aqueous solution of *Amanita argentea* polysaccharide to the oil phase without cross-linking ions is 1:3 to 1:6.

[0011] Furthermore, the volume ratio of the sodium alginate aqueous solution to the oil phase without cross-linking ions in step (3) is 1:3 to 1:6.

[0012] Furthermore, the droplet size of both primary emulsion A and primary emulsion B is 10~50μm.

[0013] Furthermore, in step (4), the volume ratio of the primary emulsion A, primary emulsion B and the oil phase containing crosslinked ions is 1:1:(1.5~3).

[0014] Furthermore, the crosslinking ion mentioned in step (4) is Ca. 2+ Ba 2+ 、Sr 2+ or Zn 2+At least one of them, wherein the concentration in the oil phase is 0.1~1.0 M.

[0015] Furthermore, the oil phase is corn oil, and the crosslinking ion is Ca2+. 2+ Ca 2+ The concentration in the oil phase is 0.5 M.

[0016] Furthermore, the conditions for the acoustic fusion method described in step (5) are: ultrasonic frequency of 15~40 kHz, power of 100-300 W, pulse mode of 1~4 seconds of operation and 2~6 seconds of interval, and total processing time of 1~5 minutes.

[0017] Furthermore, the washing solvent in step (6) is a low-concentration aqueous ethanol solution or deionized water.

[0018] The present invention also includes Janus particles of Agaricus argentea polysaccharide prepared by the above method. The particles have an asymmetric Janus structure, with one side being an Agaricus argentea polysaccharide enrichment region and the other side being a calcium alginate cross-linked gel region. The particle size is 20~100μm.

[0019] The present invention also includes the use of the above-mentioned Janus polysaccharide granules in the preparation of drugs for the prevention and treatment of alcoholic liver injury.

[0020] The present invention also includes the application of the above-mentioned Janus polysaccharide granules in the preparation of health products that have an auxiliary protective effect against chemically induced liver damage.

[0021] The present invention also includes the application of the above-mentioned Janus polysaccharide particles in anti-oxidation and transaminase control. Beneficial effects

[0022] 1. This invention achieves efficient and controllable preparation of natural active polysaccharide Janus particles through acoustic fusion. By introducing ultrasonic energy from a probe as the driving force, two pre-emulsified droplets with different compositions are precisely induced to undergo controllable collision and fusion in the oil phase, thereby forming gel particles with an asymmetric structure in situ.

[0023] 2. This invention utilizes the "egg-box" gelation principle of sodium alginate and divalent calcium ions. By pre-dissolving calcium chloride in the oil-phase continuous phase, the interface of the newly formed droplets undergoes instantaneous cross-linking and solidification upon contact with calcium ions, thereby locking the Janus structure and achieving a rapid and gentle phase transition from droplets to solid particles. This solidification process can be completed at room temperature, with a processing time of only 1-5 minutes, avoiding the destruction of polysaccharide activity by high temperatures. Simultaneously, the oil phase in the pre-emulsification stage does not contain cross-linking ions, ensuring the sequential control of droplet liquid state before fusion and rapid solidification after fusion. This rapid solidification effectively prevents structural relaxation and symmetry, resulting in a Janus particle yield of over 85%.

[0024] 3. This invention uses a combination of *Janus acutus* polysaccharide (active core material) and sodium alginate (functionalized cross-linking agent) as the gel matrix. Sodium alginate not only provides rapid cross-linking sites but also physically combines with the active polysaccharide, synergistically enhancing the mechanical stability and structural integrity of the particles. The prepared Janus particles have a distinct asymmetric structure: one side is a *Janus acutus* polysaccharide-rich region, providing immunomodulatory, antioxidant, and hepatoprotective activities; the other side is a calcium alginate cross-linked gel region, providing structural support, pH-responsive sustained release, and functionalized modification sites.

[0025] 4. The Janus granules prepared by this invention exhibit a significant synergistic effect in protecting against alcoholic liver injury. Animal experiments showed that, at the same dosage (400 mg / kg), the Janus granule group (HJ) increased the reduction of serum ALT and AST by 14.2% and 14.4% respectively compared with the free polysaccharide group (HP), the recovery of liver SOD and GSH-Px activities by 10.0% and 6.5% respectively, the reduction of MDA content by 20.8%, and the enhancement of ADH and ALDH activities by 9.5% and 8.3% respectively, which were significantly better than the free polysaccharide group. This is mainly because: (1) the calcium alginate gel matrix protects the active polysaccharide from gastric acid degradation and improves oral bioavailability; (2) the sustained-release properties of Janus granules prolong the effective action time of polysaccharides in vivo; (3) the asymmetric structure is conducive to retention and continuous release in the intestine, and improves liver accumulation.

[0026] 5. The entire preparation process of this invention is carried out in an aqueous phase and a vegetable oil phase, without the use of any organic solvents, thus avoiding the damage to bioactive substances caused by residual organic solvents and environmental and safety issues. The preparation conditions are mild (room temperature or low temperature, short-term ultrasound), maximizing the preservation of the bioactivity of *Amanita argentea* polysaccharides. This method uses readily available raw materials, is inexpensive, and is easy to operate, making it suitable for large-scale production and showing good prospects for industrial application. Attached Figure Description

[0027] Figure 1 The images show a schematic diagram of the structural design of Janus polysaccharide particles from the present invention and a scanning electron microscope (SEM) image of Example 1; wherein, A is a schematic diagram of the structural design; and B is a scanning electron microscope (SEM) image. Detailed Implementation

[0028] This invention proposes a method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0029] The polysaccharides from *Amanita argentea* used below were extracted and purified using existing techniques. Specifically, the crude polysaccharides were extracted using an ultrasonic-assisted hot water extraction method at an ultrasonic power of 200 W, a liquid-to-solid ratio of 1:60 (g / mL), a temperature of 60℃, and a time of 40 min. After protein removal by the Sevage method, dialysis, alcohol precipitation, DEAE Seplife FF anion exchange column chromatography, and Sephacryl S-400HR gel column chromatography, the polysaccharides were purified to a purity of 94.49%.

[0030] Example 1 A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of polysaccharide aqueous solution of *Amanita argentea* was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion A. The droplet size was measured to be 10~50μm. (3) 1L of sodium alginate aqueous solution was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion B. The droplet size was measured to be 10~50μm. (4) Mix 1L of colostrum A and 1L of colostrum B with 2L of emulsion containing 0.5 M Ca 2+ The oil phase is mixed to obtain a mixed emulsion; (5) The mixed emulsion was treated by acoustic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (6) The mixture was centrifuged to collect Janus gel particles, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid Janus polysaccharide particles.

[0031] Example 2 A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 3% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion A; (3) 1L of sodium alginate aqueous solution and 4L of corn oil were subjected to preliminary ultrasonic emulsification. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion B; (4) Mix 1L of colostrum A and 1L of colostrum B with 2L of emulsion containing 0.5 M Ca 2+ The oil phase is mixed to obtain a mixed emulsion; (5) The mixed emulsion was treated by acoustic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (6) The mixture was centrifuged to collect Janus gel particles, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid Janus polysaccharide particles.

[0032] Example 3 A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 6% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion A; (3) 1L of sodium alginate aqueous solution and 4L of corn oil were subjected to preliminary ultrasonic emulsification. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion B; (4) Mix 1L of colostrum A and 1L of colostrum B with 2L of emulsion containing 0.5 M Ca 2+ The oil phase is mixed to obtain a mixed emulsion; (5) The mixed emulsion was treated by acoustic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (6) The mixture was centrifuged to collect Janus gel particles, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid Janus polysaccharide particles.

[0033] Example 4 A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was mixed with 5L of corn oil for preliminary ultrasonic emulsification. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion A; (3) 1L of sodium alginate aqueous solution and 5L of corn oil were subjected to preliminary ultrasonic emulsification. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion B; (4) Mix 1L of colostrum A and 1L of colostrum B with 2.5L of solution containing 0.5 M Ca 2+ The oil phase is mixed to obtain a mixed emulsion; (5) The mixed emulsion was treated by acoustic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (6) The mixture was centrifuged to collect Janus gel particles, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid Janus polysaccharide particles.

[0034] Example 5 A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion A; (3) 1L of sodium alginate aqueous solution and 4L of corn oil were subjected to preliminary ultrasonic emulsification. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion B; (4) Mix 1L of colostrum A and 1L of colostrum B with 2L of emulsion containing 0.5 M Ca 2+ The oil phase is mixed to obtain a mixed emulsion; (5) The mixed emulsion was treated by sonic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 250 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (6) The mixture was centrifuged to collect Janus gel particles, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid Janus polysaccharide particles.

[0035] Example 6 A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion A; (3) 1L of sodium alginate aqueous solution and 4L of corn oil were subjected to preliminary ultrasonic emulsification. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion B; (4) Mix 1L of colostrum A and 1L of colostrum B with 2L of emulsion containing 0.8 M Ca 2+ The oil phase is mixed to obtain a mixed emulsion; (5) The mixed emulsion was treated by acoustic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (6) The mixture was centrifuged to collect Janus gel particles, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid Janus polysaccharide particles.

[0036] Comparative Example 1 (No ultrasonic fusion – mechanical stirring as a substitute) A method for preparing polysaccharide particles from *Amanita argentea* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was mixed with 4L of corn oil for preliminary ultrasonic emulsification. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion A. The droplet size was measured to be 20μm. (3) 1L of sodium alginate aqueous solution was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion B. The droplet size was measured to be 20 μm. (4) Mix 1L of colostrum A and 1L of colostrum B with 2L of emulsion containing 0.5 M Ca 2+The oil phase is mixed to obtain a mixed emulsion; (5) The emulsion was mechanically stirred under the following conditions: stirring speed of 500 rpm and stirring time of 2 minutes to obtain a mixture; (6) The mixture is centrifuged to separate the particles, the particles are collected, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid particles.

[0037] The resulting particles were randomly mixed composite particles with no obvious Janus structure; the yield of Janus particles was only 8.3%.

[0038] Comparative Example 2 (Pre-emulsified oil phase containing Ca) 2+ —Pre-crosslinking) A method for preparing polysaccharide particles from *Amanita argentea* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was mixed with 4L of corn oil for preliminary ultrasonic emulsification. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion A. The droplet size was measured to be 20μm. (3) Mix 1L of sodium alginate aqueous solution with 4L of solution containing 0.5 M Ca 2+ Corn oil was subjected to preliminary ultrasonic emulsification at an ultrasonic frequency of 20 kHz, an ultrasonic power of 200 W, an emulsification temperature of 40℃, and an emulsification time of 5 minutes to obtain primary emulsion B, with a droplet size of 20 μm. (4) Mix 1L of primary emulsion A and 1L of primary emulsion B with 2L of oil phase containing 0.5 M Ca²⁺ to obtain a mixed emulsion; (5) The mixed emulsion was treated by acoustic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (6) The mixture is centrifuged to separate the particles, the particles are collected, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid particles.

[0039] In the colostrum B, sodium alginate reacts with Ca during the emulsification process. 2+ Cross-linking and solidification occur, forming independent calcium alginate gel beads; the resulting product is a mixture of *Amanita argentea* polysaccharide droplets and calcium alginate gel beads, without Janus structure; the yield of Janus particles is 0%.

[0040] Comparative Example 3 (Premixed emulsification – No separate pre-emulsification) A method for preparing polysaccharide particles from *Amanita argentea* using sonofusion includes the following steps: (1) Prepare a mixed aqueous solution of 2% polysaccharide of *Amanita argentea* and 5% sodium alginate; (2) 1L of mixed aqueous solution and 4L of corn oil were subjected to preliminary ultrasonic emulsification. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain the primary emulsion. The droplet size was measured to be 20 μm. (3) Mix 1L of primary emulsion with 2L of oil phase containing 0.5 M Ca²⁺ to obtain a mixed emulsion; (4) The mixed emulsion was treated by acoustic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (5) The mixture is centrifuged to separate the particles, the particles are washed with deionized water to remove the oil phase, and the solid particles are obtained by freeze drying.

[0041] The resulting particles are homogeneous composite particles, with the polysaccharide of *Amanita argentea* and sodium alginate evenly distributed, and no Janus structure; therefore, the "activity-function" partitioning design cannot be achieved.

[0042] Comparative Example 4 (Continuous ultrasound mode - no pulse intervals) A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) 1L of argentea polysaccharide aqueous solution was mixed with 4L of corn oil for preliminary ultrasonic emulsification. Ultrasonic frequency: 20kHz, ultrasonic power: 200W, emulsification temperature: 40℃, emulsification time: 5 minutes, to obtain primary emulsion A. The droplet size was measured to be 20μm. (3) 1L of sodium alginate aqueous solution was subjected to preliminary ultrasonic emulsification with 4L of corn oil. Ultrasonic frequency: 20 kHz, ultrasonic power: 200 W, emulsification temperature: 40℃, emulsification time: 5 minutes to obtain primary emulsion B. The droplet size was measured to be 20 μm. (4) Mix 1L of colostrum A and 1L of colostrum B with 2L of emulsion containing 0.5 M Ca 2+ The oil phase is mixed to obtain a mixed emulsion; (5) The mixed emulsion was treated by sonic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, continuous mode (without interruption), and total treatment time of 2 minutes to obtain the mixture; (6) The mixture is centrifuged to separate the particles, the particles are collected, washed with deionized water to remove the oil phase, and then freeze-dried to obtain solid particles.

[0043] Continuous ultrasound caused excessive fragmentation of a large number of droplets, generating a large number of nanoscale fragments; the Janus particle yield decreased to 28.5%.

[0044] Comparative Example 5 (No pre-emulsification - direct mixing and ultrasound) A method for preparing Janus polysaccharide particles from *Janus argentis* using sonofusion includes the following steps: (1) Prepare a 2% aqueous solution of *Amanita argentea* polysaccharide and a 5% aqueous solution of sodium alginate, respectively; (2) Mix 1L of *Amanita argentea* polysaccharide aqueous solution, 1L of sodium alginate aqueous solution and 10L of corn oil containing 0.5 M Ca²⁺ directly to obtain a mixed emulsion; (3) The mixed emulsion was treated by sonic fusion under the following conditions: ultrasonic frequency of 20 kHz, power of 200 W, pulse mode of 2 seconds working and 4 seconds intermittent, and total processing time of 2 minutes to obtain the mixture; (4) The mixture is centrifuged to separate the particles, which are then washed with deionized water to remove the oil phase and freeze-dried to obtain solid particles.

[0045] The droplet size distribution is extremely wide (5~200μm), with a large number of ultra-large droplets and micro droplets coexisting; the Janus structure is unclear, and multiphase mixing is severe; the particle morphology is irregular and the sphericity is poor.

[0046] Test metrics (1) Janus structure observation: surface morphology was observed by scanning electron microscopy (SEM); (2) Average particle size: determined by a laser particle size analyzer (Malvern Mastersizer 3000); (3) Encapsulation efficiency: The total sugar content in the particles was determined by the phenol-sulfuric acid method, and the encapsulation efficiency of *Amanita argentea* polysaccharide was calculated. Encapsulation rate = (Mass of *Amanita acicularis* polysaccharide in granules / Total mass of *Amanita acicularis* polysaccharide in feed) × 100% (4) Cumulative release rate (in vitro release experiment): The particles were placed in simulated intestinal fluid (PBS, pH 6.8, containing 0.5% Tween 80), and shaken at 37°C (100 rpm). Samples were taken at regular intervals, and the content of released polysaccharides was determined by the phenol-sulfuric acid method. The cumulative release rate was calculated. The results are shown in Table 1 below: Table 1

[0047] As shown in Table 1, the Janus polysaccharide particles prepared in Examples 1-6 of this invention are significantly superior to those in Comparative Examples 1-5 in terms of three key indicators: average particle size, encapsulation efficiency, and cumulative release rate. Example 1 has an average particle size of 45 μm, an encapsulation efficiency of 78.5%, and a cumulative release rate of 68.7% after 12 h. The particles are also regular in shape and have a clear Janus structure (e.g., ...). Figure 1 (As shown). In Example 3, the increased sodium alginate concentration led to increased emulsion viscosity, which was detrimental to droplet collision and fusion efficiency, and the encapsulation rate decreased slightly; at the same time, the crosslinking density increased, and the release rate slowed down. In Example 4, increasing the oil phase volume ratio reduced the average particle size to 38 μm and increased the cumulative release rate to 70.5%, indicating that increasing the continuous phase volume helps to refine the droplet size; in Example 5, increasing the ultrasonic power from 200 W to 250 W increased the encapsulation rate to 83.6% and the cumulative release rate to 71.2% after 12 h, indicating that appropriately increasing the ultrasonic energy can promote droplet collision and fusion efficiency, and the overall effect is the best, making it the optimal example; in Example 6, Ca... 2+ Increasing the concentration from 0.5 M to 0.8 M reduced the cumulative release rate to 58.6% over 12 hours, indicating that increased cross-linking density can delay drug release, but excessive cross-linking may decrease the release rate. In contrast, Comparative Example 1, which used mechanical stirring instead of ultrasonic fusion, saw a sharp drop in encapsulation efficiency to 32.6%, while the cumulative release rate reached 91.2%. This demonstrates that mechanical stirring cannot provide sufficient energy to drive the directional collision and fusion of the two droplets, resulting in the failure to effectively encapsulate a large amount of *Amanita phalloides* polysaccharide, thus losing its sustained-release properties. Comparative Example 2 used Ca-containing... 2+ The oil phase caused the sodium alginate droplets to solidify prematurely, forming independent gel beads that could not fuse with the Janus polysaccharide droplets. Ultimately, the Janus particle yield was 0%, the encapsulation efficiency was only 15.2%, and the cumulative release rate was 88.5%. Comparative Example 3 involved premixing the two phases and then emulsifying them uniformly. The resulting particles had a homogeneous composite structure, lacking Janus characteristics, and an encapsulation efficiency of 41.5%. While higher than Comparative Examples 1, 2, and 5, this was significantly lower than Examples 1-6, and the "activity-function" separation design could not be achieved. Comparative Example 4 used continuous ultrasonication, resulting in excessive droplet fragmentation, with an average particle size of only 15 μm and an encapsulation efficiency reduced to 35.8%. Furthermore, the polysaccharide's bioactivity was impaired due to excessive ultrasonic degradation. Comparative Example 5 omitted the pre-emulsification step and directly mixed and ultrasonicated, resulting in an extremely wide droplet size distribution (average particle size 65 μm) and an encapsulation efficiency of only 29.4%. This indicates that pre-emulsification is a necessary prerequisite for obtaining Janus particles with uniform size and controllable structure.

[0048] Protective effect against alcoholic liver injury (using Janus polysaccharide particles prepared in Example 5) (1) Establishment of an animal model of alcoholic liver injury First, all mice were acclimatized for one week. After the acclimatization period, healthy mice (weighing 20±2g) that were robust and moved freely were randomly divided into 9 groups of 8 mice each. These groups were: normal control group (NC), alcohol model group (AM), positive control group (PC), low-dose group of Janus polysaccharide granules (LJ), medium-dose group of Janus polysaccharide granules (MJ), high-dose group of Janus polysaccharide granules (HJ), low-dose group of Janus polysaccharide granules (LP), medium-dose group of Janus polysaccharide granules (MP), and high-dose group of Janus polysaccharide granules (HP).

[0049] The normal control group was administered physiological saline by gavage at a dose of 10 mL / kg bw daily. The model group (Alcohol, AM), the positive control group, and the low, medium, and high dose groups of Janus granules and Janus polysaccharide were administered the same amount of baijiu (Chinese liquor) by gavage for 8 consecutive weeks. The normal control group was administered physiological saline by gavage daily. The alcohol model group, the positive control group, the three Janus granule dose groups, and the three polysaccharide dose groups were administered the same amount of alcohol by gavage daily. After 4 hours, physiological saline, 200 mg / kg bw silymarin, 100, 200, and 400 mg / kg bw Janus granules solution, and 100, 200, and 400 mg / kg bw Janus polysaccharide solution were administered by gavage, respectively, once daily for 2 consecutive weeks. After the last administration, the patient was fasted for 12 hours without water. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at 80 mg / kg. Blood was collected from the orbital cavity after anesthesia, and organs were harvested. Part of the liver was fixed with 4% paraformaldehyde, and the other part was stored at -80℃.

[0050] (2) Measurement of relevant indicators 2.1 Determination of organ index Before blood collection, the mice in each group were weighed. After euthanizing the mice, their livers, spleens, and thymuses were collected and weighed, and the organ indices (liver, spleen, and thymus) were calculated. The specific calculation formula is as follows: Organ index (%) = m1 / m × 100; Where m1 is the organ (liver, spleen, thymus) mass (g) and m is the mouse body weight (g).

[0051] 2.2 Serum marker measurement Collect the serum from the upper layer and measure ALT and AST in the mouse serum according to the instructions in the kit.

[0052] 2.3 Assay of enzymes related to alcohol metabolism According to the kit instructions, the levels of ADH and ALDH in liver tissue homogenate were measured.

[0053] 2.4 Determination of Oxidative Stress Indicators According to the kit instructions, the contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and MDA in liver tissue homogenate were determined.

[0054] 2.5 Data Processing All experiments were repeated three times, and the results were averaged. The data in the graphs are expressed as mean ± standard deviation. The response surface methodology was analyzed using Design-Expert 13 software, and the data were analyzed for statistical significance using SPSS 1 6.0 software.

[0055] Data Results: (1) Effects of Janus polysaccharide particles on body weight and organ indices in mice The effects of polysaccharides from *Amanita argentea* mycelium on body weight and organ indices in mice with alcohol-induced liver injury are shown in Table 2. Table 2. Effects of Janus polysaccharide granules on body weight and organ indices in mice (n=8)

[0056]

[0057] Note: Compared with the normal control group. a P<0.05; compared with the alcohol model group, b-h P<0.05; different letters indicate significant differences between groups (P<0.05).

[0058] As can be clearly seen from Table 2, compared with the normal control group, the liver index of mice in the alcohol model group was significantly increased by 42.0% (5.85 vs 4.12), the spleen index was increased by 36.8% (0.52 vs 0.38), and the thymus index was decreased by 35.7% (0.18 vs 0.28). This indicates that long-term alcohol gavage successfully induced pathological changes in the liver, spleen, and thymus of mice, and the alcoholic liver injury model was successfully established.

[0059] At the same dosage, the Janus granule group was superior to the corresponding polysaccharide group in terms of improvement in organ indices: LJ group (100mg / kg): Liver index decreased by 4.6% (5.42 vs 5.68) compared to LP group, spleen index decreased by 6.0% (0.47 vs 0.50), and thymus index increased by 10.5% (0.21 vs 0.19). In the MJ group (200 mg / kg): the liver index decreased by 7.6% (4.85 vs 5.25), the spleen index decreased by 6.5% (0.43 vs 0.46), and the thymus index increased by 9.1% (0.24 vs 0.22) compared to the MP group. In the HJ group (400 mg / kg): the liver index decreased by 6.9% (4.45 vs 4.78), the spleen index decreased by 4.8% (0.40 vs 0.42), and the thymus index increased by 4.0% (0.26 vs 0.25) compared with the HP group, indicating that the Janus structure has a significant synergistic effect on the hepatoprotective activity of *Cyclocarya acuminata* polysaccharide.

[0060] (2) Effects of Janus polysaccharide particles on ALT and AST, marker enzymes of alcoholic liver injury in mice. Table 3. Effects of Janus polysaccharide particles on ALT and AST, marker enzymes of alcoholic liver injury in mice (n=8)

[0061] Note: Compared with the normal control group. a P<0.05; compared with the alcohol model group, b-h P<0.05; different letters indicate significant differences between groups (P<0.05).

[0062] Table 3 shows that, compared with the normal control group, the serum ALT activity in the alcohol model group was significantly increased by 140.0% (68.4 vs 28.5), and the AST activity was significantly increased by 134.6% (75.3 vs 32.1), indicating that long-term alcohol intake leads to increased hepatocyte membrane permeability, release of large amounts of transaminases into the blood, and severe hepatocyte damage. The ALT (34.5 U / L) and AST (38.6 U / L) levels in the HJ group were significantly better than those in the HP group (40.2 and 45.1, respectively). This is mainly due to the unique "activity-function" separation design of Janus granules—the polysaccharide-rich region of *Amanita argentea* provides hepatoprotective activity, protecting the integrity of hepatocyte membranes through free radical scavenging and lipid peroxidation; the calcium alginate gel region prolongs the in vivo circulation time through a sustained-release effect, increasing polysaccharide accumulation in the liver. Furthermore, the asymmetric structure of Janus granules facilitates retention and sustained release in the intestine, improving oral bioavailability.

[0063] (3) Effects of Janus polysaccharide particles on the activity of alcohol metabolism-related enzymes in ALD mice Table 4. Effects of Janus polysaccharide particles on the activity of alcohol metabolism-related enzymes in ALD mice (n=8)

[0064] Note: Compared with the normal control group. a P<0.05; compared with the alcohol model group, b-h P<0.05; different letters indicate significant differences between groups (P<0.05).

[0065] The activities of ADH and ALDH in liver tissue homogenates from each group of mice are shown in Table 4. Compared with the normal control group, the liver ADH activity in the alcohol model group decreased by 45.6% (6.8 vs 12.5) and the ALDH activity decreased by 51.2% (4.2 vs 8.6), indicating that long-term alcohol intake severely inhibited the activity of key enzymes in liver ethanol metabolism. The Janus granule group showed a significantly better effect on increasing ADH and ALDH activity than the corresponding polysaccharide group, mainly because the calcium alginate gel matrix forms a protective barrier in the intestine, reducing the degradation of *Amanita argentea* polysaccharide by gastric acid; at the same time, the sustained-release properties of Janus granules allow the polysaccharide to maintain an effective concentration in the liver for a longer period of time, continuously activating the expression of alcohol-metabolizing enzymes; *Amanita argentea* polysaccharide upregulates the expression of metabolic enzyme genes by regulating the Nrf2 / Keap1 signaling pathway, and the Janus structure enhances this regulatory effect by improving bioavailability.

[0066] (4) Effects of Janus polysaccharide particles on antioxidant enzymes and oxidation products in ALD mice Table 5. Effects of Janus polysaccharide particles on antioxidant enzymes and oxidation products in ALD mice (n=8)

[0067] Note: Compared with the normal control group. a P<0.05; compared with the alcohol model group, b-h P<0.05; different letters indicate significant differences between groups (P<0.05).

[0068] As shown in Table 5, compared with the normal control group, the liver SOD activity in the alcohol model group decreased by 44.5% (158.4 vs 285.6), GSH-Px activity decreased by 42.8% (24.5 vs 42.8), and MDA content increased by 168.8% (8.6 vs 3.2). This indicates that the large amount of reactive oxygen species (ROS) produced by alcohol metabolism exceeded the body's antioxidant system's clearance capacity, leading to severe oxidative stress damage and lipid peroxidation. The Janus particle group was significantly better than the corresponding polysaccharide group in terms of antioxidant indicators. This is mainly because the polysaccharide-rich region of *Amanita argentea* contains a large number of active hydroxyl groups, which can directly scavenge free radicals and activate the endogenous antioxidant enzyme system. At the same time, the calcium alginate gel region forms a pH-responsive drug release system in the intestine, maintaining structural stability in the acidic environment of the stomach and slowly releasing active polysaccharides in the neutral environment of the intestinal fluid, thus prolonging the antioxidant effect time. The retention time of micron-sized Janus particles in the intestine is significantly longer than that of free polysaccharides, which is conducive to the full release and absorption of active ingredients in the intestine.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing Janus polysaccharide particles from *Janus argentis* using a sonofusion method, characterized in that, Includes the following steps: (1) Prepare aqueous solutions of polysaccharide from *Amanita argentea* and sodium alginate, respectively; (2) The aqueous solution of *Amanita argentea* polysaccharide was preliminarily emulsified with an oil phase that did not contain cross-linking ions to obtain primary emulsion A; (3) The sodium alginate aqueous solution was preliminarily emulsified with the oil phase without crosslinking ions to obtain the primary emulsion B; (4) Mix primary emulsion A and primary emulsion B with an oil phase containing cross-linking ions to obtain a mixed emulsion; (5) The mixed emulsion was treated by sonofusion to obtain a mixture; (6) The mixture was centrifuged to collect Janus gel particles, washed with washing solvent to remove the oil phase, and freeze-dried to obtain solid Janus polysaccharide particles.

2. The preparation method according to claim 1, characterized in that, The volume ratio of the aqueous solution of *Amanita argentea* polysaccharide in step (2) to the oil phase without cross-linking ions is 1:3 to 1:

6.

3. The preparation method according to claim 1, characterized in that, The volume ratio of the sodium alginate aqueous solution to the oil phase without cross-linking ions in step (3) is 1:3 to 1:

6.

4. The preparation method according to claim 1, characterized in that, The droplet size of both primary emulsion A and primary emulsion B is 10~50μm.

5. The preparation method according to claim 1, characterized in that, The volume ratio of the primary emulsion A, primary emulsion B and the oil phase containing crosslinking ions in step (4) is 1:1:(1.5~3).

6. The preparation method according to claim 1, characterized in that, The cross-linking ion mentioned in step (4) is Ca 2+ Ba 2+ 、Sr 2+ or Zn 2+ At least one of them, wherein the concentration in the oil phase is 0.1~1.0 M.

7. The preparation method according to claim 1, characterized in that, The conditions for the acoustic fusion method described in step (5) are: ultrasonic frequency of 15~40 kHz, power of 100-300 W, pulse mode of 1~4 seconds of operation and 2~6 seconds of interval, and total processing time of 1~5 minutes.

8. The Janus polysaccharide particles prepared by the method according to any one of claims 1-7 are characterized in that, The particles have an asymmetric Janus structure, with one side being a polysaccharide-rich region of *Amanita argentea* and the other side being a calcium alginate cross-linked gel region, and the particle size is 20~100μm.

9. The use of Janus polysaccharide granules according to claim 8 in the preparation of drugs for the prevention and treatment of alcoholic liver injury.

10. The application of Janus polysaccharide granules according to claim 8 in the preparation of health products with auxiliary protective effects against chemically induced liver injury.

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