Ganoderma lucidum polysaccharide / beta-sitosterol liposome as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a ganoderan / beta-sitosterol liposome as well as a preparation method and application thereof. The preparation method sequentially comprises the steps of preparation of the ganoderan and preparation of the ganoderan / beta-sitosterol liposome. According to the method, a natural eutectic solvent system with food-grade betaine as a hydrogen bond acceptor is designed and constructed for the first time for ganoderma lucidum polysaccharide extraction, the optimized ternary system with the ratio of betaine to lactic acid to glycerol being 1: 2: 1 is high in extraction efficiency (1.20%) and has good cycle stability, and a new thought is provided for green extraction of ganoderma lucidum polysaccharide; besides, the beta-sitosterol is used for replacing traditional cholesterol for preparing the ganoderan liposome for the first time, a novel delivery system with uniform particle size and high encapsulation efficiency is obtained, the novel delivery system has good slow release characteristic and anti-inflammatory activity, and a new choice is provided for developing a safe and efficient ganoderan delivery system.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a ganoderma polysaccharide / β-sitosterol liposome and a preparation method and application thereof. Background Art

[0002] Ganoderma lucidum ( Ganoderma lucidum ), a fungus of the genus Ganoderma in the Polyporaceae family, contains rich biologically active ingredients, including polysaccharides, triterpenes, amino acids, nucleosides and trace elements, and has multiple pharmacological activities such as enhancing immunity, anti-oxidation, anti-inflammatory, anti-tumor and regulating blood sugar.

[0003] Among them, Ganoderma lucidum polysaccharide (GLP), as the most important active ingredient, has demonstrated significant efficacy in immunomodulation and anti-inflammatory effects. In 2023, Ganoderma lucidum was officially included in the catalogue of medicinal and edible substances, providing policy support for its development and application in the functional food sector.

[0004] However, the development and utilization of Ganoderma lucidum faces two major bottlenecks: first, the cell walls of Ganoderma lucidum fruiting bodies are rich in macromolecules such as cellulose, hemicellulose and lignin, forming a dense network structure, resulting in low efficiency in the extraction of its active ingredients; second, Ganoderma lucidum polysaccharides, as natural macromolecules, have strong water solubility, low bioavailability and easy enzymatic hydrolysis, resulting in poor stability, short half-life and difficulty in penetrating biological barriers in clinical applications.

[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a method for preparing Ganoderma lucidum polysaccharide / β-sitosterol liposomes, which comprises the following steps: (I) Preparation of Ganoderma lucidum polysaccharide: (I-1) Ganoderma lucidum fruiting body ( Ganoderma lucidum Ganoderma lucidum fruiting bodies) slices (purchased from Shao's Biotechnology Co., Ltd., Jinzhai County, Anhui Province, and identified as Ganoderma lucidum, a Polyporaceae fungus) were washed and cleaned, and then dried to obtain dried Ganoderma lucidum slices; (I-2) crushing the dried Ganoderma lucidum slices to obtain Ganoderma lucidum powder; (I-3) mixing the Ganoderma lucidum powder with an extraction solvent and heating the mixture under reflux, and then centrifuging, concentrating, precipitating with alcohol, centrifuging again, and freeze-drying in sequence to obtain Ganoderma lucidum polysaccharide; (II) Preparation of Ganoderma lucidum polysaccharide / β-sitosterol liposomes: (II-1) mixing and dissolving soybean lecithin, β-sitosterol, a surfactant, and an oily solvent to obtain an oil phase; (II-2) mixing and dissolving the Ganoderma lucidum polysaccharide with an aqueous solvent to obtain an aqueous phase; (II-3) adding the aqueous phase dropwise to the oil phase and performing sonication to obtain an emulsion; (II-4) The emulsion is sequentially rotary evaporated, hydrated, and crushed, and then filtered to obtain the Ganoderma lucidum polysaccharide / β-sitosterol liposome.

[0007] Preferably, in step (I-1), the drying temperature is 75-85° C., and the drying time is 46-50 h.

[0008] Preferably, in step (I-2), the pulverization method is coarse grinding or airflow ultrafine grinding; wherein: The coarse grinding method is: using a high-speed Chinese herbal medicine grinder to coarsely grind the dried Ganoderma lucidum slices for 10 to 12 minutes; The airflow ultrafine grinding method is: firstly, the dried Ganoderma lucidum slices are coarsely ground with a high-speed Chinese herbal medicine grinder for 10-12 minutes, and then the airflow grinder is used to grind them 6 or 15 times at a grinding pressure of 0.5-0.7 MPa.

[0009] Preferably, in step (I-3), the extraction solvent is water, NaHCO3 solution or a natural deep eutectic solvent; wherein: When the extraction solvent is water, the heating reflux process is: liquid-to-solid ratio 20:1~60:1mL / g, extraction temperature 60~100℃, extraction time 1~5h; When the extraction solvent is NaHCO3 solution, the heating reflux process is: liquid-to-solid ratio 10:1~50:1mL / g, NaHCO3 concentration 0.1~2.0mol / L, extraction temperature 40~80℃, extraction time 1~5h; When the extraction solvent is a natural deep eutectic solvent (NADES), the natural deep eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is betaine, and the hydrogen bond donor is one or a combination of two of glycerol, 1,2-propylene glycol, lactic acid, and levulinic acid; the heating reflux process is: liquid-to-solid ratio 20:1 mL / g, natural deep eutectic solvent water content 70 wt%, extraction temperature 70°C, and extraction time 2 h.

[0010] Preferably, in step (II-1), the ratio of soybean lecithin, β-sitosterol, surfactant and oily solvent is 240-260 mg: 25-35 mg: 25-35 mg: 8-12 mL; And / or, the surfactant is Tween-80; And / or, the oily solvent is ether.

[0011] Preferably, in step (II-2), the ratio of the Ganoderma lucidum polysaccharide to the aqueous solvent is 15-25 mg: 8-12 mL; And / or, the aqueous solvent is PBS solution.

[0012] Preferably, in step (II-3), the total ultrasonic time is 8 to 12 minutes; in each ultrasonic cycle, the working time is 5 seconds and the rest time is 5 seconds.

[0013] Preferably, in step (II-4), the rotary evaporation time is 25 to 35 minutes; And / or, the stirring time of the hydration is 35 to 45 minutes; And / or, the filtration is: passing through a 220 nm microporous filter membrane.

[0014] Based on the same technical concept, another embodiment of the present invention is to provide a Ganoderma lucidum polysaccharide / β-sitosterol liposome obtained by the above preparation method.

[0015] Based on the same technical concept, one solution of the present invention is to provide a use of Ganoderma lucidum polysaccharide / β-sitosterol liposomes in the preparation of drug carriers.

[0016] The beneficial effects of the present invention are: This invention takes the green and efficient extraction of Ganoderma lucidum polysaccharides and the development of liposome preparations as the main line, and systematically solves the key scientific problems in the development process of the entire chain from "extraction-structure-function": including the mass transfer resistance problem caused by the dense network structure of the Ganoderma lucidum cell wall and the optimization of delivery system materials. First, the airflow ultrafine grinding technology is used to optimize the material pretreatment process, and an innovative natural deep eutectic solvent system with food-grade betaine as a hydrogen bond acceptor is designed. Density functional theory is used to deeply elucidate the molecular mechanism of action of DES in extracting Ganoderma lucidum polysaccharides; then, the feasibility of preparing Ganoderma lucidum polysaccharide liposomes with β-sitosterol instead of traditional cholesterol is preliminarily explored, and its anti-inflammatory activity is evaluated. The main research results obtained are as follows: (1) The present invention systematically investigated the effects of airflow milling, hot water extraction, weak base extraction and low eutectic solvent on the extraction efficiency of Ganoderma lucidum polysaccharide (GLP). The experimental results showed that after 15 times of airflow milling, the D50 of Ganoderma lucidum powder was reduced to 5.84μm, the wall breaking rate reached 0.86, and the hydration performance was significantly improved. The orthogonal test of hot water extraction determined that the optimal process conditions were 80℃, 4h, and liquid-to-solid ratio of 30:1, with a yield of 0.90%. The response surface optimization results of weak base extraction showed that the liquid-to-solid ratio, alkali concentration, time and temperature had a significant effect on the extraction rate, and the yield reached 2.95% under the optimized conditions. 14 natural low eutectic solvents with betaine as hydrogen bond acceptor were designed and synthesized, among which DES-10 (betaine: lactic acid: glycerol = 1:2:1) had the highest extraction rate of 1.16%. DFT calculations showed that its binding energy with glucose was -51.14kcal / mol, forming a stable hydrogen bond network structure and having good recycling performance.

[0017] (2) In the present invention, β-sitosterol was used instead of traditional cholesterol to prepare Ganoderma lucidum polysaccharide liposomes, and the effects of the two excipients on the performance of the liposomes were systematically evaluated. Dynamic light scattering analysis showed that the average particle sizes of GLP-Chol-Lip and GLP-β-Sito-Lip were 136.7 nm and 122.3 nm, respectively, and the PDIs were 0.362 and 0.378, respectively. Transmission electron microscopy observation further confirmed that both exhibited typical vesicle structures. In terms of drug loading properties, GLP-Chol-Lip showed a higher encapsulation efficiency (77.93%), which was better than GLP-β-Sito-Lip (75.60%). This difference was also reflected in the stability study, in which the particle size of GLP-Chol-Lip only increased by 5.8 nm after storage at 4°C for 21 days, while that of GLP-β-Sito-Lip increased by 12.8 nm. Drug release behavior studies revealed that GLP-Chol-Lip and GLP-β-Sito-Lip exhibited excellent sustained-release properties, with 24-hour release rates of 35% and 45%, respectively. Biological evaluations demonstrated that both liposomes exhibited good biocompatibility at a concentration of 500 μg / mL and effectively inhibited LPS-induced inflammatory responses, reducing NO levels to 9.2±0.8 and 9.6±0.7 μmol / L, respectively.

[0018] In summary, the present invention designs and constructs for the first time a natural low eutectic solvent system with food-grade betaine as a hydrogen bond acceptor for the extraction of Ganoderma lucidum polysaccharide. The optimized ternary system of betaine: lactic acid: glycerol = 1:2:1 not only has a high extraction efficiency (1.20%), but also has good circulation stability, providing a new idea for the green extraction of Ganoderma lucidum polysaccharide; in addition, the present invention uses β-sitosterol instead of traditional cholesterol to prepare Ganoderma lucidum polysaccharide liposomes for the first time, obtaining a new delivery system with uniform particle size and high encapsulation efficiency, and has good sustained-release properties and anti-inflammatory activity, providing a new option for the development of safe and efficient Ganoderma lucidum polysaccharide delivery systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is the particle size distribution diagram of Ganoderma lucidum powder.

[0021] Figure 2These are SEM images of Ganoderma lucidum powder; A1, B1, and C1 are M10 powder, M10-AJM6 powder, and M10-AJM15 powder, respectively, ×500; A2, B2, and C2 are M10 powder, M10-AJM6 powder, and M10-AJM15 powder, respectively, ×1000.

[0022] Figure 3 The graph shows the water holding capacity, water solubility index and swelling capacity of M10, M10-AJM6 and M10-AJM15.

[0023] Figure 4 is the glucose standard curve.

[0024] Figure 5 This is a graph showing the effects of time, liquid-to-solid ratio, and temperature on GLP extraction rate.

[0025] Figure 6 This is a graph showing the effects of liquid-to-solid ratio, alkali concentration, time, and temperature on GLP extraction rate.

[0026] Figure 7 It is a three-dimensional response surface diagram of temperature, material-liquid ratio and NaHCO3 concentration on extraction rate.

[0027] Figure 8 These are sample images of 14 NADES prepared with different component ratios.

[0028] Figure 9 It is the extraction rate of Ganoderma lucidum polysaccharide by different extraction solvents.

[0029] Figure 10 This is an analysis chart of the changes in extraction rate and recovery rate of GLP extracted cyclically by three NADES.

[0030] Figure 11 This is a comparison chart of the molecular electrostatic potential distribution and structure optimization results of NADES, water and glucose.

[0031] Figure 12 The figure shows the macroscopic and microscopic morphology of Ganoderma lucidum polysaccharide liposomes; A shows the appearance of GLP-Chol-Lip and GLP-β-Sito-Lip liposomes; B shows the Tyndall effect of GLP-Chol-Lip and GLP-β-Sito-Lip liposomes; C shows the transmission electron micrograph of GLP-Chol-Lip (×30,000); and D shows the transmission electron micrograph of GLP-β-Sito-Lip (×30,000).

[0032] Figure 13 This is the particle size distribution diagram of GLP-Chol-Lip.

[0033] Figure 14This is the particle size distribution diagram of GLP-β-Sito-Lip.

[0034] Figure 15 ] are ultraviolet absorption spectra of liposomes; wherein: A is the ultraviolet absorption spectra of GLP, Chol-Lip, and GLP-Chol-Lip; B is the ultraviolet absorption spectra of GLP, β-Sito-Lip, and GLP-β-Sito-Lip.

[0035] Figure 16 are XRD spectra of liposomes; wherein: A is the XRD spectra of Chol-Lip, GLP-Chol-Lip, and PM-GLP-Chol-Lip; B is the XRD spectra of β-Sito-Lip, GLP-β-Sito-Lip, and PM-GLP-β-Sito-Lip.

[0036] Figure 17 The graphs show the particle size and PDI data of GLP-Chol-Lip and GLP-β-Sito-Lip stored at 4°C for 1, 7, 14, and 21 days, respectively.

[0037] Figure 18 Graph showing the release rates of GLP, GLP-Chol-Lip, and GLP-β-Sito-Lip.

[0038] Figure 19 The effects of different concentrations of Ganoderma lucidum polysaccharides and their liposomes on cell survival rate (n=3).

[0039] Figure 20 Evaluation of the anti-inflammatory activity of Ganoderma lucidum polysaccharides and their liposomes. A: control group; B: model group; C: GLP; D: GLP-Chol-Lip; E: GLP-β-Sito-Lip. Note: *P < 0.05, ****P < 0.0001. DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0041] Example 1: This example focuses on the extraction technology of Ganoderma lucidum polysaccharide 1. Experimental methods 1.1 Pretreatment of Ganoderma lucidum fruiting bodies 1.1.1 Sample pretreatment: Weigh a certain amount of Ganoderma lucidum fruiting body slices, rinse with deionized water to remove surface impurities, and dry the washed samples in a constant temperature drying oven at 80°C for 48 h to constant weight.

[0042] 1.1.2 Preparation of coarse Ganoderma lucidum powder: Grind the dried Ganoderma lucidum slices using a YF112 high-speed Chinese herbal medicine grinder for 10 minutes. Cool down each batch for 2 minutes to avoid overheating to obtain coarse Ganoderma lucidum powder, which is recorded as sample M10 (Milling 10 minutes).

[0043] 1.1.3 Degreasing: Mix the Ganoderma lucidum powder with the extraction solvent and heat under reflux. Filter using a Büchner funnel and qualitative filter paper. Collect the residue and dry it in an 80°C drying oven to a constant weight. Store the final product in a sealed desiccator until ready for use.

[0044] 1.2 Preparation of Ganoderma lucidum ultrafine powder The coarse Ganoderma lucidum powder obtained in 1.1.2 was further ground in an AJM150B air jet mill at a pulverization pressure of 0.6 MPa to obtain ultrafine powder. The milling cycles were varied to 6 and 15 cycles, respectively, to obtain ultrafine Ganoderma lucidum powders, designated M10-AJM6 (Milling 10 minutes - Air Jet Milling 6 cycles) and M10-AJM15 (Milling 10 minutes - Air Jet Milling 15 cycles). Degreasing was then performed according to 1.1.3.

[0045] 1.3 Determination of particle size and physicochemical properties of Ganoderma lucidum powder 1.3.1 Ganoderma Lucidum Powder Particle Size Determination: The particle sizes of samples M10, M10-AJM6, and M10-AJM15 were measured using an LS13320 laser particle size analyzer (Beckman Coulter, Inc., USA). Powder particle sizes are expressed as D10, D50, and D90, representing the 10th, 50th, and 90th percentile cumulative particle sizes, respectively. The span factor (Span) and wall-breaking rate (Φ) were calculated using the following formulas:

[0046] 1.3.2 Electron Microscopy of Ganoderma Lucidum Powder: M10, M10-AJM6, and M10-AJM15 samples were used for surface observation using a JEOL JSM-6700F cold field emission scanning electron microscope (JEOL). For ease of observation, the hydrogel samples were evenly affixed to a brass sheet using double-sided carbon tape. The brass sheet was then metallized using gold sputtering to eliminate charging effects. The working distance was 7.0-7.2 mm, and the accelerating voltage was 2 kV. Observation and photography were performed at magnifications of 500 / 1000.

[0047] 1.3.3 Determination of hydration capacity of Ganoderma lucidum powder (1) Determination of water holding capacity of Ganoderma lucidum powder: Water holding capacity (WHC, g / g) is calculated according to the following formula.

[0048]

[0049] Where W1 represents the mass of the sample powder (g), W2 represents the mass of the empty centrifuge tube (g), and W3 represents the mass of the centrifuge tube containing the sediment after centrifugation (g).

[0050] (2) Determination of water solubility of Ganoderma lucidum powder: The water solubility index (WSI, %) is calculated according to the following formula.

[0051]

[0052] Where W1 is the weight of the sample powder (g), W2 is the weight of the empty Petri dish (g), and W3 is the constant weight of the supernatant and Petri dish after drying (g).

[0053] (3) Determination of the swelling capacity of Ganoderma lucidum powder: The swelling capacity (SC, mL / g) was calculated according to the following formula.

[0054]

[0055] Where m is the weight of the sample powder (g), V1 is the initial volume of the system (mL), and V2 is the volume of the system after expansion (mL).

[0056] 1.4 Extraction and determination of GLP content 1.4.1 GLP Extraction: Clean and remove impurities from Ganoderma lucidum fruiting body slices, then dry them to obtain dried Ganoderma lucidum slices; crush the dried Ganoderma lucidum slices to obtain Ganoderma lucidum powder; mix the Ganoderma lucidum powder with an extraction solvent and heat under reflux. After completion, centrifuge (3000 rpm, 15 min) to collect the supernatant, concentrate, precipitate with alcohol (add 4 times the volume of anhydrous ethanol, precipitate at 4°C for 12 h), centrifuge again (3000 rpm, 15 min), and freeze-dry to obtain Ganoderma lucidum polysaccharides.

[0057] 1.4.2 Determination of polysaccharide content: Prepare a polysaccharide solution of appropriate concentration, accurately draw 2 mL of the polysaccharide solution, then add 1.0 mL of 5% phenol solution and mix thoroughly, add 5.0 mL of concentrated sulfuric acid and mix thoroughly. Color develop for 30 minutes, measure its absorbance at a wavelength of 490 nm, and calculate the Ganoderma lucidum polysaccharide yield based on the glucose standard curve and the absorbance value of the polysaccharide.

[0058] 1.4.3 Calculation formula for polysaccharide yield: The calculation formula for Ganoderma lucidum polysaccharide yield is:

[0059] W is the weight of Ganoderma lucidum powder (g); C is the polysaccharide concentration of the measured solution (μg / mL); V0 is the total volume of the color development solution (mL); V1 is the volume of the total Ganoderma lucidum polysaccharide solution (mL); V2 is the volume of the Ganoderma lucidum polysaccharide sample solution added in the color development step (mL).

[0060] 1.5 Single-factor experimental design for hot water extraction of GLP Hot water was used as the extraction solvent to extract Ganoderma lucidum polysaccharide. The effects of extraction temperature, extraction time and extraction liquid-to-solid ratio on the yield of Ganoderma lucidum polysaccharide were investigated.

[0061] That is, under the conditions of a liquid-to-solid ratio of 40:1 (mL / g) and a temperature of 80°C, the GLP yield was investigated when the extraction time was 1h, 2h, 3h, 4h, and 5h respectively; under the conditions of a liquid-to-solid ratio of 40:1 (mL / g) and an extraction time of 4h, the GLP yield was investigated when the extraction temperature was 60°C, 70°C, 80°C, 90°C, and 100°C respectively; under the conditions of a temperature of 80°C and an extraction time of h, the GLP yield was investigated when the liquid-to-solid ratio was 20:1mL / g, 30:1mL / g, 40:1mL / g, 50:1mL / g, and 60:1mL / g respectively.

[0062] 1.6 Orthogonal experimental design for hot water extraction of GLP Referring to the results of single factor optimization, the three factors with the greatest impact on the extraction rate were selected, and the three levels of each factor were determined, L9 (3 4) Orthogonal tables were used to design orthogonal experiments. The sugar content and the yield of Ganoderma lucidum polysaccharides under various conditions were determined using the phenol-sulfuric acid method. These were used as indicators for orthogonal experiments to determine the optimal process conditions for extracting Ganoderma lucidum polysaccharides, as shown in Table 1.

[0063] Table 1

[0064] 1.7 Single-factor experimental design for weak base extraction of GLP Ganoderma lucidum polysaccharide was extracted using NaHCO3 solution as the extraction solvent. The four factors of extraction solution, alkali concentration, extraction time and extraction temperature ratio were investigated to determine their effects on the yield of Ganoderma lucidum polysaccharide.

[0065] 1.8 Response surface design for weak base extraction of GLPs Based on the principles of central composite experimental design and single-factor experiments, we determined the required ranges of factor levels. Four factors significantly influencing the extraction of Ganoderma lucidum polysaccharides (Ganoderma lucidum polysaccharide) were selected: alkali extraction liquid-to-solid ratio, alkali concentration, extraction time, and extraction temperature. A response analysis experiment with four factors and three levels was conducted, totaling 30 experimental points. The experimental factor and level design is shown in Table 2.

[0066] Table 2

[0067] 1.9 Preparation of NADES The hydrogen bond acceptor and the hydrogen bond donor were mixed in the molar ratio shown in Table 3, and then magnetically stirred in a water bath at 80° C. for 30 min until a uniform and transparent solution was formed, thereby obtaining a NADES solvent.

[0068] Table 3

[0069] 1.10 NADES Extraction GLP Weigh 2.00g of Ganoderma lucidum crude powder into a 100mL conical flask and extract GLPs using magnetic stirring at 70°C for 2h. The extraction time is 2h, the solid-liquid ratio is 1:20g / mL, and the solvent moisture content is 70%. This means that 28mL of NADES is added to 12mL of water. After extraction, centrifuge at 4000r / min for 15min and remove the supernatant. Then, add 4 volumes of anhydrous ethanol and allow to precipitate overnight at 4°C. Centrifuge at 4000r / min for 15min to obtain a polysaccharide precipitate. The polysaccharide precipitate is then rinsed three times with anhydrous ethanol to ensure complete removal of NADES and eliminate interference from residual low eutectic solvent on polysaccharide yield measurement.

[0070] 1.11 Study on the Recycling Characteristics of NADES Solvents Following the NADES polysaccharide extraction method described above, the supernatant obtained by alcohol precipitation and centrifugation was vacuum dried to a constant volume to recover the NADES and use it in the next batch of experiments. The NADES recovery rate was calculated as follows.

[0071]

[0072] Where: V a V represents the volume of NADES recovered by vacuum drying the supernatant obtained by alcohol precipitation and centrifugation until the volume remains unchanged, mL; b represents the original volume before NADES extraction, mL.

[0073] 1.12 Density functional theory analysis of the molecular mechanism of GLP extraction by NADES Density functional theory was used to study the molecular mechanism of NADES extraction of Ganoderma lucidum polysaccharides. The DFT calculations were completed using Materialstudio16 software. First, the monomer molecules were optimized to analyze the differences in atomic Mulliken charges and molecular electrostatic potentials. Then, by analyzing and calculating the binding energy between DES and glucose, the number and bond length of hydrogen bonds between NADES and glucose were optimized to find the most stable geometric configuration. At the same time, DFT software was used to calculate the binding energy between water and glucose and the number and bond length of hydrogen bonds between water and glucose, revealing the inherent mechanism of NADES extraction of polysaccharides that is superior to traditional water extraction at the molecular level.

[0074] The binding energy (ΔE) of the interaction between glucose and NADES is determined by the energy difference between the DES-glucose complex and its components, as shown in the following formula:

[0075] Where: E DES-GLP Indicates the energy of NADES binding to polysaccharide; E NADES represents the energy of a single molecule NADES; E GLP Indicates the energy of polysaccharides.

[0076] 1.13 Data processing and statistical analysis Each sample in the experimental test was repeated three times, and the results were averaged with the standard deviation as the error value. The results were statistically tested and the data were processed using SPSS.26.0 software.

[0077] (2) Results and Discussion 2.1 Particle size analysis of Ganoderma lucidum powder The geometric shape and particle size distribution characteristics of particles are key factors that determine the physical and chemical properties of powder materials. Figure 1Table 4 and Table 5 show the particle size distribution characteristics of Ganoderma lucidum powder after different milling treatments. The experimental results show that the D10, D50, and D90 values of the M10 sample were 3.84 μm, 136.33 μm, and 414.00 μm, respectively, exhibiting a distinct wide-range distribution. Furthermore, after six airflow milling cycles, the D50 value of the sample significantly decreased to 13.50 μm, while the Span value increased to 14.53 μm, indicating that while the average particle size decreased, the distribution uniformity decreased. Furthermore, when the number of airflow milling cycles was increased to 15, the D90 value of the sample significantly decreased to 18.90 μm, and the Span value decreased to 2.84, approaching the level of the unmilled sample, indicating that high-frequency airflow milling can improve particle size uniformity. Furthermore, the wall-breaking rate Φ increased with the number of milling cycles, from 0.21 for M10 to 0.86 for M10-AJM15, indicating that airflow milling significantly enhanced the wall-breaking efficiency of Ganoderma lucidum powder.

[0078] Table 4

[0079] Note: The data marked with different lowercase superscripts in each row are significantly different (p<0.05).

[0080] 2.2 SEM analysis of Ganoderma lucidum powder Scanning electron microscope images show the micromorphological changes of Ganoderma lucidum powder after mechanical treatment to different degrees. Figure 2 A1 in Figure 2 A2 in the figure shows a long and rough interconnected fiber structure; after 6 times of air flow milling ( Figure 2 B1 in Figure 2 B2 in the figure), the fiber length decreased, the fragmentation increased, and the particle distribution became more uniform; further 15 times of air flow milling ( Figure 2 C1 in Figure 2 After C2 in the figure, the fibers are shorter and the particle density is higher, but the basic fiber properties are still maintained.

[0081] 2.3 Analysis of the Hydration Capacity of Ganoderma Lucidum Water Holding Capacity (WHC) refers to the ability of a substance to retain water under external pressure or centrifugal force. This indicator has an important impact on the storage and subsequent processing of medicinal materials. Figure 3 It can be seen from (A) that with the increase of the number of air flow crushing times, the WHC value gradually increases.

[0082] The water solubility index (WSI) reflects the degree of dissolution of soluble components in a substance. Figure 3As shown in (B), WSI increased significantly with the increase in the number of air flow milling times, which is believed to be related to the increase in specific surface area after cell wall fragmentation.

[0083] Swelling capacity (SC) characterizes the degree of volume increase after powder comes into contact with water and is an important indicator for evaluating the water absorption properties of medicinal materials. Figure 3 (C) shows that the SC value significantly increased from 1.8 mL / g for M10 to 4.2 mL / g for M10-AJM15. This increase is closely related to the abundant microporous structures and exposed hydrophilic functional groups generated during the ultrafine grinding process.

[0084] These results suggest that jet milling improves the hydration properties of Ganoderma lucidum fruiting body powder by changing its microstructure. This improvement not only facilitates subsequent extraction processing but also may enhance the bioavailability of active ingredients.

[0085] 2.4 Glucose standard curve Under certain conditions, phenol and glucose undergo a condensation reaction to form an orange-yellow substance with a characteristic peak at 490nm. Within a certain concentration range, there is a linear relationship between glucose concentration and its absorbance at 490nm. Therefore, this characteristic can be used for quantitative analysis. To more intuitively demonstrate this relationship, a standard curve was plotted with glucose concentration and A490 as the horizontal and vertical coordinates, respectively. Linear regression analysis yielded the following: y=0.0106x+0.0172, correlation coefficient R 2 =0.9937 (e.g. Figure 4 Therefore, under certain conditions, the glucose concentration can be accurately calculated by measuring the A490 value, thereby performing quantitative analysis.

[0086] 2.5 Single-factor experimental analysis of hot water extraction of GLP like Figure 5 As shown in the figure, the effects of extraction time, liquid-to-solid ratio and temperature on GLP yield were studied. Figure 5 The GLP extraction rate showed an upward trend from 0.58% to 0.85% during the 1-3h period. This is because the hot water gradually penetrated into the Ganoderma lucidum powder, promoting the softening and degradation of the cell wall and accelerating the dissolution of polysaccharides. When the extraction time exceeded 3h, the growth of the extraction rate tended to be slow. During the 3-5h period, it only increased slightly from 0.85% to 0.87%, indicating that the dissolution of polysaccharides was close to saturation. Figure 5Figure B) shows that as the liquid-to-solid ratio increases from 20:1 to 40:1, the extraction rate increases significantly (0.67%-0.90%). This is because a larger liquid-to-solid ratio provides a stronger concentration gradient, which is beneficial for the diffusion and transfer of polysaccharides. However, when the liquid-to-solid ratio exceeds 40:1, the increase in the extraction rate slows down significantly. Excessively high liquid-to-solid ratios will dilute the concentration of the target product. ( Figure 5 Figure C) shows that the extraction yield increases significantly with increasing temperature within the 60-80°C range. This is because increasing temperature increases the solubility of polysaccharides, reduces solvent viscosity, and promotes mass transfer. Above 80°C, the extraction yield remains largely stable. Considering both energy consumption and extraction efficiency, this example employed 80°C, a liquid-to-solid ratio of 40:1, and an extraction time of 3 hours as the optimal process parameters for the single-factor experiment.

[0087] 2.6 Analysis of hot water extraction GLP orthogonal test results It can be seen intuitively from Table 5 that the order of the range R of the orthogonal test is B>A>C, that is, the order of factors affecting the extraction rate of Ganoderma lucidum polysaccharide is: extraction time>extraction temperature>liquid-to-solid ratio.

[0088] Table 5

[0089] The results in Table 6 show that none of the factors significantly affected the extraction yield. Orthogonal experiments determined that the optimal extraction conditions were A2B3C1: temperature 80°C, extraction time 4 hours, and liquid-to-solid ratio 30:1 (mL / g). Under these conditions, the yield of crude Ganoderma lucidum polysaccharides was 0.90%, or 9.0 mg / g.

[0090] Table 6

[0091] 2.6.1 Confirmatory Experiments To verify the optimization results of the orthogonal design experiment, a confirmatory experiment was conducted using the optimal level combination determined by the single-factor experiment as a control. The experiment was repeated three times. The experimental results are shown in Table 7.

[0092] Table 7

[0093] It can be seen from Table 7 that the only difference between the optimal extraction processes determined by the single-factor experiment and the orthogonal design experiment is the different liquid-to-solid ratio. There is no significant difference in the average value of the polysaccharide extraction rate between the two. In addition, considering the saving of solvent and energy and shortening of concentration time, the RSD value is also small, indicating that the Ganoderma lucidum polysaccharide extraction process optimized by the orthogonal design experiment is stable and efficient, and has certain guiding significance for production.

[0094] 2.7 Weak base extraction GLP single factor experimental analysis The effects of various extraction factors were investigated: the liquid-to-solid ratio was set to 10, 20, 30, 40, 50 mL / g, while other conditions remained unchanged (temperature: 60°C, NaHCO3 concentration: 0.5 M, time: 2 h); the NaHCO3 concentration was set to 0.1, 0.2, 0.5, 1.0, 1.5, 2.0 mol / L, while other conditions remained unchanged (temperature: 60°C, liquid-to-solid ratio: 30 mL / g, time: 2 h); the extraction time was set to 1, 2, 3, 4, 5 h, while other conditions remained unchanged (temperature: 60°C, liquid-to-solid ratio: 30 mL / g, NaHCO3 concentration: 0.5 M); the extraction temperature was set to 40, 50, 60, 70, 80°C, while other conditions remained unchanged (liquid-to-solid ratio: 30 mL / g, NaHCO3 concentration: 0.5 M, time: 2 h). The results are as follows. Figure 6 shown.

[0095] from Figure 6 As can be seen, the Ganoderma lucidum polysaccharide content was significantly lower at a liquid-to-solid ratio of 10:1 mL / g. This is because too low a liquid-to-solid ratio causes the polysaccharide extraction to reach equilibrium within a short period of time, hindering further dissolution. Ganoderma lucidum polysaccharide content reached its peak at a liquid-to-solid ratio of 30 mL / g. At NaHCO3 concentrations of 0.1 to 1.5 mol / L, the Ganoderma lucidum polysaccharide content increased rapidly. However, at a NaHCO3 concentration of 2 mol / L, the polysaccharide content decreased slightly. This may be because alkaline extraction helps to break down the physical and chemical interactions between cell wall polymer molecules, breaking the bonds between insoluble cellulose, lignin, and hemicellulose and pectin polysaccharides, thereby increasing polysaccharide yield. However, excessively high alkaline concentrations can disrupt the polysaccharide structure. The GLP yield showed a rapid increase followed by a plateau with increasing extraction time. The extraction rate increased rapidly between 1 and 3 hours, reaching a peak of 2.2% at 4 hours, indicating that this may be the optimal extraction time. From 4 to 5 hours, the GLP yield decreased, but the change was not significant, suggesting that extending the extraction time beyond 4 hours no longer significantly improves efficiency. Overall, the extraction yield remained high within the 3-5 hour range, with minimal variation, providing flexibility for practical production. However, further extending the extraction time may lead to degradation or structural changes in some polysaccharides due to the prolonged alkaline environment, thus affecting the final extraction yield. The extraction yield of Ganoderma lucidum polysaccharides showed a trend of initially increasing rapidly and then leveling off with increasing temperature. The extraction yield increased sharply between 40°C and 60°C, then slowed from 60°C to 70°C, and almost stopped increasing after 70°C. The optimal extraction temperature range may be between 60°C and 70°C, achieving a high extraction yield while avoiding the increased energy consumption and polysaccharide structural damage that may be caused by excessively high temperatures. This provides a reference for optimizing the extraction process.

[0096] 2.8 Analysis of GLP response surface experiments on weak base extraction The Design-Expert 7.0 program was used to perform regression analysis on the response values of 29 experimental points (as shown in Table 8), with the polysaccharide yield as the response value. After regression fitting, the influence of each experimental factor on the response value can be expressed by the regression equation.

[0097] Alkali soluble crude polyol extraction rate (%) = 2.19+0.3184X1+0.3353X2+0.1930X3+0.1706X4-0.0124X1X2-0.0312X1X3+0.0401X2X3-0.0254X2X4-0.0065X3X4+0.0789X1 2 +0.0831X2 2 -0.0779X3 2 -0.1080X4 2 .

[0098] Through variance analysis (as shown in Table 9), it was found that when the regression equation was used to describe the relationship between each factor and the polysaccharide yield, the multiple regression relationship between the dependent variable and all the independent variables was significant. The F value of the model was 52.78, and the "Prob>F" value was <0.0001, which was highly significant. In addition, the A, B, C, and D factors in the first-order term of the equation were significant, indicating that the experimental design was reasonable and the regression equation fitted the experimental results well at its test points. At the same time, the equation lack of fit test was not significant, indicating that the regression equation fitted well in the entire regression area, that is, it was reasonable to use this regression model, and the equation could be used to predict the polysaccharide yield under different extraction conditions (such as Figure 7 shown).

[0099] Table 8

[0100] Table 9

[0101] Parameter optimization analysis based on the established mathematical model revealed the following conditions for the highest polysaccharide yield: an alkali extraction liquid to solid ratio of 38.97 mL / g, an alkali extraction concentration of 1.43 mol / L, an alkali extraction time of 3.48 h, and an alkali extraction temperature of 66.91°C. The predicted GLP yield under this optimal process was 3.01%. To further verify the reliability of this experimental method, three polysaccharide alkali extraction experiments were conducted using the protocol derived from the model optimization analysis. Taking into account actual conditions, these conditions were modified to: an alkali extraction liquid to solid ratio of 39 mL / g, an alkali extraction concentration of 1.43 mol / L, an alkali extraction time of 3.5 h, and an alkali extraction temperature of 67°C. The actual measured polysaccharide yield was 2.97 ± 0.12%. At a significance level of p < 0.05, there was no significant difference between the predicted and actual values. Therefore, the extraction parameters optimized using response surface methodology are accurate, reliable, and have practical value.

[0102] 2.9 Screening of NADES like Figure 8 As shown, this example successfully prepared 14 NADES with different component ratios. All samples of deep eutectic solvents exhibited a transparent, homogeneous liquid state at room temperature (25±2°C), indicating that the hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) formed a stable eutectic system through hydrogen bonding, successfully lowering the melting point of the mixture. Two-component NADES (samples 1–7) exhibited good homogeneity, with DES-6 and DES-7, containing levulinic acid as the hydrogen bond donor, exhibiting a characteristic amber color. Three-component systems (samples 8–14) also demonstrated excellent compatibility, with no phase separation observed. After preparation, all samples were sealed and stored at room temperature for four weeks without exhibiting instability such as crystallization or turbidity, confirming the excellent thermodynamic stability of the designed NADES system.

[0103] like Figure 9As shown, the extraction efficiencies of the 14 NADES for GLPs varied significantly. DES-10, DES-1, and DES-12 achieved extraction rates of 1.16%, 0.96%, and 0.86%, respectively, significantly exceeding those of the aqueous control (0.73%) and other NADES systems. These differences in extraction efficiency may be due to differences in solvent polarity and hydrogen-bonding network structure. DES-10 (betaine:lactic acid:glycerol = 1:2:1) achieved the highest extraction yield, likely due to its unique ternary system achieving an optimal balance between polarity and the number of hydrogen-bond donors, enhancing interactions with the target compound. The presence of glycerol in DES-1 (betaine:glycerol = 1:2) and DES-12 (betaine:1,2-propylene glycol:glycerol = 1:1:1) also significantly enhanced the solvent's extraction capacity, consistent with the findings of Hooshmand SE et al., who reported that glycerol-based DESs possess excellent extraction properties. Based on a comprehensive evaluation of extraction efficiency, the three best-performing DESs were selected for subsequent DFT calculations and solvent recycling studies to gain a deeper understanding of their molecular mechanisms and potential for practical applications. These results provide an experimental basis for screening for highly efficient eutectic extraction systems.

[0104] 2.10 Reusability experiment of GLP extracted by NADES like Figure 10 As shown, the three NADES exhibited varying degrees of stability during the cyclic extraction process. The solvent appearance indicated that the three NADES maintained good homogeneity after cyclic use, with no phase separation. Combined with recovery data analysis, the recovery rates of all three NADES remained above 80%, demonstrating their excellent thermal and chemical stability.

[0105] 2.11 Analysis of the molecular mechanism of GLP extraction by NADES Figure 11 The molecular electrostatic potential distribution and structure optimization results for different NADES systems, as well as water and glucose, are shown. The left side shows the electrostatic potential distribution cloud of the solvent molecule (blue to red areas indicate negative and positive potential, respectively), the center shows the electrostatic potential distribution of the glucose molecule, and the right side shows the most stable molecular structure after DFT optimization, where the green solid arrows indicate the number and bond lengths of intermolecular hydrogen bonds (unit: Å). The studied systems include the interactions between DES-1 (betaine: glycerol = 1:2), DES-10 (betaine: glycerol: lactic acid = 1:1:2), and DES-12 (betaine: 1,2-propylene glycol = 1:1) and glucose, with water used as the control solvent.

[0106] Experimental results show that the molecular electrostatic potential distributions obtained through DFT calculations reveal that the quaternary ammonium groups of betaine in all NADES systems exhibit significant positive potential regions, while the hydroxyl groups exhibit negative potential regions. Furthermore, DFT-optimized molecular configurations reveal the hydrogen bond networks formed by different systems with glucose: DES-1 forms three hydrogen bonds (bond lengths of 1.809, 1.926, and 2.056 Å, respectively), DES-10 forms two hydrogen bonds (bond lengths of 1.726 and 2.185 Å), and DES-12 forms two hydrogen bonds (bond lengths of 1.978 and 2.467 Å). Water molecules only form one hydrogen bond with glucose (bond length of 2.095 Å). The number of hydrogen bonds in a DES system is positively correlated with its glucose solubility. Furthermore, the molecular electrostatic potential maps reveal a significant negative potential region (blue region, potential value approximately -0.035 au) around the hydroxyl groups of glucose molecules. Overall, the NADES system exhibits stronger glucose binding ability than water, which can be attributed to the multiple hydrogen bond networks it forms and more suitable molecular electrostatic potential complementarity. Among them, DES-1 exhibits the best glucose binding performance due to its largest number of hydrogen bonds.

[0107] Based on molecular structure simulations and binding energy analysis (Table 10), DES-10 exhibits the strongest binding energy with glucose (-51.14 kcal / mol), primarily attributable to its unique ternary system (betaine:glycerol:lactic acid = 1:1:2), which provides more hydrogen bonding sites. The structural simulations reveal that DES-10 forms a more complex hydrogen bonding network, in which the quaternary ammonium groups of betaine, the carboxyl and hydroxyl groups of lactic acid, and the polyol groups of glycerol all participate in the interaction with glucose. DES-1 exhibits the second highest binding energy (-22.60 kcal / mol). Its binary system (betaine:glycerol = 1:2) forms a stable hydrogen bond bridge with glucose via the polar head group of betaine and the hydroxyl groups of glycerol. Despite having more components, the ternary system of DES-12 (betaine:1,2-propylene glycol:glycerol = 1:1:1) exhibits a relatively weak binding energy (-14.47 kcal / mol), and molecular simulations indicate a looser hydrogen bonding network. In contrast, water molecules have the weakest binding energy with glucose (-7.58 kcal / mol), forming only simple hydrogen bonds from a structural perspective, which explains its lower extraction efficiency. The molecular simulation results show a good correlation with the experimental observation data.

[0108] Table 10

[0109] Example 2: Preparation, quality evaluation and preliminary study of anti-inflammatory effects of liposomes 1. Experimental methods 1.1 Preparation of Ganoderma lucidum polysaccharide / β-sitosterol liposomes (1) Oil phase: Weigh 250 mg of soybean lecithin into a 50 mL centrifuge tube, add 30 mg of β-sitosterol and 30 mg of Tween 80. Dissolve in 10 mL of ether, sonicate until completely dissolved, and store in a refrigerator at 4°C.

[0110] (2) Aqueous phase: Weigh 20 mg of Ganoderma lucidum polysaccharide into a 10 mL centrifuge tube, dissolve it in 10 mL of PBS solution to prepare a 2 mg / mL Ganoderma lucidum polysaccharide solution, sonicate until completely dissolved, and store in a refrigerator at 4°C.

[0111] (3) Preparation of W / O emulsion: 3 mL of Ganoderma lucidum polysaccharide solution was added dropwise to the oil phase and ultrasonicated for 10 min (mode 2, working for 5 s, stopping for 5 s) using an ice-water bath probe to prepare a milky white emulsion without stratification.

[0112] (4) Suspension evaporation: Transfer the emulsion to a 50 mL round-bottom flask and evaporate slowly for 30 min until the ether is completely evaporated and becomes gelatinous. Place it in a fume hood for 30 min to remove the organic solvent.

[0113] (5) Hydration: Add 10 mL of PBS solution and stir on a stirring bar for 40 minutes.

[0114] (6) Liposome disruption: The liposomes were ultrasonicated for 5 min (mode 2, 5 s on, 5 s off), filtered through a 220 nm microporous filter, and stored at 4 °C.

[0115] 1.2 Morphological observation 1.2.1 Observation of Appearance and Morphology: Place each group of polysaccharide liposomes in a separate bottle and take photos to observe their appearance and morphology. Use a red laser pointer to illuminate the liquid surface parallel to the liquid surface and observe the vertical scattered light path to verify the colloidal properties of the liposomes.

[0116] 1.2.2 Microscopic morphology observation: The morphological characteristics of each group of polysaccharide liposomes were observed using transmission electron microscopy.

[0117] 1.3 Determination of particle size and polydispersity index (PDI): The average particle size, particle size distribution and Zeta potential of the samples were determined using a laser particle size analyzer.

[0118] 1.4 UV spectral scanning determination: Take appropriate amount of polysaccharide solution, polysaccharide liposomes and blank liposomes, use deionized water as control, perform UV scanning in the range of 200-400 nm, and analyze the difference in absorbance among the three.

[0119] 1.5 XRD Analysis: The liposome suspension was converted to a solid, lyophilized powder using freeze-drying with 5% mannitol as a lyoprotectant to prolong the stability of the formulation. A certain amount of the physical mixture of GLP-Chol-Lipo (PM-GLP-Chol-Lip) and the physical mixture of GLP-β-Sito-Lip (PM-GLP-β-Sito-Lip) was evenly spread in an X-ray diffraction (XRD) sample well. Diffraction patterns were recorded over the 10–60° (2θ) range. Scan speed was 4° / min, step size was 0.02°, current was 15 mA, and voltage was 30 kV.

[0120] 1.6 Stability test: The prepared polysaccharide liposomes were packaged and sealed and placed at 4°C and room temperature respectively. Samples were taken on the 1st, 7th, 14th and 21st days to measure the changes in particle size, PDI and encapsulation efficiency to evaluate the storage stability of the polysaccharide liposomes.

[0121] 1.7 Determination of Encapsulation Efficiency and Drug Loading Rate: The encapsulation efficiency and drug loading rate were determined by low-temperature ultrafiltration centrifugation. 1 mL of liposomes was transferred to the inner tube of an ultrafiltration centrifuge tube and centrifuged at 13,000 rpm for 30 min. The filtrate from the outer tube was diluted with distilled water to an appropriate multiple. The absorbance was measured at an ultraviolet wavelength of 490 nm using the phenol-concentrated sulfuric acid method. The encapsulation efficiency and drug loading were calculated according to the following formula.

[0122]

[0123] W 投 : Ganoderma lucidum polysaccharide dosage, W 游 : The amount of free drug, W GLP : The amount of GLP encapsulated in the liposome, W lipid : Mixed lipid dosage.

[0124] 1.8 In vitro release assay: Neutral PBS (pH = 7.4) was used as the release medium. 3 mL of the corresponding liposomes were added to a dialysis bag (3000 kD). The dialysis bag was then tightly sealed at both ends and completely immersed in 30 mL of release medium. The bag was then placed in a thermostatic shaker (37°C, 100 rpm) to simulate a body fluid environment. At specific time intervals (1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 h), 3 mL of release medium was removed from the bag and placed in a 10 mL centrifuge tube. An equal amount of fresh release medium was then added to a conical flask. The polysaccharide content was quantified using the phenol-concentrated sulfuric acid method (n = 3).

[0125] 1.9 Cytotoxicity test: The MTT method was used to detect the effects of Ganoderma lucidum polysaccharide, Ganoderma lucidum polysaccharide liposomes, and blank liposomes on the viability of BV2 cells, and to determine the safe concentration range of their effects on BV2 cells. A blank control group, Ganoderma lucidum polysaccharide, and Ganoderma lucidum polysaccharide liposome groups were set up, with 6 parallel wells in each group. The experimental group was set up with samples of different concentrations of 0, 50, 100, 200, 300, 400, and 500 μg / mL. The blank liposome was set to the same experimental concentration, and different concentrations of preparations were added to the 96-well plate inoculated with BV2 cells. After harvesting the cells in the logarithmic growth phase, the concentration was adjusted to 1×10 5 Cells were seeded in a 96-well plate and filled to the brim with sterile PBS. After 12 hours of incubation, samples of varying concentrations were added and incubated for an additional 24 hours. Complete culture medium was used for the negative control group. After the incubation period, 20 μL of a 5 mg / mL thiazolyl blue tetrazolium bromide (MTT) solution was added to each well and incubated for 4 hours. The supernatant was removed, and 150 μL of dimethyl sulfoxide was added to the 96-well plate and mixed thoroughly. The absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated using the following formula.

[0126]

[0127] Where A1 is the absorbance of cells and samples of different concentrations, and A2 is the absorbance of cells and complete culture medium.

[0128] 1.10 Anti-inflammatory activity study: LPS-induced inflammatory factors produced by BV2 cells can increase the production of nitrite. The nitrite concentration in the supernatant of BV2 cells was measured by the Griess method as the amount of NO generated, thereby evaluating the efficacy and anti-inflammatory activity of anti-inflammatory drugs.

[0129] An in vitro model of neuroinflammation was established by inducing BV2 cells with 1 μg / mL LPS for 24 hours. The experiments were divided into a normal control group, a model group, a Ganoderma lucidum polysaccharide (GLP) group, a Ganoderma lucidum polysaccharide-cholesterol liposome group (GLP-Chol-Lip, a positive control), and a Ganoderma lucidum polysaccharide-β-sitosterol liposome group (GLP-β-Sito-Lip, obtained in Example 2). The model group was administered 1 μg / mL LPS; the GLP, GLP-Chol-Lip, and GLP-β-Sito-Lip groups were induced with 1 μg / mL LPS for 24 hours and then incubated with GLP, GLP-Chol-Lip, or GLP-β-Sito-Lip, respectively, for an additional 24 hours. Cell supernatants were collected, and 100 μL of the sample was reacted with an equal volume of Griess reagent. The absorbance at 540 nm was measured, and the NO content was calculated using the following formula.

[0130]

[0131] Where A 测定 is the absorbance of samples at different concentrations, A 空白 Absorbance of control group, A 标准 is the absorbance of the standard.

[0132] 1.11 Statistical Analysis: Each sample was repeated three times, and the results were averaged with the standard deviation as the standard deviation. Data processing and statistical analysis were performed using SPSS 26.0 software. One-way analysis of variance was performed for each group, and t-tests were used to compare data between two groups. A P value < 0.05 indicated statistical significance, and a P value < 0.01 indicated statistical significance.

[0133] (2) Results and Discussion 2.1 Morphological observation: Figure 12The macroscopic and microscopic morphological characterization results of Ganoderma lucidum polysaccharide-cholesterol liposomes (GLP-Chol-Lip) and Ganoderma lucidum polysaccharide-β-sitosterol liposomes (GLP-β-Sito-Lip) are presented. Panel A shows that both liposomes appear as milky white, translucent colloidal solutions, indicating a successful uniform dispersion with no apparent precipitation or stratification. Panel B further validates the colloidal properties of the liposomes using the Tyndall effect. Both exhibit distinct scattered light pathways, but the Tyndall bands of GLP-Chol-Lip are slightly stronger, suggesting a more uniform liposome size distribution within the dispersion, likely due to the enhanced membrane stability of cholesterol's rigid steroid ring structure. TEM images (Panels C and D) reveal that GLP-Chol-Lip (C) exhibits a typical unilamellar vesicle structure with a particle size range of 100-120 nm, well-defined boundaries, and good dispersion. Despite slight morphological differences, both meet the basic requirements for liposomes as drug carriers, demonstrating that both excipients are suitable for constructing Ganoderma lucidum polysaccharide delivery systems. On the other hand, the linear relationship between the Tyndall effect intensity and liposome concentration can be further used to quantitatively evaluate the colloidal stability of the formulation, but the particle size distribution needs to be verified in combination with the particle size data.

[0134] 2.2 Determination of particle size and polydispersity index (PDI): Figure 13 Figure 3 is the particle size distribution of GLP-Chol-Lip. The average particle size of GLP-Chol-Lip is 136.7 nm, and its particle size distribution is narrow and normally distributed. The PDI is 0.362, indicating that the prepared liposomes have good dispersibility and stability.

[0135] Figure 14 Figure 3 is the particle size distribution of GLP-β-Sito-Lip. The average particle size of GLP-Chol-Lip is 122.3 nm, and its particle size distribution is narrow and normally distributed. The PDI is 0.378, indicating that the prepared liposomes have good dispersibility and stability.

[0136] Figure 13 and Figure 14Particle size distribution and polydispersity index (PDI) data for Ganoderma lucidum polysaccharide-cholesterol liposomes (GLP-Chol-Lip) and Ganoderma lucidum polysaccharide-β-sitosterol liposomes (GLP-β-Sito-Lip) are presented. The results show that GLP-Chol-Lip has an average particle size of 136.7 nm and a PDI of 0.362, while GLP-β-Sito-Lip has an average particle size of 122.3 nm and a PDI of 0.378. Although both PDI values are slightly higher than 0.3 (a PDI < 0.3 is generally considered monodisperse), they remain within the acceptable range of 0.3-0.4, indicating good liposome dispersion and controllable batch-to-batch stability. Further analysis revealed that the particle size of GLP-Chol-Lip is slightly larger than that of GLP-β-Sito-Lip. This may be due to the rigid sterol ring structure of cholesterol enhancing lipid bilayer compactness and inhibiting vesicle fusion, resulting in slightly larger but uniform particles. In contrast, β-sitosterol, due to the ethyl substitution at the C-24 position and the different orientation of the hydroxyl group, may slightly increase membrane fluidity, promote a tighter arrangement of phospholipid molecules, and result in a smaller particle size. Furthermore, the slight difference in PDI values indicates that the two excipients have similar effects on liposome stability, and both can meet the basic requirements of drug delivery systems.

[0137] From a clinical application perspective, the particle sizes of both GLP-Chol-Lip and GLP-β-Sito-Lip fall within the 100-200 nm range, a size range believed to effectively avoid rapid clearance by the reticuloendothelial system (RES) while enhancing the EPR effect in tumor tissue. However, the smaller particle size of GLP-β-Sito-Lip may facilitate penetration of dense tissue barriers (such as the blood-brain barrier). Notably, despite PDI values slightly exceeding the ideal threshold, the particle size distributions of both formulations exhibited a unimodal normal distribution, indicating no significant aggregation or multimodal particle size distribution. On the other hand, the natural properties of β-sitosterol, as a plant-derived excipient, may confer enhanced biocompatibility to the liposomes, but long-term stability studies are needed to verify its particle size changes during storage. Overall, the data in this example demonstrate the feasibility of cholesterol and β-sitosterol as liposome excipients.

[0138] 2.3 UV spectrum scanning determination: Figure 15The UV absorption spectra of Ganoderma lucidum polysaccharide (GLP), blank liposomes (Chol-Lip and β-Sito-Lip), and drug-loaded liposomes (GLP-Chol-Lip and GLP-β-Sito-Lip) are shown. The results show that free GLP exhibits a significant absorption peak within a specific wavelength range (200-300 nm), likely related to the glycosidic bonds or conjugated structure within the molecule. In contrast, the UV absorption curve of the blank liposomes is relatively flat, with only weak absorption in the short-wavelength region (<250 nm). This is primarily due to the ester bonds in the phospholipid molecules or trace UV-active groups of cholesterol / β-sitosterol. In contrast, the absorption spectra of the drug-loaded liposomes (GLP-Chol-Lip and GLP-β-Sito-Lip) are highly similar to those of the blank liposomes, with no characteristic peaks of free GLP observed. This indicates that GLP is successfully encapsulated within the liposomal bilayer structure, and its UV-active groups are shielded by the liposome membrane, making them undetectable. This phenomenon is consistent with previous studies. Gubernator J et al. pointed out that the effective encapsulation of hydrophilic drugs by liposomes can significantly reduce their ultraviolet signals, while unencapsulated drugs will retain characteristic peaks in the spectrum.

[0139] Further analysis revealed that the spectra of GLP-Chol-Lip and GLP-β-Sito-Lip exhibited no new absorption peaks or peak shifts in the 250-300 nm range, indicating that no significant chemical interaction (such as covalent bond formation or charge transfer) occurred between Ganoderma lucidum polysaccharides and the liposome excipients, and that the encapsulation process was primarily physical encapsulation or surface adsorption. Furthermore, the absorption intensities of the two drug-loaded liposomes were similar to those of blank liposomes (Δabsorbance < 0.2), further supporting the high encapsulation efficiency of GLP. Notably, the β-Sito-Lip drug-loaded liposome exhibited a slightly higher absorbance at 280 nm than the Chol-Lip drug-loaded liposome, likely due to the higher membrane fluidity, which resulted in a small amount of drug exposed on the surface. However, this difference was not significant, indicating that the two excipients provided comparable shielding effects on drug encapsulation.

[0140] The absence of characteristic peaks in the UV absorption spectrum is a key indicator of drug encapsulation. If GLP is not encapsulated, its free form should retain its original peak shape after superimposing the spectrum of the drug-loaded liposomes. However, experimental data showed that the spectrum of the drug-loaded liposomes almost overlapped with that of the blank liposomes, and no characteristic absorption of GLP was detected, which strongly supports the conclusion that Ganoderma lucidum polysaccharide was effectively encapsulated. In addition, slight differences in absorbance between blank liposomes and drug-loaded liposomes (such as the slightly higher absorbance of GLP-β-Sito-Lip at 280nm) may be related to trace amounts of unencapsulated drug residues during the liposome preparation process, but the overall data indicate a high encapsulation efficiency.

[0141] 2.4 XRD analysis: Figure 16X-ray diffraction (XRD) patterns of cholesterol liposomes (Chol-Lip), Ganoderma lucidum polysaccharide-cholesterol liposomes (GLP-Chol-Lip), a physical mixture (PM-GLP-Chol-Lip), β-sitosterol liposomes (β-Sito-Lip), Ganoderma lucidum polysaccharide-β-sitosterol liposomes (GLP-β-Sito-Lip), and a physical mixture (PM-GLP-β-Sito-Lip) are shown. The XRD pattern of the blank liposomes exhibits typical amorphous characteristics, manifested by broad diffraction peaks, consistent with the amorphous structure of the liposome bilayer. The spectra of the drug-loaded liposomes (GLP-Chol-Lip and GLP-β-Sito-Lip) were highly similar to those of the blank liposomes. No crystalline diffraction peaks of free GLP (such as the typical polysaccharide crystal peak at 2θ = 15°-25°) were observed, indicating that the GLP was successfully encapsulated within the liposome bilayer. Its crystalline structure was disrupted by physical encapsulation, transforming it into an amorphous state. In contrast, the spectra of the physical mixtures (PM-GLP-Chol-Lip and PM-GLP-β-Sito-Lip) showed sharp diffraction peaks of free GLP superimposed on the broad peaks of the liposomes, confirming that the unencapsulated GLP retained its crystalline properties. This result is consistent with the conclusions of UV absorption spectroscopy analysis, further supporting the efficient encapsulation of GLP. Furthermore, no significant differences were observed in the XRD spectra of cholesterol- and β-sitosterol-modified liposomes, indicating that the two excipients had minimal impact on the overall crystalline structure of the liposomes. The main differences were reflected in physical properties such as membrane fluidity and particle size distribution.

[0142] Further analysis revealed that the XRD spectra of both GLP-Chol-Lip and GLP-β-Sito-Lip exhibited weak diffraction signals in the 2θ region of 5°-10°, likely due to the ordered short-range structure of the phospholipid molecules within the lipid bilayer. However, these signals were much less intense than the crystalline peaks observed in the physical mixture, indicating that the liposome encapsulation process effectively suppressed drug recrystallization. Notably, the diffraction peaks of the β-Sito-Lip drug carrier were slightly broader than those of the Chol-Lip drug carrier, likely due to the looser arrangement of phospholipids caused by its higher membrane fluidity. This phenomenon is consistent with the slightly higher PDI of GLP-β-Sito-Lip observed in previous particle size analysis, suggesting that the excipient type has a consistent effect on the liposome microstructure. Furthermore, the XRD data corroborated the TEM observations, demonstrating that the amorphous nature of the liposomes is directly related to the uniformity of their vesicle morphology. Overall, this study demonstrated the successful encapsulation of Ganoderma lucidum polysaccharide from a crystallographic perspective through XRD patterns, and that the excipient type had a limited effect on the overall structure of the liposomes.

[0143] 2.5 Stability test: Figure 17The changes in particle size and polydispersity index (PDI) of GLP-Chol-Lip and GLP-β-Sito-Lip after storage at 4°C for 1, 7, 14, and 21 days are shown. The results show that the initial particle size of GLP-Chol-Lip was 136.7 nm (PDI = 0.362), which slightly increased to 142.5 nm after 21 days of storage, and the PDI rose to 0.385, indicating that the dispersion system maintained high stability during long-term storage. In contrast, the initial particle size of GLP-β-Sito-Lip was 122.3 nm (PDI = 0.378), which significantly increased to 135.1 nm after 21 days, and the PDI rose to 0.415, suggesting that β-sitosterol-modified liposomes are more susceptible to aggregation or membrane relaxation during low-temperature storage. This difference may be directly related to the molecular properties of the excipients: the rigid steroid ring structure of cholesterol can effectively inhibit the lateral migration of phospholipid molecules, maintain the density of the lipid bilayer, and thus delay vesicle fusion; while β-sitosterol, due to differences in hydroxyl substituents and side chains, leads to slightly higher membrane fluidity, and the phospholipid arrangement gradually loosens during long-term storage, eventually causing the particle size to increase.

[0144] Further analysis revealed that the PDI increase of GLP-Chol-Lip was lower than that of GLP-β-Sito-Lip, indicating that cholesterol has a more pronounced protective effect on liposome dispersion uniformity. This may be attributed to cholesterol's ability to reduce the curvature tension of the lipid bilayer, thereby inhibiting nonspecific interactions between vesicles. Furthermore, the particle size growth rate of GLP-β-Sito-Lip (≈0.61 nm / day) was approximately 2.2 times that of GLP-Chol-Lip (≈0.28 nm / day), further confirming the weaker membrane stability of β-sitosterol-modified liposomes. Notably, while the PDI of both liposomes did not exceed 0.45 (a PDI <0.5 is generally considered acceptable), the PDI of GLP-β-Sito-Lip approached the critical threshold, suggesting that formulation optimization (such as increasing the excipient ratio or introducing surface modification) may be necessary to improve its storage stability. Overall, these data confirm the superiority of cholesterol as a liposome stabilizer and provide a direction for improving the stability of phytosterol-based excipients.

[0145] 2.6 Encapsulation Efficiency and Drug Loading Rate: Table 11 shows the encapsulation efficiency and drug loading rate data for GLP-Chol-Lip and GLP-β-Sito-Lip. The experimental results show that the encapsulation efficiency of GLP-Chol-Lip was 77.93%, slightly higher than the 75.60% of GLP-β-Sito-Lip. Both achieved high encapsulation levels. The drug loading rates of 2.35% and 2.28%, respectively, were generally low, with no significant difference. The slight difference in encapsulation efficiency may be related to the molecular structural properties of the excipients: Chol's rigid steroid ring structure enhances the density of the lipid bilayer and promotes the physical encapsulation of polysaccharide molecules; whereas, the positional differences in the hydroxyl substituents of β-Sito may slightly increase membrane fluidity, leading to some drug escape during the preparation process. This phenomenon is consistent with the results of the previous particle size analysis. GLP-Chol-Lip exhibits superior encapsulation performance due to its greater membrane stability. In addition, the generally low drug loading rate (all <3%) is mainly attributed to the hydrophilicity and high molecular weight of GLP, which makes it difficult to efficiently embed into the hydrophobic core of the lipid bilayer.

[0146] Further analysis revealed that the significant difference between the entrapment efficiency and the drug loading efficiency indicated that the entrapment of drugs by liposomes mainly relied on physical encapsulation rather than chemical bonding. This mechanism is consistent with the basic principle of liposome drug loading, which is to achieve drug retention through double-layer membrane wrapping or surface adsorption. It is worth noting that although the drug loading efficiencies of the two liposomes are similar, the difference in their entrapment efficiency may have a potential impact on drug release kinetics: a higher entrapment efficiency usually means less free drug residue, thereby reducing the burst effect. In addition, as a plant-derived excipient, although β-sitosterol has a slightly lower entrapment efficiency, its natural properties may give liposomes better biocompatibility and low immunogenicity, an advantage that is particularly important in targeted delivery systems.

[0147] Table 11

[0148] 2.7 In vitro release experiment: Figure 18The time-dependent cumulative release rates of GLP, GLP-Chol-Lip, and GLP-β-Sito-Li in in vitro release experiments are shown. The results indicate that free GLP exhibits rapid release in the initial phase (0-24 hours), with a cumulative release rate reaching approximately 65% within 24 hours. The release rate then slows significantly, ultimately reaching a plateau of 90% at 100 hours. In contrast, the release behavior of GLP-Chol-Lip and GLP-β-Sito-Lip exhibits a pronounced sustained-release pattern: the cumulative release rate of GLP-Chol-Lip is 35% at 24 hours and 70% at 100 hours, while the release rate of GLP-β-Sito-Lip is slightly faster, reaching 45% at 24 hours and 80% at 100 hours. This difference suggests that when cholesterol is used as a liposome excipient, its rigid steroid ring structure enhances lipid bilayer stability and slows drug diffusion, while β-sitosterol, due to differences in hydroxyl group orientation and side chain properties, results in slightly higher membrane fluidity, accelerating drug release.

[0149] Further analysis revealed that the burst release (initial 24-hour release rate) of the liposome formulation was significantly lower than that of free GLP. This was attributed to the physical encapsulation of the drug by the liposome bilayer structure, which effectively reduced rapid drug diffusion in the medium. Furthermore, the release profile of GLP-Chol-Lip plateaued after 50 hours, while that of GLP-β-Sito-Lip maintained a slow upward trend, suggesting that cholesterol-modified liposomes may have more stable drug retention. This phenomenon is related to differences in liposome membrane integrity and degradation kinetics: cholesterol can prolong liposome stability by inhibiting membrane fusion and oxidative damage, while phytosterol excipients may have weaker interactions with phospholipids, leading to gradual membrane disintegration during long-term release. Notably, although the burst release rate of β-sitosterol liposomes was slightly higher, its cumulative release rate over 100 hours was similar to that of cholesterol liposomes, indicating no significant difference in total long-term release between the two.

[0150] 2.8MTT cytotoxicity assay: Figure 19 The effects of GLP and its two liposome formulations on BV2 cell viability at concentrations ranging from 0 to 500 μg / mL were shown. The results showed that within the tested concentration range, cell viability in all groups exceeded 80%, indicating that Ganoderma lucidum polysaccharide and its liposomes exhibited no significant cytotoxicity in vitro. Cell viability decreased significantly when GLP and its liposomes were present at concentrations between 300 and 500 μg / mL. Therefore, a drug concentration of 300 μg / mL was selected for subsequent experiments.

[0151] 2.9 Anti-inflammatory Properties Study: Figure 20The effects of GLP and its liposomal formulation on NO production in BV2 microglia were demonstrated. The results showed that compared with the control group (Group A, 5.2±0.4μmol / L), the NO concentration in the model group (Group B) increased to 18.5±1.2μmol / L (P<0.001), confirming the effectiveness of the established inflammatory model. Previous studies have shown that BV2 microglia produce large amounts of inflammatory mediators such as NO upon inflammatory stimulation, making it a reliable in vitro model for evaluating neuroinflammatory responses. Following administration, NO levels decreased to 11.8±0.5μmol / L in the GLP group (Group C), while NO concentrations decreased to 9.2±0.8 and 9.6±0.7μmol / L in the GLP-Chol-Lip group (Group D) and the GLP-β-Sito-Lip group (Group E), respectively. Furthermore, both liposomal formulations exhibited similar NO-inhibiting effects, with both exhibiting superior inhibitory effects compared to free GLP.

[0152] Further analysis revealed that liposomal encapsulation improved the anti-inflammatory effects of GLP, likely due to the enhanced transport efficiency of the drug across the cell membrane. Furthermore, liposomes constructed with β-sitosterol instead of cholesterol (Group E) exhibited comparable efficacy to traditional cholesterol liposomes (Group D), providing a viable approach for the development of plant-derived liposome delivery systems. Overall, liposome encapsulation effectively enhanced the anti-inflammatory activity of GLP in BV2 cells, providing strong experimental evidence for its application in the treatment of neuroinflammation.

[0153] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing Ganoderma lucidum polysaccharide / β-sitosterol liposomes, characterized in that: The preparation method comprises the following steps: (I) Preparation of Ganoderma lucidum polysaccharide: (I-1) washing and removing impurities from the Ganoderma lucidum fruiting body slices, and then drying to obtain dried Ganoderma lucidum slices; (I-2) crushing the dried Ganoderma lucidum slices to obtain Ganoderma lucidum powder; (I-3) mixing the Ganoderma lucidum powder with an extraction solvent and heating the mixture under reflux, and then centrifuging, concentrating, precipitating with alcohol, centrifuging again, and freeze-drying in sequence to obtain Ganoderma lucidum polysaccharide; (II) Preparation of Ganoderma lucidum polysaccharide / β-sitosterol liposomes: (II-1) mixing and dissolving soybean lecithin, β-sitosterol, a surfactant, and an oily solvent to obtain an oil phase; (II-2) mixing and dissolving the Ganoderma lucidum polysaccharide with an aqueous solvent to obtain an aqueous phase; (II-3) adding the aqueous phase dropwise to the oil phase and performing sonication to obtain an emulsion; (II-4) The emulsion is sequentially rotary evaporated, hydrated, and crushed, and then filtered to obtain the Ganoderma lucidum polysaccharide / β-sitosterol liposome.

2. The method for preparing the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 1, characterized in that: In step (I-1), the drying temperature is 75-85° C., and the drying time is 46-50 hours.

3. The method for preparing the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 1, characterized in that: In step (I-2), the pulverization method is coarse grinding or air flow ultrafine grinding; wherein: The coarse grinding method is: using a high-speed Chinese herbal medicine grinder to coarsely grind the dried Ganoderma lucidum slices for 10 to 12 minutes; The airflow ultrafine grinding method is: firstly, the dried Ganoderma lucidum slices are coarsely ground with a high-speed Chinese herbal medicine grinder for 10-12 minutes, and then the airflow grinder is used to grind them 6 or 15 times at a grinding pressure of 0.5-0.7 MPa.

4. The method for preparing the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 1, characterized in that: In step (I-3), the extraction solvent is water, NaHCO3 solution or a natural deep eutectic solvent; wherein: When the extraction solvent is water, the heating reflux process is: liquid-to-solid ratio 20:1~60:1mL / g, extraction temperature 60~100℃, extraction time 1~5h; When the extraction solvent is NaHCO3 solution, the heating reflux process is: liquid-to-solid ratio 10:1~50:1mL / g, NaHCO3 concentration 0.1~2.0mol / L, extraction temperature 40~80℃, extraction time 1~5h; When the extraction solvent is a natural deep eutectic solvent, the natural deep eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is betaine, and the hydrogen bond donor is one or a combination of two of glycerol, 1,2-propylene glycol, lactic acid, and levulinic acid; the heating and reflux process is: liquid-to-solid ratio 20:1 mL / g, natural deep eutectic solvent water content 70 wt%, extraction temperature 70°C, and extraction time 2 h.

5. The method for preparing the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 1, characterized in that: In step (II-1), the ratio of soybean lecithin, β-sitosterol, surfactant, and oily solvent is 240-260 mg: 25-35 mg: 25-35 mg: 8-12 mL; And / or, the surfactant is Tween-80; And / or, the oily solvent is ether.

6. The method for preparing the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 1, characterized in that: In step (II-2), the ratio of the Ganoderma lucidum polysaccharide to the aqueous solvent is 15-25 mg: 8-12 mL; And / or, the aqueous solvent is PBS solution.

7. The method for preparing the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 1, characterized in that: In step (II-3), the total ultrasonic time is 8 to 12 minutes; in each ultrasonic cycle, the working time is 5 seconds and the rest time is 5 seconds.

8. The method for preparing the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 1, characterized in that: In step (II-4), the rotary evaporation time is 25 to 35 minutes; And / or, the stirring time of the hydration is 35 to 45 minutes; And / or, the filtration is: passing through a 220 nm microporous filter membrane.

9. Ganoderma lucidum polysaccharide / β-sitosterol liposomes obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the Ganoderma lucidum polysaccharide / β-sitosterol liposome according to claim 9 in the preparation of a drug carrier.

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