Astaxanthin and dihydroquercetin co-loaded liposome and application thereof in preparation of uric acid-lowering product

Abstract

The present application relates to the field of biotechnology, in particular to astaxanthin and dihydroquercetin co-loaded liposome and its application in preparation of uric acid-lowering products. According to the steps of "in vitro function screening - optimal ratio determination - co-loaded liposome construction - surface coating and stabilization treatment - uric acid-lowering application verification", the obtained system is verified through physicochemical characterization, stability test and animal experiment. The results show that the co-loaded liposome can reduce the serum uric acid level of high uric acid model mice, inhibit the activity of liver xanthine oxidase, and improve the related indexes of kidney function and histopathological damage.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a co-loaded liposome of astaxanthin and dihydroquercetin and its application in the preparation of uric acid-lowering products. Background Technology

[0002] Hyperuricemia is a metabolic disorder caused by an imbalance between uric acid production and excretion. Uric acid is the end product of purine metabolism, and xanthine oxidase (XOD) plays a key catalytic role in uric acid production. Therefore, inhibiting XOD activity can serve as an important basis for evaluating and screening uric acid-lowering activities.

[0003] Astaxanthin (AST) possesses strong antioxidant and anti-inflammatory activities. For example, Chinese patent CN103504301A discloses a formula and preparation method for astaxanthin oil with antihypertensive, hypoglycemic, uric acid-lowering, and antitumor effects. This formula includes a liquid astaxanthin oil formulation and a capsule formulation. In the liquid formulation, the weight ratio of edible oil to astaxanthin is 100g:80mg, and 100mg capsules can treat gout, hyperglycemia, and cardiovascular and cerebrovascular diseases. Dihydroquercetin (DHQ) has good antioxidant and metabolic regulation potential. For example, Chinese patent CN118986959A discloses a quercetin-containing composition for lowering uric acid and improving gout, and its application. This discloses that quercetin significantly promotes the growth of *Lactobacillus ornidus* CML180 in a dose-dependent manner, reduces hepatic uric acid production, and increases renal uric acid excretion, thereby improving gout.

[0004] Liposomes, as a commonly used delivery system, can improve the dispersibility, stability, and delivery efficiency of active ingredients through their phospholipid bilayer structure. For two active ingredients with different physicochemical properties, co-loaded liposomes can achieve co-encapsulation and simultaneous delivery within the same system, offering significant advantages. However, conventional liposomes exhibit limited stability under storage, heat treatment, and light exposure conditions, requiring further optimization of formulation and surface structure. Existing technologies typically employ conventional liposome preparation processes, such as thin-film dispersion or ultrasonic methods, to construct active ingredient encapsulation systems, using phospholipids as the membrane material and cholesterol as a membrane stabilizing agent. Some studies further introduce polysaccharide coating to enhance liposome stability.

[0005] While existing natural active ingredients show some potential in lowering uric acid, the following problems exist: First, astaxanthin (AST) is highly lipid-soluble and easily degraded by light, oxygen, and temperature; dihydroquercetin (DHQ) has limited solubility and insufficient stability, resulting in low oral absorption and utilization. When these two are directly added to food or oral systems, their activity is easily lost. Furthermore, astaxanthin is highly lipid-soluble, while dihydroquercetin is primarily alcohol-soluble with some water solubility. Significant differences in their physicochemical properties, such as solubility, lipophilic / hydrophilic properties, and in vivo absorption and transport behavior, lead to asynchronous distribution and action times in vivo, hindering simultaneous delivery and synergistic effects. Second, although there are reports of screening some uric acid-lowering active substances using in vitro XOD inhibition experiments, there are few studies on the combined use of AST and DHQ, and further optimization of the ratio using combination index (CI) analysis. This makes it difficult to determine whether a specific ratio has a better synergistic effect. Third, current research on co-loaded liposomes is mostly focused on drug or cosmetic delivery, while research on co-loaded liposomes targeting the combination of AST and DHQ, and for food or nutrient delivery and uric acid-lowering applications, remains relatively limited. Fourth, ordinary liposome systems are prone to particle size changes, leakage of active substances, and system instability under long-term storage, heating, or light exposure, which limits their practical applications. Summary of the Invention

[0006] The technical problem to be solved by this invention is as follows: (1) Free AST and DHQ are not stable enough in practical applications, their activity is easily lost, and they are not conducive to simultaneous delivery; (2) There are relatively few studies on the screening of the uric acid-lowering ratio of AST and DHQ in combination, and there is a lack of quantitative screening based on XOD inhibition and CI analysis; (3) There is a lack of research on co-loaded liposomes that combine AST and DHQ and are geared towards food or nutrient delivery; (4) Ordinary liposome systems have limited environmental stability and are prone to instability under storage, heat and light conditions; (5) In the prior art, the selection of membrane conditioning components and surface coating components has not been systematically optimized around the food and nutrition delivery scenarios.

[0007] To address the problems of existing technologies, this invention provides astaxanthin and dihydroquercetin co-loaded liposomes and their application in the preparation of uric acid-lowering products. The process follows the steps of "in vitro functional screening—determination of optimal ratio—construction of co-loaded liposomes—surface coating and stabilization treatment—verification of uric acid-lowering application." Specifically, firstly, an in vitro xanthine oxidase (XOD) inhibition experiment was used to evaluate the individual and combined effects of astaxanthin (AST) and dihydroquercetin (DHQ), screening for an optimal synergistic ratio suitable for lowering uric acid. Then, based on this optimal ratio, AST / DHQ co-loaded liposomes were prepared using a thin-film dispersion-ultrasound method, and the formulation parameters were optimized. Furthermore, carboxymethyl chitosan (CMCS) was used for surface coating to improve system stability. Finally, the obtained system was verified through physicochemical characterization, stability tests, and animal experiments. The results showed that the co-loaded liposomes could reduce serum uric acid (UA) levels in hyperuricemic mouse models, inhibit liver XOD activity, and simultaneously improve renal function-related indicators and histopathological damage.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing astaxanthin and dihydroquercetin co-loaded liposomes, comprising the following steps: (1) Astaxanthin (AST), dihydroquercetin (DHQ), phospholipids, sterols, and nonionic surfactants were added to anhydrous ethanol in proportion and thoroughly mixed and dissolved to form a homogeneous organic phase. The mass ratio of AST to DHQ was 1:6 to 1:12. The lipid bilayer structure of phospholipids was used to achieve co-loading and stable delivery of AST and DHQ. The backbone structure of sterols could be inserted into the lipid bilayer and play a regulatory role in the orderliness, fluidity, and stability of the membrane. Introducing them into the liposome system was beneficial to forming a more stable co-loading structure. Nonionic surfactants were used to improve the dispersibility and homogeneity of the system. Anhydrous ethanol, as an organic phase solvent, has good solubility and volatility, which can achieve uniform mixing of various lipid membrane components such as AST and DHQ, and facilitate the formation of a homogeneous lipid film after subsequent rotary evaporation. (2) Place the obtained organic phase in a rotary evaporator and remove all organic solvents by rotary evaporation to form a uniform lipid film on the inner wall of the container; (3) Then, a buffer solution was added and hydrated at room temperature, and a crude liposome suspension was formed by magnetic stirring. The hydration process caused the lipid film to swell and peel off in the aqueous phase, driving the phospholipid molecules to self-assemble into a lipid bilayer structure, thereby generating a crude liposome suspension and achieving preliminary encapsulation of the active ingredients. (4) The crude liposome suspension was subjected to probe sonication under ice bath conditions to reduce the particle size and improve the dispersion uniformity. After sonication, the obtained liposome suspension was stored in the dark to obtain astaxanthin and dihydroquercetin co-loaded liposomes.

[0009] Furthermore, the phospholipids mentioned in step (1) are a compound system of one or more of the following phospholipids: soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, and phosphatidylcholine; the sterols are a mixture of one or more of the following phytosterols: cholesterol, β-sitosterol, stigmasterol, campesterol, and sitosterol; and the nonionic surfactants are Tween 20, Tween 80, Span 60, Span 80, or other polyoxyethylene nonionic surfactants, or a compound system of two or more of the above surfactants.

[0010] Furthermore, in step (1), the phospholipid concentration is 3~10 mg / mL, the sterol / phospholipid mass ratio is 1:(8~15), the nonionic surfactant / phospholipid mass ratio is 1:(8~12), and the DHQ / phospholipid mass ratio is 1:(3~5).

[0011] Furthermore, the rotary evaporation conditions in step (2) are: rotary evaporation at 150-200 rpm under reduced pressure for 20-30 min at 30-40°C, preferably 35°C, 180 rpm, and 25 min. These conditions are advantageous in avoiding degradation of the active ingredients due to high temperatures while effectively removing the organic solvent, thereby forming a uniform and stable lipid film.

[0012] Furthermore, the hydration time in step (3) is 30~90 min, and the magnetic stirring speed is 300~700 rpm. Within the above conditions, it is beneficial for the lipid film to fully expand and peel off in the aqueous phase, so that the phospholipid molecules can fully self-assemble to form a lipid bilayer structure, thereby generating a crude liposome suspension with good dispersibility; at the same time, it avoids insufficient hydration due to too low stirring intensity, or instability of the system due to too high stirring intensity.

[0013] Furthermore, the ultrasound conditions in step (4) are as follows: ultrasound power is 10%~30%, preferably 20%; intermittent ultrasound is used, with an ultrasound time of 3~8 s and an interval time of 2~5 s; the total ultrasound time is 10~20 min, preferably 15 min. Through the above intermittent ultrasound conditions, the liposome particle size can be effectively refined while reducing heat accumulation in the system, resulting in liposomes with smaller particle size and narrower particle size distribution; at the same time, continuous high-intensity ultrasound can avoid damaging the lipid bilayer structure, thereby improving the stability of the system.

[0014] Furthermore, the preparation method further includes step (5): preparing a polysaccharide solution with a buffer solution, mixing the polysaccharide solution with an AST / DHQ co-loaded liposome suspension at a volume ratio of 1:(0.5~2), preferably 1:1, stirring at room temperature to allow the polysaccharide to adsorb onto the liposome surface to form a coating layer; the polysaccharide is carboxymethyl chitosan (CMCS), chitosan, sodium alginate, pectin, hydroxypropyl methylcellulose or its derivatives, or a compound system of two or more polysaccharides.

[0015] Furthermore, the concentration of the CMCS solution is 1~4 mg / mL, preferably 2 mg / mL, and the mixture is stirred at room temperature for 40 min. Within the above range, it is beneficial for CMCS molecules to form a uniform and stable coating layer on the liposome surface, thereby improving the stability of the system; at the same time, it avoids insufficient coating due to too low a concentration or too high a stirring time, or increased viscosity and decreased dispersibility of the system caused by too high a concentration or stirring time.

[0016] Carboxymethyl chitosan (CMCS) has good biocompatibility, film-forming properties and surface coating ability. It can form a hydrophilic protective layer on the outer layer of lipids, thereby improving the stability of the system under storage, heat treatment and light conditions.

[0017] Furthermore, the buffer in steps (3) and (5) is 0.1 M pH 7.5 PBS buffer, which realizes the functions of lipid film hydration, liposome formation and CMCS coating.

[0018] Astaxanthin and dihydroquercetin co-loaded liposomes prepared by the above method have a particle size of 105.94±1.27 nm, a PDI of 0.23±0.00, a zeta potential of -14.58±0.27 mV, an AST encapsulation efficiency of 94.57±1.99%, a DHQ encapsulation efficiency of 94.88±1.35%, an AST loading rate of approximately 1.80%, and a DHQ loading rate of approximately 15.27%. This indicates that the method of the present invention can achieve efficient co-loading of AST and DHQ, and obtain a liposome system with small particle size and good dispersibility.

[0019] The above-mentioned astaxanthin and dihydroquercetin co-loaded liposomes were used in the preparation of products for lowering uric acid, inhibiting hepatic xanthine oxidase (XOD) activity, and improving renal function. In this invention, the obtained co-loaded liposomes were used in intervention in a hyperuricemia model mouse. Results showed that the co-loaded liposomes could reduce serum uric acid (UA) levels, inhibit hepatic XOD activity, and improve renal function-related indicators and histopathological damage in hyperuricemia model mice. The medium-dose group, i.e., DHQ 50 mg / kg + AST 5.56 mg / kg, showed superior performance, with an overall effect better than the free active ingredient group and the single-drug liposome mixture group.

[0020] The beneficial effects of this invention are as follows: (1) This invention first screens the optimal ratio of astaxanthin (AST) and dihydroquercetin (DHQ) by in vitro xanthine oxidase (XOD) inhibition experiments and combined with combination index (CI) analysis, thereby determining that the preferred ratio is 1:9. Compared with empirical compounding, this technical solution provides a clear functional basis for the ratio of active ingredients, which is beneficial to improving the synergistic effect when the two components are used together.

[0021] (2) In this invention, AST and DHQ are co-loaded in the same liposome system, rather than using free mixing or separate drug delivery. Liposomes can improve the dispersibility of the active ingredients and provide a common carrier, which is beneficial to improving the stability and simultaneous delivery capability of the two components, thereby better exerting their combined effect.

[0022] (3) This invention introduces β-sitosterol as a membrane stabilizing component into the liposome system. β-sitosterol can participate in the regulation of the lipid bilayer structure, which helps to improve the stability of the liposome membrane structure and provides new formulation options for the application of this system in functional foods and nutrient delivery.

[0023] (4) By optimizing the concentration of soybean lecithin, the β-sitosterol / phospholipid mass ratio, the Tween 80 / phospholipid mass ratio, and the DHQ / phospholipid mass ratio, this invention obtained a co-loaded liposome system with smaller particle size, better dispersibility, and higher encapsulation efficiency. This demonstrates that the formulation design and preparation process adopted in this invention have good feasibility.

[0024] (5) The present invention further employs carboxymethyl chitosan (CMCS) to coat the surface of the co-loaded liposomes. CMCS can form a protective layer on the outer surface of the liposomes, which helps to improve the stability of the system under storage, heat treatment and light conditions, thereby improving the problem of insufficient environmental stability of ordinary liposomes.

[0025] (6) The AST / DHQ co-loaded liposomes and their CMCS coating system constructed in this invention showed the effects of reducing serum uric acid, inhibiting XOD activity and improving related kidney injury indicators in the hyperuricemia model, indicating that this invention not only has a clear technical implementation scheme, but also has practical application value in lowering uric acid. Attached Figure Description

[0026] Figure 1 The dose-response curves of allopurinol, AST, DHQ and their different combined ratios on the inhibition rate of XOD activity are shown.

[0027] Figure 2 This is the standard curve for the DHQ-anhydrous ethanol system.

[0028] Figure 3 This is the standard curve for the AST-trichloromethane system.

[0029] Figure 4 The effect of phospholipid concentration on the performance of co-loaded liposomes is shown in Figure A, where PDI is the particle size; DHQ encapsulation efficiency and loading rate are shown in Figure B; ζ potential is shown in Figure C; and AST encapsulation efficiency and loading rate are shown in Figure D.

[0030] Figure 5 The effect of β-sitosterol / phospholipid mass ratio on the performance of co-loaded liposomes: Figure A shows particle size and PDI; Figure B shows DHQ encapsulation efficiency and loading rate; Figure C shows zeta potential; and Figure D shows AST encapsulation efficiency and loading rate.

[0031] Figure 6 The effect of Tween 80 / phospholipid mass ratio on the performance of co-loaded liposomes: Figure A shows particle size and PDI; Figure B shows DHQ encapsulation efficiency and loading rate; Figure C shows zeta potential; and Figure D shows AST encapsulation efficiency and loading rate.

[0032] Figure 7 The effect of DHQ / phospholipid mass ratio on the performance of co-loaded liposomes: Figure A shows particle size and PDI; Figure B shows DHQ encapsulation efficiency and loading rate; Figure C shows zeta potential; and Figure D shows AST encapsulation efficiency and loading rate.

[0033] Figure 8 The appearance of co-loaded liposomes under optimal formulation.

[0034] Figure 9 This is the FRAP standard curve.

[0035] Figure 10 The effect of CMCS concentration on the performance of co-loaded liposomes: Figure A shows the relationship between particle size and PDI; Figure B shows the DHQ encapsulation efficiency; Figure C shows the zeta potential; and Figure D shows the AST encapsulation efficiency.

[0036] Figure 11 TEM images and particle size distributions of L (A~C) and L-CMCS (D~F) are shown; where A is the TEM image of L (scale bar is 500 nm), B is the TEM image of L (scale bar is 200 nm), C is the particle size distribution of L, D is the TEM image of L-CMCS (scale bar is 500 nm), E is the TEM image of L-CMCS (scale bar is 200 nm), and F is the particle size distribution of L-CMCS.

[0037] Figure 12 The FTIR spectra of CMCS, AST, DHQ, L, and L-CMCS are shown.

[0038] Figure 13 Thermogravimetric curves of AST, DHQ, L, and L-CMCS are shown.

[0039] Figure 14The DSC curves of L, L-CMCS and their corresponding blank liposomes are shown.

[0040] Figure 15 The changes in particle size (Figure A), PDI (Figure B), and zeta potential (Figure C) of L, Lc, and L-CMCS during storage are shown.

[0041] Figure 16 The changes in AST (Figure A) and DHQ (Figure B) encapsulation efficiency of L, Lc, and L-CMCS during storage are shown.

[0042] Figure 17 The appearance changes of L, Lc and L-CMCS during storage.

[0043] Figure 18 The changes in particle size (Figure A), PDI (Figure B), and zeta potential (Figure C) of L, Lc, and L-CMCS during heating are shown.

[0044] Figure 19 The changes in the encapsulation efficiencies of L, Lc, and L-CMCS during heating are shown in Figure A (AST) and Figure B (DHQ).

[0045] Figure 20 The appearance changes of L, Lc, and L-CMCS during the heating process are shown.

[0046] Figure 21 The changes in particle size (Figure A), PDI (Figure B), and zeta potential (Figure C) of L, Lc, and L-CMCS during illumination are shown.

[0047] Figure 22 The figures show the changes in AST (Figure A) and DHQ (Figure B) encapsulation efficiencies of L, Lc, and L-CMCS during illumination.

[0048] Figure 23 The appearance changes of L, Lc, and L-CMCS during illumination.

[0049] Figure 24 To investigate the effects of different treatments on serum UA levels in mice.

[0050] Figure 25 The effects of different treatments on XOD activity in mouse liver.

[0051] Figure 26 The effects of different treatments on serum UREA (Figure A), CRE (Figure B), and UREA / CRE ratio (Figure C) in mice.

[0052] Figure 27 To investigate the effects of different treatments on the appearance and histopathological changes of mouse kidneys.

[0053] Figure 28 To investigate the effects of different treatments on the serum GOT / ALT ratio in mice. Detailed Implementation

[0054] The following description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0055] The following embodiments can be understood as illustrating only a part of the structure or method of the present invention, or as a combination of embodiments explaining the broader structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are commercially available.

[0056] Unless otherwise specified, all raw materials used in the following embodiments were purchased from the market.

[0057] Example 1: Screening of the optimal ratio of AST to DHQ: This embodiment screened the optimal ratio of AST to DHQ to provide a clear basis for subsequent formulation design of co-loaded liposomes. The inhibitory effects of AST and DHQ on XOD activity were measured separately, and their half-maximal inhibitory concentrations (IC50) were calculated. Subsequently, different combination ratios were set based on the IC50 ratio of the two, and the XOD inhibitory activity of the combined samples was evaluated. The combination index (CI) was used to quantitatively analyze the combination effect.

[0058] 1. Main reagents: Table 1: Main Reagents .

[0059] 2. Experimental Methods: 2.1 Investigation into the synergistic effect of AST and DHQ: 2.1.1 Determination of the in vitro XOD inhibitory activity of AST and DHQ: The inhibitory activity of samples against XOD was determined by ultraviolet spectrophotometry, with allopurinol as a positive control. Xanthine substrate was prepared into a 0.5 mmol / L working solution using 0.01 mol / L PBS. XOD was diluted to a 0.02 U / mL working solution using 0.2 mol / L PBS. DHQ was dissolved in anhydrous ethanol and diluted to a suitable concentration using 0.01 mol / L PBS. AST was dissolved in a mixed solvent of acetone-dichloromethane-anhydrous ethanol-Tween 80 (90:5:2:3 v / v) and diluted to a suitable concentration using 0.01 mol / L PBS. Corresponding solvent control groups were established for each sample (except allopurinol) to eliminate potential interference from solvents on enzyme activity.

[0060] Add each component to the centrifuge tube in sequence according to Table 2, mix well, and react in a 37°C water bath for 30 min. Add 0.5 mol / L hydrochloric acid to terminate the reaction, and measure the absorbance value at 295 nm.

[0061] Table 2: Order of reagent addition in XOD inhibition activity assay: .

[0062] The inhibition rate of XOD by sample / allopurinol was calculated using the following formula: ; .

[0063] Plot a dose-response curve with sample concentration on the x-axis and inhibition rate on the y-axis. The sample concentration at which the inhibition rate reaches 50% is read from the curve; this is the IC50 of the sample / allopurinol. 50 .

[0064] 2.1.2 Investigation into the synergistic inhibition of XOD effect by AST / DHQ: Three combination groups with different mass ratios were designed based on the IC50 ratio of AST / DHQ inhibiting XOD activity. AST / DHQ mixed test solutions were prepared and diluted to appropriate concentrations, and the IC50 values ​​for each combination ratio were determined using the same method. 50 (Based on AST concentration).

[0065] The combination index (CI) method is used to evaluate the combined effect of AST / DHQ, and the CI value is calculated using the following formula: ; Wherein, (D)1 and (D)2 are the concentrations required for AST and DHQ to inhibit 50% of XOD activity when used in combination, (D) m )1 and (D m )2 is the IC50 value when the two drugs are used alone. 50 CI values. CI < 1, CI = 1, and CI > 1 represent synergistic, additive, and antagonistic effects, respectively. The ratio with the lowest CI value was selected as the optimal synergistic ratio for subsequent co-loaded liposome preparation.

[0066] Experimental results showed that both AST and DHQ, when used alone, had a certain inhibitory effect on XOD, but their inhibitory abilities differed significantly. Specifically, allopurinol had a lower IC50 value. 50 The concentration was 8.36 ± 1.04 μg / mL, the IC50 of AST was 36.99 ± 1.65 μg / mL, and the IC50 of DHQ was... 50The concentration was 312.88 ± 1.74 μg / mL. These results indicate that, under the same evaluation system, AST's direct inhibitory effect on XOD is significantly stronger than that of DHQ.

[0067] In the combined AST and DHQ system, different ratios all exhibited certain synergistic effects. Specifically, when the AST:DHQ ratio was 1:6, the corresponding IC of the combined group... 50 The concentrations were 19.68±0.60 μg / mL and 118.08±3.56 μg / mL, respectively, with a CI of 0.91±0.03; when the AST:DHQ ratio was 1:9, the corresponding IC50 values ​​for the combined group were... 50 The concentrations were 14.43±0.19 μg / mL and 129.77±1.69 μg / mL, respectively, with a CI of 0.80±0.01; when the AST:DHQ ratio was 1:12, the corresponding IC50 values ​​for the combined group were... 50 The values ​​were 13.02±0.07 μg / mL and 156.27±0.82 μg / mL, respectively, with a CI of 0.85±0.00. Since the CI values ​​were all less than 1 under the three ratio conditions, it indicates that AST and DHQ synergistically inhibit XOD activity within this ratio range; among them, the CI value was the lowest and the synergistic effect was the most significant when AST:DHQ=1:9.

[0068] Table 3: IC50 and CI values ​​of the inhibitory effects of AST, DHQ, and their different combined ratios on XOD: .

[0069] The results showed that as the DHQ ratio increased from 1:6 to 1:9, the CI value gradually decreased, indicating a stronger synergistic effect. However, when the DHQ ratio continued to increase to 1:12, the CI value increased again, suggesting a relatively stable synergistic range in inhibiting XOD activity. Deviating from this range could lead to an imbalance in component contributions, potentially weakening the overall synergistic effect. Based on the synergistic strength and ratio stability, AST:DHQ = 1:9 was ultimately determined as the fixed feed ratio for subsequent co-loaded liposome preparation.

[0070] like Figure 1 The results showed that both AST and DHQ had a certain inhibitory effect on XOD when used alone. Under the combined use condition, the CI values ​​of AST:DHQ=1:6, 1:9 and 1:12 were all less than 1, indicating that there was a synergistic effect at each ratio. Among them, the CI value of AST:DHQ=1:9 was the smallest and the synergistic effect was the strongest. Therefore, it was determined to be the preferred functional ratio for the subsequent construction of co-loaded liposomes.

[0071] Example 2: Composition and preparation method of co-loaded liposomes: After determining the optimal ratio of AST to DHQ, this embodiment further constructs a co-loaded liposome system. The co-loaded liposome includes the following components: AST, DHQ, soybean lecithin, β-sitosterol, Tween 80, and phosphate-buffered saline (PBS). β-sitosterol is added to the system as a membrane stability modulator to improve the stability of the lipid bilayer.

[0072] To obtain a co-loaded liposome system with superior performance, the formulation parameters were further optimized in this invention. Through single-factor and orthogonal experiments, using particle size, polydispersity index (PDI), zeta potential, AST and DHQ encapsulation efficiency, and loading rate as comprehensive evaluation indicators, the optimal formulation was determined to be: soybean lecithin concentration 5 mg / mL, β-sitosterol / phospholipid mass ratio 1:12, Tween 80 / phospholipid mass ratio 1:10, DHQ / phospholipid mass ratio 1:4, and AST:DHQ mass ratio 1:9.

[0073] 1. Main reagents: Table 4: Main Reagents .

[0074] 2. Optimization of the preparation process for AST / DHQ co-loaded liposomes: 2.1 Preparation of AST / DHQ co-loaded liposomes: Soybean lecithin, β-sitosterol, DHQ, AST, and Tween 80 were dissolved in anhydrous ethanol and mixed in a certain proportion. The organic solvent was removed by rotary evaporation under reduced pressure in a 35°C water bath, forming a uniform lipid film on the inner wall of the rotary evaporator flask. Subsequently, 0.01 mol / L PBS (pH 7.4) was added to elute the film from the flask wall by rotary elution. The hydration was carried out by magnetic stirring at room temperature for 1 h. The resulting crude liposome suspension was placed in an ice bath and then homogenized using an ultrasonic homogenizer (270 W, 15 min). After ultrasonication, the suspension was filtered through a 0.22 μm microporous membrane and stored at 4°C in the dark for later use.

[0075] 2.2 Single-factor optimization of preparation conditions for co-loaded liposomes: Using particle size, PDI, ζ-potential, DHQ, and AST encapsulation efficiency and loading rate as evaluation indicators, a comprehensive scoring method was adopted to optimize each factor. Specifically, the data for each indicator were normalized to obtain a standardized value K (0~1), and the comprehensive score was calculated according to the following formula: ; Wherein, EE is the encapsulation efficiency, LE is the loading rate, Size is the particle size, PDI is the polydispersity index, and ζ is the absolute value of the potential. The weighting is determined according to the importance of each indicator in the liposome performance evaluation. In this embodiment, a comprehensive scoring method is used to comprehensively evaluate the performance of liposomes at various concentrations based on multiple indicators. The criteria for optimal comprehensive performance are the smallest particle size and narrowest distribution (smallest possible PDI), stable charge properties (largest possible absolute value of ζ potential), and the highest encapsulation efficiency and loading rate.

[0076] Determination of soybean lecithin concentration: The mass ratio of soybean lecithin, β-sitosterol, DHQ, and Tween 80 was fixed at 1: : : Under the specified conditions, liposomes were prepared according to the method in 2.3.2.1 with final phospholipid concentrations of 5, 10, 15, 20, and 25 mg / mL, and the effect of phospholipid concentration on liposome performance was investigated.

[0077] Determination of the β-sitosterol / phospholipid mass ratio: The mass ratio of soybean lecithin, DHQ, and Tween 80 was fixed at 1: : Under the condition that the final concentration of phospholipids is fixed at 15 mg / mL, liposomes were prepared according to the method in 2.3.2.1 with the mass ratio of β-sitosterol to phospholipids set as 0, 1:14, 1:10.5, 1:7, and 1:3.5, and the effect of the amount of β-sitosterol added on the performance of liposomes was investigated.

[0078] Determination of the mass ratio of Tween 80 to phospholipids: The mass ratio of soybean lecithin, β-sitosterol, and DHQ was fixed at 1: : Under the condition that the final concentration of phospholipids is fixed at 15 mg / mL, the amount of Tween 80 added is set to 0%, 10%, 20%, 30%, and 40% of the amount of phospholipids. Liposomes are prepared according to the method in 2.3.2.1, and the effect of the amount of Tween 80 added on the performance of liposomes is investigated.

[0079] Determination of the DHQ to phospholipid mass ratio: The mass ratio of soybean lecithin, β-sitosterol, and Tween 80 was fixed at 1: : Under the condition that the final concentration of phospholipids is fixed at 15 mg / mL, liposomes were prepared according to the method in 2.3.2.1 with the mass ratio of DHQ to phospholipids set at 1:2, 1:4, 1:8, 1:12, and 1:16, and the effect of the amount of DHQ added on the performance of liposomes was investigated.

[0080] 2.3 Orthogonal experiment on the preparation conditions of co-loaded liposomes: Based on the single-factor experiments, phospholipid concentration (A), β-sitosterol / phospholipid mass ratio (B), DHQ / phospholipid mass ratio (C), and Tween 80 / phospholipid mass ratio (D) were selected as the factors to be investigated. Each factor was set with 3 levels, and the formulation was optimized using an L9(34) orthogonal experimental design. Liposomes were prepared according to the method in 2.3.2.1 for each experiment. The particle size, PDI, ζ potential, encapsulation efficiency of AST and DHQ, and drug loading were measured. A comprehensive scoring method was used for unified evaluation of multiple indicators. At the same time, a comprehensive balance analysis was conducted by combining the dispersibility and stability of liposomes, drug loading capacity, and preparation feasibility to determine the optimal formulation.

[0081] 2.4 Determination of liposome encapsulation efficiency and loading rate: Determination of DHQ encapsulation efficiency and loading rate: Accurately prepare DHQ series standard solutions (1.0 μg / mL~10.0 μg / mL, using anhydrous ethanol as solvent), and measure the absorbance at 290 nm using a UV-Vis spectrophotometer. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis, and establish a linear regression equation. The results are as follows: Figure 2 As shown.

[0082] A certain amount of liposome sample that has passed through a 0.22 μm filter membrane was taken, centrifuged at 4°C and 7000 r / min for 15 min to remove unencapsulated components, and the supernatant was collected (referred to as sample 1). Another equal volume of unencapsulated liposome stock solution was taken (referred to as sample 2), and 19 times the volume of anhydrous ethanol was added. The samples were placed in an ultrasonic cleaner for demulsification for 40 min, thoroughly mixed, and centrifuged at 4°C and 12000 × g for 20 min. The absorbance value at 290 nm was measured and substituted into the standard curve to calculate the DHQ mass.

[0083] Calculate the encapsulation efficiency (EE) and load factor (LE) of DHQ using the following formulas: ; ; In the formula, m1 is the mass of DHQ encapsulated in liposomes (measured from sample 1), m2 is the total mass of DHQ in liposomes (measured from sample 2), and m0 is the total mass of liposome membrane material.

[0084] Determination of AST encapsulation efficiency and loading rate: Accurately prepare AST series standard solutions (0.5 μg / mL~3.5 μg / mL, using chloroform as solvent), and measure the absorbance at 491 nm using a UV-Vis spectrophotometer. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis, and establish a linear regression equation. The results are as follows: Figure 3 As shown.

[0085] A certain amount of unmembrane-bound liposome sample was taken and 5 mL of petroleum ether was added. Extraction was performed by magnetic stirring in a 30°C water bath for 15 min. The extract was centrifuged at 3000 r / min for 5 min at 4°C, and the organic phase was collected. The extraction process was repeated three times. The combined organic phases were evaporated by rotary evaporation to remove the petroleum ether. The residue was dissolved and brought to a final volume with chloroform. The absorbance was measured at 491 nm, and the free AST content was calculated using the standard curve. The encapsulation efficiency (EE) and loading rate (LE) of AST were calculated using the following formulas: ; ; In the formula, m′ is the mass of free AST outside the liposome, m total m0 represents the total amount of AST used in liposome preparation, and m0 represents the total mass of the liposome membrane material.

[0086] Determination of liposome particle size, polydispersity index, and zeta potential: Liposome samples were diluted appropriately to avoid the influence of multiple scattering, and their particle size, PDI, and zeta potential were determined using a Malvern Zetasizer Nano-ZS90 laser particle size analyzer. Each sample was measured in triplicate, and the average value was taken.

[0087] Observation of liposome appearance: Freshly prepared liposome samples were aliquoted into transparent glass vials, left to stand at room temperature, and their color and state were observed with the naked eye and recorded by taking photographs.

[0088] Phospholipids are the main membrane materials constituting the lipid bilayer. Their concentration has a significant impact on liposome formation, particle size distribution, and the ability to encapsulate active ingredients. The results are as follows: Figure 4 As shown. By Figure 4 As shown in Figure A, the liposome particle size generally increases with increasing phospholipid concentration. The smallest particle size (95.21 ± 4.49 nm) is observed at a phospholipid concentration of 5 mg / mL; however, the particle size increases to 139.87 ± 9.95 nm when the concentration rises to 25 mg / mL. This phenomenon may indicate that as the number of phospholipid molecules in the system increases, more vesicles are formed during hydration, increasing the probability of collisions and fusion, thus forming larger vesicle structures. Furthermore, higher phospholipid concentrations may also increase system viscosity, reducing energy transfer efficiency during ultrasonic refinement, leading to increased liposome particle size. The PDI (Potential Intensity Dispersion) trend is similar to the particle size results, with the smallest PDI (0.24 ± 0.01) at 5 mg / mL, exhibiting good dispersibility and uniformity. As the phospholipid concentration increases, the PDI slightly increases, possibly due to the aggregation and fusion between vesicles leading to a wider particle size distribution, but overall remaining within a narrow range. Regarding the zeta potential, as... Figure 4 As shown in Figure C, the surface potential of liposomes changes little under different phospholipid concentrations. This result may be related to the fact that soybean lecithin is mainly composed of neutral phospholipids, and its concentration changes have a limited contribution to the surface charge of the system.

[0089] Regarding encapsulation performance, the encapsulation efficiency of DHQ and AST showed different trends with varying phospholipid concentrations. For example... Figure 4 As shown in Figure B, the encapsulation efficiency of DHQ gradually decreases with increasing phospholipid concentration, while... Figure 4 As shown in Figure D, the encapsulation efficiency of AST initially increased and then decreased (reaching a peak at 15 mg / mL). This difference may stem from the different solubility characteristics of the two active ingredients. DHQ, a highly hydrophilic flavonoid, is distributed in the aqueous phase or membrane / water interface region within liposomes. When the phospholipid concentration increases, leading to an increase in liposome size, the vesicle structure may become more compact, affecting DHQ encapsulation. AST, a strongly lipophilic carotenoid, primarily embeds in the hydrophobic region of the lipid bilayer. A moderate phospholipid concentration is beneficial for its full distribution within the lipid membrane. Further increases in phospholipid concentration may increase system viscosity, potentially limiting the distribution and diffusion of the active ingredient within the system. Simultaneously, the lipid membrane structure tends towards saturation, resulting in a decrease in encapsulation efficiency. Furthermore, the changes in loading rate and encapsulation efficiency tend to be consistent, indicating that under these experimental conditions, the change in loading rate mainly depends on the total amount of active ingredient encapsulated.

[0090] This embodiment uses a comprehensive scoring method to evaluate the performance of liposomes at various concentrations using multiple indicators. The criteria for optimal comprehensive performance are the smallest particle size and narrowest distribution, stable charge properties, and the highest encapsulation efficiency and loading rate. As shown in Table 5, the comprehensive score is the highest (0.93 ± 0.01) when the phospholipid concentration is 5 mg / mL, which is significantly better than other groups. This indicates that under this condition, the liposomes achieve a better balance between basic performance and encapsulation capacity, and can be used as the optimal level for this factor in subsequent process optimization.

[0091] Table 5: Effect of phospholipid concentration on the overall score of co-loaded liposomes: .

[0092] As a representative component of phytosterols, β-sitosterol's steroidal skeleton can be embedded in the phospholipid bilayer, influencing the stability and basic properties of liposomes by regulating membrane fluidity and arrangement order. For example... Figure 5As shown in Figure A, with increasing β-sitosterol addition, the liposome particle size and PDI did not change significantly in the low-ratio range. However, when the ratio increased to 1:3.5, the particle size increased sharply to 319.61 ± 26.72 nm, and the PDI increased sharply to 0.54 ± 0.06. Appropriate β-sitosterol incorporation can enhance the mechanical strength of the lipid membrane, but excessive addition may increase the liposome's resistance to particle size control, disrupt the ordered arrangement of phospholipid molecules, and promote vesicle aggregation and fusion, thus leading to a significant increase in particle size and poorer system dispersibility. Regarding the zeta potential, as... Figure 5 As shown in Figure C, the potentials of each experimental group remained between approximately -11.49 ± 0.33 and -12.46 ± 1.64 mV, with no significant difference. This may be because β-sitosterol itself does not provide significantly charged groups, and its ratio changes have little effect on the surface charge properties of liposomes.

[0093] In terms of payload performance, such as Figure 5 As shown in Figures B and D, the encapsulation efficiency of both DHQ and AST initially increased and then decreased with increasing β-sitosterol ratio, reaching peak values ​​at a ratio of 1:10.5 (90.14 ± 1.51% and 95.35 ± 0.02%). The loading rate, however, generally showed a decreasing trend. Appropriate β-sitosterol incorporation can promote the formation of a denser and more stable membrane structure in the phospholipid bilayer. Simultaneously, the hydroxyl groups in β-sitosterol may form hydrogen bonds with the carbonyl groups of AST or the phenolic hydroxyl groups of DHQ, reducing leakage of active ingredients during preparation and improving encapsulation efficiency. However, when the proportion of β-sitosterol is too high, its occupancy effect in the bilayer is enhanced, potentially altering the membrane structure's arrangement or affecting the distribution of active ingredients within the membrane, leading to a decrease in encapsulation efficiency. Regarding the loading rate, as the β-sitosterol ratio increases, the total mass of the membrane increases. Even if the total amount of encapsulated active ingredients changes, the proportion of active ingredients loaded per unit membrane is diluted, resulting in a gradual decrease in the loading rate.

[0094] Based on the above indicators, as shown in Table 8, the group with a phospholipid / β-sitosterol mass ratio of 1:10.5 had the highest overall score (0.88 ± 0.05), which was significantly better than the other groups. This ratio was selected as the basis for subsequent optimization.

[0095] Tween 80 is a nonionic surfactant with good emulsifying and dispersing abilities. It can influence the formation and stability of liposomes by reducing interfacial tension and adjusting lipid membrane curvature. The effects of different Tween 80 / phospholipid mass ratios on the basic properties of AST / DHQ co-loaded liposomes are shown below. Figure 6 As shown. By Figure 6As shown in Figure A, without the addition of Tween 80, the liposome particle size was the largest, at 253.58 ± 39.38 nm, and the PDI was also relatively high. After adding Tween 80, the particle size of each experimental group decreased significantly, mainly distributed in the range of 101.31 nm to 128.01 nm, and the PDI also decreased from 0.46 ± 0.03 to 0.25 to 0.31, indicating a significant improvement in the system's homogeneity. This phenomenon suggests that the adsorption of Tween 80 may have successfully reduced the interfacial tension of the lipid membrane, forming smaller vesicle structures by increasing the membrane curvature; simultaneously, the steric hindrance layer formed by Tween 80 molecules attached to the liposome surface can effectively inhibit the aggregation and fusion between vesicles, making the liposomes more stable. Regarding the zeta potential, as... Figure 6 As shown in Figure C, the potential of liposomes with different Tween 80 ratios gradually increased from -20.66 ± 1.48 mV without Tween 80 to -106 ± 1.25 mV. This change may be due to the hydration layer and steric hindrance formed by Tween 80 on the surface of liposomes, which reduced the effective charge density at the shear plane and shielded the negative charge carried by phospholipids to a certain extent, thereby changing the surface charge characteristics of liposomes.

[0096] In terms of payload performance, such as Figure 6 As shown in Figure B, the encapsulation efficiency of DHQ reached its highest level (91.62 ± 1.40%) at a ratio of 1:10, and then fluctuated slightly but remained at a high level overall; Figure 6 As shown in Figure D, the encapsulation efficiency of AST was highest at a ratio of 1:10 (93.26 ± 0.28%), and then dropped sharply to 68.55 ± 4.13% at a ratio of 2:5. The addition of an appropriate amount of Tween 80 can promote the uniform distribution of active ingredients in liposomes through its emulsifying effect. Simultaneously, its adsorption on the lipid bilayer surface can increase the effective membrane thickness, providing more encapsulation space for the active ingredients. When the proportion of Tween 80 increases further, according to Lichtenberg's three-step model, its molecules may disrupt the integrity of the lipid bilayer structure, leading to the dissolution of some liposomes and the formation of mixed micelles, resulting in leakage of contents. Since AST mainly embeds in the hydrophobic regions of the lipid bilayer, it is more dependent on the integrity of the membrane structure and therefore more sensitive to excessive Tween 80. The trend of loading rate change is basically consistent with the encapsulation efficiency.

[0097] Based on the above indicators, as shown in Table 6, the highest comprehensive score (0.86 ± 0.03) was achieved when the mass ratio of Tween 80 to phospholipid was 1:10, which was significantly better than the other groups. This ratio was selected as the basis for subsequent optimization.

[0098] Table 6: Effect of Tween 80 / phospholipid mass ratio on the overall score of co-loaded liposomes: .

[0099] Under the premise of a fixed synergistic ratio of AST to DHQ (1:9), the dosage of DHQ determines the overall drug-liposome ratio of the two active ingredients in the system. The effect of this factor level on the performance of co-loaded liposomes is as follows: Figure 7 As shown. By Figure 7 As shown in Figures A and C, different DHQ dosages had little effect on liposome particle size, PDI, and potential. The particle size of each experimental group ranged from 108.39 nm to 118.34 nm, the PDI remained generally between 0.24 and 0.30, and the absolute potential ranged from 12.36 mV to 11.48 mV with no significant differences between groups. These results indicate that under the experimental conditions, the dosage of the two active ingredients was not the main factor affecting liposome particle size and surface charge properties. While the active ingredients may participate in the liposome formation process, they are mainly distributed in the aqueous phase or lipid bilayer region within the liposome, contributing little to the surface charge of the system. Therefore, all experimental groups exhibited good dispersibility and stability.

[0100] In terms of payload performance, such as... Figure 7 As shown in Figure B, the encapsulation efficiency of DHQ generally increased with decreasing drug-to-lipid ratio, rising from 34.88 ± 1.05% at a drug-to-lipid ratio of 1:2 to 87.30 ± 2.35% at a drug-to-lipid ratio of 1:16; Figure 7 As shown in Figure D, the encapsulation efficiency of AST exhibits a trend of first increasing and then decreasing, reaching a peak at a ratio of 1:4 (94.33 ± 0.45%). This phenomenon may be related to the maximum encapsulation capacity of liposomes. When the DHQ dosage ratio is low, the two active ingredients are relatively dispersed in the system, and the liposomes have sufficient space to encapsulate them. As the dosage increases, the total amount of active ingredients gradually approaches and exceeds the encapsulation capacity of the liposomes, and the excess drug cannot be encapsulated and exists in a free form, leading to a decrease in encapsulation efficiency. The loading rates of DHQ and AST show basically the same trend, both reaching their maximum value at a DHQ / phospholipid mass ratio of 1:4. This phenomenon indicates that under this condition, the change in loading rate is affected by both the absolute amount of encapsulated drug and the total mass of the membrane material: within a lower dosage range, the total amount of encapsulated material increases with the increase in dosage, driving up the loading rate; when the dosage is too high, although the total amount of encapsulated drug may still increase, its increase is less than that of the increase in the total mass of the membrane material, and the loading rate decreases.

[0101] Based on the above indicators, as shown in Table 7, the DHQ to phospholipid mass ratio of 1:4 yielded the highest overall score (0.83 ± 0.03), significantly outperforming all other groups. This ratio was selected as the basis for subsequent optimization. Furthermore, although the 1:12 and 1:16 groups exhibited higher DHQ encapsulation rates, their loading rates were lower, and their AST encapsulation rates were also lower than those of the 1:4 group. While the 1:2 group had a certain loading rate, its encapsulation rate was significantly low, indicating that a large amount of active ingredients were not effectively encapsulated, resulting in decreased raw material utilization and a lower overall score.

[0102] Table 7: Effect of DHQ / phospholipid mass ratio on the overall score of co-loaded liposomes: .

[0103] Results of orthogonal experiment: Based on the single-factor experiments described above, an L9(3^4) orthogonal experiment was further used to optimize the preparation formula. Phospholipid concentration (A), β-sitosterol / phospholipid mass ratio (B), DHQ / phospholipid mass ratio (C), and Tween 80 / phospholipid mass ratio (D) were selected as the factors to be investigated, with three levels for each factor: A at 5, 10, and 15 mg / mL; B at 1:9, 1:10.5, and 1:12; C at 1:4, 1:6, and 1:8; and D at 1:10, 1:5, and 10:3. Liposomes were prepared according to the aforementioned method for each experiment, and particle size, PDI, zeta potential, AST and DHQ encapsulation efficiency, and drug loading rate were measured. Finally, a comprehensive scoring method was used for evaluation. The results showed that the comprehensive score range analysis indicated that the order of influence of each factor on the overall performance of liposomes was: β-sitosterol / phospholipid mass ratio > DHQ / phospholipid mass ratio > Tween 80 / phospholipid mass ratio > soybean lecithin concentration. The optimal formulation combination was A1B3C1D1, namely, soybean lecithin concentration 5 mg / mL, β-sitosterol / phospholipid mass ratio 1:12, DHQ / phospholipid mass ratio 1:4, and Tween 80 / phospholipid mass ratio 1:10.

[0104] Table 8: Factor Level Table for Orthogonal Experiments .

[0105] Table 9: Orthogonal experimental design and results: .

[0106] Table 10: Range Analysis of Orthogonal Experiments (Comprehensive Score): .

[0107] To verify the feasibility of the optimal formulation, this embodiment prepared AST / DHQ co-loaded liposomes according to the above optimal combination, and determined their basic physicochemical properties. The results showed that the obtained co-loaded liposomes were homogeneous, transparent red liquids with an average particle size of 105.94±1.27 nm, a PDI of 0.23±0.00, and a zeta potential of -14.58±0.27 mV; the encapsulation efficiencies of AST and DHQ were 94.57±1.99% and 94.88±1.35%, respectively, and the drug loading efficiencies were 1.80±0.04% and 15.27±1.36%, respectively. These results indicate that the optimal formulation can achieve efficient co-loading of AST and DHQ, and obtain a liposome system with small particle size, good dispersibility, and high encapsulation performance.

[0108] Table 11: Physicochemical properties of optimally formulated co-loaded liposomes: .

[0109] Example 3: CMCS Surface Coating and Its Function: After obtaining the preferred co-loaded liposomes, the present invention further employs CMCS for surface coating to improve the stability of the system under storage, heat treatment and light irradiation conditions.

[0110] 1. Main reagents: Table 12: Main Reagents .

[0111] Other reagents are the same as in the above examples.

[0112] 2. Experimental Methods: 2.1 Preparation of AST / DHQ co-loaded liposomes with different CMCS coating concentrations: AST / DHQ co-loaded liposomes were prepared using the optimal formulations obtained in Examples 1 and 2, denoted as L; after adding equal volumes of CMCS solutions of different concentrations dropwise under magnetic stirring, the mixture was stirred for 1 h and then allowed to stand at 4°C for 1 h to obtain CMCS-coated liposomes, denoted as L-CMCS.

[0113] CMCS concentrations of 0, 1, 2, 3, 4, and 5 mg / mL were set. Particle size, PDI, zeta potential, and encapsulation efficiency (AST and DHQ) were used as evaluation indicators. A comprehensive scoring method was employed to screen for the optimal CMCS coating concentration. The standardized values ​​K (0-1) of each indicator data were obtained after normalization. The comprehensive score was calculated using the following formula: ; Where EE is the encapsulation efficiency, Size is the particle size, PDI is the polydispersity index, and ζ is the absolute value of the potential. The weighting is determined based on the importance of each indicator in the evaluation of liposome performance.

[0114] 2.2 The determination of liposome encapsulation efficiency, liposome particle size, polydispersity index and zeta potential were the same as in Examples 1 and 2.

[0115] 2.3 Observation by transmission electron microscopy (TEM): The microstructure of liposomes was observed using phosphotungstic acid negative staining. Freshly prepared L and L-CMCS were diluted to an appropriate concentration and dropped onto the surface of a copper mesh coated with a carbon film. The mesh was allowed to stand at room temperature to allow for natural adsorption. Excess liquid was carefully blotted away with filter paper, and an appropriate amount of 2% (w / v) phosphotungstic acid solution was added for negative staining. After staining for 2 to 5 minutes, excess staining solution was blotted away with filter paper, and the liposomes were allowed to air dry at room temperature. The morphology of the liposomes was then observed and photographed under a transmission electron microscope.

[0116] 2.4 Fourier Transform Infrared (FTIR) Characterization: Using trehalose as a freeze-drying protectant, L, L-CMCS and their corresponding blank liposomes (denoted as blank L and blank L-CMCS) were freeze-dried under vacuum. Small amounts of freeze-dried samples, CMCS, AST and DHQ were weighed, mixed and ground evenly with dry potassium bromide powder, and then pressed into tablets. The infrared absorption spectra of each sample were recorded by scanning in the wavenumber range of 4000 cm-1 to 400 cm-1 using a Fourier transform infrared spectrometer.

[0117] 2.5 Thermogravimetric analysis (TGA): Weigh approximately 5 mg of sample (DHQ, AST, L lyophilized powder, L-CMCS lyophilized powder, blank L lyophilized powder, blank L-CMCS lyophilized powder) and place it in an alumina crucible. Heat the sample from 45°C to 600°C at a heating rate of 20°C / min under a nitrogen atmosphere (flow rate 30 mL / min). Record the mass change curve of the sample during this process.

[0118] 2.6 Differential Scanning Calorimetry (DSC) Analysis: Weigh approximately 10 mg of sample (L lyophilized powder, L-CMCS lyophilized powder, blank L lyophilized powder, blank L-CMCS lyophilized powder) and place it in an aluminum crucible. Using an empty crucible as a reference, scan from 45°C to 250°C at a heating rate of 15°C / min under a nitrogen atmosphere (30 mL / min) and record the DSC thermal characteristic curve of the sample.

[0119] 2.7 In vitro antioxidant activity assay: 2.7.1 Determination of ABTS free radical scavenging ability: Equal volumes of 7.4 mM ABTS solution and 2.6 mM potassium persulfate solution were mixed and reacted at room temperature in the dark for 12-16 h to prepare the ABTS+ stock solution. Before use, the solution was diluted with deionized water to an absorbance of 0.70 ± 0.02 at 734 nm to obtain the ABTS working solution. In a 96-well plate, 180 μL of ABTS working solution and 20 μL of sample (free AST / DHQ mixed solution, L, L-CMCS and their corresponding blank controls), 180 μL of ABTS working solution and 20 μL of water, and 180 μL of water and 20 μL of sample were added. The solutions were reacted in the dark for 6 min, and the absorbance was measured at 734 nm, denoted as A. i A0, A j The ABTS radical scavenging rate of each sample was calculated using the following formula: ; Plot a dose-response curve with sample concentration on the x-axis and clearance rate on the y-axis, and calculate IC50.50 value.

[0120] 2.7.2 FRAP reducing power determination: The FRAP working solution was prepared by mixing 0.3 M acetate buffer (pH 3.6), 10 mM tripyridine triazine solution, and 20 mM ferric chloride solution at a volume ratio of 10:1:1. 180 μL of the FRAP working solution and 20 μL of sample (free AST / DHQ mixed solution, L, L-CMCS and their corresponding blank controls) were added to a 96-well plate, with blank wells also included. After reacting at 37°C for 10 min, the absorbance was measured at 593 nm. A standard curve from 0 μM to 1000 μM was established using ferrous sulfate (FeSO4) standard solution. Figure 9 Subtract the absorbance value of the corresponding blank sample from the absorbance value of the sample reaction well and substitute it into the formula to calculate the reducing power of each sample (expressed as FeSO4 equivalent).

[0121] 2.8 Preparation of different liposome samples: AST / DHQ co-loaded liposomes were prepared according to the above-mentioned optimal process conditions and denoted as L; based on L, CMCS-coated AST / DHQ co-loaded liposomes were prepared using the optimal CMCS coating conditions obtained from the above examples and denoted as L-CMCS; cholesterol-type AST / DHQ co-loaded liposomes were prepared by replacing β-sitosterol with an equal amount of cholesterol and keeping the other preparation conditions the same.

[0122] 2.9 Storage stability: Freshly prepared L, Lc, and L-CMCS samples were aliquoted into brown glass bottles and sealed. They were stored at 4°C and 25°C in the dark for 48 days. Samples were taken on days 0, 3, 7, 14, 28, and 48 to determine the particle size, PDI, ζ potential, DHQ, and AST encapsulation efficiency of each sample. The appearance of the liposomes was also photographed and recorded.

[0123] 2.10 Thermal stability: The L, Lc, and L-CMCS samples were placed in a 60°C constant temperature water bath and heated continuously for 6 h. Samples were taken at 0, 1, 2, 4, and 6 h and immediately cooled to room temperature in an ice bath. The particle size, PDI, ζ potential, DHQ, and AST encapsulation efficiency of the samples at each time point were measured, and the appearance of the liposomes was recorded by taking pictures.

[0124] 2.11 Light stability: L, Lc, and L-CMCS samples were aliquoted into transparent and brown glass bottles and irradiated under a fluorescent lamp for 72 hours at an intensity of 4200 Lux. Samples were taken at 0, 24, 48, and 72 hours to determine the particle size, PDI, zeta potential, DHQ, and AST encapsulation efficiency, and the appearance of the liposomes was photographed.

[0125] 3. Experimental Results: 3.1 CMCS Concentration Optimization Results: AST / DHQ co-loaded liposomes prepared under optimal formulation conditions were coated with CMCS. The effects of different coating concentrations on liposome particle size, PDI, ζ-potential, and encapsulation efficiency of the two active ingredients were investigated. The results are as follows: Figure 10 As shown. By Figure 10 As shown in Figure A, as the CMCS concentration increased from 0 to 5 mg / mL, the liposome particle size and PDI gradually increased. This phenomenon may be related to the coating layer formed by CMCS on the liposome surface. CMCS is a linear polysaccharide whose molecular chains can be adsorbed onto the surface of the phospholipid bilayer through electrostatic or hydrogen bonding, resulting in a certain degree of entanglement or bridging, which increases the hydrodynamic diameter of the liposome particles and widens the particle size distribution. When the CMCS concentration increased to above 4 mg / mL, the PDI increased significantly, and the system homogeneity decreased. The zeta potential measurement results are as follows: Figure 10 As shown in Figure C, the negative charge on the surface of liposomes was significantly enhanced after coating with CMCS, reaching -23.00 ± 0.37 mV at a CMCS concentration of 2 mg / mL. This is because CMCS molecules contain negatively charged groups such as carboxyl groups, and their deposition increases the charge density on the surface of liposomes. When the concentration of CMCS continues to increase, the absolute value of the zeta potential tends to stabilize. This may be because the adsorption of CMCS on the surface of liposomes gradually reaches saturation, and excess CMCS is free in the solution without further binding.

[0126] In terms of encapsulation performance, by Figure 10 As shown in Figures B and D, with increasing CMCS concentration, the encapsulation efficiency of both active ingredients initially increased and then decreased, reaching a peak at 2 mg / mL (DHQ encapsulation efficiency 96.87 ± 1.95%, AST encapsulation efficiency 97.09 ± 0.19%). An appropriate amount of CMCS forming a protective layer on the liposome surface helps reduce leakage of the active ingredients; however, at higher concentrations, an excessively thick polymer layer may exert a compression effect on the liposome membrane, while enhanced interactions between polysaccharide chains can adversely affect the structural stability of the liposomes, leading to a decrease in encapsulation efficiency.

[0127] This embodiment uses a comprehensive scoring method to evaluate the coating effect at different CMCS concentrations. Considering that the loading rate is greatly affected by changes in the wall material quality during the coating process and cannot directly reflect the coating effect, this embodiment does not retain the loading rate index, but uses the encapsulation rate as the main evaluation parameter, and combines it with particle size, PDI and ζ potential for comprehensive analysis. According to the comprehensive scoring results (Table 13), the comprehensive score is the highest when the CMCS concentration is 2 mg / mL (0.87 ± 0.02), which is significantly better than other groups. At this time, the liposome particle size is 187.31 ± 7.25 nm, which is uniformly distributed, and the surface charge is significantly enhanced (-23.00 ± 0.37 mV). The encapsulation rates of both active ingredients reach the highest level. Therefore, 2 mg / mL is selected as the optimal CMCS coating concentration for subsequent studies.

[0128] Table 13: Effect of CMCS concentration on the overall score of co-loaded liposomes: .

[0129] 3.2 Structure and Characterization Results: The microstructure of uncoated liposomes (L) and CMCS-coated liposomes (L-CMCS) was observed using transmission electron microscopy (TEM), and the results are as follows: Figure 11 As shown.

[0130] Figure 11 As shown in Figures A and B, the particles in sample L are relatively uniformly dispersed in the field of view, and are nearly spherical or quasi-spherical. The vesicles are intact, with clear boundary outlines and certain ring-shaped light and dark contrasts. This is a typical feature of the phospholipid bilayer after negative staining, indicating that the liposome vesicle structure was successfully formed.

[0131] After being coated with CMCS, such as Figure 11 As shown in Figures D and E, L-CMCS maintains a near-spherical vesicle structure overall. However, compared to L-CMCS, some particles exhibit locally high electron density dark areas, which may be related to the local distribution of active ingredients within the vesicles or differences in electron density generated during TEM sample preparation. Simultaneously, continuous and relatively uniform low electron density regions appear at the particle edges, presumably due to adsorption and deposition of CMCS on the liposome surface. In terms of dispersion, L-CMCS samples are predominantly dispersed as single particles, with a small number of loose aggregates in some areas. However, no membrane fusion or structural collapse has occurred, possibly due to bridging effects caused by hydrogen bonding or chain entanglement between CMCS molecular chains, leading to slight contact or aggregation of some liposomes during TEM sample preparation.

[0132] In addition, by Figure 11As shown in Figures C and F, based on TEM images and scale estimates, the overall size distribution of liposome particles ranges from tens to hundreds of nanometers. The average particle size of L-type liposomes is 72.71 ± 29.76 nm, and the average particle size of L-CMCS is 83.37 ± 21.44 nm. This variation is consistent with the characteristic of CMCS forming a polymer coating layer on the liposome surface. Furthermore, it should be noted that there is a certain difference between the particle size measured by TEM and the results of dynamic light scattering in Section 3.4.1. This difference may stem from the measurement method itself: TEM observes the geometric size of particles in a dry state, while dynamic light scattering measures the hydration kinetic particle size of particles in solution, which typically results in a larger measurement value.

[0133] Fourier transform infrared spectroscopy (FTIR) was used to characterize the molecular structures of CMCS, AST, DHQ, AST / DHQ co-loaded liposomes (L), and CMCS-encapsulated AST / DHQ co-loaded liposomes (L-CMCS) to analyze the encapsulation state of the two active ingredients and the interaction between CMCS and liposomes. The results are as follows: Figure 12 As shown.

[0134] CMCS exhibits characteristic absorption peaks at 3440.1 cm⁻¹, 1642.5 cm⁻¹, and 1419.8 cm⁻¹, which are attributed to the stretching vibration of OH, the bending vibration of -NH₂, and the bending vibration of -COO, respectively. - Asymmetric stretching vibrations were observed. AST showed absorption peaks at 2926.4 cm⁻¹ and 1657.5 cm⁻¹, corresponding to CH₂ stretching and C=O stretching vibrations, respectively. DHQ exhibited characteristic peaks at 3399.4 cm⁻¹, 1642.5 cm⁻¹, and 1462.4 cm⁻¹, which are attributed to OH stretching, C=O stretching, and CC bending vibrations, respectively. In contrast, the characteristic peaks of AST and DHQ in the L infrared spectrum were weakened, overlapped, or indistinct, indicating that the two active ingredients no longer existed simply in a free state, and that liposome encapsulation was successful.

[0135] Comparing the infrared spectra of L and L-CMCS, the two are generally similar, with no significant shift in the positions of the main absorption peaks. The absorption peaks at 2929.0 cm⁻¹ and 1740.1 cm⁻¹ in L are attributed to CH₂ stretching vibrations and C=O stretching vibrations, respectively. These peak positions remain largely unchanged in L-CMCS (2928.9 cm⁻¹, 1740.1 cm⁻¹), indicating that CMCS coating did not alter the main structure of the phospholipid bilayer. The absorption peak at 1642.5 cm⁻¹ in L shifts to 1640 cm⁻¹ in L-CMCS, with a relatively small change. The OH / NH absorption peak at 3346.8 cm⁻¹ in L-CMCS is slightly narrower than that in L (3354.3 cm⁻¹), possibly related to the fine-tuning of the hydrogen bond network after the introduction of CMCS. Considering that CMCS is mainly distributed on the surface of liposomes and the coating layer is relatively thin, its influence on the overall infrared absorption characteristics of the system is limited. It is necessary to combine other results of this invention to comprehensively judge the coating state of CMCS.

[0136] To preliminarily investigate the thermal stability of AST / DHQ co-loaded liposomes (L) and CMCS-coated liposomes (L-CMCS), thermogravimetric analysis was performed on AST, DHQ, L and their blank liposomes (blank L), and L-CMCS and their blank liposomes (L-CMCS). The results are as follows: Figure 13 As shown in Table 14.

[0137] The thermal decomposition behaviors of free AST and DHQ showed significant differences. AST exhibited a sharp DTG peak at 308.4°C, with a weight loss rate of 94.41%, indicating that it almost completely decomposed under high-temperature conditions. In contrast, DHQ and various liposome samples showed two main weight loss stages. The first stage was concentrated between 50°C and 210°C, corresponding to the removal of adsorbed and bound water in the samples. Compared to DHQ, the weight loss rate of liposomes was relatively higher in this stage, presumably related to the water-retention capacity of components such as phospholipids and CMCS.

[0138] The second stage is the main decomposition phase of the sample. DHQ showed significant weight loss in the range of 239.7°C to 600°C, with a DTG peak temperature of 258.7°C and a weight loss rate of 48.13%. Blank L began to enter this stage at around 272°C, where the mass loss mainly came from the thermal degradation of membrane materials such as phospholipids and β-sitosterol. After the addition of active ingredients, the main decomposition initiation temperature of L dropped to 264.2°C, possibly because the introduction of AST and DHQ changed the arrangement of the phospholipid bilayer, resulting in a slight decrease in the thermal stability of the system. At the same time, no characteristic decomposition peaks of AST or DHQ were observed in the TGA curve of L, indicating that the two active ingredients no longer exist in free form in the liposomes, but participate in the overall thermal decomposition behavior of the liposomes. Combining the above TEM morphology observation results, it can be concluded that AST and DHQ have been successfully encapsulated inside the liposomes, rather than simply adsorbed on their surface.

[0139] After being coated with CMCS, the peak DTG temperatures of blank L-CMCS and L-CMCS in the main decomposition stage reached 297.3°C and 286.9°C, respectively, which were higher than those of blank L and L. At the same time, the residual mass of the system also increased slightly, indicating that CMCS formed an effective protective layer on the surface of liposomes, which slowed down the thermal degradation process of the system to a certain extent and improved the thermal stability of liposomes.

[0140] Table 14: Thermogravimetric analysis parameters of AST, DHQ, and various liposomes: ; Note: T1 and T2 represent the start and end temperatures of the first weightlessness stage, respectively; TDTG1 represents the peak temperature of the first stage DTG; Δm1 represents the mass loss rate of the first stage; T3 and T4 represent the start and end temperatures of the main decomposition stage, respectively; TDTG2 represents the peak temperature of the main decomposition stage DTG; Δm2 represents the mass loss rate of this stage.

[0141] Differential scanning calorimetry (DSC) results showed that blank L and L exhibited endothermic peaks at 130.7°C and 134.1°C, respectively, indicating that their phase transition behaviors were basically the same, although L's phase transition temperature was slightly higher. It is speculated that the loading of AST and DHQ affected the liposome membrane structure to some extent, but did not destroy its basic characteristics. After CMCS coating, the phase transition temperatures of blank L-CMCS and L-CMCS further increased to 136.6°C and 138.2°C, respectively. Surface modification of liposomes with polymers can reduce molecular chain mobility, making the membrane structure more compact and thus improving its thermal stability. Section 3.4.3 also observed an increase in the principal decomposition temperature of liposomes after CMCS coating, which corroborates the DSC results in this section. Furthermore, the endothermic peak of L-CMCS was broader and gentler than that of L, possibly due to the increased heterogeneity of molecular motion within the liposome membrane caused by the interactions between the components in the system.

[0142] ABTS experimental results showed that L-CMCS had the highest free radical scavenging rate (64.51 ± 1.18%) and IC50. 50 The lowest value (4.58 ± 0.08 μg / mL) indicates the strongest antioxidant activity, followed by L; the free drug has the weakest. FRAP experiments showed that the reducing power of both L and L-CMCS was higher than that of the free drug, but the difference between the two was not significant. In summary, although ABTS and FRAP are based on different antioxidant mechanisms, their overall trend is consistent: encapsulation of liposomes enhances the antioxidant activity of AST and DHQ, and furthermore, CMCS encapsulation can enhance the dispersion stability of liposomes to some extent, thus exhibiting stronger free radical scavenging ability.

[0143] Table 15: In vitro antioxidant activity of L, L-CMCS and free mixed drugs: .

[0144] 3.3 Stability evaluation results: The systems were evaluated under conditions of storage stability (4℃, 25℃), thermal stability (60℃), and light stability (4200 Lux).

[0145] The results show that: 3.3.1 Storage stability: L, Lc, and L-CMCS were stored in the dark at 4°C and 25°C for 48 days, respectively, and their stability during storage was investigated. The results are as follows: Figures 15 to 17 As shown. Overall, low-temperature storage is beneficial for maintaining liposome stability. CMCS coating can improve the physical stability of liposomes and the protection of active ingredients during long-term storage to a certain extent. Compared with cholesterol, β-sitosterol, as a membrane modifier, shows better performance in maintaining liposome stability.

[0146] like Figure 15As shown, under 4°C storage conditions, the particle size and PDI of the three liposomes remained relatively stable. After 48 days, the particle sizes of L, Lc, and L-CMCS increased to 180.15 ± 17.23 nm, 204.84 ± 7.37 nm, and 211.82 ± 1.61 nm, respectively, while the PDI remained below 0.38. Under 25°C storage conditions, the overall liposome particle size showed a more significant increasing trend, with the Lc particle size showing the most significant change, indicating that β-sitosterol may be superior to cholesterol in maintaining liposome membrane stability. L-CMCS showed the smallest increase in particle size at 25°C, indicating that CMCS coating can effectively inhibit the aggregation and fusion of liposomes during storage. The zeta potential results showed that all samples maintained negative charge throughout storage, while the absolute value of the zeta potential of L-CMCS was generally higher than that of L and Lc, indicating that CMCS coating enhanced the negative charge level on the liposome surface.

[0147] Depend on Figure 16 It was found that the encapsulation efficiency of AST and DHQ in the three types of liposomes decreased with prolonged storage time. In terms of temperature, the encapsulation efficiency decreased faster at 25°C than at 4°C, indicating that increasing temperature accelerates changes in liposome membrane structure and promotes leakage of active ingredients. In terms of sample type, Lc showed the fastest decrease in encapsulation efficiency, followed by L. L-CMCS maintained a high encapsulation efficiency at both temperatures, indicating that CMCS coating effectively slows down the leakage of active ingredients, and β-sitosterol replacing cholesterol also helps maintain the protection of active ingredients by liposomes.

[0148] Visual observation results as follows Figure 17 As shown, in the initial storage period, all samples exhibited a homogeneous and stable state with no obvious precipitation or flocculent matter. At 28 days of storage, a small amount of yellow granular precipitate began to appear in L and Lc samples at 25°C, possibly representing slightly aggregated liposome particles or active ingredient / lipid complexes. By 48 days, this precipitation had increased further, and a small amount of yellow particles also began to appear in the L-CMCS sample. In contrast, the overall appearance of the samples at 4°C showed less change, with only a very small amount of particle precipitation observed in the later stages. Overall, although some component precipitation or slight aggregation occurred in each group of samples during storage, no obvious stratification or large-scale precipitation was observed, and the system did not experience macroscopic instability, indicating that the liposome system still possessed good storage stability within the investigated time range.

[0149] 3.3.2 Effect of heat treatment on liposome stability: L, Lc, and L-CMCS were placed in a 60°C water bath and heated continuously for 6 hours to investigate the differences in their thermal stability. The results are as follows: Figures 17 to 20As shown. Overall, the physicochemical properties of each liposome under the treatment conditions all changed to some extent, but the overall magnitude was small. Among them, L-CMCS showed the most stability, indicating that the liposome system constructed in this invention has good stability under food processing-related temperature conditions. CMCS coating can further improve its heat resistance, while cholesterol has a limited effect on the thermal stability of liposomes compared to β-sitosterol.

[0150] like Figure 18 As shown, the particle size of the three liposomes fluctuated slightly during heating, with Lc showing the most significant change, increasing by 38.5%. The differences between sampling points for L and L-CMCS were not significant, with particle size increases of 7.9% and 4.5%, respectively. The PDI trend was similar to that of particle size; after 6 h of heating, the PDI of Lc increased to 0.52 ± 0.08, while that of L and L-CMCS increased to 0.33 ± 0.08 and 0.29 ± 0.02, respectively. These results indicate that Lc is more prone to structural changes under heat treatment, while the L and L-CMCS systems are relatively stable and well-dispersed. The zeta potential results showed that all samples maintained negative charge throughout the heating process, with the L-CMCS system exhibiting a consistently higher absolute value of zeta potential, which increased with prolonged heating time.

[0151] like Figure 19 As shown, the encapsulation efficiency of AST and DHQ in L, Lc, and L-CMCS gradually decreased with prolonged heating time. Increased temperature increases the fluidity of the lipid bilayer, promoting membrane structure rearrangement and leading to leakage of some active ingredients. Comparing the samples, the encapsulation efficiency of Lc decreased relatively more significantly, while L and L-CMCS maintained higher encapsulation efficiency, indicating that liposomes constructed with β-sitosterol exhibit better stability under heat treatment conditions. Furthermore, the external barrier formed by the CMCS coating layer further mitigates the diffusion and loss of active ingredients during heating. In addition, the encapsulation efficiency of AST decreased more significantly compared to DHQ, which may be due to the strong thermosensitivity of AST itself.

[0152] Visual observation results as follows Figure 20 As shown, during the heating process at 60°C, all samples remained in a uniform and stable state, with no obvious precipitation, flocculent matter, or stratification. Although some samples showed slight color changes, the overall differences were not significant, and the system maintained good macroscopic stability.

[0153] 3.3.3 Effect of light on liposome stability: L, Lc, and L-CMCS were continuously irradiated under light conditions (4200 Lux) for 72 h, while a light-protected control group was set up to examine the stability changes of different liposomes in the light environment. The results are as follows: Figures 21 to 23As shown in the figure. Overall, light exposure had some effect on all three types of liposomes. L-CMCS showed the best photostability, indicating that CMCS coating can improve the photostability of liposomes. The difference between L and Lc was small, suggesting that β-sitosterol, as a membrane modifier, may have a similar effect on photostability as cholesterol.

[0154] like Figure 21 As shown, compared with the light-protected group, the particle size and PDI changes of the three light-exposed groups were relatively gradual, indicating that the system maintained a good dispersion state under light conditions, demonstrating that the constructed liposome system has a certain resistance to light-induced structural changes. The zeta potential results showed that all samples maintained negative charge throughout the light exposure period, with no significant changes in L and Lc. However, the absolute values ​​of the zeta potential in the light-exposed and light-protected groups of L-CMCS decreased synchronously, suggesting that this change may be related to interfacial structural adjustments caused by prolonged storage time, rather than a direct effect of light. This result is consistent with the potential change trend observed in the aforementioned storage stability experiments.

[0155] like Figure 22 As shown, with prolonged illumination, the encapsulation efficiencies of AST and DHQ in L, Lc, and L-CMCS all decreased, with the decrease in the illuminated group being significantly greater than that in the shaded group, indicating that illumination accelerated the leakage and degradation of the active ingredients. After 72 hours of illumination, the encapsulation efficiencies of Lc and L decreased by similar amounts, indicating that β-sitosterol and cholesterol contributed essentially the same to the photostability of liposomes; while L-CMCS maintained the highest encapsulation efficiency throughout, suggesting that the physical barrier formed by CMCS encapsulation may delay the degradation of the active ingredients by shielding them from direct irradiation. Furthermore, similar to the thermal stability results, the decrease in the encapsulation efficiency of AST was greater than that of DHQ in all groups, further confirming that AST is more sensitive to light.

[0156] Appearance changes such as Figure 23 As shown, compared with the light-protected group, the L and Lc samples showed slight fading after light treatment, while the L-CMCS system showed less color change. This phenomenon may be related to the partial photodegradation of AST under light conditions. Although some samples showed slight color changes, the overall system remained homogeneous and stable, indicating that the liposome system has good macroscopic stability under light conditions.

[0157] Example 4: A method for preparing astaxanthin and dihydroquercetin co-loaded liposomes includes the following steps: (1) Astaxanthin (AST), dihydroquercetin (DHQ), soybean lecithin, β-sitosterol and Tween 80 were added to anhydrous ethanol in proportion and mixed thoroughly to form a homogeneous organic phase; wherein, the concentration of soybean lecithin was 5 mg / mL, the mass ratio of β-sitosterol / phospholipid was 1:12, the mass ratio of Tween 80 / phospholipid was 1:10, the mass ratio of DHQ / phospholipid was 1:4, and the mass ratio of AST:DHQ was 1:9. (2) The obtained organic phase was placed in a rotary evaporator and all organic solvents were removed by rotary evaporation to form a uniform lipid film on the inner wall of the container. The conditions for rotary evaporation were: rotary evaporation at 180 rpm under reduced pressure for about 25 min at 35°C. (3) Then add 0.1 M pH 7.5 PBS buffer and hydrate at room temperature for 1 h, and form a crude liposome suspension by magnetic stirring; (4) The crude liposome suspension was subjected to probe sonication under ice bath conditions to reduce the particle size and improve the dispersion uniformity. After sonication, the resulting liposome suspension was stored in the dark to obtain astaxanthin and dihydroquercetin co-loaded liposomes. The sonication conditions were 20% power, 5 s sonication / 3 s interval, and a total time of 15 min.

[0158] Example 5: Based on Example 4, this example also includes step (5): preparing a carboxymethyl chitosan CMCS solution with 0.1 M pH 7.5 PBS buffer, the concentration of the CMCS solution is 2 mg / mL, the CMCS solution and the AST / DHQ co-loaded liposome suspension are mixed at a volume ratio of 1:1, and stirred at room temperature to allow the CMCS to adsorb onto the surface of the liposomes to form a coating layer.

[0159] The AST / DHQ co-loaded liposomes prepared using the preferred formulation and process conditions in this embodiment exhibit good physicochemical properties. Specifically, the particle size is 105.94±1.27 nm, the PDI is 0.23±0.00, the zeta potential is -14.58±0.27 mV, the AST encapsulation efficiency is 94.57±1.99%, the DHQ encapsulation efficiency is 94.88±1.35%, the AST loading rate is approximately 1.80%, and the DHQ loading rate is approximately 15.27%. These results demonstrate that the present invention can achieve efficient co-loading of AST and DHQ, and obtain a liposome system with small particle size and good dispersibility.

[0160] Regarding stability, a comparison was made between uncoated liposomes and CMCS-coated liposomes under storage, heat treatment, and light irradiation conditions. The results showed that the stability of the system was improved after CMCS coating, indicating that the surface coating step can improve the problem of insufficient environmental stability of ordinary liposomes.

[0161] Example 6: Effect Verification: This invention utilizes the obtained co-loaded liposomes in the intervention of a hyperuricemic mouse model. Results showed that the co-loaded liposomes could reduce serum uric acid (UA) levels, inhibit hepatic XOD activity, and improve renal function-related indicators and histopathological damage in hyperuricemic mice. The medium-dose group, i.e., DHQ 50 mg / kg + AST 5 mg / kg, showed superior performance, with overall effects exceeding those of the free active ingredient group and the single-drug liposome mixture group.

[0162] 1. Experimental materials: Experimental materials included: AST / DHQ co-loaded liposomes, CMCS-coated AST / DHQ co-loaded liposomes, AST-loaded liposomes alone, DHQ-loaded liposomes alone, AST / DHQ free mixed solution, allopurinol, potassium oxonate, yeast extract, and corresponding biochemical assay kits. The experimental animals were 81 six-week-old SPF-grade male ICR mice, weighing 36.79 ± 1.75 g.

[0163] 2. Experimental methods and group design: After one week of acclimatization, the mice were randomly divided into 9 groups of 9 mice each, as follows: Normal control group (NC); Model group (Mod); Positive control group (PC, allopurinol 10 mg / kg); Free active ingredient group (Free, DHQ 50 mg / kg + AST 5.56 mg / kg); The single-drug liposome combination group (Mix-L, DHQ 50 mg / kg + AST 5.56 mg / kg); Low-dose co-loaded liposome group (LS, DHQ 25 mg / kg + AST 2.78 mg / kg); Medium-dose group with co-loaded liposomes (LM, DHQ 50 mg / kg + AST 5.56 mg / kg); High-dose group with co-loaded liposomes (LH, DHQ 100 mg / kg + AST 11.11 mg / kg); CMCS-coated liposome group (L-CMCS, DHQ 50 mg / kg + AST 5.56 mg / kg).

[0164] Except for the normal control group, all other groups used potassium oxonate combined with yeast extract to establish the HUA mouse model: potassium oxonate 300 mg / kg (dissolved in 1% sodium carboxymethyl cellulose solution) was injected intraperitoneally, and yeast extract 20 g / kg was administered by gavage simultaneously; sample intervention was carried out concurrently during model establishment, administered by gavage once daily for 14 consecutive days. All liposome samples were concentrated by lyophilization and reconstitution to ensure that the predetermined dosage was achieved within a gavage volume of 0.1 mL / 10 g body weight.

[0165] On day 0 after the end of the adaptation period, blood was collected from the orbital cavity of mice as a baseline. Blood was collected again within 1 hour after the last administration and serum was separated to measure serum uric acid (UA), urea (UREA), and creatinine (CRE). Mice were sacrificed after blood collection, and liver was used to measure xanthine oxidase (XOD) activity. Kidney was used for appearance observation and H&E staining histological analysis.

[0166] 3. Experimental Data and Results: 3.1 Serum UA levels and liver XOD activity: Serum UA level is a core indicator for evaluating the success of HUA model establishment and intervention effectiveness. Figure 24 It was found that at the beginning of the experiment, the serum UA levels of mice in all groups were within the normal range, and there was no significant difference between the groups, indicating that the baseline levels of the mice were consistent before the experiment. After 14 days of continuous drug intervention, the serum UA level of mice in the Mod group increased to 301.40 ± 66.54 μmol / L, which was significantly higher than that in the NC group, indicating that potassium oxonate combined with yeast extract successfully induced the HUA model.

[0167] Compared with the Mod group, the serum UA level in the PC group decreased to 20.00 ± 10.89 μmol / L, a reduction of 93.4%, significantly lower than that in the NC group, confirming the significant effect of positive drug intervention. The serum UA level in the Free group was 173.86 ± 49.71 μmol / L, a decrease of 42.3% compared with the Mod group, indicating that the combined administration of AST and DHQ in free state already has a certain uric acid-lowering effect. After liposome co-loading, the uric acid-lowering effect of the two active ingredients was significantly improved with the change in in vivo delivery efficiency. Serum UA levels in mice in the low-dose (LL), medium-dose (LM), and high-dose (LH) groups were 129.38 ± 29.18 μmol / L, 100.25 ± 24.54 μmol / L, and 116.00 ± 16.91 μmol / L, respectively, representing decreases of 25.6%, 42.3%, and 33.3% compared to the Free group. The LM group had the lowest UA level, significantly lower than the LL group, but no significant difference compared to the LH group, indicating that the medium dose may have reached an optimal intervention level under the experimental conditions, and further increasing the dose did not produce a significant benefit. In contrast, although the Mix-L group showed a decrease compared to the Mod group, its UA level was higher than that of the LM group, suggesting that co-loading AST and DHQ in the same liposome is more conducive to the simultaneous arrival of the two active ingredients at the site of action, continuing the synergistic effect observed in in vitro experiments. Furthermore, the serum UA level in the L-CMCS group was 118.71 ± 13.76 μmol / L, which was 31.7% lower than that in the Free group, but its effect was slightly lower than that in the LM group. Although CMCS coating showed excellent physical protective effects in in vitro stability studies, in vivo, the coating layer may delay the release rate of the active ingredient, resulting in a less effective uric acid-lowering effect than uncoated co-loaded liposomes.

[0168] Urea (UA) is mainly produced in the liver through purine metabolism, and xanthocyanin (XOD) is a key rate-limiting enzyme in this process. To investigate whether changes in UA are related to uric acid production, this invention measured the XOD activity in the livers of various groups of mice. Figure 25As shown, the liver XOD activity in the Mod group mice was 29.68 ± 2.40 U / mg prot, significantly higher than that in the NC group (23.60 ± 2.83 U / mg prot), indicating that the HUA model is accompanied by enhanced activity of liver UA-related enzymes, consistent with previous reports. The liver XOD activity in the PC group mice decreased to 21.79 ± 0.91 U / mg prot, a 26.6% decrease compared to the Mod group, demonstrating that allopurinol can exert its uric acid-lowering effect by inhibiting XOD activity. The XOD activity in the Free group decreased by 12.1% compared to the Mod group, indicating that the combined administration of AST and DHQ has a certain inhibitory effect on UA ​​production. After liposome encapsulation, the liver XOD activities in mice in the LL, LM, and LH groups were 24.09 ± 0.78 U / mgprot, 22.48 ± 2.03 U / mgprot, and 22.07 ± 0.15 U / mgprot, respectively, which were 6.7%, 12.2%, and 13.6% lower than those in the Free group, showing a dose-dependent effect. This indicates that liposome encapsulation can enhance XOD inhibition and improve the uric acid-lowering efficacy of the active ingredient. In contrast, the Mix-L group showed a decrease compared to the Mod group, but the improvement was relatively limited. The liver XOD activity in the L-CMCS group was 24.1% lower than that in the Mod group, but there was no significant difference compared to the LM group, indicating that CMCS encapsulation had a relatively small effect on XOD inhibition.

[0169] 3.2 Renal function-related indicators and renal histopathological results: Serum CRE and UREA are important biochemical indicators for evaluating renal function. CRE reflects glomerular filtration function, while UREA reflects protein metabolism and glomerular excretion capacity. Their ratio is often used to comprehensively evaluate renal function and nitrogen metabolism. This example investigated the effects of different delivery methods on renal function in HUA mice by measuring serum CRE and UREA levels in each group of mice. The results are as follows: Figure 26 As shown.

[0170] At the start of the experiment, serum CRE and UREA levels in all groups of mice were within the normal range. After 14 days of drug intervention, serum UREA in the Mod group increased to 14.58 ± 2.26 mmol / L, and CRE increased to 20.46 ± 2.92 μmol / L, both significantly higher than those in the NC group, indicating that the HUA model mice developed renal function impairment. This is consistent with the literature reports that HUA can induce renal inflammation and reduce glomerular filtration capacity. Serum CRE and UREA levels in the PC and Free groups were not significantly different from those in the Mod group, indicating that allopurinol failed to effectively alleviate HUA-induced renal dysfunction, and the regulatory effect of free AST and DHQ was also limited. In contrast, the renal function indicators showed a more significant recovery trend after co-loaded liposome treatment: CRE levels in the LL, LM, and LH groups decreased by 29.5%, 31.4%, and 36.3% respectively compared to the Mod group, and UREA levels decreased by 18.3%, 24.7%, and 36.5% respectively, with the LH group showing the largest decrease, and its CRE and UREA levels approaching those of the NC group. The renal function indicators in the Mix-L group improved compared to the Mod group, but the overall effect was lower than that in the co-loaded liposome group, indicating that simultaneous delivery of AST and DHQ is more effective in protecting renal function. The L-CMCS group was similar to some uncoated liposome groups, indicating that CMCS coating did not weaken the protective effect of liposomes on the kidneys.

[0171] The appearance and histopathological changes of the kidneys of mice in each group are as follows: Figure 27 As shown, black arrows indicate glomerular mesangial matrix proliferation, blue arrows indicate glomerular capillary dilation, orange arrows indicate vacuolar degeneration of renal tubular epithelial cells, yellow arrows indicate renal tubular dilation, red arrows indicate renal tubular atrophy, green arrows indicate lymphocyte infiltration, and silver arrows indicate glomerular necrosis.

[0172] The results showed that the kidneys in the NC group were uniformly dark red with no surface abnormalities, and the renal tissue structure was intact, with no inflammatory cell infiltration or degenerative changes. The kidneys in the Mod group were mildly enlarged with patchy whitening on the surface. Histological examination revealed glomerular mesangial matrix proliferation, vacuolar degeneration of renal tubular epithelial cells, extensive renal tubular dilation, and interstitial inflammatory infiltration, presenting typical characteristics of renal tissue damage.

[0173] The kidneys in the PC group showed slight improvement compared to the Mod group, but the overall color remained pale, indicating some residual tubular damage. The Free group still exhibited localized whitishness in the kidneys, and histological examination revealed tubular dilation and interstitial hyperplasia, with limited improvement. The Mix-L group showed occasional mesangial matrix hyperplasia in the glomeruli, with significant tubular dilation and interstitial inflammatory infiltration, indicating a worse condition than the co-loaded liposome group. The kidney morphology of all co-loaded liposome groups was closer to that of the NC group, showing a dose-dependent trend. Macroscopically, the kidneys in the LM and LH groups were normal dark red with no whitish areas. Histologically, the LL group showed occasional lesions in the glomeruli, with only localized mild tubular dilation; the LM group showed largely intact glomerular structure, significantly reduced tubular dilation and epithelial cell vacuolar degeneration; the LH group showed only occasional mild tubular dilation, demonstrating the best improvement. The kidney condition in the L-CMCS group was similar to that in the LM group. Based on comprehensive observations of kidney appearance and pathology, it was found that co-loaded liposomes can effectively reduce HUA-induced kidney damage, and the simultaneous delivery strategy has synergistic advantages in kidney protection.

[0174] 3.3 Liver function related indicators: To investigate the effects of different delivery methods on the liver safety of mice, this example measured the serum GOT / ALT ratio in each group of mice. The results are as follows: Figure 28 As shown.

[0175] At the start of the experiment, the GOT / ALT ratios of mice in each group were basically the same. After 14 days of drug intervention, except for the NC group, the GOT / ALT ratios of all other groups increased compared to baseline and were generally higher than those of the NC group, indicating that the HUA model caused fluctuations in liver function indicators to some extent. In terms of numerical trends, the GOT / ALT ratios of all sample treatment groups generally showed a decreasing trend compared to the Mod group, with some liposome-treated groups showing levels closer to those of the NC group. Although the differences between groups did not reach statistical significance, the results indicate that under the experimental conditions, the different AST / DHQ delivery systems did not impose additional burden on liver function while exerting their uric acid-lowering effects, demonstrating good liver safety.

[0176] 4. Experimental conclusions: In summary, the results of this embodiment demonstrate that the HUA mouse model can be successfully constructed using potassium oxonate combined with yeast extract. The AST / DHQ co-loaded liposomes prepared in this invention effectively reduced serum UA levels in model mice, inhibited hepatic XOD activity, and improved renal function-related indicators and renal tissue pathological damage, with the medium-dose group (DHQ 50 mg / kg + AST 5 mg / kg) showing superior performance. Compared with the free active ingredient group and the single-drug liposome mixture group, the co-loaded liposome group exhibited a better in vivo uric acid-lowering effect, indicating that encapsulating AST and DHQ in the same liposome system in an optimal ratio is beneficial for leveraging their synergistic delivery advantages and functional effects.

[0177] The above results demonstrate that the technical solution formed by the present invention has clear feasibility in preparation, stability advantages, and application value in lowering uric acid.

[0178] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0179] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0180] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for preparing astaxanthin and dihydroquercetin co-loaded liposomes, characterized in that... Includes the following steps: (1) Astaxanthin (AST), dihydroquercetin (DHQ), phospholipids, sterols and nonionic surfactants were added to anhydrous ethanol in proportion and mixed thoroughly to form a homogeneous organic phase; wherein the mass ratio of AST to DHQ was 1:6 to 1:

12. (2) Place the obtained organic phase in a rotary evaporator and remove all organic solvents by rotary evaporation to form a uniform lipid film on the inner wall of the container; (3) Then, a buffer solution was added and hydrated at room temperature, and a crude liposome suspension was formed by magnetic stirring; (4) The crude liposome suspension was subjected to probe sonication under ice bath conditions; after sonication, the obtained liposome suspension was stored in the dark to obtain astaxanthin and dihydroquercetin co-loaded liposomes.

2. The preparation method according to claim 1, characterized in that: The phospholipids mentioned in step (1) are a compound system of one or more of the following: soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, and phosphatidylcholine; the sterols are a mixture of one or more of the following: cholesterol, β-sitosterol, stigmasterol, campesterol, and sitosterol; the nonionic surfactants are Tween 20, Tween 80, Span 60, Span 80, or other polyoxyethylene nonionic surfactants, or a compound system of two or more of the above surfactants.

3. The preparation method according to claim 1, characterized in that: In step (1), the phospholipid concentration is 3~10 mg / mL, the sterol / phospholipid mass ratio is 1:(8~15), the nonionic surfactant / phospholipid mass ratio is 1:(8~12), and the DHQ / phospholipid mass ratio is 1:(3~5).

4. The preparation method according to claim 1, characterized in that: At 30-40℃, rotary evaporation under reduced pressure at 150-200 rpm for 20-30 minutes.

5. The preparation method according to claim 1, characterized in that: The hydration time in step (3) is 30~90 min, and the magnetic stirring speed is 300~700 rpm.

6. The preparation method according to claim 1, characterized in that: The conditions for ultrasound in step (4) are: ultrasound power of 10%~30%, intermittent ultrasound mode, ultrasound time of 3~8 s, interval time of 2~5 s; total ultrasound time of 10~20 min.

7. The preparation method according to claim 1, characterized in that: The preparation method further includes step (5): preparing a polysaccharide solution with a buffer solution, mixing the polysaccharide solution with an AST / DHQ co-loaded liposome suspension at a volume ratio of 1:(0.5~2), stirring at room temperature to allow the polysaccharide to adsorb onto the surface of the liposomes to form a coating layer; the polysaccharide is carboxymethyl chitosan (CMCS), chitosan, sodium alginate, pectin, hydroxypropyl methylcellulose or its derivatives, or a compound system of two or more polysaccharides.

8. The preparation method according to claim 7, characterized in that: In step (5), the concentration of the CMCS solution is 1~4 mg / mL, and the mixture is stirred for 40 min at room temperature.

9. A co-loaded liposome of astaxanthin and dihydroquercetin prepared by any one of claims 1-8.

10. The use of the astaxanthin and dihydroquercetin co-loaded liposomes according to claim 9 in the preparation of products for lowering uric acid and / or inhibiting hepatic xanthine oxidase (XOD) activity and / or improving renal function.

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