Astaxanthin-loaded proliposome and preparation method thereof

Abstract

The invention discloses a proliposome loaded with astaxanthin and a preparation method of the proliposome, and belongs to the cross technical field of food science and biological medicine application. The preparation method of the proliposome loaded with the astaxanthin comprises the following steps: forming a mixed solution from phospholipid and the astaxanthin, and then adding sugar alcohol powder into the mixed solution to obtain the proliposome loaded with the astaxanthin; the proliposome loaded with astaxanthin prepared by the method has good biocompatibility, has inhibition and alleviation effects on Lo-2 cell oxidative stress, inflammatory injury and abnormal fat accumulation caused by hydrogen peroxide, can significantly improve and alleviate the disease progress of C57 / BL6 mouse non-alcoholic steatohepatitis, and has good application prospects. The method has a very good application prospect in the industries of medicines and health care products.

Description

Technical Field

[0001] The present invention relates to an astaxanthin-loaded proliposome and a preparation method thereof, and belongs to the cross-technical field of food science and biomedicine application. Background Art

[0002] With significant improvements in living standards, modern consumers' food demands have evolved from simply satisfying hunger to a new era of pursuit of "nutrition and health." Bioactive ingredients found in food, including amino acids, peptides, proteins, functional lipids, polysaccharides, oligosaccharides, terpenes, polyphenols, carotenoids, flavonoids, probiotics, minerals, and vitamins, are increasingly becoming a focus of food science research due to their remarkable efficacy in regulating physiological functions, enhancing immunity, and preventing chronic diseases. However, these valuable bioactive ingredients generally face technical bottlenecks such as poor solubility, low stability, and suboptimal bioavailability. Furthermore, they are susceptible to a variety of environmental factors, including temperature, oxygen, pH, light, humidity, enzymes, and metal ions, severely restricting their industrial production and practical application in food.

[0003] To overcome the low solubility, stability, and bioavailability of functional factors, carriers such as nanoparticles, liposomes, microcapsules, vesicles, and emulsions have been designed to improve their utilization. Liposomes are encapsulated carrier formulations with a lipid bilayer structure. They can encapsulate water-soluble or fat-soluble functional substances within a vesicle or bilayer membrane formed by a hydrophilic head. This approach is an effective method for achieving high bioavailability and efficient physiological activity of functional substances. However, nanoliposomes often face problems such as leakage and aggregation of functional substances, as well as susceptibility to hydrolysis and oxidation in the gastrointestinal tract. To overcome these problems, liposomes can be designed as proliposomes. Proliposomes are small, dry solid particles composed of lipid material and functional substances adsorbed onto a water-soluble carrier. They lack a complete vesicular structure and are converted into liposomes upon hydration. Upon contact with aqueous solutions or biological fluids, the carrier rapidly dissolves, and the lipid portion swells to form liposomes, which then exert their effects. Proliposomes serve as intermediates that can enhance both bioavailability and the stability of functional substances. As a special type of liposome, proliposomes only swell in a specific aqueous environment to form a complete functional structure, avoiding the physicochemical stability problems of conventional liposomes that are prone to oxidation, hydrolysis, aggregation or leakage, while also reducing the denaturation and damage caused by physiological factors such as the gastrointestinal tract.

[0004] However, current research on proliposomes is still based on optimizing the encapsulation rate and loading rate of active substances, and the active substances encapsulated in proliposomes still have problems with poor stability, solubility and bioavailability. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides an astaxanthin-loaded proliposome and a method for preparing the same. The astaxanthin-loaded proliposome is primarily made from natural phospholipids. Sugar alcohol powder is adsorbed onto the phospholipid surface to encapsulate fat-soluble astaxanthin. The proliposome and bioactive ingredients are loaded onto a carrier to form dry solid particles, significantly improving the stability, solubility, and bioavailability of the functional ingredients. The proliposome remains solid during storage and transportation, and is reconstituted into intact liposomes in situ only in an aqueous environment, effectively avoiding the problems of traditional liposomes, such as oxidation, leakage, and aggregation.

[0006] In order to achieve the above objectives, the technical solutions provided are as follows:

[0007] The first object of the present invention is to provide a method for preparing astaxanthin-loaded proliposomes, the method comprising the following steps:

[0008] S1, fully stirring astaxanthin and raw material phospholipid to form a mixed solution; the mass ratio of raw material phospholipid to astaxanthin in the mixed solution is 30 to 50:1;

[0009] S2. Adding sugar alcohol powder to the mixed solution of step S1, then adding a food-grade organic reagent, magnetically stirring to allow the carrier to fully adsorb phospholipids and astaxanthin, rotary evaporation, and drying to obtain astaxanthin-loaded proliposome powder; the carrier sugar alcohol powder includes one or more of sorbitol, galactitol, and xylitol.

[0010] In one embodiment, the astaxanthin in step S1 includes one or more of Haematococcus pluvialis astaxanthin oil, Antarctic krill oil, and Phaffia rhodozyma astaxanthin oil.

[0011] In one embodiment, the raw material phospholipids in step S1 include one or more of soybean lecithin, sunflower lecithin, egg yolk lecithin, large yellow croaker lecithin, and Antarctic krill oil lecithin.

[0012] In one embodiment, the food-grade organic reagent in step S2 is ethanol or food-grade n-hexane.

[0013] In one embodiment, the mass ratio of the raw material phospholipids and astaxanthin in step S1 is 40:1.

[0014] In one embodiment, the mass ratio of the sugar alcohol carrier powder to the raw material phospholipid in step S2 is 5 to 10:1.

[0015] In one embodiment, the rotation speed of the magnetic stirring in step S2 is 30-200 rpm, the temperature of the rotary evaporation is 30-100° C., and the rotation speed is 30-200 rpm.

[0016] In an embodiment, step S2 can also be adding the sugar alcohol powder into the mixed solution of step S1, and then using an acoustic resonance mixer to resonate and mix, to obtain a flowable astaxanthin-loaded pre-liposome powder.

[0017] A second object of the present application is to provide an astaxanthin-loaded pre-liposome prepared by the method described above.

[0018] A third object of the present application is to provide a method for preparing an astaxanthin-loaded liposome, comprising the following steps:

[0019] Dissolving the astaxanthin-loaded pre-liposome described above in an aqueous phase to obtain an astaxanthin-loaded nanoliposome solution.

[0020] In an embodiment, the aqueous phase comprises one or more of deionized water, a pbs buffer, a Krebs-Ringer buffer, a DMEM cell culture medium, and a biological fluid.

[0021] A fourth object of the present application is to provide an astaxanthin-loaded nanoliposome solution prepared by the method described above.

[0022] A fifth object of the present application is to provide the use of the astaxanthin pre-liposome or the astaxanthin-loaded nanoliposome solution described above in the fields of food, health products, and medicine.

[0023] A sixth object of the present application is to provide the use of the astaxanthin-loaded pre-liposome described above in the preparation of a drug for relieving non-alcoholic fatty liver disease.

[0024] The beneficial effects of the present application are:

[0025] Firstly, the present application can produce pre-liposomes by mixing natural phospholipids with sugar alcohol powder, wherein the intermolecular hydrogen bonds increase the intermolecular interaction of the derivatives, ultimately leading to the formation of nanoparticles from these pre-liposomes. Compared with traditional liposomes, the pre-liposomes adopt a solid-state storage form, have excellent physicochemical stability, and can effectively avoid common problems of liposomes such as aggregation, fusion, and drug leakage. The unique "self-assembly in water" property of the pre-liposomes not only simplifies the use process, but also forms liposomes with uniform particle size and high encapsulation efficiency in vivo; it can not only improve the solubility and bioavailability of hydrophobic functional ingredients, but also protect sensitive ingredients from damage by factors such as light, heat, and oxygen, and can achieve controlled release by adjusting the composition of phospholipids, providing an ideal technical platform for the development of functional foods.

[0026] Secondly, the pre-liposome preparation method of the present application has mild conditions, maintains the activity of active substances, and has simple operation, low energy consumption, and environmental protection. The prepared nanoparticles are controllable and relatively uniform in size.

[0027] Furthermore, the prepared astaxanthin-loaded precursor liposome has inhibiting and relieving effects on Lo-2 cell oxidative stress and inflammatory damage caused by hydrogen peroxide and abnormal fat accumulation, can significantly improve and relieve the disease progression of non-alcoholic fatty liver disease of C57 / BL6 mice, and has a very good application prospect in the pharmaceutical and health product industries.

[0028] Finally, the technical scheme of the present application is suitable for the fields of food, health products and medicine, and can be widely applied to the delivery of various bioactive components such as amino acids, polypeptides, polyphenols, flavonoids and probiotics, has advantages such as simple preparation process, high stability and controlled release, and provides reliable technical support for the development of functional food. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is the physical picture of the astaxanthin-loaded precursor liposome and the astaxanthin-loaded nanoliposome in Example 1 of the present application;

[0030] Figure 2 It is the transmission electron microscope image of the astaxanthin-loaded nanoliposome in Example 1 of the present application;

[0031] Figure 3 It is the data graph of the loading rate and embedding rate of astaxanthin by the precursor liposome under different lipid-drug ratios in Example 1 and Example 3 of the present application;

[0032] Figure 4 It is the data graph of the bioavailability and stability of astaxanthin in the in vitro simulated digestion of the astaxanthin-loaded precursor liposome in Example 1 of the present application;

[0033] Figure 5 It is the ROS fluorescence staining image of Lo-2 cells after incubation with the astaxanthin-loaded precursor liposome in Example 1 of the present application;

[0034] Figure 6 It is the mitochondrial membrane potential fluorescence staining image of Lo-2 cells after incubation with the astaxanthin-loaded precursor liposome in Example 1 of the present application;

[0035] Figure 7 It is the data graph of the level of inflammatory factors in Lo-2 cells after incubation with the astaxanthin-loaded precursor liposome in Example 1 of the present application;

[0036] Figure 8 It is the oil red O staining image of Lo-2 cells for relieving fat accumulation by the astaxanthin-loaded precursor liposome in Example 1 of the present application;

[0037] Figure 9 It is the liver physical picture of non-alcoholic fatty liver disease mice after dietary intervention with the astaxanthin-loaded precursor liposome in Example 1 of the present application;

[0038] Figure 10 The oil red O staining image of the liver section of the non-alcoholic steatohepatitis mouse after the dietary intervention of the astaxanthin-loaded pre-liposome in the embodiment 1 of the present application;

[0039] Figure 11 The H&E staining image of the liver section of the non-alcoholic steatohepatitis mouse after the dietary intervention of the astaxanthin-loaded pre-liposome in the embodiment 1 of the present application;

[0040] Figure 12 The Masson staining image of the liver section of the non-alcoholic steatohepatitis mouse after the dietary intervention of the astaxanthin-loaded pre-liposome in the embodiment 1 of the present application. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application. The specific embodiments below further describe the present application.

[0042] Embodiment 1

[0043] A preparation method of an astaxanthin-loaded pre-liposome and a liposome thereof, comprising the following steps:

[0044] S1, sunflower phospholipid and astaxanthin are mixed and dissolved in food-grade ethanol at a mass ratio of 40:1 (w / w), ultrasonic acceleration dissolution is carried out under room temperature conditions, a phospholipid-astaxanthin / ethanol solution is obtained, wherein the phospholipid concentration is 20 mg / mL, and the astaxanthin concentration is 0.5 mg / mL;

[0045] S2, a galactitol carrier is added to the phospholipid-astaxanthin / ethanol solution of step S1 at 5 times the mass of the sunflower phospholipid (w / w), and the carrier is fully adsorbed with the phospholipid and the astaxanthin by magnetic stirring at 100 rpm under room temperature conditions; and then drying treatment is carried out by rotary evaporation at 65℃ and 100 rpm, to obtain a pre-liposome powder loaded with astaxanthin with good fluidity;

[0046] S3, the pre-liposome powder loaded with astaxanthin obtained in step S2 is dissolved in distilled water, and stirring is carried out at 150 rpm under room temperature conditions for 5 min, to obtain a nanoliposome solution loaded with astaxanthin.

[0047] Embodiment 2

[0048] A preparation method of an astaxanthin-loaded pre-liposome and a liposome thereof, comprising the following steps:

[0049] S1, sunflower lecithin and astaxanthin are mixed and dissolved in food-grade ethanol in a mass ratio of 10:1, 20:1, 30:1, 50:1, 60:1, and ultrasonic accelerated dissolution is performed at room temperature to obtain a lecithin-astaxanthin / ethanol solution with lecithin concentrations of 5 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, and 30 mg / mL, respectively, and an astaxanthin concentration of 0.5 mg / mL;

[0050] S2, adding a galactitol carrier at a weight (w / w) of 5 times that of sunflower lecithin to the phospholipid-astaxanthin / ethanol solution of step S1, and then resonating and mixing using an acoustic resonance mixer to obtain a pro-liposome powder loaded with astaxanthin with good fluidity;

[0051] S3, dissolving the astaxanthin-loaded proliposome powder obtained in step S2 in the aqueous phase, stirring at 150 rpm for 5 minutes at room temperature to obtain an astaxanthin-loaded nanoliposome solution.

[0052] Performance measurement

[0053] 1. Appearance of astaxanthin-loaded proliposomes and nanoliposomes

[0054] The astaxanthin-loaded proliposome powder and astaxanthin-loaded nanoliposome suspension prepared in Example 1 were photographed in high definition. Figure 1 As shown, the sugar alcohol carrier has good adsorption capacity for astaxanthin, and the nanoliposome suspension loaded with astaxanthin appears translucent red, which proves that the water solubility and dispersibility of astaxanthin are effectively improved.

[0055] 2. TEM images of astaxanthin-loaded nanoliposomes

[0056] The astaxanthin-loaded proliposomes obtained in Example 1 were hydrated and observed by transmission electron microscopy. An appropriate amount of the astaxanthin-loaded nanoliposome suspension was dispersed on the surface of a carbon film-covered copper mesh. After drying for 5 minutes, the excess liquid was absorbed with filter paper, and then 10 μL of a 20 mg / mL uranyl acetate solution was added dropwise for negative staining. After drying for 5 minutes, the excess dye was removed by absorption. Finally, the microscopic morphology of the hydrated nanoliposomes was observed by transmission electron microscopy at an accelerating voltage of 200 kV.

[0057] The results are as follows Figure 2 As shown, after hydration, the astaxanthin-loaded pre-liposome powder presents a classic multi-chamber liposome structure under the microscopic view, and the particle size is kept below 100 nm, proving that the preparation method described in Example 1 can obtain an astaxanthin-loaded nanoliposome suspension.

[0058] 3. Effect of lipid-drug ratio (Examples 1 and 2) on astaxanthin loading rate and entrapment efficiency in proliposomes

[0059] The effects of different lipid-drug ratios (10:1, 20:1, 30:1, 40:1, 50:1, and 60:1, w / w) on the astaxanthin entrapment efficiency (EE) and loading rate (LC) in proliposomes were determined. 50 mg / mL astaxanthin-loaded nanoliposome suspensions were prepared and centrifuged at 5000 rpm for 3 minutes at 4°C. 0.5 mL of the supernatant was extracted in batches with 5 mL of ethyl acetate. The absorbance of astaxanthin in the solution was measured at 25°C and 475 nm using a microplate reader. The EE and LC of astaxanthin in the astaxanthin-loaded proliposomes were determined by substituting the concentration-absorbance curve into the corresponding formula.

[0060] EE(%)=m A,L / m A,I ×100

[0061] LC(%)=m A,L / (m A +m PC90 )×100

[0062] where m A,L represents the mass of astaxanthin encapsulated in nanoliposomes, m A,I represents the initial mass of astaxanthin in the entire pre-liposomal system, m PC90 It represents the mass of phospholipids in the entire astaxanthin-loaded pre-liposome system.

[0063] The results are as follows Figure 3 As shown in the figure, with the increase of the lipid-drug ratio, the astaxanthin encapsulation efficiency and loading rate of the proliposomes showed a trend of first increasing and then decreasing. When the PC90:AXT (w / w) ratio was 30:1, the loading rate reached a peak of 4.13±0.05%, but the astaxanthin encapsulation efficiency at this time was only 54.47±14.41%. Continuing to increase the lipid-drug ratio to 40:1, the astaxanthin encapsulation efficiency of the liposomes reached saturation, reaching 81.65±11.54%, while 3.70±0.53% of the astaxanthin could still be loaded by PC90.

[0064] 4. Bioaccessibility and stability of astaxanthin

[0065] First, a simulated in vitro digestion model was established. After the small intestinal digestion was completed, the digestive fluid was diluted to 50 mL with PBS buffer (pH 7.0). At this time, the suspension system was defined as the digestive phase. Secondly, in order to remove the internal chyme, a portion of the digestive phase suspension was centrifuged at 18,000 rpm for 30 minutes, and the resulting supernatant was defined as the micellar phase. Then, equal amounts of astaxanthin in the digestive phase and micellar phase suspensions were extracted with ethyl acetate, and their absorbance was measured at 475 nm. Finally, the bioaccessibility and stability of astaxanthin in the sample were calculated by substituting the standard curve and the following formula.

[0066] Bioaccessibility (%) = (m micelle / m digesta )×100

[0067] Stability (%) = (m micelle / m original )×100

[0068] Where: m digesta and m micelle are the astaxanthin contents in the digestion phase and micelle phase, respectively, m original Indicates the astaxanthin content in the system at the initial stage of digestion.

[0069] The results are as follows Figure 4 As shown in the results, the bioaccessibility of astaxanthin in free astaxanthin (AXT group) and astaxanthin in a physical mixture of phospholipids and astaxanthin (mix group) was low, at 0.25±0.02% and 0.39±0.02%, respectively, proving that both groups of samples were difficult to digest and decompose. However, the bioaccessibility of astaxanthin in pro-AXT loaded astaxanthin liposomes was significantly increased to 73.13±0.52%, an increase of approximately 292.52 times compared to the free astaxanthin group. This significant difference in absorption can be attributed to the excellent stability of the nanoliposomes formed when the pro-AXT liposomes are hydrated during digestion. The resulting astaxanthin-loaded nanoliposomes are difficult to emulsify and decompose, effectively preventing the leakage of astaxanthin. At this time, 81.08±2.14% of astaxanthin can still be stably encapsulated in the liposomes and reach the small intestine.

[0070] In summary, astaxanthin-loaded proliposomes will be transformed into astaxanthin-encapsulated nanoliposomes during the digestion stage, which can significantly improve the solubility and dispersibility of astaxanthin in the digestive environment and is an effective means to increase the bioaccessibility of astaxanthin.

[0071] 5. ROS fluorescence staining images of Lo-2 cells after incubation with astaxanthin-loaded proliposomes

[0072] A reactive oxygen species (ROS) fluorescence staining kit was used to detect the regulatory effects of astaxanthin group (AXT), blank carrier group (i.e., proliposomes without astaxanthin loading, referred to as pro), and proliposomes loaded with astaxanthin (pro-AXT) on the ROS level in Lo-2 cells. The DCFH-DA probe was diluted 1000-fold with pure water according to the instructions. Lo-2 cells were cultured at a concentration of 1×10 5The cells were seeded at a density of 100 μg / well in a 6-well plate and incubated at 37°C and 5% CO2 for 24 hours. The culture medium was replaced with fresh culture medium containing 250 μM H2O2 to stimulate Lo-2 cells to produce oxidative stress. The culture was continued for 24 hours under the same conditions. After that, AXT, pro and pro-AXT were dispersed in the culture medium to ensure that the final concentration of astaxanthin was 0.625 μg / mL, the corresponding pro concentration was 8.74 mg / mL, and the pro-AXT concentration was 8.75 mg / mL. After continuing to culture for 24 hours, 1 mL of DCFH-DA probe working solution was added to each well and incubated for 30 minutes in the dark for probe loading. After discarding the culture medium, the well plate was washed 1-2 times with PBS and examined under a microscope at the working wavelength (E x =494nm, E m =517 nm) to analyze the ROS level in each well.

[0073] The results are as follows Figure 5 As shown, a large area of ​​bright green fluorescence appeared in the model group treated with 250 μM H₂O₂, demonstrating that H₂O₂ stimulates oxidative stress in Lo-2 cells. However, the large size of AXT crystals in the free state makes it difficult for cells to absorb and utilize them. Therefore, a small amount of green fluorescence was still observed in the AXT group, indicating that its effect on alleviating oxidative stress in Lo-2 cells was relatively limited. A small amount of dim green fluorescence was still observed in the pro group, which may be due to the high degree of unsaturation of the raw material phospholipids, which plays a certain antioxidant role. However, almost no green fluorescence was observed in the pro-AXT field, indicating its superior ROS scavenging effect. Lo-2 cells have good fusion and uptake capacity for the astaxanthin-loaded nanoliposomes formed after pro-AXT hydration, which can effectively alleviate oxidative stress in Lo-2 cells.

[0074] 6. Fluorescence staining images of mitochondrial membrane potential in Lo-2 cells after incubation with astaxanthin-loaded proliposomes

[0075] A mitochondrial membrane potential (MMP) fluorescence staining kit was used to detect the regulatory effects of AXT, pro, and pro-AXT on changes in mitochondrial membrane potential in Lo-2 cells. JC-1 working solution was prepared according to the instructions. Lo-2 cells were plated, modeled, and dosed according to the method described in Performance Assay 5. The JC-1 probe was loaded and incubated in the dark for 30 minutes. The levels of JC-1 aggregates and JC-1 monomers in each well were analyzed at the working wavelength (JC-1 monomer E x =514nm, E m =529nm; JC-1 aggregate E x =585nm, E m =590nm).

[0076] The results are as follows Figure 6As shown, after treatment with 250 μM H2O2, the red fluorescence of Lo-2 cells under the field of view was significantly faded and the green fluorescence intensity was enhanced, and the red / green fluorescence ratio was significantly reduced. This may be due to the fact that exogenous H2O2 can cause a significant increase in intracellular ROS levels through multiple mechanisms such as direct accumulation, conversion to highly active ROS, activation of ROS generating enzymes, inhibition of antioxidant systems, and interference with mitochondrial function, thereby causing an increase in mitochondrial inner membrane permeability and outflow of membrane protons, further leading to a decrease in mitochondrial membrane potential. After treatment with AXT, pro, and pro-AXT, the mitochondrial membrane potential was protected. Among them, the red fluorescence intensity of the pro-AXT group was significantly increased, and its effect was the most obvious, indicating that pro-AXT can promote the endocytosis and utilization of astaxanthin by Lo-2 cells through the hydration of astaxanthin-loaded nanoliposomes, thereby enhancing the antioxidant capacity of the cells and effectively alleviating the cell damage caused by oxidative stress.

[0077] 7. Inflammatory factor levels in Lo-2 cells after incubation with astaxanthin-loaded pre-liposomes

[0078] Lo-2 cells were plated, modeled, and dosed according to the method described in Performance Determination 5. According to the method described in the instructions, the tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 10 (IL-10), and interleukin 1 beta (IL-1β) kits were used to analyze the regulatory effect of AXT, pro, and pro-AXT on the levels of related inflammatory factors in Lo-2 cells.

[0079] The results are shown in Figure 7 As shown, compared with free AXT, pro-AXT can significantly reduce the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in Lo-2 cells, and increase the level of anti-inflammatory factor IL-10, showing a better anti-inflammatory effect on Lo-2 cells.

[0080] 8. Oil red O staining images of astaxanthin-loaded pre-liposomes alleviating fat accumulation in Lo-2 cells

[0081] Lo-2 cells were plated at a density of 1×10 5Cells were seeded at a density of 100 μg / well in a 6-well plate and incubated at 37°C and 5% CO2 for 24 hours. Fresh culture medium containing 5 mM oleic acid (OA) was used to replace the culture medium to induce Lo-2 cells to establish a metabolic-related fatty liver disease cell injury model. The cells were incubated for 12 hours under the same conditions, after which AXT, pro, and pro-AXT were dispersed in the culture medium to ensure that the final concentration of astaxanthin was 0.625 μg / mL, the corresponding pro concentration was 8.74 mg / mL, and the pro-AXT concentration was 8.75 mg / mL. After a further 24 hours of incubation, the cell samples were stained using the Oil Red O staining kit according to the instructions, and the fat staining images of the Lo-2 cells were observed and captured under a bright field microscope.

[0082] The results are as follows Figure 8 As shown, in the control group, Lo-2 cells had clear edges, and the cytoplasmic fat was lightly stained with small, uniformly dispersed particles, consistent with the characteristic that cells maintain a small amount of lipid droplets as energy reserves through lipid metabolism under normal physiological conditions. After oleic acid induction, the model group displayed large, bright red lipid droplets within and around the Lo-2 cells, clearly demonstrating abnormal fat accumulation. In the AXT and pro groups, fat accumulation was slightly reduced, while in the Lo-2 cells treated with pro-AXT, the lipid droplets were lightly stained, with small, uniformly dispersed particles. This demonstrates that astaxanthin-loaded proliposomes can effectively alleviate abnormal fat accumulation in Lo-2 cells.

[0083] 9. Actual image of the liver of mice with non-alcoholic steatohepatitis after dietary intervention with astaxanthin-loaded proliposomes

[0084] Fifty 7-week-old male C57 / BL6 mice (weighing 20±3g, SPF) were housed in a standard animal facility (room temperature: 23±2°C, air humidity: 50±10%, 12 / 12h light / dark cycle) and provided with a normal maintenance diet and purified water for a transition period. After 1 week of acclimation, the mice were randomly divided into five groups of 10: control, model, free AXT, blank vector pro, and pro-AXT. This was designated week 0. The model, AXT, pro, and pro-AXT groups were fed a high-fat, choline-deficient model diet (CDAA model diet), while the control group was fed a control diet. The NASH disease model was established after 12 weeks of feeding. Simultaneously with model establishment, dietary intervention (AXT 150mg / kg·w) was administered by gavage once daily at 8pm to minimize stress and maintain metabolic activity. The mice were killed after 12 weeks, and liver tissue samples were collected. The appearance was first recorded with a high-definition camera.

[0085] The results are as followsFigure 9 As shown, the healthy livers of the control group mice were rosy in color, soft in texture, and smooth in surface. The livers of the model group were yellowish in color, with a tight capsule and blunt edges, and a large accumulation of yellow fat particles visible to the naked eye, and poor softness and elasticity. The AXT and pro groups showed some improvement, but still showed grayish color, rough surface, and slight swelling compared to the control group. The livers of the pro-AXT group showed significant improvement, almost indistinguishable from healthy livers.

[0086] 10. Oil Red O staining of liver sections from mice with non-alcoholic steatohepatitis after dietary intervention with astaxanthin-loaded proliposomes

[0087] Cut an appropriate amount of mouse liver tissue, rinse with 0.1M PBS, and then fix with 4% paraformaldehyde for more than 24 hours. Stain the liver slices using the principle that Oil Red O has a strong affinity for triglycerides. Observe the fat accumulation and degeneration degree of the liver slices under a microscope.

[0088] The results are as follows Figure 10 As shown in the results, in a healthy state, liver parenchymal cells are closely arranged with clear edges, while in the Model group, there are a large number of red-stained lipid droplets and severe fat vacuolation; after intervention with AXT and pro, the vacuolation phenomenon is alleviated, and after intervention with pro-AXT, the number and area of ​​lipid droplets and vacuoles are significantly reduced, showing a significant effect.

[0089] 11. H&E staining of liver sections from mice with nonalcoholic steatohepatitis after dietary intervention with astaxanthin-loaded proliposomes

[0090] Cut an appropriate amount of mouse liver tissue, rinse with 0.1M PBS, and then fix with 4% paraformaldehyde for more than 24 hours. Perform H&E staining on the mouse liver tissue, and observe the cell structure and inflammation-related pathology of the liver tissue sections under a microscope.

[0091] The results are as follows Figure 11 As shown, microscopic examination of healthy livers revealed densely packed hepatocytes, uniform cytoplasm, and large, round, centrally located nuclei. Microscopic examination of the model group revealed extensive fat vacuoles, pale cytoplasm and nuclei, small, peripherally located nucleoli, and significant hepatocyte edema and inflammatory infiltration. AXT, pro, and pro-AXT alleviated these symptoms to varying degrees, with pro-AXT having the most pronounced effect.

[0092] 12. Masson staining of liver sections from mice with nonalcoholic steatohepatitis after dietary intervention with astaxanthin-loaded proliposomes

[0093] The mouse liver tissue was cut into appropriate sizes, rinsed with 0.1M PBS, and then fixed with 4% paraformaldehyde for more than 24 hours. The mouse liver tissue was subjected to Masson staining, and the liver fibrosis and cirrhosis in the liver tissue sections were observed under a microscope.

[0094] The results are as follows Figure 12 As shown, the healthy liver has a complete structure, with only a very small amount of blue-stained collagen fibers in the portal vein area; while the Model group showed mild fibrous hyperplasia in the portal vein area, with fibrous cords extending to the surrounding areas; the collagen fibers in the portal vein area of ​​the AXT group and the pro group were thicker than those in the Control group, but significantly less than those in the Model group, which can be considered as the early stage of fibrous hyperplasia; collagen fibers were almost invisible under the microscope in the pro-AXT group, which significantly alleviated the process of liver fibrosis.

[0095] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing astaxanthin-loaded proliposomes, characterized in that: The method comprises the following steps: S1, fully stirring astaxanthin and raw material phospholipid to form a mixed solution; the mass ratio of raw material phospholipid to astaxanthin in the mixed solution is 30 to 50:1; S2. Adding sugar alcohol powder to the mixed solution of step S1, then adding a food-grade organic reagent, magnetically stirring to allow the carrier to fully adsorb phospholipids and astaxanthin, rotary evaporation, and drying to obtain astaxanthin-loaded proliposome powder; the carrier sugar alcohol powder includes one or more of sorbitol, galactitol, and xylitol.

2. The method according to claim 1, characterized in that The astaxanthin in step S1 includes one or more of Haematococcus pluvialis astaxanthin oil, Antarctic krill oil, and Phaffia rhodozyma astaxanthin oil.

3. The method according to claim 1, characterized in that The raw material lecithin in step S1 includes one or more of soybean lecithin, sunflower lecithin, egg yolk lecithin, large yellow croaker lecithin, and Antarctic krill oil lecithin.

4. The method according to claim 1, wherein The mass ratio of the sugar alcohol carrier powder to the raw material phospholipid in step S2 is 5 to 10:

1.

5. The method according to claim 1, wherein Step S2 may also be to add sugar alcohol powder to the mixed solution of step S1, and then resonate and mix using an acoustic resonance mixer to obtain astaxanthin-loaded proliposome powder with good fluidity.

6. The astaxanthin-loaded proliposome prepared by the method according to any one of claims 1 to 5.

7. A method for preparing astaxanthin-loaded liposomes, characterized in that: The method comprises the following: The astaxanthin-loaded proliposomes according to claim 6 are dissolved in an aqueous phase to obtain an astaxanthin-loaded nanoliposome solution.

8. The nanoliposome solution prepared by the method according to claim 7.

9. Use of the astaxanthin proliposome according to claim 6 or the nanoliposome solution according to claim 8 in the fields of food, health food and medicine.

10. Use of the astaxanthin-loaded proliposome according to claim 6 in the preparation of a drug for alleviating non-alcoholic steatohepatitis.

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