A method for preparing recombinant astaxanthin vesicles derived from Haematococcus pluvialis with gut-protecting and lipid-regulating properties.

Abstract

This invention discloses a method for preparing recombinant astaxanthin vesicles derived from Haematococcus pluvialis with gut-protective and lipid-regulating effects, belonging to the field of bioactive substance delivery. This system uses Haematococcus pluvialis as raw material, without adding any exogenous carriers, directly extracting natural astaxanthin and algal lipids, which are then recombined and self-assembled to form a stable nano-delivery system. Algal lipids serve as the carrier framework, astaxanthin as the active ingredient, phospholipids participate in the formation of the lipid bilayer, and astaxanthin is embedded in the hydrophobic region, achieving efficient delivery and oral administration. This invention utilizes the synergistic effect of the algal components themselves to solve the problems of poor water solubility and low bioavailability of astaxanthin. Combining the lipid-regulating effects of algal lipids with the gut-protective and antioxidant effects of astaxanthin, it achieves a three-in-one effect of "delivery, protection, gut protection, and lipid regulation." It can be used in functional foods, health products, and pharmaceutical preparations to improve intestinal microecology and regulate lipid metabolism, possessing advantages such as natural safety, simple process, low cost, and broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance transport and functional food / pharmaceutical technology, specifically relating to a method for preparing recombinant astaxanthin vesicles derived from Haematococcus pluvialis with gut-protecting and lipid-regulating properties. Background Technology

[0002] Astaxanthin is a natural ketocarotenoid with extremely strong antioxidant activity. It also exhibits excellent physiological functions in regulating lipid metabolism, protecting the intestinal mucosal barrier, and improving the balance of the intestinal microecology, showing broad application prospects in health foods and pharmaceutical preparations. In nature, Haematococcus pluvialis is one of the highest natural sources of astaxanthin, with an astaxanthin content reaching 4% of cell dry weight. It mainly exists in the more bioactive 3S, 3′S configuration, primarily in diesterized and monoesterified forms. Compared to astaxanthin extracted from shrimp and crab shells, yeast, etc., it has advantages such as high purity, good biocompatibility, and fewer side effects, making it one of the optimal raw materials for extracting natural astaxanthin.

[0003] It is worth noting that Haematococcus pluvialis cells are not only rich in astaxanthin, but also contain abundant natural algal lipids (mainly including triglycerides, phospholipids, and glycolipids). These algal lipids are important components for Haematococcus pluvialis to store energy and maintain cell structure. Among them, phospholipids can participate in the formation of lipid bilayer structure and have certain lipid-regulating and intestinal-protecting auxiliary effects. They have natural molecular compatibility with astaxanthin, and astaxanthin can embed into the hydrophobic region of the lipid bilayer to form a natural antioxidant barrier. The two can achieve synergistic function, which provides a unique advantage for constructing a natural and efficient astaxanthin delivery system. However, natural astaxanthin has inherent defects such as poor stability, strong lipid solubility, and extremely poor water solubility. It is easily oxidized when exposed to oxygen and is easily degraded under acid, alkali, high temperature, and ultraviolet light irradiation. Moreover, it is difficult to be absorbed by the intestine after oral administration, resulting in extremely low bioavailability, which seriously limits its intestinal-protecting and lipid-regulating physiological functions. Meanwhile, the effects of single astaxanthin preparations or single algal lipids on gut protection and lipid regulation are limited, making it difficult to achieve synergistic effects of "gut protection" and "lipid regulation," and failing to meet the comprehensive needs for gut health and lipid metabolism regulation in clinical and daily health care.

[0004] Currently, existing technologies for astaxanthin extraction and delivery systems suffer from two major drawbacks: First, most technologies focus solely on the extraction of astaxanthin from Haematococcus pluvialis, neglecting the utilization value of its own algal lipids. The algal lipids extracted from astaxanthin are discarded as waste, resulting in raw material waste and increased environmental pressure. Furthermore, the extraction processes often suffer from insufficient cell disruption, low extraction rates, high solvent residues, and severe damage to the activity of astaxanthin and lipids. Second, existing astaxanthin delivery systems often employ exogenous chemically synthesized carriers or exogenous lipids, leading to poor biocompatibility and insufficient biodegradability. Some carriers may even irritate the intestines, failing to effectively protect astaxanthin activity and hindering synergistic effects between astaxanthin and the carrier. Moreover, they fail to utilize the natural compatibility between Haematococcus pluvialis astaxanthin and its own lipids, preventing the formation of stable and efficient delivery structures through lipid recombination and self-assembly. This results in poor overall efficacy and high production costs for the formulation. Furthermore, patent CN 113081869 A discloses a technical solution for obtaining Haematococcus pluvialis astaxanthin microcapsules by hot blanching, homogenization, enzymatic hydrolysis and cell wall breaking, and finally spray drying. However, this technical solution is actually a simple cell wall breaking and drying process for all components of Haematococcus pluvialis, without the targeted extraction and separation of algal lipids in Haematococcus pluvialis. It neither explores the carrier value of algal lipids nor achieves the precise matching of astaxanthin with natural lipids. Moreover, the microcapsules prepared by it are macroscopic powder structures of all components of Haematococcus pluvialis, with a particle size that does not reach the nanoscale. Micron-sized cells cannot effectively take them up, resulting in extremely low bioavailability. Summary of the Invention

[0005] Technical issues Existing astaxanthin extraction and utilization technologies face technical challenges such as raw material waste, poor carrier safety, insufficient synergistic effects, and unreasonable self-assembly system design, which have become key issues that urgently need to be addressed in this field.

[0006] Technical solution To address the aforementioned issues, this invention develops a method that uses Haematococcus pluvialis as raw material to simultaneously extract astaxanthin and algal lipids from the algae. The phospholipids contained in the algal lipids of Haematococcus pluvialis can be recombined and self-assembled to form nanoscale lipid vesicle structures, which can precisely encapsulate lipid-soluble astaxanthin. This not only solves the problem of low water utilization of astaxanthin, but also leverages the physiological activity of the algal lipids to achieve a synergistic effect with astaxanthin in protecting the intestines and regulating lipids. At the same time, it achieves full utilization of raw materials and reduces production costs.

[0007] This invention provides an astaxanthin self-assembly delivery system based on Haematococcus pluvialis for intestinal protection and lipid regulation. The self-assembly delivery system uses Haematococcus pluvialis as the sole active ingredient. Natural astaxanthin extracted from Haematococcus pluvialis and its own algal lipids are recombined and self-assembled to form nanoscale lipid particles, without the need for exogenous carriers, emulsifiers, or stabilizers. The preparation method of the self-assembly delivery system includes the following steps: S1. Dissolve Haematococcus pluvialis in water and add cellulase and pectinase for enzymatic hydrolysis, then cool, centrifuge and collect the precipitate; S2. Redissolve the precipitate obtained in step S1, then sonicate it, and then centrifuge to collect the precipitate. S3. Redissolve the precipitate obtained in step S2, then add an extraction solvent composed of dichloromethane and methanol for extraction, centrifuge, collect the dichloromethane layer solution and dry it to obtain the extract; S4. Add the extract obtained in step S3 to water for hydration, then centrifuge to collect the supernatant, and filter the supernatant using a microfiltration membrane to obtain astaxanthin self-assembled lipid vesicle particles.

[0008] Furthermore, in step S1, the Haematococcus pluvialis includes fresh Haematococcus pluvialis or dried Haematococcus pluvialis powder.

[0009] Furthermore, in step S1, Haematococcus pluvialis contains 1-8 wt% astaxanthin.

[0010] Furthermore, in step S1, the concentration of Haematococcus pluvialis dissolved in water is 1~10 mg / mL.

[0011] Furthermore, in step S1, the amount of cellulase added is 500~20000 U / g substrate, and the substrate is Haematococcus pluvialis.

[0012] Furthermore, in step S1, the amount of pectinase added is 10,000~200,000 U / g substrate, and the substrate is Haematococcus pluvialis.

[0013] Furthermore, the enzymatic hydrolysis temperature in step S1 is 40~60°C. o C, time is 30~90 min.

[0014] Furthermore, the centrifugation conditions in step S1 are 0~5. o C, 6000~8000 rpm, 5~15 min.

[0015] Furthermore, the concentration of the precipitate after reconstitution in step S2 is 1~10 mg / mL.

[0016] Furthermore, in step S2, the crushing power is 200~800 W, and the time is 20~60 min.

[0017] Furthermore, the centrifugation conditions in step S2 are 0~5. o C, 6000~8000 rpm, 5~15 min.

[0018] Furthermore, the concentration of the precipitate after reconstitution in step S3 is 1~10 mg / mL.

[0019] Furthermore, in step S3, the volume ratio of the solvent dichloromethane to methanol is 1:1.5~2.5.

[0020] Furthermore, in step S3, the amount of solvent used for extraction is 2 to 5 times the volume of the solution after the precipitate is redissolved.

[0021] Furthermore, the extraction conditions in step S3 are: protection from light and stirring at 20-30°C for 12-24 hours.

[0022] Furthermore, the centrifugation conditions in step S3 are 0~5. o C, 6000~8000 rpm, 5~15 min.

[0023] Furthermore, in step S3, the drying process can be selected from rotary evaporation, freeze drying, or heat drying.

[0024] Furthermore, in step S4, the concentration of the hydrated extract is 0.1~2 mg / mL.

[0025] Furthermore, the hydration conditions in step S4 are hydration at 20~30℃ for 24~48 h.

[0026] Furthermore, the centrifugation conditions in step S4 are 0~5. o C, 8000~10000 rpm, 10~20 min.

[0027] Furthermore, in step S4, the microfiltration membrane is a filter membrane with a pore size of 0.22~0.45 μm.

[0028] The present invention provides a recombinant vesicle-like particle of Haematococcus pluvialis-derived astaxanthin with intestinal protection and lipid regulation prepared by the method described above.

[0029] Furthermore, the intestinal protection refers to improving the balance of the intestinal microecology, repairing intestinal mucosal damage, or inhibiting intestinal inflammation.

[0030] This invention provides the application of the above-mentioned Haematococcus pluvialis-derived astaxanthin recombinant vesicle particles with intestinal protection and lipid regulation in the preparation of food, health products or pharmaceuticals.

[0031] Beneficial effects (1) The delivery system of this invention uses Haematococcus pluvialis as the sole source of active components. It does not require the addition of exogenous carriers, emulsifiers, stabilizers and other chemical reagents. It is formed by the recombination and self-assembly of astaxanthin and algal lipids of Haematococcus pluvialis itself, thus avoiding the safety hazards caused by exogenous substances. The product is natural and non-toxic, and is suitable for long-term consumption and medicinal use.

[0032] (2) This invention fully utilizes the resource advantages of Haematococcus pluvialis, extracts and utilizes astaxanthin and algal lipids simultaneously, achieving "multiple uses of one product" and avoiding resource waste; algal lipids not only serve as a natural carrier of astaxanthin, protecting it from oxidation and degradation and improving its bioavailability, but also exert lipid-regulating activity on their own, working synergistically with the gut-protecting and antioxidant effects of astaxanthin to significantly enhance the gut-protecting and lipid-regulating effects, solving the problem of the single efficacy of existing products. By regulating the gut-liver axis, it can effectively improve the intestinal microecological imbalance and lipid metabolism disorder caused by a high-fat diet.

[0033] (3) The preparation process of the present invention involves only simple steps such as enzymatic hydrolysis, co-extraction, and recombinant self-assembly. It is easy to operate and does not require complex equipment or harsh reaction conditions. At the same time, it does not require the addition of exogenous carriers and chemical reagents, which significantly reduces the preparation cost and is suitable for large-scale production.

[0034] (4) The astaxanthin carrier particles prepared by this invention have uniform particle size (50~200nm) and good dispersibility, realizing oral targeted delivery of astaxanthin, significantly improving its absorption efficiency and bioavailability in the intestine, and solving the technical problems of poor water solubility, easy degradation and low bioavailability of natural astaxanthin.

[0035] (5) The astaxanthin transport system prepared by the present invention can be widely used in functional foods, health products, pharmaceutical preparations and other fields. It can be prepared into various product forms such as oral liquids and capsules to meet the needs of different groups of people. It is especially suitable for people with high-fat diets and people with intestinal dysfunction. It has extremely high market application value. Attached Figure Description

[0036] Figure 1 These are the aqueous solution of recombinant astaxanthin vesicles prepared according to embodiments of the present invention and its transmission electron microscopy image, wherein... Figure 1 (A) is an aqueous solution of recombinant astaxanthin lipid vesicles. Figure 1 (B) is a transmission electron microscope image. Figure 1 (C) shows the measurement results from the laser particle size analyzer. Figure 1 (D) is the Zeta potential diagram; Figure 2 This is a graph showing the in vivo digestion and retention rate of recombinant astaxanthin vesicles prepared in the embodiments of the present invention in mice; Figure 3 This is a permeation diagram of the recombinant astaxanthin vesicles prepared in an embodiment of the present invention in porcine intestinal mucus; Figure 4 This is a permeation diagram of the intestinal barrier in Caco-2 cells of recombinant astaxanthin vesicles prepared in an embodiment of the present invention. Figure 5 This is a fluorescence staining image of astaxanthin recombinant vesicles prepared according to an embodiment of the present invention to alleviate lipid droplet fluorescence induced by oleic acid; Figure 6 This is an image of oil red O staining of oleic acid-induced lipid droplets, obtained from recombinant astaxanthin vesicles prepared according to embodiments of the present invention. Figure 7 This is a graph showing the changes in the weight and physical condition of mice with recombinant astaxanthin vesicles prepared according to embodiments of the present invention, which alleviated the effects of a high-fat diet. Figure 8 This is an image of the recombinant astaxanthin vesicles prepared according to an embodiment of the present invention, which alleviates fat in various tissues of mice induced by a high-fat diet. Figure 9 This is an HE staining image of recombinant astaxanthin-based vesicles prepared according to an embodiment of the present invention to alleviate the epididymal fat of mice induced by a high-fat diet. Figure 10 This is an image of the recombinant astaxanthin vesicles prepared according to an embodiment of the present invention, which alleviate the colonic symptoms induced by a high-fat diet in mice. Figure 11 This is an HE staining image of the mouse colon, prepared by recombinant astaxanthin vesicles in an embodiment of the present invention, which alleviates the effects of a high-fat diet. Figure 12 This is a fluorescent staining image of recombinant astaxanthin vesicles prepared according to an embodiment of the present invention to alleviate the tight junction protein in the mouse colon induced by a high-fat diet. Figure 13 This is a transmission electron microscope image of the astaxanthin lipid complex aqueous solution prepared in the comparative example of this invention. Detailed Implementation

[0037] The astaxanthin content of the Haematococcus pluvialis powder used in this invention is 5%, and it was purchased from Kunming Baiou Microalgae Technology Co., Ltd.

[0038] The room temperature mentioned in this invention is 25°C.

[0039] The cellulase used in this invention has an activity of 1000 U / g and was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; the pectinase cellulase has an activity of 100000 U / g and was purchased from Jiangsu Ruiyang Biotechnology Co., Ltd.

[0040] Example 1 The preparation of a solution of astaxanthin self-assembled lipid vesicle particles derived from Haematococcus pluvialis includes the following steps: S1. Dissolve Haematococcus pluvialis powder in deionized water to a concentration of 4 mg / mL, add cellulase to a concentration of 4 mg / mL, and pectinase to a concentration of 4 mg / mL, and heat in a water bath for 50 minutes. oC, stir for 60 min, after enzymatic hydrolysis, cool to room temperature, and centrifuge (4) o Collect the precipitate at C, 8000 rpm, for 10 min for later use; S2. Add 400 mg of the Haematococcus pluvialis enzymatic precipitate obtained in step S1 to 100 mL of deionized water for reconstitution, and then perform ultrasonic disruption at a power of 600 W for 30 min. After disruption, [the solution is then placed in a 4-channel system]. o Centrifuge at 8000 rpm for 10 min and collect the precipitate for later use; S3. Dissolve 200 mg of the precipitate collected in step S2 in 100 mL of deionized water. Add 400 mL of an extraction solvent (dichloromethane and methanol in a 1:2 volume ratio) and stir for 12 h under dark conditions. After extraction, [the solution is then removed from the heat]. o Centrifuge at 8000 rpm for 10 min and collect the dichloromethane layer solution for later use; S4. The dichloromethane layer solution extracted in step S3 is subjected to a process at 37°C. o C. The extractant was removed by rotary distillation under reduced pressure at 50 rpm to obtain a simultaneous extract of astaxanthin and algal lipids for later use. S5. Take 20 mg of the simultaneous extract of astaxanthin and algal lipids obtained in step S4, add 200 mL of deionized water and hydrate for 24 h, then... o Centrifuge at 10000 rpm for 10 min to collect the supernatant. Filter the supernatant through a 0.22 μm filter membrane to obtain astaxanthin self-assembled lipid vesicle particles.

[0041] Figure 1 (A, B) show the aqueous solution of recombinant astaxanthin lipid vesicles prepared from Haematococcus pluvialis. The figure shows that the recombinant astaxanthin transport system exhibits good solubility in the aqueous solution, appearing deep red. Transmission electron microscopy reveals that the recombinant astaxanthin lipid vesicles have a spherical structure with a double-membrane structure similar to vesicles, and the particles are uniform in size. Figure 1 (C) The hydrated particle size of the recombinant astaxanthin lipid vesicles was measured using a laser particle size analyzer to be 130 ± 0.2172 nm, with a PDI of 0.1696 ± 0.0079. The particle size falls within the range of 100–200 nm, which is consistent with the ideal particle size range for nanodelivery systems. This is beneficial for achieving enrichment at lesion sites through enhanced permeability and retention effects, and for improving cellular uptake efficiency. A PDI less than 0.2 indicates that the nanoparticles have a uniform particle size distribution and good system dispersion, with no obvious agglomeration or large particle aggregation, further demonstrating the stability and reproducibility of the preparation process. Figure 1(D) shows that the Zeta potential of the astaxanthin recombinant lipid vesicles was -48.27 ± 0.8514 mV, exhibiting a significant negative charge. This high absolute value indicates that the nanoparticles have a high density of negative charges on their surface, resulting in strong electrostatic repulsion between particles, which effectively inhibits aggregation and contributes to the system's good dispersion stability in aqueous solution. The high absolute value of the Zeta potential also suggests that the astaxanthin recombinant lipid vesicles are not prone to rapid aggregation or sedimentation in physiological environments, possessing the basic colloidal stability required for their use as a delivery carrier.

[0042] Figure 2 Astaxanthin recombinant lipid vesicles prepared for this example were orally administered to mice via gavage. Astaxanthin content in the stomach, small intestine, and colon was measured at 1, 2, 4, and 6 hours. The results showed that, compared to free astaxanthin, the gastric retention rate was significantly lower after 2 hours of gavage, reaching only 5.95 ± 2.67 μg / mL, and astaxanthin color was not observable in the intestine after 4 hours. In contrast, the gastric retention rate of the recombinant lipid vesicles was 8.01 ± 0.57 μg / mL after 2 hours of gavage, representing an approximately 1.4-fold increase. Astaxanthin color was still observable in the intestine after 6 hours. This indicates that the gastrointestinal stability of the recombinant lipid vesicles after oral administration was significantly higher than that of free astaxanthin, thus improving the in vivo utilization potential of astaxanthin.

[0043] Figure 3 The recombinant astaxanthin lipid vesicles prepared for this example were infiltrated in an in vitro porcine mucus model for 12 hours. The results showed that after 12 hours of incubation, free astaxanthin remained largely retained on the mucus surface, making diffusion to deeper layers difficult. In contrast, the recombinant astaxanthin lipid vesicle delivery system prepared in this example exhibited significantly lower residual levels on the mucus surface, demonstrating stronger mucus penetration behavior. Quantitative analysis of the drug content in the lower permeate further indicated that the permeability of the recombinant astaxanthin lipid vesicles far exceeded 40%, while the permeability of the free astaxanthin group was less than 20%. These results confirm that the recombinant astaxanthin lipid vesicle delivery system can effectively improve the diffusion and permeation behavior of astaxanthin in the mucus layer, reduce non-specific retention of astaxanthin on the mucus surface, and significantly enhance its ability to penetrate the mucus barrier, providing an important prerequisite for astaxanthin to reach its target site and exert its efficacy.

[0044] Figure 4 To construct Caco using Transwell cells A single-cell intestinal barrier model was used to evaluate the transmembrane transport capacity of the astaxanthin recombinant lipid vesicle delivery system prepared in the examples. The results showed that, compared with free astaxanthin, the astaxanthin recombinant lipid vesicle delivery system could penetrate Caco more efficiently. The two-cell monolayer barrier exhibits superior intestinal absorption and transmembrane transport efficiency. This result indicates that recombinant astaxanthin lipid vesicles can overcome the limitations of the intestinal epithelial barrier by improving the intracellular and extracellular transport behavior of astaxanthin, enhancing cellular uptake, or regulating transcellular transport pathways, thereby significantly enhancing the absorption and transport potential of astaxanthin in the intestine.

[0045] Figure 5 To construct Caco in vitro using Transwell chambers 2. A co-culture model with HepG2 cells was established, and a lipid accumulation model was created by oleic acid induction to further evaluate the regulatory effect of the astaxanthin recombinant lipid vesicle delivery system prepared in the examples on cellular lipid metabolism. The results of lipid droplet fluorescence staining clearly showed that, compared with the control group, oleic acid induction significantly increased the intracellular fluorescence signal in HepG2 cells from 23.55±2.61 to 51.79±11.48, indicating significant lipid accumulation and fatty degeneration in the cells. After treatment with the astaxanthin recombinant lipid vesicles prepared in the examples, the fluorescence intensity of intracellular lipid droplets (38.84±6.59) was significantly reduced. Compared with the free astaxanthin group (49.13±9.36), the recombinant astaxanthin lipid vesicle delivery system showed a more significant lipid regulation effect, indicating that the recombinant astaxanthin lipid vesicles can enhance the regulatory ability of astaxanthin on lipid metabolism by optimizing cellular uptake and increasing local effective concentration, effectively alleviating oleic acid-induced lipid accumulation in hepatocytes. This provides a direct cellular basis for the system to improve lipid metabolism disorders and prevent and treat related metabolic diseases.

[0046] Figure 6 To construct Caco using Transwell cells 2. Co-culture model with HepG2 cells to alleviate Oil Red O staining of HepG2 cells induced by oleic acid. After oleic acid induction, a large number of red-stained lipid droplets appeared in HepG2 cells, indicating significant lipid accumulation. After treatment with the astaxanthin recombinant lipid vesicle delivery system prepared in the examples, the red-stained cell area was significantly reduced. Compared with the lipid droplet area ratio of 17.62±4.89% in the oleic acid group, it was significantly reduced to 4.90±2.10%, indicating a significant improvement in lipid accumulation. The lipid droplet area ratio in the free astaxanthin group was only reduced to 12.32±1.56%, indicating that the astaxanthin recombinant lipid vesicle delivery system prepared in the examples has a more prominent effect on alleviating the excessive formation of lipid droplets. This further confirms that the astaxanthin recombinant lipid vesicle delivery system prepared in the examples can effectively enhance the accumulation and efficacy of astaxanthin in cells. By regulating the cellular lipid metabolism process, it significantly alleviates oleic acid-induced hepatocyte steatosis, providing intuitive morphological evidence for the system's ability to improve lipid metabolism disorders.

[0047] Figure 7To establish an obesity and lipid metabolism disorder model, 6–7-week-old male C57BL / 6 mice were selected and fed a high-fat diet for 12 weeks. Simultaneously, the mice were treated with a recombinant astaxanthin lipid vesicle formulation prepared in the examples via gavage for 12 weeks. The in vivo effect on regulating lipid metabolism was systematically evaluated. Gross morphological observations showed that the high-fat model group mice were significantly larger with substantial fat accumulation; while after intervention with the recombinant astaxanthin lipid vesicle formulation prepared in the examples, the overall body size of the mice was significantly reduced, and the excessive fat accumulation was significantly improved. During weekly weight monitoring over 12 weeks, the body weight of the model group mice showed a continuous and significant upward trend, while the weight gain trend of the mice in the oral astaxanthin recombinant lipid vesicle intervention group was significantly slower, and the weight gain was significantly lower than that of the model group. These results, from multiple aspects including overall morphology, fat distribution, and body weight changes, collectively demonstrate that the recombinant astaxanthin lipid vesicles prepared in the examples can effectively alleviate abnormal fat accumulation.

[0048] Figure 8 To establish an obesity and lipid metabolism disorder model, 6–7-week-old male C57BL / 6 mice were selected and fed a high-fat diet for 12 weeks. Simultaneously, the mice were treated with a recombinant astaxanthin lipid vesicle formulation prepared in the previous example via gavage for 12 weeks. Anatomical observation of different adipose tissue sites in the mice revealed that the high-fat model group showed a significant increase in adipose tissue volume across all sites, exhibiting marked adipocyte hypertrophy and tissue proliferation. After intervention with the recombinant astaxanthin lipid vesicle delivery system prepared in the previous example, the size of adipose tissue in all sites of the mice was significantly reduced, and the excessive proliferation and hypertrophy of adipose tissue were significantly improved, with a greater than 20% increase in lipid metabolism capacity. These results further confirm from a histological perspective that the prepared recombinant astaxanthin lipid vesicle delivery system can effectively inhibit abnormal accumulation of fat in multiple sites induced by high-fat diets, reduce adipose tissue hypertrophy, and has a significant effect on improving lipid metabolism disorders and inhibiting excessive fat deposition, providing direct histological evidence for its lipid-lowering and weight-loss effects in vivo.

[0049] Figure 9 To further investigate the effects of hematoxylin on adipose tissue from different parts of mice Eosin (HE) staining of sections revealed pathological morphological results showing that adipocytes in the high-fat diet model group mice were significantly enlarged, with plump and unevenly sized cells, exhibiting typical hypertrophic changes and disordered adipose tissue structure, suggesting that a long-term high-fat diet induces abnormal proliferation and hypertrophy of adipocytes. After intervention with the astaxanthin recombinant lipid vesicle delivery system prepared in the examples, adipocyte volume was significantly reduced, cell size became more uniform, hypertrophy was significantly improved, and tissue morphology and structure tended to normalize. These pathological results directly confirm at the cellular level that the astaxanthin recombinant lipid vesicle delivery system prepared in the examples can effectively improve adipocyte hypertrophy induced by a high-fat diet and inhibit abnormal adipose tissue proliferation, further providing important histopathological evidence for its ability to improve lipid deposition and regulate lipid metabolism disorders in vivo.

[0050] Figure 10 To establish an obesity and lipid metabolism disorder model, 6–7-week-old male C57BL / 6 mice were selected and fed a high-fat diet for 12 weeks. Simultaneously, the astaxanthin recombinant lipid vesicle formulation prepared in the examples was administered via gavage for 12 weeks. The effects of the astaxanthin recombinant lipid vesicle delivery system prepared in the examples on intestinal homeostasis and intestinal barrier function were investigated. The results showed that, compared with the normal group, a long-term high-fat diet significantly shortened the colon length in mice, suggesting that high-fat status induces intestinal structural damage and chronic inflammatory response. However, after intervention with the astaxanthin recombinant lipid vesicle system prepared in the examples, the colon length in mice was effectively restored, and the effect was significantly better than that of the free astaxanthin group. This indicates that the astaxanthin recombinant lipid vesicle carrier prepared in the examples can more effectively improve high-fat-induced intestinal structural abnormalities.

[0051] Figure 11 To establish an obesity and lipid metabolism disorder model, 6-7 week old male C57BL / 6 mice were selected and fed a high-fat diet for 12 weeks. Simultaneously, the astaxanthin recombinant lipid vesicle preparation prepared in the example was administered via gavage for 12 weeks. HE staining was then performed on the mouse colon for observation. The results showed that the high-fat model group exhibited significant pathological changes in the colon tissue, including inflammatory cell infiltration, mucosal damage, and structural disorder. After intervention with the astaxanthin recombinant lipid vesicle prepared in the example, the pathological morphology of the intestinal tissue was significantly improved, inflammatory infiltration was significantly reduced, and the integrity of the intestinal structure was significantly restored. Histologically, this confirmed that the astaxanthin recombinant lipid vesicle system prepared in the example can effectively alleviate intestinal inflammatory damage induced by a high-fat diet.

[0052] Figure 12 To establish an obesity and lipid metabolism disorder model, 6–7-week-old male C57BL / 6 mice were selected and fed a high-fat diet for 12 weeks. Simultaneously, after 12 weeks of intervention with the astaxanthin recombinant lipid vesicle preparation prepared in the previous example via gavage, the tight junction protein fluorescence staining of the mouse colon was observed. The results showed that the high-fat model group ZO 1. The significantly decreased fluorescence intensity indicates disruption of the intestinal tight junction structure and increased barrier permeability; while the astaxanthin recombinant lipid vesicles prepared in the examples significantly upregulated Z0. The expression and fluorescence intensity of 1 effectively maintain the integrity of tight junctions in the intestinal epithelium and enhance the intestinal barrier function.

[0053] Comparative Example 1 The preparation of a solution of astaxanthin self-assembled lipid vesicle particles derived from Haematococcus pluvialis includes the following steps: S1. Dissolve Haematococcus pluvialis powder in deionized water to a concentration of 4 mg / mL, add cellulase to a concentration of 4 mg / mL, and pectinase to a concentration of 4 mg / mL, and heat in a water bath for 50 minutes. o C, stir for 60 min, after enzymatic hydrolysis, cool to room temperature, and centrifuge (4) o Collect the precipitate at C, 8000 rpm, for 10 min for later use; S2. Add 400 mg of the Haematococcus pluvialis enzymatic precipitate obtained in step S1 to 100 mL of deionized water for reconstitution, and then perform ultrasonic disruption at a power of 600 W for 30 min. After disruption, [the solution is then placed in a 4-channel system]. o Centrifuge at 8000 rpm for 10 min and collect the precipitate for later use; S3. Dissolve 200 mg of the precipitate collected in step S2 in 100 mL of deionized water. Add 400 mL of an extraction solvent (dichloromethane and methanol in a 1:2 volume ratio) and stir for 12 h under dark conditions. After extraction, [the solution is then removed from the heat]. o Centrifuge at 8000 rpm for 10 min and collect the dichloromethane layer solution for later use; S4. The dichloromethane layer solution extracted in step S3 is subjected to a process at 37°C. o C. The extractant was removed by rotary distillation under reduced pressure at 50 rpm to obtain a simultaneous extract of astaxanthin and algal lipids for later use. S5. Take 20 mg of the simultaneous extract of astaxanthin and algal lipids obtained in step S4, add 200 mL of deionized water for reconstitution, and then... o Centrifuge at 10000 rpm for 10 min to collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain an astaxanthin lipid complex aqueous solution.

[0054] The astaxanthin lipid extract obtained in Comparative Example 1 was subjected to rotary evaporation with dichloromethane, followed by reconstitution with an aqueous solution. The resulting aqueous solution, after centrifugation and membrane filtration, was observed under a transmission electron microscope (TEM). The results showed that the obtained astaxanthin lipid extract, without hydration self-assembly, yielded a TEM image... Figure 13 As shown, it is impossible to form a uniform nanoscale spherical structure; the structure presented is an irregular shape.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make some modifications or equivalent substitutions to the above-disclosed technical content without departing from the scope of the technical solution of the present invention, and all such modifications or substitutions shall fall within the protection scope of the present invention.

Claims

1. A method for preparing an astaxanthin self-assembly loading and transport system based on Haematococcus pluvialis, characterized in that, Includes the following steps: S1. Dissolve Haematococcus pluvialis in water and add cellulase and pectinase for enzymatic hydrolysis, then cool, centrifuge and collect the precipitate; S2. Redissolve the precipitate obtained in step S1, then sonicate it, and then centrifuge to collect the precipitate. S3. Redissolve the precipitate obtained in step S2, then add an extraction solvent composed of dichloromethane and methanol for extraction, centrifuge, collect the dichloromethane layer solution and dry it to obtain the extract; S4. Add the extract obtained in step S3 to water for hydration, then centrifuge to collect the supernatant, and filter the supernatant using a microfiltration membrane to obtain astaxanthin self-assembled lipid vesicle particles.

2. The preparation method according to claim 1, characterized in that, In step S1, the Haematococcus pluvialis includes fresh Haematococcus pluvialis or dried Haematococcus pluvialis powder; the Haematococcus pluvialis contains 1-8 wt% astaxanthin.

3. The preparation method according to claim 1, characterized in that, In step S1, the amount of cellulase added is 500~20000 U / g substrate, and the amount of pectinase added is 10000~200000 U / g substrate, and the substrate is Haematococcus pluvialis.

4. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis temperature in step S1 is 40~60°C. o C, time: 30-90 min; centrifugation conditions: 0-5 o C, 6000~8000 rpm, 5~15 min.

5. The preparation method according to claim 1, characterized in that, In step S2, the concentration of the precipitate after redissolution is 1–10 mg / mL; the power of the crushing process is 200–800 W, and the time is 20–60 min; the centrifugation conditions are 0–5 °C. o C, 6000~8000 rpm, 5~15 min.

6. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the precipitate after redissolved is 1-10 mg / mL; the volume ratio of the extraction solvent dichloromethane to methanol is 1:1.5-2.5; the volume of the extraction solvent is 2-5 times the volume of the solution after redissolved precipitate; the extraction conditions are: stirring at 20-30℃ for 12-24 h in the dark; and the centrifugation conditions are 0-5... o C, 6000~8000rpm, 5~15 min.

7. The preparation method according to claim 1, characterized in that, In step S4, the concentration of the extract during hydration is 0.1–2 mg / mL; the hydration conditions are 20–30 °C for 24–48 h; and the centrifugation conditions are 0–5 °C. o C, 8000~10000 rpm, 10~20 min; the microfiltration membrane is a filter membrane with a pore size of 0.22~0.45 μm.

8. A recombinant vesicle-like particle containing astaxanthin derived from Haematococcus pluvialis with gut-protecting and lipid-regulating properties, characterized in that... The Haematococcus pluvialis-derived astaxanthin recombinant vesicle particles are prepared according to the preparation method described in any one of claims 1 to 7.

9. The Haematococcus pluvialis-derived astaxanthin recombinant vesicle particles according to claim 8, characterized in that, The term "intestinal protection" refers to improving the balance of the intestinal microecology, repairing intestinal mucosal damage, or inhibiting intestinal inflammation.

10. The use of the Haematococcus pluvialis-derived astaxanthin recombinant vesicle particles with intestinal protection and lipid regulation as described in claim 8 in the preparation of food, health products or pharmaceuticals.

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