Chlorella exosome-loaded astaxanthin anti-inflammatory composition as well as preparation method and application thereof

Abstract

The invention discloses an anti-inflammatory composition with chlorella exosome loaded with astaxanthin as well as a preparation method and application of the anti-inflammatory composition, and relates to the technical field of natural product delivery. The anti-inflammatory composition comprises the exosome derived from chlorella and the astaxanthin entrapped in the exosome, so that the water solubility and the stability of the astaxanthin are remarkably improved, and the technical problem that the astaxanthin is easy to degrade in the storage and application processes is solved. Experiments prove that the exosome carrier can efficiently deliver the astaxanthin into target cells, and the uptake efficiency of the astaxanthin of the macrophages is remarkably improved. The preparation method of the anti-inflammatory composition is simple in process and mild in condition, the composition with the encapsulation efficiency not lower than 50% can be obtained by optimizing the feed ratio and the ultrasonic treatment condition, and the anti-inflammatory composition has a good large-scale production prospect. In conclusion, the composition provided by the invention can be widely applied to development of anti-inflammatory functional food, health care products, immunomodulatory preparations and intestinal inflammation intervention medicines, and has good industrial application value.

Description

Technical Field

[0001] This invention relates to the field of natural product delivery and functional food / drug development technology, and in particular to an anti-inflammatory composition of Chlorella exosomes loaded with astaxanthin, its preparation method and application. Background Technology

[0002] Astaxanthin is a fat-soluble ketocarotenoid whose molecular structure contains multiple conjugated double bonds and unsaturated ketone groups, endowing it with extremely strong antioxidant activity. Studies have shown that astaxanthin can significantly inhibit the release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by regulating macrophage polarization and inhibiting the activation of the nuclear factor-κB (NF-κB) signaling pathway, demonstrating broad application prospects in alleviating chronic inflammation and regulating immune responses. However, the highly unsaturated nature of the astaxanthin molecular structure also leads to its extremely poor water solubility and chemical instability. It is sensitive to light, heat, and acid-base conditions in the environment and is prone to degradation, resulting in low oral bioavailability, which severely limits its practical application in functional foods, health products, and pharmaceuticals.

[0003] To address the issue of low delivery efficiency of active ingredients, the use of nanocarriers for encapsulation delivery has become a research hotspot in recent years. Among them, exosomes, as natural nanoscale vesicles (typically 30-200 nm in diameter) secreted by cells, have advantages such as natural origin, stable membrane structure, low immunogenicity, and excellent biocompatibility. They can protect the contents from damage by the external environment and can efficiently deliver the contents to recipient cells through membrane fusion or endocytosis, making them an ideal carrier for the delivery of active ingredients.

[0004] Chlorella ( Chlorella Chlorella, belonging to the genus Chlorella in the phylum Chlorophyta, is a single-celled freshwater microalga characterized by its short growth cycle, rapid reproduction, ease of artificial cultivation, and large-scale production, making it an ideal raw material for preparing naturally derived exosomes. Previous studies have shown that exosomes derived from Chlorella can be used as carriers for the delivery of fucoxanthin and exhibit certain antioxidant activity. However, fucoxanthin and astaxanthin differ significantly in molecular structure, physicochemical properties, and pharmacological mechanisms of action.

[0005] Currently, there are no publicly available technologies for using Chlorella-derived exosomes to load astaxanthin for anti-inflammatory purposes. Improving the bioavailability of astaxanthin has significant application value and clinical significance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to develop a delivery system that can effectively improve the stability of astaxanthin, improve its solubility, and enhance its anti-inflammatory effect.

[0007] To address the above problems, the present invention proposes the following technical solution: In a first aspect, the present invention provides an anti-inflammatory composition of Chlorella exosomes loaded with astaxanthin, comprising exosomes derived from Chlorella and astaxanthin loaded in the exosomes.

[0008] Furthermore, the exosomes have a particle size of 100-200 nm and a zeta potential of -5 mV to -15 mV.

[0009] Furthermore, the encapsulation rate of the astaxanthin is not less than 50%.

[0010] Secondly, the present invention provides a method for preparing the anti-inflammatory composition, comprising the steps of: dissolving astaxanthin in an organic solvent to obtain an astaxanthin solution; mixing the astaxanthin solution with an exosome solution derived from Chlorella vulgaris, and subjecting the mixture to ultrasonic treatment under ice bath conditions to encapsulate the astaxanthin in the exosomes.

[0011] Furthermore, the mass ratio of astaxanthin to exosomes (based on protein content) is 1:5 to 1:20.

[0012] Furthermore, it also includes allowing the mixture to stand at room temperature for 1-3 hours after ultrasonic treatment.

[0013] Furthermore, the method also includes the steps of centrifuging the settled mixture to remove free astaxanthin and collecting astaxanthin-loaded exosomes.

[0014] Thirdly, the present invention also provides the use of the anti-inflammatory composition described herein or the anti-inflammatory composition prepared by the method in the preparation of products for treating or alleviating macrophage-mediated inflammation or intestinal inflammation.

[0015] Furthermore, the product is a drug, functional food, or health product.

[0016] Furthermore, the intestinal inflammation includes colitis.

[0017] Compared with the prior art, the technical effects achieved by the present invention include: This invention provides an anti-inflammatory composition of Chlorella exosomes loaded with astaxanthin, comprising Chlorella-derived exosomes and astaxanthin encapsulated within the exosomes. By encapsulating astaxanthin in Chlorella-derived exosomes, this invention significantly improves the water solubility and stability of astaxanthin, solving the technical problem of its easy degradation during storage and application. Cellular uptake experiments confirmed that the anti-inflammatory composition of this invention can be effectively internalized by macrophages, indicating that the exosome carrier can efficiently deliver astaxanthin to target cells, significantly improving the uptake efficiency of astaxanthin by macrophages. In a lipopolysaccharide-induced macrophage inflammation model, the anti-inflammatory composition of this invention effectively inhibits the expression of pro-inflammatory factors and promotes the production of anti-inflammatory factors, exhibiting significant anti-inflammatory activity. Furthermore, in a mouse colitis model, the anti-inflammatory composition of this invention effectively alleviates colitis symptoms and significantly reduces serum pro-inflammatory factor levels.

[0018] The method for preparing the anti-inflammatory composition provided by this invention is simple and mild. By optimizing the feed ratio and ultrasonic treatment conditions, a composition with an encapsulation rate of not less than 50% can be obtained, which has good prospects for large-scale production.

[0019] In summary, this invention solves the technical challenges of poor astaxanthin solubility, low stability, and low delivery efficiency by loading astaxanthin onto Chlorella exosomes, and verifies its synergistic anti-inflammatory effect at the cellular and animal levels. This composition has broad application potential in the development of anti-inflammatory functional foods, health products, immunomodulatory agents, and drugs for intervening intestinal inflammation, demonstrating significant industrial application value. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the Chlorella exosome extraction and astaxanthin loading process provided in Embodiment 1 of the present invention.

[0022] Figure 2 The figures show the physicochemical properties of Chlorella exosomes before and after loading astaxanthin, as provided in Example 4 of this invention. In the figures: A is the result of transmission electron microscopy observation of the morphology of exosomes before and after loading astaxanthin; B is the result of dynamic light scattering method to determine the particle size distribution of exosomes before and after loading astaxanthin; C is the result of Zeta potential measurement of exosomes before and after loading astaxanthin; and D is the result of astaxanthin encapsulation efficiency measurement.

[0023] Figure 3The figure shows the stability evaluation results of the Chlorella exosome-loaded astaxanthin composition provided in Example 3 of the present invention. In the figure: A is the result of the determination of the retention rate of free astaxanthin under different temperature and pH conditions; B is the result of the determination of the retention rate of astaxanthin after loading exosomes with astaxanthin under different temperature and pH conditions; C is the result of the comparison determination of the retention rates of free astaxanthin and astaxanthin loaded in exosomes under light conditions.

[0024] Figure 4 The results of laser confocal microscopy observation of macrophage uptake of the astaxanthin composition loaded on Chlorella exosomes provided in Example 5 of the present invention.

[0025] Figure 5 The figure shows the comparison results of the improvement of lipopolysaccharide-induced inflammatory factor release before and after loading astaxanthin into Chlorella exosomes provided in Example 6 of the present invention. In the figure: A is the result of RAW264.7 macrophage activity measurement in different treatment groups; B is the result of tumor necrosis factor-α level measurement in different treatment groups; C is the result of interleukin-6 level measurement in different treatment groups; D is the result of interleukin-10 level measurement in different treatment groups.

[0026] Figure 6 The figure shows the comparison results of the relief of sulfasalazine-induced colitis symptoms in mice before and after loading astaxanthin onto Chlorella exosomes provided in Example 7 of the present invention. In the figure: A is the result of colon length measurement in mice in different treatment groups; B is the result of weight change monitoring in mice in different treatment groups; C is the result of interleukin-6 level measurement in serum of mice in different treatment groups; D is the result of tumor necrosis factor-α level measurement in serum of mice in different treatment groups. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Similar component reference numerals in the drawings represent similar components. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0029] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0030] In this invention, room temperature refers to 20-38℃, preferably 25-37℃.

[0031] Example 1: Extraction of Chlorella exosomes See Figure 1 This is a schematic diagram of the Chlorella exosome extraction and astaxanthin loading process provided in Embodiment 1 of the present invention.

[0032] The Chlorella culture medium in its plateau phase was subjected to differential centrifugation at 4°C: first, centrifugation at 500 g for 5 min removed large cell debris, and the supernatant was collected; then, centrifugation at 2000 g for 30 min removed residual cells; the resulting supernatant was further centrifuged at 10000 g for 60 min to remove macromolecular impurities. Subsequently, the supernatant was filtered through a 0.22 μm filter membrane to further remove residual large particles. The filtrate was ultracentrifuged at 100000 g for 90 min at 4°C, the supernatant was discarded, and the precipitate was the Chlorella exosome (hereinafter referred to as C-Exo). The obtained exosome precipitate was resuspended in an appropriate amount of phosphate-buffered saline (PBS), and its protein concentration was detected using a BCA protein quantification kit. It was then stored at -80°C for later use.

[0033] The morphology of the obtained exosomes was observed using transmission electron microscopy (TEM), and the results showed that they had typical exosome membrane structures and intact morphology. The particle size distribution was determined by dynamic light scattering (DLS), and the results showed that the average particle size was about 152 nm and the polydispersity index (PDI) was less than 0.2, indicating that the exosomes obtained in this example had uniform particle size and high purity.

[0034] It is important to note that the particle size of exosomes is one of the key parameters for their use as nanocarriers. Particles that are too small (e.g., less than 50 nm) may be rapidly cleared from the body during circulation, and the drug-carrying space may be limited; particles that are too large (e.g., greater than 300 nm) may be recognized and captured by the reticuloendothelial system, making it difficult to reach the target tissue. Suitable exosome particle sizes are between 100 and 200 nm, such as 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, and 200 nm. The exosomes obtained in this embodiment have a particle size of approximately 152 nm, which is within the ideal range for nanocarrier delivery systems. This facilitates prolonged circulation time in vivo and passively targets inflammatory sites through enhanced permeation-retention effect (EPR effect).

[0035] Example 2: Encapsulation and parameter optimization of astaxanthin Astaxanthin was dissolved in dimethyl sulfoxide (DMSO) to prepare an astaxanthin solution of a certain concentration. The Chlorella exosome solution prepared in Example 1 was mixed with the astaxanthin solution at different ratios to investigate the effect of the mass ratio of astaxanthin to exosome protein (1:5, 1:10, and 1:20, respectively) on the encapsulation effect.

[0036] It should be noted that after mixing the astaxanthin solution with the exosome solution, sonication was performed under ice bath conditions to encapsulate the astaxanthin within the exosomes. The cavitation effect generated by sonication creates localized high pressure and microjets in the liquid, causing transiently reversible pores in the lipid bilayer membrane of the exosomes or increasing membrane permeability, thereby allowing astaxanthin molecules to enter the exosome interior or embed themselves in the membrane layer. The choice of ice bath conditions is crucial because: sonication may generate localized heat, and astaxanthin molecules contain multiple conjugated double bonds, making them heat-sensitive and prone to degradation; an ice bath effectively dissipates the heat generated by sonication, maintaining the system at a low temperature, preventing astaxanthin from being deactivated by thermal effects, and also helping to maintain the integrity of the exosome membrane structure.

[0037] The mixture was subjected to ultrasonic treatment under ice bath conditions. The ultrasonic power was set to 100 W, 200 W, and 300 W, and the ultrasonic time was set to 2 min, 3 min, and 5 min, respectively. The ultrasonic mode was intermittent (15 s operation followed by 15 s pause). The intermittent mode was also designed for temperature control to avoid a continuous rise in system temperature caused by continuous ultrasonication, thereby further protecting the thermal stability of astaxanthin.

[0038] In other embodiments, the mixture is further subjected to ultrasonic treatment followed by standing at room temperature for 1-3 hours. After ultrasonic treatment creates transient pores in the exosome membrane, sufficient time is required for the membrane structure to naturally repair itself and reform a complete lipid bilayer, thus encapsulating the astaxanthin molecules that have entered the exosomes. The choice of standing temperature and time affects the efficiency of membrane repair; 37°C is close to physiological temperature, which is beneficial for restoring the fluidity of membrane lipids; standing for 1-3 hours ensures sufficient repair of the membrane structure and avoids leakage of encapsulated astaxanthin due to incomplete closure of membrane pores. In this embodiment, after ultrasonic treatment, the mixture is placed in a 37°C constant temperature water bath for 2 hours to restore the stability of the exosome membrane.

[0039] Furthermore, the method includes centrifuging the settled mixture to remove free astaxanthin and collecting astaxanthin-loaded exosomes. In this embodiment, the settled mixture is centrifuged at 5000 g for 5 min to remove unloaded free astaxanthin, while the exosomes, due to their small particle size, remain in the supernatant, which is collected. The supernatant is then ultracentrifuged at 100000 g for 90 min, and the precipitate is collected to obtain astaxanthin-loaded Chlorella exosomes (hereinafter referred to as C-Exo-AST). The obtained C-Exo-AST precipitate is resuspended in an appropriate amount of PBS and stored at -80℃ for later use. A portion of the C-Exo-AST precipitate is resuspended in DMSO and sonicated at 200 W for 3 min (50 s working, 10 s pause) under ice bath conditions to release the loaded astaxanthin. The characteristic absorption peak at 478 nm is measured using a UV-Vis spectrophotometer, and the encapsulation efficiency and drug loading are calculated using the astaxanthin standard curve. The encapsulation efficiency is calculated as follows: (Amount of astaxanthin encapsulated in exosomes / Total amount of astaxanthin fed) × 100%; the drug loading is calculated as follows: (Amount of astaxanthin encapsulated in exosomes / Amount of exosome protein) × 100%.

[0040] The results showed that the encapsulation efficiency was approximately 41% when the feed ratio was 1:5; the highest encapsulation efficiency (approximately 56%) was achieved at a feed ratio of 1:10, with a drug loading of approximately 6.8%; and the encapsulation efficiency decreased to approximately 48% due to partial astaxanthin exudation caused by an excessive feed ratio of 1:20. The highest encapsulation efficiency and most uniform particle size distribution were achieved with an ultrasonic power of 200 W and an ultrasonic time of 3 min. Therefore, the optimal preparation conditions were determined to be: feed ratio of 1:10, ultrasonic power of 200 W, and ultrasonic time of 3 min (intermittent mode).

[0041] Example 3: Stability Analysis Experiment Free astaxanthin (AST) and C-Exo-AST prepared under the optimal conditions in Example 2 were treated under different conditions for 48 hours to investigate their stability. Temperature stability experiments were conducted at three temperatures: 4℃, 25℃, and 37℃; acid-base stability experiments were conducted at three pH conditions: pH 2.0, pH 5.0, and pH 7.4; and photostable stability experiments were conducted under both natural light and light-protected conditions. After treatment, the residual astaxanthin content in each group of samples was measured, and the retention rate was calculated.

[0042] Figure 3The figure shows the stability evaluation results of the Chlorella exosome-loaded astaxanthin composition provided in Example 3 of this invention. Figure A shows the retention rate of free astaxanthin under different temperatures and pH conditions; Figure B shows the retention rate of astaxanthin loaded onto exosomes under different temperatures and pH conditions; Figure C shows the comparison of the retention rates of free astaxanthin and exosome-loaded astaxanthin under light conditions. The results show that at 37℃ and pH 2.0, the degradation rate of free astaxanthin is greater than 50%, while the degradation rate of C-Exo-AST is reduced to below 35%. Under natural light conditions, the degradation rate of free astaxanthin is greater than 35%, while the degradation rate of C-Exo-AST is reduced to below 15%. These results indicate that Chlorella exosome loading can significantly improve the thermal stability, acid resistance, and photodegradation resistance of astaxanthin, solving the technical problem of easy degradation of astaxanthin during storage and application.

[0043] Example 4: Detection of Particle Size, Zeta Potential and Encapsulation Efficiency The C-Exo prepared in Example 1 and the C-Exo-AST prepared under optimal conditions in Example 2 were subjected to physicochemical characterization. Morphology was observed using transmission electron microscopy (TEM); average particle size and polydispersity index (PDI) were determined using dynamic light scattering (DLS); Zeta potential was measured using a Zeta potential analyzer; and the encapsulation efficiency and drug loading of C-Exo-AST were determined using the UV-Vis method described in Example 2.

[0044] Figure 2 The figures show the physicochemical properties of Chlorella exosomes before and after loading with astaxanthin. In the figure: A is the result of transmission electron microscopy observation of exosome morphology before and after loading with astaxanthin; B is the result of dynamic light scattering method to determine the particle size distribution of exosomes before and after loading with astaxanthin; C is the result of Zeta potential measurement of exosomes before and after loading with astaxanthin; D is the result of astaxanthin encapsulation efficiency measurement.

[0045] The results showed that both C-Exo and C-Exo-AST exhibited typical exosome membrane structures with intact morphology; the particle size distribution was uniform, with an average particle size of approximately 152 nm for C-Exo and an average particle size of 150–170 nm for C-Exo-AST, and a PDI of less than 0.2 for both. It is evident that the exosome particle size increased slightly after loading with astaxanthin, which is in line with expectations, indicating that astaxanthin was successfully encapsulated within the exosome or in the membrane layer, rather than simply adsorbed onto the surface.

[0046] Zeta potential is an important parameter reflecting the surface charge state of exosomes, directly affecting their dispersion stability and cell interactions. Exosome membrane surfaces typically carry a negative charge, mainly originating from the carboxyl and sulfate functional groups of phospholipids and glycoproteins. In this embodiment, the Zeta potential of C-Exo is approximately -12.8 mV, and that of C-Exo-AST is approximately -9.8 mV. This negative potential generates electrostatic repulsion, effectively preventing exosome aggregation and maintaining their dispersion stability in solution. Simultaneously, a moderate negative charge helps reduce non-specific binding to serum proteins, prolonging in vivo circulation time. The Zeta potential slightly increased after loading astaxanthin, possibly related to the partial embedding of astaxanthin molecules into the lipid bilayer altering the surface charge distribution, but it remained within the stable range, indicating good stability of the complex system.

[0047] Encapsulation efficiency and drug loading are key indicators for evaluating the drug loading efficiency of exosomes. In this embodiment, under optimal preparation conditions, the encapsulation efficiency of C-Exo-AST is not less than 50%, and can reach up to about 56%, with a drug loading of about 6.8%, indicating that the ultrasonic method can achieve efficient encapsulation of astaxanthin in Chlorella exosomes.

[0048] It is understood that the physicochemical parameters of exosomes, such as particle size and zeta potential, are affected by various factors, including the culture conditions of Chlorella, the extraction method of exosomes, and the astaxanthin loading process. Those skilled in the art can adjust process parameters such as centrifugation force, centrifugation time, ultrasonic power, ultrasonic time, and feed ratio to ensure that the particle size and zeta potential of the resulting composition fall within the range described in this invention. The specific values ​​given in the embodiments of this invention (such as a particle size of approximately 152 nm and a zeta potential of approximately -12.8 mV) are merely illustrative examples used to demonstrate the feasibility of the technical solution of this invention and do not constitute a limitation of this invention. Any anti-inflammatory composition of Chlorella exosomes loaded with astaxanthin with a particle size of 100-200 nm and a zeta potential of -5 mV to -15 mV that can be achieved through conventional process adjustments falls within the protection scope of this invention.

[0049] Example 5: Evaluation of cellular phagocytic capacity RAW264.7 mouse macrophages were used as model cells and cultured in DMEM medium containing 10% fetal bovine serum. The experiment included a blank control group, a lipopolysaccharide (LPS) model group, a free astaxanthin group, a simple exosome group, and the composition group of this invention. The free astaxanthin group received 10 μM free astaxanthin, the simple exosome group received C-Exo at a concentration equal to the protein concentration of C-Exo-AST, and the composition group received an equal amount of astaxanthin (10 μM) of C-Exo-AST. Except for the blank control group, all other groups were treated with 1 μg / mL LPS for 24 h to induce an inflammatory response. The CCK-8 assay was used to detect the effect of different treatments on cell viability; laser confocal microscopy was used to observe the uptake of C-Exo-AST by RAW264.7 cells.

[0050] Figure 4 The results of laser confocal microscopy observation of macrophage uptake of the astaxanthin composition loaded on Chlorella exosomes provided in Example 5 of this invention are shown. The results indicate that within the 0-10 μM dose range, each treatment group had no significant effect on the viability of RAW264.7 cells, indicating no cytotoxicity within this dose range. Laser confocal microscopy images show that RAW264.7 cells can effectively uptake the fluorescently labeled C-Exo-AST complex, indicating that Chlorella exosomes can efficiently deliver astaxanthin into macrophages, solving the problem that free astaxanthin is difficult for cells to uptake due to its strong hydrophobicity.

[0051] Example 6: Anti-inflammatory activity evaluation experiment Following the grouping and treatment methods described in Example 5, cell culture supernatant was collected 24 h after cell intervention. The levels of pro-inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory factor interleukin-10 (IL-10) in the supernatant were detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0052] Figure 5This document presents a comparative study of the effects of loading astaxanthin onto Chlorella exosomes before and after treatment to improve lipopolysaccharide-induced inflammatory cytokine release, as provided in Example 6 of this invention. The results showed that compared to the LPS model group, the TNF-α level decreased by approximately 15% in the free astaxanthin group and by approximately 10% in the exosome-only group (no statistical significance, P>0.05), while the TNF-α level in the composition group of this invention decreased by approximately 20%, significantly better than the free astaxanthin group (P<0.01). The detection results for IL-6 and IL-10 showed similar trends: the level of pro-inflammatory cytokine IL-6 in the composition of this invention was significantly lower than in other treatment groups, while the level of anti-inflammatory cytokine IL-10 was significantly higher than in other treatment groups. These results indicate that exosomes alone have no significant anti-inflammatory effect, while the composition of this invention has a synergistic anti-inflammatory effect, and its anti-inflammatory effect is significantly better than that of free astaxanthin.

[0053] Example 7: Evaluation Experiment on Relief of Colitis in Mice Six- to eight-week-old male C57BL / 6J mice were randomly divided into five groups: a blank control group, a DSS model group, a free astaxanthin group, an exosome-only group, and the composition group of this invention, with ten mice in each group. For the first 14 days, the blank control group and the DSS model group were administered an equal volume of PBS by gavage, the free astaxanthin group was administered 1 mg / kg of free astaxanthin by gavage, the exosome-only group was administered C-Exo at a protein concentration equivalent to C-Exo-AST by gavage, and the composition group of this invention was administered an equal volume of astaxanthin (1 mg / kg) of C-Exo-AST by gavage. From day 15 onwards, except for the blank control group, 3% DSS was added to the drinking water of the other groups for 7 consecutive days to induce a colitis model. Mouse weight changes were monitored daily during the experiment. After the experiment, mice were sacrificed, colon tissue was collected to measure length, and serum was collected for ELISA testing of TNF-α and IL-6 levels.

[0054] Figure 6 This is a comparison of the effects of loading astaxanthin onto Chlorella exosomes before and after the onset of symptoms of dextran sulfate-induced colitis in mice, as provided in Example 7 of this invention.

[0055] The results showed that, compared with the blank control group, the DSS model group mice had significantly shorter colon length and significantly lower body weight (more than 20% reduction). Compared with the DSS model group, the mice in the composition group of the present invention had significantly increased colon length, significantly alleviated body weight loss, and significantly reduced serum levels of pro-inflammatory factors TNF-α and IL-6, with better improvement effects than the free astaxanthin group and the exosome group alone. These results indicate that the composition of the present invention can effectively alleviate intestinal inflammation in vivo and has good application prospects.

[0056] This invention fully demonstrates the preparation method, physicochemical characterization, and functional verification of an anti-inflammatory composition of Chlorella exosomes loaded with astaxanthin. The astaxanthin encapsulation process was systematically optimized, clarifying that ultrasonic treatment is the key step for achieving efficient encapsulation. Process parameters such as the feed mass ratio, ultrasonic power, and ultrasonic time were determined. Under these conditions, the encapsulation efficiency reached 56%, and the drug loading was approximately 6.8%. Stability studies confirmed that encapsulating astaxanthin in Chlorella exosomes significantly improves its tolerance to heat, acids, alkalis, and light, effectively solving the technical challenge of easy degradation of free astaxanthin and laying the foundation for its storage and application in complex environments.

[0057] Characterization of the physicochemical properties of the obtained composition showed that the exosomes loaded with astaxanthin maintained an intact membrane structure, with a slightly increased particle size still within a suitable range. The zeta potential remained in the negative range, and the polydispersity index was less than 0.2, demonstrating that the composition possesses good dispersion stability and structural integrity. The suitable range of particle size and potential is crucial for prolonging in vivo circulation time, reducing non-specific adsorption, and promoting cellular uptake. Cell experiments confirmed that the exosome carrier successfully delivered astaxanthin into target cells, overcoming the limitation of free astaxanthin being difficult for cells to take up due to its strong hydrophobicity, and improving delivery efficiency.

[0058] In a macrophage inflammation model, the composition of this invention significantly inhibited the expression of pro-inflammatory factors while promoting the production of anti-inflammatory factors, with effects significantly superior to free astaxanthin and exosomes alone. Exosomes alone did not show significant anti-inflammatory effects, confirming a synergistic relationship between the exosome carrier and astaxanthin, rather than a simple additive effect. The in vivo anti-inflammatory effect of the composition of this invention was further verified in a dextran sulfate-induced mouse colitis model. Results showed that the composition of this invention effectively alleviated colitis symptoms, manifested as increased colon length, improved weight loss, and a significant reduction in serum pro-inflammatory factor levels, validating its practical application potential in intestinal inflammation intervention.

[0059] In summary, the anti-inflammatory composition of Chlorella exosomes loaded with astaxanthin provided by this invention systematically solves the technical problems of poor solubility, low stability, and insufficient delivery efficiency of astaxanthin through the encapsulation effect of exosome carriers, and its synergistic anti-inflammatory effect has been verified at the cellular and animal levels. This composition has a simple preparation process, mild conditions, and high encapsulation efficiency, showing good prospects for large-scale production. It can be widely used in the development of anti-inflammatory functional foods, health products, immunomodulatory agents, and drugs for intervening intestinal inflammation, and has significant industrial application value.

[0060] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0061] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An anti-inflammatory composition of Chlorella exosomes loaded with astaxanthin, characterized in that, It contains exosomes derived from Chlorella and astaxanthin loaded in the exosomes.

2. The anti-inflammatory composition according to claim 1, characterized in that, The exosomes have a particle size of 100-200 nm and a zeta potential of -5 mV to -15 mV.

3. The anti-inflammatory composition according to claim 1, characterized in that, The encapsulation rate of the astaxanthin is not less than 50%.

4. A method for preparing the anti-inflammatory composition according to any one of claims 1-3, characterized in that, The steps include: dissolving astaxanthin in an organic solvent to obtain an astaxanthin solution; mixing the astaxanthin solution with an exosome solution derived from Chlorella vulgaris, and then subjecting the mixture to ultrasonic treatment under ice bath conditions to encapsulate the astaxanthin in the exosomes.

5. The method as described in claim 4, characterized in that, The mass ratio of astaxanthin to exosomes (based on protein content) is 1:5 to 1:

20.

6. The method as described in claim 4, characterized in that, It also includes ultrasonic treatment followed by standing the mixture at room temperature for 1-3 hours.

7. The method as described in claim 6, characterized in that, It also includes the steps of centrifuging the settled mixture to remove free astaxanthin and collecting astaxanthin-loaded exosomes.

8. The use of the anti-inflammatory composition of any one of claims 1 to 3 or the anti-inflammatory composition prepared by the method of claims 4 to 7 in the preparation of products for treating or alleviating macrophage-mediated inflammation or intestinal inflammation.

9. The application as described in claim 8, characterized in that, The product is a drug, functional food, or health product.

10. The application as described in claim 9, characterized in that, The intestinal inflammation includes colitis.

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