Preparation method and application of astaxanthin-loaded microalgae with antioxidant and intestinal delivery properties

Abstract

This invention discloses a method for preparing and applying an antioxidant and intestinal-delivered astaxanthin-loaded microalgae. First, hyaluronic acid-modified astaxanthin liposomes with controlled intestinal release are synthesized. Then, the carboxyl groups on the astaxanthin liposomes are activated using EDC / NHS reagents to couple with the amino groups on Chlorella. After oral administration, this drug-loaded microalgae can effectively remain in the intestinal tract and gradually degrade, slowly releasing astaxanthin. This significantly increases the astaxanthin concentration in the intestine, maximizing astaxanthin absorption and utilization while minimizing astaxanthin loss through the digestive tract. The astaxanthin released by this drug-loaded microalgae after simulated gastrointestinal digestion exhibits strong free radical scavenging and antioxidant capabilities in its polar ketone structure, showing broad application prospects in health and nutrition aspects such as anti-inflammation, photoprotection, immune enhancement, and anti-aging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying an antioxidant and intestinal-delivered astaxanthin-loaded microalgae. Background Technology

[0002] Astaxanthin is a non-vitamin A ketone carotenoid and a natural antioxidant with high economic and application value. Haematococcus pluvialis is recognized as one of the best natural sources of astaxanthin and has become an important method for producing natural astaxanthin internationally. Haematococcus pluvialis not only has an astaxanthin content as high as 1.5%-3.0% of cell dry weight, serving as a "concentrated" natural astaxanthin, but its astaxanthin composition is mainly esterified (approximately 70% monoesters, 25% diesters, and 5% monomers), which is beneficial for liposome synthesis—an advantage not found in astaxanthin extracted through chemical synthesis or using yeasts like Pharfogel's red yeast. Although astaxanthin extracted from Haematococcus pluvialis has strong free radical scavenging and antioxidant capabilities, showing broad application prospects in anti-inflammatory, photoprotective, immune-enhancing, and anti-aging health and nutrition aspects, its poor water solubility and stability result in relatively low bioavailability, significantly limiting its application.

[0003] Liposomes, as classic nanocarriers, possess excellent biocompatibility and drug encapsulation capabilities. Utilizing the high esterification state of astaxanthin extracted from Haematococcus pluvialis as a raw material, the preparation of astaxanthin liposomes can significantly improve the stability and dispersibility of astaxanthin. Simultaneously, the introduction of hyaluronic acid, which has high oral safety, into this liposome system not only protects the phospholipid molecular structure of the liposomes, preventing the generation of peroxides and hydroxyl radicals and effectively inhibiting lipid peroxidation, but also, thanks to the swelling properties of hyaluronic acid in a near-neutral pH environment, endows the astaxanthin liposomes with controllable release properties in neutral intestinal digestive fluids. Furthermore, Chlorella, as a natural single-celled green algae, exhibits unique advantages in simulated gastrointestinal digestion: its cell wall is rich in polysaccharides and active groups, which can resist gastric acid erosion, inhibit digestive enzyme degradation, and protect the structural integrity of liposomes; at the same time, its natural nanoscale size can promote intestinal cell uptake, improve drug absorption efficiency, and synergistically enhance the carrier's ability to protect and sustain drug release.

[0004] Oral liposome-based gastrointestinal digestion simulation experiments are a key method for evaluating the in vitro behavior of this composite drug delivery system, accurately simulating the gastrointestinal physiological environment. Therefore, conducting research on a semi-artificial microalgae controlled-release drug delivery platform covalently linked to Chlorella and liposomes, and its application in the intestinal delivery and controlled release of astaxanthin, will clarify the delivery characteristics of antioxidant active substances by Chlorella in simulated gastrointestinal digestion. This research has significant theoretical and experimental implications for optimizing formulation processes and improving the bioavailability of oral drugs, laying a solid foundation for potential future clinical translations. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying astaxanthin-loaded microalgae with antioxidant and intestinal delivery capabilities. The astaxanthin liposomes loaded on the surface of *Chlorella vulgaris* prepared by this method can, after oral administration, protect most of the active ingredients as they enter the intestines and gradually lyse in intestinal digestive fluids, thereby slowly releasing highly antioxidant astaxanthin. This significantly increases the astaxanthin concentration in the intestines, maximizes astaxanthin absorption and utilization, minimizes the loss of astaxanthin's antioxidant capacity, and enhances the antioxidant therapeutic effect. This provides a new approach and method for constructing multifunctional oral drug delivery systems.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: (1) Weigh 0.005-0.05 g of paste-like astaxanthin, 0.05 g of hyaluronic acid and 0.05 g of soybean lecithin and transfer them to a distillation flask. Then add 10 mL of chloroform and 2 mL of methanol and mix thoroughly in an ultrasonic cleaner to dissolve the astaxanthin. (2) Connect the distillation flask from step (1) to a rotary evaporator for rotary evaporation; (3) After a uniform orange-red film forms on the wall of the distillation flask in step (2), stop rotary evaporation, remove the distillation flask from the rotary evaporator, add 10 mL of ultrapure water and hydrate for 25-35 min. (4) Transfer the hydrated astaxanthin liposome solution from step (3) into a centrifuge tube, add 20 mL of 0.03 mol / L PBS buffer solution with pH=7, and mix well; (5) The astaxanthin liposomes from step (4) were ultrasonically broken down using an ultrasonic disruptor to form astaxanthin liposomes with smaller particle size. (6) Dissolve 0.04g of N-hydroxysuccinimide (NHS) in 2 mL of dimethyl sulfoxide and add 1 mL of astaxanthin liposomes from step (6). Stir on a magnetic stirrer for 0.5-2 h. Then add 0.08g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and stir magnetically for 20-24 h. (7) Centrifuge at 4000 rpm for 10 minutes to collect the logarithmic-stage Chlorella cultured in the laboratory and obtain wet algal pellet. Resuspend the wet algal pellet with 0.9% NaCl aqueous solution and obtain a suspension containing about 1 g of wet algal pellet per milliliter. Add 1 mL of Chlorella suspension to the solution in step (6) and shake at a constant speed for 30 min to allow the activated carboxyl groups on the astaxanthin liposomes to couple with the amino groups on the Chlorella, so that the astaxanthin liposomes can be successfully attached to the Chlorella.

[0007] The specific steps of step (2) are as follows: First, turn on the low-temperature cooling liquid circulation pump to cool the liquid. Once the required temperature is reached, turn on the water pump to circulate the water. Then, connect the distillation flask from step (1) to the rotary evaporator, secure it with clamps, and close the vent valve. Turn on the vacuum pump and set the value to 285 Pa. Set the water bath temperature to 40 ℃. When the water bath reaches the preset temperature, adjust the height of the distillation flask so that the flask containing the reagent is immersed in the water. Set the rotation speed to 60 rpm for rotary evaporation.

[0008] The centrifuge tubes used in step (4) are not limited in size. The appropriate size of the centrifuge tubes is selected according to the actual amount of medicine to be processed, such as the commonly used 50 mL or 100 mL.

[0009] The specific steps of step (5) can be referred to as follows: place the centrifuge tube containing the astaxanthin liposome solution into a large beaker containing crushed ice, place the probe of the ultrasonic disruptor below the liquid surface of the centrifuge tube, and sonicate the astaxanthin liposomes in a 200 W ultrasonic disruptor (4℃, 40% power, four seconds on and eight seconds off) to make the particle size of the astaxanthin liposomes more uniform.

[0010] Preferably, the paste-like astaxanthin in step (1) is extracted from Haematococcus pluvialis, and the molecular weight of the hyaluronic acid is 80 kDa-2000 kDa.

[0011] Preferably, the ultrasonic disruption time in step (5) is 10-20 min, more preferably 15 min, and the particle size of the astaxanthin liposomes after ultrasonic disruption is 110-120 nm, with an average particle size of about 115 nm.

[0012] Preferably, in step (6), the pH of the reaction solution is adjusted to 6.0-7.0.

[0013] Preferably, in step (7), the pH of the reaction solution is adjusted to 7.0-7.5.

[0014] The astaxanthin-loaded microalgae prepared according to the above preparation method also fall within the protection scope of this invention.

[0015] Furthermore, experimental verification shows that the astaxanthin-loaded microalgae prepared in this invention exhibit the highest astaxanthin release rate in the intestines after simulated gastrointestinal digestion, significantly improving the absorption and utilization rate of active ingredients and reducing their loss. The astaxanthin absorbed and utilized in the intestines also demonstrates strong antioxidant activity and a high free radical scavenging rate. This invention uses Chlorella as a carrier to load hyaluronic acid-modified astaxanthin liposomes, fully integrating the advantages of carrier material biocompatibility and active ingredient efficacy. It can be widely applied in bio-health fields such as anti-inflammation, immune regulation, and anti-aging. This composite system possesses excellent biological stability and targeted delivery characteristics, effectively improving the bioavailability of astaxanthin and addressing issues such as insufficient stability of active ingredients and low in vivo delivery efficiency. Leveraging the natural carrier advantages of Chlorella, a safe and efficient oral delivery system can be constructed, suitable for the conditioning and intervention of inflammation, metabolic disorders, and aging-related health problems. In practical applications, the application plan can be flexibly optimized by combining factors such as lesion distribution characteristics, body health status, and individual differences. Relying on the existing oral delivery technology system, the appropriate dosage and concentration can be easily determined. The technology is highly practical and has good prospects for industrial application and practical promotion value.

[0016] Advantages of this invention: 1. The astaxanthin-loaded microalgae of the present invention have high drug loading efficiency, high drug loading capacity, and high biocompatibility, which greatly improves the utilization efficiency and biocompatibility of the drug delivery system in the body. It can be used as a functional oral drug delivery system for disease treatment, including anti-cancer, anti-inflammatory, immune and antioxidant effects of astaxanthin liposomes.

[0017] 2. Using Chlorella as a carrier for oral drugs has great potential for commercialization and clinical translation. In this invention, EDC / NHS reagents are used to activate the carboxyl groups on astaxanthin liposomes, causing a coupling reaction with the amino groups on Chlorella, successfully linking the astaxanthin liposomes to Chlorella. This overcomes the limitation of microalgae relying solely on electrostatic adsorption and provides a new method for constructing microalgal drug delivery systems. Furthermore, the raw materials are readily available, environmentally friendly, and safe, and the preparation method is simple.

[0018] 3. In terms of oral therapeutic applications, the Chlorella loaded with astaxanthin liposomes in this invention can promote intestinal cell uptake, improve astaxanthin absorption efficiency, and also work synergistically with liposomes to enhance the carrier's protection and sustained-release ability for astaxanthin.

[0019] 4. The astaxanthin liposomes loaded by the drug-loaded microalgae are nanoscale in size. After oral administration, they are gradually degraded in the intestine and slowly release astaxanthin, which significantly increases the concentration of active substances in the intestine, maximizes the absorption and utilization rate of active substances, and reduces the loss of antioxidant activity. Attached Figure Description

[0020] Figure 1Flowchart of the preparation method of astaxanthin-loaded microalgae for antioxidant and intestinal delivery; Figure 2 Encapsulation efficiency of hyaluronic acid-modified astaxanthin liposomes at drug-liposome ratios of 1 / 1 and 1 / 10 (astaxanthin / soy lecithin); Figure 3 UV absorption spectra of astaxanthin and hyaluronic acid-modified astaxanthin liposomes; Figure 4 Hyaluronic acid-modified astaxanthin liposome potential diagram; Figure 5 Transmission electron microscopy image of hyaluronic acid-modified astaxanthin liposomes; Figure 6 The particle size distribution of hyaluronic acid-modified astaxanthin liposomes; Figure 7 Scanning electron micrograph of Chlorella vulgaris without astaxanthin liposomes; Figure 8 Scanning electron microscope image of Chlorella vulgaris loaded with astaxanthin liposomes; Figure 9 A comparison of the total reducing power of astaxanthin liposomes and vitamin C; Figure 10 A comparison of the DPPH clearance rates of astaxanthin liposomes and vitamin C. Figure 11 This is a graph showing the release rate of astaxanthin after simulated gastrointestinal digestion. Figure 12 This is a graph showing the total reducing power of astaxanthin released after simulated gastrointestinal digestion. Figure 13 This is a graph showing the DPPH clearance rate of astaxanthin released after simulated gastrointestinal digestion. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0022] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0024] Example 1: A method for preparing Chlorella vulgaris loaded with astaxanthin liposomes, comprising the following steps: (1) Weigh 0.005-0.05 g of paste-like astaxanthin extracted from Haematococcus pluvialis, 0.05 g of hyaluronic acid (80kDa-2000kDa) and 0.05 g of soybean lecithin and transfer them to a distillation flask. Then add 10 mL of chloroform and 2 mL of methanol and thoroughly mix and dissolve the astaxanthin in an ultrasonic cleaner.

[0025] (2) Turn on the low-temperature coolant circulation pump in advance for cooling. After reaching the required temperature, turn on the water pump to circulate water. Connect the distillation flask from step (1) to the rotary evaporator, secure it with clamps, and close the vent valve. Turn on the vacuum pump and set the value to 285 Pa. Set the water bath temperature to 40 ℃. When the water bath reaches the preset temperature, adjust the height of the distillation flask so that the distillation flask containing the medicine is immersed in the water. Set the rotation speed to 60 rpm and perform rotary evaporation.

[0026] (3) After a uniform orange-red film forms on the wall of the distillation flask in step (2), stop the rotary evaporation, and turn off the water bath, vacuum pump, and low-temperature cooling liquid circulation pump. After opening the vent valve, remove the distillation flask from the rotary evaporator and add 10 mL of ultrapure water to hydrate for 25-35 min.

[0027] (4) Transfer the hydrated astaxanthin liposome solution from step (3) into a 50 mL centrifuge tube. After rinsing with 20 mL of 0.03 mol / L PBS buffer solution (pH 7) in a distillation flask, transfer the solution into a 50 mL centrifuge tube and mix thoroughly.

[0028] (5) Place the 50 mL centrifuge tube containing the astaxanthin liposome solution in step (4) into a large beaker containing crushed ice, place the probe of the ultrasonic disruptor below the liquid surface of the centrifuge tube, and ultrasonically disrupt the astaxanthin liposomes for 10-20 min in a 200 W ultrasonic disruptor (4℃, 40% power, four seconds on and eight seconds off) to make the particle size of the astaxanthin liposomes uniform.

[0029] (6) Dissolve 0.04 g of N-hydroxysuccinimide (NHS) in 2 mL of dimethyl sulfoxide and add 1 mL of astaxanthin liposomes from step (5). Stir on a magnetic stirrer for 0.5-2 h. Then add 0.08 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and stir magnetically for 20-24 h. Activate the carboxyl groups on the liposomes at pH 6.0-7.0.

[0030] (7) At pH 7.0-7.5, add 1 mL of Chlorella solution and shake at a constant speed for 30 min to allow the activated carboxyl group on the liposome in step (6) to undergo a coupling reaction with the amino group on Chlorella, and the astaxanthin liposome is successfully linked to Chlorella.

[0031] according to Figure 1 The process of constructing a semi-artificial microalgae controlled-release drug delivery platform covalently linked to Chlorella and astaxanthin liposomes was implemented. A control experiment was conducted to investigate the effect of different drug-liposome ratios (astaxanthin / soy lecithin) on the encapsulation efficiency. Figure 2 As shown, when the drug-to-lipoprotein ratio was 1:10, the encapsulation efficiency reached 86.66%, significantly higher than the 8.62% under the 1:1 condition. The results indicate that a drug-to-lipoprotein ratio (astaxanthin / soybean lecithin) of 1:10 is the optimal synthesis method for hyaluronic acid-modified astaxanthin liposomes. The optimal reaction conditions are pH 7.0-7.5, which allows the activated carboxyl groups on the hyaluronic acid-modified astaxanthin liposomes to couple with the amino groups on Chlorella vulgaris. The UV-Vis absorption spectrum shows an absorption peak at 475 nm, consistent with the typical maximum absorption peak of astaxanthin. Figure 3 The zeta potential measurement result was -41.05 mV. Figure 4 This indicates that the particle surface carries a strong negative charge, which helps enhance the electrostatic repulsion of the colloidal system, prevents particle aggregation, and ensures the physical stability of hyaluronic acid-modified astaxanthin liposomes during storage. Figure 5 As shown, transmission electron microscopy (TEM) further confirmed that the hyaluronic acid-modified astaxanthin liposomes exhibited irregular ellipsoidal or rod-shaped structures with relatively uniform distribution, and no obvious fusion or precipitation was observed, indicating that the preparation process was controllable and had good reproducibility. Figure 6 As shown, dynamic light scattering (DLS) analysis revealed that the hyaluronic acid-modified astaxanthin liposomes had an average particle size of approximately 115 nm. This size range is favorable for the penetration and retention of nanoparticles in the intestinal mucosa. Scanning electron microscopy confirmed that... Figure 7 Comparison of scanning electron micrographs of Chlorella vulgaris without astaxanthin liposomes. Figure 8 Astaxanthin liposomes are effectively but unevenly loaded onto the surface of Chlorella cells.

[0032] The experiment also determined the DPPH scavenging rate and total reducing power of astaxanthin and vitamin C. The specific steps are as follows: Total reducing power determination. In a 1 mL centrifuge tube, add 50 µL of 0.2 mol / L, pH 6.6 phosphate buffer, 50 µL of sample solution, and 50 µL of 1% potassium ferricyanide solution, respectively. Mix well and incubate in a 50℃ water bath for 20 min. Then, add 50 µL of 10% trichloroacetic acid to terminate the reaction, shake well, and centrifuge (5000 rpm, 10 min). Take 150 µL of the supernatant, add 150 µL of distilled water and 20 µL of 0.1% FeCl3 solution, mix well, and incubate in the dark for 10 min. Measure the absorbance at 700 nm. Vc is used as a positive control.

[0033] Determination of DPPH scavenging rate. A certain amount of DPPH was weighed and prepared into a 0.04 g / L solution with anhydrous ethanol. 150 µL of the sample solution was taken and 150 µL of DPPH solution was added. After mixing, the solution was allowed to stand at room temperature in the dark for 30 min, followed by centrifugation at 5000 rpm for 10 min. The absorbance of the supernatant at 517 nm was measured. Vitamin C was used as a positive control. The DPPH free radical scavenging rate of the sample was calculated using the following formula: DPPH removal rate = 1 - (A1 - A2) / A0 × 100% A0—Absorbance of 150 µL anhydrous ethanol + 150 µL LPPH solution; A1—Absorbance of 150 µL sample solution + 150 µL LPPH solution; A2—Absorbance of 150 µL sample solution + 150 µL anhydrous ethanol.

[0034] The comparison of the total reducing power of astaxanthin and vitamin C shows that the total reducing power of 0.04 g / L astaxanthin liposomes is 0.6982, while the total reducing power of 4 g / L vitamin C (as a positive control) is 2.29. Figure 9 The comparison chart of DPPH clearance rates between astaxanthin liposomes and vitamin C shows that 0.04 g / L astaxanthin liposomes had a DPPH clearance rate of 95%, while the positive control, 4 g / L vitamin C, had a DPPH clearance rate of 96%. Figure 10 This shows that astaxanthin has strong reducing power and the ability to scavenge free radicals.

[0035] The astaxanthin release rate and antioxidant capacity after simulated gastrointestinal digestion were also measured. The specific steps are as follows: Simulated gastric digestion: Weigh 5 g pepsin and 1 g NaCl, add them to a mixed solution containing 450 mL deionized water and 3.50 mL concentrated hydrochloric acid, dissolve thoroughly, adjust the pH to 2, and bring the volume to 500 mL. Mix 6 mL of simulated gastric digestion fluid with 6 mL of astaxanthin liposome sample solution in a centrifuge tube, adjust the pH to 2-3, and simulate gastric digestion for 2 h in a 37℃ constant temperature water bath shaker in the dark. At 0.5, 1, 1.5, and 2 h of simulated gastric digestion, transfer a certain volume of the suspension to a centrifuge tube and place it on ice to terminate gastric digestion. Centrifuge at 12,000 rpm, 4℃ for 15 min, and collect the supernatant. Determine the astaxanthin content, total reducing power, and DPPH free radical scavenging rate released after simulated gastric digestion.

[0036] Simulated intestinal fluid: Weigh 3.40 g KH2PO4 and dissolve it in 350 mL deionized water. Add 38.50 mL of 0.20 mol / L NaOH solution and mix thoroughly. Then add 5 g trypsin and 30 g bile salts, and after thorough dissolution, centrifuge at 5000 rpm for 10 min. Take the supernatant, adjust the pH to 7.6, and bring the volume to 500 mL for later use. Mix the remaining sample suspension after simulated gastric digestion with an equal volume of simulated digested intestinal fluid. Incubate at 37℃ in a constant temperature water bath in the dark for 6 h to simulate intestinal digestion. After 1, 2, 3, 4, 5, and 6 h of simulated intestinal digestion, transfer a certain volume of the solution to centrifuge tubes and place them on ice to terminate intestinal digestion. Centrifuge at 12,000 rpm at 4℃ for 15 min and collect the supernatant. Measure the astaxanthin content, total reducing power, and DPPH free radical scavenging rate released after simulated intestinal digestion.

[0037] Observe the astaxanthin release rate after simulated gastrointestinal digestion. Figure 11 It can be seen that during the 0-2 hour simulated gastric digestion stage, the astaxanthin release rate is below 5%. From the 3rd hour of the simulated intestinal digestion stage, the astaxanthin release rate increases to 68%, and steadily increases with the duration of the simulated intestinal digestion stage. Therefore, astaxanthin liposomes loaded on Chlorella have the highest release rate in the intestine, which can significantly improve drug absorption and utilization and reduce drug loss. The total reducing power of astaxanthin released after simulated gastrointestinal digestion is also discussed. Figure 12 and the DPPH clearance rate of astaxanthin released after simulated gastrointestinal digestion Figure 13It was later determined that during the first 0-2 hours of simulated gastric digestion, astaxanthin release was low, with total reducing power below 0.2 and DPPH clearance below 10%. Starting from the 3rd hour of simulated intestinal digestion, astaxanthin release gradually increased with the duration of this stage. After 6 hours of intestinal digestion, total reducing power increased to approximately 1.2, and DPPH clearance exceeded 80%. This indicates that astaxanthin digested and utilized in the intestines possesses strong antioxidant activity and free radical scavenging capabilities, thus it can be applied to biological health aspects such as cancer prevention, anti-inflammation, immunity enhancement, and anti-aging.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A method for preparing astaxanthin-loaded microalgae with antioxidant and intestinal delivery properties, characterized in that, The preparation method includes the following steps: (1) Weigh 0.005-0.05 g of paste-like astaxanthin, 0.05 g of hyaluronic acid and 0.05 g of soybean lecithin and transfer them to a distillation flask. Then add 10 mL of chloroform and 2 mL of methanol and mix thoroughly in an ultrasonic cleaner to dissolve the astaxanthin. (2) Connect the distillation flask from step (1) to a rotary evaporator for rotary evaporation; (3) After a uniform orange-red film forms on the wall of the distillation flask in step (2), stop rotary evaporation, remove the distillation flask from the rotary evaporator, add 10 mL of ultrapure water and hydrate for 25-35 min. (4) Transfer the hydrated astaxanthin liposome solution from step (3) into a centrifuge tube, add 20 mL of 0.03 mol / L PBS buffer solution with pH=7, and mix well; (5) The astaxanthin liposomes from step (4) were ultrasonically broken down using an ultrasonic disruptor to form astaxanthin liposomes with smaller particle size. (6) Dissolve 0.04g of N-hydroxysuccinimide in 2 mL of dimethyl sulfoxide and add 1 mL of astaxanthin liposomes from step (5). Stir on a magnetic stirrer for 0.5-2 h. Then add 0.08g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and stir magnetically for 20-24 h. (7) Centrifuge at 4000 rpm for 10 minutes to collect the cultured Chlorella in the logarithmic stage and obtain wet algal pellet. Resuspend the wet algal pellet with 0.9% NaCl aqueous solution to obtain a suspension containing 1 g of wet algal pellet per milliliter. Add 1 mL of Chlorella suspension to the solution in step (6) and shake at a constant speed for 30 min to allow the activated carboxyl groups on the astaxanthin liposomes to undergo a coupling reaction with the amino groups on the Chlorella, so that the astaxanthin liposomes can be successfully attached to the Chlorella.

2. The preparation method according to claim 1, characterized in that, The paste-like astaxanthin mentioned in step (1) is extracted from Haematococcus pluvialis, and the molecular weight of the hyaluronic acid is 80kDa-2000kDa.

3. The preparation method according to claim 1, characterized in that, In step (5), the ultrasonic disruption time is 10-20 min, and the particle size of the astaxanthin liposomes after ultrasonic disruption is 110-120 nm.

4. The preparation method according to claim 1, characterized in that, In step (6), the pH of the reaction solution is adjusted to 6.0-7.

0.

5. The preparation method according to claim 1, characterized in that, In step (7), the pH of the reaction solution is adjusted to 7.0-7.

5.

6. Astaxanthin-loaded microalgae prepared according to any one of claims 1-5.

7. The use of the astaxanthin-loaded microalgae of claim 6 in the preparation of antioxidant and enteric-delivered pharmaceutical products.

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