Astaxanthin liposome and preparation method and application thereof

Abstract

This invention belongs to the field of biopharmaceutical technology, and relates to an astaxanthin liposome, its preparation method, and its application. The invention provides a method for preparing astaxanthin liposomes, comprising: heating a eutectic solvent and adding astaxanthin and tanshinone, reacting to obtain a solution containing a complex astaxanthin; adding CORM2 and soybean lecithin to the solution containing the complex astaxanthin, maintaining the heating temperature to continue the reaction, and after the reaction is completed, forming a transparent film through rotary evaporation; then adding a PBS solution containing a surfactant, fully hydrating, and finally obtaining the astaxanthin liposomes through rotary evaporation under vacuum. The invention also provides the astaxanthin liposomes obtained by the above method and their applications. The astaxanthin liposomes provided by this invention have good antioxidant properties and intestinal targeting properties.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to astaxanthin liposomes, and further to the preparation method and application of the aforementioned astaxanthin liposomes. Background Technology

[0002] Astaxanthin, a natural fat-soluble carotenoid, possesses extremely strong antioxidant, anti-inflammatory, anti-aging, and intestinal protective activities, making it highly valuable in functional foods, biomedicine, and health products. However, astaxanthin has extremely poor water solubility, is sensitive to light, heat, and oxygen, and is easily degraded by gastric acid and pepsin after oral administration, directly resulting in poor gastrointestinal stability and extremely low oral bioavailability, severely limiting the development and application of its oral formulations.

[0003] Liposomes are biomimetic delivery carriers composed of phospholipid bilayers, which can effectively encapsulate lipid-soluble active ingredients, improving drug stability and bioavailability, making them an ideal carrier for oral delivery of astaxanthin. However, traditional liposome preparation processes have significant drawbacks: First, the preparation process requires the use of volatile and toxic organic solvents such as chloroform and dichloromethane, posing a risk of solvent residue and failing to meet the requirements of green chemistry and food and drug safety. Second, ordinary phospholipid liposomes have a loose and poorly compact membrane structure, making them prone to rupture in the highly acidic environment of the stomach, leading to premature release and degradation of astaxanthin, thus failing to achieve targeted intestinal delivery. Third, traditional processes are prone to drug crystallization, resulting in low astaxanthin encapsulation efficiency, poor liposome dispersibility, and significantly reduced delivery efficiency.

[0004] In summary, current oral delivery systems for astaxanthin generally suffer from technical bottlenecks such as residual organic solvents, poor gastric stability, insufficient intestinal targeting, low encapsulation efficiency, and limited antioxidant effects, making it difficult to meet the actual needs of efficient oral delivery. There is an urgent need to develop an astaxanthin liposome technology that is green and environmentally friendly, easy to prepare, and combines high antioxidant activity with precise intestinal targeting performance. Summary of the Invention

[0005] The first objective of this invention is to provide astaxanthin liposomes, which solves the problems of poor gastric stability and insufficient intestinal targeting of existing oral astaxanthin products.

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned astaxanthin liposomes.

[0007] A third objective of this invention is to provide applications of the aforementioned astaxanthin liposomes.

[0008] The first technical solution adopted in this invention is: a method for preparing astaxanthin liposomes, comprising: heating a eutectic solvent and adding astaxanthin and tanshinone, reacting to obtain a solution containing complex astaxanthin; adding CORM2 and soybean lecithin to the solution containing complex astaxanthin, maintaining the heating temperature to continue the reaction, forming a transparent film by rotary evaporation after the reaction is completed, adding a PBS solution containing surfactant, fully hydrating, and then obtaining astaxanthin liposomes by rotary evaporation under vacuum conditions.

[0009] The first technical solution adopted in this invention is further characterized by: The eutectic solvent is composed of malic acid, citric acid, and betaine in a molar ratio of 0.5~1:0.5~1:1.

[0010] The mass ratio of astaxanthin to tanshinone is 1:0.2~0.8.

[0011] The mass ratio of soybean lecithin to surfactant is 1:0.5~0.6.

[0012] The mass ratio of astaxanthin to CORM2 is 1:0.05~0.1.

[0013] The surfactant is Tween-80.

[0014] The heating temperature is 40~50℃; the temperature for any rotary evaporator is 40~50℃.

[0015] In fully hydrated PBS solutions containing surfactants, the volume ratio of PBS solvent to eutectic solvent is 1:3~4, and the concentration of PBS is 10mM.

[0016] The second technical solution adopted in this invention is astaxanthin liposomes, which are prepared by the above-mentioned method for preparing astaxanthin liposomes.

[0017] The third technical solution adopted in this invention is the application of the above-mentioned astaxanthin liposomes in the rapid release of drugs.

[0018] The beneficial effects of this invention are: (1) The astaxanthin liposomes of the present invention significantly improve the gastric stability and intestinal targeted delivery efficiency of liposomes: a ternary eutectic solvent (DES) composed of malic acid, citric acid and betaine is used as the co-solvent of the active ingredient and the liposome film-forming component. Experiments show that the DES system can form a dense hydrogen bond network with the phospholipid bilayer, effectively resisting the destruction of gastric acid and pepsin. At the same time, the liposomes achieve precise intestinal targeted release. The technical principle may be that the malic acid and citric acid in DES provide abundant carboxyl and hydroxyl groups, which form strong hydrogen bond interactions with the quaternary ammonium groups of betaine. This hydrogen bond network is embedded in the phospholipid bilayer during the film-forming process, enhancing the order and density of the membrane; after entering the gastric environment, the hydrogen bond combination of the DES component with the phospholipid can reduce the attack of protons on the lipid bilayer, thereby significantly improving gastric stability; after entering the intestine, the increase in pH promotes the deprotonation of carboxyl groups, the disintegration of the hydrogen bond network, the increase in membrane fluidity, and the rapid release of the drug.

[0019] (2) The astaxanthin liposomes of the present invention significantly improve the drug encapsulation efficiency and liposome dispersibility: by pre-dissolving astaxanthin and tanshinone with DES, the problem of drug crystallization in the traditional organic solvent method is avoided. The high polarity and hydrogen bond donor / acceptor properties of DES can form intermolecular hydrogen bonds or π-π stacking interactions with astaxanthin and tanshinone, so that the two drugs are uniformly dispersed in the lipid precursor solution in a molecular state. After rotary evaporation to form a film, the drug is evenly distributed in the lipid film, and after hydration, liposomes with high encapsulation efficiency are formed.

[0020] (3) The astaxanthin liposomes of the present invention achieve the synergistic effect of antioxidant activity of astaxanthin and tanshinone: Tanshinone and astaxanthin are co-encapsulated in liposomes, and the two exhibit a significant synergistic effect in terms of antioxidant activity.

[0021] (4) The astaxanthin liposome CORM2 of the present invention synergistically enhances gastric stability and imparts CO sustained-release function: CORM2 is added to the liposome formulation. The CO molecules released by CORM2 can have a weak coordination with the hydrophobic tail of the phospholipid bilayer, increasing the rigidity and density of the membrane and reducing the damage of gastric acid and pepsin to the liposome.

[0022] (5) The astaxanthin liposomes of the present invention avoid the use of toxic organic solvents, making them green and environmentally friendly: they use eutectic solvents instead of volatile toxic organic solvents such as chloroform and dichloromethane commonly used in the preparation of traditional liposomes. The components of DES (malic acid, citric acid, and betaine) are all biocompatible natural products that are non-toxic, biodegradable, and inexpensive. The entire preparation process does not require the use of toxic organic solvents, avoiding the safety risks caused by solvent residues and meeting the environmental protection requirements of green chemistry and pharmaceutical formulations. Attached Figure Description

[0023] Figure 1This is a TEM image of the astaxanthin liposomes prepared in Example 1 of this invention; Figure 2 This is a comparison chart of the test results of the astaxanthin liposomes of the present invention and the DPPH free radical scavenging rate of the comparative example; Figure 3 This is a comparison chart of the free radical scavenging rate test results of the astaxanthin liposomes of the present invention and the comparative example ABTS; Figure 4 This is a comparison chart showing the encapsulation and retention rates of the astaxanthin liposomes of the present invention with those of the comparative example after 90 minutes of gastric digestion. Figure 5 This is a comparison chart showing the encapsulation and release rates of the astaxanthin liposomes of the present invention and those of the comparative example after intestinal digestion for 120 min. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0025] This invention provides a method for preparing astaxanthin liposomes, comprising: heating a eutectic solvent and adding astaxanthin and tanshinone, reacting to obtain a solution containing complex astaxanthin; adding CORM2 and soybean lecithin to the solution containing complex astaxanthin, maintaining the heating temperature to continue the reaction, forming a transparent film by rotary evaporation after the reaction is completed, adding a PBS solution containing surfactant, fully hydrating, and then obtaining astaxanthin liposomes by rotary evaporation under vacuum conditions.

[0026] The eutectic solvent is composed of malic acid, citric acid, and betaine in a molar ratio of 0.5~1:0.5~1:1.

[0027] The mass ratio of astaxanthin to tanshinone is 1:0.2~0.8.

[0028] The mass ratio of soybean lecithin to surfactant is 1:0.5~0.6.

[0029] The mass ratio of astaxanthin to CORM2 is 1:0.05~0.1.

[0030] The surfactant is Tween-80.

[0031] The heating temperature is 40~50℃.

[0032] The temperature for the rotary evaporation described above is 40~50℃.

[0033] In the above-mentioned full hydration, the volume ratio of PBS solvent to eutectic solvent used to prepare the PBS solution containing surfactant is 1:3~4, and the concentration of PBS is 10mM.

[0034] The present invention also provides an astaxanthin liposome, which is prepared by the above-described method for preparing astaxanthin liposomes.

[0035] This invention also provides the application of the above-mentioned astaxanthin liposomes in the rapid release of drugs.

[0036] The technical solution of the present invention will be described below with reference to embodiments; however, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods in each embodiment are conventional methods; the reagents and materials used are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments are weight percentages. Unless otherwise specified, all ratios in the following embodiments are weight ratios. In the following embodiments, each experiment was repeated three times, and the average value of the experimental results was taken.

[0037] In the following examples, the PBS solvent used was 10mM phosphate buffer with a pH of 7.4; the CORM2 used was a carbon monoxide-releasing molecule with a purity of 98% (CAS: 22594-69-0); the soybean lecithin used had a purity of 98% (CAS: 8030-76-0); the astaxanthin used had a purity of 96% (CAS: 472-61-7); the tanshinone used had a purity of 98% (CAS: 27210-57-7); and all other reagents were of analytical grade.

[0038] Example 1 This embodiment provides the preparation process of astaxanthin liposomes.

[0039] Step 1: Accurately weigh malic acid, citric acid and betaine in a molar ratio of 1:1:1, mix them and heat them in a water bath at a constant temperature of 50°C. Turn on the magnetic stirrer (stirring speed of about 350 r / min) and continue stirring for 25 min until a uniform, transparent eutectic solvent is formed. Reserve about 90 mL for subsequent steps.

[0040] Step 2: Maintain the heating temperature (50℃) and stirring rate (350r / min) of the above eutectic solvent, add 0.2g astaxanthin and 0.16g tanshinone, and continue stirring for 5min to obtain a solution containing compound astaxanthin.

[0041] Step 3: While maintaining the heating temperature and stirring rate, add 0.02g CORM2 and then 5.0g soybean lecithin in sequence, react for 5 minutes to obtain the liposome precursor solution.

[0042] Step 4: Perform rotary evaporation on the liposome precursor solution. Set the rotary evaporation temperature to 50℃ and the rotation speed to 130 r / min. Continue rotary evaporation until a uniform and transparent film is formed on the inner wall of the container, then stop evaporation.

[0043] Step 5: Add a PBS solution containing Tween-80 (obtained by mixing Tween-80 and 10mM PBS solvent, with 3g of Tween-80 and 22.5mL of 10mM PBS solvent) to a container containing a transparent film. Continue to rotate and evaporate the solution under a vacuum of 0.05MPa to fully hydrate the product, resulting in a light yellow, loose powdery solid, which is the astaxanthin liposome product.

[0044] Encapsulation ratio calculation formula: Encapsulation efficiency = (1 - free astaxanthin / total astaxanthin) × 100%.

[0045] Figure 1 The image shown is a TEM image of the astaxanthin liposomes prepared in this embodiment. The astaxanthin liposomes prepared in this embodiment have an average particle size of about 93.3 nm, a regular and nearly spherical morphology, and good dispersibility. The encapsulation efficiency of astaxanthin was detected by high performance liquid chromatography (HPLC), and the encapsulation efficiency was found to be 91.2%.

[0046] Example 2 This embodiment provides the preparation process of astaxanthin liposomes.

[0047] Step 1: Accurately weigh malic acid, citric acid and betaine in a molar ratio of 0.5:0.5:1, mix them and heat them in a water bath at a constant temperature of 50°C. Turn on the magnetic stirrer (stirring speed of about 350 r / min) and continue stirring for 25 min until a uniform, transparent eutectic solvent is formed. Reserve about 90 mL for subsequent steps.

[0048] Step 2: Maintain the heating temperature (50℃) and stirring rate (350r / min) of the above eutectic solvent, add 0.2g astaxanthin and 0.04g tanshinone, and continue stirring for 5min to obtain a solution containing compound astaxanthin.

[0049] Step 3: While maintaining the heating temperature and stirring rate, add 0.01g CORM2 and then 5.0g soybean lecithin in sequence, react for 5 minutes to obtain the liposome precursor solution.

[0050] Step 4: Perform rotary evaporation on the liposome precursor solution. Set the rotary evaporation temperature to 50℃ and the rotation speed to 130 r / min. Continue rotary evaporation until a uniform and transparent film is formed on the inner wall of the container, then stop evaporation.

[0051] Step 5: Add a PBS solution containing Tween-80 (obtained by mixing Tween-80 and 10mM PBS solvent, with 2.5g of Tween-80 and 30mL of 10mM PBS solvent) to a container containing a transparent film. Continue to rotate and evaporate the solution under a vacuum of 0.05MPa to fully hydrate the product, resulting in a light yellow, loose powdery solid, which is the astaxanthin liposome product.

[0052] The astaxanthin liposomes prepared in this embodiment had an average particle size of approximately 106.2 nm and an encapsulation efficiency of 90.8%.

[0053] Example 3 This embodiment provides the preparation process of astaxanthin liposomes.

[0054] Step 1: Accurately weigh malic acid, citric acid and betaine in a molar ratio of 0.5:1:1, mix them and heat them in a water bath at a constant temperature of 50°C. Turn on the magnetic stirrer (stirring speed of about 350 r / min) and continue stirring for 25 min until a uniform, transparent eutectic solvent is formed. Reserve about 90 mL for subsequent steps.

[0055] Step 2: Maintain the heating temperature (50℃) and stirring rate (350r / min) of the above eutectic solvent, add 0.2g astaxanthin and 0.10g tanshinone, and continue stirring for 5min to obtain a solution containing compound astaxanthin.

[0056] Step 3: While maintaining the heating temperature and stirring rate, add 0.014g CORM2 and then 5.0g soybean lecithin in sequence, react for 5 minutes to obtain the liposome precursor solution.

[0057] Step 4: Perform rotary evaporation on the liposome precursor solution. Set the rotary evaporation temperature to 50℃ and the rotation speed to 130r / min. Continue rotary evaporation until a uniform and transparent film forms on the inner wall of the container, then stop evaporation.

[0058] Step 5: Add a PBS solution containing Tween-80 (obtained by mixing Tween-80 and 10mM PBS solvent, with 2.75g of Tween-80 and 30mL of 10mM PBS solvent) to a container containing a transparent film. Continue to rotate and evaporate the solution under a vacuum of 0.05MPa to fully hydrate the product, resulting in a light yellow, loose powdery solid, which is the astaxanthin liposome product.

[0059] The astaxanthin liposomes prepared in this embodiment had an average particle size of approximately 98.2 nm and an encapsulation efficiency of 87.5%.

[0060] Example 4 This embodiment provides the preparation process of astaxanthin liposomes.

[0061] Step 1: Accurately weigh malic acid, citric acid and betaine in a molar ratio of 1:0.5:1, mix them and heat them in a water bath at a constant temperature of 50°C. Turn on the magnetic stirrer (stirring speed of about 350 r / min) and continue stirring for 25 min until a uniform, transparent eutectic solvent is formed. Reserve about 90 mL for subsequent steps.

[0062] Step 2: Maintain the heating temperature (50℃) and stirring rate (350r / min) of the above eutectic solvent, add 0.2g astaxanthin and 0.12g tanshinone, and continue stirring for 5min to obtain a solution containing compound astaxanthin.

[0063] Step 3: While maintaining the heating temperature and stirring rate, add 0.016g CORM2 and then 5.0g soybean lecithin in sequence, react for 5 minutes to obtain the liposome precursor solution.

[0064] Step 4: Perform rotary evaporation on the liposome precursor solution. Set the rotary evaporation temperature to 50℃ and the rotation speed to 130 r / min. Continue rotary evaporation until a uniform and transparent film is formed on the inner wall of the container, then stop evaporation.

[0065] Step 5: Add a PBS solution containing Tween-80 (obtained by mixing Tween-80 and 10mM PBS solvent, with 2.5g of Tween-80 and 22.5mL of 10mM PBS solvent) to a container containing a transparent film. Continue to rotate and evaporate the solution under a vacuum of 0.05MPa to fully hydrate the product, resulting in a light yellow, loose powdery solid, which is the astaxanthin liposome product.

[0066] The astaxanthin liposomes prepared in this embodiment had an average particle size of approximately 96.7 nm and an encapsulation efficiency of 89.3%.

[0067] Example 5 This embodiment provides the preparation process of astaxanthin liposomes.

[0068] Step 1: Accurately weigh malic acid, citric acid and betaine in a molar ratio of 0.6:0.6:1, mix them and heat them in a water bath at a constant temperature of 40°C. Turn on the magnetic stirrer (stirring speed of about 350 r / min) and continue stirring for 25 min until a uniform, transparent eutectic solvent is formed. Reserve about 90 mL for subsequent steps.

[0069] Step 2: Maintain the heating temperature (40℃) and stirring rate (350r / min) of the eutectic solvent, add 0.2g astaxanthin and 0.12g tanshinone to the eutectic solvent, and continue stirring for 5min to obtain a solution containing compound astaxanthin.

[0070] Step 3: While maintaining the heating temperature and stirring rate, add 0.008g CORM2 and then 5.0g soybean lecithin in sequence, react for 5 minutes to obtain the liposome precursor solution.

[0071] Step 4: Perform rotary evaporation on the liposome precursor solution. Set the rotary evaporation temperature to 50℃ and the rotation speed to 130 r / min. Continue rotary evaporation until a uniform and transparent film is formed on the inner wall of the container, then stop evaporation.

[0072] Step 5: Add a PBS solution containing Tween-80 (obtained by mixing Tween-80 and 10mM PBS solvent, with 2g of Tween-80 and 22.5mL of 10mM PBS solvent) to a container containing a transparent film. Continue to rotate and evaporate the solution under a vacuum of 0.05MPa until fully hydrated, and finally obtain a light yellow, loose powdery solid, which is the astaxanthin liposome product.

[0073] Example 6 This embodiment provides the preparation process of astaxanthin liposomes.

[0074] Step 1: Accurately weigh malic acid, citric acid and betaine in a molar ratio of 0.8:0.8:1, mix them and heat them in a water bath at a constant temperature of 45°C. Turn on the magnetic stirrer (stirring speed of about 350 r / min) and continue stirring for 25 min until a uniform, transparent eutectic solvent is formed. Reserve about 90 mL for subsequent steps.

[0075] Step 2: Maintain the heating temperature (45℃) and stirring rate (350r / min) of the eutectic solvent, add 0.2g of astaxanthin and 0.08g of tanshinone to the eutectic solvent, and continue stirring for 5min to obtain a solution containing compound astaxanthin.

[0076] Step 3: While maintaining the heating temperature and stirring rate, add 0.016g CORM2 and then 5.0g soybean lecithin in sequence, react for 5 minutes to obtain the liposome precursor solution.

[0077] Step 4: Perform rotary evaporation on the liposome precursor solution. Set the rotary evaporation temperature to 45℃ and the rotation speed to 130r / min. Continue rotary evaporation until a uniform and transparent film is formed on the inner wall of the container, then stop evaporation.

[0078] Step 5: Add a PBS solution containing Tween-80 (obtained by mixing Tween-80 and 10mM PBS solvent, with 3g of Tween-80 and 22.5mL of 10mM PBS solvent) to a container containing a transparent film. Continue to rotate and evaporate the solution under a vacuum of 0.05MPa to fully hydrate the product, resulting in a light yellow, loose powdery solid, which is the astaxanthin liposome product.

[0079] Comparative Example 1 This comparative example is the same as Example 1, except that citric acid is replaced with an equimolar amount of malic acid when preparing the eutectic solvent in step 1.

[0080] The astaxanthin liposomes prepared in this comparative example had an average particle size of approximately 128.5 nm and an encapsulation efficiency of 72.8%.

[0081] Comparative Example 2 This comparative example is the same as Example 1, except that in step 2, tanshinone is replaced with an equal mass of astaxanthin.

[0082] The astaxanthin liposomes prepared in this comparative example had an average particle size of approximately 115.3 nm and an encapsulation efficiency of 82.6%.

[0083] Comparative Example 3 This comparative example is the same as Example 1, except that CORM2 was not added in step 3.

[0084] The astaxanthin liposomes prepared in this comparative example had an average particle size of approximately 118.7 nm and an encapsulation efficiency of 86.4%.

[0085] Comparative Example 4 This comparative example is the same as Example 1, except that the eutectic solvent is replaced with chloroform.

[0086] The astaxanthin liposomes prepared in this comparative example had an average particle size of approximately 132.1 nm and an encapsulation efficiency of 70.5%.

[0087] Performance tests were conducted on astaxanthin liposomes prepared by different methods in the embodiments and comparative examples of the present invention, and the results are as follows.

[0088] (1) Antioxidant performance test The antioxidant properties of the samples were evaluated using a combination of DPPH and ABTS radical scavenging methods. The specific testing procedure is as follows: Reagent preparation: Prepare 0.1 mmol / L DPPH ethanol solution, 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution (mixed in a 1:1 volume ratio, incubated at room temperature in the dark for 12 h to obtain ABTS free radical stock solution, diluted with anhydrous ethanol to absorbance of 0.70±0.02 before use), 0.1 mol / L PBS buffer (pH=7.4), and 1 mg / mL of each test sample solution (prepared with the above PBS buffer, ultrasonically dispersed for 10 min to ensure uniform dispersion).

[0089] DPPH radical scavenging rate assay: In a 96-well plate, add 100 μL of DPPH ethanol solution to each well, followed by 100 μL of each test sample solution and a blank control solution. A blank group (100 μL DPPH ethanol solution + 100 μL anhydrous ethanol) and a control group (100 μL PBS buffer + 100 μL DPPH ethanol solution) were also prepared. Each sample was placed in three parallel wells. The 96-well plate was incubated at room temperature in the dark for 30 min, and the absorbance of each well was measured at 517 nm using a microplate reader.

[0090] ABTS radical scavenging rate assay: In a 96-well plate, add 100 μL of ABTS radical diluent to each well, followed by 10 μL of each test sample solution and a blank control solution. A blank group (100 μL ABTS radical diluent + 10 μL anhydrous ethanol) and a control group (100 μL ABTS radical diluent + 10 μL PBS buffer) were also prepared. Each sample was recorded in triplicate. After reacting at room temperature in the dark for 10 min, the absorbance of each well was measured at 734 nm using a microplate reader.

[0091] Results calculation: Free radical scavenging rate (%) = (absorbance of control group - absorbance of sample group) / (absorbance of control group - absorbance of blank group) × 100%. The average value of 3 parallel wells is taken as the final result. The higher the scavenging rate, the stronger the antioxidant performance of the sample.

[0092] The results of the DPPH free radical scavenging rate test are as follows: Figure 2 As shown, the results of the ABTS free radical scavenging rate test are as follows: Figure 3 As shown.

[0093] (2) Intestinal targeting performance test An in vitro simulated gastrointestinal digestion method was used to mimic the human stomach and intestinal environment. The intestinal targeting performance of liposomes was evaluated by measuring the changes in astaxanthin encapsulation efficiency at different digestion stages. The specific testing process is as follows: Preparation of simulated digestive fluids: Simulated gastric juice (pH=1.2): Weigh 2.0g NaCl and 3.2g pepsin, add distilled water to a final volume of 1000mL, stir magnetically until completely dissolved, and filter aseptically for later use; Simulated intestinal juice (pH=7.4): Weigh 6.8g KH2PO4, 0.76g pancreatin, and 10.0g bile salts, add distilled water to a final volume of 1000mL, stir magnetically until completely dissolved, and filter aseptically for later use.

[0094] Sample preparation: Take 0.1g of each test sample, add 10mL of PBS buffer (pH=7.4), sonicate for 10min to prepare a uniform liposome suspension for later use.

[0095] In vitro simulated gastric digestion: Take 5 mL of the above liposome suspension, add 5 mL of simulated gastric juice, place in a 37℃ constant temperature water bath shaker, shake at a rate of 100 r / min, and take 1 mL samples at 0 min, 30 min, 60 min and 90 min respectively. Immediately add 0.5 mL of 1 mol / L NaOH solution to terminate digestion, centrifuge (8000 r / min, 4℃, 10 min), take the supernatant, and determine the content of free astaxanthin in the supernatant by HPLC. Calculate the astaxanthin encapsulation efficiency of the liposomes at this time.

[0096] In vitro simulated intestinal digestion: After 90 min of gastric digestion, the remaining sample was adjusted to pH 7.4, and 10 mL of simulated intestinal fluid was added. The sample was then placed in a 37℃ constant temperature water bath shaker at a shaking rate of 100 r / min. 1 mL samples were taken at 0 min, 30 min, 60 min, and 120 min of digestion, and 0.5 mL of 1 mol / L HCl solution was added immediately to terminate the digestion. The samples were centrifuged (8000 r / min, 4℃, 10 min), and the supernatant was collected. The content of free astaxanthin in the supernatant was determined by HPLC, and the astaxanthin encapsulation efficiency of the liposomes was calculated.

[0097] In this embodiment, the HPLC conditions for detecting astaxanthin content were as follows: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase methanol:acetonitrile = 1:1 (v / v), flow rate 1.0 mL / min, detection wavelength 474 nm, column temperature 30 °C, and injection volume 20 μL; all tests were performed in triplicate, and the average value of the results was taken.

[0098] Intestinal targeting performance was evaluated using the encapsulation retention rate after 90 minutes of gastric digestion and the encapsulation release rate after 120 minutes of intestinal digestion. The retention rate was calculated as (encapsulation rate after 90 minutes of gastric digestion / initial encapsulation rate) × 100%. A higher retention rate indicates better stability of the liposomes in the gastric environment. The release rate was calculated as (encapsulation rate before entering the in vitro simulated intestinal digestion - encapsulation rate after 120 minutes of intestinal digestion) / encapsulation rate before entering the in vitro simulated intestinal digestion × 100%. A higher release rate indicates better release performance of the liposomes in the intestinal environment and stronger intestinal targeting.

[0099] The results of the encapsulation retention rate test after 90 minutes of gastric digestion are as follows: Figure 4 As shown, the encapsulation efficiency and release rate test results after 120 minutes of intestinal digestion are as follows: Figure 5 As shown.

[0100] Based on the above experimental results, this study successfully developed a method for preparing astaxanthin liposomes using a eutectic solvent (DES), and systematically investigated the effects of DES composition, drug ratio, and functional excipients on the physicochemical properties, antioxidant activity, and intestinal targeting performance of the liposomes. Experimental results showed that a ternary DES system (molar ratio 1:1:1) composed of malic acid, citric acid, and betaine effectively dissolved astaxanthin and tanshinone, and together with soybean lecithin and CORM2, formed a homogeneous liposome precursor solution. After rotary evaporation and hydration with PBS, astaxanthin liposomes with uniform particle size (93.3–106.2 nm) and high encapsulation efficiency (87.5%–91.2%) were obtained. Comparative experiments confirmed that the ternary DES system (malic acid:citric acid:betaine = 1:1:1) was significantly superior to the binary DES system (malic acid:betaine = 2:1) without citric acid and the traditional organic solvent (chloroform). The addition of citric acid increased the gastric environment retention rate of liposomes from 35.8% to 88.7% and the encapsulation efficiency from 72.8% to 91.2%. In contrast, when chloroform was used instead of DES, the gastric retention rate was only 2.4%, indicating that DES plays a crucial protective role in the gastric stability of liposomes. Furthermore, the introduction of CORM2 not only achieved sustained CO release but also increased the gastric retention rate from 48.2% to 88.7%, an increase of 84%. The combined use of tanshinone and astaxanthin exhibited a significant synergistic antioxidant effect, increasing the DPPH free radical scavenging rate from 68.9% to 82.3%.

[0101] Based on comprehensive evaluation of various indicators, Example 1 represents the optimal approach: a DES molar ratio of 1:1:1, an astaxanthin to tanshinone mass ratio of 1:0.8, an astaxanthin to CORM2 mass ratio of 1:0.1, a soybean lecithin to Tween-80 mass ratio of 1:0.6, and an aqueous to organic phase volume ratio of 1:4. The liposomes prepared using this approach had an average particle size of 93.3 nm, an encapsulation efficiency of 91.2%, and DPPH and ABTS free radical scavenging rates of 82.3% and 84.1%, respectively. After 90 minutes of gastric digestion, the encapsulation retention rate was 88.7%, and after 120 minutes of intestinal digestion, the release rate reached 99.5%, achieving highly efficient oral intestinal-targeted delivery.

[0102] In summary, the astaxanthin liposomes, their preparation method, and applications provided by this invention have comprehensive advantages such as simple preparation process, high encapsulation rate, strong gastric stability, complete intestinal release, and excellent antioxidant activity. They provide a new technical strategy for the oral delivery of poorly soluble active ingredients and have good application prospects in the fields of functional foods and pharmaceutical preparations.

Claims

1. A method for preparing astaxanthin liposomes, characterized in that, include: Astaxanthin and tanshinone were added after heating the eutectic solvent, and a solution containing complex astaxanthin was obtained after the reaction. CORM2 and soybean lecithin were added to the solution containing compound astaxanthin, and the reaction was continued at the heating temperature. After the reaction was completed, a transparent film was formed by rotary evaporation. Then, a PBS solution containing surfactant was added, and after full hydration, astaxanthin liposomes were obtained by rotary evaporation under vacuum conditions.

2. The method for preparing astaxanthin liposomes according to claim 1, characterized in that, The eutectic solvent is composed of malic acid, citric acid and betaine in a molar ratio of 0.5~1:0.5~1:

1.

3. The method for preparing astaxanthin liposomes according to claim 1, characterized in that, The mass ratio of astaxanthin to tanshinone is 1:0.2~0.

8.

4. The method for preparing astaxanthin liposomes according to claim 1, characterized in that, The mass ratio of soybean lecithin to surfactant is 1:0.5~0.

6.

5. The method for preparing astaxanthin liposomes according to claim 1, characterized in that, The mass ratio of astaxanthin to CORM2 is 1:0.05~0.

1.

6. The method for preparing astaxanthin liposomes according to claim 1, characterized in that, The surfactant is Tween-80.

7. The method for preparing astaxanthin liposomes according to claim 1, characterized in that, The heating temperature is 40~50℃; the rotary evaporation temperature is 40~50℃.

8. The method for preparing astaxanthin liposomes according to claim 1, characterized in that, In the fully hydrated state, the volume ratio of PBS solvent to eutectic solvent used to prepare the PBS solution containing the surfactant is 1:3~4, and the concentration of PBS is 10mM.

9. Astaxanthin liposomes, characterized in that, Astaxanthin liposomes were prepared using the method described in any one of claims 1 to 8.

10. The application of the astaxanthin liposomes according to claim 9 in the rapid drug release.

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