High bioavailability lutein ester-fumaric acid complex liposome and preparation method and application thereof

Abstract

The application relates to the technical field of liposomes, in particular to high-bioavailability lutein ester-fumaric acid composite liposomes as well as a preparation method and application thereof. The preparation method of the lutein ester-fumaric acid composite liposomes comprises the following steps: mixing lutein ester with oil to obtain phase A; adding phospholipid, cholesterol and vitamin E into the phase A and then performing ultrasonic dispersion treatment to obtain phase B; adding fumaric acid and water into the phase B and then sequentially performing high-pressure homogenization treatment, high-pressure microjet treatment and freeze-drying treatment to obtain the lutein ester-fumaric acid composite liposomes. The high-bioavailability lutein ester-fumaric acid composite liposomes have the advantages of small average particle size, high Zeta potential, high encapsulation rate, high bioavailability, high solubility and excellent stability, and have a potential application prospect in the preparation of eye-protecting functional food, medicine or health care products.

Description

Technical Field

[0001] This invention relates to the field of liposome technology, and in particular to a highly bioavailable lutein ester-fumaric acid complex liposome, its preparation method, and its application. Background Technology

[0002] Lutein is a globally recognized essential nutrient that plays a crucial role in eye development. Its unique ionone ring dihydroxy structure enables it to filter blue light and quench singlet oxygen, exhibiting antioxidant and retinal protective functions. The macula of the human retina is rich in lutein and its homologues, giving the macula its characteristic yellow color. However, lutein is highly sensitive to light and high temperatures, exhibiting poor stability. Lutein esters, on the other hand, are broken down into free lutein after absorption by the body, possessing the basic function of replenishing lost lutein, similar to crystalline lutein. Therefore, lutein esters are commonly used as a nutrient supplement.

[0003] Lutein esters are carotenoid fatty acid esters widely distributed in various plant tissues, including marigolds, spinach, cauliflower, cabbage, celery, and pumpkin. When ingested, lutein esters are broken down into free lutein by human lipases, replenishing lost lutein and thus improving eye function. Clinical trials have shown that both healthy individuals and patients with age-related macular degeneration (AMD) can benefit from lutein ester supplements. Research indicates that lutein ester supplementation can also regulate cellular immunity, promote antigen-stimulated lymphocyte proliferation, and affect the functional expression of cell surface molecules, thereby enhancing the body's own immunity. However, there are still some problems with lutein esters in practical applications: (1) Lutein esters are insoluble in water, slightly soluble in oil at room temperature, and have a slow dissolution rate in the gastrointestinal tract, resulting in poor oral absorption and poor solubility; (2) Lutein esters have a conjugated long chain containing 18 carbon atoms and two esterified ionone rings on both sides, which are easily affected by light, heat, oxygen, etc., and thus decompose or degrade, thereby losing their biological activity and having poor stability.

[0004] Therefore, how to improve the stability of lutein esters while increasing their solubility is an urgent problem to be solved. Summary of the Invention

[0005] This invention provides a method for preparing lutein ester-fumaric acid complex liposomes with high bioavailability. This method can prepare lutein ester-fumaric acid complex liposomes with small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, and excellent stability. The lutein ester-fumaric acid complex liposomes can effectively protect lutein esters, making them less prone to decomposition or degradation due to external environmental factors, thereby effectively improving the bioavailability of lutein esters.

[0006] The present invention also provides a lutein ester-fumaric acid complex liposome with high bioavailability, which is prepared by the above preparation method. Therefore, the lutein ester-fumaric acid complex liposome has the advantages of small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility and excellent stability. It can effectively protect lutein ester, making it less likely to decompose or degrade due to external environmental factors, and can effectively improve the bioavailability of lutein ester.

[0007] This invention also provides the application of the above-mentioned lutein ester-fumaric acid complex liposomes in the preparation of food, pharmaceuticals, or health products with eye-protecting functions. Research by the inventors shows that the lutein ester-fumaric acid complex liposomes have advantages such as small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, and excellent stability. They can effectively protect lutein esters, making them less prone to decomposition or degradation due to external environmental factors. Therefore, the lutein ester-fumaric acid complex liposomes can be used to prepare food, pharmaceuticals, or health products with eye-protecting functions.

[0008] The first aspect of this invention provides a method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes, comprising the following steps:

[0009] Lutein esters were mixed with oils to obtain phase A;

[0010] Phospholipids, cholesterol, and vitamin E were added to phase A and then subjected to ultrasonic dispersion to obtain phase B.

[0011] Fumaric acid and water were added to phase B and then subjected to high-pressure homogenization under a first gas atmosphere to obtain a homogenized liquid.

[0012] Under a second gas atmosphere, the homogenized liquid is subjected to high-pressure microfluidic treatment to obtain a liposome solution;

[0013] The liposome solution was freeze-dried to obtain the lutein ester-fumaric acid complex liposome.

[0014] The preparation method of the highly bioavailable lutein ester-fumaric acid complex liposomes described above, by mass parts, comprises the following raw material composition of the lutein ester-fumaric acid complex liposomes: 3-8 parts lutein ester, 5-15 parts oil, 20-30 parts phospholipid, 5-15 parts cholesterol, 0.1-1 part vitamin E, and 1-2 parts fumaric acid.

[0015] In the preparation method of the high bioavailability lutein ester-fumaric acid complex liposomes described above, the ultrasonic dispersion treatment has a frequency of 40 kHz, a power of 200-300 W, and a time of 5-15 min.

[0016] In the preparation method of the high bioavailability lutein ester-fumaric acid complex liposomes described above, the high-pressure homogenization process is carried out at a pressure of 30-50 MPa, a temperature of 40-70°C, and a time of 10-15 min.

[0017] The method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes as described above involves subjecting the homogenized solution to high-pressure microfluidic treatment, including:

[0018] The homogenized liquid was subjected to high-pressure microjet treatment at a pressure of 120-160 MPa and circulated 3-6 times.

[0019] The method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes as described above, wherein the lyophilization process sequentially includes a pre-lyophilization process and a main lyophilization process;

[0020] The temperature of the pre-freeze-drying treatment is higher than the temperature of the main freeze-drying treatment.

[0021] In the preparation method of the highly bioavailable lutein ester-fumaric acid complex liposomes described above, the pre-freeze drying process is carried out at a temperature of -20 to -18°C for 20 to 24 hours.

[0022] And / or, in the main freeze-drying process, the temperature is -55 to -45°C and the time is 36 to 48 hours.

[0023] The method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes as described above, wherein mixing lutein ester with oil to obtain phase A, comprises: mixing lutein ester with oil under stirring and heating in a water bath at 70-80°C to obtain a mixture, and then keeping the mixture in a water bath at 70-80°C to obtain phase A;

[0024] And / or, the oil is at least one of medium-chain triglycerides, sunflower seed oil, peanut oil, and algal oil;

[0025] And / or, the phospholipid is at least one of soybean lecithin, egg yolk lecithin, and hydrogenated lecithin.

[0026] A second aspect of the present invention provides a highly bioavailable lutein ester-fumaric acid complex liposome, which is prepared by the method for preparing the highly bioavailable lutein ester-fumaric acid complex liposome.

[0027] A third aspect of the present invention provides the application of the highly bioavailable lutein ester-fumaric acid complex liposome in the preparation of food, pharmaceutical or health products with eye-protecting functions.

[0028] In summary, the solution of the present invention has at least the following effects:

[0029] Fumaric acid is a metabolic intermediate in organisms and directly participates in the tricarboxylic acid cycle. It can not only improve the utilization rate of nutrients, but also lower the pH of the stomach, stimulate the secretion of various digestive enzymes and increase the activity of digestive enzymes, improve the function of the digestive system, and promote digestion and absorption.

[0030] The present invention provides a method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes. This method involves combining lutein esters with fumaric acid to prepare lutein ester-fumaric acid complex liposomes. These liposomes possess advantages such as small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, and excellent stability (excellent storage stability, excellent light stability, and excellent thermal stability). They effectively protect lutein esters, preventing them from decomposing or degrading due to external environmental factors, thereby effectively improving the bioavailability of lutein esters. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This study aims to measure the bioavailability of each embodiment and comparative example in the oral cavity, stomach, and intestinal stages during in vitro simulated digestive system experiments. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Where specific techniques or conditions are not specified in the embodiments of this invention, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0035] It should be noted that the use of terms such as "first" and "second" in this invention is for distinguishing similar objects and not for describing a specific order or sequence, and therefore should not be construed as a limitation of this invention.

[0036] The first aspect of this invention provides a method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes, comprising the following steps:

[0037] Lutein esters were mixed with oils to obtain phase A;

[0038] Phospholipids, cholesterol, and vitamin E were added to phase A and then subjected to ultrasonic dispersion to obtain phase B.

[0039] Fumaric acid and water were added to phase B and then subjected to high-pressure homogenization under a first gas atmosphere to obtain a homogenized liquid.

[0040] Under a second gas atmosphere, the homogenized liquid is subjected to high-pressure microfluidic treatment to obtain a liposome solution;

[0041] The liposome solution was freeze-dried to obtain the lutein ester-fumaric acid complex liposome.

[0042] The present invention does not impose any particular limitation on the specific source of the above-mentioned raw materials, which can be purchased from commercial channels.

[0043] The present invention prepares lutein ester-fumaric acid composite liposomes. Specifically, lutein ester is first mixed with oils to obtain phase A; then phospholipids, cholesterol, and vitamin E are added to phase A to obtain a first mixture. The first mixture is then subjected to ultrasonic dispersion to obtain phase B. Ultrasonic dispersion utilizes the cavitation effect generated by ultrasound to disperse the components in the liquid. The cavitation effect creates a high-temperature, high-pressure environment, which is conducive to the formation of nanoparticles. Furthermore, ultrasonic dispersion can reduce the binding force between components, effectively preventing aggregation. Fumaric acid and water are then added to phase B to obtain a second mixture. The second mixture is then subjected to high-pressure homogenization under a first gas atmosphere. The homogenized liquid is subjected to high-pressure homogenization under a first gas atmosphere to further disperse the second mixture evenly and prevent precipitation or stratification. Next, under a second gas atmosphere, the homogenized liquid is subjected to high-pressure microfluidic treatment to obtain a liposome solution. High-pressure microfluidic treatment enables precise particle size control and uniform distribution, and reduces the impact of localized high temperatures during ultrasonic dispersion. Finally, the liposome solution is freeze-dried to obtain lutein ester-fumaric acid complex liposomes. Freeze-drying effectively preserves the chemical properties and biological activity of the active substance (lutein ester) and makes the lutein ester-fumaric acid complex liposomes more stable during transportation and storage, reducing quality problems caused by temperature changes.

[0044] The present invention can prepare lutein ester-fumaric acid complex liposomes with small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, excellent storage stability, excellent light stability and excellent thermal stability through the above method. The lutein ester-fumaric acid complex liposomes can effectively protect lutein esters, making them less prone to decomposition or degradation due to external environmental factors, and can effectively improve the bioavailability of lutein esters.

[0045] In one specific embodiment, the raw material composition of the above-mentioned lutein ester-fumaric acid complex liposome, by mass parts, includes: 3-8 parts of lutein ester, 5-15 parts of oil, 20-30 parts of phospholipid, 5-15 parts of cholesterol, 0.1-1 parts of vitamin E, and 1-2 parts of fumaric acid.

[0046] Based on mass fractions, when the raw materials and their respective masses of the above-mentioned lutein ester-fumaric acid complex liposomes are within the above-mentioned range, the components can be better matched, which is beneficial to preparing lutein ester-fumaric acid complex liposomes with small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, excellent storage stability, excellent light stability, and excellent thermal stability.

[0047] In one specific embodiment, the ultrasonic dispersion process described above uses a frequency of 40 kHz, a power of 200–300 W, and a duration of 5–15 min.

[0048] When the parameters of frequency, power, and time are within the above ranges during ultrasonic dispersion, the cavitation effect generated by ultrasound can be effectively utilized to disperse the components in the liquid. The cavitation effect can generate a high temperature and high pressure environment, which is more conducive to the formation of nanoparticles and can also reduce the binding force between the components, effectively preventing agglomeration.

[0049] For example, in the above ultrasonic dispersion process, the frequency is 40kHz;

[0050] The power can be any one or any combination of 200W, 220W, 240W, 260W, 280W, and 300W;

[0051] The time can be any one of 5 min, 8 min, 10 min, 12 min, 14 min, or 15 min, or a range of any two of them.

[0052] In one specific embodiment, the pressure in the above-mentioned high-pressure homogenization process is 30-50 MPa, the temperature is 40-70°C, and the time is 10-15 min.

[0053] When the parameters of pressure, temperature, and time are within the above-mentioned ranges during high-pressure homogenization, the second mixture can be dispersed more evenly, and precipitation or stratification is less likely to occur.

[0054] For example, in the above high-pressure homogenization process, the pressure can be any one or any combination of 30MPa, 35MPa, 40MPa, 45MPa, and 50MPa.

[0055] The temperature can be any one or any combination of 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃.

[0056] The time can be any one or a range of any two of the following: 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0057] In one specific embodiment, the above-mentioned high-pressure microfluidic treatment of the homogenized liquid includes:

[0058] The homogenized liquid was subjected to high-pressure microjet treatment at a pressure of 120–160 MPa and circulated 3–6 times.

[0059] When the homogenized liquid is subjected to high-pressure microfluidic treatment using the above process, finer particle size control and more uniform distribution can be achieved, and the influence of local high temperature during ultrasonic dispersion treatment can be reduced. This is beneficial for the subsequent preparation of lutein ester-fumaric acid complex liposomes with high solubility, high lutein ester content, uniform particle size distribution, high encapsulation rate and high stability.

[0060] In one specific embodiment, the freeze-drying process includes a pre-freeze-drying process and a main freeze-drying process in sequence; the temperature of the pre-freeze-drying process is higher than the temperature of the main freeze-drying process.

[0061] Specifically, the liposome solution was subjected to pre-freeze-drying and main freeze-drying processes to obtain lutein ester-fumaric acid composite liposomes. The pre-freeze-drying temperature was higher than the main freeze-drying temperature. Pre-freeze-drying freezes the water in the liposome solution, reducing ice crystal formation and preventing localized boiling or bubbling due to unfrozen water during the subsequent main freeze-drying process, thus minimizing damage to the liposome structure. Main freeze-drying prevents recrystallization of ice crystals, thereby maintaining the bilayer structure of the liposomes. By controlling the temperatures of the pre-freeze-drying and main freeze-drying processes, solute effects can be reduced, preventing liposome membrane denaturation and fusion caused by solute concentration.

[0062] In one specific embodiment, the temperature in the above-mentioned pre-freeze-drying process is -20 to -18°C, and the time is 20 to 24 hours.

[0063] When the temperature and time parameters are within the above range during the pre-freeze-drying process, the water in the liposome solution can be frozen, reducing the formation of ice crystals and preventing local boiling or bubbling caused by unfrozen water during the subsequent main freeze-drying process, thus reducing damage to the liposome structure.

[0064] For example, in the pre-freeze drying process, the temperature can be any one of -20°C, -19°C, -18°C, or a range of any two of them;

[0065] The time can be any one of 20h, 21h, 22h, 23h, 24h, or a range of any two of them.

[0066] In one specific embodiment, the temperature in the above-mentioned main freeze-drying process is -55 to -45°C, and the time is 36 to 48 hours.

[0067] When the temperature and time parameters during the main freeze-drying process are within the above-mentioned ranges, recrystallization of ice crystals can be avoided, thereby maintaining the bilayer structure of liposomes. This results in the preparation of lutein ester-fumaric acid complex liposomes with small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, excellent storage stability, excellent photostability, and excellent thermal stability.

[0068] For example, in the main freeze-drying process, the temperature can be any one of -55°C, -50°C, -45°C, or a range of any two of them.

[0069] The time can be any one or any combination of 36h, 38h, 40h, 42h, 44h, 46h, and 48h.

[0070] In one specific embodiment, the step of mixing lutein esters with oils to obtain phase A includes: mixing lutein esters and oils uniformly under stirring and heating in a water bath at 70-80°C to obtain a mixture, and then keeping the mixture at 70-80°C in a water bath to obtain phase A.

[0071] In one specific embodiment, the oil is at least one selected from medium-chain triglycerides, sunflower seed oil, peanut oil, and algal oil, preferably a medium-chain triglyceride.

[0072] Medium-chain triglycerides are mixtures of triglycerides from saturated fatty acids, possessing physicochemical properties such as low viscosity, low surface tension, and good oxidative stability. Mixing medium-chain triglycerides with lutein esters can effectively reduce the surface tension of lutein esters in solution, thereby improving their dispersibility and solubility, exhibiting a unique solubilizing effect on lutein esters.

[0073] In one specific embodiment, the phospholipid is at least one of soybean lecithin, egg yolk lecithin, and hydrogenated lecithin.

[0074] A second aspect of this invention provides a highly bioavailable lutein ester-fumaric acid complex liposome, prepared by the aforementioned method for preparing lutein ester-fumaric acid complex liposomes. Therefore, this lutein ester-fumaric acid complex liposome possesses advantages such as small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, excellent storage stability, excellent photostability, and excellent thermal stability. It effectively protects lutein esters, preventing them from decomposing or degrading due to external environmental factors, thereby effectively improving the bioavailability of lutein esters.

[0075] A third aspect of this invention provides the application of the aforementioned highly bioavailable lutein ester-fumaric acid complex liposomes in the preparation of food, pharmaceuticals, or health products with eye-protecting functions. Research by the inventors shows that these lutein ester-fumaric acid complex liposomes possess advantages such as small average particle size, high zeta potential, high encapsulation efficiency, high bioavailability, high solubility, excellent storage stability, excellent photostability, and excellent thermal stability. They effectively protect lutein esters, preventing them from decomposing or degrading due to external environmental factors, thus effectively improving the bioavailability of lutein esters. Therefore, these lutein ester-fumaric acid complex liposomes can be used in the preparation of food, pharmaceuticals, or health products with eye-protecting functions.

[0076] The embodiments and comparative examples of the present invention will be described in detail below.

[0077] Example 1

[0078] The method for preparing lutein ester-fumaric acid complex liposomes provided in this embodiment includes the following steps:

[0079] (1) By mass, 5 parts of lutein ester and 10 parts of medium-chain triglycerides were mixed evenly in a water bath at 75°C with stirring and heating to obtain a mixture. The mixture was then kept warm in a water bath at 75°C to obtain phase A.

[0080] (2) Add 25 parts of soybean lecithin, 8 parts of cholesterol and 0.5 parts of vitamin E to phase A, and then perform ultrasonic dispersion treatment for 10 minutes at a frequency of 40 kHz and a power of 300 W to obtain phase B;

[0081] (3) Add 1.5 parts of fumaric acid and 50 parts of water to phase B, and then perform high-pressure homogenization for 15 min under nitrogen atmosphere at a pressure of 40 MPa and a temperature of 60 °C to obtain homogenized liquid.

[0082] (4) The homogenized liquid was placed in a high-pressure microjet device and subjected to high-pressure microjet treatment at 140 MPa under a nitrogen atmosphere and circulated 5 times to obtain a liposome solution.

[0083] (5) The liposome solution was transferred to a vial and pre-lyophilized at -20°C for 24 h, and then pre-lyophilized at -55°C for 36 h to obtain lutein ester-fumaric acid complex liposomes.

[0084] Example 2

[0085] The method for preparing lutein ester-fumaric acid complex liposomes provided in this embodiment includes the following steps:

[0086] (1) By mass, 5 parts of lutein ester and 15 parts of sunflower seed oil were mixed evenly under stirring and heating in a water bath at 80°C to obtain a mixture. The mixture was then kept warm in a water bath at 80°C to obtain phase A.

[0087] (2) Add 20 parts egg yolk lecithin, 12 parts cholesterol and 0.3 parts vitamin E to phase A, and then perform ultrasonic dispersion treatment for 8 minutes at a frequency of 40 kHz and a power of 250 W to obtain phase B;

[0088] (3) Add 1.2 parts of fumaric acid and 47.7 parts of water to phase B, and then perform high-pressure homogenization for 10 min under nitrogen atmosphere at a pressure of 45 MPa and a temperature of 50 °C to obtain homogenized liquid.

[0089] (4) The homogenized liquid was placed in a high-pressure microjet device and subjected to high-pressure microjet treatment at 150 MPa under a nitrogen atmosphere and circulated 4 times to obtain a liposome solution.

[0090] (5) The liposome solution was transferred to a vial and pre-lyophilized at -19℃ for 22h, and then pre-lyophilized at -50℃ for 40h to obtain lutein ester-fumaric acid complex liposomes.

[0091] Example 3

[0092] The method for preparing lutein ester-fumaric acid complex liposomes provided in this embodiment includes the following steps:

[0093] (1) By mass, 8 parts of lutein ester, 5 parts of medium-chain triglycerides and 10 parts of peanut oil are mixed evenly under stirring and heating in a water bath at 75°C to obtain a mixture. The mixture is then kept warm in a water bath at 75°C to obtain phase A.

[0094] (2) Add 15 parts of soybean lecithin, 15 parts of hydrogenated lecithin, 5 parts of cholesterol and 1 part of vitamin E to phase A, and then perform ultrasonic dispersion treatment for 5 minutes at a frequency of 40 kHz and a power of 300 W to obtain phase B.

[0095] (3) Add 1 part fumaric acid and 41 parts water to phase B, and then perform high pressure homogenization for 14 min under nitrogen atmosphere at a pressure of 30 MPa and a temperature of 70 °C to obtain homogenized liquid.

[0096] (4) The homogenized liquid was placed in a high-pressure microjet device and subjected to high-pressure microjet treatment at 160 MPa under a nitrogen atmosphere and circulated 3 times to obtain a liposome solution.

[0097] (5) The liposome solution was transferred to a vial and pre-lyophilized at -18℃ for 23h, and then pre-lyophilized at -53℃ for 42h to obtain lutein ester-fumaric acid complex liposomes.

[0098] Example 4

[0099] The method for preparing lutein ester-fumaric acid complex liposomes provided in this embodiment includes the following steps:

[0100] (1) By mass, 3 parts of lutein ester and 5 parts of algal oil were mixed evenly under stirring and heating in a water bath at 70°C to obtain a mixture. The mixture was then kept warm in a water bath at 70°C to obtain phase A.

[0101] (2) Add 10 parts egg yolk lecithin, 5 parts hydrogenated lecithin, 15 parts cholesterol and 0.1 parts vitamin E to phase A, and then perform ultrasonic dispersion treatment for 15 minutes at a frequency of 40 kHz and a power of 200 W to obtain phase B.

[0102] (3) Add 2 parts of fumaric acid and 61.9 parts of water to phase B, and then perform high-pressure homogenization for 12 min under nitrogen atmosphere at a pressure of 50 MPa and a temperature of 40 °C to obtain homogenized liquid.

[0103] (4) The homogenized liquid was placed in a high-pressure microfluidic device and subjected to high-pressure microfluidic treatment at 120 MPa under a nitrogen atmosphere and circulated 6 times to obtain a liposome solution.

[0104] (5) The liposome solution was transferred to a vial and pre-lyophilized at -20℃ for 20h, and then pre-lyophilized at -45℃ for 48h to obtain lutein ester-fumaric acid complex liposomes.

[0105] Comparative Example 1 (without fumaric acid)

[0106] The preparation method of the liposome complex provided in this comparative example is basically the same as that in Example 1, except that:

[0107] (3) Add 50 parts of water to phase B, and then perform high-pressure homogenization for 15 minutes under nitrogen atmosphere at a pressure of 40 MPa and a temperature of 60 °C to obtain homogenized liquid.

[0108] Comparative Example 2 (without added Vitamin E)

[0109] The preparation method of the liposome complex provided in this comparative example is basically the same as that in Example 1, except that:

[0110] (2) Add 25 parts of soybean lecithin and 8 parts of cholesterol to phase A, and then perform ultrasonic dispersion treatment for 10 minutes at a frequency of 40 kHz and a power of 300 W to obtain phase B.

[0111] Comparative Example 3 (without high-pressure microjets)

[0112] The method for preparing the liposome complex provided in this comparative example includes the following steps:

[0113] (1) By mass, 5 parts of lutein ester and 10 parts of medium-chain triglycerides were mixed evenly in a water bath at 75°C with stirring and heating to obtain a mixture. The mixture was then kept warm in a water bath at 75°C to obtain phase A.

[0114] (2) Add 25 parts of soybean lecithin, 8 parts of cholesterol and 0.5 parts of vitamin E to phase A, and then perform ultrasonic dispersion treatment for 10 minutes at a frequency of 40 kHz and a power of 300 W to obtain phase B;

[0115] (3) Add 1.5 parts of fumaric acid and 50 parts of water to phase B, and then perform shear emulsification treatment for 15 min under nitrogen atmosphere at a rate of 12000 r / min to obtain liposome proemulsion.

[0116] (4) Under a nitrogen atmosphere, the liposome colostrum was subjected to high-pressure homogenization for 15 min at a pressure of 40 MPa and a temperature of 60 °C to obtain a homogenized solution.

[0117] (5) The homogenized solution was transferred to a vial and pre-lyophilized at -20°C for 24 h, and then pre-lyophilized at -55°C for 36 h to obtain the liposome complex.

[0118] Comparative Example 4 (without freeze-drying treatment)

[0119] The method for preparing the liposome complex provided in this comparative example includes the following steps:

[0120] (1) By mass, 5 parts of lutein ester and 10 parts of medium-chain triglycerides were mixed evenly in a water bath at 75°C with stirring and heating to obtain a mixture. The mixture was then kept warm in a water bath at 75°C to obtain phase A.

[0121] (2) Add 25 parts of soybean lecithin, 8 parts of cholesterol and 0.5 parts of vitamin E to phase A, and then perform ultrasonic dispersion treatment for 10 minutes at a frequency of 40 kHz and a power of 300 W to obtain phase B;

[0122] (3) Add 1.5 parts of fumaric acid and 50 parts of water to phase B, and then perform high-pressure homogenization for 15 min under nitrogen atmosphere at a pressure of 40 MPa and a temperature of 60 °C to obtain homogenized liquid.

[0123] (4) The homogenized liquid was placed in a high-pressure microfluidic device and subjected to high-pressure microfluidic treatment at 140 MPa under a nitrogen atmosphere and circulated 5 times to obtain the liposome complex.

[0124] Performance testing

[0125] (1) Particle size, zeta potential and encapsulation efficiency testing

[0126] The particle size and zeta potential of the lutein ester-fumaric acid composite liposomes in Examples 1-4 and the liposome complexes in Comparative Examples 1-4 were measured using a Malvern nanoparticle size analyzer. The encapsulation efficiency of the lutein ester-fumaric acid composite liposomes in Examples 1-4 and the liposome complexes in Comparative Examples 1-4 was also measured. The test results are shown in Table 1.

[0127] Table 1 Test Results

[0128]

[0129]

[0130] As shown in Table 1, overall, the lutein ester-fumaric acid composite liposomes in Examples 1-4 are superior to the liposome complexes in Comparative Examples 1, 2, and 3. The encapsulation efficiency of Example 1 was 97.61% ± 1.15%, compared to 91.78% ± 0.36% in Comparative Example 1, indicating an increase, possibly due to the surfactant effect of fumaric acid. Compared to Comparative Examples 2-3, Examples 1-4 showed a smaller average particle size, higher zeta potential and encapsulation efficiency, and a more stable and uniformly distributed system. The test results indicate that the method provided in Example 1 of this invention offers smaller particle size and better encapsulation effect, maximizing the preservation of lutein ester activity and improving the bioavailability of lutein ester. These results demonstrate that the lutein ester-fumaric acid composite liposomes in Examples 1-4 of this invention have the advantages of small average particle size, high zeta potential, and high encapsulation efficiency.

[0131] (2) Storage stability test

[0132] The samples were placed in sealed containers and stored at 4℃, room temperature (25℃), and high temperature (60℃) for 30 days, respectively. The encapsulation efficiency of the samples was tested at room temperature at the initial state and after 30 days (d).

[0133] The encapsulation efficiency of each sample was tested using the lutein ester-fumaric acid complex liposomes in Examples 1-4 and the liposome complexes in Comparative Examples 1-4, respectively, according to the method described above. The results are shown in Table 2.

[0134] Table 2 Storage stability test results

[0135]

[0136] As shown in Table 2, the lutein ester-fumaric acid complex liposomes prepared in Example 1 exhibited the highest encapsulation efficiency, best dispersibility and stability, and the most stable storage. The lutein ester-fumaric acid complex liposomes in Examples 1-4 demonstrated stronger stability than the liposome complexes in Comparative Examples 1-4, particularly Comparative Example 4. This indicates that the method provided by the present invention better protects the active ingredient (lutein ester), reduces loss, and has a longer shelf life. Furthermore, it maintains a relatively stable encapsulation efficiency even at high temperatures, eliminating the need for strict low-temperature storage conditions. These results demonstrate the excellent storage stability of the lutein ester-fumaric acid complex liposomes in Examples 1-4 of the present invention.

[0137] (3) In vitro simulated digestive system experiment

[0138] An in vitro simulated digestive system was used, and samples were tested sequentially according to the oral cavity, stomach, and intestine stages. After each stage, the concentration of lutein esters in the solution was determined by high-performance liquid chromatography (HPLC) to obtain the mass of lutein esters in the solution, and the bioavailability was calculated. The formula for calculating bioavailability (characterized by biological accessibility) is as follows:

[0139] Bioavailability % = (mass of lutein esters in solution ÷ mass of lutein esters in sample) × 100%;

[0140] Using the lutein ester-fumaric acid complex liposomes from Examples 1-4 and the liposome complexes from Comparative Examples 1-4 as samples, the bioavailability was calculated according to the above method, and the results are as follows. Figure 1 As shown in Table 3.

[0141] Table 3 Results of in vitro simulated digestive system experiments

[0142]

[0143] Depend on Figure 1 As shown in Table 3, after the in vitro simulated digestive system experiment, the lutein ester-fumaric acid complex liposome prepared in Example 1 had the highest bioavailability, with a bioavailability of 43.2% in the intestinal stage. Using the lutein ester-fumaric acid complex liposomes in Examples 1-4, the bioavailability of lutein ester was higher than that of the liposome complexes in Comparative Examples 1-4, especially stronger than that in Comparative Example 4, where the bioavailability of the liposome complex in the intestinal stage was only 21.6%. These results demonstrate that the method provided by the embodiments of the present invention can improve the absorption efficiency and bioavailability of lutein ester, and can achieve better results in practical applications.

[0144] (4) Solubility test

[0145] The solubility of lutein ester-fumaric acid complex liposomes in Examples 1-4, liposome complexes in Comparative Examples 1-4, and lutein esters in water was tested. The test results are shown in Table 4.

[0146] Table 4 Solubility Test Results

[0147] project Solubility (%) Example 1 92.6 Example 2 91.8 Example 3 92.2 Example 4 91.2 Comparative Example 1 89.9 Comparative Example 2 90.1 Comparative Example 3 90.2 Comparative Example 4 85.4 Lutein esters It is basically insoluble in water.

[0148] As shown in Table 4, the lutein ester-fumaric acid complex liposomes in Example 1 exhibited the highest solubility, at 92.6%. The solubility of the lutein ester-fumaric acid complex liposomes in Examples 1-4 was significantly higher than that of the liposome complexes in Comparative Examples 1-4, especially compared to the liposome complex in Comparative Example 4. Lutein esters are essentially insoluble in water. The method provided by this invention can significantly improve the water solubility of lutein esters, allowing them to more effectively cross the intestinal mucosa and enter the bloodstream, thereby increasing their bioavailability in vivo. It also provides better compatibility with aqueous formulation systems, enhancing their compatibility.

[0149] (5) The samples were irradiated under ultraviolet light (UV) with wavelengths of 254 nm and 365 nm, respectively, and the lutein ester retention rate in the samples was measured after different irradiation times (3 h, 6 h, 12 h, 24 h).

[0150] The lutein ester-fumaric acid complex liposomes in Examples 1-4 and the liposome complexes in Comparative Examples 1-4 were used as samples and tested according to the above method. The test results are shown in Table 5.

[0151] Table 5. Results of Lutein Ester Retention Rate Test

[0152]

[0153] As shown in Table 5, the lutein ester-fumaric acid composite liposomes in Example 1 retained 92.9% of the lutein ester after 24 hours of UV irradiation. The lutein ester retention rates of the lutein ester-fumaric acid composite liposomes in Examples 1-4 after UV irradiation were all stronger than those of the liposome complexes in Comparative Examples 1-4, especially stronger than those in Comparative Example 4. These results demonstrate that the lutein ester-fumaric acid composite liposomes prepared using the method provided in this invention possess excellent photostability and are not easily decomposed under light irradiation.

[0154] (6) Under light-protected conditions, the lutein ester-fumaric acid complex liposomes in Examples 1-4 and the liposome complexes in Comparative Examples 1-4 were heated at 80°C for 4 hours. The lutein ester retention rate was then tested to evaluate the thermal stability. The test results are shown in Table 6.

[0155] Table 6 Results of Lutein Ester Retention Rate Test

[0156]

[0157]

[0158] As shown in Table 6, the lutein ester-fumaric acid composite liposomes in Example 1 exhibited the highest lutein ester retention rate (90.1%) after heat treatment at 80°C for 4 hours. In Examples 1-4, the lutein ester-fumaric acid composite liposomes after heat treatment all showed higher lutein ester retention rates than the liposome complexes in Comparative Examples 1-4, especially compared to the liposome complex in Comparative Example 4. These results demonstrate that the lutein ester-fumaric acid composite liposomes prepared using the method provided in this invention possess excellent thermal stability and are not easily decomposed or degraded by heat.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a highly bioavailable lutein ester-fumaric acid complex liposome, characterized in that, Includes the following steps: Lutein esters were mixed with oils to obtain phase A; Phospholipids, cholesterol, and vitamin E were added to phase A and then subjected to ultrasonic dispersion to obtain phase B. Fumaric acid and water were added to phase B and then subjected to high-pressure homogenization under a nitrogen atmosphere to obtain a homogenized liquid. Under a nitrogen atmosphere, the homogenized liquid was subjected to high-pressure microfluidic treatment to obtain a liposome solution; The liposome solution was freeze-dried to obtain the lutein ester-fumaric acid complex liposomes; Of which, by weight, lutein esters are 3-8 parts, oils are 5-15 parts, phospholipids are 20-30 parts, cholesterol is 5-15 parts, vitamin E is 0.1-1 part, and fumaric acid is 1-2 parts; The freeze-drying process includes pre-freeze-drying and main freeze-drying processes in sequence. The temperature of the pre-freeze-drying treatment is higher than the temperature of the main freeze-drying treatment; in the pre-freeze-drying treatment, the temperature is -20 to -18°C and the time is 20 to 24 hours; in the main freeze-drying treatment, the temperature is -55 to -45°C and the time is 36 to 48 hours.

2. The method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes according to claim 1, characterized in that, In the ultrasonic dispersion process, the frequency is 40kHz, the power is 200-300W, and the time is 5-15min.

3. The method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes according to claim 1, characterized in that, In the high-pressure homogenization process, the pressure is 30–50 MPa, the temperature is 40–70 °C, and the time is 10–15 min.

4. The method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes according to claim 1, characterized in that, The homogenized liquid is subjected to high-pressure microfluidic treatment, including: The homogenized liquid was subjected to high-pressure microjet treatment at a pressure of 120-160 MPa and circulated 3-6 times.

5. The method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes according to claim 1, characterized in that, The process of mixing lutein esters with oils to obtain phase A includes: mixing lutein esters and oils evenly under stirring and heating in a water bath at 70-80°C to obtain a mixture, and then keeping the mixture at 70-80°C in a water bath to obtain phase A; And / or, the oil is at least one of medium-chain triglycerides, sunflower seed oil, peanut oil, and algal oil; And / or, the phospholipid is at least one of soybean lecithin, egg yolk lecithin, and hydrogenated lecithin.

6. A highly bioavailable lutein ester-fumaric acid complex liposome, characterized in that, It is prepared by the method for preparing highly bioavailable lutein ester-fumaric acid complex liposomes as described in any one of claims 1 to 5.

7. The use of the highly bioavailable lutein ester-fumaric acid complex liposome as described in claim 6 in the preparation of pharmaceuticals or health products with eye-protecting functions.

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