A method for the preparation of a phytosteryl stabilized liposomal carrier for delivery of a trophic factor

Abstract

The application belongs to the technical field of biological materials, and provides a preparation method of a phytosterol-stabilized liposome carrier for delivering a nutritional factor, comprising the following steps: dissolving phospholipid, phytosterol and the nutritional factor in a poor solvent, rotating and evaporating the obtained mixture under vacuum until the poor solvent is completely removed, and forming a uniform phytosterol-liposome film for delivering the nutritional factor; hydrating the liposome film with a phosphate buffer solution containing TwEEn-80 to form a crude liposome; and performing low-temperature water bath ultrasonic treatment on the crude liposome carrier loaded with the nutritional factor, so as to obtain the phytosterol-liposome carrier for delivering the nutritional factor. The phytosterol-liposome carrier for loading the nutritional factor provided by the application uses phytosterol instead of cholesterol as a membrane stabilizer, can improve the stability and bioavailability of the nutritional factor in the gastrointestinal environment while loading the nutritional factor, and avoids the adverse effects of excessive intake of cholesterol on the human body.

Description

Technical Field

[0001] This invention belongs to the field of biofunctional material preparation technology, and particularly relates to a method for preparing a liposome carrier for delivering nutrient factors stabilized by phytosterols. Background Technology

[0002] Nutrients possess a variety of biological activities, and various delivery systems have been developed to package, protect, and release them. However, some nutrient factors face challenges such as strong hydrophobicity, easy oxidation, poor stability, and low oral bioavailability, which affect their application.

[0003] Nutrient carriers prepared using related technologies exhibit low bioavailability after gastrointestinal digestion, meaning that the nutrients cannot be effectively absorbed and utilized by the human body. Furthermore, some current nutrient carriers contain lipid components, such as cholesterol, and excessive cholesterol intake increases the risk of cardiovascular disease and atherosclerosis. Therefore, to improve the bioavailability of nutrient carriers and reduce health risks, there is an urgent need to develop a cholesterol-free, healthier liposome carrier for delivering nutrients. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a liposome carrier for delivering nutritional factors that is stable by phytosterols, thereby solving the problem of low bioavailability of cholesterol-containing nutritional factor carriers in the prior art after gastrointestinal digestion.

[0005] According to a first aspect of the present invention, a method for preparing a liposome carrier for delivering nutrient factors stabilized by phytosterols is provided, comprising the following steps: dissolving phospholipids, phytosterols, and nutrient factors in a poor solvent; rotary evaporating the resulting mixture under vacuum until the poor solvent is completely removed to form a uniform liposome film for delivering nutrient factors by phytosterols; hydrating the liposome film with a phosphate buffer solution containing TwEEn-80 to form crude liposomes; and sonicating the crude liposome carrier carrying nutrient factors in a low-temperature water bath to obtain a liposome carrier for delivering nutrient factors by phytosterols.

[0006] Furthermore, the phospholipids are selected from at least one of soybean lecithin and egg yolk lecithin; the phytosterols are stigmasterol, β-sitosterol, rapeseed sterol, or campesterol; and the nutritional factors are selected from at least one of lutein, lycopene, curcumin, astaxanthin, or resveratrol.

[0007] Furthermore, methanol, ethanol, or chloroform are poor solvents for dissolving phospholipids, phytosterols, and nutrients.

[0008] Furthermore, the vacuum level for rotary evaporation under vacuum to completely remove the undesirable solvent is 0.00 MPa ~ 0.02 MPa.

[0009] Furthermore, the unsuitable solvent is 40 mL to 80 mL; the concentration ratio of phospholipids, phytosterols, and nutrient factors is 9:0.5:1 to 9:3:1; the mass fraction of TwEEn-80 in the phosphate buffer solution containing TwEEn-80 is 0.1% to 0.4%; the phosphate buffer solution containing TwEEn-80 is 100 mL to 200 mL; and the temperature for hydrating liposome membranes is 40°C to 50°C.

[0010] Furthermore, when using low-temperature water bath ultrasound to sonicate crude liposomes, the ultrasound power is 250W~350W; when using low-temperature water bath ultrasound to sonicate crude liposomes, each time the ultrasound is turned on for 1s~2s, then immediately turned off for 2s~3s, then turned on again for 1s~2s, then turned off again for 2s~3s, and so on repeatedly; when using low-temperature water bath ultrasound to sonicate crude liposomes, the water bath temperature is 2~8℃, and the ultrasound time is 10~20min.

[0011] Furthermore, crude liposome carriers for delivering nutrient factors from phytosterols were prepared by thin-film hydration, ethanol injection, reverse-phase evaporation, or microfluidic methods.

[0012] According to another aspect of the present invention, a liposome carrier for delivering nutrient factors from phytosterols is provided, characterized in that the liposome carrier for delivering nutrient factors from phytosterols is prepared by the above-described method for preparing a liposome carrier for delivering nutrient factors from phytosterols.

[0013] Experimental verification has shown that the liposome carrier based on phytosterol-loaded nutritional factors provided by this invention, with phytosterol replacing cholesterol as a membrane stabilizer, endows the liposome with better stability. While loading nutritional factors, it can also improve the stability and bioavailability of nutritional factors in the gastrointestinal environment, and avoid the adverse effects of excessive cholesterol intake on the human body.

[0014] Based on the implementation methods provided in the above aspects, this application can be further combined to provide more implementation methods. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0016] Figure 1 This is a simplified schematic diagram of the liposome carrier for delivering nutritional factors using phytosterols in this invention.

[0017] Figure 2Particle size and PDI of Lu-lip with different amounts of stigmasterol;

[0018] Figure 3 Figure A shows the Zeta potential changes of Lu-lip under different amounts of stigmasterol; Figure B shows the encapsulation efficiency of Lu-lip for lutein prepared with different stigmasterol addition ratios; Figure C shows the particle size changes of stigmasterol-stabilized lutein liposomes prepared with different amounts of stigmasterol under different temperature conditions.

[0019] Figure 4 Fourier transform infrared spectrum of lutein liposomes (Lu-lip) stabilized by stigmasterol; Figure 5 X-ray diffraction pattern of lutein liposomes (Lu-lip) stabilized by stigmasterol;

[0020] Figure 6 TSI curves of multiple light changes for lutein liposomes (Lu-lip) stabilized by stigmasterol;

[0021] Figure 7 Release curves of lutein under different amounts of stigmasterol;

[0022] Figure 8 Bioaccessibility of Lu-lip with different amounts of stigmasterol. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0024] This invention provides a method for preparing a liposome carrier for delivering nutrient factors stabilized by phytosterols, comprising the following steps: dissolving phospholipids, phytosterols, and nutrient factors in a poor solvent; rotary evaporating the resulting mixture under vacuum until the poor solvent is completely removed to form a uniform liposome film for delivering nutrient factors by phytosterols; hydrating the liposome film with a phosphate buffer solution containing TwEEn-80 to form crude liposomes; and sonicating the crude liposome carrier carrying nutrient factors in a low-temperature water bath to obtain the liposome carrier for delivering nutrient factors by phytosterols.

[0025] The method for preparing liposome carriers stabilized by phytosterols provided by this invention has simple steps. The liposome carriers for delivering nutritional factors stabilized by phytosterols have good stability and uniform particle size distribution. They can avoid the adverse effects of excessive cholesterol intake on the human body. While loading nutritional factors, they can also improve the stability of nutritional factors in the gastrointestinal environment and improve the bioavailability of nutritional factors.

[0026] Liposomes are an important food delivery system due to their attractive properties. Typically composed of one or more phospholipid bilayers, they represent microscopic lipid vesicles—spherical, self-enclosed structures assembled like cell membranes. This bilayer structure reduces the side effects of the loaded substance, improves its environmental stability, allows for controlled release, and exhibits high biodegradability and biocompatibility in vivo. Liposomes are promising nanomedicine delivery systems due to their low immunogenicity, good stability, low toxicity, and high cost. However, the preparation of liposomes usually requires the addition of a certain amount of cholesterol. Excessive cholesterol intake increases the risk of cardiovascular disease and atherosclerosis. With increasing demands for food quality and health, cholesterol limits the further development of liposomes in the food industry. Phytosterols, with structures similar to cholesterol, offer healthier benefits. Phytosterols are a class of triterpenoid compounds widely found in plant cells and have attracted attention for their ability to lower serum cholesterol levels. Studies have shown that phytosterols can reduce serum cholesterol levels by decreasing cholesterol synthesis and increasing cholesterol reverse transport and excretion pathways, and possess physiological functions such as immunomodulation, anti-inflammation, anti-oxidation, and free radical scavenging. The addition of phytosterols makes the phospholipid membrane structure more compact, helping to improve the orderly arrangement of liposome membranes and increase their rigidity. Simultaneously, phytosterols bind to the phospholipid bilayer to regulate the fluidity and permeability of the phospholipid membrane. Liposome carriers that deliver nutrients stably using phytosterols can not only reduce the loss of nutrients in the gastrointestinal environment and improve their stability, but also achieve targeted release, improving the bioavailability of bioactive substances.

[0027] In this invention, since the liposome suspension delivering nutrients, stabilized by phytosterols, passes through the oral cavity in a very short time, oral digestion is not considered. Upon entering the stomach, the osmotic pressure difference between the internal aqueous phase and the external low-pH environment causes contraction, resulting in a reduction in diameter and maintaining a relatively intact structure. Furthermore, the interaction forces of the lipid bilayer (hydrophobic interactions, van der Waals forces, hydrogen bonds) prevent the release and degradation of nutrients encapsulated within the liposomes. Subsequently, the combined action of bile salts and intestinal enzymes such as phospholipase, pancreatic lipase, and cholesterol lipase leads to the hydrolysis of liposome phospholipids. Bile salts accelerate the degradation of the bilayer membrane, disrupting the liposome structure and causing the delivered nutrients to leak from the liposomes, aiding in the formation of micelles. These micelles are further absorbed by intestinal cells, achieving targeted release of nutrients and improving their bioavailability.

[0028] Optionally, the phospholipids are selected from at least one of soybean lecithin and egg yolk lecithin; the phytosterols are stigmasterol, β-sitosterol, brassosterol, or campesterol; and the nutritional factors are selected from at least one of lutein, lycopene, curcumin, astaxanthin, or resveratrol.

[0029] Optionally, methanol, ethanol, or chloroform are poor solvents for dissolving phospholipids, phytosterols, and nutrients; phospholipids, phytosterols, and nutrients are all soluble in organic solvents such as methanol, ethanol, or chloroform and have a certain degree of morphological stability.

[0030] Optionally, the vacuum degree for rotary evaporation under vacuum to completely remove the undesirable solvent is 0.00 MPa ~ 0.02 MPa. If the vacuum degree is too high or too low, the undesirable solvent cannot be completely removed, which will affect the stability of the liposome film and the encapsulation efficiency of the nutrient factors, and the nutrient factors cannot be encapsulated well, and some nutrient factors will be leaked out. Experiments have verified that when the vacuum degree is within the above range, a uniform liposome film carrying nutrient factors can be formed.

[0031] Optionally, the amount of unsuitable solvent is 40 mL to 80 mL. Unsuitable solvent promotes the dissolution of phospholipids, phytosterols and nutrients. If the amount is too low, it will not be conducive to the dissolution of phospholipids, phytosterols and nutrients, and the lipids will not be dispersed, resulting in uneven distribution of the formed liposome film. If the amount is too high, it will cause unnecessary waste of unsuitable solvent. Experiments have verified that when the amount of unsuitable solvent added is within the above range, it can ensure the formation of a uniform and stable liposome film and avoid unnecessary waste.

[0032] Optionally, the concentration ratio of phospholipids, phytosterols, and nutritional factors is 9:0.5:1 to 9:3:1. The addition of phytosterols helps to improve the orderly arrangement of liposome membranes, increase their rigidity, and make the phospholipid membrane structure more compact. If the concentration of phytosterols is too low, too few liposomes will be formed or insufficient to encapsulate the added nutritional factors. If the concentration of phytosterols is too high, too many liposomes will be formed, resulting in reduced stability and leakage of the loaded nutritional factors. Experiments have verified that when the amount of phytosterols added is within the above-mentioned range, the concentration of phytosterols is within a suitable range, which can ensure that the liposome size is within a suitable range and that the stability of the liposomes is also guaranteed.

[0033] Preferably, as verified by experiments, when the ratio of phospholipid: phytosterol: nutrient factor is 9:1:1, the addition of phytosterol makes the phospholipid bilayer structure more regularly and orderly arranged and evenly distributed, and the liposomes that deliver nutrient factors stably by phytosterol have the best loading effect on nutrient factors.

[0034] Optionally, the temperature for complete removal of undesirable solvents by rotary evaporation under vacuum is 40-50°C. Too low a temperature is not conducive to the effective removal of undesirable solvents, nor is it conducive to the formation of a uniform and stable liposome film. Since the nutrient factors are very sensitive to temperature, too high a temperature may cause partial denaturation of the nutrient factors, which is not conducive to the formation of the liposome film. Experiments have verified that when the temperature is within the above-mentioned range, a uniform liposome film carrying nutrient factors can be formed.

[0035] Optionally, the phosphate buffer solution for the hydrated liposome membrane contains 100 mL to 200 mL of TwEEn-80 phosphate buffer solution.

[0036] Optionally, the phosphate buffer solution for the hydrated liposome membrane contains 0.1% to 0.4% TwEEn-80 by mass. TwEEn-80 is a surfactant whose emulsifying effect can regulate the size of the formed liposomes. A lower TwEEn-80 mass fraction results in poor ordered arrangement and rigidity of the liposome membrane, while a higher TwEEn-80 mass fraction can solubilize the wall material components of the liposomes, disrupting the integrity of the liposome structure and leading to leakage of nutrients. Experiments have verified that when the phosphate buffer solution for the hydrated liposome membrane contains TwEEn-80 within the above-mentioned range, it can increase the effective thickness of the membrane without damaging the phospholipid membrane, thus improving the stability of the liposomes.

[0037] Optionally, the temperature for hydrating liposome membranes is 40-50℃. If the temperature is too low, the phosphate buffer solution containing TwEEn-80 cannot completely hydrate the liposome membrane, resulting in low efficiency. If the temperature is too high, some nutrients may denature, affecting the encapsulation efficiency and also hindering liposome formation. Experiments have verified that when the temperature is within the above range, uniform and stable liposomes carrying nutrients stabilized by phytosterols can be formed.

[0038] Optionally, when using low-temperature water bath ultrasound to sonicate crude liposomes, the ultrasound power is 250W~350W; during this process, the ultrasound is turned on for 1~2 seconds, then immediately turned off for 2~3 seconds, then turned on again for 1~2 seconds, then turned off again for 2~3 seconds, and so on repeatedly; the water bath temperature is 2~8℃, and the ultrasound time is 10~20 minutes. Ultrasonic energy is used to separate lipid aggregates into smaller monolayer liposomes. The ultrasound time and intensity have a decisive effect on the size of the prepared liposome particles. When the ultrasound temperature is too high, since ultrasound is an exothermic process for the crude liposome carrier carrying nutrients, a higher reaction temperature is actually detrimental to liposome formation. Excessive ultrasound power or time can cause liposome membrane rupture, flocculation, and aggregation, leading to leakage of the loaded nutrients and reduced stability. Experiments have verified that when the reaction temperature, ultrasound power, and time are set within the above-mentioned ranges, it is beneficial to regulate the fluidity and permeability of the phospholipid membrane and promote liposome formation.

[0039] Alternatively, crude liposome carriers for delivering nutrient factors from phytosterols can be prepared by thin-film hydration, ethanol injection, reverse-phase evaporation, or microfluidic methods.

[0040] Preferably, liposomes that deliver nutrients stably from phytosterols are prepared by thin-film hydration. Thin-film hydration is simple to operate, economical and practical, and is usually combined with methods such as ultrasound and homogenization to obtain single-chamber liposomes with uniform particle size and high drug encapsulation capacity.

[0041] This invention also provides a liposome carrier for delivering nutritional factors using phytosterols. The liposome carrier is prepared using the above-described method for preparing a phytosterol-stabilized liposome carrier for delivering nutritional factors. This method avoids the adverse effects of excessive cholesterol intake on the human body and improves the stability and bioavailability of nutritional factors in the gastrointestinal environment while loading them.

[0042] like Figure 1 As shown, taking lutein as an example, this invention proposes a method for preparing a liposome carrier for delivering nutrient factors stabilized by phytosterols, comprising the following steps:

[0043] (1) Soybean lecithin (SL), stigmasterol, and lutein were dissolved in an ethanol solution using a thin-film evaporation method. The resulting mixture was then rotary evaporated under vacuum until all ethanol was removed to form a uniform liposome film for delivering lutein, stabilized by stigmasterol. (2) The liposome film was hydrated with a phosphate buffer solution containing TwEEn-80 to form crude liposomes. (3) The crude liposome carrier carrying lutein was further homogenized by water bath and low-temperature ultrasonication to obtain a liposome carrier (Lutein-liposome, Lu-lip) for delivering nutrients, stabilized by phytosterol. This renewable composite material shows potential advantages in the fields of food, environmental protection, and pharmaceutical formulation. In addition, the raw materials are safe, biodegradable, readily available, inexpensive, and easy to commercialize.

[0044] In a possible embodiment of the present invention (not shown), it can be Figure 1 The lutein in the formula can be replaced with lycopene, curcumin, astaxanthin, or resveratrol.

[0045] The specific embodiments of the present invention will be described in detail below:

[0046] Example 1

[0047] Control group: Preparation of liposome carriers for delivering nutrient factors without phytosterols and carrying lutein.

[0048] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:0:1. The resulting mixture was rotary evaporated under a vacuum of 0.01 MPa until the ethanol was completely removed to form a uniform lutein liposome film.

[0049] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0050] (3) The crude liposome carrier carrying lutein was ultrasonically treated in a water bath at 4°C with a power of 300 W. The ultrasonic treatment was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This process was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein without phytosterols.

[0051] Example 2

[0052] Preparation of liposomes carrying lutein, which are stable delivery factors of phytosterols.

[0053] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:0.5:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0054] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0055] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0056] Example 3

[0057] Preparation of liposomes carrying lutein, which are stable delivery factors of phytosterols.

[0058] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0059] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0060] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0061] Example 4

[0062] Preparation of liposomes carrying lutein, which are stable delivery factors of phytosterols.

[0063] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:2:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0064] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0065] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0066] Example 5

[0067] Preparation of liposomes carrying lutein, which are stable delivery factors of phytosterols.

[0068] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:3:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0069] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0070] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0071] Example 6

[0072] (1) Soy lecithin (SL), stigmasterol and lutein were added to 10 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0073] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0074] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0075] Example 7

[0076] (1) Soy lecithin (SL), stigmasterol and lutein were added to 100 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0077] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0078] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0079] Example 8

[0080] (1) Soy lecithin (SL), stigmasterol and lutein were added to 200 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0081] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0082] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0083] Example 9

[0084] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0085] (2) Use 100 mL of phosphate buffer solution containing 0.6 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0086] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0087] Example 10

[0088] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0089] (2) Use 100 mL of phosphate buffer solution containing 1.0 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0090] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0091] Example 11

[0092] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0093] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0094] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 30 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0095] Example 12

[0096] (1) Soy lecithin (SL), stigmasterol and lutein were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0097] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0098] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 50 min to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

[0099] Example 13

[0100] (1) Soy lecithin (SL), β-sitosterol and lutein were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0101] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0102] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by β-sitosterol.

[0103] Example 14

[0104] (1) Soybean lecithin (SL), rapeseed sterol and lutein were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film.

[0105] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0106] (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by β-sitosterol.

[0107] Example 15

[0108] Preparation of liposome carriers for delivering nutrients from phytosterols and loaded with curcumin.

[0109] (1) Soy lecithin (SL), stigmasterol and curcumin were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was rotary evaporated under a vacuum of 0.01 MPa until the ethanol was completely removed to form a uniform curcumin liposome film.

[0110] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0111] (3) The crude liposome carrier carrying curcumin was ultrasonically treated in a water bath at 4°C with a power of 300 W. The ultrasonic treatment was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This process was repeated for 10 minutes to further homogenize the liposome carrier that delivers curcumin stably with stigmasterol.

[0112] Example 16

[0113] Preparation of liposomes carrying lycopene and phytosterol-stable delivery factors.

[0114] (1) Soy lecithin (SL), stigmasterol and lycopene were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until the ethanol was completely removed to form a uniform lycopene liposome film.

[0115] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0116] (3) The crude liposome carrier carrying lycopene was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lycopene stably by stigmasterol.

[0117] Example 17

[0118] Preparation of liposome carriers for delivering nutrients from phytosterols and loaded with resveratrol.

[0119] (1) Soybean lecithin (SL), stigmasterol and resveratrol were added to 50 mL of ethanol in a ratio of 9:1:1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lycopene liposome film.

[0120] (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes.

[0121] (3) The crude liposome carrier loaded with resveratrol was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers resveratrol stably with stigmasterol.

[0122] Comparing Examples 1, 2, 3, 4, and 5, it was found that the soybean lecithin (SL), stigmasterol, and lutein were most stable when the ratio was 9:1:1. Figure 3 As shown in B, the encapsulation efficiency for lutein is the best.

[0123] Comparing Examples 2, 6, 7, and 8, it was found that soybean lecithin, stigmasterol, and lutein were dissolved in different volumes of ethanol and rotary evaporated to form liposome films. Among them, the volume of the unsuitable solvent was 50 ml, which yielded the best results and formed a uniform and stable lutein liposome film.

[0124] Comparing Examples 2, 9, and 10, it was found that when the phosphate buffer solution containing TwEEn-80 had a mass fraction of 0.2 wt%, Lu-lip exhibited the highest stability, which increased the effective thickness of the membrane without damaging the phospholipid membrane.

[0125] Comparing Examples 2, 11, and 12, we found that Lu-lip obtained under different cryo-ultrasound times showed reduced vesicle fusion after cryo-ultrasound, with 10 min of cryo-ultrasound showing the best effect.

[0126] Comparing Examples 2, 13, and 14, it was found that the liposome carriers for delivering lutein, stabilized by different phytosterols, all exhibited good stability.

[0127] Example 2, Examples 15, 16, and 17 demonstrate that the liposome carrier of the present invention, which is stabilized by phytosterols and carries nutritional factors, is applicable to some other nutritional factors, which will not be listed here.

[0128] Figure 2The particle size and PDI (Polydispersity Index) of Lu-lip with different amounts of stigmasterol were investigated. The addition of stigmasterol reduced the particle size of Lu-lip. A PDI < 0.3 indicates that Lu-lip is evenly and stably distributed. The particle size was smallest and had high stability when SL, stigmasterol and Lutein were in a ratio of 9:1:1.

[0129] Depend on Figure 3 As shown in Figure A, the Zeta potential of Lu-lip varies with the content of stigmasterol, and its absolute value is consistently above 25 mV, indicating good stability and reduced aggregation due to the repulsive resistance generated by the negative charge on the liposome surface. Stigmasterol can enhance the hydrophobic interaction with the fatty acyl chains, further stabilizing the bilayer and improving the stability of Lu-lip. Figure 3 As shown in section B, considering the overall encapsulation efficiency (EE) of lutein, Lu-lip prepared with a 9:1:1 ratio of soybean lecithin (SL), stigmasterol, and lutein showed the best lutein loading effect. Lu-lip with added stigmasterol exhibited higher lutein encapsulation efficiency than Lu-lip without stigmasterol. The addition of stigmasterol helps improve the orderly arrangement of the lipid membrane, making the phospholipid membrane structure more compact. Simultaneously, stigmasterol binds to the phospholipid bilayer to regulate the fluidity and permeability of the phospholipid membrane, improving the stability of Lu-lip and achieving effective encapsulation of lutein while also promoting its effective release in the gastrointestinal tract. Figure 3 As shown in C, the lutein liposomes prepared by adding different amounts of stigmasterol showed the best thermal stability when treated at different temperatures, with a soybean lecithin:stigmasterol:lutein ratio of 9:1:1.

[0130] Depend on Figure 4 As can be seen from the Fourier transform infrared spectrum shown, Figure 4 A represents the infrared spectra of Lu-lip containing stigmasterol, soybean lecithin, empty liposomes, and stigmasterol to soybean lecithin in ratios of 0:9 and 1:9. Lu-lip is spectral in the range of 3200-3600 cm⁻¹. -1 The range shows a broad and strong peak, corresponding to the OH tensile vibration. The -OH tensile vibration of SL and Lutein ranges from 3403 cm⁻¹. -1 and 3355 cm -1 Moved to 3394 cm -1 and 3388 cm -1 This indicates a hydrogen bond interaction between lutein and SL; the stretching vibration peak of stigmasterol at C=O is observed from 1740 cm⁻¹. -1 Reduced to 1738 cm -1 and 1736 cm-1 This indicates that the ester carbonyl group may participate in the formation of hydrogen bonds. The C=O group is very sensitive to changes in the conformation and orientation of ester bonds, suggesting that the conformation of ester bonds in phospholipid molecules changes when lutein is loaded. Hydrogen bonds are formed between the hydroxyl group of stigmasterol and the oxygen group of phospholipids, thereby reducing the frequency of stretching vibrations. Stigmasterol can induce stronger molecular interactions to form a more stable interfacial film. Almost no characteristic peaks of lutein were observed in Lu-lip, indicating effective encapsulation of lutein in liposomes. Figure 4 B shows the infrared spectra of soybean lecithin and lutein liposomes with different amounts of stigmasterol. It can be concluded that the change in the -CH vibration in CH2 is mainly due to the difference in the saturation of the fatty acid chains in the phospholipids. With increasing stigmasterol content, PO... 2- The asymmetric tensile vibration absorption peak is from 1240 cm⁻¹ -1 Redshifted to 1239 and 1238 cm -1 This redshift is due to the formation of hydrogen bonds between the hydroxyl groups of stigmasterol and the oxygen groups of phospholipids, which reduces the frequency of tensile vibrations and forms a stable interfacial film.

[0131] like Figure 5 As shown, Figure 5 Figures A and B show the XRD patterns of lutein and stigmasterol, respectively, with different amounts of stigmasterol added to lutein liposomes. Powder XRD (X-ray diffraction) of figures A and B shows that lutein was successfully encapsulated in stigmasterol-stabilized lutein liposomes. In figure A, the sharp characteristic peak of lutein disappears, indicating an amorphous state. Figure 5 As shown in Figure B, the absorption peaks of lutein liposomes with different amounts of stigmasterol at 15° and 18° are the absorption peaks of stigmasterol.

[0132] like Figure 6 As shown, TSI (Turbiscan stability index) data indicate that lutein liposomes stabilized by stigmasterol exhibit good physical stability and minimal membrane fusion, demonstrating the stability of Lu-lip.

[0133] like Figure 7 As shown in the in vitro simulated release curve analysis, the cumulative release rate of lutein liposomes stabilized by stigmasterol is relatively slow. The rigidity of the liposome membrane gradually increases with the addition of stigmasterol, ensuring the stability of Lu-lip.

[0134] Figure 8 This is a schematic diagram showing the bioavailability of stable lutein liposomes with different amounts of stigmasterol. The bioavailability of Lu-lip varies with different amounts of stigmasterol, demonstrating the controllability of lutein release.

[0135] Specifically, such as Figure 8 As shown, the bioavailability of lutein liposomes with different stigmasterol additions was highest at a ratio of 21.69% for soybean lecithin:stigmasterol:lutein of 9:1:1. The appropriate addition of stigmasterol increased the stability of Lu-lip, providing better protection for the lutein load and preventing lutein from interacting with other chemicals in digestive fluids, thereby reducing its degradation before intestinal digestion. Liposomes with ratios of 9:2:1 and 9:3:1 may form non-vesicular structures within the membrane, and cross-linking between particles is likely to occur, which may cause premature release of lutein, resulting in partial loss of lutein.

[0136] In related technologies, there have been studies on encapsulating bioactive substances using liposomes. For example, CN105726482A discloses a lutein nanoliposome and its preparation method. However, the addition of cholesterol as a raw material during the preparation process, and the negative health effects of cholesterol, limit its application in liposomes. CN111718288A discloses a lutein ester and water-soluble lutein ester microcapsules and their preparation method. The high temperature during preparation increases the risk of degradation of the lutein ester, affecting its stability. CN101716144A discloses a lutein hydrogenated phospholipid liposome precursor and its preparation method, which involves adding lutein and hydrogenated phospholipid to a solution of dichloromethane and ethanol. Dichloromethane is highly toxic and prone to residues. Furthermore, the supercritical carbon dioxide antisolvent method for preparing lutein liposomes involves high operating pressure and high cost. None of the above-mentioned related technologies disclose the preparation method of phytosterol-stabilized liposomes of the present invention, nor the phytosterol-stabilized nutrient-carrying liposome carrier prepared by the present invention.

[0137] The beneficial technical effects of this invention are as follows:

[0138] The present invention provides a method for preparing liposomes stabilized by phytosterols and the liposome carriers for carrying nutrient factors stabilized by phytosterols obtained therefrom. By using phytosterols to replace cholesterol as a membrane stabilizer, phytosterols become an indispensable structural component of the complete membrane structure, thereby enhancing the bioaccessibility and controllability of nutrient factors and making them more beneficial to human health.

[0139] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a liposome carrier for delivering nutritional factors stabilized by phytosterols, characterized in that, Includes the following steps: (1) Soybean lecithin, stigmasterol and lutein were dissolved in 50 mL of ethanol in a ratio of 9:1:

1. The resulting mixture was then rotary evaporated under a vacuum of 0.01 MPa until all ethanol was removed to form a uniform lutein liposome film. (2) Use 100 mL of phosphate buffer solution containing 0.2 wt% TwEEn-80 to hydrate the liposome membrane at 50 °C to form crude liposomes; (3) The crude liposome carrier carrying lutein was ultrasonicated in a water bath at 4°C with a power of 300 W. The ultrasonic was turned on for 1 second and then turned off for 2 seconds. Then it was turned on again for 1 second and then turned off for 2 seconds. This cycle was repeated for 10 minutes to further homogenize the liposome carrier that delivers lutein stably by stigmasterol.

2. A liposome carrier for delivering nutritional factors stabilized by phytosterols, characterized in that, The liposome carrier for delivering nutrient factors stabilized by phytosterols was prepared using the method described in claim 1.

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