Preparation method of lipidosome carrier for stably delivering nutritional factors by phytosterol

Abstract

The invention belongs to the technical field of biological materials, and provides a preparation method of a lipidosome carrier capable of stably delivering nutritional factors by phytosterol, which comprises the following steps: dissolving phospholipid, phytosterol and nutritional factors in a poor solvent, and carrying out rotary evaporation on the obtained mixture under vacuum until the poor solvent is completely removed to obtain the lipidosome carrier capable of stably delivering the nutritional factors by phytosterol. A uniform lipidosome film for delivering the nutritional factors by the phytosterol is formed; hydrating the lipidosome membrane with a phosphate buffer solution containing TweEEn-80 to form a crude lipidosome; the preparation method comprises the following steps: carrying out low-temperature water bath ultrasonic treatment on a crude lipidosome carrier loaded with the nutritional factor to obtain the lipidosome carrier for delivering the nutritional factor by the phytosterol. The stability and the biological accessibility of the nutritional factors in the gastrointestinal tract environment can be improved while the nutritional factors are loaded, and the adverse effect on the human body caused by excessive intake of cholesterol is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of biological functional materials, and in particular relates to a method for preparing a liposome carrier for delivering nutritional factors stably using phytosterols. Background Art

[0002] Nutritional factors have multiple biological activities, and related technologies have developed various delivery systems to package, protect and release them. However, some nutritional factors face challenges such as strong hydrophobicity, easy oxidation, poor stability and low oral bioavailability, which affect their application.

[0003] The nutritional factor carriers prepared by related technologies show low bioaccessibility after gastrointestinal digestion, which means that the nutritional factors cannot be effectively absorbed and utilized by the human body. In addition, some current nutritional factor carriers contain lipid components, such as cholesterol, and excessive intake of cholesterol increases the risk of cardiovascular and cerebrovascular diseases and atherosclerosis. Therefore, in order to improve the bioavailability of nutritional factors and reduce health risks, it is urgent to develop a cholesterol-free, healthier liposome carrier for delivering nutritional factors. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols, so as to solve the problem of low bioaccessibility of nutritional factor carriers containing cholesterol after gastrointestinal digestion in the prior art.

[0005] According to a first aspect of the present invention, there is provided a method for preparing a liposome carrier for delivering nutritional factors stably by plant sterols, comprising the following steps: dissolving phospholipids, plant sterols and nutritional 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 nutritional factors by plant sterols; hydrating the liposome film with a phosphate buffer solution containing TwEEn-80 to form a crude liposome; and sonicating the crude liposome carrier carrying the nutritional factors in a low-temperature water bath to obtain a liposome carrier for delivering nutritional factors by plant sterols.

[0006] Furthermore, the phospholipid is selected from at least one of soybean lecithin and egg yolk lecithin; the phytosterol is stigmasterol, β-sitosterol, brassicasterol or campesterol; and the nutritional factor is selected from at least one of lutein, lycopene, curcumin, astaxanthin or resveratrol.

[0007] Furthermore, the poor solvent for dissolving phospholipids, phytosterols and nutritional factors is methanol, ethanol or chloroform.

[0008] Furthermore, the vacuum degree of rotary evaporation under vacuum to completely remove the poor solvent is 0.00MPa ~ 0.02MPa.

[0009] Furthermore, the poor solvent is 40 mL ~ 80 mL; the concentration ratio of phospholipids, phytosterols and nutritional factors is 9:0.5:1~9:3:1; the mass fraction of TwEEn-80 in the phosphate buffer solution containing TwEEn-80 is 0.1%~0.4%; the phosphate buffer solution containing TwEEn-80 is 100 mL ~ 200 mL; and the temperature when hydrating the liposome film is 40℃ ~50℃.

[0010] Furthermore, when the crude liposomes are ultrasonically prepared in a low-temperature water bath, the ultrasonic power is 250W~350W; when the crude liposomes are ultrasonically prepared in a low-temperature water bath, each time the ultrasonic wave is turned on for 1s~2s, then turned off for 2s~3s, then turned on again for 1s~2s, then turned off again for 2s~3s, and this cycle is repeated; when the crude liposomes are ultrasonically prepared in a low-temperature water bath, the water bath temperature is 2~8℃, and the ultrasonic time is 10~20min.

[0011] Furthermore, crude liposome carriers for delivering nutritional factors by phytosterols are prepared by thin film hydration method, ethanol injection method, reverse phase evaporation method or microfluidics method.

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

[0013] Experimental verification shows that the liposome carrier provided by the present invention is based on nutritional factors carried by plant sterols. Plant sterols replace cholesterol as a membrane stabilizer, which gives the liposome better stability. While loading the nutritional factors, it can also improve the stability and bioaccessibility of the nutritional factors in the gastrointestinal environment, and avoid the adverse effects of excessive cholesterol intake on the human body.

[0014] Based on the implementations provided in the above aspects, this application can also be further combined to provide more implementations. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 is a simplified schematic diagram of a liposome carrier for delivering nutritional factors by phytosterols in the present invention; Figure 2The particle size and PDI of Lu-lip with different stigmasterol addition amounts; Figure 3 Figure A in the middle shows the change of Zeta potential of Lu-lip with different stigmasterol addition amounts; Figure B shows the encapsulation efficiency of Lu-lip prepared with different stigmasterol addition ratios for lutein; Figure C shows the change of particle size of lutein liposomes stabilized by stigmasterol prepared with different stigmasterol addition amounts under different temperature conditions; Figure 4 is the Fourier transform infrared spectrum of lutein liposomes (Lu-lip) stabilized by stigmasterol; Figure 5 is the X-ray diffraction spectrum of lutein liposomes (Lu-lip) stabilized by stigmasterol; Figure 6 The multiple light-variable TSI curves of lutein liposomes (Lu-lip) stabilized by stigmasterol; Figure 7 The release curve of lutein under different stigmasterol addition amounts; Figure 8 The bioaccessibility diagram of Lu-lip with different stigmasterol addition amounts. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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.

[0018] The invention provides a method for preparing a liposome carrier for delivering nutritional factors stably by plant sterols, comprising the following steps: dissolving phospholipids, plant sterols and nutritional factors in a poor solvent, rotary evaporating the obtained mixture under vacuum until the poor solvent is completely removed to form a uniform liposome film for delivering nutritional factors by plant sterols; hydrating the liposome film with a phosphate buffer solution containing TwEEn-80 to form a crude liposome; and subjecting the crude liposome carrier carrying the nutritional factors to low-temperature water bath ultrasound to obtain the liposome carrier for delivering nutritional factors by plant sterols.

[0019] The preparation method of the liposome carrier stabilized by plant sterols provided by the present invention has simple steps. The prepared liposome carrier for delivering nutritional factors stabilized by plant sterols has good stability and uniform particle size distribution, can avoid the adverse effects of excessive cholesterol intake on the human body, and can improve the stability of the nutritional factors in the gastrointestinal environment while loading the nutritional factors, thereby improving the biological accessibility of the nutritional factors.

[0020] Among them, liposomes are one of the important food delivery systems with attractive properties. They are usually composed of one or more phospholipid bilayers. They represent microparticle lipid vesicles, i.e., spherical self-enclosed structures, bilayer structures assembled like cell membranes, which can reduce the side effects of the loaded substances and improve their environmental stability, control release, and have high biodegradability and biocompatibility in vivo. Liposomes are promising nano drug delivery systems due to their low immunogenicity, good stability, low toxicity, and high cost. The preparation of liposomes usually requires the addition of a certain amount of cholesterol. However, excessive cholesterol intake increases the risk of cardiovascular and cerebrovascular diseases and atherosclerosis. As people's demand for food quality and health increases, cholesterol limits the further development of liposomes in the food industry. Phytosterols have a similar structure to cholesterol and have healthier effects. Phytosterols are a class of triterpene compounds that are widely present in plant cells and have attracted much attention for their ability to reduce serum cholesterol levels. Studies have shown that phytosterols can reduce the cholesterol content in serum by reducing the synthesis of cholesterol and increasing the reverse transport and excretion of cholesterol. They have physiological functions such as immunomodulation, anti-inflammatory, anti-oxidation, and free radical scavenging. The addition of phytosterols makes the structure of the phospholipid membrane more compact, helps to improve the orderly arrangement of the liposome membrane, and increases its rigidity. At the same time, phytosterols are bound to the bilayer of phospholipids to regulate the fluidity and permeability of the phospholipid membrane. Liposome carriers that deliver nutritional factors stably by phytosterols can not only reduce the loss of nutritional factors in the gastrointestinal environment and improve their stability, but also achieve directional release and improve the bioavailability of bioactive substances.

[0021] In the present invention, because the liposome suspension for delivering nutritional factors stabilized by plant sterols passes through the oral cavity in a very short time, its oral digestion is not considered. When it enters the stomach, the contraction caused by the osmotic pressure difference between the aqueous phase inside the liposome and the external low pH environment causes its diameter to decrease and maintain a relatively complete structure. Due to the interaction force of the lipid bilayer (hydrophobic interaction, van der Waals force, hydrogen bond), the release and degradation of nutrients encapsulated in the liposome can also be prevented. Afterwards, under the combined action of bile salts and intestinal enzymes such as phospholipases, pancreatic lipases and cholesterol lipases, the hydrolysis of liposome phospholipids is caused. Bile salts accelerate the degradation of the bilayer membrane to destroy the liposome structure and cause the delivered nutritional factors to leak from the liposome and help form micelles, which are further absorbed by intestinal cells, achieving the directional release of the nutritional factors and improving their bioavailability.

[0022] Optionally, the phospholipid is selected from at least one of soybean lecithin and egg yolk lecithin; the phytosterol is stigmasterol, β-sitosterol, brassica sterol or campesterol; and the nutritional factor is selected from at least one of lutein, lycopene, curcumin, astaxanthin or resveratrol.

[0023] Optionally, the poor solvent for dissolving phospholipids, phytosterols and nutritional factors is methanol, ethanol or chloroform; phospholipids, phytosterols and nutritional factors are all soluble in organic solvents such as methanol, ethanol, or chloroform and have a certain morphological stability.

[0024] Optionally, the vacuum degree of rotary evaporation under vacuum to completely remove the poor solvent is 0.00MPa ~ 0.02 MPa; if the vacuum degree is too high or too low, the poor solvent cannot be completely removed, which affects the stability of the liposome film and the encapsulation efficiency of the nutritional factors, and the nutritional factors cannot be better encapsulated, and some nutritional factors will be missed; it has been verified by experiments that when the vacuum degree is within the above numerical range, a uniform liposome film carrying nutritional factors can be formed.

[0025] Optionally, the poor solvent is 40 mL ~ 80 mL; the poor solvent promotes the dissolution of phospholipids, phytosterols and nutritional factors. If it is too low, it is not conducive to the dissolution of phospholipids, phytosterols and nutritional factors, and the lipids cannot be dispersed, resulting in uneven distribution of the formed liposome film; if it is too high, it causes unnecessary waste of the poor solvent; it has been verified by experiments that when the addition amount of the poor solvent is within the above numerical range, it can not only ensure the formation of a uniform and stable liposome film, but also avoid unnecessary waste.

[0026] Optionally, the concentration ratio of phospholipids, phytosterols and nutritional factors is 9:0.5:1~9:3:1; the addition of phytosterols helps to improve the orderly arrangement of the liposome membrane, increase its rigidity, and make the phospholipid membrane structure more compact. If the concentration of phytosterols is too low, the formed liposomes are too few or insufficient to encapsulate the added nutritional factors. If the concentration of phytosterols is too high, too many liposomes are formed, resulting in reduced stability and causing the loaded nutritional factors to leak. Experimental verification shows that when the amount of phytosterols added is within the above numerical range and the concentration of phytosterols is within an appropriate range, the liposome size can be guaranteed to be within an appropriate range and the stability of the liposomes can be guaranteed.

[0027] Preferably, it has been experimentally verified that when the ratio of phospholipids: phytosterols: nutritional factors is 9:1:1, the addition of phytosterols makes the phospholipid bilayer structure more regularly arranged and evenly distributed, and the liposomes that stably deliver nutritional factors by phytosterols have the best loading effect on nutritional factors.

[0028] Optionally, the temperature for rotary evaporation under vacuum to completely remove the poor solvent is 40-50°C; too low a temperature is not conducive to the effective removal of the poor solvent, 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; it has been experimentally verified that when the temperature is within the above numerical range, a uniform liposome film carrying the nutrient factors can be formed.

[0029] Optionally, the phosphate buffer solution for hydrating the liposome film contains 100 mL to 200 mL of TwEEn-80.

[0030] Optionally, the mass fraction of TwEEn-80 contained in the phosphate buffer solution of the hydrated liposome film is: 0.1%~0.4%; TwEEn-80 is a surfactant, and its emulsification effect can adjust the size of the formed liposomes. When the mass fraction of TwEEn-80 is low, the ordered arrangement and rigidity of the liposome membrane are poor. When the mass fraction of TwEEn-80 is high, it will solubilize the wall material components of the liposome, destroy the integrity of the liposome structure, and then cause the leakage of nutritional factors; experimental verification shows that when the mass fraction of TwEEn-80 contained in the phosphate buffer solution of the hydrated liposome film is within the above numerical range, it can increase the effective thickness of the membrane without destroying the phospholipid membrane, thereby improving the stability of the liposome.

[0031] Optionally, the temperature during hydration of the liposome film is 40-50°C; if the temperature is too low, the phosphate buffer solution containing TwEEn-80 cannot completely hydrate the liposome film, and the efficiency is low; if the temperature is too high, some nutritional factors may denature and affect the encapsulation efficiency, and it is also not conducive to the formation of liposomes; experiments have shown that when the temperature is within the above numerical range, uniform and stable liposomes loaded with nutritional factors stabilized by plant sterols can be formed.

[0032] Optionally, when the crude liposomes are ultrasonically treated in a low-temperature water bath, the ultrasonic power is 250W~350W; when the crude liposomes are ultrasonically treated in a low-temperature water bath, each time it is turned on for 1s~2s, then turned off for 2s~3s, then turned on again for 1s~2s, then turned off again for 2s~3s, and this cycle is repeated; when the crude liposomes are ultrasonically treated in a low-temperature water bath, the water bath temperature is 2~8°C, and the ultrasonic time is 10~20 min; the lipid aggregates are separated into smaller single-layer liposomes by ultrasonic sound energy, and the ultrasonic time and ultrasonic intensity have a decisive effect on the size of the prepared liposome particles. When the ultrasonic temperature is too high, since the ultrasonic treatment of the crude liposome carrier carrying the nutritional factor is an exothermic process, a higher reaction temperature is not conducive to the formation of the liposome; excessive ultrasonic power or too long ultrasonic time will cause the rupture, flocculation and aggregation of the liposome membrane, resulting in the leakage of the loaded nutritional factor and reduced stability; experimental verification shows that when the reaction temperature, ultrasonic power and time are set within the above numerical range, it is beneficial to regulate the fluidity and permeability of the phospholipid membrane and promote the formation of the liposome.

[0033] Alternatively, crude liposome carriers for delivering nutritional factors by phytosterols are prepared by thin film hydration method, ethanol injection method, reverse phase evaporation method or microfluidics method.

[0034] Preferably, liposomes for delivering nutritional factors stabilized by plant sterols are prepared by thin film hydration method. Thin film hydration method is simple to operate, economical and practical, and is usually combined with ultrasound, homogenization and other methods to obtain single-chamber liposomes with uniform particle size and high drug encapsulation capacity.

[0035] The present invention also provides a liposome carrier for delivering nutritional factors by plant sterols, characterized in that the liposome carrier for delivering nutritional factors stably delivered by plant sterols is prepared by the above-mentioned preparation method of the liposome carrier for delivering nutritional factors stably delivered by plant sterols, which can avoid the adverse effects of excessive cholesterol intake on the human body, and at the same time as loading the nutritional factors, it can also improve the stability and bioaccessibility of the nutritional factors in the gastrointestinal environment.

[0036] like Figure 1 As shown, taking lutein as an example, the present invention proposes a method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols, comprising the following steps: (1) Soybean lecithin (SL), stigmasterol, and lutein were dissolved in ethanol solution by thin-film evaporation method, and the resulting mixture was rotary evaporated under vacuum until the ethanol was completely removed to form a uniform liposome film for delivering lutein stabilized by stigmasterol; (2) The liposome film was hydrated with phosphate buffer solution containing TwEEn-80 to form crude liposomes; (3) The crude liposome carrier loaded with lutein was further homogenized by water bath and low-temperature ultrasound to obtain a liposome carrier for delivering nutritional factors stabilized by plant sterols (Lutein-liposome, Lu-lip). This renewable composite material shows potential advantages in the fields of food, environmental protection and drug formulation. In addition, due to the safety, biodegradability and easy availability of raw materials, it is inexpensive and easy to commercialize.

[0037] In an embodiment of the present invention not shown in the figure, Figure 1 Replace the lutein in vitamin C with lycopene, curcumin, astaxanthin or resveratrol.

[0038] The specific embodiments of the present invention are described in detail below: Example 1 Control group: Preparation of liposome carriers for delivering nutritional factors stabilized by lutein but without plant sterols.

[0039] (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at 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.

[0040] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0041] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein without plant sterols was obtained.

[0042] Example 2 Preparation of lutein-loaded liposome carriers stabilized by phytosterols for the delivery of nutritional factors.

[0043] (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:0.5:1. 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.

[0044] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0045] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0046] Example 3 Preparation of lutein-loaded liposome carriers stabilized by phytosterols for the delivery of nutritional factors.

[0047] (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and 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.

[0048] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0049] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0050] Example 4 Preparation of lutein-loaded liposome carriers stabilized by phytosterols for the delivery of nutritional factors.

[0051] (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:2:1. 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.

[0052] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0053] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0054] Example 5 Preparation of lutein-loaded liposome carriers stabilized by phytosterols for the delivery of nutritional factors.

[0055] (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:3:1, and 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.

[0056] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0057] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0058] Example 6 (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 10 mL of ethanol at a ratio of 9:1:1, and 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.

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

[0060] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0061] Example 7 (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 100 mL of ethanol at a ratio of 9:1:1, and the resulting mixture was rotary evaporated under a vacuum degree of 0.01 MPa until the ethanol was completely removed to form a uniform lutein liposome film.

[0062] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0063] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0064] Example 8 (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 200 mL of ethanol at a ratio of 9:1:1, and the resulting mixture was rotary evaporated under a vacuum degree of 0.01 MPa until the ethanol was completely removed to form a uniform lutein liposome film.

[0065] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0066] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0067] Example 9 (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and 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.

[0068] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.6 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0069] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 s each time, then turned off for 2 s, then turned on again for 1 s, and then turned off again for 2 s. This cycle was repeated for 10 min. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0070] Example 10 (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and 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.

[0071] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 1.0 wt% TwEEn-80 at 50 °C to form crude liposomes.

[0072] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0073] Embodiment 11 (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and 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.

[0074] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0075] (3) The crude liposome carrier loaded with lutein was sonicated in a water bath at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 30 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0076] Example 12 (1) Soy lecithin (SL), stigmasterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and 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.

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

[0078] (3) The crude liposome carrier loaded with lutein was sonicated in a water bath at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 50 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by stigmasterol was obtained.

[0079] Example 13 (1) Soy lecithin (SL), β-sitosterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:1:1. The resulting mixture was rotary evaporated under a vacuum degree of 0.01 MPa until the ethanol was completely removed to form a uniform lutein liposome film.

[0080] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0081] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by β-sitosterol was obtained.

[0082] Embodiment 14 (1) Soy lecithin (SL), brassica sterol and lutein were dissolved in 50 mL of ethanol at a ratio of 9:1:1. The resulting mixture was rotary evaporated under a vacuum degree of 0.01 MPa until the ethanol was completely removed to form a uniform lutein liposome film.

[0083] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0084] (3) The crude liposome carrier loaded with lutein was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lutein stabilized by β-sitosterol was obtained.

[0085] Embodiment 15 Preparation of curcumin-loaded liposome carriers stabilized by phytosterols for the delivery of nutritional factors.

[0086] (1) Soy lecithin (SL), stigmasterol and curcumin were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and the resulting mixture was rotary evaporated under a vacuum degree of 0.01 MPa until the ethanol was completely removed to form a uniform curcumin liposome film.

[0087] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0088] (3) The crude liposome carrier loaded with curcumin was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering curcumin stabilized by stigmasterol was obtained.

[0089] Example 16 Preparation of lycopene-loaded liposome carriers stabilized by phytosterols for the delivery of nutritional factors.

[0090] (1) Soy lecithin (SL), stigmasterol and lycopene were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and the resulting mixture was rotary evaporated under a vacuum degree of 0.01 MPa until the ethanol was completely removed to form a uniform lycopene liposome film.

[0091] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0092] (3) The crude liposome carrier loaded with lycopene was subjected to water bath sonication at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier for delivering lycopene stabilized by stigmasterol was obtained.

[0093] Embodiment 17 Preparation of resveratrol-loaded liposome carriers stabilized by plant sterols for the delivery of nutritional factors.

[0094] (1) Soy lecithin (SL), stigmasterol and resveratrol were dissolved in 50 mL of ethanol at a ratio of 9:1:1, and the resulting mixture was rotary evaporated under a vacuum degree of 0.01 MPa until the ethanol was completely removed to form a uniform lycopene liposome film.

[0095] (2) The liposome membrane was hydrated with 100 mL of phosphate buffer solution containing 0.2 wt % TwEEn-80 at 50 °C to form crude liposomes.

[0096] (3) The crude liposome carrier loaded with resveratrol was sonicated in a water bath at 4°C with a power of 300 W. The ultrasound was turned on for 1 second, then turned off for 2 seconds, then turned on again for 1 second, and then turned off again for 2 seconds. This cycle was repeated for 10 minutes. After further homogenization, a liposome carrier stably delivering resveratrol by stigmasterol was obtained.

[0097] Comparing Example 1, Example 2, Example 3, Example 4, and Example 5, it was found that the most stable ratio of soybean lecithin (SL), stigmasterol, and lutein was 9:1:1. Figure 3 As shown in B, the encapsulation efficiency of lutein is the best.

[0098] By comparing Example 2, Example 6, Example 7 and Example 8, it is obtained that soybean lecithin, stigmasterol and lutein are dissolved in different volumes of ethanol and rotary evaporated into liposome films, among which the volume of the poor solvent is 50 ml, and the effect is best, forming a uniform and stable lutein liposome film.

[0099] By comparing Example 2, Example 9 and Example 10, it is found that when the mass fraction of TwEEn-80 in the phosphate buffer solution of the hydrated liposome film is 0.2 wt%, Lu-lip has the highest stability, which increases the effective thickness of the film without destroying the phospholipid membrane.

[0100] By comparing Example 2, Example 11, and Example 12, Lu-lip obtained under different low-temperature ultrasound times was obtained. The vesicle fusion was reduced after low-temperature ultrasound, and the low-temperature ultrasound for 10 min had the best effect.

[0101] By comparing Example 2, Example 13 and Example 14, it was found that the liposome carriers for delivering lutein stabilized by different phytosterols all had good stability.

[0102] Example 2, Example 15, Example 16 and Example 17 show that the liposome carrier of nutritional factors stabilized by plant sterols of the present invention is applicable to some other nutritional factors, which are not listed one by one here.

[0103] Figure 2 The particle size and PDI (Polydispersity Index) of Lu-lip with different addition amounts of stigmasterol are shown in Figure 2. The addition of stigmasterol reduces the particle size of Lu-lip. PDI < 0.3 indicates that Lu-lip is evenly distributed and stable, and the particle size is the smallest when the ratio of SL, stigmasterol and Lutein is 9:1:1, which shows high stability.

[0104] Depend on Figure 3A shows that the Zeta potential of Lu-lip changes with the content of stigmasterol, and its absolute value is higher than 25 mV, indicating good stability and repulsive resistance due to the negative charge on the surface of liposomes and reduced aggregation. Stigmasterol can enhance the hydrophobic interaction with fatty acyl chains, further stabilize the bilayer and improve the stability of Lu-lip. Figure 3 B shows that the encapsulation rate EE (%) of Lutein is generally considered to be the best when the ratio of soybean lecithin (SL), stigmasterol and lutein is 9:1:1. The encapsulation rate of lutein in Lu-lip with stigmasterol added is higher than that in Lu-lip without stigmasterol. The addition of stigmasterol helps to improve the orderly arrangement of lipid membranes and make the phospholipid membrane structure more compact. At the same time, stigmasterol is incorporated into the phospholipid bilayer to regulate the fluidity and permeability of the phospholipid membrane, improve the stability of Lu-lip, and achieve effective encapsulation of lutein while promoting its effective release in the gastrointestinal tract. Figure 3 C shows that the lutein liposomes stabilized by stigmasterol prepared by adding different amounts of stigmasterol are treated at different temperatures, and the thermal stability is best when the ratio of soybean lecithin: stigmasterol: lutein is 9:1:1.

[0105] Depend on Figure 4 From the Fourier infrared transform spectrum shown, we can see that Figure 4 A is the infrared spectra of stigmasterol, soybean lecithin, empty liposomes and Lu-lip with a ratio of stigmasterol to soybean lecithin of 0:9 and 1:9. Lu-Lip has a wavelength of 3200-3600 cm -1 The -OH stretching vibration of SL and Lutein ranges from 3403 cm -1 and 3355 cm -1 Move to 3394 cm -1 and 3388 cm -1 , indicating that there is a hydrogen bond interaction between Lutein and SL; the stretching vibration peak of C=O of stigmasterol increases from 1740 cm -1 Reduced to 1738 cm -1 and 1736 cm -1 , indicating that the ester carbonyl group may be involved in the formation of hydrogen bonds. The C=O group is very sensitive to changes in the conformation and orientation of the ester bond, indicating that the conformation of the ester bond in the phospholipid molecule changes when lutein is loaded; hydrogen bonds are formed between the hydroxyl groups of stigmasterol and the oxygen groups of phospholipids, thereby reducing the frequency of stretching vibrations; stigmasterol can induce stronger molecular interactions to form a more stable interfacial film. The characteristic peaks of Lutein were almost not observed in Lu-lip, indicating the effective encapsulation of lutein in liposomes. Figure 4 B is the infrared spectra of soybean lecithin and lutein liposomes with different amounts of stigmasterol added. It can be concluded that the vibration shift of -CH in CH2 is mainly due to the difference in the saturation of fatty acid chains in phospholipids. 2- The asymmetric stretching vibration absorption peak of -1 Red shifted to 1239 and 1238 cm -1 This red shift 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 stretching vibrations and forms a stable interfacial film.

[0106] like Figure 5 As shown, Figure 5 A and B in the figure are the XRD spectra of lutein and stigmasterol with different stigmasterol addition amounts of lutein liposomes, respectively. The powder XRD (X-ray Diffraction) of Figures A and B shows that lutein is successfully encapsulated in the lutein liposomes stabilized by stigmasterol. In Figure A, the sharp characteristic peak of lutein disappears, and it is in an amorphous state. Figure 5 As shown in B, the absorption peaks at 15° and 18° of lutein liposomes with different stigmasterol additions are the absorption peaks of stigmasterol.

[0107] like Figure 6 As shown in the figure, TSI (multiple light scattering technology, Turbiscan stability index) data shows that the lutein liposomes stabilized by stigmasterol show good physical stability and minimal membrane fusion, reflecting the stability of Lu-lip.

[0108] like Figure 7 As shown, from the analysis of the in vitro simulated release curve, it can be seen that the cumulative release rate of lutein liposomes stabilized by stigmasterol is slow, and the rigidity of the liposome membrane gradually increases with the addition of stigmasterol, ensuring the stability of Lu-lip.

[0109] Figure 8 This is a schematic diagram of the bioaccessibility of lutein liposomes stabilized by different amounts of stigmasterol. The bioaccessibility of Lu-lip with different amounts of stigmasterol added is different, which proves the controllability of lutein release.

[0110] Specifically, Figure 8As shown in the figure, the bioaccessibility of lutein liposomes with different stigmasterol addition amounts, the highest bioaccessibility of 21.69% was observed when the ratio of soy lecithin: stigmasterol: lutein was 9:1:1, the addition of appropriate amount of stigmasterol increased the stability of Lu-lip, provided better protection for the loading of lutein, prevented lutein from interacting with other chemicals in the digestive juice, thereby reducing its degradation before intestinal digestion; liposomes with ratios of 9:2:1 and 9:3:1 may form non-vesicular structures in the membrane, and cross-linking is prone to occur between particles, which may cause premature release of lutein and lead to partial loss of lutein.

[0111] In the related art, there have been studies on encapsulating bioactive substances by liposomes. For example, the publication number CN105726482A discloses a lutein nanoliposome and a preparation method thereof. Cholesterol is added as a raw material during the preparation process. The negative impact of cholesterol on human health limits its application in liposomes; the publication number CN111718288A discloses a lutein ester and a water-soluble lutein ester microcapsule and a preparation method thereof. The temperature during the preparation process is high, which increases the degradation risk of the lutein ester and affects the stability of the lutein ester. The lutein hydrogenated phospholipid liposome precursor and its preparation method disclosed in the publication number CN101716144A add lutein and hydrogenated phospholipids to dichloromethane and ethanol solutions. Dichloromethane is highly toxic and easy to remain. At the same time, the supercritical carbon dioxide antisolvent method is used to prepare lutein liposomes, which has high operating pressure and high cost. None of the above related arts discloses the preparation method of the liposome stabilized by plant sterols of the present invention and the liposome carrier of the nutritional factor stabilized by plant sterols prepared by the present invention.

[0112] The beneficial technical effects of the present invention are: The method for preparing liposomes stabilized by plant sterols and the prepared liposome carriers carrying nutritional factors stabilized by plant sterols utilize plant sterols to replace cholesterol as a membrane stabilizer, making it an indispensable structural component in a complete membrane structure, thereby enhancing the bioaccessibility of the nutritional factors and making the release characteristics more controllable, and being more beneficial to human health.

[0113] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols, characterized in that: The following steps are involved: Dissolving phospholipids, phytosterols and nutritional factors in a poor solvent, and rotary evaporating the resulting mixture under vacuum until the poor solvent is completely removed to form a uniform liposome film in which the nutritional factors are delivered by phytosterols; hydrating the liposome membrane with a phosphate buffer solution containing TwEEn-80 to form crude liposomes; The crude liposome carriers loaded with nutritional factors were sonicated in a low-temperature water bath to obtain liposome carriers for delivering nutritional factors by phytosterols.

2. The method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols according to claim 1, characterized in that: The phospholipid is selected from at least one of soybean lecithin and egg yolk lecithin; The plant sterol is stigmasterol, β-sitosterol, brassicasterol or campesterol; The nutritional factor is selected from at least one of lutein, lycopene, curcumin, astaxanthin or resveratrol.

3. The method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols according to claim 1, characterized in that: The poor solvent for dissolving phospholipids, phytosterols and nutritional factors is methanol, ethanol or chloroform.

4. The method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols according to claim 1, characterized in that: The vacuum degree of the rotary evaporation under vacuum to completely remove the poor solvent is 0.00 MPa to 0.02 MPa.

5. The method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols according to claim 1, characterized in that: The poor solvent is 40 mL to 80 mL; The concentration ratio of the phospholipids, phytosterols and nutritional 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; The temperature of the hydrated liposome film is 40°C to 50°C.

6. The method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols according to claim 1, characterized in that: When the crude liposomes are ultrasonicated in a low-temperature water bath, the ultrasonic power is 250W~350 W; When the crude liposomes are ultrasonicated in the low-temperature water bath, the ultrasonic wave is turned on for 1s to 2s each time, and then turned off for 2s to 3s; then turned on again for 1s to 2s, and then turned off again for 2s to 3s, and this cycle is repeated; When the crude liposomes are ultrasonicated in a low-temperature water bath, the water bath temperature is 2-8° C. and the ultrasonication time is 10-20 min.

7. The method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols according to claim 1, characterized in that: Crude liposome carriers for delivering nutritional factors via phytosterols were prepared by thin film hydration method, ethanol injection method, reverse phase evaporation method or microfluidics method.

8. A liposome carrier for delivering nutritional factors stabilized by phytosterols, characterized in that: The liposome carrier for delivering nutritional factors stabilized by plant sterols is prepared by the method for preparing a liposome carrier for delivering nutritional factors stabilized by plant sterols as described in any one of claims 1 to 7.

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