Preparation method of tea pigment photoresponsive cationic nanoliposome

Abstract

The application discloses a preparation method of tea pigment light response cationic nanoliposome. The method comprises the following steps: taking black tea branch and leaf waste as raw materials, and preparing an extract with a tea pigment content of greater than or equal to 80% by performing enzymatic oxidation under optimized pH and temperature conditions and combining with macroporous resin purification; taking the extract and a photosensitizer demagnified chlorophyll acid as active ingredients, taking soybean lecithin and cholesterol with a mass ratio of (5:1) to (8:1) as a capsule material, and preparing primary liposomes by a reverse evaporation method; then, the primary liposomes are coated and modified by using a cationic chitosan acetic acid solution with a concentration of 0.05% to 0.15%; finally, the final product is obtained through homogenization and purification treatment. The nanoliposome has a tea pigment encapsulation rate of greater than or equal to 80%, an average particle size of 80 nm to 200 nm, a Zeta potential of +30 mV to +50 mV, and can realize release of active ingredients under 400 nm to 480 nm blue light irradiation.

Description

Technical Field

[0001] This invention belongs to the field of nanoparticle technology, specifically relating to a method for preparing tea pigment photoresponsive cationic nanoliposomes. Background Technology

[0002] Tea pigments are a class of natural polyphenol oxidized polymers with multiple biological activities extracted from tea leaves. They mainly include theaflavins, thearubigins, and theabrownins, and have excellent antioxidant, antibacterial, and anti-inflammatory effects, showing great potential in the food, pharmaceutical, and agricultural fields. However, their industrial application faces two major technical bottlenecks: (1) The raw material preparation process is backward, and the product quality is uneven. Traditional extraction methods for tea pigments include boiling water decoction and organic solvent extraction. The former has the problem of high heating temperature, which leads to the polymerization of polyphenols in the tea pigments and the resulting darkening of the color. The latter uses ethanol aqueous solution to extract tea pigments, which improves the extraction rate, but has the problems of high production cost, low content, and unevenness. (2) Poor physicochemical stability and low bioavailability. Tea pigments contain phenolic hydroxyl active groups, which are easily oxidized or hydrolyzed by light, oxygen, pH value, and enzymes in the in vivo and in vitro environment, resulting in low utilization and poor stability, which seriously limits their efficacy. At the same time, a large number of tea tree pruning branches and leaves have not been utilized in a high-value manner as agricultural waste, resulting in resource waste.

[0003] To improve the stability of tea pigments, liposomes are commonly used for encapsulation. Liposomes are completely encapsulated multilayer vesicles formed by a lipid bilayer, such as phospholipids, and can encapsulate both water-soluble and lipid-soluble substances, offering controlled, sustained release and safety. However, conventional liposomes still suffer from insufficient gastrointestinal stability and a lack of responsive release capabilities. In recent years, photoresponsive nanocarrier systems have attracted considerable attention due to their ability to achieve controlled release of active ingredients. However, existing technologies often employ photosensitizers excited by ultraviolet light, such as azobenzene, which suffer from weak tissue penetration and low biocompatibility.

[0004] In summary, the existing technology has not yet disclosed a method for obtaining high-purity, stable, blue light-controlled release, and charge-targeting tea pigment nanoliposomes using agricultural waste such as black tea tree branches and leaves as raw materials through enzymatic oxidation, macroporous resin purification, reverse evaporation, and cationic chitosan coating, as described in claim 1. Summary of the Invention

[0005] This invention provides a method for preparing tea pigment photoresponsive cationic nanoliposomes.

[0006] This invention can be achieved through the following technical solutions: A method for preparing tea pigment photoresponsive cationic nanoliposomes includes the following steps: S1. Using black tea tree branches and leaves as raw materials, tea pigment extract is prepared by enzymatic oxidation and macroporous resin purification technology; wherein the enzymatic oxidation reaction is carried out under the conditions of pH 4.5-5.5 and temperature 40-50℃; S2. The soybean lecithin, cholesterol, photosensitizer, and tea pigment extract prepared in step S1 are dissolved together in an organic solvent. The organic solvent is removed under reduced pressure to form a lipid film. The lipid film is hydrated with a buffer solution of pH 6.5-7.5 to obtain a primary liposome suspension loaded with tea pigment and pheophytic chlorophyll. The mass ratio of soybean lecithin to cholesterol is (5:1)-(8:1). S3. Prepare a cationic chitosan acetate solution with a mass-volume concentration of 0.05%-0.15%, and add it to the primary liposome suspension obtained in step S2 under stirring conditions. Use electrostatic interaction to coat the liposome shell with chitosan to form cationic nanoliposomes. S4. The product obtained in step S3 is homogenized and purified to obtain tea pigment photoresponsive cationic nanoliposomes.

[0007] Preferably, in step S1, the raw material is pruning waste from black tea tree branches and leaves, which is a reuse of agricultural waste, reducing environmental pollution and effectively saving costs. The enzymatic oxidation method uses polyphenols in the raw material as substrates, and under the specific catalysis of enzymes, directional oxidation, coupling, and polymerization reactions occur, efficiently converting them into tea pigments. The macroporous resin purification utilizes the adsorption properties of the resin to selectively enrich the tea pigments; by using a gradient elution with a specific concentration of ethanol aqueous solution, impurities such as proteins and polysaccharides can be effectively removed, thereby increasing the content of the obtained tea pigment extract.

[0008] Preferably, in step S1, the content of tea pigments accounts for ≥80% of the total content of the tea pigment extract. The tea pigments mainly include theaflavins, thearubigins, and theabrownins. Theaflavins exhibit the most significant bioactivity and are the key component endowing tea pigments with antioxidant, anti-inflammatory, and antibacterial effects; thearubigins are next, while theabrownins have relatively weaker bioactivity. Based on this, the combination of enzymatic oxidation and macroporous resin purification processes aims to regulate the reaction pathway to promote the formation of theaflavins and thearubigins and inhibit their excessive conversion to theabrownins, thereby obtaining an extract with a higher proportion of highly active components.

[0009] Preferably, in step S1, the enzyme used in the enzymatic oxidation reaction is polyphenol oxidase and / or peroxidase, and the amount of enzyme added is 0.1%-0.5% of the raw material mass. This range is determined based on the balance between reaction efficiency and product composition: if the amount of enzyme added is less than 0.1%, the reaction kinetics are insufficient, resulting in incomplete substrate conversion and a significant decrease in the yield of tea pigments; if the amount added is more than 0.5%, it will promote the excessive polymerization of intermediate quinones, leading to an increase in the proportion of high molecular weight theabrownins, which in turn reduces the relative content of highly active theaflavins and thearubigins.

[0010] Preferably, in step S1, the macroporous resin purification uses HPD-100 or AB-8 type macroporous resin, and gradient elution is performed using an ethanol-water solution with a concentration of 30%-60%. Both HPD-100 and AB-8 resins are moderately polar adsorption resins, and their surface pore size and specific surface area are suitable for adsorbing natural products such as tea pigments, which have polyphenol structures and medium molecular weights. Compared to strongly polar or non-polar resins, these two types of resins have higher adsorption capacity and more suitable selectivity for tea pigments, effectively adsorbing the target analyte while allowing strongly polar impurities such as inorganic salts and some sugars to permeate. The principle of setting the gradient elution concentration is to utilize the polarity difference between tea pigments and impurities to achieve selective separation and enrichment. First, elution is performed using approximately 30% ethanol to remove strongly polar impurities that are not firmly adsorbed by the resin; theaflavins and thearubigins in tea pigments have the best desorption efficiency at a concentration of approximately 60% ethanol. This concentration of ethanol is sufficient to disrupt the hydrophobic interactions and hydrogen bonds between tea pigment molecules and the resin skeleton, allowing them to be concentrated and eluted efficiently, thus achieving high-purity enrichment.

[0011] Preferably, the concentrations of the ethanol-water solution used for gradient elution are 30%, 40%, 50%, and 60% respectively.

[0012] Preferably, in step S2, the tea pigment extract accounts for 10%-20% of the total lipid mass, based on the total mass of soybean lecithin and cholesterol; the mass ratio of the photosensitizer to the tea pigment extract is (1:8)-(1:12). Controlling the amount of tea pigment extract added to 10%-20% of the total lipid mass aims to balance drug loading and liposome structural stability. Too low a ratio results in insufficient drug loading, while too high a ratio damages the lipid membrane structure, leading to a decrease in encapsulation efficiency. Controlling the ratio of the photosensitizer to (1:8)-(1:12) aims to synergistically improve photosensitivity and carrier stability. Too low a ratio results in insufficient phototriggered release, while too high a ratio affects liposome stability and increases potential impacts.

[0013] Preferably, the photosensitizer is pheophoric acid. It is excited by visible blue light at a wavelength of 400-480 nm, generating reactive oxygen species such as singlet oxygen, which then disrupt the integrity of the lipid bilayer through lipid peroxidation, thereby achieving the controlled release of nanoliposomes. This photoresponsive system exhibits better tissue penetration and biocompatibility compared to ultraviolet light-excited systems.

[0014] Preferably, in step S2, the organic solvent is at least one of chloroform, ethanol, methanol, and dichloromethane; and the buffer solution is a phosphate buffer solution.

[0015] Preferably, in step S3, the solvent for the cationic chitosan acetic acid solution is an aqueous acetic acid solution with a concentration of 0.5%-1.5%. Chitosan requires protonation under acidic conditions to dissolve and exert its cationic properties. When the acetic acid concentration is below 0.5%, the acidity is insufficient to completely dissolve it, easily producing insoluble particles and resulting in uneven coating. When the concentration is above 1.5%, excessively high acidity will exacerbate the hydrolysis of chitosan molecular chains, leading to a decrease in its molecular weight and viscosity, thereby weakening the stability of the formed coating layer. In addition, excessively high acetic acid concentrations will increase the burden and potential impact of subsequent purification.

[0016] Preferably, in step S4, the homogenization process is performed by probe ultrasound, with an ultrasound power of 200-400W and a processing time of 3-10 minutes; the purification process is performed by dialysis or ultracentrifugation.

[0017] Dialysis is based on the principle of molecular diffusion through a semipermeable membrane in solution. A cationized nanoliposome suspension is placed in a dialysis bag with a molecular weight cutoff of 10 kDa and immersed in a large amount of deionized water or buffer solution for dialysis. Free small molecules diffuse through the membrane pores to the external aqueous phase, while nanoliposomes encapsulated inside the liposomes and bound to the surface are retained inside the bag. This process is gentle, requires no high-speed shear forces, and preserves the integrity of the nanoliposome structure to the greatest extent.

[0018] Ultracentrifugation is based on the difference in sedimentation rates of different particles under a strong centrifugal force field. Centrifugation at a relative centrifugal force of ≥100,000 × g causes nanoliposomes to precipitate due to their larger particle size and density, while free active ingredient molecules and small molecule impurities remain in the supernatant. Separation is achieved by removing the supernatant. This method is efficient and rapid, suitable for the purification and concentration of batch samples.

[0019] The present invention also provides photoresponsive cationic nanoliposomes of tea pigments prepared by the above method.

[0020] A photoresponsive cationic nanoliposome of tea pigment is composed of the following components: tea pigment extract, soybean lecithin, cholesterol, photosensitizer, and a cationic chitosan layer coated on the surface of the liposome.

[0021] Preferably, the tea pigment extract serves simultaneously as a structural component forming the liposome bilayer and as the encapsulated active ingredient. The nanoliposomes achieve controlled release of the active ingredient under blue light irradiation at a wavelength of 400-480 nm.

[0022] The beneficial effects of this invention are: 1. High-value utilization of resources is achieved by using waste materials from black tea tree branches and leaves. By optimizing process parameters such as pH and temperature of the enzymatic reaction and concentration of the eluent for macroporous resin purification, tea pigment extract with a purity of ≥80% is obtained, providing a uniform raw material for the preparation of nanoliposomes.

[0023] 2. By controlling the mass ratio of soybean lecithin to cholesterol and the drug-lipid ratio in the encapsulation material, the stability of the liposome structure and the encapsulation efficiency are fundamentally ensured to be ≥80%. By optimizing the concentration of cationic chitosan coating, the zeta potential on the surface of the nanoliposomes is stabilized at ≥+30 mV, endowing them with charge targeting ability and enriching them in negatively charged bacteria or inflammatory sites.

[0024] 3. This invention encapsulates blue light-responsive natural photosensitizer pheophytic acid with tea pigments and combines it with cationic modification to achieve a delivery system that combines light-controlled targeting and charge targeting, thus solving the problems of poor stability and weak targeting of tea pigments.

[0025] 4. The key parameters of each step in the preparation method of this invention are clearly defined, the process is stable, it is easy to scale up production, the obtained nanoliposomes have uniform particle size and good dispersibility, the key performance indicators have small batch-to-batch differences, and it is easy to establish quality standards. Attached Figure Description

[0026] Figure 1 This is a graph showing the effect of different pH values ​​on the yield of tea pigments in Example 1.

[0027] Figure 2 This is a bar chart showing the effect of different lecithin / cholesterol mass ratios on the encapsulation efficiency of tea pigments in Example 2.

[0028] Figure 3 This is a schematic diagram of the structure of the tea pigment photoresponsive cationic nanoliposomes of the present invention.

[0029] Figure 4 This is a process flow diagram of the preparation method described in this invention.

[0030] Figure 5 This is a comparison of the in vitro cumulative release curves of the nanoliposomes prepared in Example 4 under blue light irradiation. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to specific embodiments. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0033] Example 1 Example 1: Optimization of pH and temperature in tea pigment extraction process To optimize the enzymatic oxidation process, the effects of pH and temperature on the yield and composition of tea pigments were investigated. Five 50g portions of dried and pulverized Yunnan black tea leaves were placed in citrate-disodium hydrogen phosphate buffer solutions at pH 4.0, 4.5, 5.0, 5.5, and 6.0, respectively, with a liquid-to-solid ratio of 10:1 (mL / g). Polyphenol oxidase (PPO) at 0.3% of the raw material mass was added to each portion, and the mixtures were reacted at 40℃, 45℃, and 50℃ with constant temperature shaking for 2 hours. After the reaction, the enzyme was inactivated in a boiling water bath for 10 minutes, filtered, and the filtrate was extracted three times with an equal volume of ethyl acetate. The organic phases were combined, concentrated under reduced pressure, and freeze-dried. The yield of tea pigments was calculated, and the ratios of theaflavins, thearubigins, and theabrownins were analyzed by high-performance liquid chromatography (HPLC).

[0034] The results are as follows Figure 1 As shown, when the reaction system pH is 5.0 and the temperature is 45℃, the tea pigment yield reaches its highest value of 7.2%, and the ratio of theaflavins to thearubigins is optimal. When the pH is below 4.5 or above 5.5, the yield decreases significantly; the reaction is incomplete below 40℃, and enzyme activity decreases above 50℃. Therefore, the optimal conditions for enzymatic oxidation are determined to be pH 4.5-5.5 and temperature 40-50℃.

[0035] Example 2: Optimization of the proportion of encapsulation material in nanoliposome formulations To optimize the formulation of nanoliposomes, the effect of encapsulation material composition on encapsulation efficiency was investigated. The mass of tea pigment extract was fixed at 20 mg, and the tea pigment mass ratio was set to 12.5% ​​of the total lipids (i.e., a drug-lipid ratio of 1:8), resulting in a total lipid content of 160 mg. Four experimental groups were set up with soybean lecithin to cholesterol mass ratios of 3:1, 5:1, 8:1, and 10:1. Materials were weighed according to each ratio and dissolved together with 2.0 mg of pheophoric acid in 20 mL of chloroform and ethanol (volume ratio 1:1). The organic solvent was removed by rotary evaporation in a 40℃ water bath using a reverse evaporation method to form a lipid film. The film was then hydrated with 15 mL of pH 7.4 phosphate-buffered saline (PBS) at 45℃ for 60 minutes to obtain a primary liposome suspension. After separation by ultracentrifugation, the free tea pigment content in the supernatant was determined by HPLC, and the encapsulation efficiency was calculated.

[0036] The results are as follows Figure 2 As shown, when the mass ratio of lecithin to cholesterol is 5:1, the encapsulation efficiency of tea pigments reaches a peak of 88.5%. At a ratio of 3:1, the encapsulation efficiency is 82.1%, but the system viscosity increases; at a ratio of 8:1, the encapsulation efficiency is 85.3%; and at a ratio of 10:1, the encapsulation efficiency is 77.8%, but the liposome stability deteriorates. In summary, the optimal mass ratio range for the encapsulation material is (5:1)-(8:1).

[0037] Example 3: Optimization of cationic chitosan coating concentration To optimize the cationic modification process, the effect of cationic chitosan acetate solution concentration on the surface potential and stability of nanoliposomes was investigated. Four 10 mL aliquots of the primary liposome suspension prepared in Example 2 at the optimal ratio (5:1) were taken. 5.0 mL of cationic chitosan acetate solution (1% acetic acid aqueous solution) with a mass / volume concentration of 0.02%, 0.05%, 0.10%, and 0.20% respectively were added to each aliquot, and the mixture was stirred for 30 minutes for coating. Subsequently, the mixture was sonicated in an ice-water bath for 6 minutes, and then transferred to a dialysis bag with a molecular weight cutoff of 10 kDa. The solution was purified by dialyzing with deionized water at 4°C for 12 hours. The zeta potential of each group of nanoliposomes was measured using a nanoparticle size analyzer and a zeta potential analyzer, and their physical stability after standing at room temperature for 7 days was observed.

[0038] Results: When the cationic chitosan acetate solution concentration was 0.10%, the zeta potential of the nanoliposomes was +36.5 mV, and no visible aggregation or precipitation was observed after 7 days, with stable particle size distribution. At a concentration of 0.05%, the zeta potential was +25.1 mV; at a concentration of 0.20%, the zeta potential was +39.2 mV, but slight flocculation occurred in some samples. Therefore, the optimal concentration range for cationic chitosan acetate was determined to be 0.05%–0.15%.

[0039] Example 4: Complete preparation and characterization Based on the optimized parameters determined in the aforementioned embodiments, tea pigment photoresponsive cationic nanoliposomes were prepared and characterized: (1) Take 1 kg of black tea tree branches and leaves, and carry out enzymatic oxidation under the optimal conditions determined in Example 1 (pH 5.0, 45℃, PPO enzyme 0.3%). The reaction solution is purified by HPD-100 macroporous resin column (column bed volume 2L), and eluted sequentially with 5 column volumes (BV) of water, 3 BV of 30% ethanol, and 5 BV of 60% ethanol. Collect the 60% ethanol eluent, concentrate under reduced pressure, and freeze-dry to obtain 76 g of tea pigment extract powder. The total tea pigment content was determined by HPLC to be 83.5%.

[0040] (2) Weigh 200 mg of soybean lecithin, 40 mg of cholesterol (mass ratio 5:1), 30 mg of tea pigment extract obtained in step (1) (accounting for 12.5% ​​of the total lipid mass), and 3.0 mg of pheophytic acid (mass ratio of 1:10 to tea pigment), and dissolve them together in 20 mL of a mixed solvent of chloroform and ethanol (volume ratio 1:1). Form a lipid film using the reverse evaporation method, and hydrate it with 15 mL of pH 7.4 PBS at 45℃ to obtain a primary liposome suspension.

[0041] (3) 10 mL of cationic chitosan acetate (solvent: 1% acetic acid) with a mass-volume concentration of 0.10% was added to the above primary liposomes and stirred for 30 minutes for coating. Subsequently, the mixture was sonicated in an ice-water bath for 6 minutes, and then the solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and purified by dialyzing with deionized water at 4°C for 12 hours to obtain tea pigment photoresponsive cationic nanoliposomes.

[0042] Figure 3 This is a schematic diagram of the structure of tea pigment photoresponsive cationic nanoliposomes; Figure 4 This is a flowchart of the entire preparation process.

[0043] Characterization results: Encapsulation efficiency: The encapsulation efficiency of tea pigments was 88.7% and that of pheophytic acid was 91.2%, determined by ultracentrifugation combined with HPLC.

[0044] Particle size and potential: The average hydrodynamic particle size is 162 nm, the polydispersity index (PDI) is 0.16, and the zeta potential is +37.8 mV.

[0045] Photoresponsive release: In vitro release experiment ( Figure 5The results showed that under periodic irradiation with 450 nm blue light (50 mW / cm²), the cumulative release rate of the active ingredient reached 89.3% within 48 hours; while the release rate of the control group that was protected from light throughout the process was only 61.8%.

[0046] Comparative Example 1 Except for changing the mass ratio of soybean lecithin to cholesterol to 10:1, the other steps and parameters are exactly the same as in Example 4.

[0047] Results: The encapsulation efficiency of tea pigments in the prepared nanoliposomes was 77.5%, with an average particle size of 172 nm and a PDI of 0.22. The in vitro release curves showed that, under the same blue light irradiation conditions, the release rate and cumulative release amount of the active ingredient were significantly lower than those of the sample in Example 4. This result indicates that deviations from the optimized range of the encapsulation material ratio in this invention will lead to a decrease in encapsulation efficiency and a deterioration in stability.

[0048] Comparative Example 2 Except for changing the proportion of tea pigment extract to 25% of the total lipid mass, the other steps and parameters are exactly the same as in Example 4.

[0049] Results: The encapsulation efficiency of tea pigments in the prepared nanoliposomes was 70.2%, and visible precipitation appeared in the liposome suspension within several hours after preparation, indicating poor physicochemical stability. These results suggest that deviations from the optimized range of drug-liposome ratio in this invention lead to decreased encapsulation efficiency and deteriorated stability.

[0050] Comparative Example 3 Except for changing the concentration of the cationic chitosan acetate solution to 0.01%, the other steps and parameters are exactly the same as in Example 4.

[0051] Results: The zeta potential of the prepared nanoliposomes was only +18.3 mV, and significant aggregation and precipitation occurred after 3 days at room temperature. Insufficient surface positive charge affected their active targeting ability based on electrostatic interactions. These results indicate that deviations from the optimized range of chitosan solution concentration lead to a decrease in zeta potential and deterioration of stability.

[0052] Comparative Example 4 Except for the absence of the photosensitizer pheophytic acid, the remaining steps and parameters were exactly the same as in Example 4, and conventional tea pigment cationic liposomes were prepared.

[0053] Results: The encapsulation efficiency, particle size, and zeta potential of the prepared nanoliposomes were not significantly different from those of the sample in Example 4. However, their in vitro release curves showed that, under the same blue light irradiation conditions, the release behavior of the active ingredient was no different from that of the light-shielded control group. This result indicates that the absence of the photosensitizer pheophytic acid resulted in the obtained liposomes lacking blue light-responsive release characteristics.

[0054] The above embodiments demonstrate that by optimizing key process parameters in the extraction, nano-assembly, and surface modification processes, this invention prepares tea pigment nanoliposomes with high encapsulation efficiency, strong positive charge, and excellent blue light-responsive release performance.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing tea pigment photoresponsive cationic nanoliposomes, characterized in that, Includes the following steps: S1. Using black tea tree branches and leaves as raw materials, tea pigment extract is prepared by enzymatic oxidation and macroporous resin purification technology; wherein the enzymatic oxidation reaction is carried out under the conditions of pH 4.5-5.5 and temperature 40-50℃; S2. The soybean lecithin, cholesterol, photosensitizer, and tea pigment extract prepared in step S1 are dissolved together in an organic solvent. The organic solvent is removed under reduced pressure to form a lipid film. The lipid film is hydrated with a buffer solution of pH 6.5-7.5 to obtain a primary liposome suspension loaded with tea pigment and photosensitizer. The mass ratio of soybean lecithin to cholesterol is (5:1)-(8:1). S3. Prepare a cationic chitosan acetate solution with a mass-volume concentration of 0.05%-0.15%, and add it to the primary liposome suspension obtained in step S2 under stirring conditions. Use electrostatic interaction to coat the liposome shell with chitosan to form cationic nanoliposomes. S4. The product obtained in step S3 is homogenized and purified to obtain tea pigment photoresponsive cationic nanoliposomes.

2. The preparation method according to claim 1, characterized in that, In step S1, the enzyme used in the enzymatic oxidation reaction is polyphenol oxidase and / or peroxidase, and the amount of enzyme added is 0.1%-0.5% of the raw material mass; the macroporous resin purification uses HPD-100 or AB-8 macroporous resin, and gradient elution is performed using an ethanol aqueous solution with a concentration of 30%-60%.

3. The preparation method according to claim 1, characterized in that, In step S2, the total lipid mass is calculated based on the total mass of soybean lecithin and cholesterol, and the mass of the tea pigment extract accounts for 10%-20% of the total lipid mass.

4. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of the photosensitizer to the tea pigment extract is (1:8)-(1:12); the photosensitizer is pheophytic acid.

5. The preparation method according to claim 1, characterized in that, In step S2, the organic solvent is one or more mixed solvents selected from chloroform, ethanol, methanol, and dichloromethane; the buffer solution is phosphate buffer.

6. The preparation method according to claim 1, characterized in that, In step S3, the solvent for the cationic chitosan acetate solution is an aqueous acetic acid solution with a concentration of 0.5%-1.5%.

7. The preparation method according to claim 1, characterized in that, In step S4, the homogenization process is performed by probe ultrasound with an ultrasound power of 200-400W and a processing time of 3-10 minutes; the purification process is performed by dialysis or ultracentrifugation.

8. A tea pigment photoresponsive cationic nanoliposome, characterized in that, It consists of the following components: tea pigment extract, soybean lecithin, cholesterol, photosensitizer, and a cationic chitosan layer coated on the surface of the liposomes.

9. The tea pigment photoresponsive cationic nanoliposome according to claim 8, characterized in that, The tea pigment extract serves as both a structural component of the lipid bilayer and a photoresponsive active ingredient; the nanoliposomes achieve controlled release of the active ingredient under blue light irradiation at a wavelength of 400-480 nm.

10. The tea pigment photoresponsive cationic nanoliposomes according to claim 9, characterized in that, Its average particle size is 80-200 nm, and its Zeta potential is +30 mV to +50 mV.

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