Lucid ganoderma lipid exosome containing liver protection component as well as preparation method and application of lucid ganoderma lipid exosome

Abstract

The invention discloses a ganoderma lucidum lipid exosome containing a liver protection component and a preparation method and application thereof.The preparation method comprises the steps that the liver protection component is wrapped with a coarsely extracted ganoderma lucidum exosome, then the liver protection component is wrapped with lipidosome, and therefore a lipidosome-exosome-liver protection component three-layer structure is formed from outside to inside; in the three-layer structure, the outer layer liposome can protect the storage of the liver protection component and can improve the transmembrane transport efficiency, the middle layer exosome serves as a carrier and can reach a designated position to be released, and the inner layer contains the liver protection component and can improve the transmembrane transport efficiency. Cell cycle arrest can be prevented, antioxidant enzyme activity can be improved, cell membranes can be protected from oxidative damage, and redox steady state can be maintained to relieve cell aging. The prepared ganoderma lucidum lipid exosome containing the liver protection component realizes slow release synergy through a three-layer structure, and the action time of the active component is remarkably prolonged.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical preparation, and in particular to a Ganoderma lucidum lipid exosome containing liver-protecting components, and a preparation method and application thereof. Background Art

[0002] Exosomes are nanoscale (30-150nm) vesicles secreted by cells and are a type of extracellular vesicle. They are released extracellularly after the fusion of intracellular multivesicular bodies with the cell membrane. Almost all cell types can secrete exosomes. These vesicles are rich in cholesterol, sphingomyelin, and specific transmembrane proteins, and carry a variety of bioactive molecules such as proteins, lipids, and RNA (including mRNA and miRNA). By transferring these substances between cells, exosomes can regulate gene expression in recipient cells, participate in immune regulation, and promote tissue repair, demonstrating their important research value in protecting liver damage, promoting liver recovery, and treating diseases.

[0003] Exosomes act as natural nanocarriers to transport drugs to their target sites. They can carry substances such as proteins, lipids, and nucleic acids to mediate intercellular communication and have low immunogenicity, natural targeting, and biocompatibility. Currently, drugs can be loaded into exosomes through endogenous (genetically engineered donor cells) or exogenous (such as electroporation) methods, while surface modification techniques (such as antibody and peptide modification) can be used to enhance their targeting to specific cells. In the treatment of tumors, neurodegenerative diseases, cardiovascular diseases, and autoimmune diseases, exosomes have shown significant therapeutic effects as drug carriers in clinical treatment.

[0004] Ganoderma lucidum polysaccharides, the main active ingredient in Ganoderma lucidum, possess significant antioxidant properties. They can directly eliminate harmful free radicals in the liver. They also enhance the activity of key enzymes such as superoxide dismutase and glutathione peroxidase, as well as glutathione levels, thereby strengthening the liver's antioxidant defense system. They effectively inhibit lipid peroxidation and protect liver cell membrane structure. Furthermore, Ganoderma lucidum polysaccharides bidirectionally regulate immune function, suppressing liver damage caused by excessive immune responses while also enhancing the body's ability to eliminate pathogens. They can inhibit hepatocyte apoptosis, primarily by regulating apoptosis-related proteins, such as balancing the Bcl-2 / Bax ratio, inhibiting Caspase-3 activity, and protecting mitochondrial function, thereby reducing hepatocyte death. Some studies have also suggested that they may promote hepatocyte regeneration and accelerate tissue repair.

[0005] Liposomes are nano- or micron-sized vesicles composed of a phospholipid bilayer. They can encapsulate water-soluble or lipid-soluble substances and are widely used in drug delivery, gene therapy, cosmetics, and vaccine development. Their structure is similar to that of a cell membrane, composed of amphiphilic phospholipid molecules. They can contain cholesterol to enhance membrane stability and can be surface-modified to extend blood circulation or attached to targeting ligands for precise delivery. The main advantages of liposomes include high biocompatibility, the ability to carry both hydrophilic and hydrophobic drugs, sustained-release and protective properties, and the ability to achieve targeted delivery through modification.

[0006] In related technologies, drug molecules are usually simply encapsulated in liposomes or exosomes and then transported to the target site, facing problems such as limited drug loading efficiency, incomplete controllable drug release, and stability during the transport pathway. Summary of the Invention

[0007] In order to solve the problems in the prior art, the present invention provides a Ganoderma lucidum lipid exosome containing liver-protecting ingredients, and a preparation method and application thereof. The preparation method encapsulates the liver-protecting ingredients with exosomes, and then encapsulates them with liposomes to form a three-layer structure of liposomes-exosomes-liver-protecting ingredients. The outer layer liposomes can protect the storage of the liver-protecting ingredients and improve the transmembrane transport efficiency. The middle layer exosomes act as carriers and can reach the designated location for release, thereby achieving the purposes of high loading capacity, targeted cell uptake, biocompatibility, controlled release, high stability and low immunogenicity.

[0008] In order to achieve the above objectives, the present invention provides a method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting ingredients, comprising the following steps:

[0009] (1) Exosome extraction: Ganoderma lucidum was washed and added into PBS solution. After homogenization, enzymatic hydrolysis and centrifugation, the precipitate was collected to obtain crude extracted Ganoderma lucidum exosomes;

[0010] (2) Encapsulation of liver-protecting ingredients: The crudely extracted Ganoderma lucidum exosomes obtained in step (1) were subjected to ultrasonic treatment to temporarily break the membrane, and a PBS solution containing liver-protecting ingredients was added. After standing, a solution containing Ca was added. 2 + PBS solution for repair, and collect the precipitate to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients;

[0011] (3) Liposome encapsulation: Cholesterol, DPPC, sphingomyelin, and PEG-ceramide are dissolved to form a phospholipid film, and the Ganoderma lucidum exosomes containing the liver-protecting component obtained in step (2) are added, and the sample is vortexed to form a three-layer structure of liposomes-exosomes-liver-protecting component;

[0012] (4) Separation and purification: The sample obtained in step (3) is purified by a purification column to collect the target component, thereby obtaining Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0013] Preferably, the centrifugation step in step (1) comprises centrifugation at 400-600×g for 10-30 minutes, 800-1000×g for 10-30 minutes, 13000-15000×g for 10-30 minutes, and then at 10000-12000×g for 30-90 minutes.

[0014] Preferably, the PBS solution containing liver-protecting ingredients in step (2) comprises, by mass percentage, 0.4-0.6% Ganoderma lucidum polysaccharide, 0.2-0.3% carotenoids, 0.05-0.1% vitamin C, 0.05-0.1% vitamin E, 0.1-0.2% flavonoids, 0.01-0.05% trace elements, and the remainder PBS.

[0015] Preferably, the power of ultrasonic treatment in step (2) is 20 to 40 W and the time is 30 to 120 seconds; 2 +Ca in PBS solution 2 +The concentration is 1-2 mM, the repair temperature is 35-40°C, and the repair time is 30-120 minutes.

[0016] Preferably, in step (3), 0.4-0.5% cholesterol, 0.2-0.3% DPPC, 0.15-0.3% sphingomyelin and 0.05-0.1% PEG-ceramide are dissolved in the remaining solvent and evaporated to prepare the phospholipid film.

[0017] Preferably, the temperature of the vortex treatment in step (3) is 40 to 60° C., and the time is 15 to 30 minutes.

[0018] Preferably, the filler of the purification column in step (4) is a molecular exclusion chromatography medium, and PBS solution is used as the mobile phase during purification until no target peak appears in the ultraviolet detector.

[0019] The present invention also provides a Ganoderma lucidum lipid exosome containing a liver-protecting component, which is prepared using the above-mentioned method for preparing a Ganoderma lucidum lipid exosome containing a liver-protecting component. The exosome has a three-layer structure of liposome-exosome-liver-protecting component from the outside to the inside, a particle size of 80 to 150 nm, and a T value of 12 to 24 hours.

[0020] The present invention also provides a use of the above-mentioned Ganoderma lucidum lipid exosomes containing liver-protecting components in the preparation of a liver-protecting preparation.

[0021] Preferably, the liver protection preparation is used for transdermal delivery, and the liver protection preparation is in the form of an external cream, essence or patch.

[0022] Compared with the prior art, the preparation method of the present invention is to firstly collect the crude extracted Ganoderma lucidum exosomes after homogenization, enzymolysis and centrifugation in PBS solution; then temporarily break the membrane by ultrasonic treatment, add PBS solution containing liver protection ingredients, let it stand and then add Ca-containing solution. 2 + PBS solution for repair, and the precipitate is collected to obtain Ganoderma lucidum exosomes wrapping the liver-protecting ingredients; finally, the phospholipid film and the Ganoderma lucidum exosomes wrapping the liver-protecting ingredients are vortexed, and after purification, a three-layer structure of liposomes-exosomes-liver-protecting ingredients is obtained from the outside to the inside. In the three-layer structure, the outer layer liposomes can protect the storage of liver-protecting ingredients and improve the efficiency of transmembrane transport. The middle layer exosomes act as carriers and can reach the designated position for release. The inner layer is the liver-protecting ingredient, which can prevent cell cycle arrest, increase the activity of antioxidant enzymes, protect cell membranes from oxidative damage, and maintain redox homeostasis to alleviate cell aging. The prepared Ganoderma lucidum lipid exosomes containing liver-protecting ingredients are used to encapsulate the liver-protecting ingredients with extracted Ganoderma lucidum exosomes, and then the exosomes containing the liver-protecting ingredients are encapsulated with liposomes and used in the preparation of liver-protecting preparations. The liver-protecting preparations are used for transdermal delivery, and achieve liver-protecting effects by inhibiting the activity of β-galactosidase, a marker of cell aging, increasing collagen synthesis, and scavenging reactive oxygen species (ROS). The composite exosomes achieve sustained-release synergy through the lipid-exosome composite structure, significantly prolonging the action time of the active ingredients, thereby achieving the goals of high loading capacity, targeted cell uptake, biocompatibility, controlled release, high stability, and low immunogenicity. DETAILED DESCRIPTION

[0023] The present invention will be further explained below in conjunction with the specific embodiments of the specification. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present application. In addition, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods of the following examples that do not specify specific experimental conditions are usually in accordance with conventional experimental conditions. Unless otherwise specified, reagents and raw materials used in the present invention all can be obtained commercially.

[0024] The preparation method of the present invention specifically comprises the following steps:

[0025] Step 1: Wash fresh Ganoderma lucidum with double distilled water and add phosphate buffer solution (PBS), then place it in a homogenizer and grind it into a homogenate. After filtering, add one or more enzymes including but not limited to cellulase, pectinase, papain, etc., and adjust the pH value to 7.0-7.4 with tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl). Centrifuge the homogenate at 400-600, 800-1000, and 13000-15000 × g for 10-30 minutes, and then centrifuge at 10000-12000 × g for 30-90 minutes. Collect the precipitate, add Tris-HCl, and mix it with a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0026] Step 2: The exosomes were treated with ultrasound at a power of 20-40W for 30-120s to temporarily break the exosome membrane. A solution of liver-protecting ingredients such as Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids, and trace elements dissolved in PBS was added. Due to the concentration gradient, the liver-protecting ingredients efficiently entered the exosomes. A solution containing 1-2 mMCa was added. 2+ The cells were fixed with PBS solution at 35-40°C for 30-120 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0027] Preferably, the liver-protecting component solution dissolved in PBS includes, by mass percentage, 0.4-0.6% of Ganoderma lucidum polysaccharide, 0.2-0.3% of carotenoids, 0.05-0.1% of vitamin C, 0.05-0.1% of vitamin E, 0.1-0.2% of flavonoids, 0.01-0.05% of trace elements, and the balance PBS.

[0028] Step 3: After dissolving cholesterol, DPPC, sphingomyelin, and PEG-ceramide in ethanol, the solvent is removed using a rotary evaporator to form a uniform phospholipid film in the container. The above-mentioned buffer solution containing Ganoderma lucidum exosomes is added to the container and vortexed at 40-60°C for 15-30 minutes to convert the outer layer of liposomes into unilamellar liposomes, forming a three-layer structure with liposomes on the outside, exosomes in the middle layer, and liver-protecting ingredients in the inner layer.

[0029] Preferably, the following mass percentages are dissolved in ethanol solvent: 0.4-0.5% cholesterol, 0.2-0.3% DPPC, 0.15-0.3% sphingomyelin, and 0.05-0.1% PEG-ceramide, followed by evaporation to prepare a phospholipid film. The addition of PEG-ceramide can effectively prevent particles larger than 150 nm from being recognized and eliminated by the immune system.

[0030] Step 4: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 5 to 15 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0031] The Ganoderma lucidum lipid exosomes containing liver-protecting ingredients of the present invention are obtained by the above-mentioned preparation method. Ganoderma lucidum polysaccharides and other liver-protecting ingredients are wrapped in exosomes, and then wrapped in liposomes to form a three-layer structure of liposomes-exosomes-liver-protecting ingredients from the outside to the inside. The particle size is 80-150nm, and the T value is 12-24 hours. In the three-layer structure, the outer layer liposomes can protect the storage of liver-protecting ingredients and improve the transmembrane transport efficiency. The middle layer exosomes act as carriers and can reach the designated location for release. The inner layer is Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids and trace elements, which can prevent cell cycle arrest, increase antioxidant enzyme activity, protect cell membranes from oxidative damage and maintain redox homeostasis to alleviate cell aging. It is used to prepare liver protection preparations, which are used for transdermal delivery. They achieve liver protection effects by inhibiting the activity of β-galactosidase, a marker of cell aging, increasing collagen synthesis and scavenging reactive oxygen species (ROS). Liver protection preparations are in the form of topical creams, essences or patches, and maintain liver protection effects within 72 hours after a single application.

[0032] The present invention is described in more detail below with reference to specific embodiments.

[0033] Example 1:

[0034] Preparation of Ganoderma lucidum lipid exosomes containing liver-protecting ingredients

[0035] Exosome extraction: Fresh Ganoderma lucidum was washed with double-distilled water and added with phosphate buffered saline (PBS), then ground into a homogenate in a homogenizer. After filtration, one or more enzymes including but not limited to cellulase, pectinase, and papain were added. The pH was adjusted to 7 with tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl). The homogenate was centrifuged at 400, 800, and 13,000 × g for 10 min, and then at 10,000 × g for 30 min. The precipitate was collected, Tris-HCl was added, and mixed using a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0036] Encapsulating liver protection ingredients: The exosomes were treated with ultrasound at a power of 20-40W for 30-120s to temporarily break the exosome membrane. A solution containing 0.4% Ganoderma lucidum polysaccharide, 0.2% carotenoids, 0.05% vitamin C, 0.05% vitamin E, 0.1% flavonoids, and 0.01% trace elements dissolved in PBS was added. After standing, a solution containing 1mM Ca was added. 2+ The cells were fixed with PBS solution at 35°C for 30 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0037] Liposome encapsulation: 0.4% cholesterol, 0.2% DPPC, 0.15% sphingomyelin, and 0.05% PEG-ceramide were dissolved in ethanol and the solvent was removed using a rotary evaporator to form a uniform phospholipid film in the container. The buffer containing the Ganoderma lucidum exosomes was added to the container and vortexed at 40°C for 15 minutes to obtain the sample buffer.

[0038] Separation and purification: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 5 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0039] Example 2:

[0040] Preparation of Ganoderma lucidum lipid exosomes containing liver-protecting ingredients

[0041] Exosome extraction: Fresh Ganoderma lucidum was washed with double-distilled water and added with phosphate buffered saline (PBS). The slurry was then ground into a homogenate in a homogenizer. After filtration, one or more enzymes including but not limited to cellulase, pectinase, and papain were added. The pH was adjusted to 7 with tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl). The homogenate was centrifuged at 500, 900, and 14,000 × g for 20 min, and then at 11,000 × g for 60 min. The precipitate was collected, Tris-HCl was added, and mixed using a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0042] Encapsulating liver protection ingredients: The exosomes were treated with ultrasound at 30W for 75s to temporarily break the exosome membrane. A solution containing 0.5% Ganoderma lucidum polysaccharide, 0.25% carotenoids, 0.075% vitamin C, 0.075% vitamin E, 0.15% flavonoids, and 0.025% trace elements dissolved in PBS was added. After standing, a solution containing 1.5mM Ca was added. 2+The cells were fixed with PBS solution at 40°C for 75 minutes, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0043] Liposome encapsulation: 0.45% cholesterol, 0.25% DPPC, 0.2% sphingomyelin, and 0.05% PEG-ceramide were dissolved in ethanol, and the solvent was removed using a rotary evaporator to form a uniform phospholipid film in the container. The buffer containing the Ganoderma lucidum exosomes was added to the container and vortexed at 50°C for 20 minutes to obtain the sample buffer.

[0044] Separation and purification: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 10 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0045] Example 3:

[0046] Preparation of Ganoderma lucidum lipid exosomes containing liver-protecting ingredients

[0047] Exosome extraction: Fresh Ganoderma lucidum was washed with double-distilled water and added with phosphate buffered saline (PBS). The mixture was then ground into a homogenate in a homogenizer. After filtration, one or more enzymes including but not limited to cellulase, pectinase, and papain were added. The pH was adjusted to 7 with tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl). The homogenate was centrifuged at 600, 1000, and 15000 × g for 30 min, and then at 12000 × g for 90 min. The precipitate was collected, Tris-HCl was added, and mixed using a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0048] Encapsulating liver protection ingredients: The exosomes were treated with 40W ultrasonic wave for 120s to temporarily break the exosome membrane. A solution containing 0.6% Ganoderma lucidum polysaccharide, 0.3% carotenoids, 0.1% vitamin C, 0.1% vitamin E, 0.2% flavonoids, and 0.05% trace elements dissolved in PBS was added. After standing, a solution containing 2mM Ca was added. 2+ The cells were fixed with PBS solution at 40°C for 120 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0049] Liposome encapsulation: 0.5% cholesterol, 0.3% DPPC, 0.3% sphingomyelin, and 0.1% PEG-ceramide were dissolved in ethanol, and the solvent was removed using a rotary evaporator to form a uniform phospholipid film in the container. The buffer containing the Ganoderma lucidum exosomes was added to the container and vortexed at 60°C for 30 minutes to obtain the sample buffer.

[0050] Separation and purification: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 15 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0051] Experimental part:

[0052] Ganoderma lucidum polysaccharide is one of the main active ingredients of Ganoderma lucidum. It is mainly composed of β-glucan and various monosaccharides. It can improve the liver's reactive oxygen species (ROS) clearance rate and achieve antioxidant effects. Therefore, it can be used as a functional food or a potential natural liver protection substance in medicine bottles.

[0053] In vitro sustained-release performance test:

[0054] The sustained-release properties of the Ganoderma lucidum lipid exosomes and Ganoderma lucidum exosomes containing liver-protecting ingredients described in the present invention were verified by the following method: Example 1 and Ganoderma lucidum exosomes loaded with an equal amount of Ganoderma lucidum polysaccharide were respectively loaded into dialysis bags with a molecular weight cutoff of 80-120 kDa (soaked in deionized water for 12-20 hours in advance), immersed in a phosphate buffer solution of pH 5.0-6.0 (200±10 mL, 37±1° C.), and shaken at 50-150 rpm for 72 hours. The external release liquid was collected every 0.5-72 hours and supplemented with an equal volume of pre-warmed medium; using Ganoderma lucidum polysaccharide as an indicator, the phenol-sulfuric acid method (5% phenol solution to release liquid volume ratio of 1:2, concentrated sulfuric acid added in an amount of 2.5 times) was used in combination with an ultraviolet spectrophotometer to detect and calculate the cumulative release rate. The cumulative release rate (Q) was calculated according to formula (1):

[0055]

[0056] where Ct is the concentration at the current time point, Vt is the total volume of the release medium, ΣCiVi is the cumulative amount of samples taken at all previous time points, and M is the initial total drug dose.

[0057] The cumulative release rate of Example 1 is Q1, and the cumulative release rate of Ganoderma lucidum exosomes loaded with the same amount of Ganoderma lucidum polysaccharide is Q2. The measured cumulative release rates are shown in the following table:

[0058] Time (h) <![CDATA[Q1]]> <![CDATA[Q2]]> 0.5 8.2±1.3 32.5±2.6 1 12.6±1.5 42.8±1.9 2 18.4±1.8 58.3±2.3 4 24.7±1.4 68.9±2.4 6 30.1±2.2 75.4±2.9 12 41.5±1.5 82.6±2.0 24 53.2±2.8 88.1±2.7 48 66.8±2.1 91.5±1.5 72 78.3±1.7 93.7±1.6

[0059] The results showed that the composite exosomes T prepared in Example 1 50 The time required for the release rate of active ingredients to reach 50% is 12 to 24 hours, while the T 50 The value was 1 to 2 h, which confirmed that the composite exosomes achieved sustained release synergy through the lipid-exosome complex structure and significantly prolonged the action time of the active ingredients.

[0060] Example 4:

[0061] The preparation method specifically comprises:

[0062] Step 1: Wash fresh Ganoderma lucidum with double-distilled water and add phosphate buffer solution (PBS), then grind it into a homogenate in a homogenizer. After filtering, add one or more enzymes including but not limited to cellulase, pectinase, papain, etc., and adjust the pH value to 7.0 with trishydroxymethylaminomethane hydrochloride (Tris-HCl). The homogenate prepared above is centrifuged at 400, 800, and 13000 × g for 10 minutes, and then at 10000 × g for 30 minutes. The precipitate is collected, Tris-HCl is added, and mixed with a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0063] Step 2: The exosomes were treated with ultrasound at a power of 20W for 30s to temporarily break the exosome membrane. A solution of liver-protecting ingredients such as Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids, and trace elements dissolved in PBS was added. The mass percentage ratio included: Ganoderma lucidum polysaccharides 0.4%, carotenoids 0.2%, vitamin C 0.05%, vitamin E 0.05%, flavonoids 0.1%, trace elements 0.01%, and the remaining PBS. Due to its concentration gradient, the liver-protecting ingredients efficiently entered the exosomes. A solution containing 1mM Ca was added. 2+ The cells were fixed with PBS solution at 35°C for 30 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0064] Step 3: After dissolving 0.4% cholesterol, 0.2% DPPC, 0.15% sphingomyelin and 0.05% PEG-ceramide in the remaining ethanol according to mass percentage, the solvent was removed by rotary evaporator to form a uniform phospholipid film in the container, and the above-mentioned buffer solution containing Ganoderma lucidum exosomes was added to the container and vortexed at 40°C for 15 minutes to convert the outer layer of liposomes into unilamellar liposomes, forming a three-layer structure with liposomes on the outside, exosomes in the middle layer, and liver-protecting components in the inner layer.

[0065] Step 4: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 5 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0066] Example 5:

[0067] The preparation method specifically comprises the following steps:

[0068] Step 1: Wash fresh Ganoderma lucidum with double-distilled water and add phosphate buffer solution (PBS), then grind it into a homogenate in a homogenizer. After filtering, add one or more enzymes including but not limited to cellulase, pectinase, papain, etc., and adjust the pH value to 7.1 with tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl). The homogenate prepared above is centrifuged at 450, 850, and 13500 × g for 15 minutes, and then centrifuged at 10500 × g for 40 minutes. The precipitate is collected, Tris-HCl is added, and mixed with a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0069] Step 2: The exosomes were treated with ultrasound at a power of 25W for 50s to temporarily break the exosome membrane. A solution of liver-protecting ingredients such as Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids, and trace elements dissolved in PBS was added. The mass percentage ratio included: Ganoderma lucidum polysaccharides 0.45%, carotenoids 0.23%, vitamin C 0.07%, vitamin E 0.07%, flavonoids 0.12%, trace elements 0.02%, and the remaining PBS. Due to its concentration gradient, the liver-protecting ingredients efficiently entered the exosomes. A solution containing 1.2mM Ca 2+ The cells were fixed with PBS solution at 36°C for 50 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0070] Step 3: After dissolving 0.42% cholesterol, 0.22% DPPC, 0.17% sphingomyelin and 0.06% PEG-ceramide in the remaining ethanol, the solvent was removed by rotary evaporator to form a uniform phospholipid film in the container. The above-mentioned buffer containing Ganoderma lucidum exosomes was added to the container and vortexed at 45°C for 18 minutes to convert the outer layer of liposomes into unilamellar liposomes, forming a three-layer structure with liposomes on the outside, exosomes in the middle layer, and liver-protecting components in the inner layer.

[0071] Step 4: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 7 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0072] Example 6:

[0073] The preparation method specifically comprises the following steps:

[0074] Step 1: Wash fresh Ganoderma lucidum with double-distilled water and add phosphate buffer solution (PBS), then place it in a homogenizer and grind it into a homogenate. After filtering, add one or more enzymes including but not limited to cellulase, pectinase, papain, etc., and adjust the pH value to 7.2 with trishydroxymethylaminomethane hydrochloride (Tris-HCl). The homogenate prepared above is centrifuged at 500, 900, and 14000×g for 20 minutes, and then at 11000×g for 60 minutes. The precipitate is collected, Tris-HCl is added, and mixed with a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0075] Step 2: The exosomes were treated with ultrasound at a power of 30W for 75s to temporarily break the exosome membrane. A solution of liver-protecting ingredients such as Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids, and trace elements dissolved in PBS was added. The mass percentage ratio included: Ganoderma lucidum polysaccharides 0.5%, carotenoids 0.25%, vitamin C 0.075%, vitamin E 0.075%, flavonoids 0.15%, trace elements 0.03%, and the remaining PBS. Due to its concentration gradient, the liver-protecting ingredients efficiently entered the exosomes. A solution containing 1.5mM Ca 2+ The cells were fixed with PBS solution at 38°C for 75 minutes, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0076] Step 3: After dissolving 0.45% cholesterol, 0.25% DPPC, 0.225% sphingomyelin and 0.075% PEG-ceramide in the remaining ethanol, the solvent was removed by rotary evaporator to form a uniform phospholipid film in the container. The above-mentioned buffer solution containing Ganoderma lucidum exosomes was added to the container and vortexed at 50°C for 25 minutes to convert the outer layer of liposomes into unilamellar liposomes, forming a three-layer structure with liposomes on the outside, exosomes in the middle layer, and liver-protecting components in the inner layer.

[0077] Step 4: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 10 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0078] Example 7:

[0079] The preparation method specifically comprises the following steps:

[0080] Step 1: Wash fresh Ganoderma lucidum with double-distilled water and add phosphate buffer solution (PBS), then grind it into a homogenate in a homogenizer. After filtering, add one or more enzymes including but not limited to cellulase, pectinase, papain, etc., and adjust the pH value to 7.3 with tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl). Centrifuge the homogenate at 550, 950, and 14500 × g for 25 minutes, and then at 11500 × g for 80 minutes. Collect the precipitate, add Tris-HCl, and mix it with a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0081] Step 2: The exosomes were treated with ultrasound at a power of 35W for 110s to temporarily break the exosome membrane. A solution of liver-protecting ingredients such as Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids, and trace elements dissolved in PBS was added. The mass percentage ratio included: Ganoderma lucidum polysaccharides 0.55%, carotenoids 0.27%, vitamin C 0.08%, vitamin E 0.07%, flavonoids 0.18%, trace elements 0.04%, and the remaining PBS. Due to its concentration gradient, the liver-protecting ingredients efficiently entered the exosomes. A solution containing 1.7mM Ca 2+ The cells were fixed with PBS solution at 38°C for 90 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0082] Step 3: After dissolving 0.47% cholesterol, 0.28% DPPC, 0.27% sphingomyelin and 0.08% PEG-ceramide in the remaining ethanol, the solvent was removed by rotary evaporator to form a uniform phospholipid film in the container. The above-mentioned buffer containing Ganoderma lucidum exosomes was added to the container and vortexed at 55°C for 25 minutes to convert the outer layer of liposomes into unilamellar liposomes, forming a three-layer structure with liposomes on the outside, exosomes in the middle layer, and liver-protecting components in the inner layer.

[0083] Step 4: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 12 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0084] Example 8:

[0085] The preparation method specifically comprises the following steps:

[0086] Step 1: Wash fresh Ganoderma lucidum with double-distilled water and add phosphate buffer solution (PBS), then place it in a homogenizer and grind it into a homogenate. After filtering, add one or more enzymes including but not limited to cellulase, pectinase, papain, etc., and adjust the pH value to 7.4 with trishydroxymethylaminomethane hydrochloride (Tris-HCl). The homogenate prepared above is centrifuged at 600, 1000, and 15000×g for 30 minutes, and then at 12000×g for 90 minutes. The precipitate is collected, Tris-HCl is added, and mixed with a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0087] Step 2: The exosomes were treated with ultrasound at a power of 40W for 120s to temporarily break the exosome membrane. A solution of liver-protecting ingredients such as Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids, and trace elements dissolved in PBS was added. The mass percentage ratio included: Ganoderma lucidum polysaccharides 0.6%, carotenoids 0.3%, vitamin C 0.1%, vitamin E 0.1%, flavonoids 0.2%, trace elements 0.05%, and the remaining PBS. Due to its concentration gradient, the liver-protecting ingredients efficiently entered the exosomes. A solution containing 2mM Ca was added. 2+ The cells were fixed with PBS solution at 40°C for 120 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0088] Step 3: After dissolving 0.5% cholesterol, 0.3% DPPC, 0.3% sphingomyelin and 0.1% PEG-ceramide in the remaining ethanol, the solvent was removed by rotary evaporator to form a uniform phospholipid film in the container. The above-mentioned buffer containing Ganoderma lucidum exosomes was added to the container and vortexed at 60°C for 30 minutes to convert the outer layer of liposomes into unilamellar liposomes, forming a three-layer structure with liposomes on the outside, exosomes in the middle layer, and liver-protecting components in the inner layer.

[0089] Step 4: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 15 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0090] Example 9:

[0091] The preparation method specifically comprises the following steps:

[0092] Step 1: Wash fresh Ganoderma lucidum with double-distilled water and add phosphate buffer solution (PBS), then place it in a homogenizer and grind it into a homogenate. After filtering, add one or more enzymes including but not limited to cellulase, pectinase, papain, etc., and adjust the pH value to 7.4 with trishydroxymethylaminomethane hydrochloride (Tris-HCl). The homogenate prepared above is centrifuged at 400, 1000, and 15000 × g for 10 minutes, and then centrifuged at 10000 × g for 90 minutes. The precipitate is collected, Tris-HCl is added, and mixed with a vortex oscillator to obtain crudely extracted Ganoderma lucidum exosomes.

[0093] Step 2: The exosomes were treated with ultrasound at a power of 20W for 120s to temporarily break the exosome membrane. A solution of liver-protecting ingredients such as Ganoderma lucidum polysaccharides, carotenoids, vitamin C, vitamin E, flavonoids, and trace elements dissolved in PBS was added. The mass percentage ratio included: Ganoderma lucidum polysaccharides 0.6%, carotenoids 0.2%, vitamin C 0.05%, vitamin E 0.1%, flavonoids 0.2%, trace elements 0.05%, and the remaining PBS. Due to its concentration gradient, the liver-protecting ingredients efficiently entered the exosomes. A solution containing 2mM Ca was added. 2+ The cells were fixed with PBS solution at 40°C for 30 min, and the precipitate was collected after standing to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients.

[0094] Step 3: After dissolving 0.5% cholesterol, 0.2% DPPC, 0.15% sphingomyelin and 0.1% PEG-ceramide in the remaining ethanol, the solvent was removed by rotary evaporator to form a uniform phospholipid film in the container. The above-mentioned buffer containing Ganoderma lucidum exosomes was added to the container and vortexed at 60°C for 15 minutes to convert the outer layer of liposomes into unilamellar liposomes, forming a three-layer structure with liposomes on the outside, exosomes in the middle layer, and liver-protecting components in the inner layer.

[0095] Step 4: Fix the purification column with an iron stand, add filler that is greater than 2 / 3 of the column length, soak it with PBS for 5 minutes, add the above-mentioned sample buffer, open the piston, and collect the washing liquid with a test tube. During this period, PBS is continuously added to ensure that the liquid in the column always covers the filler until no peak appears in the ultraviolet-visible (UV-Vis) detector. Collect the washing liquid corresponding to the protein molecular weight peak to obtain the purified Ganoderma lucidum lipid exosomes containing liver-protecting components.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting ingredients, characterized in that: The following steps are involved: (1) Exosome extraction: Ganoderma lucidum was washed and added into PBS solution. After homogenization, enzymatic hydrolysis and centrifugation, the precipitate was collected to obtain crude extracted Ganoderma lucidum exosomes; (2) Encapsulation of liver-protecting components: The crudely extracted liver-protecting exosomes obtained in step (1) were subjected to ultrasonic treatment to temporarily break the membrane, and a PBS solution containing liver-protecting components was added. After standing, a solution containing Ca was added. 2 + PBS solution for repair, and collect the precipitate to obtain Ganoderma lucidum exosomes containing liver-protecting ingredients; (3) Liposome encapsulation: Cholesterol, DPPC, sphingomyelin, and PEG-ceramide are dissolved to form a phospholipid film, and the Ganoderma lucidum exosomes containing the liver-protecting component obtained in step (2) are added, and the sample is vortexed to form a three-layer structure of liposomes-exosomes-liver-protecting component; (4) Separation and purification: The sample obtained in step (3) is purified by a purification column to collect the target component, thereby obtaining Ganoderma lucidum lipid exosomes containing liver-protecting components.

2. The method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting ingredients according to claim 1, characterized in that: The centrifugation step in step (1) comprises centrifugation at 400-600×g for 10-30 minutes, centrifugation at 800-1000×g for 10-30 minutes, centrifugation at 13000-15000×g for 10-30 minutes, and centrifugation at 10000-12000×g for 30-90 minutes.

3. The method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting components according to claim 1, characterized in that: The PBS solution containing liver-protecting ingredients in step (2) comprises, by mass percentage, 0.4-0.6% of Ganoderma lucidum polysaccharide, 0.2-0.3% of carotenoids, 0.05-0.1% of vitamin C, 0.05-0.1% of vitamin E, 0.1-0.2% of flavonoids, 0.01-0.05% of trace elements, and the remainder of PBS.

4. The method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting components according to claim 3, characterized in that: The power of ultrasonic treatment in step (2) is 20-40W and the time is 30-120 seconds; 2 +Ca in PBS solution 2 +The concentration is 1-2 mM, the repair temperature is 35-40°C, and the repair time is 30-120 minutes.

5. The method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting components according to claim 1, characterized in that: In the step (3), 0.4-0.5% cholesterol, 0.2-0.3% DPPC, 0.15-0.3% sphingomyelin and 0.05-0.1% PEG-ceramide are dissolved in the remaining solvent and evaporated to prepare a phospholipid film.

6. The method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting components according to claim 5, characterized in that: The vortex treatment in step (3) is performed at a temperature of 40 to 60° C. and for a time of 15 to 30 minutes.

7. The method for preparing Ganoderma lucidum lipid exosomes containing liver-protecting components according to claim 1, characterized in that: The filler of the purification column in step (4) is a molecular exclusion chromatography medium, and PBS solution is used as the mobile phase during purification until no target peak appears in the ultraviolet detector.

8. A Ganoderma lucidum lipid exosome containing liver-protecting ingredients, characterized in that: The method for preparing a Ganoderma lucidum lipid exosome containing a liver-protecting component according to any one of claims 1 to 7 is used, and the lipid exosome has a three-layer structure of liposomes-exosomes-liver-protecting components from the outside to the inside, a particle size of 80 to 150 nm, and a T value of 12 to 24 hours.

9. Use of the Ganoderma lucidum lipid exosomes containing liver-protecting components as claimed in claim 8 in the preparation of a liver-protecting preparation.

10. The use of Ganoderma lucidum lipid exosomes containing liver-protecting components in the preparation of a liver-protecting preparation according to claim 9, characterized in that: The liver protection preparation is used for transdermal delivery and is in the form of an external cream, essence or patch.

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