Plant exosome as well as extraction method and application thereof in cosmetics

Abstract

The invention provides a plant exosome as well as an extraction method and application thereof in cosmetics, and belongs to the technical field of exosome extraction. The preparation method of the plant exosome comprises the following steps: mixing plant tissues with a solvent, carrying out high-pressure homogenization treatment, adding immobilized compound enzyme for enzymolysis, filtering, carrying out differential centrifugation on enzymatic hydrolysate, and carrying out size exclusion chromatography and ultrafiltration purification to obtain the plant exosome. Wherein exosome release and protection are considered, and the influence of agglomeration on the exosome extraction effect is avoided; high-pressure homogenization and immobilized compound enzyme enzymolysis are adopted, physical wall breaking is combined with chemical wall breaking, and the yield of the exosome is increased; the immobilized compound enzyme can avoid free enzyme residues, and the subsequent purification step is simplified; the plant exosome can be further purified through size exclusion chromatography and ultrafiltration. The exosome prepared by the method disclosed by the invention can retain the active ingredients of the exosome to the greatest extent, has excellent antioxidant, anti-inflammatory and whitening effects, and is suitable for the field of cosmetics.

Description

Technical Field

[0001] This invention relates to the field of exosome extraction technology, specifically to a plant exosome, its extraction method, and its application in cosmetics. Background Technology

[0002] With the rapid development of biotechnology and cosmetic science, exosomes, as a novel bioactive carrier, have shown broad application prospects in the fields of medicine, skincare, and regenerative medicine. Exosomes are nanoscale vesicles secreted by cells, rich in bioactive components such as proteins, lipids, and nucleic acids (e.g., mRNA, miRNA), and possess excellent intercellular communication and bioregulatory functions. Studies have shown that plant-derived exosomes, similar to animal exosomes, can participate in cellular metabolic regulation, immune regulation, and tissue repair, while also possessing advantages such as wide availability, high safety, and ease of large-scale production, thus becoming a research hotspot in the cosmetic and biomedical fields.

[0003] Traditional exosome research has primarily focused on animal cells (such as mesenchymal stem cells and immune cells), but their application is limited due to potential immunogenicity, ethical controversies, and high costs associated with animal exosomes. In contrast, plant exosomes are naturally non-toxic, have low immunogenicity, and are environmentally friendly. Furthermore, plant cell culture conditions are simple, allowing for stable production through large-scale cultivation. Currently, plant exosome extraction methods generally suffer from low efficiency, susceptibility to damage to exosome integrity, and the presence of impurities.

[0004] In the cosmetics industry, consumers are increasingly demanding natural, safe, and highly effective active ingredients. Plant exosomes, with their natural origin and multiple biological functions, hold promise as a new generation of skincare active ingredients. However, current research on plant exosome extraction remains insufficient, necessitating the development of more efficient extraction technologies and in-depth verification of their specific efficacy in skincare. Therefore, exploring an efficient method for extracting plant exosomes and studying their application in cosmetics has significant scientific and market value. Summary of the Invention

[0005] The purpose of this invention is to provide a plant exosome, its extraction method, and its application in cosmetics. The plant exosomes prepared by this invention have antioxidant, anti-inflammatory, and whitening effects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing plant exosomes, comprising: mixing plant tissue with a solvent, homogenizing under high pressure, adding an immobilized complex enzyme for enzymatic hydrolysis, filtering, centrifuging the enzymatic hydrolysate at differential speed, and then purifying it by size exclusion chromatography and ultrafiltration to obtain plant exosomes; wherein the solvent includes sodium chloride, trehalose, mannitol, glycerol, glutathione, L-cysteine ​​and Tween 80.

[0008] Preferably, the solvent comprises, by concentration, 80-120 mM sodium chloride, 50-100 mg / mL trehalose, 0.5-1 mol / L mannitol, 40-60 mg / mL glycerol, 5-10 mM glutathione, 10-20 mM L-cysteine, and 15-25 mg / mL Tween 80.

[0009] Preferably, the pressure of the high-pressure homogenization process is 50-70 MPa, the temperature is 2-5℃, the number of cycles is 3-5, and each cycle lasts 30-40 seconds.

[0010] Preferably, the immobilized complex enzyme is obtained by immobilizing cellulase, hemicellulase and pectinase using an immobilization carrier.

[0011] More preferably, the enzymatic hydrolysis temperature is 37-42℃, the pH value is 5-5.5, the rotation speed is 40-80 rpm, and the time is 40-50 min.

[0012] Preferably, the weight ratio of the plant tissue to the solvent is 1:5-10 g / mL.

[0013] Preferably, the differential centrifugation includes: centrifuging the enzymatic hydrolysate sequentially at 300-700g for 8-12 min, at 1800-2200g for 18-22 min, at 10000-14000g for 28-32 min, and at 120000-150000g for 45-65 min.

[0014] Preferably, the plant is one or more of the following: orchid, tea, lemon, chlorella, turmeric, ginseng, coix seed, dendrobium officinale, black truffle, and ganoderma.

[0015] The present invention also provides plant exosomes obtained by the above preparation method.

[0016] This invention also provides the application of the above-mentioned plant exosomes in the preparation of cosmetics.

[0017] The present invention also provides a composition of plant exosomes, which is composed of Phalaenopsis exosomes, ginseng exosomes, coix seed exosomes and Dendrobium officinale exosomes in a weight ratio of 1-3:2-4:1-3:2-4.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention provides a method for preparing plant exosomes, comprising: mixing plant tissue with a solvent, homogenizing under high pressure, adding an immobilized complex enzyme for enzymatic hydrolysis, filtering, centrifuging the hydrolysate differentially, and then purifying it by size exclusion chromatography and ultrafiltration to obtain plant exosomes; the solvent includes sodium chloride, trehalose, mannitol, glycerol, glutathione, L-cysteine, and Tween 80. The solvent balances osmotic pressure, provides antioxidant protection, and stabilizes the membrane, balancing exosome release and protection while preventing exosome aggregation that could affect extraction efficiency; high-pressure homogenization and immobilized complex enzyme synergistically disrupt the cell wall using a combination of physical and chemical methods, improving exosome yield; the immobilized complex enzyme improves enzyme stability, avoids free enzyme residue, and simplifies subsequent purification steps; differential centrifugation avoids high-speed centrifugation that could directly cause exosome aggregation or damage; size exclusion chromatography and ultrafiltration further purify the plant exosomes. Exosomes prepared using this method retain the most active components.

[0020] This invention also provides a composition of plant exosomes, comprising Phalaenopsis orchid exosomes, ginseng exosomes, Job's tears exosomes, and Dendrobium officinale exosomes. This exosome composition, when combined, can enhance free radical scavenging, inhibit tyrosinase activity, and reduce inflammatory factor levels, exhibiting excellent antioxidant, anti-inflammatory, and whitening effects, making it suitable for the cosmetics industry. Detailed Implementation

[0021] This invention provides a method for preparing plant exosomes, comprising: mixing plant tissue with a solvent, homogenizing under high pressure, adding an immobilized complex enzyme for enzymatic hydrolysis, filtering, centrifuging the enzymatic hydrolysate at differential speed, and then purifying it by size exclusion chromatography and ultrafiltration to obtain plant exosomes; wherein the solvent includes sodium chloride, trehalose, mannitol, glycerol, glutathione, L-cysteine ​​and Tween 80.

[0022] The solvent of the present invention preferably comprises, by concentration, 80-120 mM sodium chloride, 50-100 mg / mL trehalose, 0.5-1 mol / L mannitol, 40-60 mg / mL glycerol, 5-10 mM glutathione, 10-20 mM L-cysteine, and 15-25 mg / mL Tween 80; more preferably, it comprises 100 mM sodium chloride, 80 mg / mL trehalose, 0.8 mol / L mannitol, 50 mg / mL glycerol, 8 mM glutathione, 15 mM L-cysteine, and 20 mg / mL Tween 80. In this invention, sodium chloride in the solvent maintains an isotonic environment, protecting cell integrity; trehalose, a natural protective agent, stabilizes the exosome membrane structure and prevents degradation; mannitol, an osmotic regulator, synergistically protects cell membrane integrity; glycerol increases solvent viscosity, reducing mechanical damage to exosomes; glutathione and L-cysteine ​​act as antioxidants, inhibiting oxidative stress-induced damage to plant exosomes; and Tween 80, a surfactant, promotes cell wall rupture and exosome release. The solvent in this invention stabilizes the osmotic environment, reduces oxidative stress during exosome preparation, assists in disrupting plant tissue cell walls, promotes exosome release, and provides a suitable stable environment for subsequent enzymatic hydrolysis and centrifugation.

[0023] The preferred weight ratio of plant tissue to solvent in this invention is 1:5-10 g / mL, more preferably 1:8 g / mL.

[0024] The pressure of the high-pressure homogenization process described in this invention is preferably 50-70 MPa, more preferably 60 MPa; the temperature is preferably 2-5℃, more preferably 4℃; the number of cycles is preferably 3-5 times, more preferably 4 times, with each cycle lasting 35 seconds. High-pressure homogenization can disrupt the cell wall structure of cell tissues, promoting the release of exosomes. Processing at low temperatures can reduce thermal damage and minimize the destruction of active ingredients.

[0025] The immobilized composite enzyme of this invention is obtained by immobilizing cellulase, hemicellulase, and pectinase using an immobilization carrier. A preferred method for preparing the immobilized composite enzyme of this invention includes: selecting activated carbon particles with an average particle size of 180-220 mesh, activating them with high-temperature steam at 400-500℃ for 1.5-2.5 h, cooling to 55-65℃, immersing in 0.8-1.2M hydrochloric acid solution for 1-2 h, filtering, mixing the precipitate with 0.25-0.35 g / mL nitric acid solution at a ratio of 1:8-12 g / mL, microwave-assisted oxidation at 80-90℃ and 600-800 W for 20-30 min, cooling to 23-27℃, and washing 2-4 times with deionized water. The immobilized carrier was dried at 55-65℃ to constant weight. Cellulase, hemicellulase, pectinase, and the immobilized carrier were added to PBS buffer and immobilized at 33-38℃ for 3 hours. After filtration, the mixture was washed 2-4 times with PBS buffer and filtered again to obtain the immobilized complex enzyme. The preferred enzymatic hydrolysis temperature was 37-42℃, more preferably 40℃; the preferred pH value was 5-5.5, more preferably 5.2; the preferred rotation speed was 40-80 rpm, more preferably 50 rpm; and the preferred time was 40-50 min, more preferably 45 min. Cellulase, hemicellulase, and pectinase can synergistically degrade plant cell wall polysaccharides, further promoting the release of exosomes. During the preparation of the immobilized complex enzyme, high-temperature activation and acid treatment can increase the carrier porosity, thereby increasing the enzyme loading; microwave-assisted oxidation can introduce functional groups such as carboxyl groups, further enhancing the binding ability between the enzyme and the carrier. Immobilizing the complex enzyme can improve its stability, avoid free enzyme residue, simplify subsequent purification steps, and allow for rapid separation from the enzyme hydrolysate through filtration, enabling repeated recycling and reducing production costs.

[0026] The centrifugation speed of the present invention is preferably 4000-5000g, and the centrifugation time is preferably 8-10min, more preferably 9min.

[0027] The differential centrifugation method of the present invention preferably includes: centrifuging the enzymatic hydrolysate sequentially at 300-700g for 8-12 min (to remove large particulate impurities such as cell debris and unbroken cells), at 1800-2200g for 18-22 min (to precipitate larger organelles (such as mitochondria and chloroplasts) and cell debris), and at 10000-14000g for 28-32 min (to collect smaller organelle debris and some vesicles), discarding the bottom precipitate, taking the supernatant, and centrifuging the supernatant at 120000-150000g for 45-65 min, and taking the precipitate. More preferably, it includes: centrifuging the enzymatic hydrolysate sequentially at 500g for 10 min, at 2000g for 20 min, and at 9000g for 30 min, then centrifuging the supernatant at 130000g for 50 min, and taking the precipitate.

[0028] The plant used in this invention is preferably one or more of the following: orchid, tea, lemon, chlorella, turmeric, ginseng, coix seed, dendrobium officinale, black truffle, and ganoderma.

[0029] The present invention also provides plant exosomes obtained by the above preparation method.

[0030] This invention also provides the application of the above-mentioned plant exosomes in the preparation of cosmetics.

[0031] This invention also provides a composition of plant exosomes, comprising Phalaenopsis orchid exosomes, ginseng exosomes, Job's tears exosomes, and Dendrobium officinale exosomes in a weight ratio of 1-3:2-4:1-3:2-4. This composition exhibits synergistic effects in antioxidation, anti-inflammation, and whitening through multi-component and multi-pathway synergy. Experimental results show that the exosome composition enhances free radical scavenging, inhibits tyrosinase activity, and reduces inflammatory factor levels, demonstrating excellent antioxidant, anti-inflammatory, and whitening effects, making it suitable for the development of multifunctional cosmetics.

[0032] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, the following embodiments are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Cellulase was purchased from Nanba (Shanxi) Biomedical Technology Co., Ltd., with an enzyme activity of 20,000 U / g; hemicellulase was purchased from Xi'an Juntian Biotechnology Co., Ltd., with an enzyme activity of 10,000 U / g; pectinase was purchased from Nanjing Deju Biotechnology Co., Ltd., with an enzyme activity of 10,000 U / g; the DPPH free radical scavenging rate assay kit and the total antioxidant capacity (ABTS method) test kit were purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.; the TNF-α ELISA assay kit and the IL-6 ELISA assay kit were purchased from Shanghai Keaibo Biotechnology Co., Ltd.

[0036] Example 1

[0037] Extraction of Phalaenopsis exosomes

[0038] (1) Preparation of immobilized complex enzymes

[0039] Activated carbon made from 200-mesh cedar wood particles was activated by high-temperature steam at 450℃ for 2 hours, cooled to 60℃, immersed in 1M hydrochloric acid solution for 2 hours, filtered, and the solid residue was mixed with 0.3mol / L nitric acid solution at a ratio of 1:10 g / mL. The mixture was then microwave-assisted oxidized at 85℃ and 700W for 25 minutes, cooled to 25℃, washed three times with deionized water, and dried at 60℃ to constant weight to obtain the immobilized carrier.

[0040] Cellulase, hemicellulase, pectinase and immobilization carrier were added to 1×PBS buffer (pH 7.4) at a weight ratio of 3:1.5:1:45, with a material-to-liquid ratio of 1:12 g / mL. The mixture was reacted at 40°C for 3 h, filtered, and washed 3 times with 1×PBS to obtain the immobilized complex enzyme.

[0041] (2) Enzymatic hydrolysis

[0042] Fresh Phalaenopsis orchids were mixed with solvent at a ratio of 1:8 g / mL, and homogenized under high pressure at 60 MPa and 4℃ for 4 cycles (35 s each time). The mixture was then centrifuged at 5000 g for 15 min at 4℃, and the supernatant was collected.

[0043] Solvent composition: Water-based, containing 100mM sodium chloride, 80mg / mL trehalose, 0.8mol / L mannitol, 50mg / mL glycerol, 8mM glutathione, 15mM L-cysteine, and 20mg / mL Tween 80, diluted to volume with deionized water;

[0044] Add immobilized complex enzyme (3% of the weight of fresh Phalaenopsis orchid) to the supernatant, and enzymatically hydrolyze for 45 min at 40℃, pH 5.0, and 50 rpm. Filter to recover the immobilized enzyme and obtain the enzymatic hydrolysate.

[0045] (3) Differential centrifugation

[0046] The enzymatic hydrolysate was centrifuged sequentially at 4°C at 500g for 10 min, 2000g for 20 min, and 12000g for 30 min. The supernatant was then centrifuged at 130000g for 60 min, and the precipitate was collected.

[0047] (4) Size exclusion chromatography and ultrafiltration purification

[0048] The precipitate was resuspended in 1×PBS buffer (pH 7.4) at a ratio of 1:3 g / mL, and filtered through a 0.22 μm filter membrane to obtain the resuspended solution.

[0049] The Sephacryl S-500HR chromatography column was equilibrated with 1×PBS. The resuspension was injected at a flow rate of 0.5 mL / min, and the eluent was collected at a rate of 1 mL / tube. The UV values ​​at 280 nm and 260 nm were monitored, and the flow-through liquid in tubes 5-7 of the main peak was collected.

[0050] Phalaenopsis exosomes were obtained by retaining the convection fluid using a 100 kDa regenerated cellulose ultrafiltration membrane.

[0051] Example 2

[0052] Extraction of ginseng exosomes

[0053] (1) Preparation of immobilized complex enzymes

[0054] 180-mesh cedar granules of activated carbon were activated by high-temperature steam at 400℃ for 2.5h, cooled to 55℃, immersed in 0.8M hydrochloric acid solution for 2h, filtered, and the solid residue was mixed with 0.25mol / L nitric acid solution at a ratio of 1:12g / mL. The mixture was then microwave-assisted oxidized at 80℃ and 600W for 30min, cooled to 23℃, washed 4 times with deionized water, and dried at 55℃ to constant weight to obtain the immobilized carrier.

[0055] Cellulase, hemicellulase, pectinase and immobilization carrier were added to 1×PBS buffer (pH 7.4) at a weight ratio of 2:1:1:20, with a material-to-liquid ratio of 1:10 g / mL. The mixture was reacted at 35°C for 3.5 h, filtered, and washed 4 times with 1×PBS to obtain the immobilized complex enzyme.

[0056] (2) Enzymatic hydrolysis

[0057] Mix human ginseng with solvent at a ratio of 1:5 g / mL, homogenize under high pressure at 70 MPa and 5℃ for 3 cycles (40 s each time), centrifuge at 3000 g for 12 min at 4℃, and collect the supernatant.

[0058] Solvent composition: 120mM sodium chloride, 50mg / mL trehalose, 1mol / L mannitol, 40mg / mL glycerol, 10mM glutathione, 10mM L-cysteine ​​and 15mg / mL Tween 80, diluted to volume with deionized water;

[0059] Add immobilized complex enzyme (4% of the weight of ginseng) to the supernatant, and enzymatically hydrolyze for 50 min at 42℃, pH 5.5 and 40 rpm. Filter to recover the immobilized enzyme and obtain the enzymatic hydrolysate.

[0060] (3) Differential centrifugation

[0061] The enzymatic hydrolysate was centrifuged sequentially at 4°C at 300g for 12 min, 1800g for 22 min, and 10000g for 32 min. The supernatant was then centrifuged at 120000g for 65 min, and the precipitate was collected.

[0062] (4) Size exclusion chromatography and ultrafiltration purification

[0063] The precipitate was resuspended in 1×PBS buffer (pH 7.4) at a ratio of 1:3 g / mL, and filtered through a 0.22 μm filter membrane to obtain the resuspended solution.

[0064] The Sephacryl S-500HR chromatography column was equilibrated with 1×PBS. The resuspension was injected at a flow rate of 0.4 mL / min, and the eluent was collected at a rate of 1 mL / tube. The UV values ​​at 280 nm and 260 nm were monitored, and the flow-through liquid in tubes 5-7 of the main peak was collected.

[0065] Ginseng exosomes were obtained by retaining the convection fluid using a 100 kDa regenerated cellulose ultrafiltration membrane.

[0066] Example 3

[0067] Extraction of Job's tears exosomes

[0068] (1) Preparation of immobilized complex enzymes

[0069] Activated carbon granules of Chinese fir with an average particle size of 220 mesh were activated by high-temperature steam at 500℃ for 1.5h, cooled to 65℃, immersed in 1.2M hydrochloric acid solution for 1.5h, filtered, and the solid residue was mixed with 0.35mol / L nitric acid solution at a ratio of 1:12g / mL. The mixture was then microwave-assisted oxidized at 90℃ and 800W for 20min, cooled to 27℃, washed twice with deionized water, and dried at 65℃ to constant weight to obtain the immobilized carrier.

[0070] Cellulase, hemicellulase, pectinase and immobilization carrier were added to 1×PBS buffer (pH 7.4) at a weight ratio of 5:2:1:65, with a material-to-liquid ratio of 1:15 g / mL. The mixture was reacted at 37°C for 3.5 h, filtered, and washed twice with 1×PBS to obtain the immobilized complex enzyme.

[0071] (2) Enzymatic hydrolysis

[0072] Fresh Job's tears were mixed with solvent at a ratio of 1:8 g / mL, and homogenized under high pressure at 60 MPa and 4℃ for 4 cycles (35 s each time). The mixture was then centrifuged at 5000 g for 15 min at 4℃, and the supernatant was collected.

[0073] Solvent composition: Water-based, containing 100mM sodium chloride, 80mg / mL trehalose, 0.8mol / L mannitol, 50mg / mL glycerol, 8mM glutathione, 15mL M cysteine, and 20mg / mL Tween 80, diluted to volume with deionized water;

[0074] Add immobilized complex enzyme (3% of the weight of fresh coix seed) to the supernatant, and enzymatically hydrolyze for 40 min at 38℃, pH 5.2 and 80 rpm. Filter and recover the immobilized enzyme to obtain the enzymatic hydrolysate.

[0075] (3) Differential centrifugation

[0076] The enzymatic hydrolysate was centrifuged sequentially at 700g for 8 min, 2200g for 18 min, and 14000g for 28 min at 4℃. The supernatant was then centrifuged at 150000g for 45 min, and the precipitate was collected.

[0077] (4) Size exclusion chromatography and ultrafiltration purification

[0078] The precipitate was resuspended in 1×PBS buffer (pH 7.4) at a ratio of 1:3 g / mL, and filtered through a 0.22 μm filter membrane to obtain the resuspended solution.

[0079] The Sephacryl S-500HR chromatography column was equilibrated with 1×PBS. The resuspension was injected at a flow rate of 0.6 mL / min, and the eluent was collected at a rate of 1 mL / tube. The UV values ​​at 280 nm and 260 nm were monitored, and the flow-through liquid in tubes 5-7 of the main peak was collected.

[0080] Coix seed exosomes were obtained by retaining the convection fluid using a 100 kDa regenerated cellulose ultrafiltration membrane.

[0081] Example 4

[0082] Extraction of Dendrobium officinale exosomes

[0083] (1) Preparation of immobilized complex enzymes

[0084] Activated carbon granules of cedar wood with an average particle size of 200 mesh were activated by high-temperature steam at 480℃ for 1.8h, cooled to 58℃, immersed in 1M hydrochloric acid solution for 2h, filtered, and the solid residue was mixed with 0.32mol / L nitric acid solution at a ratio of 1:9g / mL. The mixture was then microwave-assisted oxidized at 82℃ and 720W for 23min, cooled to 24℃, washed 3 times with deionized water, and dried at 58℃ to constant weight to obtain the immobilized carrier.

[0085] Cellulase, hemicellulase, pectinase and immobilization carrier were added to 1×PBS buffer (pH 7.4) at a weight ratio of 4:2:1:50, with a material-to-liquid ratio of 1:11 g / mL. The mixture was reacted at 40°C for 3 h, filtered, and washed 3 times with 1×PBS to obtain the immobilized complex enzyme.

[0086] (2) Enzymatic hydrolysis

[0087] Fresh Dendrobium officinale stems were mixed with solvent at a ratio of 1:8 g / mL, and homogenized under high pressure at 60 MPa and 4℃ for 4 cycles (35 s each time). The mixture was then centrifuged at 5000 g for 15 min at 4℃, and the supernatant was collected.

[0088] Solvent composition: Water-based, containing 90 mM sodium chloride, 70 mg / mL trehalose, 0.9 mol / L mannitol, 52 mg / mL glycerol, 7 mM glutathione, 18 mM cysteine, and 22 mg / mL Tween 80, diluted to volume with deionized water;

[0089] Immobilized complex enzyme (4% of the weight of fresh Dendrobium officinale stems) was added to the supernatant and enzymatically hydrolyzed at 42℃, pH 5.2, and 60 rpm for 42 min. The immobilized enzyme was then recovered by filtration to obtain the enzymatic hydrolysate.

[0090] (3) Differential centrifugation

[0091] The enzymatic hydrolysate was centrifuged sequentially at 4°C at 600g for 8 min, 2000g for 20 min, and 11000g for 32 min. The supernatant was then centrifuged at 140000g for 58 min, and the precipitate was collected.

[0092] (4) Size exclusion chromatography and ultrafiltration purification

[0093] The precipitate was resuspended in 1×PBS buffer (pH 7.4) at a ratio of 1:3 g / mL, and filtered through a 0.22 μm filter membrane to obtain the resuspended solution.

[0094] The Sephacryl S-500HR chromatography column was equilibrated with 1×PBS. The resuspension was injected at a flow rate of 0.5 mL / min, and the eluent was collected at a rate of 1 mL / tube. The UV values ​​at 280 nm and 260 nm were monitored, and the flow-through liquid in tubes 5-7 of the main peak was collected.

[0095] Dendrobium officinale exosomes were obtained by retaining the convection fluid using a 100 kDa regenerated cellulose ultrafiltration membrane.

[0096] Example 5

[0097] Exosome composition with antioxidant, anti-inflammatory and skin-whitening effects

[0098] The exosome composition was obtained by mixing Phalaenopsis exosomes (Example 1), ginseng exosomes (Example 2), coix seed exosomes (Example 3), and Dendrobium officinale exosomes (Example 4) in a weight ratio of 2:3:2:3.

[0099] Example 6

[0100] Exosome composition with antioxidant, anti-inflammatory and skin-whitening effects

[0101] The exosome composition was obtained by mixing Phalaenopsis exosomes (Example 1), ginseng exosomes (Example 2), coix seed exosomes (Example 3), and Dendrobium officinale exosomes (Example 4) in a weight ratio of 1:2:1:2.

[0102] Example 7

[0103] Exosome composition with antioxidant, anti-inflammatory and skin-whitening effects

[0104] The exosome composition was obtained by mixing Phalaenopsis exosomes (Example 1), ginseng exosomes (Example 2), coix seed exosomes (Example 3), and Dendrobium officinale exosomes (Example 4) in a weight ratio of 3:4:3:4.

[0105] Comparative Example 1

[0106] The specific implementation method is the same as in Example 1, except that step (1) is deleted and the immobilized complex enzyme is replaced with a complex enzyme. The complex enzyme is composed of cellulase, hemicellulase and pectinase in a weight ratio of 3:1.5:1. The "filtration and recovery of immobilized enzyme" in step (2) is replaced with "filtration".

[0107] Comparative Example 2

[0108] The specific implementation method is the same as that in Example 2, except that the immobilization carrier in step (1) is activated carbon made of fir wood particles with an average particle size of 180 mesh.

[0109] Comparative Example 3

[0110] The specific implementation method is the same as in Example 3, except that the solvent in step (2) is replaced with 1×PBS buffer (pH 7.4).

[0111] Comparative Example 4

[0112] The specific implementation method is the same as that in Example 4, except that the Sephacryl S-500HR chromatography column in step (3) is replaced with a Sepharose CL-2B chromatography column.

[0113] Comparative Example 5

[0114] The specific implementation method is the same as that in Example 5, except that "Examination 1 Phalaenopsis exosomes" is replaced with "Comparative Example 1 Phalaenopsis exosomes".

[0115] Comparative Example 6

[0116] The specific implementation method is the same as that in Example 5, except that "Example 2 Ginseng Exosomes" is replaced with "Comparative Example 2 Ginseng Exosomes".

[0117] Comparative Example 7

[0118] The specific implementation method is the same as that in Example 5, except that "Exosomes of Coix Seed in Example 3" is replaced with "Exosomes of Coix Seed in Comparative Example 3".

[0119] Comparative Example 8

[0120] The specific implementation method is the same as that in Example 5, except that "Exosomes of Dendrobium officinale in Example 4" is replaced with "Exosomes of Dendrobium officinale in Comparative Example 4".

[0121] Comparative Example 9

[0122] The exosome composition was obtained by mixing Phalaenopsis exosomes from Example 1, ginseng exosomes from Example 2, and Dendrobium officinale exosomes from Example 4 in a weight ratio of 2:5:3.

[0123] Comparative Example 10

[0124] The exosome composition was obtained by mixing Phalaenopsis exosomes from Example 1, Coix seed exosomes from Example 3, and Dendrobium officinale exosomes from Example 4 in a weight ratio of 2:5:3.

[0125] Experimental Example 1

[0126] Antioxidant test

[0127] The exosome compositions of Examples 5-7 and Comparative Examples 5-10 were mixed with 1×PBS buffer (pH 7.4) at a volume ratio of 3:97 and resuspended to obtain the test samples.

[0128] Using vitamin C as a positive control, vitamin C was mixed with 1×PBS buffer (pH 7.4) at a concentration of 0.03 g / mL and resuspended to obtain the sample to be tested.

[0129] The DPPH radical scavenging rate and ABTS of the test samples were measured respectively. + Free radical scavenging rate: three replicates per group, average value was taken, and the specific results are shown in Table 1.

[0130] DPPH and ABTS + Free radical scavenging rate was tested according to the kit instructions.

[0131] Table 1. DPPH radical scavenging rate and ABTS of different exosome compositions + Free radical scavenging rate

[0132]

[0133] As shown in Table 1, the exosome compositions of Examples 5-7 exhibit significantly better antioxidant effects than the comparative examples, approaching the positive control. A comparison between Example 5 and Comparative Examples 5-6 reveals that the immobilized complex enzyme stabilizes enzyme activity through the carrier, reduces enzyme inactivation, improves enzymatic hydrolysis efficiency, and increases cell wall disruption efficiency. Furthermore, the immobilization of the complex enzyme enhances the purity of exosomes and strengthens their antioxidant capacity. A comparison between Example 5 and Comparative Example 7 shows that during high-pressure homogenization and enzymatic hydrolysis, the solvent reduces oxidative damage to exosomes during extraction, stabilizes the membrane structure of exosomes, and indirectly enhances their antioxidant properties. A comparison between Example 5 and Comparative Example 8 shows that the selection of size exclusion chromatography packing material affects the antioxidant properties of exosomes. A comparison of data from Example 5 and Comparative Examples 9-10 indicates that the combined use of ginseng exosomes and coix seed exosomes significantly improves the antioxidant activity of the exosome composition. It is evident that optimizing the immobilized enzyme process, solvent composition, size exclusion chromatography packing material, and the synergistic effect of multiple components can significantly improve free radical scavenging efficiency and enhance antioxidant properties.

[0134] Experimental Example 2

[0135] Whitening test

[0136] Mouse melanoma B16-F10 cells were cultured to confluence, then dispersed by dispersing with 1.5 mL of 0.25% trypsin to obtain a B16-F10 cell suspension. Logarithmic-phase B16-F10 cells (1×10⁶ cells) were collected. 5 Add 100 μL of cfu / mL to each well of a 96-well plate and incubate for 24 h.

[0137] The samples were divided into a blank control group, Example 5-7 groups, and Comparative Example 5-10 groups.

[0138] The exosome compositions of Examples 5-7 and Comparative Examples 5-10 were diluted with culture medium to a concentration of 0.4 mg / mL. After culturing for 24 h, the supernatant was aspirated, and 100 μL of culture medium containing the test sample was added to each well. The blank control group was added with only an equal amount of culture medium. Each group was repeated 5 times and incubated in an incubator for 72 h. The culture medium was discarded, and the sample was washed twice with PBS solution. After centrifugation, the supernatant was discarded, and 1 mL of 1% Triton X-100 solution was added to each well. The sample was frozen at -80℃ for 30 min, thawed at room temperature for 20 min, centrifuged, and the supernatant was collected. 60 μL of 0.5% L-L-DOPA solution was added and mixed well. The sample was incubated at 37℃ for 2 h, and the OD490 value was measured. Each group was repeated 3 times, and the tyrosinase activity inhibition rate was calculated. The average value was taken. The specific results are shown in Table 2.

[0139] Tyrosinase activity inhibition rate (%) = (Absorbance of blank group - Absorbance of sample) / Absorbance of blank group × 100%.

[0140] Table 2. Tyrosinase activity inhibition rate of different exosome compositions

[0141]

[0142]

[0143] As shown in Table 2, the examples exhibit a stronger tyrosinase inhibition rate compared to the comparative examples. A comparison of the data from Example 5 and Comparative Examples 6-7 shows that using immobilized complex enzymes and modifying activated carbon can improve the purity of exosomes, thereby increasing the tyrosinase inhibition rate. A comparison of Example 5 and Comparative Example 8 shows that the solvent can protect the stability of exosomes and protect the whitening ingredients through solubilization and reduction, thus improving the tyrosinase inhibition rate. A comparison of Example 5 and Comparative Examples 9-10 shows that ginseng exosomes and coix seed exosomes are key whitening components.

[0144] Experimental Example 3

[0145] Anti-inflammatory test

[0146] RAW264.7 cells in the logarithmic growth phase (6 × 10⁻⁶) 4 100 μL of cfu / mL was added to each well of a 96-well plate and incubated for 36 h. The plates were divided into a blank control group, a model group, Examples 5-7, and Comparative Examples 5-10.

[0147] The exosome compositions of Examples 5-7 and Comparative Examples 5-10 were diluted with culture medium to a concentration of 0.03 g / mL to obtain the test samples. After culturing for 36 h, the supernatant was removed, and 100 μL of culture medium containing the test samples was added to each well. The blank control group and the model group were added with the same amount of culture medium. After incubation for 2 h, except for the blank control group, the other groups were treated with 1 μg / mL lipopolysaccharide for 24 h. The blank control group was treated with the same weight of culture medium. After 24 h, the contents of TNF-α and IL-6 in each group were detected according to the ELISA kit. Each group was repeated 3 times, and the average value was taken. The specific results are shown in Table 3.

[0148] Table 3. Effects of different exosome compositions on TNF-α and IL-6 levels.

[0149]

[0150]

[0151] Table 3 shows that the levels of inflammatory factors in the model group were significantly higher than those in the blank control group, indicating that the inflammation model was successfully constructed. The examples in this case study showed better inhibition of inflammatory factors and superior anti-inflammatory effects compared to the comparative examples. A comparison between Example 5 and Comparative Examples 5-8 shows that immobilizing the complex enzyme, modifying it with activated carbon, optimizing the solvent composition, and screening the size exclusion chromatography column packing material can significantly improve the anti-inflammatory effect of the exosome composition. A comparison between Example 5 and Comparative Examples 9-10 shows that the absence of ginseng exosomes and coix seed exosomes affects the overall anti-inflammatory effect.

[0152] In summary, this invention enhances the activity of exosomes by immobilizing complex enzymes, optimizing solvent components, and screening size exclusion chromatography column packing materials. The combined use of Phalaenopsis orchid exosomes, ginseng exosomes, coix seed exosomes, and Dendrobium officinale exosomes achieves optimal effects through multi-component and multi-pathway synergy, significantly improving the antioxidant, whitening, and anti-inflammatory efficacy of the exosome composition.

[0153] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing plant exosomes, characterized in that, include: Plant tissues were mixed with solvents, homogenized under high pressure, and then enzymatically hydrolyzed with immobilized complex enzymes. After filtration, the hydrolysate was centrifuged at differential speed and then purified by size exclusion chromatography and ultrafiltration to obtain plant exosomes. The solvents include sodium chloride, trehalose, mannitol, glycerol, glutathione, L-cysteine, and Tween 80.

2. The preparation method according to claim 1, characterized in that, The solvent comprises, by concentration, 80-120 mM sodium chloride, 50-100 mg / mL trehalose, 0.5-1 mol / L mannitol, 40-60 mg / mL glycerol, 5-10 mM glutathione, 10-20 mM L-cysteine, and 15-25 mg / mL Tween 80.

3. The preparation method according to claim 1, characterized in that, The high-pressure homogenization process is carried out at a pressure of 50-70 MPa, a temperature of 2-5℃, and 3-5 cycles, each lasting 30-40 seconds.

4. The preparation method according to claim 1, characterized in that, The immobilized complex enzyme is obtained by immobilizing cellulase, hemicellulase and pectinase using an immobilization carrier.

5. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis was performed at a temperature of 37-42℃, a pH of 5-5.5, a rotation speed of 40-80 rpm, and a time of 40-50 min.

6. The preparation method according to claim 1, characterized in that, The weight ratio of the plant tissue to the solvent is 1:5-10 g / mL.

7. The preparation method according to claim 1, characterized in that, The differential centrifugation includes: centrifuging the enzymatic hydrolysate sequentially at 300-700g for 8-12 min, at 1800-2200g for 18-22 min, at 10000-14000g for 28-32 min, and at 120000-150000g for 45-65 min.

8. The preparation method according to claim 1, characterized in that, The plant is one or more of the following: orchid, tea, lemon, chlorella, turmeric, ginseng, coix seed, dendrobium officinale, black truffle, and ganoderma.

9. A plant exosome obtained by the preparation method according to any one of claims 1-8.

10. The use of the plant exosomes of claim 9 in the preparation of cosmetics.

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