Plant exosome with anti-acne effect as well as preparation method and application of plant exosome

Abstract

The invention belongs to the technical field of exosome preparation, and discloses a plant exosome with an anti-acne effect and a preparation method and application of the plant exosome with the anti-acne effect. According to the application, an integrated technical route of microbial regulation and induction, complexing-agent-free green extraction and synergistic acne resistance is established: a microbial strain spore suspension is used as a biological inducer, a plant active precursor is released by dynamic countercurrent of ethanol, and PEG / CaCl2 residues are completely eradicated through physical separation in the whole process; finally, the microorganism-induced plant exosome with high yield, high activity and high safety is obtained.

Description

Technical Field

[0001] This application belongs to the field of exosome preparation technology, specifically relating to a plant exosome with anti-acne effect, its preparation method and application. Background Technology

[0002] Plant-derived nanovesicles (PNVs), with their natural lipid bilayer structure, possess both targeted delivery and metabolic regulation functions. Traditional Chinese medicines such as Gynostemma pentaphyllum, Panax notoginseng, and Polygonatum sibiricum are rich in active ingredients such as saponins and polysaccharides, and have been shown to be able to obtain exosome-like nanoparticles (Gp-NVs) through ultracentrifugation. Existing studies have shown that Gp-NVs can significantly inhibit abnormal proliferation of keratinocytes and inflammatory cytokine-mediated migration in in vitro cell models, indicating that these plants possess endogenous exosome secretion capabilities and demonstrating their potential application in the field of anti-acne treatment.

[0003] Acne is a chronic inflammatory skin disease driven by the overgrowth of Propionibacterium acnes (C. acnes), excessive sebum secretion, and abnormal follicular keratinization. Plant nanovesicles (PNVs), due to their natural biocompatibility and synergistic advantages with multiple components, have become the most promising novel delivery platform in the field of "acne-sebum regulation." However, current technologies in this area still face five major bottlenecks: 1. Low extraction efficiency and limited vesicle yield: Traditional enzymatic hydrolysis or physical cell wall disruption methods do not sufficiently destroy plant cell walls, resulting in a vesicle release rate of <15%, which cannot meet the industrialization demand for high-dose PNVs in topical / oral preparations.

[0004] 2. The lack of induction mechanism and the fact that secondary metabolism is at the "background" level, coupled with the lack of targeted biological induction, means that key pathways such as saponins and flavonoids are not fully activated. This results in insufficient abundance of anti-lipase and anti-inflammatory signaling molecules in Gp-NVs, making it difficult to achieve efficient oil control and rapid anti-inflammation.

[0005] 3. Single function, lack of synergistic anti-acne components. Pure plant PNVs only carry the primary metabolites of the parent plant and lack a synergistic module that simultaneously intervenes in the three pathological links of "C. acnes-sebum-keratinization", resulting in limited clinical efficacy.

[0006] 4. The potential of fermentation strains has not been fully explored. Conventional fermentation with Bifidobacterium and Lactobacillus only increases soluble components, without significantly improving vesicle yield or functional modification. Furthermore, it cannot introduce synergistic "oil-controlling and anti-inflammatory" factors such as bacterial β-glucan and ferulic acid esterase.

[0007] 5. Risk of chemical additive residues: Existing enrichment processes generally use PEG-6000 or CaCl2 complexation. Incomplete removal of complexing agents can lead to skin irritation and oral safety concerns, severely limiting their compliant application in cosmetics and functional foods.

[0008] Therefore, there is an urgent need for a new strategy for preparing highly active and safe plant exosomes for acne indications: to achieve efficient release and functional loading of plant vesicles without introducing any chemical chelating agents, to simultaneously integrate a synergistic "antibacterial-lipid-anti-inflammatory" three-effect module, and to meet the industrialization needs of both topical and oral dosage forms. Summary of the Invention

[0009] The purpose of this application is to address the problems of the prior art by providing a plant exosome with anti-acne effects, its preparation method, and its application.

[0010] To solve the technical problem, the technical solution of this application is: a method for preparing plant exosomes with anti-acne effect, comprising the following steps: S1: Activation of microbial strains and preparation of spore suspensions; Microbial strains stored at -80℃ are streaked onto PDA solid medium to obtain activated single colonies. The microbial strains are Aspergillus elii, Aspergillus Amsterdam, or Aspergillus intercalans. Activated single colonies with a diameter ≥2mm are picked and transferred twice to purify the strain. The spores are scraped off with sterile water from the third generation plate medium and transferred into an Erlenmeyer flask containing glass beads. The spores are shaken to obtain a spore suspension, which is stored at 4℃ in the dark. S2: Preparation of plant GIE; the dried plant is pulverized and sieved, and ethanol is added at a material-to-liquid ratio of 1g:20~30mL. Dynamic countercurrent extraction is performed to obtain an extract; the extract is filtered, centrifuged, and the supernatant is concentrated under reduced pressure to a solid content of 8%~12%, and then filtered through a microporous membrane for sterilization to obtain GIE extract, which is stored at 4℃ in the dark; the plant is Gynostemma pentaphyllum, Panax notoginseng, or Polygonatum sibiricum. S3: Induced fermentation; Prepare liquid fermentation medium, adjust the initial pH to 5.5-6.5, and sterilize it; After cooling the liquid fermentation medium, add 10%-20% of GIE extract and inducer to the liquid fermentation medium, which is either cytochalasin B with a final concentration of 0.01-0.1 mM or plant growth hormone with a final concentration of 0.1-1.0 mg / L, and induce fermentation; then inoculate the spore suspension from S1 at an inoculum rate of 3%-8%, shake and ferment to obtain the fermentation broth; S4: Exosome isolation and purification; After fermentation, the fermentation broth was centrifuged to remove mycelium; The supernatant was immediately transferred to an autoclaved ultracentrifuge tube and then centrifuged; The filtrate was discarded, the precipitate was collected and resuspended in pre-cooled sterile PBS, and centrifuged again in the same tube; Finally, the precipitate was collected, and the volume was adjusted with sterile PBS to obtain a microbial-induced plant exosome suspension, which was stored at 4°C in the dark. S5: Stabilization treatment and formulation collection; Microbial-induced plant exosome suspensions are freeze-dried or resuspended in buffer solution according to application requirements to obtain exosome preparations.

[0011] Preferably, step S1 specifically involves: streaking microbial strains stored at -80℃ onto PDA solid culture medium and culturing them at 28-35℃ and 85%-90% relative humidity for 72-80 hours to obtain activated single colonies; picking activated single colonies with a diameter ≥2mm and subculturing them twice to purify the strain; taking third-generation plate culture medium and scraping spores with sterile water containing 0.05% Tween-20, transferring them into Erlenmeyer flasks containing glass beads, and shaking and dispersing them at 28-35℃ and 200-300 rpm for 60-80 minutes; counting the spores using a hemocytometer and adjusting the spore concentration to 2.0 × 10⁻⁶. 8 CFU / mL was used to obtain a spore suspension, which was then stored at 4°C in the dark.

[0012] Preferably, the spore suspension obtained in S1 is irradiated under an ultraviolet lamp for 10-60 seconds. The ultraviolet lamp has a power of 15W and the distance between the ultraviolet lamp and the spore suspension is 25-35cm.

[0013] Preferably, step S2 specifically involves: pulverizing the dried plant material and passing it through a 70-90 mesh sieve; adding 60%-80% ethanol at a material-to-liquid ratio of 1g:20-30mL; and dynamically countercurrent extraction at 55-70℃ for 60-120 minutes to obtain an extract; filtering the extract and centrifuging at 3000-5000r / min for 10-20 minutes; concentrating the supernatant under reduced pressure at 50-60℃ until the solid content is 8%-12%; and then sterilizing the extract by filtering it through a 0.22-0.45μm microporous membrane to obtain a GIE extract, which is stored at 4℃ in the dark.

[0014] Preferably, step S3 specifically involves: preparing a liquid fermentation medium, initially adjusting the pH to 5.5-6.5, and sterilizing it at 115-121℃ for 15-25 minutes; after cooling the liquid fermentation medium, adding 10%-20% of GIE extract and 0.01-0.1 mM cytochalasin B or 0.1-1.0 mg / L plant growth hormone to the liquid fermentation medium for induction treatment; then inoculating the spore suspension from step S1 at an inoculum rate of 3%-8%, and fermenting at 26-30℃ and 150-180 rpm for 60-84 hours with shaking to obtain the fermentation broth.

[0015] Preferably, the liquid fermentation medium in S3 comprises a carbon source, a nitrogen source, inorganic salts, and growth factors. The carbon source is selected from one or more of glucose, sucrose, maltose, and soluble starch, and its concentration in the liquid fermentation medium is 20-50 g / L. The nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source, wherein the organic nitrogen source is selected from one or more of peptone, yeast extract, beef extract, and soybean meal, and its concentration in the liquid fermentation medium is 5-20 g / L. The inorganic nitrogen source is selected from one or more of sodium nitrate, ammonium sulfate, and urea, and its concentration in the liquid fermentation medium is 1-5 g / L. / L; the inorganic salt is potassium dihydrogen phosphate, magnesium sulfate heptahydrate, or trace elements. The concentration of potassium dihydrogen phosphate in the liquid fermentation medium is 0.5~2g / L, the concentration of magnesium sulfate heptahydrate in the liquid fermentation medium is 0.5~1g / L, and the concentration of trace elements in the liquid fermentation medium is 0.01~0.1g / L. The trace elements are selected from one or more of ferrous sulfate heptahydrate, copper sulfate pentahydrate, manganese sulfate monohydrate, and zinc sulfate heptahydrate. The growth factor is selected from one or more of biotin, vitamin B1, and inositol. The concentration of the growth factor in the liquid fermentation medium is 0.001~0.01g / L.

[0016] Preferably, step S4 specifically involves: after fermentation, centrifuging the fermentation broth at 3000×g for 15-30 min at 4°C to remove mycelia; immediately transferring the supernatant to a 0.22-0.45μm sterile filter-grade ultracentrifuge tube that has been autoclaved at 120-130°C for 30-60 min, followed by centrifugation at 100000×g at 4°C for 80-100 min; discarding the filtrate, resuspending the precipitate in pre-cooled sterile PBS, and centrifuging again at 100000×g for 80-100 min in the same tube; finally collecting the precipitate, making up the volume with sterile PBS to obtain the microbial-induced plant exosome suspension, which is stored at 4°C in the dark.

[0017] Preferably, an anti-acne plant exosome is prepared by the above-mentioned method for preparing an anti-acne plant exosome, wherein the plant exosome is a spherical / cup-shaped nanovesicle with a double membrane structure.

[0018] Preferably, the diameter of the spherical / cup-shaped nanovesicles is 40~150nm.

[0019] Preferably, an application of an anti-acne plant exosome, wherein the plant exosome prepared by the above-described method for preparing an anti-acne plant exosome is used to treat acne.

[0020] Compared with the prior art, the advantages of this application are: (1) The plant exosomes of this application have a synergistic anti-acne mechanism of antibacterial, lipid-lowering, and anti-inflammatory effects. The antibacterial mechanism is as follows: The microbial-induced plant exosomes of this application serve as natural vesicle carriers, which jointly carry lovastatin synthesis precursors (including acetyl-CoA, malonyl-CoA, and monacolin) produced by the metabolism of microbial strains and lipid-lowering active saponins derived from Gynostemma pentaphyllum, Panax notoginseng, and Polygonatum sibiricum (including Gynostemma pentaphyllum saponins, Panax notoginseng saponins, and Polygonatum sibiricum saponins). In the microenvironment inside the vesicles, the precursors and saponin molecules can self-assemble into a stable "statin-saponin" supramolecular complex through hydrophobic interactions and intermolecular forces such as hydrogen bonds. This complex structure not only effectively improves the solubility and stability of the active components, but also significantly enhances the anti-acne effect through multiple molecular synergistic mechanisms. Enhanced targeted penetration and destruction efficiency of Propionibacterium acnes cell membrane, resulting in a synergistic antibacterial effect far exceeding that of a single component; Lipid-suppressing mechanism: After the plant exosomes induced by the microorganisms of this application are taken up by sebaceous cells, the saponin components they carry can specifically inhibit the activity of HMG-CoA reductase in the cells, thereby reducing the excessive synthesis of cholesterol and other sebum components from the source and playing a fundamental lipid-suppressing role; Anti-inflammatory mechanism: The membrane surface of the plant exosomes induced by the microorganisms of this application is enriched with high abundance of β-glucan. This polysaccharide can specifically bind to the Dectin-1 receptor on the surface of immune cells such as host macrophages, thereby negatively regulating the downstream NF-κB inflammatory signaling pathway and effectively alleviating the inflammatory response at the acne site; (2) This application induces microbial fermentation, and the strains secrete a rich extracellular enzyme system during the growth process, including but not limited to pectin lyase, pectin methyl esterase, exoglucanase, xylanase, mannanase, protease and cutinase. These enzymes can work synergistically on key components of plant cell walls and cell membranes (such as pectin, cellulose, hemicellulose, and proteins), and significantly enhance the secretion pathway of vesicles by efficiently degrading the extracellular matrix, relaxing the cell wall structure, and stimulating cell membrane activation, thereby ultimately achieving an overall improvement in the yield of plant exosomes. (3) The microbial-induced plant exosomes of this application achieve a “closed-loop” synergistic intervention of three key links from pathogens (bacteriostasis), sebum secretion (lipid inhibition) to immune response (anti-inflammatory), demonstrating a comprehensive anti-acne advantage that cannot be matched by a single component; (4) This application establishes an integrated technical route of “microbial regulation and induction - green extraction without complexing agent - synergistic anti-acne”: using microbial strain spore suspension as biological inducer, ethanol dynamically countercurrently releases plant active precursors, physical separation throughout the process eliminates PEG / CaCl2 residues, and finally obtains high-yield, high-activity, and high-safety microbial-plant complex exosomes; (4) This application does not require chemical complexing agents: physical methods are used to extract exosomes throughout the process, avoiding problems such as protein residue and membrane damage caused by polymer precipitation, thereby improving the purity and biocompatibility of exosomes. Attached Figure Description

[0021] Figure 1 This is a transmission electron microscope (TEM) image of plant exosomes induced by Aspergillus eliifolia-Gynostemma pentaphyllum microorganisms prepared in Example 1 of this application. Detailed Implementation

[0022] The present application is described in detail below with reference to the accompanying drawings and specific embodiments, but the present application is not limited to these embodiments. The present application covers any alternatives, modifications, equivalent methods, and solutions made within the spirit and scope of the present application. To provide the public with a thorough understanding of the present application, specific details are described in detail in the following embodiments, but those skilled in the art will fully understand the present application even without these detailed descriptions.

[0023] This application discloses a method for preparing plant exosomes with anti-acne effects, comprising the following steps: S1: Activation of microbial strains and preparation of spore suspensions; Microbial strains stored at -80℃ are streaked onto PDA solid medium to obtain activated single colonies. The microbial strains are Aspergillus elii, Aspergillus Amsterdam, or Aspergillus intercalans. Activated single colonies with a diameter ≥2mm are picked and transferred twice to purify the strain. The spores are scraped off with sterile water from the third generation plate medium and transferred into an Erlenmeyer flask containing glass beads. The spores are shaken to obtain a spore suspension, which is stored at 4℃ in the dark. S2: Preparation of plant GIE; the dried plant is pulverized and sieved, and ethanol is added at a material-to-liquid ratio of 1g:20~30mL. Dynamic countercurrent extraction is performed to obtain an extract; the extract is filtered, centrifuged, and the supernatant is concentrated under reduced pressure to a solid content of 8%~12%, and then filtered through a microporous membrane for sterilization to obtain GIE extract, which is stored at 4℃ in the dark; the plant is Gynostemma pentaphyllum, Panax notoginseng, or Polygonatum sibiricum. S3: Induced fermentation; Prepare liquid fermentation medium, adjust the initial pH to 5.5-6.5, and sterilize it; After cooling the liquid fermentation medium, add 10%-20% of GIE extract and inducer to the liquid fermentation medium, which is either cytochalasin B with a final concentration of 0.01-0.1 mM or plant growth hormone with a final concentration of 0.1-1.0 mg / L, and induce fermentation; then inoculate the spore suspension from S1 at an inoculum rate of 3%-8%, shake and ferment to obtain the fermentation broth; S4: Exosome isolation and purification; After fermentation, the fermentation broth was centrifuged to remove mycelium; The supernatant was immediately transferred to an autoclaved ultracentrifuge tube and then centrifuged; The filtrate was discarded, the precipitate was collected and resuspended in pre-cooled sterile PBS, and centrifuged again in the same tube; Finally, the precipitate was collected, and the volume was adjusted with sterile PBS to obtain a microbial-induced plant exosome suspension, which was stored at 4°C in the dark. S5: Stabilization treatment and formulation collection; Microbial-induced plant exosome suspensions are freeze-dried or resuspended in buffer solution according to application requirements to obtain exosome preparations.

[0024] Preferably, step S1 specifically involves: streaking microbial strains stored at -80℃ onto PDA solid culture medium and culturing them at 28-35℃ and 85%-90% relative humidity for 72-80 hours to obtain activated single colonies; picking activated single colonies with a diameter ≥2mm and subculturing them twice to purify the strain; taking third-generation plate culture medium and scraping spores with sterile water containing 0.05% Tween-20, transferring them into Erlenmeyer flasks containing glass beads, and shaking and dispersing them at 28-35℃ and 200-300 rpm for 60-80 minutes; counting the spores using a hemocytometer and adjusting the spore concentration to 2.0 × 10⁻⁶. 8 CFU / mL was used to obtain a spore suspension, which was then stored at 4°C in the dark.

[0025] Preferably, the spore suspension obtained in S1 is irradiated under an ultraviolet lamp for 10-60 seconds. The ultraviolet lamp has a power of 15W and the distance between the ultraviolet lamp and the spore suspension is 25-35cm.

[0026] Preferably, step S2 specifically involves: pulverizing the dried plant material and passing it through a 70-90 mesh sieve; adding 60%-80% ethanol at a material-to-liquid ratio of 1g:20-30mL; and dynamically countercurrent extraction at 55-70℃ for 60-120 minutes to obtain an extract; filtering the extract and centrifuging at 3000-5000r / min for 10-20 minutes; concentrating the supernatant under reduced pressure at 50-60℃ until the solid content is 8%-12%; and then sterilizing the extract by filtering it through a 0.22-0.45μm microporous membrane to obtain a GIE extract, which is stored at 4℃ in the dark.

[0027] Preferably, step S3 specifically involves: preparing a liquid fermentation medium, initially adjusting the pH to 5.5-6.5, and sterilizing it at 115-121℃ for 15-25 minutes; after cooling the liquid fermentation medium, adding 10%-20% of GIE extract and 0.01-0.1 mM cytochalasin B or 0.1-1.0 mg / L plant growth hormone to the liquid fermentation medium for induction treatment; then inoculating the spore suspension from step S1 at an inoculum rate of 3%-8%, and fermenting at 26-30℃ and 150-180 rpm for 60-84 hours with shaking to obtain the fermentation broth.

[0028] In the process described in this application, the fermentation medium is first sterilized and cooled, and then GIE and inducers are added by volume. To avoid ambiguity and facilitate scale-up, all inducer / GIE values ​​should be clearly defined as the concentration or volume fraction measured at the total volume of the fermentation system after mixing.

[0029] The term "final concentration" refers to the final concentration in the liquid fermentation medium, i.e., the final fermentation broth. The term "3%–8% inoculum" refers to the inoculum volume (v / v), which is the percentage of the spore suspension volume in the total fermentation medium volume. A volume ratio of 3%–8% (v / v) is compatible with different scale-up scales and the growth characteristics of various strains. For example, in a 250 mL Erlenmeyer flask fermentation system, a 5% (v / v) inoculum volume means adding 12.5 mL of spore suspension.

[0030] Operational instructions: If the inducer is added in the process section as a stock solution, the volume to be added should be calculated according to the dilution formula.

[0031] Example of inducin volume calculation: To achieve a final concentration of 0.05 mM of cytochalasin B in a 250 mL fermentation system, and with an inducin stock solution of 10 mM, the required volume V is: C final V final / C stock =0.05mM 250mL / 10mM = 1.25mL Example of inoculation amount: If a 250mL fermentation bottle is inoculated at 5% (v / v), then the volume of spore suspension added = 250mL × 5% = 12.5mL.

[0032] The addition of inducers can upregulate secondary metabolism, promote extracellular enzyme secretion and relax plant cell walls / membranes, increase exosome release rate and yield, and achieve functional loading (such as statin precursors and saponin enrichment), which is quantitatively supported in the examples and comparative experiments of this application (yield increased by about 3.3 times, see NTA data in Example 6).

[0033] GIE extract provides plant secondary metabolic precursors (saponins, flavonoid precursors, etc.) as inducing substrates; Extracellular degradative enzymes (pectinase, cellulase, protease, etc.) secreted by microbial strains (Aspergillus) during co-culture synergistically degrade plant cell walls / matrix, relax membrane structures, and promote the release of plant-derived exosome-like nanovesicles. At low concentrations, inducible hormones (cytochalasin B / plant growth hormones) can stimulate membrane dynamics or secondary metabolic pathways, further enhancing exosome release and the accumulation of target molecules within vesicles, thereby simultaneously improving yield and functionalization levels.

[0034] Preferably, the liquid fermentation medium in S3 comprises a carbon source, a nitrogen source, inorganic salts, and growth factors. The carbon source is selected from one or more of glucose, sucrose, maltose, and soluble starch, and its concentration in the liquid fermentation medium is 20-50 g / L. The nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source, wherein the organic nitrogen source is selected from one or more of peptone, yeast extract, beef extract, and soybean meal, and its concentration in the liquid fermentation medium is 5-20 g / L. The inorganic nitrogen source is selected from one or more of sodium nitrate, ammonium sulfate, and urea, and its concentration in the liquid fermentation medium is 1-5 g / L. / L; the inorganic salt is potassium dihydrogen phosphate, magnesium sulfate heptahydrate, or trace elements. The concentration of potassium dihydrogen phosphate in the liquid fermentation medium is 0.5~2g / L, the concentration of magnesium sulfate heptahydrate in the liquid fermentation medium is 0.5~1g / L, and the concentration of trace elements in the liquid fermentation medium is 0.01~0.1g / L. The trace elements are selected from one or more of ferrous sulfate heptahydrate, copper sulfate pentahydrate, manganese sulfate monohydrate, and zinc sulfate heptahydrate. The growth factor is selected from one or more of biotin, vitamin B1, and inositol. The concentration of the growth factor in the liquid fermentation medium is 0.001~0.01g / L.

[0035] Preferably, step S4 specifically involves: after fermentation, centrifuging the fermentation broth at 3000×g for 15-30 min at 4°C to remove mycelia; immediately transferring the supernatant to a 0.22-0.45μm sterile filter-grade ultracentrifuge tube that has been autoclaved at 120-130°C for 30-60 min, followed by centrifugation at 100000×g at 4°C for 80-100 min; discarding the filtrate, resuspending the precipitate in pre-cooled sterile PBS, and centrifuging again at 100000×g for 80-100 min in the same tube; finally collecting the precipitate, making up the volume with sterile PBS to obtain the microbial-induced plant exosome suspension, which is stored at 4°C in the dark.

[0036] Preferably, an anti-acne plant exosome is prepared by the above-mentioned method for preparing an anti-acne plant exosome, wherein the plant exosome is a spherical / cup-shaped nanovesicle with a double membrane structure.

[0037] Preferably, the diameter of the spherical / cup-shaped nanovesicles is 40~150nm.

[0038] Preferably, an application of an anti-acne plant exosome, wherein the plant exosome prepared by the above-described method for preparing an anti-acne plant exosome is used to treat acne.

[0039] This application discloses a plant exosome preparation with anti-acne effects and its application.

[0040] Example 1: Preparation of Aspergillus elii-Gynostemma pentaphyllum exosomes; (1) Activation of strains and preparation of spore suspension; Aspergillus eliae stored at -80℃ was streaked onto PDA plates and incubated at 28℃ and 85% relative humidity for 72 h. Single colonies with a diameter ≥2 mm were picked and subcultured twice for purification. Spores were scraped from third-generation plates using sterile water containing 0.05% Tween-20 and placed in Erlenmeyer flasks containing glass beads. The spores were shaken at 28℃ and 200 rpm for 60 min, and counted using a hemocytometer, adjusting the concentration to 2.0 × 10⁻⁶. 8 CFU / mL was used to obtain a spore suspension, which was then stored at 4°C.

[0041] (2) Preparation of Gynostemma pentaphyllum ethanol extract (GIE); The dried leaves of Gynostemma pentaphyllum were pulverized and passed through an 80-mesh sieve. 75% (v / v) ethanol was added at a material-to-liquid ratio of 1:25 (g / mL), and the mixture was extracted under dynamic countercurrent at 60℃ for 90 min. After coarse filtration through a 200-mesh filter cloth, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was concentrated under reduced pressure at 55℃ to a solid content of 10% (w / v), and sterilized by microfiltration at 0.22 μm to obtain GIE. The product was stored at 4℃ protected from light.

[0042] (3) Induced fermentation; Liquid culture medium was prepared in a 250 mL Erlenmeyer flask: glucose 30 g / L, peptone 10 g / L, yeast extract 5 g / L, MgSO4·7H2O 0.8 g / L, CaCl2 0.4 g / L, initial pH 6.0; sterilized at 115 °C for 20 min. After cooling, 15% (v / v) GIE was added, and spore suspension was inoculated at 5% (v / v), and induced culture was carried out at 28 °C and 160 rpm for 72 h.

[0043] (4) Exosome isolation and purification; The fermentation broth was centrifuged at 3000×g for 15 min at 4℃ to remove mycelia; the supernatant was microfiltered at 0.22 μm and then ultracentrifuged at 100000×g for 80 min; the precipitate was resuspended in pre-cooled PBS and centrifuged again under the same conditions; finally, an Aspergillus elixirii-Gynostemma pentaphyllum exosome suspension was obtained, with a particle concentration of (5.4±0.3)×10⁻⁶. 10 particles / mL (NTA determination), store at 4°C protected from light, and use within 7 days.

[0044] like Figure 1 The image shown is a transmission electron microscope (TEM) image of plant exosomes induced by Aspergillus eliifolia and Gynostemma pentaphyllum microorganisms. The exosomes appear as typical spherical / cup-shaped nanovesicles with a distinct bilayer membrane structure. The scale bar in the image is 100 nm, and the vesicle diameters are mainly distributed in the range of approximately 40–150 nm (NTA measurements are consistent with these morphological results). Figure 1The nanovesicles shown represent the electron microscopic morphology of the isolated exosome-like particles. A complete lipid bilayer membrane and a relatively uniform particle size distribution can be observed, supporting the fact that the preparation method described in this application can obtain structurally intact microbial-induced plant exosomes.

[0045] Example 2: Preparation of Aspergillus Amsterdam-Gynostemma pentaphyllum exosomes; (1) Spore suspension; Aspergillus amstelodami (CGMCC 3.4382) was activated according to the method in Example 1 to prepare a concentration of 1.5 × 10⁻⁶. 8 CFU / mL spore suspension.

[0046] (2) Preparation of GIE; Same as Example 1, except that the extraction temperature was 65°C, the extraction time was 75 min, and the final solids content was 10%.

[0047] (3) Induced fermentation; Liquid culture medium: glucose 25 g / L, peptone 12 g / L, yeast extract 6 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.5 g / L, initial pH 5.5; sterilized at 115℃ for 20 min; added 20% (v / v) GIE, inoculated with 7 mL of spore suspension (7% v / v), and induced at 30℃ and 180 rpm for 84 h.

[0048] (4) Exosome purification; Following the ultracentrifugation protocol of Example 1, the final concentration of Aspergillus Amsterdam-Gynostemma pentaphyllum exosomes was (5.7±0.2)×10⁻⁶. 10 particles / mL.

[0049] Example 3: Preparation of Aspergillus mesenteroides-Gynostemma pentaphyllum exosomes; (1) Spore suspension; After activation, Aspergillus intermedius (CICC 2654) was prepared to a concentration of 2.2 × 10⁻⁶. 8 CFU / mL spore suspension.

[0050] (2) Preparation of GIE; Same as Example 3, with a material-to-liquid ratio of 1:30, an extraction time of 120 min, and a final solids content of 10%.

[0051] (3) Induced fermentation; Liquid culture medium: glucose 35 g / L, peptone 8 g / L, yeast extract 4 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.3 g / L, initial pH 6.5; sterilized at 115℃ for 20 min; added 10% (v / v) GIE, inoculated with 6 mL of spore suspension (6% v / v), and induced at 29℃ and 170 rpm for 96 h.

[0052] (4) Exosome purification; Ultracentrifugation was performed as in Example 1, ultimately yielding a concentration of (6.0 ± 0.4) × 10- Aspergillus mesoderm-Gynostemma pentaphyllum exosomes. 10 particles / mL.

[0053] Example 4: Preparation of Aspergillus elixirsus-Panax notoginseng exosomes; (1) Activation of strains and preparation of spore suspension; Same as Example 1 (1).

[0054] (2) Preparation of Panax notoginseng ethanol extract (PNE); The dried roots of Panax notoginseng were pulverized and passed through an 80-mesh sieve. 75% (v / v) ethanol was added at a material-to-liquid ratio of 1:25 (g / mL), and dynamic countercurrent extraction was performed at 60℃ for 90 min. After coarse filtration through a 200-mesh filter cloth, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was concentrated under reduced pressure at 55℃ to a solid content of 10% (w / v), and sterilized by microfiltration at 0.22 μm to obtain PNE, which was stored at 4℃ protected from light.

[0055] (3) Induced fermentation; Liquid culture medium was prepared in a 250 mL Erlenmeyer flask: glucose 30 g / L, peptone 10 g / L, yeast extract 5 g / L, MgSO4·7H2O 0.8 g / L, CaCl2 0.4 g / L, initial pH 6.0; sterilized at 115℃ for 20 min. After cooling, 15% (v / v) PNE was added, and spore suspension was inoculated at 5% (v / v), and induced culture was carried out at 28℃ and 160 rpm for 72 h.

[0056] (4) Exosome isolation and purification; Same as in Example 1 (4), a suspension of Aspergillus elixirsiflora-Panax notoginseng exosomes was finally obtained, with a particle concentration of (5.6±0.3)×10. 10 particles / mL (NTA determination), store at 4°C protected from light, and use within 7 days.

[0057] Example 5: Preparation of Aspergillus elixirsus-Polygonatum exosomes; (1) Activation of strains and preparation of spore suspension; Same as Example 1 (1).

[0058] (2) Preparation of Polygonatum odoratum ethanol extract (PCE); The dried rhizomes of Polygonatum were pulverized and passed through an 80-mesh sieve. 75% (v / v) ethanol was added at a material-to-liquid ratio of 1:25 (g / mL), and dynamic countercurrent extraction was performed at 60℃ for 90 min. After coarse filtration through a 200-mesh filter cloth, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was concentrated under reduced pressure at 55℃ to a solid content of 10% (w / v), sterilized by 0.22 μm microfiltration, and PCE was obtained. It was stored at 4℃ protected from light.

[0059] (3) Induced fermentation; Liquid culture medium was prepared in a 250 mL Erlenmeyer flask: glucose 30 g / L, peptone 10 g / L, yeast extract 5 g / L, MgSO4·7H2O 0.8 g / L, CaCl2 0.4 g / L, initial pH 6.0; sterilized at 115℃ for 20 min. After cooling, 15% (v / v) PCE was added, and spore suspension was inoculated at 5% (v / v), and induced culture was carried out at 28℃ and 160 rpm for 72 h.

[0060] (4) Exosome isolation and purification; Same as in Example 1 (4), the final Aspergillus elixirii-Polygonatum exosome suspension was obtained, with a particle concentration of (5.2±0.4)×10⁻⁶. 10 particles / mL (NTA determination), store at 4°C protected from light, and use within 7 days.

[0061] Application Example 1: 1. Antibacterial zone test against Propionibacterium acnes (Oxford cup method, anaerobic at 37 ℃ for 48 h), the diameter of the inhibition zone was ≥13 mm; The activated Propionibacterium acnes (ATCC 6919, anaerobic blood agar plate, 37℃, 48h) was adjusted to 0.5 McFarland (≈1.5×10⁻⁶) with physiological saline. 8 CFU / mL), spread evenly at 100 μL / plate onto the surface of Brucella agar containing 5% defibrinated sheep blood. Place four sterile Oxford cups (outer diameter 8 mm × inner diameter 6 mm × height 10 mm) at equal intervals on each plate, and inject less than 200 μL of sample into each cup. ① PBS negative control; ② Erythromycin positive control (0.5 mg / mL); ③ Aspergillus elii-Gynostemma pentaphyllum exosome suspension (protein concentration 1.0 mg / mL, PBS solvent); ④ Gynostemma pentaphyllum ethanol extract (1.0 mg / mL, PBS solvent).

[0062] After anaerobic culture at 37℃ for 48 h, the diameter of the inhibition zone (including the outer diameter of the cup) was measured using a digital vernier caliper (accuracy 0.01 mm) via the cross-cross method. The results are shown in the table below: In three independent experiments (n=6), the diameter of the inhibition zone in the exosome group was 13.2~15.7 mm, with an average of 14.5 mm, all ≥13 mm, significantly larger than the negative control (0 mm, P<0.01), and consistent with the trend of the positive control (29.4 mm); the diameter of the inhibition zone in the Gynostemma pentaphyllum ethanol extract group was 8.1~9.3 mm, with an average of 8.7 mm, significantly lower than the exosome group (P<0.01), but still better than the negative control. This indicates that the Aspergillus elixirii-Gynostemma pentaphyllum exosomes prepared in this application have significant inhibitory activity against Propionibacterium acnes.

[0063] Note: An inhibition zone ≥13mm is considered to have a significant inhibitory effect on Propionibacterium acnes.

[0064] Application Example 2: Pancreatic lipase inhibition experiment (orlistat as positive control); (1) Substrate: 2 mmol / L p-nitrophenol laurate (p-NPL) dissolved in 50 mmol / L Tris-HCl (pH 8.2, 100 mmol / L NaCl) containing 5% Triton X-100.

[0065] (2) Enzyme solution: porcine pancreatic lipase (PPL, Type II) was diluted with the above buffer to 0.2 mg protein / mL.

[0066] (3) Sample preparation: ① The plant exosome lyophilized powder of this application was first dissolved in anhydrous DMSO to prepare a 50 mg / mL stock solution, and then serially diluted with buffer to make the final DMSO concentration 1% (v / v). ② The positive control orlistat was dissolved in DMSO in the same way and then diluted to 0.01~0.5 mg / mL, and the final DMSO concentration was also kept at 1%. ③ The lyophilized powder of Gynostemma pentaphyllum ethanol extract was prepared in the same way with anhydrous DMSO to prepare a 50 mg / mL stock solution, and then serially diluted with buffer to make the final DMSO concentration 1% (v / v). (4) Solvent control: A "1% DMSO solvent blank" group (without any inhibitors) was added to subtract the basic inhibition of enzyme activity by DMSO. (5) Reaction system (96-well plate): 80 μL buffer + 10 μL enzyme solution + 10 μL sample (or solvent control), pre-incubated at 37℃ for 10 min; 100 μL substrate was added to start the reaction, shaken at 37℃ for 20 min, and the absorbance A was measured immediately at 405 nm. (6) Inhibition rate calculation (background deducted): Inhibition rate % = [1 - (A sample - A1% DMSO blank) / (A solvent - A1% DMSO blank)] × 100 (7) Results: After deducting the 1% DMSO background, the exosomes still meet the "high potency" standard of the "Technical Specifications for Inspection and Evaluation of Health Foods" (IC50 for plant-derived samples). 50 (≤0.5 mg / mL), the results are shown in the table below: The above examples demonstrate that different microbial strains can be used to induce plant exosomes to obtain anti-acne preparations with high yield, high activity, and consistent function. These preparations are not only stable in process and suitable for large-scale production, but can also be further formulated into topical or oral dosage forms to meet various clinical and daily skin care needs.

[0067] Example 6: Effect of microbial induction on the production of Gynostemma pentaphyllum exosomes (comparative experiment); 1. Experimental objective: To verify the significant effect of the microbial induced co-fermentation method used in this application on increasing the yield of Gynostemma pentaphyllum exosomes compared to the simple plant extraction method.

[0068] 2. Experimental grouping and methods: Experimental group (microbial-induced co-fermentation): The method was carried out entirely according to the method described in Example 1 of this application, that is, after co-fermenting the ethanol extract of Gynostemma pentaphyllum (GIE) with Aspergillus elixirs spore suspension, the exosomes were separated and purified.

[0069] Control group (plant extract only): Except for not inoculating with Aspergillus elixirii spore suspension, the other steps were exactly the same as in Example 1. The same amount of Gynostemma pentaphyllum ethanol extract (GIE) was added to a sterile fermentation medium and incubated statically for 72 hours under the same conditions (28°C, 160 rpm). The fermentation broth was then treated with the same separation and purification method as in Example 1.

[0070] 3. Detection and Results: After fermentation, the fermentation broths of the two groups were collected, and the exosomes were separated and purified strictly following step (4) of Example 1. The particle concentration of the final exosome suspension was determined using nanoparticle tracking analysis (NTA), and the results are shown in the table below: 4. Experimental Conclusion: The test results clearly show that the exosome yield obtained by the microbial induced co-fermentation method of this application (experimental group) is about 3.3 times that of the traditional method without microbial induction (control group). This data strongly proves that microbial induction is a key and indispensable process step to significantly improve the yield of plant exosomes, and solves the bottleneck problem of low yield of plant exosomes and difficulty in industrialization in the prior art, reflecting the significant progress of this application.

[0071] In addition, in animal experiments, the plant exosomes of this application can cross the intestinal barrier, expanding their use for secondary intervention of metabolic syndrome and filling the gap in the indications of plant exosomes in the "skin-gut" axis.

[0072] This application presents plant exosomes that possess antioxidant, anti-inflammatory, and intestinal barrier regulating functions, making them suitable for comprehensive management of metabolic syndrome. They can be seamlessly freeze-dried or loaded into hydrogels / microcapsules, meeting the needs of various applications including topical application, oral administration, and solid beverages.

[0073] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application.

[0074] Many other changes and modifications can be made without departing from the concept and scope of this application. It should be understood that this application is not limited to the specific embodiments, and the scope of this application is defined by the appended claims.

Claims

1. A method for preparing plant exosomes with anti-acne effects, characterized in that, Includes the following steps: S1: Activation of microbial strains and preparation of spore suspensions; Microbial strains stored at -80℃ are streaked onto PDA solid medium to obtain activated single colonies. The microbial strains are Aspergillus elii, Aspergillus Amsterdam, or Aspergillus intercalans. Activated single colonies with a diameter ≥2mm are picked and transferred twice to purify the strain. The spores are scraped off with sterile water from the third generation plate medium and transferred into an Erlenmeyer flask containing glass beads. The spores are shaken to obtain a spore suspension, which is stored at 4℃ in the dark. S2: Preparation of plant GIE; the dried plant is pulverized and sieved, and ethanol is added at a material-to-liquid ratio of 1g:20~30mL. Dynamic countercurrent extraction is performed to obtain an extract; the extract is filtered, centrifuged, and the supernatant is concentrated under reduced pressure to a solid content of 8%~12%, and then filtered through a microporous membrane for sterilization to obtain GIE extract, which is stored at 4℃ in the dark; the plant is Gynostemma pentaphyllum, Panax notoginseng, or Polygonatum sibiricum. S3: Induced fermentation; Prepare liquid fermentation medium, adjust the initial pH to 5.5-6.5, and sterilize it; After cooling the liquid fermentation medium, add 10%-20% of GIE extract and inducer to the liquid fermentation medium, which is either cytochalasin B with a final concentration of 0.01-0.1 mM or plant growth hormone with a final concentration of 0.1-1.0 mg / L, and induce fermentation; then inoculate the spore suspension from S1 at an inoculum rate of 3%-8%, shake and ferment to obtain the fermentation broth; S4: Exosome isolation and purification; After fermentation, the fermentation broth was centrifuged to remove mycelium; The supernatant was immediately transferred to an autoclaved ultracentrifuge tube and then centrifuged; The filtrate was discarded, the precipitate was collected and resuspended in pre-cooled sterile PBS, and centrifuged again in the same tube; Finally, the precipitate was collected, and the volume was adjusted with sterile PBS to obtain a microbial-induced plant exosome suspension, which was stored at 4°C in the dark. S5: Stabilization treatment and formulation collection; Microbial-induced plant exosome suspensions are freeze-dried or resuspended in buffer solution according to application requirements to obtain exosome preparations.

2. The method for preparing plant exosomes with anti-acne effect according to claim 1, characterized in that, S1 specifically involves: streaking microbial strains stored at -80℃ onto PDA solid medium and culturing them at 28-35℃ and 85%-90% relative humidity for 72-80 hours to obtain activated single colonies; picking activated single colonies with a diameter ≥2mm and subculturing twice to purify the strain; taking third-generation plate medium and scraping spores with sterile water containing 0.05% Tween-20, transferring them into Erlenmeyer flasks containing glass beads, and shaking to disperse the spores for 60-80 minutes at 28-35℃ and 200-300 rpm; counting the spores using a hemocytometer and adjusting the spore concentration to 2.0 × 10⁻⁶. 8 CFU / mL was used to obtain a spore suspension, which was then stored at 4°C in the dark.

3. The method for preparing plant exosomes with anti-acne effect according to claim 1, characterized in that, The spore suspension obtained in S1 was irradiated under a UV lamp for 10-60 seconds. The UV lamp power was 15W, and the distance between the UV lamp and the spore suspension was 25-35cm.

4. The method for preparing plant exosomes with anti-acne effect according to claim 1, characterized in that, S2 specifically involves: pulverizing dried plant material and passing it through a 70-90 mesh sieve; adding 60%-80% ethanol at a material-to-liquid ratio of 1g:20-30mL; and dynamically countercurrent extraction at 55-70℃ for 60-120 minutes to obtain an extract; filtering the extract and centrifuging at 3000-5000r / min for 10-20 minutes; concentrating the supernatant under reduced pressure at 50-60℃ until the solid content is 8%-12%; and then sterilizing the extract by filtering it through a 0.22-0.45μm microporous membrane to obtain the GIE extract, which is stored at 4℃ in the dark.

5. The method for preparing plant exosomes with anti-acne effect according to claim 1, characterized in that, S3 specifically involves: preparing a liquid fermentation medium, adjusting the initial pH to 5.5-6.5, and sterilizing it at 115-121℃ for 15-25 minutes; after cooling the liquid fermentation medium, adding 10%-20% of GIE extract and 0.01-0.1mM cytochalasin B or 0.1-1.0mg / L plant growth hormone to the liquid fermentation medium for induction treatment; then inoculating the spore suspension from S1 at an inoculum of 3%-8%, and fermenting it at 26-30℃ and 150-180rpm for 60-84 hours to obtain the fermentation broth.

6. The method for preparing plant exosomes with anti-acne effect according to claim 5, characterized in that, The liquid fermentation medium in S3 contains a carbon source, a nitrogen source, inorganic salts, and growth factors. The carbon source is selected from one or more of glucose, sucrose, maltose, and soluble starch, and its concentration in the liquid fermentation medium is 20-50 g / L. The nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source. The organic nitrogen source is selected from one or more of peptone, yeast extract, beef extract, and soybean meal, and its concentration in the liquid fermentation medium is 5-20 g / L. The inorganic nitrogen source is selected from one or more of sodium nitrate, ammonium sulfate, and urea, and its concentration in the liquid fermentation medium is 1-5 g / L. The inorganic salt is potassium dihydrogen phosphate, magnesium sulfate heptahydrate, or trace elements. The concentration of potassium dihydrogen phosphate in the liquid fermentation medium is 0.5~2 g / L, the concentration of magnesium sulfate heptahydrate in the liquid fermentation medium is 0.5~1 g / L, and the concentration of trace elements in the liquid fermentation medium is 0.01~0.1 g / L. The trace elements are selected from one or more of ferrous sulfate heptahydrate, copper sulfate pentahydrate, manganese sulfate monohydrate, and zinc sulfate heptahydrate. The growth factor is selected from one or more of biotin, vitamin B1, and inositol. The concentration of the growth factor in the liquid fermentation medium is 0.001~0.01 g / L.

7. The method for preparing plant exosomes with anti-acne effect according to claim 1, characterized in that, S4 specifically involves: After fermentation, the fermentation broth is centrifuged at 3000×g for 15-30 min at 4℃ to remove mycelia; the supernatant is immediately transferred to a 0.22-0.45μm sterile filter-grade ultracentrifuge tube that has been autoclaved at 120-130℃ for 30-60 min, and then centrifuged at 100000×g at 4℃ for 80-100 min; the filtrate is discarded, the precipitate is resuspended in pre-cooled sterile PBS, and centrifuged again at 100000×g for 80-100 min in the same tube; finally, the precipitate is collected and diluted with sterile PBS to obtain the microbial-induced plant exosome suspension, which is stored at 4℃ in the dark.

8. A plant exosome with anti-acne effect, characterized in that, The plant exosomes with anti-acne effect are prepared by the method described in any one of claims 1 to 7, wherein the plant exosomes are spherical / cup-shaped nanovesicles with a double membrane structure.

9. A plant exosome with anti-acne effect according to claim 8, characterized in that, The diameter of the spherical / cup-shaped nanovesicles is 40~150nm.

10. The application of a plant exosome with anti-acne effect, characterized in that, Plant exosomes prepared by the method for preparing plant exosomes with anti-acne effect according to any one of claims 1 to 7 are used to treat acne.

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