Gynostemma pentaphylla induced eurotium cristatum exosome as well as preparation method and application thereof
The method for preparing Gynostemma pentaphyllum-induced exosomes of *Aspergillus cristatus* has solved the problems of low release rate and immature extraction methods, achieving efficient and green preparation and functional application, and expanding its application potential in lipid dysplasia diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies have low exosome release rates, immature extraction methods, lack of effective induction techniques, and insufficient functional verification, making them difficult to apply to the intervention of lipid dysplasia diseases.
A method for preparing exosomes of *Aspergillus cristatus* induced by *Gynostemma pentaphyllum* was adopted, including plant induction, fungal culture, exosome extraction and purification. The extracellular secretion system of *Aspergillus cristatus* was activated by *Gynostemma pentaphyllum* extract, and a "plant-fungus" signaling interaction mode was constructed by physical enrichment and density gradient purification.
It significantly improves the release amount and efficiency of exosomes, and the extracted exosomes have intact structures, making them suitable for drug delivery and functional development. They have the potential for large-scale preparation and are applicable to products such as lipid-regulating beverages and intestinal immune intervention.
Smart Images
Figure CN121801709A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of biomedical technology, specifically relating to a Gynostemma pentaphyllum-inducible Aspergillus cristatus exosome, its preparation method, and its application. Background Technology
[0002] Fungal exosomes (EVs) are a class of nanovesicles secreted by fungi, possessing a double-membrane structure. Rich in polysaccharides, proteins, lipids, and small molecule metabolites, they exhibit important biological activities in areas such as microbial communication, host immune regulation, anti-inflammation, and metabolic intervention. In recent years, fungal exosomes have become an emerging research hotspot in functional foods, drug delivery, and microecological intervention.
[0003] Eurotium cristatum is a dominant microorganism in dark tea and has been proven to possess abundant extracellular enzyme systems and lipid-lowering active ingredients. However, the application of its exosomes remains unexplored and faces the following bottlenecks: 1. Low release of fungal exosomes: Under natural culture conditions, the release efficiency of exosomes is low, and there is a lack of effective induction methods to stimulate their vesicle secretion pathway; 2. Lack of systematic methods for extracellular vesicle extraction: Currently, there is no standard procedure for the extraction of exosomes from fungi (such as *Aspergillus cristatus*), and traditional methods are prone to structural damage or contamination by impurities; 3. Induction mechanism not established: No activation mechanism targeting the fungal metabolic system has been constructed, making it difficult to regulate the secretory flux and functional components of extracellular vesicles; 4. Insufficient functional validation: There is no data on the intervention of Coronavirus cristatum EVs on lipid abnormal accumulation-related diseases (such as xanthomas).
[0004] Therefore, it is urgent to construct a complete technology chain of "plant induction-fungal response-physical purification-targeted application" to achieve large-scale, high-yield, and green preparation of *Aspergillus cristatus* EVs and expand its transformation pathway in lipid metabolism-related skin lesions. Summary of the Invention
[0005] The purpose of this application is to address the problems of the prior art by providing a Gynostemma pentaphyllum-induced Aspergillus cristatus exosome, its preparation method, and its application.
[0006] To solve the technical problem, the technical solution of this application is: a method for preparing *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes, comprising the following steps: S1: Preparation of induction solution; Take the pretreated dried Gynostemma pentaphyllum leaf powder, add 50-95% ethanol solution, extract and centrifuge to obtain supernatant, concentrate the supernatant to remove alcohol and obtain Gynostemma pentaphyllum ethanol extract, and store it in a sealed container at 2-8℃ for later use. S2: Fungal culture and induction; 1×10 of the suspension of *Eurotium cristatum* spores 6 ~1×10 7 CFU / mL was inoculated into liquid culture medium and cultured at 28-30℃ with shaking at 150-200 rpm for 12-24 h to obtain the culture solution; then 1-25% of the Gynostemma pentaphyllum ethanol extract of the culture solution was added, and the culture was induced for another 48-96 h to obtain the fermentation broth; during the induction phase, the dissolved oxygen was 30-60%, and the C / N ratio was controlled at 10-20:1 by online sugar supplementation to induce the exosome secretion of *Aspergillus cristatus* into the exponential phase. S3: Exosome extraction and purification; The fermentation broth of S2 was centrifuged at 2-8℃ and 1000-5000×g for 5-30 min to remove mycelia and large particulate impurities. The supernatant was then directly subjected to ultracentrifugation at 80000-120000×g and 2-8℃ for 60-120 min. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS, and ultracentrifugation was repeated once or multiple times. Finally, a high-purity exosome suspension was obtained by resuspending the precipitate in PBS. After sterilization by filtering through a 0.22μm filter membrane, the particle recovery rate was ≥50%, and the protein removal rate was ≥70%. S4: Stabilization; Add a freeze-drying protectant, pre-freeze at -70~-90℃ and then freeze-dry under vacuum to obtain a white, loose exosome powder, and store at 2~8℃ away from light.
[0007] Preferably, S1 specifically comprises: S1-1: Take the pretreated dried Gynostemma pentaphyllum leaf powder and add 50-95% ethanol solution at a material-to-liquid ratio of 1:5-50 g / mL; place the mixture in a dynamic countercurrent extraction device, set the extraction temperature to 40-80℃, and the extraction time to 20-180 min; after extraction, first filter through a 200-mesh filter cloth to remove coarse residue and collect the initial filtrate; then transfer the initial filtrate into a centrifuge tube and centrifuge at 3000-5000 r / min for 10-20 min to remove suspended fine residue and take the supernatant for later use; S1-2: Transfer the supernatant into a rotary evaporator and set the parameters: vacuum degree -0.07~-0.10MPa, water bath temperature 40~60℃; concentrate until there is no ethanol odor, then stop the concentration; then add sterile water to make up the volume so that the solid content of the extract is stable at 5~15%; finally, sterilize through a 0.22μm microporous membrane to obtain the Gynostemma pentaphyllum ethanol extract, and store it in a sealed container at 2~8℃ for later use.
[0008] Preferably, the liquid culture medium in S2 comprises glucose, peptone, yeast extract, magnesium sulfate heptahydrate, and calcium chloride, with a weight ratio of 30:10:5:0.8:0.4; and a suspension of *Aspergillus cristatus* spores is prepared at a concentration of 1×10⁻⁶. 6 ~1×107 Inoculate the liquid culture medium with CFU / mL at an inoculum rate of 1-10%.
[0009] Preferably, the induction stage in S2 further includes the addition of Zn at a concentration of 0.05~0.5 g / L. 2+ Mn with a concentration of 0.01~0.2 g / L 2+ Or plant-derived hormone analogs at a concentration of 0.1~2 mg / L.
[0010] Preferably, the plant-derived hormone analog is salicylic acid.
[0011] Preferably, step S4 specifically involves adding trehalose or mannitol as a freeze-drying protectant. The amount of trehalose added is 1-5% of the exosome suspension, and the amount of mannitol added is 0.5-2% of the exosome suspension. After pre-freezing at -80°C, the mixture is vacuum freeze-dried to obtain a white, loose exosome powder with a moisture content of ≤5%. The powder is then sealed and stored at 2-8°C for later use.
[0012] Preferably, a Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosome is prepared by any one of the preparation methods of Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosomes described in any one of the claims. The Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosome has a typical cup-shaped structure, an intact phospholipid bilayer membrane, an average particle size of 80-120 nm, and a PDI ≤ 0.25.
[0013] Preferably, the application of *Gynostemma pentaphyllum*-inducible *Aspergillus cristatus* exosomes is prepared by any of the methods described above. The *Gynostemma pentaphyllum*-inducible *Aspergillus cristatus* exosomes have the effects of regulating blood lipids, regulating immunity, antioxidation, and antitumor activity. They are used in pharmaceuticals, enzymes, beverages, and foods with health-promoting functions. The formulations of *Gynostemma pentaphyllum*-inducible *Aspergillus cristatus* exosomes include conventional formulations, easily digestible formulations, and novel advanced formulations. Conventional formulations include gels, pills, tablets, capsules, and powders. Easily digestible formulations include oral liquids, chewable tablets, orally disintegrating tablets, and oral films. Novel advanced formulations include liposomes, nanoparticles, microcapsules, and 3D-printed customized dosage forms. The gel is prepared by mixing exosome powder at a ratio of 1×10⁻⁶. 10 ~1×10 12 Particles / g were dispersed in a Carbomer 940 matrix at pH 6.0-7.0 and homogenized and degassed at 8000-12000 rpm to obtain a translucent gel. The preparation method of the pills is as follows: exosome powder is mixed with pharmaceutical excipients, and the mass ratio of exosome powder to pharmaceutical excipients is 1:2 to 1:5; the pills are made by fluidized bed coating technology to obtain micro-pills with a particle size of 0.3 to 0.8 mm, which are then filled into hard capsules or compressed into tablets.
[0014] Preferably, the carbomer 940 matrix has a mass percentage concentration of 0.5% to 1.2% in the entire gel system.
[0015] Preferably, the pharmaceutical excipient is one or more of microcrystalline cellulose and hydroxypropyl methylcellulose.
[0016] Compared with the prior art, the advantages of this application are: (1) This application is the first to propose a plant-induced fungal exosome release mechanism. By activating the extracellular secretion system of *Aspergillus cristatus* through *Gynostemma pentaphyllum* extract, a "plant-fungus" signal interaction mode is constructed for the first time, which increases the release amount of fungal exosomes and significantly improves the release efficiency of fungal exosomes. (2) This application is a green and pollution-free extraction route. The entire process adopts physical enrichment and density gradient purification to avoid potential pollutants such as PEG and complexing agents, and to meet the purity requirements of food and medicine homology products. (3) The exosomes extracted in this application have high structural integrity: microscopic observation and particle size distribution detection show that the vesicles obtained by this method have concentrated particle size (mostly distributed in 80~120nm) and intact membrane structure, which is suitable for delivery and active carrying function development; (4) This application constructs a microbial-plant synergistic functional model. The polysaccharides / lipids / signaling molecules carried in the exosomes have synergistic lipid regulation, antioxidant, and immune regulation mechanisms with the active components of Gynostemma pentaphyllum. It is suitable for developing various types of products such as "lipid-regulating drinks, intestinal immune intervention, and compound functional powders". (5) This application has the potential for large-scale preparation. The method is applicable to conventional bio-fermentation equipment, the culture medium is simple, the inducing components are naturally derived, and a green and controllable fungal exosome industrial transformation path can be realized. Attached Figure Description
[0017] Figure 1 This is a transmission electron microscope (TEM) image of the exosomes of Gynostemma pentaphyllum-induced Aspergillus cristatus in Example 1 of this application. Detailed Implementation
[0018] 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.
[0019] This application discloses a method for preparing Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosomes, comprising the following steps: S1: Preparation of induction solution; Take the pretreated dried Gynostemma pentaphyllum leaf powder, add 50-95% ethanol solution, extract and centrifuge to obtain supernatant, concentrate the supernatant to remove alcohol and obtain Gynostemma pentaphyllum ethanol extract, and store it in a sealed container at 2-8℃ for later use. S2: Fungal culture and induction; 1×10 of the suspension of *Eurotium cristatum* spores 6 ~1×10 7 CFU / mL was inoculated into liquid culture medium and cultured at 28-30℃ with shaking at 150-200 rpm for 12-24 h to obtain the culture solution; then 1-25% of the Gynostemma pentaphyllum ethanol extract of the culture solution was added, and the culture was induced for another 48-96 h to obtain the fermentation broth; during the induction phase, the dissolved oxygen was 30-60%, and the C / N ratio was controlled at 10-20:1 by online sugar supplementation to induce the exosome secretion of *Aspergillus cristatus* into the exponential phase. S3: Exosome extraction and purification; The fermentation broth of S2 was centrifuged at 2-8℃ and 1000-5000×g for 5-30 min to remove mycelia and large particulate impurities. The supernatant was then directly subjected to ultracentrifugation at 80000-120000×g and 2-8℃ for 60-120 min. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS, and ultracentrifugation was repeated once or multiple times. Finally, a high-purity exosome suspension was obtained by resuspending the precipitate in PBS. After sterilization by filtering through a 0.22μm filter membrane, the particle recovery rate was ≥50%, and the protein removal rate was ≥70%. S4: Stabilization; Add a freeze-drying protectant, pre-freeze at -70~-90℃ and then freeze-dry under vacuum to obtain a white, loose exosome powder, and store at 2~8℃ away from light.
[0020] Preferably, S1 specifically comprises: S1-1: Take the pretreated dried Gynostemma pentaphyllum leaf powder and add 50-95% ethanol solution at a material-to-liquid ratio of 1:5-50 g / mL; place the mixture in a dynamic countercurrent extraction device, set the extraction temperature to 40-80℃, and the extraction time to 20-180 min; after extraction, first filter through a 200-mesh filter cloth to remove coarse residue and collect the initial filtrate; then transfer the initial filtrate into a centrifuge tube and centrifuge at 3000-5000 r / min for 10-20 min to remove suspended fine residue and take the supernatant for later use; S1-2: Transfer the supernatant into a rotary evaporator and set the parameters: vacuum degree -0.07~-0.10MPa, water bath temperature 40~60℃; concentrate until there is no ethanol odor, then stop the concentration; then add sterile water to make up the volume so that the solid content of the extract is stable at 5~15%; finally, sterilize through a 0.22μm microporous membrane to obtain the Gynostemma pentaphyllum ethanol extract, and store it in a sealed container at 2~8℃ for later use.
[0021] Preferably, the liquid culture medium in S2 comprises glucose, peptone, yeast extract, magnesium sulfate heptahydrate, and calcium chloride, with a weight ratio of 30:10:5:0.8:0.4; and a suspension of *Aspergillus cristatus* spores is prepared at a concentration of 1×10⁻⁶. 6 ~1×10 7 Inoculate the liquid culture medium with CFU / mL at an inoculum rate of 1-10%.
[0022] Preferably, the induction stage in S2 further includes the addition of Zn at a concentration of 0.05~0.5 g / L. 2+ Mn with a concentration of 0.01~0.2 g / L 2+ Or plant-derived hormone analogs at a concentration of 0.1~2 mg / L.
[0023] The Zn 2+ It can promote the activity of metabolic enzymes, Mn 2+ It can increase the flux of lipids related to vesicle formation, and plant-derived hormone analogs can further enhance the fungal response to Gynostemma pentaphyllum saponins.
[0024] Preferably, the plant-derived hormone analog is salicylic acid.
[0025] Preferably, step S4 specifically involves adding trehalose or mannitol as a freeze-drying protectant. The amount of trehalose added is 1-5% of the exosome suspension, and the amount of mannitol added is 0.5-2% of the exosome suspension. After pre-freezing at -80°C, the mixture is vacuum freeze-dried to obtain a white, loose exosome powder with a moisture content of ≤5%. The powder is then sealed and stored at 2-8°C for later use.
[0026] Preferably, a Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosome (GIE-induced EVs) is prepared by any one of the preparation methods of Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosomes described above. The Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosomes have a typical cup-shaped structure, an intact phospholipid bilayer membrane, an average particle size of 80-120 nm, and a PDI ≤ 0.25.
[0027] The exosomes obtained in this application are "functionalized exosomes modified with Gynostemma pentaphyllum components" or "biosynthetic complex exosomes", which are essentially products of the interaction between Aspergillus cristatus and Gynostemma pentaphyllum, rather than a simple physical mixture of the two substances.
[0028] Preferably, the application of *Gynostemma pentaphyllum*-inducible *Aspergillus cristatus* exosomes is prepared by any of the methods described above. The *Gynostemma pentaphyllum*-inducible *Aspergillus cristatus* exosomes have the effects of regulating blood lipids, regulating immunity, antioxidation, and antitumor activity. They are used in pharmaceuticals, enzymes, beverages, and foods with health-promoting functions. The formulations of *Gynostemma pentaphyllum*-inducible *Aspergillus cristatus* exosomes include conventional formulations, easily digestible formulations, and novel advanced formulations. Conventional formulations include gels, pills, tablets, capsules, and powders. Easily digestible formulations include oral liquids, chewable tablets, orally disintegrating tablets, and oral films. Novel advanced formulations include liposomes, nanoparticles, microcapsules, and 3D-printed customized dosage forms. The gel is prepared by mixing exosome powder at a ratio of 1×10⁻⁶. 10 ~1×10 12 Particles / g were dispersed in a Carbomer 940 matrix at pH 6.0-7.0 and homogenized and degassed at 8000-12000 rpm to obtain a translucent gel. The preparation method of the pills is as follows: exosome powder is mixed with pharmaceutical excipients, and the mass ratio of exosome powder to pharmaceutical excipients is 1:2 to 1:5; the pills are made by fluidized bed coating technology to obtain micro-pills with a particle size of 0.3 to 0.8 mm, which are then filled into hard capsules or compressed into tablets.
[0029] Preferably, the carbomer 940 matrix has a mass percentage concentration of 0.5% to 1.2% in the entire gel system.
[0030] Preferably, the pharmaceutical excipient is one or more of microcrystalline cellulose and hydroxypropyl methylcellulose.
[0031] Example 1 (1) Preparation of induction solution (plant source): Take the pretreated dried Gynostemma pentaphyllum leaf powder and add 75% ethanol solution at a material-to-liquid ratio of 1:20 (g / mL); perform dynamic countercurrent extraction at 60℃ for 90 min; after coarse filtration through a 200-mesh filter cloth, centrifuge at 4000 r / min for 15 min; concentrate the supernatant under reduced pressure at -0.085 MPa and 50℃ until no alcohol odor is detected, add sterile water to bring the volume to 10% (w / v) solids content, sterilize with a 0.22 μm microporous membrane, and store at 4℃ to obtain "Gynostemma pentaphyllum ethanol extract" (GIE).
[0032] (2) Fungal culture and induction: A suspension of *Eurotium cristatum* spores (1×10) 7Inoculate with 5% (v / v) of GIE into liquid medium (30 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 0.8 g / L MgSO4·7H2O, 0.4 g / L CaCl2, initial pH 6.0); pre-culture at 28℃ and 160 rpm for 12 h; then add 15% (v / v) GIE and continue induction for 72 h; maintain dissolved oxygen at 40-50% and supplement glucose online to maintain C / N≈15:1.
[0033] (3) Exosome extraction and purification (without complexing agent): After induction, the fermentation broth was centrifuged at 3000×g for 15 min at 4℃ to remove mycelia; the supernatant was directly ultracentrifuged at 100000×g at 4℃ for 80 min; the supernatant was discarded, the precipitate was resuspended in PBS, and ultracentrifugation was repeated once; finally, the precipitate was resuspended in PBS and sterilized at 0.22 μm to obtain a high-purity exosome suspension with a particle concentration of (6.1±0.2)×10⁻⁶. 10 particles / mL, protein removal rate ≥70%, no PEG and no gradient media throughout the process.
[0034] (4) Stabilization: Add 3% trehalose and 1% mannitol to the exosome suspension, pre-freeze at -80℃ and then freeze-dry under vacuum to obtain a white loose powder with a moisture content of ≤5%. Store at 4℃ in the dark for 12 months with a particle recovery rate of >90%. The powder was dispersed at 1×10¹¹ particles / g in a carbomer 940 matrix (the carbomer 940 matrix was 0.8% by mass in the whole gel system), at pH 6.5, homogenized and degassed to obtain a translucent external gel.
[0035] like Figure 1 The image shown is a transmission electron microscope (TEM) image of the exosomes of *Gynostemma pentaphyllum* induced by *Aspergillus cristatus* in Example 1. Under the TEM, they exhibit a typical cup-shaped structure with an intact phospholipid bilayer membrane, an average particle size of 80-120 nm, and a PDI ≤ 0.25.
[0036] Example 2 (1) The preparation of the induction solution is the same as in Example 1, except that the material-liquid ratio is changed to 1:50, 95% ethanol solution, extraction temperature is 40℃, extraction time is 180min, centrifugation at 5000r / min speed for 20min, and the final solids are still adjusted to 10%.
[0037] (2) Fungal culture: The inoculum of *Aspergillus cristatus* spore suspension was 7%, GIE 25% (v / v), induction temperature 30℃, induction time 96h, and other conditions were the same as in Example 1.
[0038] (3)–(4) The ultracentrifugation and formulation steps are the same as in Example 1, and the final exosome particle concentration is (5.9±0.3)×1010 The particles / mL gel properties are consistent with those of Example 1.
[0039] Example 3 (1) The preparation of the induction solution is the same as in Example 1, except that the material-liquid ratio is changed to 1:5, 50% ethanol solution, extraction temperature is 80℃, extraction time is 20min, centrifugation at 3000r / min speed for 10min, and the final solids are still adjusted to 10%.
[0040] (2) Fungal culture: The inoculum of *Aspergillus cristatus* spore suspension was 10%, GIE 2% (v / v), the induction temperature was 28℃, the induction time was 48h, and the other conditions were the same as in Example 1.
[0041] (3)–(4) The ultracentrifugation and formulation steps are the same as in Example 1, and the final exosome particle concentration is (2.2±0.1)×10⁻⁶. 9 The particles / mL gel properties are consistent with those of Example 1.
[0042] Example 4 Effects of different concentrations of Gynostemma pentaphyllum on exosome production: The basal culture medium (30 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 0.8 g / L MgSO4·7H2O, 0.4 g / L CaCl2, initial pH 6.0) and culture parameters (28℃, 160 rpm, inoculum size 5%, 72 h) were fixed, with only the GIE addition amount (v / v) changed: 0%, 5%, 10%, 15%, 20%, 25%. Exosomes were collected by S3 ultracentrifugation, and the particle number was determined by NTA. As can be seen from the table above, 15% GIE is the optimal induction concentration, and the yield is about 51 times higher than that of the control group.
[0043] Example 5 Single-factor screening of optimal culture medium and culture conditions: Under the optimal combination, the peak exosome concentration stabilized at (6.1±0.3)×10⁻⁶. 10 particles / mL.
[0044] Example 6 Xanthomonas mouse intervention experiment: Animals: ApoE - / - Male mice, 6 weeks old, fed a high-fat diet for 10 weeks, developed eyelid xanthoma.
[0045] Grouping (n=10); ① Model group (blank gel); ② Example 1: Low-dose exosome gel (1×10) 10 (particles / mL) ③Example 1 High-dose exosome gel (1×10 11 (particles / mL) ④ Positive gel (0.1% atorvastatin).
[0046] Administration: Apply 0.05 mL to both eyelids twice daily for 4 consecutive weeks.
[0047] Image acquisition: The same stereomicroscope (×10) was used to take pictures before the experiment and on day 28. The boundaries of the tumor were delineated and the area was calculated using ImageJ software.
[0048] The results are shown in the table below. In the high-dose group, the initial skin area was 5.0 ± 0.5 mm², which decreased to 1.9 ± 0.4 mm² after 4 weeks, a reduction of 62% on average (paired t-test, P < 0.001), with no statistically significant difference compared to the positive control (P = 0.78). Serum TC decreased by 28%, foam cell infiltration decreased by 55%, and ABCA1 expression increased by 58% (all P < 0.01), with no skin irritation throughout the treatment.
[0049] Effect of exosome gel on xanthoma area (mean ± SD, n = 10) Example 7 Macrophage foaming inhibition experiment: Cells: RAW264.7 cells were stimulated with 80 μg / mL ox-LDL for 24 h to establish a foaming model.
[0050] Intervention: Add 0–1 × 10 10 particles / mL, incubated for 24 hours.
[0051] The results showed that intracellular cholesterol decreased by 51%, cholesterol efflux rate increased to 55%, ABCA1 protein was upregulated by 2.0 times, SR-A was downregulated by 57%, and inflammatory factors TNF-α and IL-6 decreased by 59% and 52%, respectively (P<0.01).
[0052] Conclusion: Gynostemma pentaphyllum-induced Aspergillus cristatus exosomes significantly inhibit macrophage foaming via the ABCA1 / SR-A axis, providing cytological evidence for in vivo anti-xanthoma therapy.
[0053] This application constructs a complete technology chain of "plant induction-fungal response-physical purification-targeted application" to achieve large-scale, high-yield, and green preparation of *Aspergillus cristatus* EVs, and expands its transformation pathway in lipid metabolism-related skin lesions.
[0054] This application pioneers a plant-induced fungal exosome release mechanism, which activates the extracellular secretion system of *Aspergillus cristatus* through *Gynostemma pentaphyllum* extract and constructs a "plant-fungus" signaling interaction mode for the first time, significantly improving the release efficiency of fungal exosomes.
[0055] This application presents a green and pollution-free extraction route, employing physical enrichment and density gradient purification throughout the entire process to avoid potential contaminants such as PEG and complexing agents, thus meeting the purity requirements for food and medicine homologous products.
[0056] The exosomes extracted in this application have high structural integrity: microscopic observation and particle size distribution detection show that the vesicles obtained by this method have concentrated particle size (mostly distributed in 80~120nm) and intact membrane structure, which is suitable for the development of delivery and active carrying functions.
[0057] This application constructs a microbial-plant synergistic functional model. The polysaccharides / lipids / signaling molecules carried in exosomes have synergistic mechanisms with the active components of Gynostemma pentaphyllum, such as lipid regulation, anti-oxidation, and immune regulation. This model is suitable for developing various types of products, such as "lipid-regulating drinks, intestinal immune intervention, and compound functional powders".
[0058] This application has the potential for large-scale preparation. The method is applicable to conventional bio-fermentation equipment, the culture medium is simple, the inducing components are naturally derived, and a green and controllable industrial transformation path for fungal exosomes can be realized.
[0059] 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.
[0060] 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 Gynostemma pentaphyllum-induced Aspergillus cristatus exosomes, characterized in that, Includes the following steps: S1: Preparation of induction solution; Take the pretreated dried Gynostemma pentaphyllum leaf powder, add 50-95% ethanol solution, extract and centrifuge to obtain supernatant, concentrate the supernatant to remove alcohol and obtain Gynostemma pentaphyllum ethanol extract, and store it in a sealed container at 2-8℃ for later use. S2: Fungal culture and induction; 1×10 of the suspension of *Eurotium cristatum* spores 6 ~1×10 7 CFU / mL was inoculated into liquid culture medium and cultured at 28-30℃ with shaking at 150-200 rpm for 12-24 h to obtain the culture solution; then 1-25% of the Gynostemma pentaphyllum ethanol extract of the culture solution was added, and the culture was induced for another 48-96 h to obtain the fermentation broth; during the induction phase, the dissolved oxygen was 30-60%, and the C / N ratio was controlled at 10-20:1 by online sugar supplementation to induce the exosome secretion of *Aspergillus cristatus* into the exponential phase. S3: Exosome extraction and purification; The fermentation broth of S2 was centrifuged at 2-8℃ and 1000-5000×g for 5-30 min to remove mycelia and large particulate impurities. The supernatant was then directly subjected to ultracentrifugation at 80000-120000×g and 2-8℃ for 60-120 min. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS, and ultracentrifugation was repeated once or multiple times. Finally, a high-purity exosome suspension was obtained by resuspending the precipitate in PBS. After sterilization by filtering through a 0.22μm filter membrane, the particle recovery rate was ≥50%, and the protein removal rate was ≥70%. S4: Stabilization; Add a freeze-drying protectant, pre-freeze at -70~-90℃ and then freeze-dry under vacuum to obtain a white, loose exosome powder, and store at 2~8℃ away from light.
2. The method for preparing *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes according to claim 1, characterized in that, Specifically, S1 is: S1-1: Take the pretreated dried Gynostemma pentaphyllum leaf powder and add 50-95% ethanol solution at a material-to-liquid ratio of 1:5-50g / mL; place the mixture in a dynamic countercurrent extraction device, set the extraction temperature to 40-80℃ and the extraction time to 20-180min; after extraction, filter through a 200-mesh filter cloth to remove coarse residue and collect the initial filtrate. Transfer the initial filtrate into a centrifuge tube and centrifuge at 3000-5000 r / min for 10-20 min to remove suspended fine residue. Take the supernatant for later use. S1-2: Transfer the supernatant into a rotary evaporator and set the parameters: vacuum degree -0.07~-0.10MPa, water bath temperature 40~60℃; concentrate until there is no ethanol odor, then stop the concentration; then add sterile water to make up the volume so that the solid content of the extract is stable at 5~15%; finally, sterilize through a 0.22μm microporous membrane to obtain the Gynostemma pentaphyllum ethanol extract, and store it in a sealed container at 2~8℃ for later use.
3. The method for preparing *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes according to claim 1, characterized in that, The liquid culture medium in S2 includes glucose, peptone, yeast extract, magnesium sulfate heptahydrate, and calcium chloride, with a weight ratio of 30:10:5:0.8:0.4; a suspension of *Aspergillus cristatus* spores is added at a concentration of 1×10⁻⁶. 6 ~1×10 7 Inoculate the liquid culture medium with CFU / mL at an inoculum rate of 1-10%.
4. The method for preparing *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes according to claim 1, characterized in that, The induction phase in S2 also involves the addition of Zn at a concentration of 0.05~0.5 g / L. 2+ Mn with a concentration of 0.01~0.2 g / L 2+ Or plant-derived hormone analogs at a concentration of 0.1~2 mg / L.
5. The method for preparing *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes according to claim 1, characterized in that, The plant-derived hormone analogue is salicylic acid.
6. The method for preparing *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes according to claim 1, characterized in that, S4 specifically involves adding trehalose or mannitol as a freeze-drying protectant. The amount of trehalose added is 1-5% of the exosome suspension, and the amount of mannitol added is 0.5-2% of the exosome suspension. After pre-freezing at -80℃, the mixture is freeze-dried under vacuum to obtain a white, loose exosome powder with a moisture content of ≤5%. The powder is then sealed and stored at 2-8℃ for later use.
7. A type of Gynostemma pentaphyllum-inducible *Aspergillus cristatus* exosome, characterized in that, The exosomes are prepared by the method of preparing Gynostemma pentaphyllum-inducible Ascorbicula fructus exosomes according to any one of claims 1 to 6. The exosomes are characterized by a typical cup-shaped structure, with an intact phospholipid bilayer membrane, an average particle size of 80-120 nm, and a PDI ≤ 0.
25.
8. The application of a Gynostemma pentaphyllum-induced Aspergillus cristatus exosome, characterized in that, The exosomes are prepared by a method according to any one of claims 1 to 6. The exosomes have the effects of regulating blood lipids, regulating immunity, antioxidation, and antitumor. They are used in pharmaceuticals, enzymes, beverages, and foods with health care functions. The preparations of the exosomes include conventional preparations, edible preparations, and novel advanced preparations. Conventional preparations include gels, pills, tablets, capsules, and powders. Edible preparations include oral liquids, chewable tablets, orally disintegrating tablets, and oral films. Novel advanced preparations include liposomes, nanoparticles, microcapsules, and 3D printed customized dosage forms. The gel is prepared by mixing exosome powder at a ratio of 1×10⁻⁶. 10 ~1×10 12 Particles / g were dispersed in a Carbomer 940 matrix at pH 6.0-7.0 and homogenized and degassed at 8000-12000 rpm to obtain a translucent gel. The preparation method of the pills is as follows: exosome powder is mixed with pharmaceutical excipients, and the mass ratio of exosome powder to pharmaceutical excipients is 1:2 to 1:5; the pills are made by fluidized bed coating technology to obtain micro-pills with a particle size of 0.3 to 0.8 mm, which are then filled into hard capsules or compressed into tablets.
9. The application of the *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes according to claim 8, characterized in that: The carbomer 940 matrix has a mass percentage concentration of 0.5% to 1.2% in the entire gel system.
10. The application of the *Gynostemma pentaphyllum*-induced *Aspergillus cristatus* exosomes according to claim 8, characterized in that: The pharmaceutical excipient is one or more of microcrystalline cellulose and hydroxypropyl methylcellulose.