An extracellular vesicle composition for promoting sleep, its preparation method and application

Abstract

The present invention relates to an extracellular vesicle composition for promoting sleep, its preparation method and application. The composition includes two or more of passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles. The extracellular vesicle composition of the present invention adopts a combined formula, and through the multi-dimensional combination of passionflower and extracellular vesicles of various Chinese herbal medicines, it can achieve a multi-faceted and highly efficient sleep-promoting effect from aspects such as increasing the cellular CL- level, increasing the expression level of GABA-related proteins, promoting the expression of cellular brain-derived neurotrophic factor (BDNF), promoting the growth of neuronal cell synapses, and inhibiting the level of microglial inflammatory factors, etc., and can reduce the sleep induction time and improve the sleep quality. At the same time, the extracellular vesicle composition of the present invention uses nano-scale raw materials, has good permeability, and is more easily absorbed by cells and the skin to exert its efficacy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an extracellular vesicle composition for promoting sleep, a preparation method thereof, and applications thereof. Background Art

[0002] Extracellular vesicles are membrane-bound vesicles with diameters ranging from approximately 20 nm to 5 μm. Extracellular vesicles can be derived not only from animal cells but also from plant cells. Plant extracellular vesicles and animal-derived extracellular vesicles have similar size distributions, surface charges, surface morphologies, and contents. Plant extracellular vesicles are widely sourced, safe, non-toxic, have low immunogenicity, can be produced on a large scale, and are inexpensive, with good biocompatibility and environmental friendliness. Plant extracellular vesicles can be directly consumed and absorbed through the gastrointestinal tract, and play a role in interspecies communication between plants and mammalian cells. Currently, there are few specific applications of membrane-bound vesicles of extracellular vesicles derived from plant cells.

[0003] Existing insomnia treatment products (such as chemical drugs and traditional herbal preparations) have many deficiencies, including obvious side effects, high dependence, unstable efficacy, etc. Long-term or incorrect use may also lead to dependence or other health risks. In contrast, traditional Chinese herbs have a long history and rich clinical experience, and their natural ingredients show unique advantages in regulating the nervous system and improving sleep quality. By extracting extracellular vesicles from traditional Chinese herbs, new sleep-promoting products with better safety and effectiveness can be developed using their characteristics of good biological safety and high permeability. This innovative method can not only overcome the limitations of existing products but also fully utilize the multiple effects of traditional Chinese herbs, providing a more reliable and sustainable solution for insomnia patients. Summary of the Invention

[0004] Problems to be Solved by the Invention

[0005] In view of the above problems existing in the prior art, there is an urgent need for an extracellular vesicle composition of traditional Chinese herbs with good sleep-promoting efficacy, high safety, and few side effects.

[0006] Solutions for Solving the Problems

[0007] The present invention provides an extracellular vesicle composition for promoting sleep, the composition comprising: two or more of passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0008] Preferably, the composition comprises: passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0009] Preferably, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is (1 - 20):(1 - 5):(1 - 5).

[0010] Preferably, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is (1 - 10):(1 - 2):(1 - 2).

[0011] Preferably, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 5:1:1.

[0012] The present invention also provides a method for preparing the composition, which includes: separately extracting the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles, and then compounding them in proportion.

[0013] Preferably, the extraction of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles includes: respectively taking passion fruit, chamomile, and licorice, adding a solvent to break the cell walls, and centrifuging to collect the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0014] Preferably, the solvent is PBS buffer;

[0015] And / or, the specific steps of the centrifugation include:

[0016] (1) Centrifuge at 400 - 800 g for 5 - 20 min, and take the supernatant;

[0017] (2) Centrifuge at 1000 - 3000 g for 10 - 30 min, and take the supernatant;

[0018] (3) Centrifuge at 3000 - 5000 g for 20 - 40 min, and take the supernatant;

[0019] (4) Centrifuge at 8000 - 12000 g for 40 - 70 min, and take the supernatant;

[0020] (5) Centrifuge at 100000 - 120000 g for 60 - 80 min, and take the precipitate.

[0021] The present invention also provides an application of the composition in the preparation of sleep - aid products.

[0022] Preferably, the sleep - aid products include emulsions, micro - emulsions, gels, ointments, creams, nasal sprays, inhalants, sprays.

[0023] Effects of the Invention

[0024] The extracellular vesicle composition of the present invention adopts a combined formula, and through the multi-dimensional combination method of compounding passion fruit with extracellular vesicles of various Chinese herbal medicines, it can achieve a multi-faceted and highly efficient sleep-promoting effect from aspects such as increasing the intracellular Cl- level, increasing the expression level of GABA-related proteins, promoting the expression of brain-derived neurotrophic factor (BDNF) in cells, promoting the growth of neuronal cell synapses, and inhibiting the level of microglial inflammatory factors, etc., and can reduce the sleep induction time and improve the sleep quality. At the same time, the extracellular vesicle composition of the present invention uses nano-scale raw materials, has good permeability, and is more easily absorbed and ingested by cells and the skin to exert its effects. Description of the Drawings

[0025] Figure 1 It is a transmission electron microscope (TEM) image of extracellular vesicles of passion fruit, chamomile, and licorice.

[0026] Figure 2 It shows that the extracellular vesicle composition of the present invention can rapidly penetrate into the skin of 8-week-old Bama minipigs within 2 h.

[0027] Figure 3 It shows the effect of the extracellular vesicle composition of the present invention on the GABA secretion of neuronal cells.

[0028] Figure 4 It shows the effect of the extracellular vesicle composition of the present invention on the expression level of DDC in neuronal cells.

[0029] Figure 5 It shows the effect of the extracellular vesicle composition of the present invention on the intracellular Cl- level of neuronal cells.

[0030] Figure 6 It shows the effect of the extracellular vesicle composition of the present invention on the BDNF secretion level of neuronal cells.

[0031] Figure 7 It is a micrograph of the length of neuronal cell synapses.

[0032] Figure 8 It shows the effect of the extracellular vesicle composition of the present invention on the length of neuronal cell synapses.

[0033] Figure 9 It shows the effect of the extracellular vesicle composition of the present invention on the IL-6 level of neuronal cells. Detailed Embodiments

[0034] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following provides a detailed description by listing specific embodiments. Among them, the attached drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0035] The present invention provides an extracellular vesicle composition for promoting sleep, and the composition includes two or more of passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0036] In some embodiments, the composition includes passionflower extracellular vesicles and chamomile extracellular vesicles.

[0037] In some embodiments, the mass ratio of the passionflower extracellular vesicles to the chamomile extracellular vesicles is 5:(1 - 3).

[0038] In some embodiments, the mass ratio of the passionflower extracellular vesicles to the chamomile extracellular vesicles is 5:1, or 5:2, or 5:3.

[0039] In some embodiments, the composition includes passionflower extracellular vesicles and licorice extracellular vesicles.

[0040] In some embodiments, the mass ratio of the passionflower extracellular vesicles to the licorice extracellular vesicles is 5:(1 - 3).

[0041] In some embodiments, the mass ratio of the passionflower extracellular vesicles to the licorice extracellular vesicles is 5:1, or 5:2, or 5:3.

[0042] In some embodiments, the composition includes chamomile extracellular vesicles and licorice extracellular vesicles.

[0043] In some embodiments, the mass ratio of the passionflower extracellular vesicles to the licorice extracellular vesicles is 5:(1 - 3).

[0044] In some embodiments, the mass ratio of the passionflower extracellular vesicles to the licorice extracellular vesicles is 5:1, or 5:2, or 5:3.

[0045] In some embodiments, the composition includes passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0046] In some embodiments, the mass ratio of the passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is (1 - 20):(1 - 5):(1 - 5).

[0047] In some embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is (1 to 10):(1 to 2):(1 to 2).

[0048] In some embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 1:1:1, or 1:1:2, or 1:2:1, or 1:2:2, or 2:1:1, or 2:1:2, or 2:2:1, or 2:2:2, or 3:1:1, or 3:1:2, or 3:2:1, or 3:2:2, or 4:1:1, or 4:1:2, or 4:2:1, or 4:2:2, or 5:1:1, or 5:1:2, or 5:2:1, or 5:2:2.

[0049] In some embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 1:1:1.

[0050] In some embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 5:1:1.

[0051] In some embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 5:2:2.

[0052] In some embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 10:1:1.

[0053] In some embodiments, the particle concentration of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 10 9 ~10 12 / mL.

[0054] The present invention also provides a method for preparing the composition, the preparation method comprising: separately extracting passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles, and then compounding them in proportion.

[0055] In some embodiments, the extraction method is selected from one or more of density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow filtration (TFF).

[0056] In some embodiments, the extraction of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles comprises: separately taking passion fruit, chamomile, and licorice, adding a solvent to break the cell walls, and centrifuging to collect the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0057] In some embodiments, passion flower, chamomile, and licorice are soaked in PBS buffer before cell wall breaking.

[0058] In some embodiments, the solvent is PBS buffer.

[0059] In some embodiments, the cell wall breaking is carried out for 8 - 20 min until there are no obvious massive fragments.

[0060] In some embodiments, the centrifugation is density gradient centrifugation and / or ultracentrifugation.

[0061] In some embodiments, the centrifugation is density gradient centrifugation and ultracentrifugation.

[0062] In some embodiments, the centrifugation is density gradient centrifugation.

[0063] In some embodiments, the centrifugation is ultracentrifugation.

[0064] In some embodiments, the centrifugation is performed using a low - speed centrifuge at 4°C.

[0065] In some embodiments, the specific steps of the centrifugation include:

[0066] (1) Centrifuge at 400 - 800 g for 5 - 20 min and take the supernatant;

[0067] (2) Centrifuge at 1000 - 3000 g for 10 - 30 min and take the supernatant;

[0068] (3) Centrifuge at 3000 - 5000 g for 20 - 40 min and take the supernatant;

[0069] (4) Centrifuge at 8000 - 12000 g for 40 - 70 min and take the supernatant;

[0070] (5) Centrifuge at 100000 - 120000 g for 60 - 80 min and take the precipitate.

[0071] In some embodiments, step (1) is to centrifuge at 500 g for 10 min and take the supernatant.

[0072] In some embodiments, step (2) is to centrifuge at 2000 g for 20 min and take the supernatant.

[0073] In some embodiments, step (3) is to centrifuge at 4000 g for 10 min and take the supernatant.

[0074] In some embodiments, step (4) is to centrifuge at 10000 g for 60 min and take the supernatant.

[0075] In some embodiments, step (5) is centrifugation at 110,000 g for 70 min, and the precipitate is taken.

[0076] In some embodiments, the precipitate obtained in step (5) is resuspended with the solvent.

[0077] In some embodiments, the precipitate obtained in step (5) is resuspended with PBS buffer.

[0078] In some embodiments, the particle concentrations of passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles prepared according to the method are 10 9 ~10 12 / mL.

[0079] The present invention also provides an application of the composition as described above in the preparation of sleep aid products.

[0080] In some embodiments, the sleep aid products include emulsions, microemulsions, gels, ointments, creams, nasal sprays, inhalants, sprays.

[0081] In some embodiments, the sleep aid products include sleep gels, sleep nasal sprays, and sleep sprays.

[0082] Example 1: Extraction of extracellular vesicles

[0083] 1. Weigh 100 g of passion fruit, chamomile, and licorice respectively, wash them 3 times with ultrapure water to remove impurities, place them in a clean beaker, and add 500 mL of PBS respectively, soak overnight at 4°C.

[0084] 2. Wash 3 times with sterile water, transfer to a blender, add 500 mL of PBS, tighten the lid, blend for 8 - 20 min to ensure no obvious lumpy fragments.

[0085] 3. Transfer the juice extracts to 50 mL sterile centrifuge tubes respectively, centrifuge at 500×g for 10 min using a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0086] 4. Centrifuge at 2000×g for 20 min using a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0087] 5. Centrifuge at 4000×g for 30 min using a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0088] 6. Centrifuge at 10000×g for 1 h using a high-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0089] 7. Centrifuge at 110,000 × g for 70 min at 4°C, discard the supernatant, and resuspend the precipitate with an appropriate amount of PBS to obtain extracellular vesicles of passionflower, chamomile, and licorice (labeled as PA-Exo, MC-Exo, and GU-Exo, respectively). The particle concentration per ml was 10 9 ~10 12 between.

[0090] Example 2: Preparation of extracellular vesicle compositions

[0091] The extracellular vesicles of passionflower, chamomile and licorice in Example 1 were compounded according to different mass ratios (as shown in Table 1) to obtain an extracellular vesicle composition. For short-term use, it is directly stored in a -80°C refrigerator. For long-term use and transportation, a lyophilization protective agent can be added for freeze-drying.

[0092] Table 1: Extracellular vesicle compositions with different ratios

[0093]

[0094] Example 3: Preparation of sleep aid products

[0095] The extracellular vesicle composition prepared in Example 2 was added to the sleep gel (Table 2), sleep nasal spray (Table 3), and sleep spray (Table 4). The specific formulas are shown below.

[0096] Table 2: Sleeping gel formula

[0097]

[0098] Table 3: Sleeping nasal spray formula

[0099]

[0100] Table 4: Sleep spray formula

[0101]

[0102] Example 4: Transmission Electron Microscopy (TEM) Analysis of Extracellular Vesicles

[0103] The shapes of the extracellular vesicle particles of each Chinese herbal medicine prepared in Example 1 were analyzed by transmission electron microscopy (TEM).

[0104] According to the sample conditions, the extracellular vesicles prepared in Example 1 are adjusted to a suitable concentration or viscosity. Use a pipette to draw about 15 μL of the extracellular vesicle sample onto the copper mesh and let it stand for 1 min. Use filter paper to blot the extracellular vesicle sample on the copper mesh, and draw about 15 μL of 2% uranyl acetate staining solution to stain at room temperature for 1 min. If obvious adsorbents are visible on the copper mesh, pure water can be dripped onto the surface, quickly blotted off, and repeatedly washed several times. Use filter paper to blot the extracellular vesicle sample on the copper mesh. Observe and take pictures, and save the pictures. The results are as follows. Figure 1 As shown, saucer-shaped vesicles can be observed in extracellular vesicles derived from passion flower (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo).

[0105] Example 5: Skin penetration of extracellular vesicle compositions

[0106] The back or abdominal skin of 8-week-old Bama miniature pigs was cut into 1 cm × 1 cm pieces for use. Take 1 mg of YHSM-YL0012 prepared in Example 2, and use diluent C in the kit (Sigma, PKH26 red fluorescent cell labeling kit MINI26-1KT) to make the sample volume dilute to 1 mL. Take 6 μL of PKH26 dye in the kit and add it to a test tube containing 1 mL of diluent C. Use a pipette to blow gently and mix continuously for 30 seconds. Let stand at room temperature for 5 minutes. Add 2 mL of 10% BSA in PBS (Sigma-Aldrich, D8537) for quenching. Use serum-free medium to make the volume dilute to 30 mL. Centrifuge at 110,000 g for 2 hours at 2-8°C. Use a pipette to blow gently and resuspend the extracellular vesicle pellet in 1 ml of serum-free medium for use.

[0107] Place 1cm×1cm 8-week-old Bama miniature pig skin in a 32℃ environment, take 50μL 1mg / ml PKH26-labeled YHSM-YL0012 and drip it onto 1cm×1cm 8-week-old Bama miniature pig skin, evenly cover the entire epidermis, and incubate for 2h. After incubation, cryosections were performed with a thickness of 10 μm. After DAPI staining the cell nucleus, the cells were observed and photographed under a fluorescence microscope. The results are shown in Figure 2 As shown, the extracellular vesicle composition of the present invention can quickly penetrate into the skin of 8-week-old Bama miniature pigs within 2 hours.

[0108] Example 6: Effect of the extracellular vesicle composition on the secretion level of γ-aminobutyric acid (GABA) in neuronal cells (SH-SY5Y)

[0109] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 h. The cell supernatants were discarded, and 100 μL of negative control reagent (NC: DMEM medium), experimental group reagents (Passiflora edulis, Matricaria chamomilla, Glycyrrhiza glabra extracellular vesicles prepared in Example 1 and YHSM-YL0011, YHSM-YL0012 prepared in Example 2 (diluted to a protein concentration of 20 μg / mL with DMEM medium)) were added respectively, and cultured for 24 h. The supernatants were taken, and the secretion of GABA was detected by the Pumei Biotechnology γ-aminobutyric acid (GABA) detection kit.

[0110] The results are as Figure 3 shown that extracellular vesicle particles derived from Passiflora edulis (PA-Exo), Matricaria chamomilla (MC-Exo), and Glycyrrhiza glabra (GU-Exo) can significantly promote the secretion of GABA by neuronal cells. YHSM-YL0012 of the present invention can also significantly promote the secretion of GABA by neuronal cells, and the effect is better than that of YHSM-YL0011 and the extracellular vesicles of Passiflora edulis, Matricaria chamomilla, and Glycyrrhiza glabra used alone.

[0111] Example 7: Effect of extracellular vesicle composition on the RNA level of GABA-related genes in neuronal cells

[0112] Neuronal cells were seeded into 12-well plates at a density of 200,000 cells / 1000 μL / well. The cells were cultured for 24 h. The cell supernatants were discarded. 1000 μL of negative control reagent (NC: DMEM medium), experimental group reagents (Passiflora edulis, Matricaria chamomilla, Glycyrrhiza glabra extracellular vesicles prepared in Example 1 and YHSM-YL0011, YHSM-YL0012 prepared in Example 2 (diluted to a protein concentration of 20 μg / mL with DMEM medium)) were added respectively, and cultured for 24 h. The supernatants were discarded, the cells were collected, and the RNA of the cells was extracted to detect the expression of GABA-related genes DDC, GAD1, GABRA1, GABRA4, and GABRA6.

[0113] The results are as Figure 4As shown, extracellular vesicle particles derived from passion fruit (PA-Exo) can significantly promote the expression level of DDC in neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly promote the expression level of DDC in neuronal cells, and the effect is better than that of YHSM-YL0011 and the extracellular vesicles of passion fruit, chamomile, and licorice used alone. Extracellular vesicles derived from passion fruit (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) can significantly promote the expression level of GAD1 in neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly promote the expression level of GAD1 in neuronal cells, and the effect is better than that of YHSM-YL0011 and the extracellular vesicles of passion fruit, chamomile, and licorice used alone. Extracellular vesicles derived from passion fruit (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) can significantly promote the expression level of GABRA1 in neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly promote the expression level of GABRA1 in neuronal cells, and the effect is better than that of YHSM-YL0011 and the extracellular vesicles of passion fruit, chamomile, and licorice used alone.

[0114] Example 8: Effect of extracellular vesicle composition on the Cl- level in neuronal cells

[0115] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 h. The cell supernatant was discarded, and 100 μL of negative control reagent (NC: DMEM medium), positive control group reagent (PC: 5 mg / mL GABA, diluted with DMEM medium), and experimental group reagent (extracellular vesicles of passion fruit, chamomile, and licorice prepared in Example 1 and YHSM-YL0011 and YHSM-YL0012 prepared in Example 2 (diluted with DMEM medium to a protein concentration of 20 μg / mL)) were added respectively, and cultured for 24 h. The supernatant was taken, and the MAQE fluorescent probe was used to measure the effect of different samples on the Cl- level in neuronal cells.

[0116] The results are shown as Figure 5 shown, extracellular vesicles derived from passion fruit (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) can significantly increase the Cl- level in neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly increase the Cl- level in neuronal cells, and the effect is better than that of YHSM-YL0011 and the extracellular vesicles of passion fruit, chamomile, and licorice used alone.

[0117] Example 9: Effect of extracellular vesicle composition on the brain-derived neurotrophic factor BDNF level in neuronal cells

[0118] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 h. The cell supernatant was discarded, and 100 μL of negative control reagent (NC: DMEM medium), positive control group reagent (PC: 10 μM GABA, diluted with DMEM medium), and experimental group reagent (Passiflora edulis, Matricaria chamomilla, Glycyrrhiza glabra extracellular vesicles prepared in Example 1 and YHSM-YL0011, YHSM-YL0012 prepared in Example 2 (diluted with DMEM medium to a protein concentration of 20 μg / mL)) were added respectively, and cultured for 24 h. The supernatant was taken, and the secretion of BDNF was detected by the BDNF ELISA detection kit of Linke Biotechnology Co., Ltd.

[0119] The results showed that Figure 6 as shown, extracellular vesicles derived from Passiflora edulis (PA-Exo) and Matricaria chamomilla (MC-Exo) could significantly promote the BDNF secretion level of neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention could also significantly promote the BDNF secretion level of neuronal cells, and the effect was better than that of YHSM-YL0011 and the extracellular vesicles of Passiflora edulis, Matricaria chamomilla, and Glycyrrhiza glabra used alone.

[0120] Example 10: Effect of extracellular vesicle composition on the neuronal length of neuronal cells

[0121] Neuronal cells were seeded into 24-well plates at a density of 100,000 cells / 100 μL / well. The cells were cultured for 24 h. The cell supernatant was discarded, and 500 μL of negative control reagent (NC: DMEM medium), positive control group reagent (PC: 10 μM RA, diluted with DMEM medium), and experimental group reagent (Passiflora edulis, Matricaria chamomilla, Glycyrrhiza glabra extracellular vesicles prepared in Example 1 and YHSM-YL0011, YHSM-YL0012 prepared in Example 2 (diluted with DMEM medium to a protein concentration of 20 μg / mL)) were added respectively, and cultured for 48 h. Photographs were taken through a microscope (as Figure 7 shown), and the length of nerve synapses was statistically analyzed using Image J.

[0122] The results showed that Figure 8 as shown, extracellular vesicles derived from Passiflora edulis (PA-Exo), Matricaria chamomilla (MC-Exo), and Glycyrrhiza glabra (GU-Exo) could significantly promote the growth of nerve synapse length of neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention could also significantly promote the growth of nerve synapse length of neuronal cells, and the effect was better than that of YHSM-YL0011 and the extracellular vesicles of Passiflora edulis, Matricaria chamomilla, and Glycyrrhiza glabra used alone.

[0123] Example 11: Effect of Extracellular Vesicle Composition on Inflammatory Factor Levels in Microglia

[0124] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 h. The cell supernatants were discarded, and 100 μL of negative control reagent (NC: MEM complete medium), positive control group reagent (PC: 100 μg / ml dexamethasone, diluted with MEM complete medium), and experimental group reagent (passion fruit, chamomile, and licorice extracellular vesicles prepared in Example 1 and YHSM-YL0011 and YHSM-YL0012 prepared in Example 2 (diluted with MEM complete medium to a protein concentration of 20 μg / mL)) were added respectively, and cultured for 24 h. The supernatants were taken, and the secretion of the pro-inflammatory factor IL-6 was detected by the Lianke Biotech Inflammatory Factor Interleukin-6 (IL-6) Elisa Detection Kit.

[0125] The results showed that, as Figure 9 shown, extracellular vesicles derived from passion fruit (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) could significantly inhibit the IL-6 level in neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention could also significantly inhibit the IL-6 level in neuronal cells, and the effect was better than that of YHSM-YL0011 and the extracellular vesicles of passion fruit, chamomile, and licorice used alone.

[0126] Example 12: Human Treatment of Insomnia Test with Sleep Gel

[0127] 210 experiencers aged 30 - 50 with insomnia problems and who had not used insomnia treatment drugs recently were enrolled and randomly divided into 7 groups. In the experimental group, the sleep gel prepared in Example 3 (extracellular vesicle composition content 30%) was applied behind the ears 30 minutes before going to bed every night. In the control group, a blank sleep gel (i.e., a sleep gel without extracellular vesicle composition) was applied once every night. A sleep diary was filled out every day, and the PSQI questionnaire was filled out before use and after continuous use for one week. The effective rate was calculated as follows: (((number of significantly improved people × 100%) + (number of slightly improved people × 50%)) / 30) × 100%. The results of the human treatment of insomnia test with the sleep gel are shown in Table 5.

[0128] Table 5: Results of Human Treatment of Insomnia Test with Sleep Gel

[0129]

[0130] Example 13: Human Treatment of Insomnia Test with Sleep Nasal Spray

[0131] 60 subjects aged 30 - 50 years old who suffered from insomnia and had not used insomnia treatment drugs recently were recruited and randomly divided into two groups. The experimental group sprayed the sleep nasal spray prepared in Example 3 (the content of extracellular vesicle composition YHSM - YL0012 was 90%) into the nasal cavity 30 minutes before going to bed every night, and the control group sprayed a blank sleep nasal spray (i.e., a nasal spray without extracellular vesicle composition) every night. The subjects filled in the sleep diary every day and filled in the PSQI questionnaire before use and after continuous use for one week. The calculation of the effective rate was: (((the number of significantly improved subjects × 100%) + (the number of slightly improved subjects × 50%)) / 30) × 100%. The results of the human treatment of insomnia test for the sleep nasal spray are shown in Table 6.

[0132] Table 6: Results of the human treatment of insomnia test for the sleep nasal spray

[0133]

[0134] Example 14: Human treatment of insomnia test for the sleep spray

[0135] 60 subjects aged 30 - 50 years old who suffered from insomnia and had not used insomnia treatment drugs recently were recruited and randomly divided into two groups. The experimental group sprayed the sleep spray prepared in Example 3 (the content of extracellular vesicle composition YHSM - YL0012 was 5%) on the bedside 30 minutes before going to bed every night, and the control group sprayed a blank sleep spray (i.e., a spray without extracellular vesicle composition) every night. The subjects filled in the sleep diary every day and filled in the PSQI questionnaire before use and after continuous use for one week. The calculation of the effective rate was: (((the number of significantly improved subjects × 100%) + (the number of slightly improved subjects × 50%)) / 30) × 100%. The results of the human treatment of insomnia test for the sleep spray are shown in Table 7.

[0136] Table 7: Results of the human treatment of insomnia test for the sleep spray

[0137]

[0138] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can be made on the basis of the above embodiments. Similarly, various technical features of the above embodiments can be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. An extracellular vesicle composition for promoting sleep, characterized in that, The composition comprises: passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles, and the mass ratio of the passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is (5-10):(1-2):(1-2).

2. The composition according to claim 1, wherein The mass ratio of the passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is 5:1:

1.

3. A method for preparing the composition according to any one of claims 1-2, characterized in that, The preparation method comprises: separately extracting passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles, and then compounding them in proportion.

4. The preparation method according to claim 3, characterized in that, The extraction of the passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles comprises: separately taking passionflower, chamomile, and licorice, adding PBS buffer solution and then breaking the cell walls, and centrifuging to collect the passionflower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

5. The preparation method according to claim 4, wherein The specific steps of the centrifugation include: (1) Centrifuge at 400-800 g for 5-20 min and take the supernatant; (2) Centrifuge at 1000-3000 g for 10-30 min and take the supernatant; (3) Centrifuge at 3000-5000 g for 20-40 min and take the supernatant; (4) Centrifuge at 8000-12000 g for 40-70 min and take the supernatant; (5) Centrifuge at 100000-120000 g for 60-80 min and take the precipitate.

6. Use of the composition according to any one of claims 1-2 in the preparation of a sleep aid product.

7. The application according to claim 6, characterized in that, The sleep aid product includes lotion, gel, ointment, inhalant, or spray.

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