Extracellular vesicle composition for aiding sleep as well as preparation method and application of extracellular vesicle composition

Abstract

The invention relates to an extracellular vesicle composition for aiding sleep as well as a preparation method and application thereof. The composition comprises two or more of passion flower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles. According to the extracellular vesicle composition, a combined formula is adopted, and the CL-level of cells is improved, the expression level of GABA related proteins is improved, the expression of brain-derived neurotrophic factors (BDNF) of the cells is promoted, and the synaptic growth of neuronal cells is promoted in a multi-dimensional combined mode that the passiflora edulis is compounded with multiple Chinese herbal medicine extracellular vesicles; the sleep-aiding pillow has the advantages that multi-aspect efficient sleep-aiding effects can be realized, sleep time can be shortened, and sleep quality can be improved. Meanwhile, the extracellular vesicle composition disclosed by the invention adopts nano-scale raw materials, has good permeability, and is more easily absorbed and ingested by cells and skin to play a role.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an extracellular vesicle composition for aiding sleep, and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles are membrane-structured vesicles with a diameter of about 20 nm-5 μm. Extracellular vesicles can be derived not only from animal cells but also from plant cells. Plant extracellular vesicles have similar size distribution, surface charge, surface morphology, content composition, etc. to animal-derived extracellular vesicles. Plant extracellular vesicles are widely available, safe and non-toxic, have low immunogenicity, can be mass-produced, and are low in cost. They have good biocompatibility and environmental friendliness. Plant extracellular vesicles can be eaten directly, absorbed through the gastrointestinal tract, and play a role in interspecies communication between plant and mammalian cells. At present, there are few specific applications of membrane-structured extracellular vesicles derived from plant cells.

[0003] Existing insomnia treatment products (such as chemical drugs and traditional herbal preparations) have many shortcomings, including obvious side effects, high dependence, unstable efficacy, etc. Long-term or improper use may also lead to dependence or other health risks. In contrast, Chinese herbal medicine has a long history and rich clinical experience, and its natural ingredients show unique advantages in regulating the nervous system and improving sleep quality. By extracting extracellular vesicles from Chinese herbal medicines, we can take advantage of their good biosafety and high permeability to develop new, safer and more effective sleep aid products. This innovative method can not only overcome the limitations of existing products, but also give full play to the multiple functions of Chinese herbal medicines, providing more reliable and sustainable solutions for insomnia patients. Summary of the invention

[0004] Problem that the invention aims to solve In view of the above problems existing in the prior art, there is an urgent need for a Chinese herbal extracellular vesicle composition with good sleep-aiding effect, high safety and few side effects.

[0005] Solutions for solving problems

[0006] The invention provides an extracellular vesicle composition for aiding sleep, the composition comprising: two or more of passionflower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles.

[0007] Preferably, the composition comprises: passionflower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles.

[0008] Preferably, the mass ratio of the passion fruit extracellular vesicles, the chamomile extracellular vesicles and the liquorice extracellular vesicles is (1-20):(1-5):(1-5).

[0009] Preferably, the mass ratio of the passion fruit extracellular vesicles, the chamomile extracellular vesicles and the liquorice extracellular vesicles is (1-10):(1-2):(1-2).

[0010] Preferably, the mass ratio of the passionflower extracellular vesicles, the chamomile extracellular vesicles and the liquorice extracellular vesicles is 5:1:1.

[0011] The present invention also provides a method for preparing the composition, which comprises: extracting passionflower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles respectively, and then compounding them according to a proportion.

[0012] Preferably, the extraction of the passionflower extracellular vesicles, chamomile extracellular vesicles and licorice extracellular vesicles comprises: taking passionflower, chamomile and licorice respectively, breaking the walls after adding a solvent, and collecting the passionflower extracellular vesicles, chamomile extracellular vesicles and licorice extracellular vesicles by centrifugation.

[0013] Preferably, the solvent is PBS buffer; And / or, the specific steps of the centrifugation include: (1) Centrifuge at 400-800 g for 5-20 min and collect the supernatant; (2) Centrifuge at 1000-3000 g for 10-30 min and collect the supernatant; (3) Centrifuge at 3000-5000g for 20-40min and collect the supernatant; (4) Centrifuge at 8000-12000 g for 40-70 min and collect the supernatant; (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

[0014] The present invention also provides an application of the composition in preparing a sleep-aiding product.

[0015] Preferably, the sleep aid product includes emulsion, microemulsion, gel, ointment, cream, nasal spray, inhalant, and spray.

[0016] Effects of the Invention

[0017] The extracellular vesicle composition of the present invention adopts a combined formula, and through the multi-dimensional combination of passion fruit and a variety of Chinese herbal extracellular vesicles, it can improve the cell CL-level, improve the expression level of GABA-related proteins, promote the expression of brain-derived neurotrophic factor (BDNF), promote the growth of neuronal cell synapses, inhibit the level of microglial inflammatory factors, etc., to achieve a multi-faceted and efficient sleep-aiding effect, which can reduce the time to fall asleep and improve the quality of sleep. At the same time, the extracellular vesicle composition of the present invention adopts nano-scale raw materials, has good permeability, and is more easily absorbed and ingested by cells and skin to exert its efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Transmission electron microscopy (TEM) images of extracellular vesicles from passion flower, chamomile, and licorice.

[0019] Figure 2 It was shown that the extracellular vesicle composition of the present invention can quickly penetrate into the skin of 8-week-old Bama miniature pigs within 2 hours.

[0020] Figure 3 The effect of the extracellular vesicle composition of the present invention on GABA secretion of neuronal cells is shown.

[0021] Figure 4 The effect of the extracellular vesicle composition of the present invention on the expression level of DDC in neuronal cells is shown.

[0022] Figure 5 The effect of the extracellular vesicle composition of the present invention on the Cl- level of neuronal cells is shown.

[0023] Figure 6 The effect of the extracellular vesicle composition of the present invention on the BNDF secretion level of neuronal cells is shown.

[0024] Figure 7 This is a microscope photo of the length of a neuron synapse.

[0025] Figure 8 The effect of the extracellular vesicle composition of the present invention on the length of neuronal synapses of neuronal cells is shown.

[0026] Fig. 9 The effect of the extracellular vesicle composition of the present invention on the IL-6 level of neuronal cells is shown. DETAILED DESCRIPTION

[0027] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may 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 field to which this application belongs.

[0028] The invention provides an extracellular vesicle composition for aiding sleep, the composition comprising: two or more of passionflower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles.

[0029] In certain embodiments, the composition comprises: passion flower extracellular vesicles and chamomile extracellular vesicles.

[0030] In certain embodiments, the mass ratio of the passion fruit extracellular vesicles to the chamomile extracellular vesicles is 5:(1-3).

[0031] In certain embodiments, the mass ratio of the passion flower extracellular vesicles to the chamomile extracellular vesicles is 5:1, or 5:2, or 5:3.

[0032] In certain embodiments, the composition comprises: passion flower extracellular vesicles and liquorice extracellular vesicles.

[0033] In certain embodiments, the mass ratio of the passion fruit extracellular vesicles to the chamomile extracellular vesicles is 5:(1-3).

[0034] In certain embodiments, the mass ratio of the passion flower extracellular vesicles to the chamomile extracellular vesicles is 5:1, or 5:2, or 5:3.

[0035] In certain embodiments, the composition comprises: chamomile extracellular vesicles and licorice extracellular vesicles.

[0036] In certain embodiments, the mass ratio of the passion fruit extracellular vesicles to the liquorice extracellular vesicles is 5:(1-3).

[0037] In certain embodiments, the mass ratio of the passion fruit extracellular vesicles to the liquorice extracellular vesicles is 5:1, or 5:2, or 5:3.

[0038] In certain embodiments, the composition comprises: passion flower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0039] In certain embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles is (1-20): (1-5): (1-5).

[0040] In certain embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles is (1-10): (1-2): (1-2).

[0041] In certain embodiments, the mass ratio of the passion fruit extracellular vesicles, chamomile extracellular vesicles and liquorice 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.

[0042] In certain embodiments, the mass ratio of the passionflower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles is 1:1:1.

[0043] In certain embodiments, the mass ratio of the passionflower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles is 5:1:1.

[0044] In certain embodiments, the mass ratio of the passionflower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles is 5:2:2.

[0045] In certain embodiments, the mass ratio of the passion flower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles is 10:1:1.

[0046] In certain embodiments, the particle concentration of passionflower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles is 10 9 ~10 12 / mL.

[0047] The present invention also provides a method for preparing the composition, which comprises: extracting passionflower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles respectively, and then compounding them according to a proportion.

[0048] In certain embodiments, the extraction method is selected from one or more of density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow filtration (TFF).

[0049] In certain embodiments, the extraction of passion flower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles comprises: taking passion flower, chamomile, and licorice respectively, adding a solvent to break the cell walls, and centrifuging to collect the passion flower extracellular vesicles, chamomile extracellular vesicles, and licorice extracellular vesicles.

[0050] In certain embodiments, passion flower, chamomile, and licorice are soaked in PBS buffer before cell wall breaking.

[0051] In certain embodiments, the solvent is PBS buffer.

[0052] In certain embodiments, the cell wall is broken for 8 to 20 minutes until there are no obvious block fragments.

[0053] In certain embodiments, the centrifugation is density gradient centrifugation and / or ultracentrifugation.

[0054] In certain embodiments, the centrifugation is density gradient centrifugation and ultracentrifugation.

[0055] In certain embodiments, the centrifugation is density gradient centrifugation.

[0056] In certain embodiments, the centrifugation is ultracentrifugation.

[0057] In certain embodiments, the centrifugation uses a 4°C low-speed centrifuge.

[0058] In certain embodiments, the specific steps of centrifugation include: (1) Centrifuge at 400-800 g for 5-20 min and collect the supernatant; (2) Centrifuge at 1000-3000 g for 10-30 min and collect the supernatant; (3) Centrifuge at 3000-5000g for 20-40min and collect the supernatant; (4) Centrifuge at 8000-12000 g for 40-70 min and collect the supernatant; (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

[0059] In certain embodiments, the step (1) is to centrifuge at 500 g for 10 min and take the supernatant.

[0060] In certain embodiments, the step (2) is centrifuging at 2000 g for 20 min and taking the supernatant.

[0061] In certain embodiments, the step (3) is to centrifuge at 4000 g for 10 min and collect the supernatant.

[0062] In certain embodiments, the step (4) is centrifugation at 10,000 g for 60 min and taking the supernatant.

[0063] In certain embodiments, the step (5) is to centrifuge at 110,000 g for 70 min and collect the precipitate.

[0064] In certain embodiments, the precipitate in step (5) is resuspended using the solvent.

[0065] In certain embodiments, the precipitate in step (5) is resuspended using PBS buffer.

[0066] In certain embodiments, the particle concentration of passionflower extracellular vesicles, chamomile extracellular vesicles, and liquorice extracellular vesicles prepared according to the method is 10 9 ~10 12 / mL.

[0067] The present invention also provides an application of the composition in preparing a sleep-aiding product.

[0068] In certain embodiments, the sleep aid product comprises an emulsion, a microemulsion, a gel, an ointment, a cream, a nasal spray, an inhalant, or a spray.

[0069] In certain embodiments, the sleep aid product includes a sleep gel, a sleep nasal spray, or a sleep spray.

[0070] Example 1: Extraction of extracellular vesicles

[0071] 1. Weigh 100g of passionflower, chamomile and licorice respectively, wash them three times with ultrapure water to remove impurities, place them in a clean beaker, add 500mL of PBS respectively, and soak them at 4℃ overnight.

[0072] 2. Wash three times with sterile water, transfer to a wall-breaking machine, add 500 mL of PBS, cover tightly, and break the wall for 8-20 minutes to ensure that there are no obvious lumps and fragments.

[0073] 3. Transfer the squeezed juice to 50mL sterile centrifuge tubes, centrifuge at 500×g for 10min at 4℃ low speed centrifuge, discard the precipitate and take the supernatant.

[0074] 4. Centrifuge at 2000×g for 20 min at 4℃, discard the precipitate and take the supernatant.

[0075] 5. Centrifuge at 4000×g for 30 min at 4℃, discard the precipitate and take the supernatant.

[0076] 6. Centrifuge at 10,000 × g for 1 h at 4°C, discard the precipitate and take the supernatant.

[0077] 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.

[0078] Example 2: Preparation of extracellular vesicle compositions

[0079] 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.

[0080] Table 1: Extracellular vesicle compositions with different ratios

[0081] Example 3: Preparation of sleep aid products

[0082] 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.

[0083] Table 2: Sleeping gel formula

[0084] Table 3: Sleeping nasal spray formula

[0085] Table 4: Sleep spray formula

[0086] Example 4: Transmission Electron Microscopy (TEM) Analysis of Extracellular Vesicles

[0087] The shapes of the extracellular vesicle particles of each Chinese herbal medicine prepared in Example 1 were analyzed by transmission electron microscopy (TEM).

[0088] 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).

[0089] Example 5: Skin penetration of extracellular vesicle compositions

[0090] 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.

[0091] 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.

[0092] Example 6: Effect of the extracellular vesicle composition on the secretion level of γ-aminobutyric acid (GABA) in neuronal cells (SH-SY5Y)

[0093] 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), experimental group reagents (passionflower, chamomile, licorice 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 GABA was detected by the Pumei Bio γ-aminobutyric acid (GABA) detection kit.

[0094] The results are as follows Figure 3 As shown, the extracellular vesicle particles derived from passion flower (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) can significantly promote the secretion of GABA in neuronal cells. The YHSM-YL0012 of the present invention can also significantly promote the secretion of GABA in neuronal cells, and the effect is better than that of YHSM-YL0011 and passion flower, chamomile, and licorice extracellular vesicles used alone.

[0095] Example 7: Effect of the extracellular vesicle composition on the RNA level of GABA-related genes in neuronal cells

[0096] Neuronal cells were seeded into 12-well plates at a density of 200,000 cells / 1000 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded. 1000 μL of negative control reagent (NC: DMEM medium), experimental group reagents (passionflower, chamomile, licorice extracellular vesicles 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 hours. The supernatant was discarded, the cells were collected, the RNA of the cells was extracted, and the expression of GABA-related genes DDC, GAD1, GABRA1, GABRA4, and GABRA6 was detected.

[0097] The results are as follows Figure 4 As shown, the 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 YHSM-YL0011 and passion fruit, chamomile, and licorice extracellular vesicles 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 YHSM-YL0011 and passion fruit, chamomile, and licorice extracellular vesicles 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 passionflower, chamomile and licorice used alone.

[0098] Example 8: Effect of the extracellular vesicle composition on the Cl- level of neuronal cells

[0099] 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 reagent (PC: 5 mg / mL GABA, diluted with DMEM medium), and experimental group reagent (passionflower, chamomile, and licorice extracellular vesicles 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 effects of different samples on the Cl- level of neuronal cells were determined using the MAQE fluorescent probe.

[0100] The results show Figure 5 As shown, the extracellular vesicles derived from passion flower (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) can significantly increase the Cl- level of neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly increase the Cl- level of neuronal cells, and the effect is better than YHSM-YL0011 and passion flower, chamomile, and licorice extracellular vesicles used alone.

[0101] Example 9: Effect of the extracellular vesicle composition on the level of brain-derived neurotrophic factor (BDNF) in neuronal cells

[0102] Neuronal cells were inoculated 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 reagent (PC: 10 μM GABA, diluted with DMEM medium), and experimental group reagent (passionflower, chamomile, and licorice extracellular vesicles 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 secretion of BDNF was detected by the Lianke Biotechnology Brain-derived Neurotrophic Factor (BDNF) Elisa detection kit.

[0103] The results show Figure 6 As shown, the extracellular vesicles derived from passion fruit (PA-Exo) and chamomile (MC-Exo) can significantly promote the secretion level of BNDF in neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly promote the secretion level of BNDF in neuronal cells, and the effect is better than YHSM-YL0011 and passion fruit, chamomile, and licorice extracellular vesicles used alone.

[0104] Example 10: Effect of the extracellular vesicle composition on neuronal cell length

[0105] 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 reagent (PC: 10 μM RA, diluted with DMEM medium), and experimental group reagent (passionflower, chamomile, and licorice extracellular vesicles 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 48 h. Photos were taken under a microscope (such as Figure 7 The synaptic length was calculated using Image J.

[0106] The results show Figure 8 As shown, the extracellular vesicles derived from passion flower (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) can significantly promote the growth of neuronal synapse length. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly promote the growth of neuronal synapse length, and the effect is better than YHSM-YL0011 and passion flower, chamomile, and licorice extracellular vesicles used alone.

[0107] Example 11: Effects of the extracellular vesicle composition on the levels of microglial inflammatory factors

[0108] Neuronal cells were inoculated 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: MEM complete medium), positive control reagent (PC: 100 μg / ml dexamethasone, diluted with MEM complete medium), and experimental group reagents (passionflower, 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 supernatant was taken and the secretion of the pro-inflammatory factor IL-6 was detected by the Lianke Biotechnology Inflammatory Factor Interleukin-6 (IL-6) Elisa Detection Kit.

[0109] The results show Fig. 9As shown, the extracellular vesicles derived from passion flower (PA-Exo), chamomile (MC-Exo), and licorice (GU-Exo) can significantly inhibit the IL-6 level of neuronal cells. The extracellular vesicle composition YHSM-YL0012 of the present invention can also significantly inhibit the IL-6 level of neuronal cells, and the effect is better than YHSM-YL0011 and passion flower, chamomile, and licorice extracellular vesicles used alone.

[0110] Example 12: Sleep gel human treatment insomnia test

[0111] 210 participants aged 30-50 years with insomnia who had not used any insomnia treatment drugs recently were enrolled and randomly divided into 7 groups. The experimental group applied the sleep gel prepared in Example 3 (with an extracellular vesicle composition content of 30%) behind the ear 30 minutes before going to bed every night, and the control group applied a blank sleep gel (i.e., a sleep gel without an extracellular vesicle composition) once a night. They filled in a sleep diary every day and filled in a PSQI questionnaire before use and after one week of continuous use. The effective rate was calculated as follows: ((number of people with significant improvement × 100% + number of people with slight improvement × 50%) / 30) × 100%. The test results of the sleep gel for treating insomnia in humans are shown in Table 5.

[0112] Table 5: Results of human insomnia treatment test of sleep gel

[0113] Example 13: Sleep nasal spray human body treatment insomnia test 60 participants aged 30-50 years with insomnia who had not used any insomnia treatment drugs recently were enrolled and randomly divided into two groups. The experimental group sprayed the sleep nasal spray prepared in Example 3 (with an extracellular vesicle composition YHSM-YL0012 content of 90%) in 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 the extracellular vesicle composition) every night. They filled in a sleep diary every day and filled in a PSQI questionnaire before use and after one week of continuous use. The effective rate was calculated as: ((number of people with significant improvement × 100% + number of people with slight improvement × 50%) / 30) × 100%. The test results of the sleep nasal spray for the treatment of insomnia in humans are shown in Table 6.

[0114] Table 6: Results of human test on sleep nasal spray for treating insomnia

[0115] Example 14: Sleep spray human body treatment insomnia test 60 participants aged 30-50 years with insomnia who had not used any insomnia treatment drugs recently were enrolled and randomly divided into two groups. The experimental group sprayed the sleep spray prepared in Example 3 (with an extracellular vesicle composition YHSM-YL0012 content of 5%) at the bedside 30 minutes before going to bed every night, and the control group sprayed a blank sleep spray (i.e., a spray without an extracellular vesicle composition) every night. They filled in a sleep diary every day and filled in a PSQI questionnaire before use and after one week of continuous use. The effective rate was calculated as follows: ((number of people with significant improvement × 100% + number of people with slight improvement × 50%) / 30) × 100%. The test results of the sleep spray for treating insomnia in humans are shown in Table 7.

[0116] Table 7: Results of human test on sleep spray for treating insomnia

[0117] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. An extracellular vesicle composition for aiding sleep, characterized in that: The composition comprises: two or more of passionflower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles.

2. The composition according to claim 1, characterized in that The composition comprises passion flower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles.

3. The composition according to claim 2, characterized in that The mass ratio of the passion fruit extracellular vesicles, the chamomile extracellular vesicles and the liquorice extracellular vesicles is (1-20): (1-5): (1-5).

4. The composition according to claim 3, characterized in that The mass ratio of the passion fruit extracellular vesicles, the chamomile extracellular vesicles and the liquorice extracellular vesicles is (1-10): (1-2): (1-2).

5. The composition according to claim 4, characterized in that The mass ratio of the passionflower extracellular vesicles, the chamomile extracellular vesicles and the liquorice extracellular vesicles is 5:1:

1.

6. A method for preparing the composition according to any one of claims 1 to 5, characterized in that: The preparation method comprises: extracting passionflower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles respectively, and then compounding them according to a proportion.

7. The preparation method according to claim 6, characterized in that: The extraction of passionflower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles comprises: taking passionflower, chamomile and liquorice respectively, breaking the walls after adding solvent, and collecting the passionflower extracellular vesicles, chamomile extracellular vesicles and liquorice extracellular vesicles by centrifugation.

8. The preparation method according to claim 7, characterized in that: The solvent is PBS buffer; And / or, the specific steps of the centrifugation include: (1) Centrifuge at 400-800 g for 5-20 min and collect the supernatant; (2) Centrifuge at 1000-3000 g for 10-30 min and collect the supernatant; (3) Centrifuge at 3000-5000g for 20-40min and collect the supernatant; (4) Centrifuge at 8000-12000 g for 40-70 min and collect the supernatant; (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

9. Use of the composition according to any one of claims 1 to 5 in preparing a sleep-aiding product.

10. The use according to claim 9, characterized in that: The sleep-aiding products include emulsions, microemulsions, gels, ointments, creams, nasal sprays, inhalants, and sprays.

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