Composition with sleep aiding effect and application thereof

Abstract

The invention discloses a composition with a sleep aiding effect and application thereof, and particularly, the composition with the sleep aiding effect comprises extracellular vesicles derived from schisandra chinensis and extracellular vesicles derived from spina date seeds. The extracellular vesicle composition provided by the invention has the efficacy of helping sleep and soothing nerves, and is safe and free of toxic and side effects.

Description

Technical Field

[0001] The present invention relates to the technical field of extracellular vesicles, and particularly to a composition with sleep-aiding efficacy and its application. Background Art

[0002] Currently, the mainstream sleep-aiding means include drug intervention (such as benzodiazepines, melatonin), physical therapy (such as intelligent sleep-aiding devices), and traditional Chinese herbal compound preparations. However, long-term use of chemical drugs is prone to cause drug resistance, dependence, and withdrawal reactions, and melatonin has limited effects on chronic insomnia accompanied by anxiety or depression. Physical sleep-aiding devices (such as intelligent mattresses, sleep monitoring bracelets) can provide data feedback, but it is difficult to directly intervene in the sleep physiological mechanism. Although traditional Chinese herbal extracts (such as wild jujube seeds, Schisandra chinensis) have the effect of calming the mind, their active ingredients are easily affected by gastrointestinal metabolism, with low bioavailability, and traditional dosage forms (such as oral liquids, capsules) have problems such as slow onset and poor targeting.

[0003] Extracellular vesicles of traditional Chinese herbs are nano-sized vesicles extracted from traditional Chinese herbs, with a size of about 50 - 200 nm, rich in active substances such as lipids, proteins, nucleic acids, etc., and having both natural medicinal effects and high-efficiency delivery characteristics. Compared with traditional Chinese herbal extracts, it has the following core advantages: the phospholipid bilayer structure of extracellular vesicles of traditional Chinese herbs is highly similar to the human cell membrane, can cross the blood-brain barrier, and directly act on the central nervous system, improving the brain distribution efficiency of drugs; plant-derived extracellular vesicles avoid the immunogenic risk of animal-derived exosomes, and there is no need for chemical modification in the production process, and the natural ingredients have higher safety. Currently, there is less development of plant-derived extracellular vesicles, especially there is no report on using extracellular vesicles of Schisandra chinensis and wild jujube seeds as sleep-aiding products.

[0004] In view of this, there is an urgent need for a traditional Chinese herbal extracellular vesicle composition with good sleep-aiding efficacy, high safety, and few side effects. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a traditional Chinese herbal extracellular vesicle that can effectively improve sleep efficacy, has high safety, and has few side effects on the human body.

[0006] Solution for Solving the Problem: On the one hand, the present invention provides a composition with sleep-aiding efficacy, and the composition includes extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from wild jujube seeds.

[0007] Preferably, the composition is composed of extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from wild jujube seeds.

[0008] Preferably, the concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from wild jujube seeds is 1:0.1 - 10.

[0009] Preferably, the concentration ratio of the Schisandra chinensis-derived extracellular vesicles to the Ziziphus jujuba var. spinosa-derived extracellular vesicles is 1:0.5 - 5.

[0010] On the one hand, the present invention provides a method for preparing the composition according to any one of the above, comprising separately taking Schisandra chinensis or Ziziphus jujuba var. spinosa, soaking, breaking the cell wall, centrifuging, and taking the supernatant; filtering the obtained supernatant through a tangential flow system to obtain Schisandra chinensis-derived extracellular vesicles and Ziziphus jujuba var. spinosa-derived extracellular vesicles, and mixing the obtained extracellular vesicles to obtain the composition.

[0011] On the one hand, the present invention provides a method for preparing the composition according to any one of the above, comprising separately taking Schisandra chinensis or Ziziphus jujuba var. spinosa, soaking, breaking the cell wall, centrifuging, and taking the supernatant; performing ultracentrifugation on the obtained supernatant, taking the precipitate, resuspending it with a buffer, and mixing the obtained extracellular vesicles to obtain the composition.

[0012] On the one hand, the present invention provides a pharmaceutical composition with a sleep-aiding effect, which comprises the composition according to any one of the above or the composition prepared by the method as described above and a pharmaceutically acceptable carrier.

[0013] Preferably, the mass content of the composition in the composition with a sleep-aiding effect is 50 - 95%.

[0014] On the one hand, the present invention provides the application of the composition according to any one of the above, the composition prepared by the method as described above, or the pharmaceutical composition according to any one of the above in the preparation of a product with a sleep-aiding effect.

[0015] Preferably, the composition or the pharmaceutical composition is formulated as a nasal spray for administration.

[0016] Effects of the invention: Compared with the prior art, the present invention adopts a combined formula of Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicles, and there is a synergistic effect between the two, which enables it to increase the expression level of GABA-related proteins, promote the expression of brain-derived neurotrophic factor (BDNF) in cells, promote the growth of neuronal cell synapses, and inhibit the level of microglial cell inflammatory factors, thereby achieving a multi-faceted and highly efficient sleep-aiding effect, and the effect is significantly better than that of single-component extracellular vesicles. Description of the drawings

[0017] Figure 1 It is a transmission electron microscope (TEM) observation diagram of Schisandra chinensis extracellular vesicles (SCB-Exo) and Ziziphus jujuba var. spinosa extracellular vesicles (ZJV-Exo) of the present invention (the red arrow indicates extracellular vesicles); Figure 2 It is a test result diagram of the cytotoxicity of Schisandra chinensis extracellular vesicles (SCB-Exo) and Ziziphus jujuba var. spinosa extracellular vesicles (ZJV-Exo) of the present invention on neuronal cells; Figure 3 Schematic diagram of the expression of GABA-related genes GAD1, GABRA1, and GABRA4 by the Schisandra chinensis extracellular vesicles (SCB-Exo), Ziziphus jujuba var. spinosa extracellular vesicles (ZJV-Exo), and their combination (SZ-Mix2) of the present invention; Figure 4 Schematic diagram of the effect of the Schisandra chinensis extracellular vesicles (SCB-Exo), Ziziphus jujuba var. spinosa extracellular vesicles (ZJV-Exo), and their combination (SZ-Mix2) of the present invention on the expression level of brain-derived neurotrophic factor BDNF in neuronal cells; Figure 5 Effect of the Schisandra chinensis extracellular vesicles (SCB-Exo), Ziziphus jujuba var. spinosa extracellular vesicles (ZJV-Exo), and their combination (SZ-Mix2) of the present invention on the length of nerve synapses; Figure 6 Schematic diagram of the effect of the Schisandra chinensis extracellular vesicles (SCB-Exo), Ziziphus jujuba var. spinosa extracellular vesicles (ZJV-Exo), and their combination (SZ-Mix2) of the present invention on the expression level of inflammatory factor IL-6 in microglial cells; Figure 7 Schematic diagram of the effect of the Schisandra chinensis extracellular vesicles (SCB-Exo), Ziziphus jujuba var. spinosa extracellular vesicles (ZJV-Exo), and their combination (SZ-Mix2) of the present invention on the expression level of inflammatory factor TNF-α in microglial cells. Detailed implementation manners

[0018] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following will be described in detail by listing specific embodiments. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of 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 the present application belongs.

[0019] Currently, for drugs with sleep-aiding effects, whether they are chemical drugs or traditional Chinese medicines, there will be certain toxic and side effects, including discomfort symptoms such as feeling groggy the next morning after taking the medicine, decreased memory or attention, dizziness, dry mouth, constipation or diarrhea, etc. Even, long-term or incorrect use may lead to dependence or other health risks. The inventors of the present invention have found through a large number of experimental studies that the exosome-like vesicle composition derived from Schisandra chinensis and Ziziphus jujuba var. spinosa has a significant effect of helping sleep and calming the nerves, and has low immunogenicity and better biocompatibility. Especially by the way of nasal spray administration, it can bypass the blood-brain barrier and directly deliver the drug to the target area in the brain through the olfactory nerve and trigeminal nerve pathways, significantly improving the bioavailability, and then significantly improving the insomnia problem of the subjects. Based on this, the present invention is proposed.

[0020] On the one hand, the present invention provides a composition having a sleep-aiding effect, and the composition comprises extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from Ziziphus jujuba var. spinosa.

[0021] In one embodiment, the composition consists of extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from Ziziphus jujuba var. spinosa.

[0022] In one embodiment, the concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from Ziziphus jujuba var. spinosa is 1:0.1 to 10, such as 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, etc.

[0023] In one embodiment, the concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from Ziziphus jujuba var. spinosa is 1:0.5 to 5.

[0024] In one embodiment, the concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from Ziziphus jujuba var. spinosa is 1:0.67 to 4.

[0025] In one embodiment, the concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from Ziziphus jujuba var. spinosa is 1:0.67.

[0026] In one embodiment, the concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from Ziziphus jujuba var. spinosa is 1:4.

[0027] On the one hand, the present invention provides a method for preparing the composition according to any one of the above, respectively taking Schisandra chinensis or Ziziphus jujuba var. spinosa, soaking, breaking the wall, centrifuging, and taking the supernatant; filtering the obtained supernatant through a tangential flow system to obtain extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from Ziziphus jujuba var. spinosa, and mixing the prepared extracellular vesicles to obtain the composition.

[0028] In a preferred and specific embodiment, the centrifugation is carried out at a rotational speed of 2000 to 6000 g for 10 to 30 min, and the supernatant is collected.

[0029] On the one hand, the present invention provides a method for preparing the composition according to any one of the above, respectively taking Schisandra chinensis or Ziziphus jujuba var. spinosa, soaking, breaking the wall, centrifuging, and taking the supernatant; performing ultracentrifugation on the obtained supernatant, taking the precipitate and resuspending it with a buffer solution, and mixing the prepared extracellular vesicles to obtain the composition.

[0030] In a preferred and specific embodiment, the centrifugation steps are as follows: Centrifuge the liquid after cell wall breaking at a rotational speed of 400 - 600 g for 5 - 15 min, collect the supernatant, centrifuge the supernatant at a rotational speed of 1500 - 2500 g for 10 - 30 min, collect the supernatant, centrifuge the supernatant at a rotational speed of 3000 - 5000 g for 10 - 30 min, collect the supernatant, and centrifuge the supernatant at a rotational speed of 9000 - 11000 g for 40 - 70 min, then collect the supernatant.

[0031] In a specific and preferred embodiment, the ultra - centrifugation is carried out at a rotational speed of 100000 - 120000 g for 80 - 100 min.

[0032] In a preferred and specific embodiment, the soaking is to mix Schisandra chinensis or Ziziphus jujuba var. spinosa with PBS and soak for 6 - 10 hours.

[0033] On the one hand, the present invention provides a pharmaceutical composition with a sleep - aiding effect, which includes the composition described in any one of the above or the composition prepared by the method described above and a pharmaceutically acceptable carrier.

[0034] In a certain embodiment, the mass content of the composition in the composition with a sleep - aiding effect is 50 - 95%.

[0035] On the one hand, the present invention provides the application of the composition described in any one of the above, or the composition prepared by the method described above, or the pharmaceutical composition described in any one of the above in the preparation of a product with a sleep - aiding effect.

[0036] In a certain embodiment, the composition or the pharmaceutical composition is formulated as a nasal spray for administration.

[0037] The nose - to - brain delivery is a non - invasive drug delivery method, which can bypass the blood - brain barrier and directly deliver drugs to the target area in the brain through the olfactory nerve and trigeminal nerve pathways, significantly improving the bioavailability. Delivering the Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicles composition in the form of a nasal spray can not only shorten the onset time (more than 30 minutes for traditional oral administration, only 5 - 10 minutes for nasal spray), but also reduce the side effects caused by systemic exposure. Moreover, the rich capillary and lymphatic network in the nasal mucosa can promote the rapid absorption of extracellular vesicles, and their nano - scale particle size (50 - 200 nm) can further optimize the mucosal penetration efficiency, significantly enhancing the sleep - aiding effect.

[0038] In a certain embodiment, the application is the application of the composition in the preparation of a product for increasing the expression level of GABA - related proteins in neuronal cells.

[0039] In a preferred and specific embodiment, the neuron cell GABA-related proteins include GAD1, GABRA1, and GABRA4.

[0040] In one embodiment, the application is the application of the composition in the preparation of a product for promoting the expression of brain-derived neurotrophic factor (BDNF) in neuron cells.

[0041] In one embodiment, the application is the application of the composition in the preparation of a product for promoting the synapse growth of neuron cells.

[0042] In one embodiment, the application is the application of the composition in the preparation of a product for inhibiting the level of inflammatory factors in microglia.

[0043] In one embodiment, the inflammatory factors include IL-6 and TNF-α.

[0044] The present invention will be further described below through specific examples. Unless otherwise specified in this article, "%" represents mass / volume percentage. The materials and reagents in the following examples, unless otherwise specified, are commonly used materials or reagents in the art, and can all be obtained from commercial channels or synthesized by known methods. The experimental methods without specified conditions in the following implementation cases are usually carried out according to conventional experimental conditions or the conditions recommended by the manufacturers of relevant reagents (kits).

[0045] Example 1 Extraction of Schisandra chinensis and Ziziphus jujuba Mill. var. spinosa Cell-derived Extracellular Vesicle Particles by Differential Centrifugation Weigh 100 g of Schisandra chinensis and Ziziphus jujuba Mill. var. spinosa Chinese herbal medicines respectively, wash them 3 times with purified water to remove impurities, transfer them to a sterile receiving bottle, add 500 mL of PBS, and soak at 4°C for 8 h; after the soaking is completed, wash them 3 times with sterile water again, transfer them to a blender, add 800 mL of PBS, seal and break the wall for 10 min to ensure that there are no obvious lumpy fragments; transfer the juice to a 50 mL sterile centrifuge tube, centrifuge at 500×g for 10 min with a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant; centrifuge at 2000×g for 20 min with a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant; centrifuge at 4000×g for 30 min with a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant; centrifuge at 10000×g for 1 h with a high-speed centrifuge at 4°C, discard the precipitate, and take the supernatant; centrifuge at 110000×g for 70 min with an ultra-high-speed centrifuge at 4°C, discard the supernatant, and resuspend the precipitate with an appropriate amount of PBS to obtain Schisandra chinensis and Ziziphus jujuba Mill. var. spinosa cell-derived extracellular vesicle particles (labeled as SCB-Exo and ZJV-Exo respectively); the particle concentrations detected by nano-flow cytometry are 4.57E+12 particles / mL and 6.17E+11 particles / mL respectively.

[0046] Example 2 Extraction of Schisandra chinensis and Ziziphus jujuba Mill. var. spinosa Cell-derived Extracellular Vesicle Particles by Tangential Flow Weigh 100 g of Schisandra chinensis and Ziziphus jujuba Chinese herbal medicines separately, wash them 3 times with purified water to remove impurities, transfer them to a sterile receiving bottle, add 500 mL of PBS, and soak at 4°C for 8 h. After soaking, wash them 3 times with sterilized water again, transfer them to a blender, add 800 mL of PBS, seal and break the cells for 10 min to ensure no obvious lumpy fragments. Centrifuge at 4000×g for 15 min using a high-speed centrifuge at 4°C to remove large particles and cell debris, discard the precipitate, and take the supernatant. Use a tangential flow system for fractional filtration and sterile filtration to obtain Schisandra chinensis and Ziziphus jujuba extracellular vesicle particles (labeled as SCB-Exo and ZJV-Exo respectively). The particle concentrations measured by nano-flow cytometry are 1.08E+12 particles / mL and 2.19E+11 particles / mL respectively.

[0047] Example 3 Preparation of a compound Chinese herbal medicine extracellular vesicle composition The extracellular vesicle particles of Schisandra chinensis and Ziziphus jujuba from Example 1 or 2 were compounded according to different particle concentration ratios to obtain different Chinese herbal medicine extracellular vesicle compositions with sleep-promoting effects, as shown in Table 1. If used short-term, store them in a -80°C refrigerator. To facilitate long-term use and transportation, preservatives can be added.

[0048] Table 1

[0049] Example 4 Preparation of a sleep nasal spray preparation Add the Chinese herbal medicine extracellular vesicle composition from Example 3 to the sleep nasal spray preparation. The specific formula is shown in Table 2.

[0050] Table 2

[0051] Detection Example 1 Identification of Chinese herbal medicine extracellular vesicle particles Use a transmission electron microscope (TEM) to observe the morphological characteristics of the Chinese herbal medicine extracellular vesicle particles prepared in Example 1. According to the sample situation, dilute the extracellular vesicle particles from Example 1 to an appropriate concentration. Use a pipette to aspirate about 15 μL of the extracellular vesicle sample and let it stand on the copper mesh for 1 min. Blot the remaining sample from one side with filter paper, aspirate about 15 μL of 2% uranyl acetate staining solution and stain it at room temperature for 1 min. If obvious adsorbed substances are visible on the copper mesh, drop pure water on the surface, quickly aspirate it, and wash it several times repeatedly. Observe the morphological structure of the extracellular vesicles using a 120 kV transmission electron microscope (TEM), observe and take pictures, and save the data. The results show ( Figure 1 ) that cup-shaped structures can be observed in both Schisandra chinensis and Ziziphus jujuba extracellular vesicles.

[0052] Detection Example 2 Safety evaluation of Chinese herbal medicine extracellular vesicle particles on cells The exosomes of SCB-Exo and ZJV-Exo prepared in Example 1 were respectively diluted to 5E+8 particles / mL, 1E+9 particles / mL, 5E+9 particles / mL, 1E+10 particles / mL, 2E+10 particles / mL, 5E+10 particles / mL, and 1E+11 particles / mL to detect their effects on cell viability.

[0053] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. After culturing 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 reagents (SCB-Exo and ZJV-Exo of Example 1) were added respectively. After culturing for 24 h, the supernatant was discarded, and the cells were washed 3 times with PBS. 100 μL of diluted CCK8 was added to each well, and the absorbance at 450 nm was measured using a microplate reader after incubation for 1 h to evaluate the safety of the Chinese herbal medicine extracellular vesicle particles.

[0054] The results showed ( Figure 2 ): The extracellular vesicle particles derived from Schisandra chinensis (SCB-Exo) and Ziziphus jujuba Mill. var. spinosa (ZJV-Exo) were not toxic to neuronal cells at concentrations of 5E+8 - 5E+11 particles / mL, indicating their good safety.

[0055] Detection Example 3 The Chinese herbal medicine extracellular vesicle composition can increase the RNA levels of GABA-related genes in neuronal cells Neuronal cells were seeded into 12-well plates at a density of 200,000 cells / 1000 μL / well. After culturing for 24 h, the supernatant was discarded. 1000 μL of negative control reagent (NC: DMEM medium) and experimental group reagents [SCB-Exo and ZJV-Exo of Example 1 and SZ-Mix2 in Example 3 (diluted to a particle concentration of 5E+10 particles / mL with DMEM medium)] were added respectively and cultured for 24 h. The supernatant was discarded, the cells were collected, and the RNA of the cells was extracted to detect the expression of GABA-related genes GAD1, GABRA1, and GABRA4.

[0056] The results showed ( Figure 3): Extracellular vesicle particles derived from single Schisandra chinensis (SCB-Exo) can significantly promote the expression level of GAD1 in neuronal cells. The Chinese herbal medicine extracellular vesicle composition of the present invention can also significantly promote the expression level of GAD1 in neuronal cells, with a 2242% increase, and the effect is significantly better than that of single Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicle particles. Extracellular vesicle particles derived from single Ziziphus jujuba var. spinosa (ZJV-Exo) can significantly promote the expression level of GABRA1 in neuronal cells; the Chinese herbal medicine extracellular vesicle composition of the present invention can also significantly promote the expression level of GABRA1 in neuronal cells, with a 4471% increase, and the effect is significantly better than that of single Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicle particles. Extracellular vesicle particles derived from single Ziziphus jujuba var. spinosa (ZJV-Exo) can promote the expression level of GABRA4 in neuronal cells; the Chinese herbal medicine extracellular vesicle composition of the present invention can also significantly promote the expression level of GABRA4 in neuronal cells, with a 406.5% increase, and the effect is significantly better than that of single Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicle particles.

[0057] Detection Example 4: The Chinese herbal medicine extracellular vesicle composition can increase the level of brain-derived neurotrophic factor BDNF in neuronal cells Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. After culturing for 24 h, the cell supernatant was discarded, and 100 μL of blank control reagent (BC: DMEM medium), positive control group reagent (PC: 10 μM GABA, diluted with DMEM medium), and experimental group reagent [SCB-Exo and ZJV-Exo of Example 1 and SZ-Mix2 in Example 3 (diluted with DMEM medium to a particle concentration of 5E+10 particles / 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 United Bio.

[0058] The results showed ( Figure 4 ): Extracellular vesicle particles derived from Schisandra chinensis (SCB-Exo) and Ziziphus jujuba var. spinosa (ZJV-Exo) can significantly promote the secretion level of BDNF in neuronal cells. The Chinese herbal medicine extracellular vesicle composition (SZ-Mix2) of the present invention can also significantly promote the secretion level of BDNF in neuronal cells, with a 277.59% increase, and the effect is better than that of single Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicle particles.

[0059] Detection Example 5: The Chinese herbal medicine extracellular vesicle composition can increase the neuronal length of neuronal cells Neuronal cells were seeded into 24-well plates at a density of 100,000 cells / 100 μL / well. After culturing for 24 h, the supernatant was discarded, and 500 μL of blank control reagent (BC: DMEM medium), positive control group reagent (PC: 10 μM RA, diluted with DMEM medium), and experimental group reagent [SCB-Exo and ZJV-Exo of Example 1 and SZ-Mix2 of Example 3 (diluted with DMEM medium to a particle concentration of 5E+10 particles / mL)] were added respectively, and then cultured for 48 h. Photographs were taken by microscope and the length of nerve synapses was counted using Image J.

[0060] The results showed ( Figure 5 ): Extracellular vesicle particles derived from Schisandra chinensis (SCB-Exo) and Ziziphus jujuba var. spinosa (ZJV-Exo) could promote the growth of nerve synapse length in neuronal cells, but there was no significant difference. The Chinese herbal medicine extracellular vesicle composition (SZ-Exo) of the present invention could significantly promote the growth of nerve synapse length in neuronal cells, and the effect was better than that of single Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicle particles.

[0061] Detection Example 6 The Chinese herbal medicine extracellular vesicle composition can inhibit the levels of inflammatory factors in microglial cells Microglial cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. After culturing for 24 h, the cell supernatant was discarded, and 100 μL of blank control reagent (BC: MEM complete medium), negative control reagent (NC: MEM complete medium containing 1 μg / mL LPS), positive control group reagent (PC: 100 μg / ml dexamethasone, diluted with MEM complete medium), and experimental group reagent [SCB-Exo and ZJV-Exo of Example 1 and SZ-Mix2 of Example 3 (diluted with MEM complete medium to a particle concentration of 5E+10 particles / mL)] were added respectively, and then cultured for 24 h. The supernatant was taken, and the secretion of pro-inflammatory factors TNF-α and IL-6 was detected by ELISA kits for tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) of Bioteke Corporation.

[0062] The results showed ( Figure 6 and Figure 7 ): Extracellular vesicle particles derived from Schisandra chinensis (SCB-Exo) and Ziziphus jujuba var. spinosa (ZJV-Exo) could significantly inhibit the levels of TNF-α and IL-6 in microglial cells. The Chinese herbal medicine extracellular vesicle composition (SZ-Mix2) of the present invention could also significantly inhibit the levels of TNF-α and IL-6 in microglial cells, and the effect was better than that of single Schisandra chinensis and Ziziphus jujuba var. spinosa extracellular vesicle particles.

[0063] Detection Example 7 Human treatment of insomnia test with the Chinese herbal medicine extracellular vesicle composition 100 experiencers aged 30 - 50 with insomnia problems and who have not used insomnia medications recently were recruited and randomly divided into 5 groups. In the experimental group, the sleep nasal spray (formulation 1 in Example 4) was used 1 hour before bedtime every night, with one spray in each of the left and right nostrils; in the control group, the sleep nasal spray matrix (i.e., the nasal spray without the extracellular vesicle composition) was used 1 hour before bedtime every day, with one spray in each of the left and right nostrils; a sleep diary was filled out daily, and the PSQI questionnaire was filled out before use and after continuous use for one week. The formula for the effective rate is: effective rate = ((number of significantly improved people * 100% + number of slightly improved people * 50%) / 20) * 100%. The test results of the traditional Chinese medicine extracellular vesicle composition for the treatment of insomnia in humans are shown in Table 3.

[0064] Table 3

[0065] As can be seen from Table 3, after the traditional Chinese medicine extracellular vesicle composition of the present invention is formulated into a nasal spray, it can effectively improve the sleep quality of the subjects, and the effective rate reaches 62 - 85%. In particular, the sleep - promoting effect of nasal spray formulation 2 is more significant.

[0066] It should be understood that the above - mentioned 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 also be made on the basis of the above - mentioned embodiments. Similarly, any combination of the technical features of the above - mentioned embodiments can also be made to form additional embodiments of the present invention that may not be clearly described. Therefore, the above - mentioned embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. A composition with sleep-aiding efficacy, characterized in that, The composition comprises extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from Ziziphus jujuba var. spinosa.

2. The composition according to claim 1, characterized in that, The composition consists of extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from Ziziphus jujuba var. spinosa.

3. The composition according to claim 1 or 2, characterized in that, The concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from Ziziphus jujuba var. spinosa is 1:0.1 - 10.

4. The composition according to claim 3, characterized in that, The concentration ratio of the extracellular vesicles derived from Schisandra chinensis to the extracellular vesicles derived from Ziziphus jujuba var. spinosa is 1:0.5 - 5.

5. A preparation method of the composition according to any one of claims 1 to 4, characterized in that, Take Schisandra chinensis or Ziziphus jujuba var. spinosa respectively, soak, break the cell wall, centrifuge, and take the supernatant; filter the obtained supernatant through a tangential flow system to obtain extracellular vesicles derived from Schisandra chinensis and extracellular vesicles derived from Ziziphus jujuba var. spinosa, and mix the prepared extracellular vesicles to obtain the composition.

6. A preparation method of the composition according to any one of claims 1 to 4, characterized in that, Take Schisandra chinensis or Ziziphus jujuba var. spinosa respectively, soak, break the cell wall, centrifuge, and take the supernatant; perform ultracentrifugation on the obtained supernatant, take the precipitate and resuspend it with a buffer, and mix the prepared extracellular vesicles to obtain the composition.

7. A pharmaceutical composition with sleep-aiding efficacy, characterized in that, The pharmaceutical composition comprises the composition according to any one of claims 1 - 4 or the composition prepared by the method according to claim 5 or 6 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The mass content of the composition in the composition having the efficacy of promoting sleep is 50 - 95%.

9. Use of the composition according to any one of claims 1 to 4, or the composition prepared by the method according to claim 5 or 6, or the pharmaceutical composition according to any one of claims 7 to 8 in the preparation of a product with sleep-aiding efficacy.

10. The use according to claim 9, characterized in that, The composition or the pharmaceutical composition is formulated as a nasal spray for administration.

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