Exosome composition for improving sleep quality and preparation method and application thereof

Abstract

The invention relates to an exosome composition for improving sleep quality. The exosome composition comprises a lavender exosome, a chamomile exosome and a saffron crocus exosome. The exosome composition disclosed by the invention can be used for improving the expression level of GABA (gamma-aminobutyric acid) related proteins of neuronal cells, promoting the expression of brain-derived neurotrophic factors (BDNF) of the neuronal cells, promoting the synaptic growth of the neuronal cells, improving the CL <-> level of the neuronal cells and inhibiting the inflammatory factor level of microglial cells. The exosome composition disclosed by the invention can be used for improving sleep quality reduction caused by various body discomfort, and has a more obvious using effect than that of the same Chinese herbal medicine extract.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to an exosome composition for improving sleep quality, its preparation method and application. Background Art

[0002] Traditional Chinese herbs such as lavender, chamomile and saffron have been widely studied and applied due to their natural sedative and sleep-aiding properties. In the prior art, multiple patents (such as CN202211198489.9, CN104800774B, CN119184231A) have reported the application of extracts of these Chinese herbs or their single components in improving sleep. However, these prior arts mainly rely on traditional extracts of Chinese herbs, and their effects are limited by factors such as extraction methods, component stability and bioavailability.

[0003] Although the Chinese herb extracts in the prior art have certain sleep-aiding effects, their mechanisms of action are mostly based on traditional pharmacological active ingredients such as volatile oils and flavonoid compounds. The metabolism and distribution of these ingredients in the body are often not ideal, resulting in unstable effects or the need for higher doses to achieve the desired effects. Moreover, there is a lack of research and application of Chinese herb exosomes in the field related to sleep. As an important medium for intercellular communication, exosomes can carry bioactive molecules such as proteins, lipids, nucleic acids, etc., and have higher biocompatibility and targeting, so they show great potential in therapeutic applications and drug delivery. However, there are currently no reports on the research and application of lavender, chamomile and saffron exosomes in improving sleep quality. Summary of the Invention

[0004] Problems to be Solved by the Invention In view of the above problems existing in the prior art, the object of the present invention is to provide a composition of Chinese herb exosomes, so as to provide a more effective and stable solution for improving sleep quality.

[0005] Solutions for Solving the Problems The present invention provides an exosome composition for improving sleep quality, and the composition includes: lavender exosomes, chamomile exosomes and saffron exosomes.

[0006] Preferably, the composition consists of lavender exosomes, chamomile exosomes and saffron exosomes.

[0007] Preferably, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is (1~5):(1~5):(1~5).

[0008] Preferably, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is (1-3):(1-3):(1-3).

[0009] Preferably, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is 1:1:1.

[0010] The present invention also provides a preparation method of the composition, and the preparation method includes: separately extracting lavender exosomes, chamomile exosomes and saffron exosomes, and then compounding them in proportion.

[0011] Preferably, the extraction of the lavender exosomes, chamomile exosomes and saffron exosomes includes: separately taking lavender, chamomile and saffron, adding a solvent and then breaking the cell walls, and centrifuging to collect the lavender exosomes, chamomile exosomes and saffron exosomes.

[0012] 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 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.

[0013] The present invention also provides an application of the composition in the preparation of sleep aid products.

[0014] Preferably, the sleep aid products include lotion, microemulsion, gel, ointment, cream, nasal spray, inhalant, spray.

[0015] Effects of the Invention The exosome composition of the present invention can increase the expression level of GABA-related proteins in neuronal cells, promote the expression of brain-derived neurotrophic factor (BDNF) in neuronal cells, promote the growth of neuronal cell synapses, increase the CL- level in neuronal cells, and inhibit the level of inflammatory factors in microglial cells. The exosome composition of the present invention can improve the sleep quality decline caused by various physical discomforts, and has a more obvious effect than the use of the same kind of Chinese herbal medicine extract. The exosome composition of the present invention has a synergistic effect through compatibility, and has a more obvious effect than using a single exosome in the composition alone. Brief Description of the Drawings

[0016] Figure 1Transmission electron microscope (TEM) images of extracellular vesicles from lavender, chamomile and saffron.

[0017] Figure 2 It shows that the exosome composition of the present invention can rapidly penetrate into the skin of 8-week-old Bama mini-pigs within 2 h. Among them, the exosomes of MLC-YH0021 labeled with PKH26 are indicated by the yellow arrow; the cell nucleus is indicated by the green arrow. Detailed implementation manners

[0018] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following is a detailed description by listing specific examples. 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 field to which this application belongs.

[0019] The present invention provides an exosome composition for improving sleep quality, and the composition includes: lavender exosomes, chamomile exosomes and saffron exosomes.

[0020] In some embodiments, the composition consists of lavender exosomes, chamomile exosomes and saffron exosomes.

[0021] In some embodiments, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is (1-5):(1-5):(1-5).

[0022] In some embodiments, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is 1:1:1, or 1:1:2, or 1:1:3, or 1:1:4, or 1:1:5, or 1:2:1, or 1:2:2, or 1:2:3, or 1:2:4, or 1:2:5, or 1:3:1, or 1:3:2, or 1:3:3, or 1:3:4, or 1:3:5, or 1:4:1, or 1:4:2, or 1:4:3, or 1:4:4, or 1:4:5, or 1:5:1, or 1:5:2, or 1:5:3, or 1:5:4, or 1:5:5, or 2:1:1, or 2:1:2, or 2:1:3, or 2:1:4, or 2:1:5, or 2:2:1, or 2:2:2, or 2:2:3, or 2:2:4, or 2:2:5, or 2:3:1, or 2:3:2, or 2:3:3, or 2:3:4, or 2:3:5, or 2:4:1, or 2:4:2, or 2:4:3, or 2:4:4, or 2:4:5, or 2:5:1, or 2:5:2, or 2:5:3, or 2:5:4, or 2:5:5, or 3:1:1, or 3:1:2, or 3:1:3, or 3:1:4, or 3:1:5, or 3:2:1, or 3:2:2, or 3:2:3, or 3:2:4, or 3:2:5, or 3:3:1, or 3:3:2, or 3:3:3, or 3:3:4, or 3:3:5, or 3:4:1, or 3:4:2, or 3:4:3, or 3:4:4, or 3:4:5, or 3:5:1, or 3:5:2, or 3:5:3, or 3:5:4, or 3:5:5, or 4:1:1, or 4:1:2, or 4:1:3, or 4:1:4, or 4:1:5, or 4:2:1, or 4:2:2, or 4:2:3, or 4:2:4, or 4:2:5, or 4:3:1, or 4:3:2, or 4:3:3, or 4:3:4, or 4:3:5, or 4:4:1, or 4:4:2, or 4:4:3, or 4:4:4, or 4:4:5, or 4:5:1, or 4:5:2, or 4:5:3, or 4:5:4, or 4:5:5, or 5:1:1, or 5:1:2, or 5:1:3, or 5:1:4, or 5:1:5, or 5:2:1, or 5:2:2, or 5:2:3, or 5:2:4, or 5:2:5, or 5:3:1, or 5:3:2, or 5:3:3, or 5:3:4, or 5:3:5, or 5:4:1, or 5:4:2, or 5:4:3, or 5:4:4, or 5:4:5, or 5:5:1, or 5:5:2, or 5:5:3, or 5:5:4, or 5:5:5.

[0023] In some embodiments, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is (1-3):(1-3):(1-3).

[0024] In some embodiments, the mass ratio of the lavender exosomes, chamomile exosomes, and saffron exosomes is 1:1:1.

[0025] The present invention also provides a method for preparing the composition, which includes: separately extracting lavender exosomes, chamomile exosomes, and saffron exosomes, and then compounding them in proportion.

[0026] In some embodiments, the extraction methods of the lavender, chamomile, and saffron exosomes include, but are not limited to, density gradient centrifugation, ultracentrifugation, tangential flow filtration, fractional filtration, precipitation method, etc.

[0027] In some embodiments, the extraction of the lavender exosomes, chamomile exosomes, and saffron exosomes includes: separately taking lavender, chamomile, and saffron, adding a solvent to break the cell walls, and centrifuging to collect the lavender exosomes, chamomile exosomes, and saffron exosomes.

[0028] In some embodiments, the lavender, chamomile, and saffron are soaked in PBS buffer before cell wall breaking.

[0029] In some embodiments, the solvent is PBS buffer.

[0030] In some embodiments, the cell wall breaking is carried out for 8 - 10 minutes until there are no obvious large pieces of debris.

[0031] In some embodiments, the centrifugation is density gradient centrifugation and / or ultracentrifugation.

[0032] In some embodiments, the centrifugation is density gradient centrifugation and ultracentrifugation.

[0033] In some embodiments, the centrifugation is density gradient centrifugation.

[0034] In some embodiments, the centrifugation is ultracentrifugation.

[0035] In some embodiments, the centrifugation is carried out using a low-speed centrifuge at 4°C.

[0036] In some embodiments, the specific steps of the centrifugation include.

[0037] (1) Centrifuge at 400 - 800 g for 5 - 20 minutes and take the supernatant; (2) Centrifuge at 1000 - 3000 g for 10 - 30 minutes and take the supernatant; (3) Centrifuge at 3000 - 5000 g for 20 - 40 minutes and take the supernatant; (4) Centrifuge at 8000 - 12000 g for 40 - 70 minutes and take the supernatant; (5) Centrifuge at 100,000 - 120,000 g for 60 - 80 min and collect the precipitate.

[0038] In certain embodiments, step (1) is to centrifuge at 500 g for 10 min and collect the supernatant.

[0039] In certain embodiments, step (2) is to centrifuge at 2000 g for 20 min and collect the supernatant.

[0040] In certain embodiments, step (3) is to centrifuge at 4000 g for 30 min and collect the supernatant.

[0041] In certain embodiments, step (4) is to centrifuge at 10,000 g for 60 min and collect the supernatant.

[0042] In certain embodiments, step (5) is to centrifuge at 110,000 g for 70 min and collect the precipitate.

[0043] In certain embodiments, resuspend the precipitate obtained in step (5) with the solvent.

[0044] In certain embodiments, resuspend the precipitate obtained in step (5) with PBS buffer.

[0045] The present invention also provides an application of the composition in the preparation of sleep aid products.

[0046] In certain embodiments, the sleep aid products include emulsions, microemulsions, gels, ointments, creams, nasal sprays, inhalants, sprays.

[0047] In certain embodiments, the sleep aid product is a sleep gel.

[0048] In certain embodiments, the addition ratio of the exosome composition in the sleep gel is 0.1% - 30%.

[0049] In certain embodiments, the addition ratio of the exosome composition in the sleep gel is 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30%.

[0050] In certain embodiments, the concentration of the exosome composition in the sleep gel is 1.0E+8 particles / ml - 3.0E+10 particles / ml.

[0051] Example 1: Extraction of exosomes Weigh 100g of lavender, chamomile and saffron respectively, wash them three times with ultrapure water to remove impurities; place them in a clean, UV-irradiated beaker; add 500mL of sterile PBS respectively, soak them at 4℃ overnight; discard the soaking water, wash them three times with sterilized water, and add 500mL of sterile PBS. Transfer them to a wall-breaking machine, cover them tightly, and break them for 8-10 minutes to ensure that there are no obvious lumps. The squeezed juice was transferred to 50 ml sterile centrifuge tubes, centrifuged at 500 × g for 10 min at 4 °C low speed centrifuge, discarded the precipitate and took the supernatant; centrifuged at 2000 × g for 20 min at 4 °C low speed centrifuge, discarded the precipitate and took the supernatant; centrifuged at 4000 × g for 30 min at 4 °C low speed centrifuge, discarded the precipitate and took the supernatant; centrifuged at 10000 × g for 1 h at 4 °C high speed centrifuge, discarded the precipitate and took the supernatant; centrifuged at 110000 × g for 70 min at 4 °C ultracentrifuge, discarded the supernatant, and resuspended the precipitate with an appropriate amount of PBS to obtain the exosomes of lavender, chamomile, and saffron (labeled as MC-Exo, LAF-Exo, and CS-Exo, respectively).

[0052] Example 2: Configuration of exosome composition The lavender, chamomile and saffron exosomes prepared in Example 1 were compounded according to different mass ratios to obtain different types of Chinese herbal exosome compositions with sleep-aiding effects (as shown in Table 1). The volume of one portion of exosomes was 10 ml, and the exosome concentration was 1.0E+11 particles / ml.

[0053] Table 1: Exosome composition ratio

[0054] Example 3: Preparation of sleeping gel The Chinese herbal exosome composition prepared in Example 2 was added to the sleeping gel at a ratio of 30%. The specific formula is shown in Table 2.

[0055] Table 2: Sleeping gel formula

[0056] Example 4: Transmission electron microscopy (TEM) analysis of exosome particles The shape of each exosome prepared in Example 1 was analyzed by transmission electron microscopy (TEM).

[0057] According to the sample conditions, the exosomes prepared in Example 1 are adjusted to a suitable concentration or viscosity. Use a pipette to draw about 15 μL of the exosome sample onto the copper mesh and let it stand for 1 min. Use filter paper to blot the exosome sample on the copper mesh, and draw about 15 μL of 2% uranyl acetate staining solution and 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 exosome sample on the copper mesh. Observe and take pictures, and save the pictures. The results are as follows Figure 1 As shown, saucer-cup-shaped vesicles can be observed in chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) exosomes.

[0058] Example 5: Skin penetration of exosome compositions 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 the exosome composition MLC-YH0021 prepared in Example 2, and 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 exosome pellet in 1 ml of serum-free medium for use.

[0059] Place 1cm×1cm 8-week-old Bama miniature pig skin in a 32℃ environment, take 50μL 1mg / ml PKH26-labeled exosome composition MLC-YH0021 and drop it onto 1cm×1cm 8-week-old Bama miniature pig skin, evenly cover the entire epidermis, and incubate for 2 hours. 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 exosome composition of the present invention can quickly penetrate into the skin of 8-week-old Bama miniature pigs within 2 hours.

[0060] Example 6: Effect of exosome composition on the secretion level of γ-aminobutyric acid (GABA) in neuronal cells (SH-SY5Y) 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) and experimental group reagent (chamomile, lavender, saffron exosomes prepared in Example 1 and MLC-YH0021 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 supernatant was taken, and the secretion of GABA was detected by Pumei Biotechnology γ-aminobutyric acid (GABA) detection kit. The results are shown in Table 3.

[0061] Table 3: Effects of exosome composition on the secretion level of γ-aminobutyric acid (GABA) in neuronal cells (SH-SY5Y)

[0062] The results showed that exosomes of chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) could significantly promote the secretion of GABA in neuronal cells. The exosome composition MLC-YH0021 of the present invention could also significantly promote the secretion of GABA in neuronal cells, and the effect was better than that of each individual use of chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) exosomes.

[0063] Example 7: Effects of exosome composition on the expression level 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. The cells were cultured for 24 h. The cell supernatant was discarded. 1000 μL of negative control reagent (NC: DMEM medium) and experimental group reagent (chamomile, lavender, saffron exosomes prepared in Example 1 and MLC-YH0021 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 supernatant was discarded, the cells were collected, the RNA of the cells was extracted, and the expression of GABA-related genes DDC (dopa decarboxylase), GAD1 (glutamic acid decarboxylase 1), GABRA1 (γ-aminobutyric acid type A receptor subunit α1), GABRA4 (γ-aminobutyric acid type A receptor subunit α4), and GABRA6 (γ-aminobutyric acid type A receptor subunit α6) was detected. The results are shown in Table 4.

[0064] Table 4: Expression results of γ-aminobutyric acid (GABA)-related genes in vitro neuronal cells (SH-SY5Y)

[0065] The results showed that exosomes from chamomile (MC-Exo) and lavender (LAF-Exo) could significantly promote the expression level of DDC in neuronal cells, while saffron exosomes (CS-Exo) did not show a significant promoting effect on the expression level of DDC. The exosome composition MLC-YH0021 of the present invention could significantly promote the expression level of DDC in neuronal cells, and the effect was better than that of chamomile, lavender, and saffron exosomes used alone. Exosomes from chamomile (MC-Exo) and saffron (CS-Exo) could significantly promote the expression level of GAD1 in neuronal cells. The exosome composition MLC-YH0021 of the present invention could significantly promote the expression level of GAD1 in neuronal cells, and the effect was better than that of chamomile, lavender, and saffron exosomes used alone. Exosomes from chamomile (MC-Exo) and saffron (CS-Exo) could significantly promote the expression level of GABRA1 in neuronal cells. The exosome composition MLC-YH0021 of the present invention could significantly promote the expression level of GABRA1 in neuronal cells, and the effect was better than that of chamomile, lavender, and saffron exosomes used alone. Exosomes from chamomile (MC-Exo) could significantly promote the expression level of GABRA4 in neuronal cells. The exosome composition MLC-YH0021 of the present invention could significantly promote the expression level of GABRA4 in neuronal cells, and the effect was better than that of chamomile, lavender, and saffron exosomes used alone. Exosomes from chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) could significantly promote the expression level of GABRA6 in neuronal cells. The exosome composition MLC-YH0021 of the present invention could significantly promote the expression level of GABRA6 in neuronal cells, and the effect was better than that of chamomile, lavender, and saffron exosomes used alone.

[0066] Example 8: Effect of exosome composition on Cl- level in neuronal cells 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 culture supernatant was aspirated and discarded, and 50 μL of basal medium containing 5 mM MAQE was added to each well and incubated for 30 min. The cell supernatant was discarded, and 100 μL of negative control reagent (NC: DMEM basal medium) and experimental group reagent (chamomile, lavender, and saffron exosomes prepared in Example 1 and MLC-YH0021 prepared in Example 2 (diluted to a protein concentration of 20 μg / mL with DMEM basal medium)) were added respectively and cultured for 15 min. After the incubation, the plate was placed on the detection stage of a fluorescence microplate reader, the incident light wavelength was set at 350 nm, the excitation light wavelength was set at 460 nm, and the readings were taken. The effects of different samples on the Cl- level in neuronal cells were measured, and the results are shown in Table 5.

[0067] Table 5: Results of measuring Cl- levels in vitro in neuronal cells (SH-SY5Y)

[0068] The results showed that exosome particles of chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) could significantly increase the Cl- levels in neuronal cells. The exosome composition MLC-YH0021 of the present invention could also significantly increase the Cl- levels in neuronal cells, and the effect was better than that of using chamomile, lavender, and saffron exosomes alone.

[0069] Example 9: Effect of the exosome composition on 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. The cells were cultured for 24 h. The cell supernatants were discarded, and 100 μL of negative control reagent (NC: DMEM complete medium) and experimental group reagents (chamomile, lavender, and saffron exosomes prepared in Example 1 and MLC-YH0021 prepared in Example 2 (diluted to a protein concentration of 20 μg / mL with DMEM complete medium)) were added respectively, and cultured for 24 h. The supernatants were taken, and the secretion of BDNF was detected by the Brain-derived Neurotrophic Factor (BDNF) ELISA detection kit of Linkage Biotechnology. The results are shown in Table 6.

[0070] Table 6: Results of measuring BDNF levels in vitro in neuronal cells (SH-SY5Y)

[0071] The results showed that exosome particles of chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) could significantly promote the secretion level of BDNF in neuronal cells. The exosome composition MLC-YH0021 of the present invention could also significantly promote the secretion level of BDNF in neuronal cells, and the effect was better than that of using chamomile, lavender, and saffron exosomes alone.

[0072] Example 10: Effect of the exosome composition on the length of neurons in neuronal cells 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 supernatants were discarded, and 500 μL of negative control reagent (NC: 1% serum DMEM medium) and experimental group reagents (chamomile, lavender, and saffron exosomes prepared in Example 1 and MLC-YH0021 prepared in Example 2 (diluted to a protein concentration of 20 μg / mL with 1% serum DMEM medium)) were added respectively, and cultured for 48 h. Photographs were taken by microscope and the length of nerve synapses was statistically analyzed using Image J. The results are shown in Table 7.

[0073] Table 7: Results of determination of neuronal synapse length in vitro neuronal cells (SH-SY5Y)

[0074] The results showed that exosomes of chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) could significantly promote the growth of neuronal synapses in neuronal cells. The exosome composition MLC-YH0021 of the present invention could also significantly promote the growth of neuronal synapses in neuronal cells, and the effect was better than that of using chamomile, lavender, and saffron exosomes alone.

[0075] Example 11: Effect of exosome composition on the levels of inflammatory factors in microglial cells HMC3 Microglial cells HMC3 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 blank control reagent (BC: MEM complete medium), negative control reagent (NC: MEM complete medium), and experimental group reagent (chamomile, lavender, and saffron exosomes prepared in Example 1 and MLC-YH0021 prepared in Example 2 (diluted to a protein concentration of 20 μg / mL with MEM complete medium)) were added respectively, and cultured for 20 h. The cell supernatants were discarded. 200 μL of MEM complete medium was added to each well of the blank control group, and 200 μL of MEM complete medium containing 1 μg / mL LPS (the storage concentration of LPS was 1 mg / mL, and it was used at a ratio of 1:1000, i.e., 1 μg / mL LPS) was added to each well of the negative control group and the experimental group, and cultured for 6 h ± 2 h. The supernatants were taken, and the secretion of the pro-inflammatory factor IL-6 was detected by the inflammation factor interleukin-6 (IL-6) Elisa detection kit of Lianke Biotech. The results are shown in Table 8.

[0076] Table 8: Results of determination of the secretion levels of inflammatory factors in vitro microglial cells (HMC3)

[0077] The results showed that exosomes of chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) could significantly inhibit the level of IL-6 in microglial cells. The exosome composition MLC-YH0021 of the present invention could also significantly inhibit the level of IL-6 in microglial cells, and the effect was better than that of using chamomile, lavender, and saffron exosomes alone.

[0078] Example 11: Human treatment of insomnia test with sleep gel 120 experiencers aged 30 - 50 with insomnia problems and who had not used insomnia medications recently were enrolled and randomly divided into 4 groups. In the experimental group, the sleep gel prepared in Example 3 was applied behind the ears 30 minutes before going to bed every night. In the control group, a blank matrix sleep gel (i.e., a gel matrix without the exosome composition) was applied once in the morning and once in the evening every day. A sleep diary was filled out daily, and the PSQI questionnaire was filled out before use and after continuous use for one week. The calculation of the effective rate was: (((number of significantly improved people * 100%) + (number of slightly improved people * 50%)) / 30) * 100%. The test results of the human treatment of insomnia with the Chinese herbal medicine exosome composition are shown in Table 9.

[0079] Table 9: Test Results of the Human Treatment of Insomnia with the Sleep Gel

[0080] The results showed that the MLC - YH0021 exosome sleep gel prepared with the sleep formula of Example 3 could significantly improve the sleep quality of insomnia patients, and the MLC - YH0022 and MLC - YH0023 exosome sleep gels also had varying degrees of improvement effects on the sleep quality of insomnia patients.

[0081] 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 also be made based on the above embodiments. Similarly, any combination of the technical features of the above embodiments can also be made to form additional embodiments of the present invention that may not be clearly 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 exosome composition for improving sleep quality, characterized in that: The composition comprises: lavender exosomes, chamomile exosomes and saffron exosomes.

2. The composition according to claim 1, characterized in that The composition consists of lavender exosomes, chamomile exosomes and saffron exosomes.

3. The composition according to claim 2, characterized in that The mass ratio of the lavender exosomes, the chamomile exosomes and the saffron exosomes is (1-5): (1-5): (1-5).

4. The composition according to claim 3, characterized in that The mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is (1~3):(1~3):(1~3).

5. The composition according to claim 4, characterized in that The mass ratio of the lavender exosomes, the chamomile exosomes and the saffron exosomes is 1:1:

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 lavender exosomes, chamomile exosomes, and saffron exosomes respectively, and then compounding them according to a proportion.

7. The preparation method according to claim 6, characterized in that: The extraction of the lavender exosomes, chamomile exosomes and saffron exosomes comprises: taking lavender, chamomile and saffron respectively, adding a solvent to break the wall, and centrifuging to collect the lavender exosomes, chamomile exosomes and saffron exosomes.

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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