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

Abstract

The present invention relates to an exosome composition for improving sleep quality, comprising lavender exosomes, chamomile exosomes, and saffron exosomes. The exosome composition can increase the expression of GABA-related proteins in neurons, promote brain-derived neurotrophic factor (BDNF) expression in neurons, promote synaptic growth in neurons, increase CL levels in neurons, and inhibit the levels of inflammatory factors in microglia. The exosome composition can improve sleep quality caused by various physical discomforts, achieving a more pronounced effect than extracts of the same herbal remedies.

Description

Technical Field

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

[0002] Traditional Chinese herbal medicines such as lavender, chamomile, and saffron have been widely studied and used for their natural sedative and sleep-inducing properties. Several patents (e.g., CN202211198489.9, CN104800774B, and CN119184231A) report on the use of extracts or individual components of these herbs to improve sleep. However, these existing technologies primarily rely on traditional herbal extracts, whose effectiveness is limited by factors such as extraction methods, component stability, and bioavailability.

[0003] While existing Chinese herbal extracts have some sleep-inducing effects, their mechanisms of action are mostly based on traditional pharmacologically active ingredients, such as volatile oils and flavonoids. The metabolism and distribution of these ingredients in the body are often suboptimal, resulting in unstable effects or requiring higher doses to achieve the desired effect. Furthermore, existing research lacks the research and application of Chinese herbal exosomes in sleep-related fields. Exosomes, as important mediators of intercellular communication, can carry bioactive molecules such as proteins, lipids, and nucleic acids, and have higher biocompatibility and targeting, thus showing 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

[0005] In view of the above problems existing in the prior art, the purpose of the present invention is to provide a composition of Chinese herbal exosomes to provide a more effective and stable solution for improving sleep quality.

[0006] Solutions for solving problems

[0007] The present invention provides an exosome composition for improving sleep quality, comprising: lavender exosomes, chamomile exosomes and saffron exosomes.

[0008] Preferably, the composition consists of lavender exosomes, chamomile exosomes and saffron exosomes.

[0009] Preferably, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is (1-5):(1-5):(1-5).

[0010] Preferably, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is (1-3):(1-3):(1-3).

[0011] Preferably, the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is 1:1:1.

[0012] The present invention also provides a method for preparing the composition, which comprises extracting lavender exosomes, chamomile exosomes, and saffron exosomes respectively, and then compounding them according to proportion.

[0013] Preferably, the extraction of the lavender exosomes, chamomile exosomes, and saffron exosomes comprises: taking lavender, chamomile, and saffron respectively, adding a solvent to break the walls, and centrifuging to collect the lavender exosomes, chamomile exosomes, and saffron exosomes.

[0014] Preferably, the solvent is PBS buffer;

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

[0016] (1) Centrifuge at 400-800g for 5-20 minutes and collect the supernatant;

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

[0018] (3) Centrifuge at 3000-5000g for 20-40 minutes and collect the supernatant;

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

[0020] (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

[0021] The present invention also provides an application of the composition in preparing a sleep-aiding product.

[0022] Preferably, the sleep aid product includes an emulsion, a microemulsion, a gel, an ointment, a cream, a nasal spray, an inhaler, or a spray.

[0023] Effects of the Invention

[0024] The exosome composition of the present invention can increase the expression of GABA-related proteins in neurons, promote brain-derived neurotrophic factor (BDNF) expression in neurons, promote synaptic growth in neurons, increase CL- levels in neurons, and inhibit the levels of inflammatory factors in microglia. The exosome composition of the present invention can improve sleep quality caused by various physical discomforts, with a more pronounced effect than using extracts of the same herbal remedy. The exosome composition of the present invention, when used in combination, exhibits a synergistic effect, more pronounced than using any single exosome in the composition alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Transmission electron microscopy (TEM) images of extracellular vesicles from lavender, chamomile, and saffron.

[0026] Figure 2 This demonstrates that the exosome composition of the present invention can rapidly penetrate into the skin of 8-week-old Bama miniature pigs within 2 hours. The yellow arrows indicate the exosomes of MLC-YH0021 labeled with PKH26, and the green arrows indicate the cell nuclei. DETAILED DESCRIPTION

[0027] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate 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 present invention provides an exosome composition for improving sleep quality, comprising: lavender exosomes, chamomile exosomes and saffron exosomes.

[0029] In certain embodiments, the composition consists of lavender exosomes, chamomile exosomes, and saffron exosomes.

[0030] In certain embodiments, the mass ratio of the lavender exosomes, chamomile exosomes, and saffron exosomes is (1-5):(1-5):(1-5).

[0031] In certain 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: 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.

[0032] In certain embodiments, the mass ratio of the lavender exosomes, chamomile exosomes, and saffron exosomes is (1-3):(1-3):(1-3).

[0033] In certain embodiments, the mass ratio of the lavender exosomes, chamomile exosomes, and saffron exosomes is 1:1:1.

[0034] The present invention also provides a method for preparing the composition, which comprises extracting lavender exosomes, chamomile exosomes, and saffron exosomes respectively, and then compounding them according to proportion.

[0035] In certain 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.

[0036] In certain embodiments, the extraction of lavender exosomes, chamomile exosomes, and saffron exosomes comprises: taking lavender, chamomile, and saffron, respectively, adding a solvent to break the walls of the flowers, and centrifuging to collect the lavender exosomes, chamomile exosomes, and saffron exosomes.

[0037] In certain embodiments, the lavender, chamomile, and saffron are soaked in PBS buffer before cell wall breaking.

[0038] In certain embodiments, the solvent is PBS buffer.

[0039] In certain embodiments, the cell wall is broken for 8 to 10 minutes until there are no obvious block fragments.

[0040] In certain embodiments, the centrifugation is density gradient centrifugation and / or ultracentrifugation.

[0041] In certain embodiments, the centrifugation is density gradient centrifugation and ultracentrifugation.

[0042] In certain embodiments, the centrifugation is density gradient centrifugation.

[0043] In certain embodiments, the centrifugation is ultracentrifugation.

[0044] In certain embodiments, the centrifugation uses a 4°C low-speed centrifuge.

[0045] In certain embodiments, the specific step of centrifugation includes.

[0046] (1) Centrifuge at 400-800g for 5-20 minutes and collect the supernatant;

[0047] (2) Centrifuge at 1000-3000 g for 10-30 min and collect the supernatant;

[0048] (3) Centrifuge at 3000-5000g for 20-40 minutes and collect the supernatant;

[0049] (4) Centrifuge at 8000-12000g for 40-70min and collect the supernatant;

[0050] (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

[0051] In certain embodiments, the step (1) is centrifugation at 500 g for 10 min and taking the supernatant.

[0052] In certain embodiments, the step (2) is centrifugation at 2000 g for 20 min and taking the supernatant.

[0053] In certain embodiments, the step (3) is centrifugation at 4000 g for 30 min, and the supernatant is collected.

[0054] In certain embodiments, the step (4) is centrifugation at 10,000 g for 60 min, and the supernatant is collected.

[0055] In certain embodiments, the step (5) is centrifugation at 110,000 g for 70 min to obtain a precipitate.

[0056] In certain embodiments, the precipitate of step (5) is resuspended using the solvent.

[0057] In certain embodiments, the precipitate of step (5) is resuspended using PBS buffer.

[0058] The present invention also provides an application of the composition in preparing a sleep-aiding product.

[0059] In certain embodiments, the sleep aid product comprises an emulsion, a microemulsion, a gel, an ointment, a cream, a nasal spray, an inhaler, or a spray.

[0060] In certain embodiments, the sleep aid product is a sleep gel.

[0061] In certain embodiments, the exosome composition is added to the sleeping gel in a ratio of 0.1% to 30%.

[0062] In certain embodiments, the exosome composition is added to the sleeping gel at a ratio of 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%.

[0063] In certain embodiments, the concentration of the exosome composition in the sleeping gel is 1.0E+8 particles / ml-3.0E+10 particles / ml.

[0064] Example 1: Extraction of exosomes

[0065] Weigh 100g each of lavender, chamomile, and saffron and wash each three times with ultrapure water to remove impurities. Place in a clean, UV-irradiated beaker and add 500ml of sterile PBS to each beaker. Soak overnight at 4°C. Discard the soaking water, rinse three times with sterile water, and add 500ml of sterile PBS. Transfer to a wall-breaking machine, secure the lid, and break for 8-10 minutes, ensuring no visible fragments remain. The juice was transferred to a 50 ml sterile centrifuge tube, centrifuged at 500 × g for 10 min at 4 °C, the precipitate was discarded, and the supernatant was taken; the juice was centrifuged at 2000 × g for 20 min at 4 °C, the precipitate was discarded, and the supernatant was taken; the juice was centrifuged at 4000 × g for 30 min at 4 °C, the precipitate was discarded, and the supernatant was taken; the juice was centrifuged at 10000 × g for 1 h at 4 °C, the precipitate was discarded, and the supernatant was taken; the juice was centrifuged at 110000 × g for 70 min at 4 °C ultracentrifuge, the supernatant was discarded, and the precipitate was resuspended 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).

[0066] Example 2: Configuration of exosome composition

[0067] The lavender, chamomile, and saffron exosomes prepared in Example 1 were compounded in varying weight ratios to produce various Chinese herbal exosome compositions with sleep-inducing properties (as shown in Table 1). The volume of one aliquot of exosomes was 10 ml, and the exosome concentration was 1.0E+11 particles / ml.

[0068] Table 1: Exosome composition ratio

[0069]

[0070] Example 3: Preparation of Sleep Gel

[0071] 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.

[0072] Table 2: Sleeping gel formula

[0073]

[0074] Example 4: Transmission electron microscopy (TEM) analysis of exosome particles

[0075] The shape of each exosome prepared in Example 1 was analyzed by transmission electron microscopy (TEM).

[0076] According to the sample conditions, the exosomes prepared in Example 1 were adjusted to an appropriate 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 dry the exosome 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 absorbed, and washed repeatedly. Use filter paper to dry the exosome sample on the copper mesh. Observe, take pictures, and save the pictures. The results are as follows Figure 1 As shown in the figure, saucer-shaped vesicles can be observed in chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) exosomes.

[0077] Example 5: Skin penetration of exosome compositions

[0078] Cut the skin of the back or abdomen of an 8-week-old Bama miniature pig into 1 cm x 1 cm sections for later use. Take 1 mg of the exosome composition MLC-YH0021 prepared in Example 2 and adjust the sample volume to 1 mL using Diluent C from the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit MINI26-1KT). Add 6 μL of the PKH26 dye from the kit to a test tube containing 1 mL of Diluent C. Gently pipette 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. Adjust the volume to 30 mL using serum-free medium. Centrifuge at 110,000 g for 2 hours at 2-8°C. Gently pipette and resuspend the exosome pellet in 1 mL of serum-free medium for later use.

[0079] A 1cm×1cm piece of 8-week-old Bama miniature pig skin was placed in a 32°C environment. 50 μL of 1mg / ml PKH26-labeled exosome composition MLC-YH0021 was added dropwise to the 1cm×1cm piece of 8-week-old Bama miniature pig skin, evenly covering the entire epidermis. The skin was incubated for 2 hours. After incubation, frozen sections were taken with a thickness of 10 μm. After DAPI staining of cell nuclei, the cells were observed and photographed using a fluorescence microscope. The results are shown in Figure 2. 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.

[0080] Example 6: Effect of exosome composition on the secretion level of γ-aminobutyric acid (GABA) in neuronal cells (SH-SY5Y)

[0081] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 100 μL of the negative control reagent (NC:DMEM medium) and the experimental group reagents (chamomile, lavender, and saffron exosomes prepared in Example 1, and MLC-YH0021 prepared in Example 2 (diluted in DMEM medium to a protein concentration of 20 μg / mL)) were added, respectively, and cultured for 24 hours. The supernatant was collected and GABA secretion was measured using the PUMEC Biotechnology γ-aminobutyric acid (GABA) detection kit. The results are shown in Table 3.

[0082] Table 3: Effects of exosome composition on the secretion level of γ-aminobutyric acid (GABA) in neuronal cells (SH-SY5Y)

[0083]

[0084] The results showed that exosomes from chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) significantly promoted GABA secretion in neurons. The exosome composition MLC-YH0021 of the present invention also significantly promoted GABA secretion in neurons, with superior effects compared to the individual exosomes from chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo).

[0085] Example 7: Effect of exosome composition on the expression level of GABA-related genes in neuronal cells

[0086] 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) and experimental reagents (chamomile, lavender, and saffron exosomes prepared in Example 1, and MLC-YH0021 prepared in Example 2 (diluted in DMEM medium to a protein concentration of 20 μg / mL)) were added, and the cells were cultured for 24 hours. The supernatant was discarded, and the cells were harvested. RNA was extracted and the expression of GABA-related genes DDC (dopa decarboxylase), GAD1 (glutamate 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 measured. The results are shown in Table 4.

[0087] Table 4: GABA-related gene expression results in in vitro neuronal cells (SH-SY5Y)

[0088]

[0089] The results showed that chamomile (MC-Exo) and lavender (LAF-Exo) exosomes significantly promoted the expression of DDC in neuronal cells, while saffron exosomes (CS-Exo) did not show a significant promoting effect on DDC expression. The exosome composition MLC-YH0021 of the present invention significantly promoted the expression of DDC in neuronal cells, and its effect was superior to that of chamomile, lavender, and saffron exosomes used alone. Chamomile (MC-Exo) and saffron (CS-Exo) exosomes significantly promoted the expression of GAD1 in neuronal cells. The exosome composition MLC-YH0021 of the present invention significantly promoted the expression of GAD1 in neuronal cells, and its effect was superior to that of chamomile, lavender, and saffron exosomes used alone. Chamomile (MC-Exo) and saffron (CS-Exo) exosomes significantly promoted the expression of GABRA1 in neuronal cells. The exosome composition MLC-YH0021 of the present invention can significantly increase the expression level of GABRA1 in neuronal cells, with a greater effect than using chamomile, lavender, or saffron exosomes alone. Chamomile (MC-Exo) exosomes can significantly increase the expression level of GABRA4 in neuronal cells. The exosome combination MLC-YH0021 of the present invention can significantly increase the expression level of GABRA4 in neuronal cells, with a greater effect than using chamomile, lavender, or saffron exosomes alone. Chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) exosomes can significantly increase the expression level of GABRA6 in neuronal cells. The exosome combination MLC-YH0021 of the present invention can significantly increase the expression level of GABRA6 in neuronal cells, with a greater effect than using chamomile, lavender, or saffron exosomes alone.

[0090] Example 8: Effect of exosome composition on Cl- level of neuronal cells

[0091] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 hours. The cell culture supernatant was aspirated, and 50 μL of basal medium containing 5 mM MAQE was added to each well and incubated for 30 minutes. The cell supernatant was discarded, and 100 μL of the negative control reagent (NC:DMEM basal medium) and the 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 in DMEM basal medium)) were added, respectively, and incubated for 15 minutes. After the incubation period, the plates were placed on the fluorescence microplate reader, set to an incident light wavelength of 350 nm and an excitation light wavelength of 460 nm, and the readings were taken. The effects of different samples on the Cl- levels of neuronal cells were measured, and the results are shown in Table 5.

[0092] Table 5: Results of Cl- level determination in neuronal cells (SH-SY5Y) in vitro

[0093]

[0094] The results showed that chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) exosome particles significantly increased Cl- levels in neuronal cells. The exosome composition MLC-YH0021 of the present invention also significantly increased Cl- levels in neuronal cells, with an effect superior to that of chamomile, lavender, or saffron exosomes alone.

[0095] Example 9: Effect of exosome composition on brain-derived neurotrophic factor (BDNF) levels in neuronal cells

[0096] Neuronal cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 100 μL of each of the negative control reagent (NC: complete DMEM) and the experimental reagents (chamomile, lavender, and saffron exosomes prepared in Example 1, and MLC-YH0021 prepared in Example 2 (diluted in complete DMEM to a protein concentration of 20 μg / mL)) were added and cultured for 24 hours. The supernatant was collected and assayed for BDNF secretion using a Lianke Biotechnology Brain-Derived Neurotrophic Factor (BDNF) ELISA kit. The results are shown in Table 6.

[0097] Table 6: Results of in vitro BDNF level determination in neuronal cells (SH-SY5Y)

[0098]

[0099] The results showed that chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) exosome particles significantly promoted BNDF secretion in neuronal cells. The exosome composition MLC-YH0021 of the present invention also significantly promoted BNDF secretion in neuronal cells, with an effect superior to that of chamomile, lavender, or saffron exosomes alone.

[0100] Example 10: Effect of exosome composition on neuronal cell length

[0101] Neuronal cells were seeded into 24-well plates at a density of 100,000 cells / 100 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 500 μL of the negative control reagent (NC: 1% serum DMEM medium) and the 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 in 1% serum DMEM medium)) were added, and cultured for 48 hours. Microscopic images were taken, and synapse lengths were calculated using Image J. The results are shown in Table 7.

[0102] Table 7: Results of in vitro neuronal cell (SH-SY5Y) synapse length determination

[0103]

[0104] The results showed that exosomes from chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) significantly promoted the growth of neuronal synapses. The exosome composition MLC-YH0021 of the present invention also significantly promoted the growth of neuronal synapses, with an effect superior to that of chamomile, lavender, or saffron exosomes alone.

[0105] Example 11: Effect of exosome composition on the level of HMC3 inflammatory factors in microglia

[0106] HMC3 microglial cells were seeded into 96-well plates at a density of 50,000 cells / 100 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 100 μL of each blank control reagent (BC:MEM complete medium), negative control reagent (NC:MEM complete medium), and experimental group reagents (chamomile, lavender, and saffron exosomes prepared in Example 1, and MLC-YH0021 prepared in Example 2 (diluted with MEM complete medium to a protein concentration of 20 μg / mL)) were added. The cells were cultured for 20 hours. The cell supernatant was discarded, and 200 μL of MEM complete medium was added to each well of the blank control group. 200 μL of MEM complete medium containing 1 μg / mL LPS (LPS stock concentration is 1 mg / mL, used at a 1:1000 ratio, i.e., 1 μg / mL LPS) was added to each well of the negative control and experimental groups. The cells were cultured for 6 hours ± 2 hours. The supernatant was taken and the secretion of the pro-inflammatory factor IL-6 was detected using the Lianke Bio inflammatory factor interleukin-6 (IL-6) Elisa detection kit. The results are shown in Table 8.

[0107] Table 8: In vitro microglial cell (HMC3) inflammatory factor secretion level determination results

[0108]

[0109] The results showed that chamomile (MC-Exo), lavender (LAF-Exo), and saffron (CS-Exo) exosomes significantly inhibited microglial IL-6 levels. The exosome composition MLC-YH0021 of the present invention also significantly inhibited microglial IL-6 levels, and the effect was better than using chamomile, lavender, or saffron exosomes alone.

[0110] Example 11: Sleep Gel Human Treatment of Insomnia Test

[0111] One hundred and twenty participants aged 30-50 years with insomnia who had not recently used insomnia medication were randomly divided into four groups. The experimental group applied the sleep gel prepared in Example 3 behind their ears 30 minutes before bedtime each night. The control group applied a blank matrix sleep gel (i.e., a gel matrix without the exosome composition) once every morning and evening. The participants completed a daily sleep diary and completed the PSQI questionnaire before 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 results of the Chinese herbal exosome composition treatment of insomnia in humans are shown in Table 9.

[0112] Table 9: Test results of sleep gel in treating insomnia in humans

[0113]

[0114] The results showed that the MLC-YH0021 exosome sleep gel prepared by the sleep formula of Example 3 can significantly improve the sleep quality of insomnia patients, and the MLC-YH0022 and MLC-YH0023 exosome sleep gels also have different degrees of improvement on the sleep quality of insomnia patients.

[0115] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. An exosome composition for improving sleep quality, characterized in that: The composition consists of lavender exosomes, chamomile exosomes and saffron exosomes; the mass ratio of the lavender exosomes, chamomile exosomes and saffron exosomes is 1:1:

1.

2. A method for preparing the composition according to claim 1, characterized in that: The preparation method comprises: extracting lavender exosomes, chamomile exosomes, and saffron exosomes respectively, and then compounding them according to proportion.

3. The preparation method according to claim 2, 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 walls, and centrifuging to collect the lavender exosomes, chamomile exosomes, and saffron exosomes.

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

5. Use of the composition according to claim 1 in preparing a sleep-aiding product.

6. The use according to claim 5, characterized in that The sleep-aid products include lotions, gels, ointments, inhalants, and sprays.

Images