COMPOSITIONS CONTAINING EXOSOMES FROM ANIMAL PLACENTA, METHODS FOR PRODUCING THE SAME AND USES THEREOF

Abstract

Disclosed are a method for preparing exosomes from bovine placenta, lyophilized powder of the exosomes, as well as a composition containing exosomes from animal placenta and a preparation method and use thereof in anti-wrinkle on skin.

Claims

1. A composition containing exosomes from animal placenta, wherein by mass percentage, the composition comprises: TABLE-US-00023 lyophilized powder of exosomes from animal placenta 0.05-5%; humectant    1-20%; material for microencapsulation 0.05-5%; vitamin E derivative 0.01-2%; water the balance; wherein, the material for microencapsulation is selected from the group consisting of soybean lecithin, hydrogenated soybean phospholipid, hydroxylated lecithin, cyclodextrin, hydroxypropyl cyclodextrin and a combination thereof.

2. The composition according to claim 1, wherein the lyophilized powder of exosomes from animal placenta is selected from the group consisting of lyophilized powder of exosomes from horse placenta, lyophilized powder of exosomes from porcine placenta, lyophilized powder of exosomes from ovine placenta, lyophilized powder of exosomes from bovine placenta, lyophilized powder of exosomes from deer placenta and a combination thereof.

3. The composition according to claim 1, wherein the humectant is selected from the group consisting of polyol, plant polysaccharide, sodium hyaluronate, sodium PCA, ceramide and a combination thereof; and the vitamin E derivative is selected from the group consisting of tocopherol acetate, tocopherol phosphate, tocopherol glucoside, tocopherol polyether and a combination thereof.

4. The composition according to claim 1, wherein the composition further comprises amino acid; and the mass percentage of the amino acid is 0.1-5%.

5. The composition according to claim 4, wherein the amino acid is selected from the group consisting of glycine, tryptophan, threonine, arginine, glutamic acid, polyglutamic acid, proline, lysine, citrulline, valine, methionine, leucine, serine and a combination thereof.

6. The composition according to claim 1, wherein by mass percentage, the composition comprises: TABLE-US-00024 lyophilized powder of exosomes from animal placenta 0.05-2%;  humectant   2-15%; material for microencapsulation 0.1-3%; amino acid 0.5-3%; vitamin E derivative  0.05-1.5%; water the balance.

7. The composition according to claim 1, wherein by mass percentage, the composition comprises: TABLE-US-00025 lyophilized powder of exosomes from animal placenta 0.1-1%; humectant   5-10%; material for microencapsulation 0.5-2%; amino acid .sup. 1-2%; vitamin E derivative 0.1-1%; water the balance.

8. A method for preparing the composition according to claim 1, comprising: heating, dissolving and mixing the humectant, the material for microencapsulation and water, and cooling to 20-50° C. to prepare a first mixture; mixing the lyophilized powder of exosomes from animal placenta and the vitamin E derivative to prepare a second mixture; and mixing the second mixture and the first mixture to obtain the composition containing exosomes from animal placenta.

9. The method according to claim 8, wherein the lyophilized powder of exosomes from animal placenta is lyophilized powder of exosomes from bovine placenta.

10. The method according to claim 9, wherein the lyophilized powder of exosomes from bovine placenta is prepared by: mixing the exosomes from bovine placenta with an excipient to obtain a mixture, and pre-freezing, freezing, vacuumizing, and vacuum drying the mixture; wherein the excipient is composed of mannitol, trehalose, glycine, glycerol, Tween, albumin and water; the excipient is composed of the following components by mass percentage: 2.0%-10.0% mannitol, 0.1%-1.0% trehalose, 0.05%-1.0% glycine, 1.0%-5.0% glycerol, 0.01%-0.05% Tween, 0.1%-0.5% albumin, and the balance is water; and the volume ratio of the exosomes from bovine placenta to the excipient is 1:1.

11. The method according to claim 9, wherein the exosomes from bovine placenta is prepared by: homogenizing bovine placenta to obtain a homogenate, and subjecting the homogenate to freeze-thaw; performing a first round of ultrasonic treatment on the thawed homogenate and filtering, and freezing the filtrate at −80° C.; and thawing the filtrate at 4° C. and centrifuging to obtain a supernatant, performing a second round of ultrasonic treatment on the supernatant, and performing multiple filtration through filters to obtain the exosomes from bovine placenta; wherein the second round of ultrasonic treatment is performed once at an interval of 8 s, each ultrasonic treatment is lasted for 8 s, the power of the second round of ultrasonic treatment is 100-700 w, and the total time of ultrasonic treatment is 30-60 min.

12. The method according to claim 11, wherein the freeze-thaw comprises freezing at −80° C. for 12-24 h and then thawing at room temperature; and the number of freeze-thaw is 1-5 times.

13. The method according to claim 11, wherein the power of the first round of ultrasonic treatment is 990 w and the time is 30 min.

14. The method according to claim 11, wherein the second round of ultrasonic treatment is performed once at an interval of 8 s, each ultrasonic treatment is lasted for 8 s, the total time of ultrasonic treatment is 30 min, and the power of ultrasonic treatment is 500 w.

15. The method according to claim 11, wherein the multiple filtration is four times of filtration.

16. The method according to claim 15, wherein the four times of filtration is to filter through filter meshes with pore sizes of 5 μm, 1 μm, 0.45 μm and 0.22 μm in sequence.

17. The method according to claim 11, wherein the centrifugation is differential centrifugation.

18. The method according to claim 17, wherein the differential centrifugation comprises: centrifuging at 300 g for 30 min, taking supernatant for centrifuging at 3,000 g for 30 min, and taking supernatant again for centrifuging at 10,000 g for 30 min.

19. An anti-wrinkle essence comprising the composition containing exosomes from animal placenta according to claim 1.

20. A method for treating skin wrinkle, comprising applying the composition containing exosomes from animal placenta according to claim 1 to the skin of a subject in need thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0096] FIG. 1 shows the effect of different ultrasound parameters on the protein concentration of exosomes from bovine placenta;

[0097] FIG. 2 shows the results of particle size analysis of exosomes from bovine placenta prepared in Example 1 and Comparative Example 1;

[0098] FIG. 3 shows the electron microscope picture of exosomes from bovine placenta prepared in Example 1;

[0099] FIG. 4 shows the results of western blotting analysis of exosomes from bovine placenta prepared in Example 1;

[0100] FIG. 5 shows the efficacy assessment of the exosomes from bovine placenta prepared in Example 1 on the proliferation of human skin fibroblasts;

[0101] FIG. 6 shows the preparation process flow chart of the lyophilized powder of exosomes of the present disclosure;

[0102] FIG. 7 shows the electron microscope picture of the exosomes from bovine placenta after reconstitution in Example 8;

[0103] FIG. 8 shows the western blotting detection results of exosomes in Example 10;

[0104] FIG. 9 shows the detection result of promoting cell proliferation activity by exosomes in Example 11;

[0105] FIG. 10 shows the detection result of promoting cell proliferation activity by exosomes in Example 12.

DETAILED DESCRIPTION

[0106] Those skilled in the art can implement the present disclosure by learning from the content herein and appropriately improving the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present disclosure. The method and use of the present disclosure have been described through the preferred embodiments, and it is apparent that relevant persons can make changes or appropriate modifications and combinations of the methods and use herein without departing from the content, spirit and scope of the present disclosure to implement and apply the present disclosure.

[0107] The materials used in the present disclosure are all commercially available. The sodium PCA is sodium pyrrolidone carboxylate; the UV spectrophotometer involved is Japan Shimadzu UV-2700, the transmission electron microscope is Japan JEM-3200FS, and the skin tester is American Visia.

[0108] Bovine placenta, purchased from dairy farms, was bovine placenta naturally delivered by healthy cows. After being qualified by testing virus and bacteria, the bovine placenta was washed repeatedly with water, cut into pieces, packaged and stored frozen at −80° C. The lyophilized powder of exosomes from horse placenta, lyophilized powder of exosomes from porcine placenta, lyophilized powder of exosomes from ovine placenta and lyophilized powder of exosomes from deer placenta were commercially available general products.

[0109] The present disclosure is further illustrated below in conjunction with examples.

Example 1 Preparation of Exosomes from Bovine Placenta

[0110] 1) 500 g of bovine placenta tissue frozen at −80° C. was weighed and thawed at room temperature.

[0111] 2) After the tissue was completely thawed, it was minced with an automatic meat grinder.

[0112] 3) The tissue was homogenized using a tissue homogenizer to prepare a uniform homogenate.

[0113] 4) The homogenate was frozen at −80° C. for 24 h and then thawed at room temperature. Such freeze-thaw was repeated 3 times to fully broken the placental cells.

[0114] 5) The thawed homogenate was subjected to a first round of ultrasonic treatment (990 w, 30 min) to further break cells and release exosomes from bovine placenta.

[0115] 6) The homogenate after the first round of ultrasonic treatment was filtered through stainless steel filters with different pore sizes (20 mesh and 80 mesh) to remove insoluble substances, and the filtrate was collected and frozen at −80° C.

[0116] 7) Centrifugation: The filtrate of the bovine placenta extract obtained in step 6) was thawed at 4° C. and then centrifuged under different centrifugal forces (300 g, 30 min; 3000 g, 30 min; 10,000 g, 30 min) in sequence. The supernatant from the previous centrifugation was further centrifuged to fully remove insoluble substances in the filtrate.

[0117] 8) The supernatant obtained in the last centrifugation (10,000 g, 30 min) of step 7) was subjected to a second round of ultrasonic treatment. The second round of ultrasonic treatment was performed once at an interval of 8 s, each ultrasonic treatment was lasted for 8 s, the power of ultrasonic treatment was 500 w and the total time of ultrasonic treatment was 30 min.

[0118] 9) Filtration: The mixture after ultrasonic treatment was subjected to multiple filtration (the pore size of the filter was 5 μm, 1 μm, 0.45 μm and 0.22 μm in sequence) to obtain exosomes from bovine placenta (labeled as Exo-00).

Example 2

[0119] In step 8), the second round of ultrasonic treatment was performed once at an interval of 8 s, each ultrasonic treatment was lasted for 8 s, the power of ultrasonic treatment was 700 w and the total time of ultrasonic treatment was 30 min.

[0120] Other steps were the same as in Example 1.

Example 3

[0121] In step 8), the second round of ultrasonic treatment was performed once at an interval of 8 s, each ultrasonic treatment was lasted for 8 s, the power of ultrasonic treatment was 500 w and the total time of ultrasonic treatment was 60 min.

[0122] Other steps were the same as in Example 1.

Example 4

[0123] In step 8), the second round of ultrasonic treatment was performed once at an interval of 8 s, each ultrasonic treatment was lasted for 8 s, the power of ultrasonic treatment was 700 w and the total time of ultrasonic treatment was 60 min.

[0124] Other steps were the same as in Example 1.

Example 5

[0125] Comparative Examples 1˜4 were set up, wherein the ultrasonic parameters of step 8) in the preparation method of Comparative Examples 1˜4 are shown in Table 1, and other steps were the same as in Example 1.

TABLE-US-00005 TABLE 1 Total Ultrasound Ultrasound Running/Stopping Experiment Group Power Time (min) Time (s/s) Group 1 (Comparative 100 w 30 8/8 Example 1) Group 2 (Comparative 300 w 30 8/8 Example 2) Group 3 (Example 1) 500 w 30 8/8 Group 4 (Example 2) 700 w 30 8/8 Group 5 (Comparative 100 w 60 8/8 Example 3) Group 6 (Comparative 300 w 60 8/8 Example 4) Group 7 (Example 3) 500 w 60 8/8 Group 8 (Example 4) 700 w 60 8/8
Identification of Exosomes from Bovine Placenta

[0126] 1) Protein Concentration Assay:

[0127] The prepared exosomes from bovine placenta were measured for protein concentration using the BCA method. The results showed that the protein concentration of Examples 1˜4 was significantly higher than that of Comparative Examples 1-4, with the protein concentration of exosomes from bovine placenta prepared in Example 1 being 11.33±2.09 (mg/ml) (group 3, n=6) (as shown in FIG. 1).

[0128] 2) Exosome Identification

[0129] The products obtained in Example 1 and Comparative Example 1 were subjected to particle size analysis using Nanosight NS300. As shown in FIG. 2, the distribution of particle sizes are mainly in the range of 30-150 nm, which is consistent with the size range of exosomes. Compared with the Comparative Example 1, Example 1 contained more particles with the sizes in the range of exosomes.

[0130] The product obtained in Example 1 was observed by electron microscope. As shown in FIG. 3, there are vesicles in the obtained product, which have round pie structure with low refractive index in the middle and high around, in line with the classical structure of exosomes.

[0131] The product obtained in Example 1 was subjected to western blotting analysis to detect the biomarkers of exosomes, CD63, TSG101 and CD9, and GAPDH was the loading control. As shown in FIG. 4, CD63, TSG101 and CD9 were detected in the product.

[0132] The above results show that the main component of the product obtained by the preparation method of the present disclosure is exosomes.

Efficacy Assessment of Exosomes from Bovine Placenta

[0133] The product obtained in Example 1 was assessed for the proliferative effect on human skin fibroblasts. CCK8 method was used to detect the activity of exosomes from bovine placental prepared in Examples 1. Human skin fibroblasts cells (HSF) were cultured for 24 hours, exosomes were added to the culture medium according to a concentration gradient (0, 25, 50, 100, 200 μg/ml), and NC was a negative control without exosomes. After culturing for 48 hours, CCK8 reagent was added to the cells and incubated for 2 hours, and then the OD450 was measured by using a microplate reader, and the cell viability was calculated. As shown in FIG. 5, the exosomes from bovine placenta prepared by the present disclosure had a significant effect of promoting the proliferation of human skin fibroblasts in the concentration range of 25-200 μg/mL.

Example 6 Preparation of Lyophilized Powder of Exosomes from Bovine Placenta

[0134] The extract of exosomes from bovine placenta obtained by the method of Example 1 was thawed at room temperature and mixed with excipients (3.0% mannitol, 0.4% trehalose, 0.3% glycine, 2.0% glycerol, 0.02% Tween, 0.2% albumin, and the balance is water) in a volume ratio of 1:1 to obtain a mixture. The mixture was subjected to lyophilization in a sterile environment using a Tofflon's lyophilizer according to the procedure in Table 2 (including pre-freezing, freezing, vacuumizing, primary sublimation and vacuum drying), and the maximum temperature during the lyophilization process should not exceed 40° C. The lyophilized powder of exosomes from bovine placenta was prepared, labeled as Exo-01.

[0135] Preparation process is with reference to FIG. 6 and parameter setting to Table 2. The specific steps are as follows:

[0136] (1) Filling: The prepared and compounded mixture of exosomes from bovine placenta was poured into the charging basket of a filling machine. Before starting the filling machine, the filling weight should be calibrated and recorded. During the filling process, a monitoring record of the filling process was required.

[0137] (2) Pre-freezing and vacuumizing: the product after filling was placed on the shelf of the lyophilizer, and the door of the lyophilizer was closed; after checking that the water pressure, current and air pressure met the conditions for starting the lyophilizer, the lyophilizer was started, the product was pre-frozen to below −40° C. in the front box, and the vacuum was controlled to 0.19 mbar;

[0138] (3) Primary sublimation: The product was pre-frozen to −40° C. and maintained for 190 min, and then the oil temperature for cooling was turned off and turned to the rear box for cooling. The condensing coil of the rear box was frozen to −50° C., and then the partition valve was opened, the vacuum pump and the vacuum control program were turned on to rise and maintain temperature in sequence. The moisture in the lyophilized powder was evaporated to the condensing coil for water capture and condensation.

[0139] (4) Secondary vacuum drying: after the primary drying was completed, the vacuum adjustment program of the lyophilizer was closed, and secondary vacuum drying was carried out. The residual moisture in the lyophilized powder was continued to be drained. The bottle temperature of the lyophilized powder should not exceed 32° C., and the process time should be kept for 6 h.

[0140] (5) Pressing the plug: After the secondary vacuum drying was completed, the hydraulic system of the lyophilizer was started to press the plug. After the plug is pressed, whether there is a jumping plug is observed. If there is no abnormality, release the vacuum in the lyophilizer.

TABLE-US-00006 TABLE 2 Set temperature Set time Duration Step (° C.) (min) (min) Pre-freezing −40 1 190 Set temperature Duration (° C.) (min) Freezing −50 1 with condenser Pre-vacuumizing Alarm vacuum Alarm vacuum (mbar) (mbar) duration (s) Vacuumizing 0.15 0.5 3000 Set Set Vacuum temperature time Duration controlled (° C.) (min) (min) (mbar) Primary −15 70 300 0.19 drying −10 60 300 0.19 −5 60 300 0.19 0 60 220 0.19 Set Set Vacuum temperature time Duration controlled (° C.) (min) (min) (mbar) Secondary 30 40 500 0.19 drying 30 1 120 0.0002 Total time 38 h

Example 7

[0141] The extract of exosomes from bovine placenta obtained by the method of Example 1 was thawed at room temperature and mixed with an excipient (2.0% mannitol, 1.0% trehalose, 1.0% glycine, 1.0% glycerol, 0.05% Tween, 0.5% albumin, and the balance is water) in a volume ratio of 1:1 to obtain a uniform mixture. The mixture was subjected to lyophilization in a sterile environment using a lyophilizer according to the procedure in Table 2 to obtain the lyophilized powder of exosomes from bovine placenta (Exo-02).

[0142] The extract of exosomes from bovine placenta obtained by the method of Example 1 was thawed at room temperature and mixed with an excipient (10.0% mannitol, 0.1% trehalose, 0.05% glycine, 5.0% glycerol, 0.01% Tween, 0.1% albumin, and the balance is water) in a volume ratio of 1:1 to obtain a uniform mixture. The mixture was subjected to lyophilization in a sterile environment using a lyophilizer according to the procedure in Table 2 to obtain the lyophilized powder of exosomes from bovine placenta (Exo-03).

[0143] The extract of exosomes from bovine placenta obtained by the method of Example 1 was thawed at room temperature and mixed with an excipient (5.0% mannitol, 0.4% trehalose, 0.06% glycine, 4.0% glycerol, 0.02% Tween, 0.2% albumin, and the balance is water) in a volume ratio of 1:1 to obtain a uniform mixture. The mixture was subjected to lyophilization in a sterile environment using a lyophilizer according to the procedure in Table 2 to obtain the lyophilized powder of exosomes from bovine placenta (Exo-04).

Example 8 Morphological Identification of Exosomes from Bovine Placenta

[0144] The lyophilized powder of exosomes from bovine placenta prepared in Example 6 was resuspended in 500-1,000 μL of PBS. 10 μL was added dropwise to the copper grid of a transmission electron microscope for 1 min of precipitation, and the suspension was removed with filter paper. 10 μL of uranyl acetate was added dropwise to the copper grid of the transmission electron microscope for 1 min of precipitation, and the suspension was removed with filter paper. After drying at room temperature for several minutes, it was observed on the machine, and the results are shown in FIG. 7.

[0145] The results show that the exosomes reconstituted from the lyophilized powder of exosomes from bovine placenta had a diameter of about 100 nm and a round-cake structure with low refraction in the middle and high refraction around, which was a complete structure and conformed to the classical structure of exosomes.

Example 9 Exosomes Concentration and Nanoparticle Tracking Analysis (NTA) for Particle Size Detection

[0146] The particle size and concentration of exosomes were analyzes by a nanoparticle tracking analyzer. The lyophilized powder of exosomes obtained in Examples 6 and 7 was dissolved in 1 mL of PBS which had been filtered through 0.22 μm, sealed and placed on ice. A disposable clean sample pool was used and wiped with a clean paper facing the light to ensure that no particles adhered to the outer tube wall on the light path. The exosome solution was slowly transferred to avoid air bubbles. The sample pool may be tilted moderately if required and was sealed with a lid. Then the sample pool was put into the instrument and detected according to the standard operating procedures. The data were saved and analyzed for the particle size range and concentration of exosomes, and the results are shown in Table 3, which show that the exosomes reconstituted from the lyophilized powder of the present disclosure had a relatively uniform particle size (30-150 nm) and a large number of particles.

TABLE-US-00007 TABLE 3 Average particle size Concentration Exosome sample (nm) (Particles/mL) Exo-00 85.28 2.02E+10 Exo-01 88.56 1.94E+10 Exo-02 84.54 1.82E+10 Exo-03 85.92 1.74E+10 Exo-04 87.28 1.62E+10

Example 10 Western Blot Analysis of Exosomes

[0147] The lyophilized powder prepared in Examples 6 and 7 was reconstituted, added with the protein lysis solution, placed on ice for 5-15 min and shaken 3-4 times during this period, each for 30 s. Then, centrifugation was carried out at 12,000 rpm for 5 min at 4° C., and the supernatant was transferred to a new centrifuge tube for Western blot in order to detect the expression of CD63 and TSG101 proteins. The results are shown in FIG. 8 that the exosomes reconstituted from the lyophilized powder prepared in Examples 6 and 7 of the present disclosure can detect the expression of CD63 and TSG101 proteins.

Example 11 Exosomes Promote Cell Proliferation

[0148] The exosomes reconstituted from the lyophilized powder prepared in Example 6 was detected for activity by CCK8 method. The exosomes were added in the skin fibroblast medium according to the concentration gradient (0, 25, 50, 100 μg/ml) with a control without adding the lyophilized powder of exosomes. 48 hours later, CCK8 reagent was added to incubate for 2 h, and then the OD450 was detected by a microplate reader to calculate the cell viability. The results are shown in FIG. 9 that exosomes had a significant concentration-dependent effect on promoting cell proliferation.

Example 12 Comparison of Different Processes for Preparing Lyophilized Powder of Exosomes

[0149] Lyophilized powder of exosomes was prepared from the placental exosomes and different excipients by different lyophilization processes. Control groups were set up for the experiment: the control without adding the lyophilized powder of exosomes and Comparative Examples 1-3, and the experimental groups: Examples 1 and 6.

[0150] Comparative Example A: exosomes from bovine placenta of Example 1 preserved in liquid (Exo-05);

[0151] Comparative Example B: the lyophilized powder prepared by the method of Example 1 in patent application No. 201910553008.3 “Exosome freeze-dried powder and preparation method and application thereof”, labeled as Exo-06.

[0152] Comparative Example C: the lyophilized powder prepared by the method of Example 2 in the patent application No. 201410705636.6 “Method for preparing lyophilized powder of exosomes from human amniotic membrane mesenchymal stem cells”, labeled as Exo-07.

[0153] Experimental group: lyophilized powder of Example 6 (Exo-01) and lyophilized powder of Example 7 (Exo-02).

[0154] Storage conditions: room temperature 25° C.

[0155] Comparative Examples A-C and the lyophilized powder of exosomes prepared in Examples 6 and 7 (Exo-01, Exo-02) were respectively placed at 25° C. for 6 months, reconstituted and subjected to exosome identification (nanoparticle size detection by NTA, Table 4).

TABLE-US-00008 TABLE 4 Average particle size Concentration Exosome sample (nm) (Particles/mL) Exo-05 74.56 0.16E+10 Exo-06 86.54 1.05E+10 Exo-07 79.92 1.14E+10 Exo-08 87.45 1.82E+10 Exo-09 91.28 1.64E+10

[0156] Their biological activity was tested in a skin fibroblast proliferation model (CCK8 method) and results are shown in FIG. 10. Among them, the lyophilized powder of exosomes numbered Exo-01 and Exo-02 were re-numbered as Exo-08 and Exo-09 respectively after reconstitution. The results show that the lyophilized powder of Examples 6 and 7 of the present disclosure can maintain the integrity and biological activity of exosomes after reconstitution (Exo-08, Exo-09), and the result was significantly better than that of Comparative Examples A-C.

Comparative Example I

[0157] According to the component of each composition in Table 5, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 1.

[0158] Detected by an ultraviolet spectrophotometer, the transmittance of composition 1 was 79.13%.

TABLE-US-00009 TABLE 5 Phase Component Mass % A Water The balance Glycerol 2 Sodium hyaluronate 0.2 Hydroxylated lecithin 1.5 B Polyglutamic acid 0.8 Tocopherol acetate 0.05 Total 100

Example 13

[0159] According to the component of each composition in Table 6, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 2.

[0160] Detected by an ultraviolet spectrophotometer, the transmittance of composition 2 was 78.79%.

TABLE-US-00010 TABLE 6 Phase Component Mass % A Water The balance Glycerol 2 Sodium hyaluronate 0.2 Hydroxylated lecithin 1.5 B Polyglutamic acid 0.8 Tocopherol acetate 0.05 Lyophilized powder of exosomes 0.5 from bovine placenta Total 100

Comparative Example II

[0161] According to the component of each composition in Table 7, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 3.

[0162] Detected by an ultraviolet spectrophotometer, the transmittance of composition 3 was 69.79%.

TABLE-US-00011 TABLE 7 Phase Component Mass % A Water The balance Glycerol 17 Sodium hyaluronate 0.02 B Glycine 0.8 Tocopherol acetate 0.15 Lyophilized powder of exosomes 0.5 from bovine placenta Total 100

Example 14

[0163] According to the component of each composition in Table 8, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 4.

[0164] Detected by an ultraviolet spectrophotometer, the transmittance of composition 4 was 85.16%.

TABLE-US-00012 TABLE 8 Phase Component Mass % A Water The balance Butanediol 15 Trehalose 2 Hydroxylated lecithin 0.5 B Glycine 0.8 Tocopherol acetate 0.05 Lyophilized powder of exosomes 0.5 from bovine placenta Total 100

Example 15

[0165] According to the component of each composition in Table 9, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 5.

[0166] Detected by an ultraviolet spectrophotometer, the transmittance of composition 5 was 82.73%.

TABLE-US-00013 TABLE 9 Phase Component Mass % A Water The balance 1,3-Propanediol 10 Sodium PCA 1 Hydroxylated lecithin 1 B Arginine 0.8 Tocopherol acetate 0.05 Lyophilized powder of exosomes 0.5 from bovine placenta Total 100

Example 16

[0167] According to the component of each composition in Table 10, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 6.

[0168] Detected by an ultraviolet spectrophotometer, the transmittance of composition 6 was 81.48%.

TABLE-US-00014 TABLE 10 Phase Component Mass % A Water The balance Glycerol 10 Butanediol 8 Hydroxylated lecithin 1.5 Tocopherol acetate 0.05 B Lyophilized powder of exosomes 0.5 from bovine placenta Total 100

Example 17

[0169] According to the component of each composition in Table 11, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 7.

[0170] Detected by an ultraviolet spectrophotometer, the transmittance of composition 7 was 80.99%.

TABLE-US-00015 TABLE 11 Phase Component Mass % A Water The balance Glycerol 16 Trehalose 2 Hydroxylated lecithin 1.5 B Polyglutamic acid 0.2 Tocopherol acetate 0.05 Lyophilized powder of exosomes 0.5 from bovine placenta Total 100

Example 18

[0171] According to the component of each composition in Table 12, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 8.

[0172] Detected by an ultraviolet spectrophotometer, the transmittance of composition 8 was 96.48%.

TABLE-US-00016 TABLE 12 Phase Component Mass % A Water The balance Butanediol 10 Sodium hyaluronate 0.2 Hydroxypropyl cyclodextrin 4.8 B Arginine 0.8 Tocopherol glucoside 2 Lyophilized powder of exosomes 0.5 from ovine placenta Total 100

Example 19

[0173] According to the component of each composition in Table 13, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 9.

[0174] Detected by an ultraviolet spectrophotometer, the transmittance of composition 9 was 63.81%.

TABLE-US-00017 TABLE 13 Phase Component Mass % A Water The balance Tremella polysaccharide 18 Soybean lecithin 0.05 B Proline 1 Tocopherol polyether 0.02 Lyophilized powder of exosomes 1.5 from porcine placenta Total 100

Example 20

[0175] According to the component of each composition in Table 14, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 10.

[0176] Detected by an ultraviolet spectrophotometer, the transmittance of composition 10 was 92.85%.

TABLE-US-00018 TABLE 14 Phase Component Mass % A Water The balance Sodium PCA 1 Cyclodextrin 0.05 B Methionine 2.5 Tocopherol phosphate 1.5 Lyophilized powder of exosomes 1 from horse placenta Total 100

Example 21

[0177] According to the component of each composition in Table 15, phase A was mixed, heated to 80±2° C., stirred until completely dissolved, and then cooled to 40±2° C.; and phase B was mixed well, added to phase A that had been cooled, and stirred uniformly to obtain composition 11.

[0178] Detected by an ultraviolet spectrophotometer, the transmittance of composition 11 was 74.83%.

TABLE-US-00019 TABLE 15 Phase Component Mass % A Water The balance Glycerol 20 Hydroxylated lecithin 4 B Valine 4.5 Tocopherol acetate 0.1 Lyophilized powder of exosomes 4.5 from horse placenta Total 100

Example 22

[0179] This example of the present disclosure provides the detection of the number of exosome vesicles in compositions 1-11, and the steps are as follows:

[0180] After the compositions 1-11 prepared above were sealed and stored in the dark for 15 days at room temperature, they were observed for morphology under a transmission electron microscope at 5000× magnification, and the intact vesicles were counted. The mean value of the number of intact vesicles observed in three observations was summarized and shown in Table 16.

TABLE-US-00020 TABLE 16 Sample Number of intact vesicles Composition 1 0 Composition 2 18.33 Composition 3 6 Composition 4 12.67 Composition 5 15.67 Composition 6 8.67 Composition 7 16.33 Composition 8 19.67 Composition 9 9 Composition 10 10.33 Composition 11 21.67

[0181] As can be seen from Table 16, the number of intact vesicles in composition 1 (containing no exosomes from bovine placenta) was 0; as for compositions 2-5, the order of the number of intact vesicles was composition 2>composition 5>composition 4>composition 3, which corresponded to the content of hydroxylated lecithin in the composition (composition 2>composition 5>composition 4>composition 3), indicating that hydroxylated lecithin had a protective effect on the vesicle structure of exosomes from bovine placenta. As for compositions 2, 6 and 7, the order of the number of intact vesicles was composition 2>composition 7>composition 6, which corresponded to the content of amino acids in the composition (composition 2>composition 7>composition 6), indicating that amino acids have a protective effect on the vesicle structure of exosomes from bovine placenta.

Example 23

[0182] This example of the present disclosure provides the essence product prepared from composition 1, and the specific preparation method is as follows:

[0183] According to the component of each composition in Table 17, phase A was mixed, heated to 85±2° C., stirred until completely dissolved, maintained at this temperature for 30 min, and then cooled to 40±2° C.; and phase B was added to phase A that had been cooled, stirred uniformly and then cooled to room temperature to obtain Essence I.

TABLE-US-00021 TABLE 17 Phase No. Raw material Weight ratio (%) A 1 Water The balance 2 Glycerol 5 3 Butanediol 4 4 Disodium EDTA 0.05 5 Ammonium acryloyldimethyltaurate/ 0.3 VP copolymer 6 Methylparaben 0.2 7 Allantoin 0.2 B 8 Composition 1 20 9 Leaf extract of Aloe Barbadensis 1 10 Phenoxyethanol 0.4 11 Ethylhexylglycerol 0.3

Example 24

[0184] This example of the present disclosure provides the essence product prepared from composition 2. The specific preparation method is with reference to Example 23, and the difference is that in the present example, the No. 8 raw material in phase B was replaced by composition 2, and the remaining steps are the same as Example 23. Essence II was obtained.

Example 25

[0185] This example of the present disclosure provides the essence product prepared from composition 3. The specific preparation method is with reference to Example 23, and the difference is that in the present example, the No. 8 raw material in phase B was replaced by composition 3, and the remaining steps are the same as Example 23. Essence III was obtained.

Example 26

[0186] This example of the present disclosure provides the essence product prepared from composition 4. The specific preparation method is with reference to Example 23, and the difference is that in the present example, the No. 8 raw material in phase B was replaced by composition 4, and the remaining steps are the same as Example 23. Essence IV was obtained.

Example 27

[0187] This example of the present disclosure provides the anti-skin aging observation test of the above four essences, and the specific steps are as follows:

[0188] 1. Essences I, II, III and IV prepared in the above examples were served as cosmetic to be tested.

[0189] 2. 80 female volunteers with wrinkled skin, aged 29-45 years old, with an average age of 34 years old, were under test, and they were all indoor clerks in the same office building. The volunteers did not use other anti-wrinkle products during the test period. The area within the lines connected by three points, the inner corner of the eyes, corners of the mouth and the temple, was selected as the test area.

[0190] 3. The skin wrinkle and smoothness of the volunteers' face test area before applying cosmetics were measured with a Visia skin tester.

[0191] 4. The volunteers were randomly divided into 4 groups with 20 in each group. The essences of Examples 23-26 were used in the test area respectively, and each essence was used in an amount of about 0.8 g each night from 20:00 to 22:00, and stayed for at least 8 hours.

[0192] 5. After 10, 20 and 30 days of continuous use of cosmetic products, the skin texture was tested with a Visia skin tester.

[0193] 6. The numerical value measured in the test area of the subject each time was recorded, and day 0 was used as a benchmark to compare and analyze skin wrinkles and smoothness, and evaluate the improvement of the essence on the skin texture. After using Essences I, II, III and IV respectively for 30 days, the statistics results are shown in Table 18.

TABLE-US-00022 TABLE 18 Wrinkle improvement rate (%) Smoothness improvement rate (%) Sample Day 10 Day 20 Day 30 Day 10 Day 20 Day 30 Essence I 5.3 7.1 8.6 5.3 7.7 8.3 Essence II 7.7 20.2 25.6 7.6 19.9 25.3 Essence III 6.2 13.8 15.3 5.7 13.2 14.6 Essence IV 6.8 15.6 19.5 6.6 15.3 20.0

[0194] As can be seen from the test results in Table 18, the wrinkle improvement rate and smoothness improvement rate of Essence II and Essence III on skin were better than that of Essence I, indicating that the composition containing exosomes from bovine placenta of the present disclosure had obvious anti-wrinkle effect. The wrinkle improvement rate and smoothness improvement rate of Essence II were better than that of Essence III, indicating that hydroxylated lecithin can improve the anti-wrinkle effect of the system. The wrinkle improvement rate and smoothness improvement rate of Essence II, Essence III and Essence IV on skin were in the order of Essence II>Essence IV>Essence III, among which Essence II had more hydroxylated lecithin than Essence IV and Essence III did not contain hydroxylated lecithin, indicating that hydroxylated lecithin can improve the anti-wrinkle effect of the system.

[0195] To sum up, the compositions provided by the examples of the present disclosure had excellent light transmittance that was not lower than 60% of the transmittance of pure water under the same test conditions, and can be directly used in skin care products of transparent systems without turbidity. Compared with composition 1, compositions 2-11 contained exosome vesicles structure; compared with composition 3, composition 2 and compositions 4-11 had intact exosome vesicle structure, and the retention rate of exosome vesicles was significantly improved; compared with Essence I, Essence II, Essence III and Essence IV had a dramatic effect on wrinkle improvement and smoothing.

[0196] The above is only the preferred embodiment of the present disclosure, it should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principle of the present disclosure, and these improvements and modifications should also be considered within the scope of protection of the present disclosure.