Exosome compound as well as preparation method and application thereof

Abstract

The invention relates to an exosome compound as well as a preparation method and application thereof, and belongs to the technical field of plant extract processing. The preparation method of the exosome compound comprises the following steps: (1) respectively crushing and mixing highland barley and sunflower seeds, and sequentially carrying out two-stage enzymolysis by adopting alkaline protease and papain to obtain a polypeptide solution; (2) crushing olive leaves, homogenizing with a buffer solution, and then separating and purifying to obtain an olive leaf exosome; and (3) entrapping the polypeptide solution in the olive leaf exosome at 0-4 DEG C by a segmented ultrasonic method to obtain the exosome compound. A polypeptide solution can be efficiently and stably entrapped in the exosome; meanwhile, the olive leaf exosome is matched with a polypeptide solution extracted by compounding highland barley and sunflower seeds, so that the long-acting moisturizing, repairing, soothing and anti-aging effects are achieved.

Description

Technical Field

[0001] The present application relates to the technical field of plant extract processing, and in particular to an exosome complex and a preparation method and application thereof. Background Art

[0002] With the continuous development of biomedicine, food science and agriculture, plant exosomes, as an emerging biomacromolecule carrier, have gradually attracted widespread attention. Plant exosomes not only play an important role in plant growth and development and intercellular communication, but also become one of the research hotspots because of their unique advantages in drug delivery, nutrient carriers and antioxidant, anti-inflammatory and other biological activities. As natural nanoparticles, exosomes have good biocompatibility, low immunogenicity and the ability to carry a variety of biomolecules (such as proteins, lipids, nucleic acids, etc.), making them a biomaterial with great potential, especially in the fields of disease treatment, functional food and health care product development. At present, the sources of plant exosomes are mainly concentrated in some common plant species, such as tea, grapes, apples, etc. However, in recent years, olive leaf exosomes have gradually become the focus of research due to their significant biological activities in antioxidant, anti-inflammatory, anti-tumor, and immunomodulation. Olive leaves are rich in polyphenol compounds and have strong biological activities, which can be used as one of the preferred sources of exosomes.

[0003] At the same time, plant peptides, as bioactive substances, have a wide range of application potential. Peptides can not only regulate the body's immune function, but also improve human health through antioxidant, antibacterial, and anti-inflammatory effects. Sunflower seeds and highland barley, as natural plant sources, are rich in a variety of functional peptide components and have been widely studied in recent years. Sunflower seeds are rich in protein, peptides, and unsaturated fatty acids, which can play a role in a variety of biologically active fields; highland barley is rich in protein, dietary fiber, minerals, and a variety of bioactive small molecules, and has strong antioxidant, anti-inflammatory, and immunomodulatory effects.

[0004] At present, there are relatively few studies on the introduction of active molecules such as peptides into plant exosomes, and existing technologies mainly focus on the binding mode of exogenous substances to exosome membranes and the optimization of carrier systems. Although some studies have explored the application of functional carriers of plant exosomes, how to efficiently load plant peptides from different sources into plant exosomes and maintain their activity remains an unresolved problem. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the embodiments of the present application includes providing an exosome complex and a preparation method and application thereof, which can efficiently and stably encapsulate plant polypeptides in plant exosomes and have long-lasting moisturizing, repairing, soothing and anti-aging effects.

[0006] In a first aspect, the present invention provides a method for preparing an exosome complex, comprising:

[0007] (1) Highland barley and sunflower seeds are crushed and mixed respectively, and then enzymatically hydrolyzed in two stages using alkaline protease and papain in sequence to obtain a polypeptide solution;

[0008] (2) crushing the olive leaves, homogenizing with a buffer solution, and then separating and purifying to obtain olive leaf exosomes;

[0009] (3) Encapsulating the polypeptide solution into olive leaf exosomes by segmented ultrasound at 0 to -4°C to obtain an exosome complex.

[0010] The present application obtains an exosome complex by encapsulating a polypeptide solution extracted from a combination of highland barley and sunflower seeds into olive leaf exosomes. At the same time, the polypeptide solution is efficiently and stably encapsulated in the exosomes through a low-temperature segmented ultrasonic method. At the same time, since the olive leaf exosomes and the polypeptide solution extracted from a combination of highland barley and sunflower seeds are coordinated, they have long-lasting moisturizing, repairing, soothing and anti-aging effects.

[0011] In some embodiments of the present application, highland barley flour obtained by crushing highland barley and sunflower seed flour obtained by crushing sunflower seeds are mixed in a mass ratio of (6:4) to (8:2).

[0012] The present application uses highland barley powder and sunflower seed powder to mix in a suitable mass ratio to extract a polypeptide solution, thereby extracting a polypeptide solution with richer polypeptide types.

[0013] In some embodiments of the present application, the amount of alkaline protease added is 0.3% to 0.7% of the substrate mass, and the enzyme activity is ≥150 U / mg.

[0014] The present application uses an appropriate amount of alkaline protease to perform the first-stage enzymatic hydrolysis on highland barley flour and sunflower seed flour, thereby efficiently extracting a polypeptide solution.

[0015] In some embodiments of the present application, the amount of papain added is 0.1% to 0.3% of the substrate mass, and the enzyme activity is ≥ 2000 U / mg.

[0016] The present application uses an appropriate amount of papain to perform a second-stage enzymatic hydrolysis on highland barley flour and sunflower seed flour, and cooperates with the first-stage enzymatic hydrolysis of alkaline protease to efficiently extract and obtain a polypeptide solution with richer polypeptide types.

[0017] In some embodiments of the present application, the enzymatic hydrolysis of alkaline protease is carried out at a pH value of 7.5-8.5, a temperature of 40-50°C, and a stirring rate of 150-250 rpm for 25-35 min; the enzymatic hydrolysis of papain is carried out at a pH value of 6.3-6.7, a temperature of 50-60°C, and a stirring rate of 120-180 rpm for 80-100 min.

[0018] The present application adopts appropriate enzymatic hydrolysis conditions to sequentially perform enzymatic hydrolysis of alkaline protease and enzymatic hydrolysis of papain to support the coordination of the first stage enzymatic hydrolysis of alkaline protease and the second stage enzymatic hydrolysis of papain, so as to obtain a polypeptide solution with richer polypeptide types through efficient extraction.

[0019] In some embodiments of the present application, the average particle size of olive leaf exosomes is 50 to 200 nm.

[0020] The present application controls the average particle size of the olive leaf exosomes extracted to be 50-200 nm, which is convenient for subsequent mixing with the polypeptide solution to encapsulate the polypeptide solution.

[0021] In some embodiments of the present application, the separation and purification in step (2) includes centrifugation at 3000-5000 rpm for 10-25 min, pre-filtration on a 0.1-0.22 μm filter membrane, and tangential flow filtration in a tangential flow filtration system with a membrane pore size of 0.1-0.2 μm, a pump flow rate of 50-200 mL / min, and a pressure of 0.2-2 bar.

[0022] The present application adopts appropriate separation and purification conditions to facilitate obtaining olive leaf exosomes with high purity, high activity and appropriate average particle size.

[0023] In some embodiments of the present application, the concentration of the polypeptide solution is 1-10 mg / mL and the concentration of the olive leaf exosomes is 1-5×10 9 / mL for mixing.

[0024] The present application adopts a polypeptide solution of appropriate concentration and an olive leaf exosome of appropriate concentration for mixed encapsulation, so that the polypeptide solution can be efficiently and stably encapsulated in the olive leaf exosomes, and an exosome complex with high activity and good moisturizing, repairing, soothing and anti-aging effects is obtained.

[0025] In some embodiments of the present application, the segmented ultrasound method includes ultrasound at 30-100W for 1-5 minutes, standing for 3-10 minutes, and then ultrasound at 10-50W for 1-5 minutes; and the ultrasound power of the front section is greater than the ultrasound power of the back section.

[0026] The present application adopts segmented ultrasound under appropriate conditions to first quickly open the membrane pores of the exosomes with high power to improve the drug loading efficiency, and then reduces the power to maintain the open state of the exosome membrane and reduce membrane damage, so that the polypeptide solution can be efficiently and stably encapsulated in the olive leaf exosomes.

[0027] In a second aspect, an embodiment of the present application provides an exosome complex prepared by the preparation method provided in the first aspect.

[0028] The exosome complex prepared by the above-mentioned preparation method in the present application has high activity and stability, and has good moisturizing, repairing, soothing and anti-aging effects.

[0029] In a third aspect, an embodiment of the present application provides a use of the exosome complex provided in the second aspect in the preparation of a skin care product.

[0030] The exosome complex prepared by the above-mentioned preparation method in the present application has good moisturizing, repairing, soothing and anti-aging effects, so it is applied to skin care products, and the skin care products also have good moisturizing, repairing, soothing and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is a graph showing the results of skin moisture content provided in Experimental Example 6 of this application.

[0033] Figure 2 This is a result graph of the transepidermal water loss rate of the skin provided in Experimental Example 6 of this application.

[0034] Figure 3 This is a result graph of the skin erythema value provided in Test Example 7 of this application.

[0035] Figure 4 This is a result graph of the transepidermal water loss rate of the skin provided in Experimental Example 7 of this application.

[0036] Figure 5 This is a result diagram of the skin elasticity parameter R2 provided in Test Example 8 of the present application. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0038] The present application provides a method for preparing an exosome complex, comprising:

[0039] (1) Highland barley and sunflower seeds are crushed separately, highland barley powder obtained by crushing highland barley and sunflower seed powder obtained by crushing sunflower seeds are mixed in a mass ratio of (6:4) to (8:2), and alkaline protease with an enzyme activity of ≥150 U / mg is used at a pH value of 7.5 to 8.5, a temperature of 40 to 50° C., and a stirring rate of 150 to 250 rpm for a first-stage enzymolysis, and papain with an enzyme activity of ≥2000 U / mg is used at a pH value of 6.3 to 6.7, a temperature of 50 to 60° C., and a stirring rate of 120 to 180 rpm for a second-stage enzymolysis to obtain a polypeptide solution;

[0040] (2) crushing the olive leaves, homogenizing with a buffer solution, centrifuging at 3000-5000 rpm for 10-25 min, pre-filtering with a 0.1-0.22 μm filter membrane, and performing tangential flow filtration in a tangential flow filtration system with a membrane pore size of 0.1-0.2 μm, a pump flow rate of 50-200 mL / min, and a pressure of 0.2-2 bar to obtain olive leaf exosomes;

[0041] (3) The polypeptide solution with a concentration of 1 to 10 mg / mL was loaded into the solution with a concentration of 1 to 5 × 10 9 / mL of olive leaf exosomes to obtain exosome complexes.

[0042] The present application obtains an exosome complex by encapsulating a polypeptide solution extracted from a combination of highland barley and sunflower seeds into olive leaf exosomes. At the same time, the polypeptide solution is efficiently and stably encapsulated in the exosomes through a low-temperature segmented ultrasonic method. At the same time, since the olive leaf exosomes and the polypeptide solution extracted from a combination of highland barley and sunflower seeds are coordinated, they have long-lasting moisturizing, repairing, soothing and anti-aging effects.

[0043] As an example, highland barley flour obtained by crushing highland barley and sunflower seed flour obtained by crushing sunflower seeds can be mixed in a mass ratio of 6:4, 6:3, 6:2, 7:4, 7:3, 7:2, 8:4, 8:3, 8:2, but not limited to. By mixing highland barley flour and sunflower seed flour in a suitable mass ratio to extract a polypeptide solution, a polypeptide solution with richer polypeptide types can be obtained.

[0044] In some embodiments of the present application, highland barley is husked and crushed to a particle size of 50-100 mesh, and the bran is removed to obtain highland barley flour. As an example, the particle size of highland barley flour can be, but is not limited to, 50 mesh, 60 mesh, 70 mesh, 80 mesh, 90 mesh, and 100 mesh.

[0045] In some embodiments of the present application, sunflower seeds are defatted by low temperature pressing and crushed to a particle size of 80-120 meshes to obtain sunflower seed defatted protein powder. As an example, the particle size of the sunflower seed defatted protein powder can be, but is not limited to, 80 mesh, 90 mesh, 100 mesh, 110 mesh, and 120 mesh.

[0046] In some embodiments of the present application, after highland barley flour and sunflower seed flour are mixed, 8-12 times the volume of deionized water is added for subsequent two-stage enzymatic hydrolysis.

[0047] As an example, the amount of alkaline protease added can be, but is not limited to, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7% of the substrate mass. The first-stage enzymolysis of highland barley flour and sunflower seed flour using an appropriate amount of alkaline protease can efficiently extract the polypeptide solution.

[0048] As an example, the amount of papain added can be, but is not limited to, 0.1%, 0.2%, or 0.3% of the substrate mass. A suitable amount of papain is used to perform a second-stage enzymolysis on highland barley flour and sunflower seed flour, and combined with the first-stage enzymolysis of alkaline protease, a polypeptide solution with richer polypeptide types is efficiently extracted.

[0049] In some embodiments of the present application, after the two-stage enzymatic hydrolysis, the temperature is raised to 80-90°C and maintained for 10-15 minutes to inactivate the enzyme, the enzymatic hydrolyzate is centrifuged at 3000-5000 rpm for 15-2 minutes, the solid matter is removed, and the supernatant is collected to obtain a polypeptide solution.

[0050] In some embodiments of the present application, the molecular weight of the polypeptide solution is less than 5000 Daltons.

[0051] In some embodiments of the present application, the average particle size of olive leaf exosomes is 50-200 nm. As an example, the average particle size of olive leaf exosomes can be, but is not limited to, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm. The extracted olive leaf exosomes are controlled to have an average particle size of 50-200 nm, which is conducive to subsequent mixing with the polypeptide solution and encapsulation of the polypeptide solution.

[0052] As an example, the concentration of the mixed entrapped polypeptide solution can be, but is not limited to, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL. The concentration of the mixed entrapped olive leaf exosomes can be, but is not limited to, 1×10 9 / mL, 2×10 9 / mL, 3×10 9 / mL, 4×10 9 / mL, 5×10 9 / mL. A peptide solution of appropriate concentration and an olive leaf exosome of appropriate concentration are mixed and encapsulated, so that the peptide solution can be efficiently and stably encapsulated in the olive leaf exosomes, and an exosome complex with high activity, good moisturizing, repairing, soothing and anti-aging effects is obtained.

[0053] The present application embodiment provides an exosome complex prepared by the above-mentioned preparation method.

[0054] The exosome complex prepared by the above-mentioned preparation method in the present application has high activity and stability, and has good moisturizing, repairing, soothing and anti-aging effects.

[0055] The present application embodiment provides an application of the above-mentioned exosome complex in the preparation of skin care products.

[0056] The exosome complex prepared by the above-mentioned preparation method in the present application has good moisturizing, repairing, soothing and anti-aging effects, so it is applied to skin care products, and the skin care products also have good moisturizing, repairing, soothing and anti-aging effects.

[0057] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] This embodiment provides a method for preparing an exosome complex, comprising:

[0060] (1) After shelling highland barley, the highland barley is crushed to a particle size of 80 mesh, and the bran is removed to obtain highland barley flour; sunflower seeds are defatted by low-temperature pressing and crushed to a particle size of 100 mesh to obtain sunflower seed defatted protein powder; highland barley flour and sunflower seed defatted protein powder are mixed in a mass ratio of 7:3, and 10 volumes of deionized water are added; the pH value of the suspension is adjusted to 8.0, the temperature is controlled to 40° C., 0.5% of the substrate mass and alkaline protease with an enzyme activity of ≥150 U / mg are added, and the stirring speed is 200 rpm for 30 min to carry out the first stage of enzymolysis; then the system pH is maintained at 6.5, the temperature is controlled to 55° C., 0.2% of the substrate mass and papain with an enzyme activity of ≥2000 U / mg are added, and the stirring speed is 150 rpm for 90 min to carry out the second stage of enzymolysis; the temperature is raised to 85° C., maintained for 10 min to inactivate the enzyme, the enzymolysis solution is centrifuged at 4000 rpm for 20 min, the solid matter is removed, and the supernatant is collected to obtain a polypeptide solution;

[0061] (2) Wash and chop the olive leaves, suspend them in a buffer solution PBS, homogenize them, centrifuge them at 3000 rpm for 25 min, pre-filter them through a 0.22 μm filter membrane to obtain a supernatant, add the supernatant to a tangential flow filtration system with a membrane pore size of 0.2 μm, a pump flow rate of 100 mL / min, and a pressure of 1 bar to obtain olive leaf exosomes;

[0062] (3) The peptide solution with a concentration of 5 mg / mL was mixed with a solution with a concentration of 3×10 9 / mL of olive leaf exosomes were mixed evenly, put into an ice bath, and ultrasonicated at 50W for 3 minutes by segmented ultrasonication, allowed to stand for 5 minutes, and then ultrasonicated at 30W for 2 minutes to encapsulate the polypeptide solution in the olive leaf exosomes to obtain the exosome complex.

[0063] Example 2

[0064] This embodiment provides a method for preparing an exosome complex, comprising:

[0065] (1) After shelling highland barley, the highland barley is crushed to a particle size of 80 mesh, and the bran is removed to obtain highland barley flour; sunflower seeds are defatted by low-temperature pressing and crushed to a particle size of 100 mesh to obtain sunflower seed defatted protein powder; highland barley flour and sunflower seed defatted protein powder are mixed in a mass ratio of 7:3, and 10 volumes of deionized water are added; the pH value of the suspension is adjusted to 8.0, the temperature is controlled to 40° C., 0.5% of the substrate mass and alkaline protease with an enzyme activity of ≥150 U / mg are added, and the stirring speed is 200 rpm for 30 min to carry out the first stage of enzymolysis; then the system pH is maintained at 6.5, the temperature is controlled to 55° C., 0.2% of the substrate mass and papain with an enzyme activity of ≥2000 U / mg are added, and the stirring speed is 150 rpm for 90 min to carry out the second stage of enzymolysis; the temperature is raised to 85° C., maintained for 10 min to inactivate the enzyme, the enzymolysis solution is centrifuged at 4000 rpm for 20 min, the solid matter is removed, and the supernatant is collected to obtain a polypeptide solution;

[0066] (2) Wash and chop the olive leaves, suspend them in a buffer solution PBS, homogenize them, centrifuge them at 3000 rpm for 25 min, pre-filter them through a 0.22 μm filter membrane to obtain a supernatant, add the supernatant to a tangential flow filtration system with a membrane pore size of 0.2 μm, a pump flow rate of 100 mL / min, and a pressure of 1 bar to obtain olive leaf exosomes;

[0067] (3) The peptide solution with a concentration of 5 mg / mL was mixed with a solution with a concentration of 3×10 9 / mL of olive leaf exosomes were mixed evenly, put into an ice bath, and ultrasonicated at 50W for 3 minutes by segmented ultrasonication, allowed to stand for 5 minutes, and then ultrasonicated at 50W for 2 minutes to encapsulate the polypeptide solution in the olive leaf exosomes to obtain the exosome complex.

[0068] Example 3

[0069] This embodiment provides a method for preparing an exosome complex, comprising:

[0070] (1) After shelling highland barley, the highland barley is crushed to a particle size of 80 mesh, and the bran is removed to obtain highland barley flour; sunflower seeds are defatted by low-temperature pressing and crushed to a particle size of 100 mesh to obtain sunflower seed defatted protein powder; highland barley flour and sunflower seed defatted protein powder are mixed in a mass ratio of 7:3, and 10 volumes of deionized water are added; the pH value of the suspension is adjusted to 8.0, the temperature is controlled to 40° C., 0.5% of the substrate mass and alkaline protease with an enzyme activity of ≥150 U / mg are added, and the stirring speed is 200 rpm for 30 min to carry out the first stage of enzymolysis; then the system pH is maintained at 6.5, the temperature is controlled to 55° C., 0.2% of the substrate mass and papain with an enzyme activity of ≥2000 U / mg are added, and the stirring speed is 150 rpm for 90 min to carry out the second stage of enzymolysis; the temperature is raised to 85° C., maintained for 10 min to inactivate the enzyme, the enzymolysis solution is centrifuged at 4000 rpm for 20 min, the solid matter is removed, and the supernatant is collected to obtain a polypeptide solution;

[0071] (2) Wash and chop the olive leaves, suspend them in a buffer solution PBS, homogenize them, centrifuge them at 3000 rpm for 25 min, pre-filter them through a 0.22 μm filter membrane to obtain a supernatant, add the supernatant to a tangential flow filtration system with a membrane pore size of 0.2 μm, a pump flow rate of 100 mL / min, and a pressure of 1 bar to obtain olive leaf exosomes;

[0072] (3) The peptide solution with a concentration of 5 mg / mL was mixed with a solution with a concentration of 3×10 9 / mL of olive leaf exosomes were mixed evenly, put into an ice bath, and ultrasonicated at 30W for 3 minutes by segmented ultrasonic method, let it stand for 5 minutes, and then ultrasonicated at 50W for 2 minutes to encapsulate the polypeptide solution in the olive leaf exosomes to obtain the exosome complex.

[0073] Comparative Example 1

[0074] This comparative example provides a method for preparing an exosome complex, which is different from Example 1 only in that highland barley flour and sunflower seed defatted protein powder are hydrolyzed at a pH of 8.5 and a temperature of 50° C. for 120 min using only 0.7% of the substrate mass of alkaline protease.

[0075] Comparative Example 2

[0076] This comparative example provides a method for preparing an exosome complex, which is different from Example 1 only in that highland barley and sunflower seed raw materials are replaced with soybeans and wheat.

[0077] Comparative Example 3

[0078] This comparative example provides a method for preparing an exosome complex, which is different from Example 1 only in that the olive leaf raw material is replaced by grape leaves.

[0079] Comparative Example 4

[0080] This comparative example provides a method for preparing an exosome complex, which is different from Example 1 only in that when the polypeptide solution is encapsulated in olive leaf exosomes, continuous ultrasound is applied at room temperature and 100 W for 5 min.

[0081] Table 1 Preparation methods of Examples 1-3 and Comparative Examples 1-4

[0082]

[0083]

[0084]

[0085]

[0086] Note: “ / ” in Table 1 means that the component or step is not included.

[0087] Test Example 1

[0088] In this test example, the polypeptide solutions extracted from Examples 1-3 and Comparative Examples 1-2 were tested for polypeptide extraction rate, polypeptide solution molecular weight, and the number of polypeptide species. The specific method is as follows:

[0089] (1) Take the undigested raw material suspension, centrifuge at 10000 rpm for 10 min, take the supernatant, and operate according to the instructions of the BCA protein quantification kit (Thermo Fisher) to prepare a bovine serum albumin (BSA) standard curve (0-2000 μg / mL), measure the sample absorbance, and calculate the total protein concentration (C) of the raw material; the soluble polypeptide concentration (C) of the polypeptide solution is determined in the same way; polypeptide extraction rate (%) = (C×V) / (C×W)×100, where V is the volume of the polypeptide solution (mL) and W is the mass of the raw material (g).

[0090] (2) Determination of average molecular weight of peptide solution: The peptides were separated by size exclusion chromatography (SEC-HPLC) and the molecular weight was estimated based on the retention time.

[0091] (3) Quantitative analysis of peptide species: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to identify peptide sequences.

[0092] The drug loading capacity of the exosome complexes prepared in Examples 1-3 and Comparative Examples 1-4 was determined. The specific method was as follows: after ultrafiltration to remove unencapsulated polypeptides, the exosome concentration was determined using NanoSight NS300; drug loading capacity = total polypeptide amount (μg) / number of exosomes (10 10 EVs).

[0093] The results are shown in Table 2.

[0094] Table 2 Analysis of peptide solutions and drug loading of exosome complexes

[0095]

[0096]

[0097] As shown in Table 2, Example 1-3 uses highland barley flour and sunflower seed defatted protein powder, and uses alkaline protease and papain for two-stage enzymolysis in sequence, and the obtained polypeptide solution has a high polypeptide extraction rate, rich polypeptide species, and a molecular weight of less than 5000 Daltons. And Example 1-3 uses olive leaf exosomes to encapsulate polypeptides extracted from highland barley flour and sunflower seed powder by segmented ultrasound at low temperature, and its drug loading capacity is high, which can make the polypeptide solution efficiently and stably encapsulated in exosomes. Comparing Example 1 and Example 2-3, when performing segmented ultrasound in Example 1, high-power ultrasound can quickly open the membrane pores of the exosomes, improve the drug loading efficiency, and subsequently reduce the ultrasonic power to maintain the membrane open state of the exosomes, reduce membrane damage, and thus more conducive to the efficient and stable encapsulation of the polypeptide solution in olive leaf exosomes.

[0098] Test Example 2

[0099] In this test example, the DPPH clearance rate of the exosome complexes prepared in Examples 1-3 and Comparative Examples 1-4 was measured. The results are shown in Table 3.

[0100] Table 3 DPPH clearance rate of exosome complexes

[0101]

[0102]

[0103] From the results in Table 3, it can be seen that the exosome complex prepared in Examples 1-3 has a high DPPH scavenging rate, which indicates that the exosome complex prepared by the preparation method provided in the present application has a good antioxidant effect.

[0104] Test Example 3

[0105] This test example uses the MTT method to test the effect of the exosome complex prepared in Examples 1-3 and Comparative Examples 1-4 on the proliferation activity of human immortalized keratinocytes (HaCaT), and the specific method is: HaCaT cells at a certain concentration are inoculated in a 96-well plate, incubated for 24 hours, and the solution in the well is removed. The exosome complex is diluted to a concentration of 1% with DMEM, and placed in an incubator for 24 hours. The culture solution without the exosome complex sample is used as a control, and 20 μL of MTT solution is added. Continue to culture for 2 hours, remove the culture medium, add 100 μL of isopropanol to each well, shake at 100 rpm for 30 minutes under 25 ° C light-shielding conditions, and use an enzyme marker to measure the absorbance at 570 nm to detect cell survival rate; wherein, the MTT solution is 250 mg of thiazolyl blue (CAS: 298-93-1) dissolved in 50 mL of DPBS and then filtered and sterilized through a 0.22 μm filter. The results are shown in Table 4.

[0106] Table 4 Effects of exosome complexes on the proliferation activity of HaCaT cells

[0107] Group HaCaT cell viability (%) Blank control group 100 Example 1 145.6 Example 2 132.2 Example 3 130.7 Comparative Example 1 119.2 Comparative Example 2 124.6 Comparative Example 3 123.5 Comparative Example 4 121.3

[0108] From the results in Table 4, it can be seen that the exosome complex prepared in Examples 1-3 has a significant improvement on the proliferation activity of human immortalized keratinocytes. HaCat cells are an important component of the epidermis of the skin, indicating that the exosome complex prepared by the preparation method provided in this application has a good repair and improvement effect on the skin.

[0109] Test Example 4

[0110] In this test example, the exosome complexes prepared in Examples 1-3 and Comparative Examples 1-4 were tested for soothing and anti-inflammatory activity. The specific method was as follows: Raw264.7 cells were inoculated in a 24-well plate at a certain concentration and incubated at 37°C and 5% CO 2 After culturing for 24 hours under the conditions, the culture medium in the wells was removed and then DMEM culture medium containing LPS (sigma, 025M4040V, 10,000 units / mL) and each exosome complex sample at a concentration of 5% was added, and dexamethasone (DEX, 10 μM) was added as a positive control group, and the culture was continued for 24 hours. The inflammatory factors PGE2 and NO were detected using a kit purchased from Cayman. The results are shown in Table 5.

[0111] Table 5 Inhibition rate of NO by exosome complexes

[0112] Group NO inhibition rate (%) Positive control group (DEX) 35.2±2.8 Example 1 74.3±2.5 Example 2 72.2±2.1 Example 3 71.5±1.9 Comparative Example 1 46.5±3.0 Comparative Example 2 58.1±2.8 Comparative Example 3 64.2±2.4 Comparative Example 4 67.8±2.2

[0113] From the results in Table 5, it can be seen that the exosome complex prepared in Examples 1-3 has a high inhibition rate on NO produced by cells, which indicates that the exosome complex prepared by the preparation method provided in the present application has good soothing and anti-inflammatory activity.

[0114] Test Example 5

[0115] In this test example, the exosome complexes prepared in Examples 1-3 and Comparative Examples 1-4 were tested for anti-aging efficacy. The specific method was as follows: a certain concentration of HSF cells were inoculated in a 24-well plate, incubated for 24 hours, the culture medium in the wells was removed, PBS was added, and UVA (365nm) irradiation dose was 10J / cm 2 After removing PBS, DMEM medium containing 5% of each exosome complex sample was added, and the non-irradiated group was used as a blank control, and the medium without exosome complex sample was added as a model group. The cells were placed in an incubator and cultured for 24 hours. The qPCR method was used to detect the effect on the mRNA expression levels of HSF cell aging marker genes COL-1 and MMP-1; the mRNA expression level of the blank control group was set as 1, and the relative mRNA expression level of each test group was calculated. The results are shown in Table 6.

[0116] Table 6 Effect of exosome complexes on mRNA expression levels of senescence marker genes in HSF cells

[0117]

[0118] As shown in Table 6, compared with the model group, the exosome complex prepared in Examples 1-3 significantly increased the mRNA expression level of COL-1 in cells and significantly reduced the mRNA expression level of MMP-1, indicating that the exosome complex prepared by the preparation method provided in the present application has a good anti-aging effect.

[0119] Implementation Group

[0120] This implementation group added the exosome complex provided in Example 1 to the basic cream at a mass percentage of 1% to obtain a skin care product containing 1% of the exosome complex (the exosome complex provided in Example 1 replaced the glycerin in the basic cream). The basic cream includes the components shown in Table 7.

[0121] Table 7 Basic facial cream

[0122]

[0123] Test Example 6

[0124] In this test example, the skin care products containing 1% exosome complex were tested for clinical moisturizing and repairing ability in the implementation group, and the basic face cream was used in the control group. The TTest method was used to calculate the skin moisture content and transepidermal water loss rate (TEWL) of the skin care products containing 1% exosome complex and the basic face cream in the control group on the 0th, 7th and 14th days after use. The results are as follows: Figure 1 and Figure 2 shown.

[0125] Depend on Figure 1 The results show that there was no significant change in the skin moisture content of the control group after 7d and 14d of use, indicating that the basic cream does not have a significant moisturizing effect. The skin care product sample containing 1% exosome complex can significantly increase the skin moisture content after 7d of continuous use (**P<0.01), and the skin moisture content continues to increase after 14d of use (***P<0.001), which has a continuous moisturizing effect. Compared with day 0, the skin moisture content of the skin care product sample containing 1% exosome complex increased by 36.45% at most, indicating that the exosome complex provided by this application is used in skin care products and has a significant moisturizing effect.

[0126] Depend on Figure 2 The results show that there was no significant change in the transepidermal water loss rate of the control group after 7d and 14d of use, indicating that the basic cream does not have an obvious repair effect. The skin care product sample containing 1% exosome complex can significantly reduce the transepidermal water loss rate of the skin after 7d of continuous use (**P<0.01), and the transepidermal water loss rate of the skin after 14d of use continues to decrease (***P<0.001). Compared with the 0th day, the transepidermal water loss rate of the skin on the 7th and 14th days of the skin care product sample containing 1% exosome complex decreased by 9.81% and 24.98% respectively, indicating that the exosome complex provided by this application is used in skin care products and has a good repair effect.

[0127] Test Example 7

[0128] In this test example, the skin care products containing 1% exosome complex were used in the implementation group to test the clinical soothing effect. The control group used basic cream, and the model group used capsaicin to stimulate the skin. Then, the skin care product samples containing 1% exosome complex were used, and the erythema value and transepidermal water loss rate of the skin were tested. The results are as follows Figure 3 and Figure 4 shown.

[0129] Depend on Figure 3The results show that the control group does not stimulate the skin to cause erythema. Compared with the control group, the erythema value of the skin in the model group increased significantly after capsaicin was used to stimulate the skin, and the erythema value was 40.9, indicating that the skin barrier was obviously damaged under the stimulation of external conditions. Compared with the model group, the use of skin care products containing 1% exosome complexes can reduce the increase in erythema values ​​caused by capsaicin stimulation, and the erythema value is 34.1, which is 16.63% lower than that of the model group (P<0.01). It shows that the exosome complex provided in this application is used in skin care products, which can alleviate the increase in skin erythema values ​​caused by capsaicin stimulation and has the effect of repairing skin irritation.

[0130] Depend on Figure 4 The results show that compared with the control group, the transepidermal water loss rate of the model group was significantly increased after capsaicin was used to stimulate the skin (P < 0.001), indicating that the stimulation conditions were effective. Compared with the model group, the use of skin care products containing 1% exosome complexes can reduce the increase in transepidermal water loss rate caused by capsaicin stimulation. The transepidermal water loss rate was 15.3, which was 28.39% lower than that of the model group (P < 0.01). It shows that the exosome complex provided in this application is used in skin care products, which can alleviate the increase in transepidermal water loss rate of the skin caused by capsaicin stimulation and has the effect of repairing skin irritation.

[0131] Test Example 8

[0132] In this test example, the skin care products containing 1% exosome complex were tested for their anti-wrinkle and firming efficacy in the implementation group, and the basic cream was used in the control group. The skin elasticity of the skin care products containing 1% exosome complex and the basic cream in the control group was tested before use, 2 weeks after use, and 4 weeks after use. The results are as follows: Figure 5 shown.

[0133] Depend on Figure 5 The results show that compared with before use, the skin elasticity parameter R2 of the basic cream did not change significantly after 2 and 4 weeks of use; while the skin elasticity parameter R2 of the skin care product sample containing 1% exosome complex increased significantly after 2 and 4 weeks of use (P<0.05). This shows that the exosome complex provided by the present application has anti-wrinkle and firming effects when used in skin care products.

[0134] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A method for preparing an exosome complex, characterized in that: include: (1) Highland barley and sunflower seeds are crushed and mixed respectively, and then enzymatically hydrolyzed in two stages using alkaline protease and papain in sequence to obtain a polypeptide solution; (2) crushing the olive leaves, homogenizing with a buffer solution, and then separating and purifying to obtain olive leaf exosomes; (3) Encapsulating the polypeptide solution in the olive leaf exosomes by segmented ultrasound at 0 to -4°C to obtain an exosome complex.

2. The preparation method according to claim 1, characterized in that: The highland barley flour obtained by crushing the highland barley and the sunflower seed flour obtained by crushing the sunflower seeds are mixed in a mass ratio of (6:4) to (8:2).

3. The preparation method according to claim 1, characterized in that: The amount of alkaline protease added is 0.3% to 0.7% of the substrate mass, and the enzyme activity is ≥150U / mg; And / or, the added amount of papain is 0.1% to 0.3% of the substrate mass, and the enzyme activity is ≥2000U / mg.

4. The preparation method according to claim 3, characterized in that: The enzymatic hydrolysis of the alkaline protease is carried out at a pH value of 7.5 to 8.5, a temperature of 40 to 50° C., and a stirring rate of 150 to 250 rpm for 25 to 35 minutes; the enzymatic hydrolysis of the papain is carried out at a pH value of 6.3 to 6.7, a temperature of 50 to 60° C., and a stirring rate of 120 to 180 rpm for 80 to 100 minutes.

5. The preparation method according to claim 1, characterized in that: The average particle size of the olive leaf exosomes is 50 to 200 nm.

6. The preparation method according to claim 1, characterized in that: The separation and purification in step (2) includes centrifugation at 3000-5000 rpm for 10-25 min, pre-filtration on a 0.1-0.22 μm filter membrane, and tangential flow filtration in a tangential flow filtration system with a membrane pore size of 0.1-0.2 μm, a pump flow rate of 50-200 mL / min, and a pressure of 0.2-2 bar.

7. The preparation method according to claim 1, characterized in that: The concentration of the polypeptide solution is 1-10 mg / mL and the concentration of the olive leaf exosomes is 1-5×10 9 / mL for mixing.

8. The preparation method according to claim 7, characterized in that: The segmented ultrasonic method includes ultrasonic treatment at 30-100W for 1-5 minutes, standing for 3-10 minutes, and then ultrasonic treatment at 10-50W for 1-5 minutes; and the ultrasonic power of the front section is greater than that of the back section.

9. An exosome complex prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the exosome complex according to claim 9 in preparing skin care products.

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