A preparation method of exosomes with antioxidant and whitening effects, and its products and applications

Abstract

The present invention relates to a method for preparing exosomes with antioxidant and whitening effects, and its products and applications. The preparation method comprises: (1) crushing plant raw materials, then mixing them in water, adding a composite enzyme for enzymolysis, and obtaining an enzymolysis solution; (2) performing solid-liquid separation on the enzymolysis solution and obtaining a supernatant; (3) mixing the supernatant of step (2) with polyglycerol esters, standing and performing solid-liquid separation, obtaining a supernatant, and then freeze-drying to obtain the exosomes; the plant raw materials include Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus. Compared with the traditional method for preparing plant extracts, the preparation method of the present invention uses a composite enzyme to enzymolyze the plant raw materials, and then uses a specific separation procedure to maximize the extraction of active ingredients, thereby making the whitening and antioxidant effects of the final product better.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and specifically relates to a preparation method of exosomes with antioxidant and whitening effects, and a product and application thereof. Background Art

[0002] Plant exosomes are a type of membrane vesicles isolated from plant tissues, containing proteins, lipids, nucleic acids and other components. They are similar in structure to animal exosomes, and many common plants in life, such as algae, ginger, and Centella asiatica, contain a large number of exosomes. Plant exosomes have the effects of enhancing cell vitality, anti-aging, moisturizing, whitening, and promoting skin repair and regeneration. Compared with conventional cosmetic components, they are milder, safer for the skin, and have fewer side effects. Therefore, they are increasingly used in the cosmetics field.

[0003] CN117327153A discloses a method for preparing an active peptide and plant exosome anti-aging cosmetic. The present invention provides for the first time a polypeptide having the activity of stimulating fibroblast proliferation. After the active polypeptide is introduced into pomegranate exosomes, it can significantly promote the proliferation of fibroblasts and the secretion of collagen, delay skin aging, and promote wound healing.

[0004] CN117414327A discloses an exosome composition, which is obtained by mixing cucurbitacin exosomes, cnidium monnieri exosomes and mesenchymal stem cell exosomes in a mass ratio of 2-5:3-8:0.5-1. Cucurbitacin exosomes can significantly reduce the concentration of inflammatory factors, thereby inhibiting excessive sebum secretion and improving pores; cnidium monnieri exosomes also synergize with cucurbitacin exosomes to strengthen the normal differentiation of keratinocytes, effectively alleviate the excessive stratum corneum multilayering caused by excessive sebum secretion, thereby inhibiting the increase in hair follicle volume. After the combination of mesenchymal stem cell exosomes and plant exosomes, it can significantly improve the elasticity of the skin and improve the problem of skin sagging, thereby making the pores more delicate and visually significantly reducing the pores. The exosome composition of the present invention has the effect of significantly improving enlarged pores and increasing skin elasticity, can be made into a variety of product forms, and has a wide range of application prospects.

[0005] However, although the above-mentioned prior art discloses the application of plant exosomes in skin care and also discloses a method for preparing exosomes, the plant exosomes obtained by the method have fewer active ingredients and lower purity, and therefore cannot fully exert the efficacy of the plant exosomes. Therefore, it is very meaningful to develop a method for preparing plant exosomes with simple steps and more significant product efficacy. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing exosomes with antioxidant and whitening effects, as well as a product and application thereof.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing exosomes with antioxidant and whitening effects, the preparation method comprising:

[0009] (1) crushing the plant raw material, mixing it in water, adding a composite enzyme to perform enzymolysis, and obtaining an enzymolysis solution;

[0010] (2) separating the enzymatic hydrolyzate into solid and liquid and taking the supernatant;

[0011] (3) mixing the supernatant of step (2) with polyglycerol esters, allowing the mixture to stand for solid-liquid separation, taking the supernatant, and freeze-drying the mixture to obtain the exosomes;

[0012] The plant raw materials include microscopic chlorella, black truffle, white truffle and Agaricus bisporus.

[0013] Preferably, the mass ratio of the microscopic chlorella, black truffle, white truffle and Agaricus bisporus is (1-5):(0.5-1.5):(1-2):(2-4).

[0014] Wherein "1-5" may be 1.1, 1.3, 1.5, 1.7, 2, 2.3, 2.5, 2.7, 3, 3.1, 3.3, 3.5, 3.7, 3.9, 4, 4.2, 4.5, 4.7 or 4.9, etc.;

[0015] “0.5-1.5” can mean 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3 or 1.4, etc.;

[0016] "1-2" can be 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9, etc.;

[0017] “2-4” can be 2.1, 2.3, 2.5, 2.7, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9, etc.

[0018] Preferably, the material-liquid ratio of the plant raw material to water is 0.1-0.5 g / mL, for example, it can be 0.15 g / mL, .2 g / mL, 0.25 g / mL, .3 g / mL, 0.35 g / mL, 0.4 g / mL or 0.45 g / mL, etc.

[0019] Preferably, the added amount of the complex enzyme is 1-5% by weight of the plant raw material, for example, it can be 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5% or 4.8%, etc.

[0020] Preferably, the pH of the enzymatic hydrolysis treatment is 5-7, for example, it can be 5.2, 5.5, 5.8, 6, 6.2, 6.5 or 6.8.

[0021] Preferably, the temperature of the enzymatic hydrolysis treatment is 25-40°C, for example, it can be 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C or 39°C.

[0022] Preferably, the enzymatic treatment time is 3-6 h, for example, it can be 3.2 h, 3.5 h, 3.8 h, 4 h, 4.2 h, 4.5 h, 4.8 h, 5 h, 5.2 h, 5.5 h or 5.8 h.

[0023] Preferably, the complex enzyme comprises a combination of at least two of protease, amylase, cellulase, lipase, deaminase, β-glucanase, xylanase or pectinase.

[0024] Preferably, the complex enzyme is a combination of protease, amylase, deaminase and pectinase.

[0025] Preferably, the mass ratio of the protease, amylase, deaminase and pectinase is (1-4):(1-5):(1-2):(1-3).

[0026] Wherein "1-4" may be 1.1, 1.3, 1.5, 1.7, 1.9, 2, 2.2, 2.5, 2.7, 2.9, 3, 3.1, 3.3, 3.5, 3.7 or 3.9, etc.;

[0027] “1-5” can be 1.1, 1.3, 1.5, 1.7, 2, 2.3, 2.5, 2.7, 3, 3.1, 3.3, 3.5, 3.7, 3.9, 4, 4.2, 4.5, 4.7 or 4.9, etc.;

[0028] "1-2" can be 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9, etc.;

[0029] “1-3” can be 1.1, 1.3, 1.5, 1.7, 1.9, 2, 2.2, 2.5, 2.7 or 2.9, etc.

[0030] Preferably, the solid-liquid separation in step (2) is performed 1-3 times, for example, once, twice or 3 times.

[0031] Preferably, the solid-liquid separation in step (2) is centrifugal separation, and the temperature during each solid-liquid separation is independently 5-15°C (for example, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C or 14°C, etc.), and the centrifugal speed is independently 1000-2000rpm (for example, 1100rpm, 1300rpm, 1500rpm, 1700rpm or 1900rpm, etc.).

[0032] Preferably, the solid-liquid separation in step (3) is centrifugal separation, the temperature during the solid-liquid separation is 15-25°C (for example, it can be 16°C, 18°C, 20°C, 22°C or 24°C, etc.), and the centrifugal speed is 1000-2000rpm (for example, it can be 1100rpm, 1300rpm, 1500rpm, 1700rpm or 1900rpm, etc.).

[0033] Preferably, the polyglycerol esters include any one of polyglyceryl-2 laurate, polyglyceryl-3 methylsaccharide distearate, polyglyceryl-6 laurate, polyglyceryl-10 laurate, polyglyceryl-6 oleate or polyglyceryl-6 caprylate, or a combination of at least two thereof.

[0034] Preferably, the polyglycerol esters are polyglycerol-6 oleate.

[0035] Preferably, the volume ratio of the polyglycerol esters to the supernatant of step (2) is 1:(5-15), for example, it can be 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 or 1:14, etc.

[0036] Preferably, the standing time in step (3) is 8-15 hours.

[0037] Preferably, the freeze-drying step in step (2) is: first cooling the supernatant after the third solid-liquid separation, and then drying it by gradient heating under vacuum.

[0038] Preferably, the temperature is lowered to -30 to -35°C, for example, -31°C, -32°C, -33°C or -34°C.

[0039] Preferably, the cooling rate is 0.5-1°C / min, for example, 0.6°C / min, 0.7°C / min, 0.8°C / min or 0.9°C / min.

[0040] Preferably, the step of gradient temperature increase is: heating from -30 to -35°C to -25 to -15°C (for example, -16°C, -18°C, -20°C, -22°C or -24°C, etc.), maintaining for 1-3h (for example, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, etc.); heating from -25 to -15°C to -10 to -5°C (for example, -6°C, -7°C, -8°C or -9°C, etc.), maintaining for 1-3h (for example, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, etc.); heating from -10 to -5°C to 0-5°C (for example, For example, it may be 1°C, 2°C, 3°C or 4°C, etc.), maintained for 1-3h (for example, it may be 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, etc.); the temperature is increased from 0-5°C to 20-30°C (for example, it may be 21°C, 23°C, 25°C, 27°C or 29°C, etc.), maintained for 1-3h (for example, it may be 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, etc.), and the heating rates are independently 1-1.5°C / min (for example, it may be 1.1°C / min, 1.2°C / min, 1.3°C / min or 1.4°C / min, etc.).

[0041] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0042] In a second aspect, the present invention provides an exosome with antioxidant and whitening effects, wherein the exosome with antioxidant and whitening effects is prepared by the method described in the first aspect.

[0043] In a third aspect, the present invention provides a use of the exosomes with antioxidant and whitening effects as described in the second aspect in the preparation of cosmetics.

[0044] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) Compared with the traditional method for preparing plant extracts, the preparation method of the present invention uses a composite enzyme to enzymatically hydrolyze the plant raw materials, and then uses a specific separation procedure to maximize the extraction of active ingredients, thereby making the whitening and antioxidant effects of the final product better.

[0047] (2) The plant raw materials selected by the present invention have a high content of polyphenols, which can inhibit pigment synthesis and transport, and there is a synergistic effect between the microscopic algae, black truffle, white truffle and Agaricus bisporus, so that the final product has excellent antioxidant and whitening effects.

[0048] (3) The components of the complex enzyme selected in the present invention can synergize with each other, which can change the polysaccharides and polyphenols in the plant raw materials, enhance their biological activity, and reduce the active ingredients from high molecular weight to low molecular weight, thereby making them easier to be absorbed by the human body.

[0049] (4) The preparation method of the present invention has simple steps and does not use organic solvents during the preparation process, which is more environmentally friendly and suitable for industrial production. DETAILED DESCRIPTION

[0050] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0051] The following materials and comparative examples are as follows:

[0052]

[0053] The rest of the raw materials can be used as long as they are purchased from regular dealers.

[0054] Example 1

[0055] This embodiment provides an exosome with antioxidant and whitening effects, wherein the exosome is prepared by the following method:

[0056] (1) Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus in a mass ratio of 4:4:1.5:3 are crushed respectively, then mixed in water (the solid-liquid ratio to the raw material is 0.3 g / mL), a composite enzyme of 3% of the total amount of the plant raw material is added, the pH of the system is adjusted to 6, and enzymolysis is performed at 30° C. for 4 hours to obtain an enzymolysis solution, wherein the composite enzyme is a protease, an amylase, a deaminase and a pectinase in a mass ratio of 3:3:1.5:2;

[0057] (2) subjecting the enzymatic hydrolysate to a first solid-liquid separation at 8° C. and 1500 rpm to remove the solids, and then subjecting the enzymatic hydrolysate to a second solid-liquid separation at 12° C. and 1800 rpm to obtain the supernatant;

[0058] (3) The supernatant of step (2) was mixed with polyglycerol-6 oleate in a volume ratio of 10:1, and after standing for 12 hours, solid-liquid separation was performed for the third time at 20° C. and 1500 rpm, and the supernatant was taken;

[0059] (4) The supernatant obtained in step (3) is cooled to -35°C at a rate of 0.8°C / min, and then heated from -35 to -20°C under vacuum and maintained for 2h; heated from -20°C to -5°C and maintained for 2h; heated from -5°C to 5°C and maintained for 2h; heated from 5°C to 25°C and maintained for 2h, wherein the heating rates are independently 1.2°C / min, to obtain the exosomes.

[0060] Example 2

[0061] This embodiment provides an exosome with antioxidant and whitening effects, wherein the exosome is prepared by the following method:

[0062] (1) Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus in a mass ratio of 5:0.5:2:2 are crushed respectively, then mixed in water (the solid-liquid ratio to the raw material is 0.5 g / mL), a composite enzyme of 2% of the total amount of the plant raw material is added, the pH of the system is adjusted to 6.5, and enzymolysis is performed at 35° C. for 3 h to obtain an enzymatic solution, wherein the composite enzyme is a protease, an amylase, a deaminase and a pectinase in a mass ratio of 4:1:2:1;

[0063] (2) subjecting the enzymatic hydrolysate to a first solid-liquid separation at 10° C. and 2000 rpm to remove the solids, and then subjecting the enzymatic hydrolysate to a second solid-liquid separation at 15° C. and 1000 rpm to obtain the supernatant;

[0064] (3) mixing the supernatant of step (2) with polyglycerol-6 oleate in a volume ratio of 5:1, standing for 8 hours, and performing a third solid-liquid separation at 15° C. and 2000 rpm to obtain the supernatant;

[0065] (4) The supernatant obtained in step (3) is cooled to -33°C at a rate of 1°C / min, and then heated from -33°C to -15°C under vacuum and maintained for 3 hours; heated from -15°C to -10°C and maintained for 1 hour; heated from -10°C to 3°C and maintained for 1 hour; heated from 3°C to 28°C and maintained for 1 hour, wherein the heating rates are independently 1.5°C / min, to obtain the exosomes.

[0066] Example 3

[0067] This embodiment provides an exosome with antioxidant and whitening effects, wherein the exosome is prepared by the following method:

[0068] (1) Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus in a mass ratio of 1:1.5:1:4 are crushed respectively, then mixed in water (the solid-liquid ratio to the raw material is 0.2 g / mL), a composite enzyme of 5% of the total amount of the plant raw material is added, the pH of the system is adjusted to 5, and enzymolysis is performed at 25° C. for 5 h to obtain an enzymolysis solution, wherein the composite enzyme is a protease, an amylase, a deaminase and a pectinase in a mass ratio of 1:5:1:3;

[0069] (2) subjecting the enzymatic hydrolysate to a first solid-liquid separation at 5°C and 1000 rpm to remove the solids, and then subjecting the enzymatic hydrolysate to a second solid-liquid separation at 10°C and 2000 rpm to obtain the supernatant;

[0070] (3) mixing the supernatant of step (2) with polyglycerol-6 oleate in a volume ratio of 15:1, standing for 15 hours, performing a third solid-liquid separation at 25° C. and 1000° C., and taking the supernatant;

[0071] (4) The supernatant obtained in step (3) is cooled to -30°C at a rate of 0.5°C / min, and then heated from -30 to -22°C under vacuum and maintained for 1 hour; heated from -22°C to -12°C and maintained for 3 hours; heated from -12°C to 0°C and maintained for 3 hours; heated from 0°C to 20°C and maintained for 3 hours, wherein the heating rates are independently 1°C / min, to obtain the exosomes.

[0072] Example 4

[0073] This embodiment provides an exosome with antioxidant and whitening effects. The exosome is different from that in Example 1 only in that the plant raw materials are microscopic chlorella, black truffle and white truffle in a mass ratio of 4:4:1.5, and the amount of the plant raw materials is kept unchanged. The remaining steps and raw material amounts are consistent with those in Example 1.

[0074] Example 5

[0075] This embodiment provides an exosome with antioxidant and whitening effects. The exosome is different from that in Example 1 only in that the plant raw materials are Chlorella microsporus, black truffle and Agaricus bisporus in a mass ratio of 4:4:3, and the amount of the plant raw materials is kept unchanged. The remaining steps and raw material amounts are consistent with those in Example 1.

[0076] Example 6

[0077] This embodiment provides an exosome with antioxidant and whitening effects. The exosome is different from that in Example 1 only in that the plant raw materials are Chlorella vulgaris, white truffle and Agaricus bisporus in a mass ratio of 4:1.5:3, and the amount of the plant raw materials is kept unchanged. The remaining steps and raw material amounts are consistent with those in Example 1.

[0078] Example 7

[0079] This embodiment provides an exosome with antioxidant and whitening effects. The exosome is different from that in Example 1 only in that the plant raw materials are black truffle, white truffle and Agaricus bisporus in a mass ratio of 4:1.5:3, and the amount of the plant raw materials is kept unchanged. The remaining steps and raw material amounts are consistent with those in Example 1.

[0080] Example 8

[0081] This embodiment provides an exosome with antioxidant and whitening effects. The difference between the exosome and Example 1 is that the composite enzyme is a protease, an amylase and a deaminase in a mass ratio of 3:3:1.5, and the amount of the composite enzyme is kept unchanged. The remaining steps and the amount of raw materials are consistent with Example 1.

[0082] Example 9

[0083] This embodiment provides an exosome with antioxidant and whitening effects. The exosome is different from that in Example 1 only in that the compound enzyme is a protease, an amylase and a pectinase in a mass ratio of 3:3:2, and the dosage of the compound enzyme is kept unchanged. The remaining steps and the dosage of raw materials are consistent with those in Example 1.

[0084] Example 10

[0085] This embodiment provides an exosome with antioxidant and whitening effects. The exosome is different from that in Example 1 only in that the composite enzyme is a protease, a deaminase and a pectinase in a mass ratio of 3:1.5:2, and the amount of the composite enzyme is kept unchanged. The remaining steps and the amount of raw materials are consistent with those in Example 1.

[0086] Embodiment 11

[0087] This embodiment provides an exosome with antioxidant and whitening effects. The exosome is different from that in Example 1 only in that the compound enzyme is amylase, deaminase and pectinase in a mass ratio of 3:1.5:2, and the amount of the compound enzyme is kept unchanged. The remaining steps and the amount of raw materials are consistent with those in Example 1.

[0088] Example 12

[0089] This embodiment provides an exosome with antioxidant and whitening effects. The difference between the exosome and Example 1 is that step (3) is: the supernatant of step (2) is allowed to stand for 12 hours and then subjected to a third solid-liquid separation at 20° C. and 1500 rpm to obtain the supernatant. The remaining steps and raw material amounts are the same as those in Example 1.

[0090] Comparative Example 1

[0091] This comparative example provides an exosome with antioxidant and whitening effects, and the exosome is prepared by the following method:

[0092] (1) Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus in a mass ratio of 4:4:1.5:3 are crushed respectively, and then mixed in water (the material-liquid ratio to the raw material is 0.3 g / mL) to obtain a raw material liquid;

[0093] (2) subjecting the raw liquid to solid-liquid separation at 8° C. and 1500 rpm to remove the solid, and then subjecting the raw liquid to a second solid-liquid separation at 12° C. and 1800 rpm to obtain the supernatant;

[0094] (3) The supernatant of step (2) was mixed with polyglycerol-6 oleate in a volume ratio of 10:1, and after standing for 12 hours, solid-liquid separation was performed for the third time at 20° C. and 1500 rpm, and the supernatant was taken;

[0095] (4) The supernatant obtained in step (3) is cooled to -35°C at a rate of 0.8°C / min, and then heated from -35 to -20°C under vacuum and maintained for 2h; heated from -20°C to -5°C and maintained for 2h; heated from -5°C to 5°C and maintained for 2h; heated from 5°C to 25°C and maintained for 2h, wherein the heating rates are independently 1.2°C / min, to obtain the exosomes.

[0096] Comparative Example 2

[0097] This comparative example provides an exosome with antioxidant and whitening effects, and the exosome is prepared by the following method:

[0098] (1) Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus in a mass ratio of 4:4:1.5:3 are crushed respectively, then mixed in water (the solid-liquid ratio to the raw material is 0.3 g / mL), a composite enzyme of 3% of the total amount of the plant raw material is added, the pH of the system is adjusted to 6, and enzymolysis is performed for 4 hours to obtain an enzymolysis solution, wherein the composite enzyme is a protease, an amylase, a deaminase and a pectinase in a mass ratio of 3:3:1.5:2;

[0099] (2) subjecting the enzymatic hydrolysate to solid-liquid separation at 8° C. and 1500 rpm, removing the solid and obtaining the supernatant;

[0100] (3) The supernatant of step (2) was mixed with polyglycerol-6 oleate in a volume ratio of 10:1, and after standing for 12 hours, a second solid-liquid separation was performed at 20° C. and 1500 rpm to obtain the supernatant;

[0101] (4) The supernatant obtained in step (3) is cooled to -35°C at a rate of 0.8°C / min, and then heated from -35 to -20°C under vacuum and maintained for 2h; heated from -20°C to -5°C and maintained for 2h; heated from -5°C to 5°C and maintained for 2h; heated from 5°C to 25°C and maintained for 2h, wherein the heating rates are independently 1.2°C / min, to obtain the exosomes.

[0102] Comparative Example 3

[0103] This comparative example provides an exosome with antioxidant and whitening effects, and the exosome is prepared by the following method:

[0104] (1) Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus in a mass ratio of 4:4:1.5:3 are crushed respectively, then mixed in water (the solid-liquid ratio to the raw material is 0.3 g / mL), a composite enzyme of 3% of the total amount of the plant raw material is added, the pH of the system is adjusted to 6, and enzymolysis is performed for 4 hours to obtain an enzymolysis solution, wherein the composite enzyme is a protease, an amylase, a deaminase and a pectinase in a mass ratio of 3:3:1.5:2;

[0105] (3) mixing the enzymatic hydrolysate of step (1) with polyglycerol-6 oleate in a volume ratio of 10:1, standing for 12 h, performing solid-liquid separation at 20° C. and 1500 rpm, and taking the supernatant;

[0106] (4) The supernatant obtained in step (3) is cooled to -35°C at a rate of 0.8°C / min, and then heated from -35 to -20°C under vacuum and maintained for 2h; heated from -20°C to -5°C and maintained for 2h; heated from -5°C to 5°C and maintained for 2h; heated from 5°C to 25°C and maintained for 2h, wherein the heating rates are independently 1.2°C / min, to obtain the exosomes.

[0107] Comparative Example 4

[0108] The present invention provides a plant extract, and the preparation method of the plant extract is as follows:

[0109] Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus in a mass ratio of 4:4:1.5:3 are crushed respectively, mixed with 10 times the mass of an ethanol aqueous solution with a volume fraction of 70%, heated to boiling, refluxed and extracted three times, each time for 30 minutes, and the extract is concentrated and dried to obtain the plant extract.

[0110] Test Example 1 Antioxidant Efficacy Study - DPPH Free Radical Scavenging Experiment

[0111] Test samples: samples provided in Examples 1-12 and Comparative Examples 1-4;

[0112] Test method:

[0113] Accurately weigh 47.7 mg of DPPH reagent, dilute to 100 ml brown volumetric flask, take 5 ml of it and dilute to 50 ml brown volumetric flask with anhydrous ethanol to obtain DPPH test solution (47.7 mg / L). Pipette 2 mL of DPPH test solution respectively, add 0.1 mL of exosomes (aqueous solution, concentration of 0.3 g / mL) prepared in Examples 1-12 and Comparative Examples 1-4 respectively as shown in the reaction solution composition table, and then add an appropriate amount of anhydrous ethanol to make the total mass equal. After fully mixing, let it stand in the dark for 30 minutes, use anhydrous ethanol as a blank control, measure its absorbance at 517 nm, and set vitamin C as a control, and calculate the scavenging rate of each extract for DPPH according to the following formula:

[0114] DPPH removal rate (%) = [1-(As-Ar) / A0] × 100%

[0115] Where: As is the absorbance of the extract and DPPH reaction system; Ar is the absorbance of the extract-anhydrous ethanol; A0 is the absorbance of the blank control anhydrous ethanol.

[0116] Reaction solution composition table

[0117]

[0118]

[0119] The DPPH free radical scavenging rate reflects its antioxidant capacity to a certain extent. The higher the scavenging rate, the better the antioxidant degree and the better the anti-aging effect. Therefore, the anti-aging effect of the essence can be judged by studying its ability to scavenge DPPH free radicals. Vitamin C is a highly effective antioxidant recognized by the cosmetics industry. This experiment used vitamin C as a control to examine its antioxidant capacity. The test results are shown in Table 1.

[0120] Table 1

[0121]

[0122]

[0123] As can be seen from Table 1, compared with the plant extract prepared by the conventional method in Comparative Example 4, the exosomes prepared by the specific method of the present invention in Examples 1-3 all have better scavenging effects on DPPH, and the scavenging rates are all above 88%.

[0124] Among them, the removal rates of Examples 1-3 are higher than those of Examples 4-7, indicating that the microscopic chlorella, black truffle, white truffle and Agaricus bisporus of the present invention have a direct synergistic effect, and their combined use can make the antioxidant capacity of the final product better, verifying the rationality of the raw material selection.

[0125] Compared with Examples 8-11, Examples 1-3 also have higher clearance rates, indicating that the selection of the composite enzyme in the preparation method of the present invention also affects the antioxidant capacity of the final product.

[0126] Compared with Example 12, Example 1 has a higher clearance rate, indicating that the exosomes can form a packaging structure by mixing with polyglycerol esters, thereby improving the stability of the active ingredients and ultimately improving the antioxidant performance.

[0127] It can be seen from Example 1 and Comparative Example 1 that the active ingredients of the plant raw materials can be better extracted by enzymatic hydrolysis with a specific composite enzyme, and thus the oxidation performance can be greatly improved;

[0128] It can be seen from Example 1 and Comparative Examples 2 and 3 that in the preparation method of the present invention, the number of centrifugal separations also affects the antioxidant effect of the final product.

[0129] Test Example 2 Whitening effect - in vitro tyrosinase inhibition test

[0130] (1) Sample preparation

[0131] The exosomes prepared in Example 1-12 and Comparative Example 1-4 were diluted with serum-free culture medium to a concentration of 0.45 mg / ml;

[0132] (2) Cell culture

[0133] Mouse B16 melanoma cells were grown to a confluent state, digested with trypsin-EDTA for 5 minutes, and then dispersed by blowing. The cell suspension was aspirated into a centrifuge tube and centrifuged at 1000r / min for 5 minutes. The supernatant was discarded and culture medium was added to obtain a cell suspension. The cell suspension was diluted to a certain concentration with culture medium. B16 cells in the logarithmic growth phase were inoculated in a 96-well plate at a cell density of 7×10 4 cfu / ml, 100 μL per well, mark the top cover of the cell plate after laying the plate, and culture in the incubator for 24 hours.

[0134] After 24 hours of culture, the cell plate was carefully aspirated with a pipette to remove the supernatant and divided into a blank group, a positive control group, and a sample group. The prepared sample was added to the sample group, the culture solution containing α-arbutin was added to the positive control group, and the serum-free culture medium without drugs was added to the blank group. The addition amount was 100 μL / well, and 4 replicates were made for each concentration. After addition, the cells were placed in an incubator for 72 hours of culture.

[0135] (3) Determination of intracellular tyrosinase inhibition rate

[0136] After incubation for 72 hours, the supernatant was discarded, 150 μL of 1% TritonX-100 solution was added to each well, and the cells were quickly frozen at -80°C for 30 minutes, then thawed at room temperature to completely rupture the cells, and 60 μL of 0.5% L-DOPA solution was added after preheating at 37°C for 5 minutes, and reacted at 37°C for 2 hours. The absorbance (OD) value was measured at 490 nm on a microplate reader. The tyrosinase activity inhibition rate was calculated.

[0137] Tyrosinase activity inhibition rate (%) = (1-average absorbance value of sample / average absorbance value of blank) × 100%. The results are shown in Table 2.

[0138] Table 2

[0139]

[0140]

[0141] As shown in Table 2, compared with the plant extract obtained by the conventional method in Comparative Example 4, the exosomes involved in the present invention have a better inhibitory effect on tyrosinase, with an inhibition rate of more than 45%. Among them, the inhibition rate of Example 1 is higher than that of Examples 4-11, indicating that the selection of plant raw materials and complex enzymes significantly affects the efficacy of the final product; the inhibition rate of Example 1 is higher than that of Comparative Examples 1-3, indicating that the process steps of the method of the present invention have a certain influence on the whitening effect of the product, verifying the rationality of the process method.

[0142] Test Case 3 Safety Evaluation

[0143] The specific method is as follows: 40 females aged 18-35 years old with no history of allergies were selected, and each subject was given the sample (aqueous solution, concentration of 0.3 g / mL) prepared in Examples 1-12. After cleaning the back of the subject, the spot tester with the sample was applied to the selected position on the back with a non-irritating adhesive tape. After pasting, it was lightly pressed with fingers to make it evenly attached to the skin for 48 hours. The subject kept the patch site dry within 48 hours, and avoided intense exercise, scratching the patch site, long-term sunlight exposure, etc. After 48 hours, the tester was removed and marked, and after 30 minutes, the indentation disappeared and the judgment was made under sufficient light.

[0144] The grading standards for skin adverse reactions are shown in Table 3:

[0145] Table 3

[0146]

[0147] The results are shown in Table 4:

[0148] Table 4

[0149]

[0150]

[0151] It can be seen from the data in Table 4 that after removing the spot tester, no adverse reactions were observed on the skin of the volunteers, which shows that the raw materials used for the exosomes of the present invention are safe and non-irritating, have no adverse reactions to the human body, and are highly safe.

[0152] The applicant declares that the present invention illustrates the preparation method of exosomes with antioxidant and whitening effects and its products and applications through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0153] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0154] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing exosomes with antioxidant and whitening effects, characterized in that: The preparation method comprises: (1) The plant raw material is crushed, mixed in water, and then a composite enzyme is added for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; (2) separating the enzymatic hydrolysate into solid and liquid and taking the supernatant; (3) mixing the supernatant of step (2) with polyglycerol esters, allowing the mixture to stand for solid-liquid separation, taking the supernatant, and freeze-drying the mixture to obtain the exosomes; The plant raw materials consist of Chlorella microcarpa, black truffle, white truffle and Agaricus bisporus; The mass ratio of the microscopic chlorella, black truffle, white truffle and Agaricus bisporus is (1-5): (0.5-1.5): (1-2): (2-4); The complex enzyme is a combination of protease, amylase, deaminase and pectinase; The mass ratio of the protease, amylase, deaminase and pectinase is (1-4): (1-5): (1-2): (1-3).

2. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The solid-liquid ratio of the plant raw material to water is 0.1-0.5 g / mL.

3. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The added amount of the complex enzyme is 1-5% of the weight of the plant raw material.

4. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The pH of the enzymatic hydrolysis treatment is 5-7.

5. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The temperature of the enzymatic hydrolysis treatment is 25-40°C and the time is 3-6h.

6. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The solid-liquid separation in step (2) is performed 1-3 times.

7. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The solid-liquid separation in step (2) is centrifugal separation, and the temperature during each solid-liquid separation is independently 5-15° C., and the centrifugal speed is independently 1000-2000 rpm.

8. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The solid-liquid separation in step (3) is performed by centrifugation, the temperature during the solid-liquid separation is 15-25° C., and the centrifugal speed is 1000-2000 rpm.

9. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The polyglycerol esters include any one of polyglyceryl-2 laurate, polyglyceryl-3 methylsaccharide distearate, polyglyceryl-6 laurate, polyglyceryl-10 laurate, polyglyceryl-6 oleate or polyglyceryl-6 caprylate, or a combination of at least two thereof.

10. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The volume ratio of the polyglycerol esters to the supernatant of step (2) is 1:(5-15).

11. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The standing time in step (3) is 8-15 hours.

12. The method for preparing exosomes with antioxidant and whitening effects according to claim 1, characterized in that: The freeze-drying step in step (3) is as follows: the supernatant after solid-liquid separation is first cooled and then dried at a gradient temperature under vacuum.

13. The method for preparing exosomes with antioxidant and whitening effects according to claim 12, characterized in that: The temperature is lowered to -30~-35°C.

14. The method for preparing exosomes with antioxidant and whitening effects according to claim 12, characterized in that: The cooling rate is 0.5-1°C / min.

15. The method for preparing exosomes with antioxidant and whitening effects according to claim 12, characterized in that: The steps of the gradient temperature increase are: heating from -30~-35 to -25~-15°C, maintaining for 1-3h; heating from -25~-15°C to -10~-5°C, maintaining for 1-3h; heating from -10~-5°C to 0-5°C, maintaining for 1-3h; heating from 0-5°C to 20-30°C, maintaining for 1-3h, and the heating rates are independently 1-1.5°C / min.

16. An exosome with antioxidant and whitening effects, characterized in that: The exosomes with antioxidant and whitening effects are prepared by the method according to any one of claims 1 to 15.

17. Use of the exosomes with antioxidant and whitening effects as claimed in claim 16 in the preparation of cosmetics.

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