CN118207149A - Exosomes derived from birch material and method for preparing same - Google Patents

Exosomes derived from birch material and method for preparing same Download PDF

Info

Publication number
CN118207149A
CN118207149A CN202211623138.8A CN202211623138A CN118207149A CN 118207149 A CN118207149 A CN 118207149A CN 202211623138 A CN202211623138 A CN 202211623138A CN 118207149 A CN118207149 A CN 118207149A
Authority
CN
China
Prior art keywords
birch
exosomes
supernatant
centrifuging
precipitate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211623138.8A
Other languages
Chinese (zh)
Inventor
王倩
王莎莎
温轶
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Yangshengtang Institute of Natural Medication Co Ltd
Original Assignee
Zhejiang Yangshengtang Institute of Natural Medication Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Yangshengtang Institute of Natural Medication Co Ltd filed Critical Zhejiang Yangshengtang Institute of Natural Medication Co Ltd
Priority to CN202211623138.8A priority Critical patent/CN118207149A/en
Priority to TW112147183A priority patent/TW202426630A/en
Priority to PCT/CN2023/136376 priority patent/WO2024125343A1/en
Publication of CN118207149A publication Critical patent/CN118207149A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Dermatology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Birds (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Medical Informatics (AREA)
  • Mycology (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Cosmetics (AREA)
  • Steroid Compounds (AREA)

Abstract

An exosome derived from birch material, said exosome being nanovesicles having a particle size of about 40-400nm, preferably about 85-350nm, more preferably about 100-250nm, with an irregular hemispherical or disc-shaped morphology. A method for preparing exosomes from birch material comprises (1) pretreating birch material to obtain pretreatment solution; (2) Carrying out centrifugal treatment on the pretreatment liquid for one or more times to obtain crude extract; and (3) carrying out enrichment treatment on the crude extract to obtain exosomes.

Description

Exosomes derived from birch material and method for preparing same
Technical Field
The invention relates to the field of plant exosomes, in particular to an exosome derived from birch raw materials, a preparation method and application thereof.
Background
The birch is a tree of Betulaceae, and is obtained from birch such as birch juice, birch leaf, birch root, birch bark, birch trunk, birch bud, etc., which has abundant application. In particular, the birch juice is colorless transparent, precipitation-free and impurity-free juice obtained by manually drilling holes in the trunk base of birch between thawing and early spring leaf, contains saccharides, amino acids, vitamins, biotin, trace mineral elements, aromatic oil, betulin, saponin and other compounds, and has good moisturizing, anti-inflammatory, anti-wrinkle, whitening and other skin care effects.
The exosomes may be of animal or plant origin, the species of the two sources being very different and the efficacy of the exosomes of different plant origin being totally different even if they are of plant origin. The exosome has a variety of special effects.
It is reported that the ginger exosome-like nano-particles (gingerexosome-l ike nanopart icles, GELN) can be ingested by intestinal bacteria, are easily absorbed by lactobacillus, and the MIRNA MIRNA ATH-miR167a contained in the ginger can down regulate the expression of lactobacillus rhamnosus SpaC and inhibit colonitis. Wheat exosomes have a surprising proliferation and migration promoting effect on endothelial cells, epithelial cells and dermal fibroblasts, increase the formation of tubular structures of endothelial cells, enhance the expression of genes related to wound healing, modify and coordinate the formation of blood vessels, and promote wound healing.
The exosomes derived from the mesenchymal stem cells can reduce the expression of macrophage chemotactic protein (CX 3CL 1) and tumor necrosis factor, up-regulate the expression of interleukin 10, reduce local inflammatory reaction and play a role in immunoregulation; the exosomes secreted by the bone marrow mesenchymal stem cells have the function of protecting kidney functions, and the repair capability of the exosomes is not significantly different from that of the maternal cells.
However, the existing technology for obtaining exosomes, whether of plant or animal origin, always faces the problems of difficult tissue culture, high extraction cost and poor stability, and it is difficult to realize the final industrialization and clinical application. For example, in the process of obtaining exosomes by immunoaffinity capture, reagents are costly, low in yield, and only suitable for free cell samples. In the process of obtaining exosomes by ultrafiltration, the exosomes are significantly lost, and special instruments are required, with a long extraction period.
At present, exosomes derived from birch raw materials and extraction processes thereof have not been reported yet.
Drawings
FIG. 1 shows the morphology of birch juice exosomes prepared by ultracentrifugation in example 1.
FIG. 2 morphology of birch leaf exosomes prepared by ultracentrifugation in example 2.
FIG. 3 morphology of birch root exosomes prepared by ultracentrifugation in example 3.
FIG. 4 shows the morphology of birch bark exosomes prepared by ultracentrifugation in example 4.
FIG. 5 morphology of callus exosomes prepared by ultracentrifugation in example 5.
FIG. 6 morphology of birch juice exosomes prepared by PEG precipitation in example 6.
FIG. 7 morphology of birch leaf exosomes prepared by PEG precipitation in example 7.
FIG. 8 morphology of birch callus exosomes prepared by PEG precipitation in example 8.
FIG. 9 morphology of birch sap exosomes prepared by PEG/DEX biphasic precipitation in example 9.
FIG. 10 morphology of birch leaf exosomes prepared by PEG/DEX biphasic precipitation in example 10.
FIG. 11 morphology of birch callus exosomes prepared by PEG/DEX biphasic precipitation in example 11.
FIG. 12 morphology of birch leaf exosomes prepared by density gradient centrifugation in example 12.
Disclosure of Invention
In one aspect, the invention provides an exosome derived from birch material, the exosome being nanovesicles having a particle size of about 40-400nm, preferably about 85-350nm, more preferably about 100-250 nm. The nanovesicles have an irregular hemispherical or disc-shaped morphology.
In another aspect, the invention provides a method of preparing exosomes from birch material comprising the steps of:
(1) Pretreating birch raw materials to obtain pretreatment liquid;
(2) Carrying out centrifugal treatment on the pretreatment liquid for one or more times to obtain a crude extract;
(3) And (3) carrying out enrichment treatment on the crude extract to obtain exosomes.
The birch material in the above step (1) is selected from the group consisting of birch juice, birch leaves, birch roots, birch bark, birch trunks, birch buds, birch flowers and birch calli, preferably from the group consisting of birch juice, birch leaves, birch roots, birch bark and birch calli.
The birch juice may be obtained, for example, from finland birch, great Khingan birch, or lesser Khingan birch, etc., and the birch juice is commercially available, for example, from great Khingan beyond wild berry development liability company.
The birch leaves, birch bark, birch roots, birch flowers, birch trunks and birch buds may be obtained, for example, from finland birch, great or lesser Khingan birch, etc.
The birch callus is plant culture material obtained by performing in vitro culture of cells and tissues derived from birch as explants, and inducing, dividing and differentiating. The birch callus is commercially available, for example, from the university of northeast forestry, professor laboratory.
The pretreatment of (1) above is conventional with the aim of converting the non-liquid form of the birch material into a liquid mixture, which generally comprises optionally washing the birch material to obtain a clean birch material, crushing the birch material to obtain a liquid crushed mixture, and filtering the liquid crushed mixture to remove insoluble matter and birch material residues therein to obtain a pretreatment liquid in the form of a filtrate. When the birch material is birch juice, no pretreatment step is required.
The above washing step is optional, and is not required when birch juice or birch callus is used. But when birch leaves, birch bark, birch roots, birch trunks, birch buds and birch flowers are used as birch raw materials, a washing step may be required. The washing step is conventional, for example, washing the birch material one or more times with water and/or ultrapure water to thoroughly remove impurities such as dirt and dust from the surface.
The above-mentioned step of crushing the birch raw material is also conventional, and is usually performed by using a crusher, for example, putting the washed birch raw material into a wall breaking machine, adding an equal volume of deionized water and/or buffer solution such as PBS (phosphate buffer solution) to the wall breaking machine, and crushing until the raw material does not have a distinct form, but is in the form of a mixture of particulate matter and liquid, thereby obtaining a liquid crushed mixture.
The above filtration step is conventional, for example, filtering the liquid disruption mixture using a membrane cloth of about 9 to 2000 mesh, preferably about 9 to 1000 mesh, to remove insoluble matter therein and larger solid matters such as birch raw material residues, to obtain a pretreatment liquid in the form of a filtrate.
The purpose of the centrifugation step (2) above is to further remove fibers, cells and debris in the pretreatment liquid described above, which generally comprises first centrifuging the pretreatment liquid at a centrifugal force of 400-3000g and a temperature of about 0-30 ℃ for about 5-30 minutes; and optionally, further centrifuging the supernatant at a centrifugal force of about 10,000-18,000g and a temperature of about 4-30deg.C for about 10-30 minutes to obtain a crude extract. Optionally, the supernatant may be filtered through a filter of about 0.2-0.5um to remove smaller contaminants prior to performing the second centrifugation.
The enrichment treatment in the step (3) is to obtain relatively pure exosomes, which can be performed by ultracentrifugation, PEG (polyethylene glycol) precipitation, PEG/DEX (polyethylene glycol/dextran) biphasic precipitation or density gradient centrifugation.
The enrichment treatment process by the ultracentrifugation method includes:
(a) Centrifuging the crude extract at a temperature of about 0-30deg.C, preferably about 4-25deg.C, and a centrifugal force of 100,000-200,000g, preferably about 100,000-150,000g for about 15-90 minutes, and then removing the supernatant, leaving a centrifugal precipitate;
(b) Optionally, mixing the precipitate with a heavy suspension (which is typically water or other buffer such as PBS) at a mass/volume ratio of about 0.1-99%, preferably about 1-50% (mass of precipitate to volume of heavy suspension, the same applies below), further centrifuging at a temperature of about 0-30deg.C, preferably about 4-25deg.C, and a centrifugal force of 100,000-200,000g, preferably about 100,000-15,000g for about 15-90 minutes, and then removing the supernatant to give a purer precipitate; and
(C) The precipitate is mixed with a heavy suspension (which is typically water or other buffer such as PBS) at a mass/volume ratio of about 0.1-99%, preferably about 1-50%, to give the exosomes.
The enrichment treatment process by PEG precipitation method comprises:
(a) Mixing about 10-50%, preferably about 15-40%, of a PEG solution with the crude extract at a volume ratio of 1:1-10:1, preferably about 1:1-4:1, and settling at a temperature of about 0-30 ℃, preferably about 0-20 ℃, most preferably 2-12 ℃ overnight to obtain a settled solution; wherein the PEG solution is typically an aqueous PEG solution or a PEG salt solution, such as a sodium chloride solution that may be PEG, such as a PEG salt solution obtained by dissolving about 15-40% PEG in about 3-30% sodium chloride solution.
(B) Centrifuging the above-mentioned sediments at a temperature of about 0-30deg.C, preferably about 2-25deg.C, most preferably 2-12deg.C, and a centrifugal force of about 9,000-20,000g, preferably about 10,000-15,000g for about 20-60 minutes, and then removing the supernatant to obtain a centrifugal precipitate;
(c) Optionally, the centrifuged precipitate is mixed with a heavy suspension (which is typically water or other buffer such as PBS) at a mass/volume ratio of about 1-70%, preferably about 5-50%, and then about 10-40%, preferably about 15-35% sucrose solution is added to the mixture and centrifuged at a temperature of about 0-30 ℃, preferably about 2-25 ℃, most preferably 2-12 ℃ and a centrifugal force of about 80,000-150,000g, preferably about 100,000-120,000g, for about 30-90 minutes; then removing about 70-90% of the supernatant, supplementing with heavy suspension, and further centrifuging at a centrifugal force of about 80,000-150,000g, preferably about 100,000-120,000g, for about 30-90 minutes, and then removing the supernatant to obtain a centrifugal precipitate; and
(D) The centrifugation pellet is mixed with a heavy suspension (which is typically water or other buffer such as PBS) at a mass/volume ratio of about 1-70%, preferably about 5-50%, to give the exosomes.
The enrichment treatment process by the PEG/DEX biphase precipitation method comprises the following steps:
(a) Dissolving about 2-20%, preferably about 3-15%, of PEG and about 1-10%, preferably about 1.5-6%, of DEX in water to obtain a PEG/DEX solution, mixing the PEG/DEX solution with the crude extract at a volume ratio of about 1:1-20:1, preferably about 1:1-5:1, and centrifuging at a temperature of about 0-30deg.C, preferably about 4-25deg.C, and a centrifugal force of about 400-1,500g, preferably about 400-1000g for about 5-30 minutes to separate the two phases;
(b) About 60-95% by volume, preferably about 80-95% by volume, of the liquid in the upper phase is removed, then made up with a PEG/DEX dilution (which is obtained by diluting the PEG/DEX solution to about 20-80%, preferably 20-60% with water), centrifuged at a temperature of about 0-30deg.C, preferably about 4-25deg.C, and a centrifugal force of about 400-1,500g, preferably about 800-1000g for about 5-30 minutes;
(c) Then 75-95% by volume of the supernatant is removed and the remaining 5-25% by volume of the liquid is filtered through a filter of about 0.2-0.5um, preferably about 0.22-0.45um, at a temperature of about 0-30 c, preferably about 4-25 c, to give the exosomes.
The enrichment treatment process by the density gradient centrifugation method comprises the following steps:
(a) Mixing about 10-50%, preferably about 15-40%, of heavy water sucrose with the crude extract in a volume ratio of about 1:1-1:15, preferably about 1:3-1:10, and centrifuging at a temperature of about 0-30 ℃, preferably about 4-25 ℃, and a centrifugal force of about 80,000-150,000g, preferably about 90,000-120,000 g for about 30-90 minutes, and then removing the supernatant to obtain a centrifuged precipitate;
(b) Mixing the centrifuged precipitate and the resuspended liquid (which is typically water or other buffer such as PBS) at a mass/volume ratio of 20-90%, and centrifuging at a temperature of about 0-30deg.C, preferably about 4-25deg.C, and a centrifugal force of about 80,000-150,000g, preferably about 90,000-120,000g for about 30-90 minutes, then removing the supernatant, leaving a centrifuged precipitate;
(c) The above-mentioned centrifugal pellet is mixed with a heavy suspension (which is usually water or other buffer such as PBS) at a mass/volume ratio of 20-90% to obtain exosomes.
The exosomes obtained by the above method are nanovesicles having a particle size of about 40-400nm, preferably about 85-350nm, more preferably about 100-250 nm. The nanovesicles have an irregular hemispherical or disc-shaped morphology.
The exosomes have a variety of uses including, for example, promoting wound healing, promoting barrier repair, anti-aging, and can modulate signaling pathways associated with neurodegeneration such as synapse generation signaling pathways, modulate signaling pathways associated with inflammation such as IL-1 (interleukin 1), 1L-6 (interleukin 6), IL-8 (interleukin 8), TGF- β (transforming growth factor- β) signaling pathways, modulate signaling pathways associated with repair of ultraviolet injury such as MAPK (mitogen activated protein kinase) signaling pathways, and modulate adipogenesis related signaling pathways.
In yet another aspect, the present invention relates to a skin external composition comprising the exosomes derived from birch material as described above.
The exosomes are present in an amount of about 0.1-99%, preferably about 0.5-98%, more preferably about 2-95%, based on the total weight of the skin external composition.
The skin external composition includes a pharmaceutical composition or a cosmetic composition, particularly a cosmetic composition, such as a skin care cosmetic composition and an anti-aging cosmetic composition.
In addition to the birch material-derived exosomes, the skin external composition may optionally comprise (B) ingredients commonly used in skin external compositions, including but not limited to vehicles, active ingredients, excipients, and the like. Component (B) is known in the art, and the type and amount thereof may be selected by those skilled in the art as desired, for example, the content of component (B) is about 2 to 82% based on the total weight of the skin external composition.
Such vehicles include, for example, diluents, dispersants or carriers, and the like, examples of which include, but are not limited to, ethanol, dipropylene glycol, butylene glycol, and the like. The content of the vehicle in the skin external composition is known in the art, and for example, it generally accounts for 0.5 to 20% of the total weight of component (B).
The active ingredients include, for example, emollients, moisturizers, whitening actives, anti-aging actives, and the like.
Examples of such emollients include, but are not limited to, one or more of olive oil, macadamia nut oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, white pool seed oil, safflower seed oil, dog rose fruit oil, argan tree seed oil, jojoba seed oil, sunflower seed oil, mao Ruilv fruit oil, squalane, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutene, isohexadecane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroyl sarcosinate, isononyl isononanoate, hydrogenated polydecene, tri (ethylhexanoate), cetyl ethyl hexanoate, bis-diethoxydiglycol cyclohexane 1, 4-dicarboxylate, caprylic/capric triglyceride, oleyl erucate, octyl dodecyl myristate, octyl dodecanol, polydimethylsiloxane, cetyl dimethicone, cyclopentadimethicone, and the like. Examples of solid emollients include, but are not limited to, one or more of cetyl alcohol, stearyl alcohol, cetostearyl alcohol, behenyl alcohol, squalane, lauric acid, myristic acid, palmitic acid, stearic acid, beeswax, candelilla wax, carnauba wax, lanolin, ozokerite wax, jojoba seed wax, paraffin wax, microcrystalline wax, hydrogenated rice bran wax, hydrogenated cocoglycerides, glyceryl behenate/eicosanoate, myristyl alcohol myristate, di-diglycerol polyacyl adipate-2, shea butter, wood Lu Xingguo palm seed butter, and the like. The amount of said emollient in said skin external composition is known in the art, for example it generally represents 1-50% of the total weight of component (B).
Examples of such humectants include, but are not limited to, one or more of glycerin, diglycerin, butylene glycol, propylene glycol, 1, 3-propanediol, dipropylene glycol, 1, 2-pentanediol, polyethylene glycol-8, polyethylene glycol-32, methyl glucitol polyether-10, methyl glucitol polyether-20, PEG/PPG-17/6 copolymer, glycerin polyether-7, glycerin polyether-26, glycerin glucoside, PPG-10 methyl glucose ether, PPG-20 methyl glucose ether, PEG/PPG/polytetramethylene glycol-8/5/3 glycerin, sucrose, trehalose, rhamnose, mannose, raffinose, betaine, erythritol, xylitol, urea, glycerin polyether-5 lactate, sodium hyaluronate, hydrolyzed sodium hyaluronate, sodium polyglutamate, hydrolyzed sclerotium gum, pullulanase polysaccharide, tremella polysaccharide, acid bean seed polysaccharide, and the like. The humectant is known in the art in an amount of, for example, 1 to 30% by weight based on the total weight of component (B).
The whitening active ingredients include, but are not limited to, one or more of kojic acid, ascorbyl glucoside, arbutin, tranexamic acid, nicotinamide, phytosterol/behenyl/octyldecanol lauroyl glutamate, phenethyl resorcinol, turmeric root extract, birch bark extract, ceramide 2, ceramide 3, acetylphytosphingosine, resveratrol, pterocarpus marsupium bark extract, coleus forskohlii root extract, piper seed extract, ubiquinone, cholesterol stearate, ascorbic acid, ascorbyl dipalmitate, tocopherol (vitamin E), tocopheryl acetate, bisabolol, ascorbyl tetraisopalmitate, pyridoxine dicaprylate, pyridoxine dipalmitate, retinol palmitate, phytosterol/octyldodecanol lauroyl glutamate, bis-behenyl alcohol/isostearyl alcohol/phytosterol dimer linoleate, phytosterol australite, various peptides, various plant extracts, and the like. The content of the whitening active ingredient in the skin external composition is known in the art, and for example, it generally accounts for 0.01 to 30% of the total weight of component (B).
Examples of such anti-aging actives include, but are not limited to, tocopherol (vitamin E), retinol palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydrocurcumin, ellagic acid, ubiquinone, whey protein, polypeptides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicyl phytosphingosine, concentrated birch juice, silymarin, serin, sodium tocopheryl phosphate, ribonucleic acid (RNA), dipeptidyl diamino Ding Xianbian-amide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptides, palmitoyl tetrapeptide-7, grape (VITIS VINIFERA) seed extract, flower (PTEROCARPUS MARSUPIUM) bark extract, tea (CAMELLIA SINENSIS) polyphenol wine extract, apple seed extract, european cyclobalanopsis glabra (FAGUS SYLVATICA) bud extract, hydrolyzed monkey bread tree (ADANSONIA DIGITATA) extract, ARTEMIA (ARTEMIA) extract, iris (IRIS FLORENTINA) root extract, hesperidin, ginsenoside, red sage (SALVIA MILTIORRHIZA) extract, nicotinamide, ursolic acid, sodium hyaluronate, hydrolyzed sodium hyaluronate, lycopene, coffee (cofea araica) extract, dipeptide-2, lactic acid, superoxide dismutase (SOD), evening primrose (OENOTHERA BIENNIS) oil, ceramide, dipalmitoyl hydroxyproline, hydroxystearic acid, salicylic acid, ergothioneine, lysolecithin, carnosine, decarboxylated carnosine HCL, lipoic acid, adenosine, glycogen, resveratrol, ferulic acid, yeast fermentation lysate, lactobacillus fermentation lysate, etc. The content of the anti-aging active ingredient in the skin external composition is known in the art, and for example, it generally accounts for 0.01 to 10% of the total weight of component (B).
Such adjuvants include, for example, emulsifiers, thickeners, preservatives, fragrances, and the like.
Examples of such emulsifiers include, but are not limited to, cetostearyl olive oleate, sorbitan olive oleate, polysorbate-60, polysorbate-80, methyl glucose sesquistearate, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-butanol polyether-26, PEG-4 polyglyceryl-2 stearate, PEG-60 hydrogenated castor oil, stearyl polyether-2, stearyl polyether-21, PPG-13-decyltetradecyl polyether-24, cetostearyl glucoside, PEG-100 stearate, glyceryl stearate SE, cocoyl glucoside, cetostearyl polyether-25, PEG-40 stearate, polyglyceryl-3 methyl glucose distearate, glyceryl stearate citrate, polyglyceryl-10 stearate, polyglyceryl-10 myristate, polyglyceryl-10 dioleate, polyglyceryl-10 laurate, polyglyceryl-10 isostearate, polyglyceryl-10 oleate, polyglyceryl-10 diisostearate, polyglyceryl-6 myristate, sucrose stearate, sucrose or the like. The content of the emulsifier in the skin external composition is known in the art, and for example, it generally accounts for 0.5 to 10% of the total weight of component (B).
Examples of the thickener include, but are not limited to, one or more of Yu Kabo mers, acrylic acid (esters) and derivatives thereof, xanthan gum, acacia, polyethylene glycol-14M, polyethylene glycol-90M, succinoglycan, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and the like. The content of the thickener in the skin external composition is known in the art, and for example, it generally accounts for 0.1 to 10% of the total weight of component (B).
Examples of such preservatives include, but are not limited to, one or more of methylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis (hydroxymethyl) imidazolidinyl urea, potassium sorbate, sodium benzoate, chlorophenylglycol, sodium dehydroacetate, octanoyl hydroxamic acid, 1, 2-hexanediol, 1, 2-pentanediol, p-hydroxyacetophenone, octanoyl glycol, glyceryl caprylate, glyceryl undecylenate, sorbitan caprylate, ethylhexyl glycerol, peony root extract, and the like. The content of the preservative in the skin external composition is known in the art, and for example, it generally accounts for 0.01 to 2% of the total weight of component (B).
The birch tissue exosome-like vesicles may be mixed with other pharmaceutical or cosmetic ingredients according to any method known in the skin external composition (pharmaceutical or cosmetic composition) industry to obtain a pharmaceutical or cosmetic composition. Wherein the other pharmaceutical or cosmetic ingredients are ingredients commonly used in skin external compositions as described above.
The skin external composition may be formulated into various dosage forms such as a solution, suspension, cream, emulsion, gel, powder, spray, or the like, as required.
Examples
The present invention will be described in further detail with reference to examples. It should be understood that these examples, comparative examples, are merely intended to illustrate the invention in more detail and should not be construed as limiting the scope of the invention in any way as set forth in the appended claims. The specific conditions not specified in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. The materials used were not identified to the manufacturer and were all conventional products commercially available.
EXAMPLE 1 preparation of exosomes by ultracentrifugation
In this example, the exosomes were prepared by ultracentrifugation using finnish birch juice as starting material, the preparation steps being as follows.
(1) Pretreating birch material to obtain pretreatment solution
Thawing the Finnish birch juice to obtain pretreatment solution.
(2) Centrifuging the pretreatment liquid to obtain crude extract
Dispensing the pretreatment liquid into 50ml centrifuge tubes, centrifuging at 25deg.C and 800g for 20 min by using a centrifuge (Eppendorf, 5910R) to remove cells and debris, transferring the supernatant into another 50ml centrifuge tube, and balancing;
Using a centrifuge (Eppendorf, 5810R), further centrifugation was performed at 15000g of centrifugal force and 25℃for 20 minutes to further remove cell debris, and then the supernatant was transferred to another 50ml centrifuge tube to obtain a crude extract.
(3) Enriching the crude extract to obtain exosomes
(A) Adding the crude extract to Ul tra-CLEAR CENTR ifuge tubes, placing into a bucket and tightening the lid of the bucket, centrifuging at 4deg.C and 150,000g for 60 min, and removing supernatant to leave a centrifugal precipitate;
(b) Adding PBS buffer solution into the centrifugal sediment according to the mass/volume ratio of 0.1%, repeating the ultracentrifugation step, and removing supernatant to obtain purer centrifugal sediment;
(c) 0.01g of the centrifugal pellet was added to 1ml of PBS for resuspension, and collected into 1.5ml of Safe-Lock Tubes and wound around a sealing film, and then vortexed and shaken for 10 minutes to thoroughly mix and break up the aggregated exosomes, and stored at-80 ℃.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 1.
The average particle size of the exosomes was 151.1nm as determined by nanoparticle tracking analysis.
EXAMPLE 2 preparation of birch-derived exosomes by ultracentrifugation
In this example, birch leaves from great Khingan, northeast, were used as raw materials, and exosomes were prepared by ultracentrifugation, as follows.
(1) Pretreating birch material to obtain pretreatment solution
Cleaning, namely putting 50g of birch leaves into a clean container, cleaning with water for 2 times, and then cleaning with ultrapure water for 1 time to thoroughly clean soil and dust on the surface.
Crushing: putting the washed birch leaves into a wall breaking machine, adding 200ml of deionized water and PBS into the wall breaking machine, and breaking until the raw materials do not have obvious forms, but are in the form of a mixture of particles and liquid, thereby obtaining a liquid broken mixture.
And (3) filtering: the mixture was broken up by filtering the liquid through a 9 mesh membrane cloth, and the filtrate was collected into a 500ml clean reagent bottle to obtain a pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 1.
(3) Enriching the crude extract to obtain exosomes
The same as in example 1.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 2.
The average particle size of the exosomes was 201.5nm as determined by nanoparticle tracking analysis.
EXAMPLE 3 preparation of birch-derived exosomes by ultracentrifugation
In this example, experimental birch bark from the university of northeast forestry professor laboratory was used as a starting material to prepare exosomes by ultracentrifugation, and the preparation procedure is as follows.
(1) Pretreating birch material to obtain pretreatment solution
Cleaning: 10g of birch bark is put into a clean container, firstly washed with water for 2 times and then with ultrapure water for 1 time so as to thoroughly wash out soil and dust on the surface.
Crushing: putting 10g of birch bark into a wall breaking machine, adding deionized water and 60ml of PBS into the wall breaking machine, and breaking until the raw materials do not have obvious forms, but are in the form of a mixture of particles and liquid, thus obtaining a liquid broken mixture.
Filtering, namely filtering the solution by using 9-mesh membrane cloth, and collecting filtrate into a 500ml clean reagent bottle to obtain pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 1.
(3) Enriching the crude extract to obtain exosomes
The same as in example 1.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 3.
The average particle size of the exosomes was 153.5nm as determined by nanoparticle tracking analysis.
EXAMPLE 4 preparation of birch-derived exosomes by ultracentrifugation
In this example, experimental birch roots from the university of northeast forestry professor laboratory were used as starting material to prepare exosomes by ultracentrifugation, as follows.
(1) Pretreating birch material to obtain pretreatment solution
Cleaning: putting 5g of birch roots into a clean container, washing with water for 2 times and then with ultrapure water for 1 time to thoroughly clean out soil and dust on the surface.
Crushing: putting 5g of birch roots into a wall breaking machine, adding deionized water and PBS (phosphate buffer solution) into the wall breaking machine for breaking until no obvious flaky birch roots exist, and obtaining a liquid breaking mixture in the form of a mixture of particles and liquid.
And (3) filtering: the liquid broken mixture was filtered with a 9 mesh membrane cloth, and the filtrate was collected into a 500ml clean reagent bottle to obtain a pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 1.
(4) Enriching the crude extract to obtain exosomes
The same as in example 1.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 4.
The average particle size of the exosomes was 152.2nm as determined by nanoparticle tracking analysis.
EXAMPLE 5 preparation of birch-derived exosomes by ultracentrifugation
In this example, birch callus from the university of northeast forestry professor laboratory was used as a raw material to prepare exosomes by ultracentrifugation, as follows.
(1) Pretreating birch material to obtain pretreatment solution
Crushing: putting 8g of callus into a wall breaking machine, adding 60ml of deionized water and PBS into the wall breaking machine, and breaking until the raw materials have no obvious form, but are in the form of a mixture of particulate matters and liquid, thus obtaining a liquid breaking mixture.
And (3) filtering: the liquid broken mixture was filtered with a 9 mesh membrane cloth, and the filtrate was collected into a 500ml clean reagent bottle to obtain a pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 1.
(3) Enriching the crude extract to obtain exosomes
The same as in example 1.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 5.
The average particle size of the exosomes was 150.4nm as determined by nanoparticle tracking analysis.
Example 6 preparation of birch-derived exosomes by PEG precipitation
In this example, birch juice from great Khingan, northeast, was used as a starting material to prepare exosomes by PEG precipitation, the preparation steps were as follows.
(1) Pretreating birch material to obtain pretreatment solution
Thawing birch juice to obtain pretreatment solution.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The pretreatment solution was dispensed into 50ml centrifuge tubes, centrifuged at 25℃and 1000g for 20 minutes using a centrifuge (Eppendorf, 5910R) to remove cells and debris, and the supernatant was transferred into another 50ml centrifuge tube and leveled.
Using a centrifuge (Eppendorf, 5810R), the mixture was centrifuged at 10000g for 20 minutes at 25℃to further remove cell debris, and then the supernatant was transferred to another 50ml centrifuge tube to obtain a crude extract.
(3) Enriching the crude extract to obtain exosomes
(A) Dissolving 20g of PEG6000 (S igma 81260) in 100ml of 1M sodium chloride solution to obtain a PEG solution, mixing the PEG solution and the crude extract according to the volume ratio of 1:1, and settling at 4 ℃ overnight to obtain a settled solution;
(b) Centrifuging the sediment solution at 4 ℃ and 10000g centrifugal force for 60 minutes, and removing supernatant to obtain centrifugal sediment;
(c) Mixing the centrifugal precipitate with PBS heavy suspension according to the mass/volume ratio of 10%, obtaining exosomes, and storing in a refrigerator at-80 ℃.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 6.
The average particle size of the exosomes was 177.1nm as determined by nanoparticle tracking analysis.
Example 7 preparation of birch-derived exosomes by PEG precipitation
In this example, birch leaves from great Khingan, northeast, were used as raw materials to prepare exosomes by PEG precipitation, the preparation steps were as follows.
(1) Pretreating birch material to obtain pretreatment solution
Cleaning: 20g of birch leaves were put into a clean container, which was washed with water for 2 times and then with ultrapure water for 1 time to thoroughly clean the soil and dust on the surface.
Crushing, namely putting the cleaned birch leaves into a wall breaking machine, adding 50ml of deionized water and PBS (phosphate buffer solution) into the wall breaking machine, and crushing until the raw materials do not have obvious forms, but are in the form of a mixture of particles and liquid, so as to obtain a liquid crushed mixture.
And (3) filtering: the crushed mixture was filtered with a 9-mesh membrane cloth, and the filtrate was collected into a 500ml clean reagent bottle to obtain a pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 6.
(3) Enriching the crude extract to obtain exosomes
The same as in example 6.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 7.
The average particle size of the exosomes was 338.4nm as determined by nanoparticle tracking analysis.
Example 8 preparation of birch-derived exosomes by PEG precipitation
In this example, birch callus from the university of northeast forestry professor laboratory was used as a starting material to prepare exosomes by PEG precipitation, the preparation procedure being as follows.
(1) Pretreating birch material to obtain pretreatment solution
Crushing: putting 8g of birch callus into a wall breaking machine, adding deionized water and 60ml of PBS into the wall breaking machine, and breaking until the raw materials have no obvious form, but are in the form of a mixture of particulate matters and liquid, thus obtaining a liquid breaking mixture.
And (3) filtering: the above liquid disruption mixture was filtered with a 9-mesh membrane cloth, and the filtrate was collected into a 50ml centrifuge tube to obtain a pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 6.
(3) Enriching the crude extract to obtain exosomes
The same as in example 6.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 8.
The average particle size of the exosomes was 179.0nm as determined by nanoparticle tracking analysis.
Example 9 preparation of birch-derived exosomes by PEG/DEX biphasic precipitation
In this example, birch juice purchased from greater Khingan beyond wild berry development liability company was used as a starting material to prepare exosomes by PEG/DEX biphasic precipitation, the preparation procedure being as follows.
(1) Pretreating birch material to obtain pretreatment solution
Thawing birch juice to obtain pretreatment solution.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The pretreatment solution was dispensed into 50ml centrifuge tubes, centrifuged at 25℃and 800g for 20 minutes using a centrifuge (Eppendorf, 5910R) to remove cells and debris, and the supernatant was transferred into another 50ml centrifuge tube and leveled.
Using a centrifuge (Eppendorf, 5810R), the mixture was centrifuged at 12000g for 20 minutes at4℃to further remove cell debris, and then the supernatant was transferred to another 50ml centrifuge tube to obtain a crude extract.
(3) Enriching the crude extract to obtain exosomes
(A) Dissolving 7% PEG and 3% DEX in water to obtain PEG/DEX solution, mixing with the crude extract at a volume ratio of 1:1, and centrifuging at 4deg.C and 1000g for 10 min to separate the two phases;
(b) The upper phase was removed of 80% by volume of liquid, then made up with a PEG/DEX dilution (which was obtained by diluting the PEG/DEX solution to 50% with water), and centrifuged at 4 ℃ and a centrifugal force of 1000g for 10 minutes;
(c) 90% by volume of the supernatant was removed and the remaining 10% by volume of the liquid was filtered through a 0.22um filter at 25℃to give the exosomes.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 9.
The average particle size of the exosomes was 192.8nm as determined by nanoparticle tracking analysis.
Example 10 preparation of birch-derived exosomes by PEG/DEX biphasic precipitation
In this example, birch leaves from the northeast lesser Khingan were used as starting materials to prepare exosomes by PEG/DEX biphasic precipitation, the preparation steps were as follows.
(1) Pretreating birch material to obtain pretreatment solution
Cleaning: 20g of birch leaves were put into a clean container, which was washed with water for 2 times and then with ultrapure water for 1 time to thoroughly clean the soil and dust on the surface.
Crushing, namely putting the cleaned birch leaves into a wall breaking machine, adding 50ml of deionized water and PBS (phosphate buffer solution) into the wall breaking machine, and crushing until the raw materials do not have obvious forms, but are in the form of a mixture of particles and liquid, so as to obtain a liquid crushed mixture.
And (3) filtering: the liquid disruption mixture was filtered with a 9-mesh membrane cloth, and the filtrate was collected into a 500ml clean reagent bottle to obtain a pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 9.
(3) Enriching the crude extract to obtain exosomes
The same as in example 9.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 10.
The average particle size of the exosomes was 209.3nm as determined by nanoparticle tracking analysis.
Example 11 preparation of birch-derived exosomes by PEG/DEX biphasic precipitation
In this example, birch callus from the university of northeast forestry professor laboratory was used as a starting material to prepare exosomes by the PEG/DEX biphasic precipitation method, the preparation procedure being as follows.
(1) Pretreating birch material to obtain pretreatment solution
Crushing: putting 5g of birch callus into a wall breaking machine, adding 60ml of deionized water and PBS into the wall breaking machine, and breaking until the raw materials have no obvious form, but are in the form of a mixture of particles and liquid, thus obtaining a liquid breaking mixture.
And (3) filtering: the broken mixture was filtered through a 9-mesh membrane cloth and transferred to a 500ml clean reagent bottle to obtain a pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The same as in example 9.
(3) Enriching the crude extract to obtain exosomes
The same as in example 9.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 11.
The average particle size of the exosomes was 123.3nm as determined by nanoparticle tracking analysis.
Example 12 preparation of birch-derived exosomes by Density gradient centrifugation
In this example, birch leaves from finland were used as starting materials, and exosomes were prepared by density gradient centrifugation, the preparation steps being as follows.
(1) Pretreating birch material to obtain pretreatment solution
Cleaning: 20g of birch leaves were put into a clean container, which was washed with water for 2 times and then with ultrapure water for 1 time to thoroughly clean the soil and dust on the surface.
Crushing, namely putting the cleaned birch leaves into a wall breaking machine, adding 50ml of deionized water and PBS (phosphate buffer solution) into the wall breaking machine, and crushing until the raw materials do not have obvious forms, but are in the form of a mixture of particles and liquid, so as to obtain a liquid crushed mixture.
Filtering, namely filtering the liquid crushed mixture by using 9-mesh membrane cloth, and collecting filtrate into a 500ml clean reagent bottle to obtain pretreatment liquid.
(2) Centrifuging the pretreatment liquid to obtain crude extract
The pretreatment solution was dispensed into 50ml centrifuge tubes, centrifuged at 25℃and 800g for 20 minutes using a centrifuge (Eppendorf, 5910R) to remove cells and debris, and the supernatant was transferred into another 50ml centrifuge tube and leveled.
Using a centrifuge (Eppendorf, 5810R), the mixture was centrifuged at 12000g for 20 minutes at4℃to further remove cell debris, and then the supernatant was transferred to another 50ml centrifuge tube to obtain a crude extract.
(3) Enriching the crude extract to obtain exosomes
(A) Mixing 30% heavy water sucrose with the crude extract at a volume ratio of 1:1, and centrifuging at a centrifugal force of 100,000g and a temperature of 4 ℃ for about 60 minutes, then removing the supernatant, leaving a centrifuged precipitate;
(b) Mixing the centrifuged precipitate with PBS at a mass/volume ratio of 20%, and centrifuging at a centrifugal force of 100,000 and a temperature of 4 ℃ for about 60 minutes, and then removing the supernatant, leaving a centrifuged precipitate;
(c) Mixing the above centrifugal precipitate with PBS according to a mass/volume ratio of 20%, to obtain exosomes.
The morphology of the resulting exosomes was observed by electron microscopy, see fig. 12.
The average particle size of the exosomes was 185.1nm as determined by nanoparticle tracking analysis.
Example 13 efficacy of exosomes in promoting wound healing and barrier repair
This example examines the role of the exosomes prepared in the above examples in promoting keratinocyte scratch healing to evaluate their efficacy in promoting wound healing and skin barrier repair.
The detection method is as follows.
Grouping is performed according to the following method:
Model group after scratch, serum-free DMEM (Dalbek's modified eagle's Medium) high sugar Medium was added
Positive drug group, after scratch, 2.5% FBS (fetal bovine serum) was added
Sample group exosome samples prepared in each example were added after scoring.
Cell plating by mixing cell suspension at 1×10 6/ml
Inoculated in 6-well plate, 2 ml/well, and cultured in a incubator at 37℃and 5% CO 2 for 24 hours after the inoculation.
And (3) scratching and sample adding, namely determining the concentration range of the tested object according to the result of the pre-test, wherein the highest concentration group of the test is not higher than the Maximum Tolerance Concentration (MTC). The 6-well plate was removed, the supernatant was discarded, 1ml of PBS was added to each well, two straight lines were drawn on the bottom of the 6-well plate (the surface on which the cells were adhered) with a 1ml gun head, the two straight lines were spaced about 1.5cm apart, and the suspended cells were washed away with PBS. The model group is added with a serum-free DMEM high-sugar culture medium, the positive medicine group is added with 2.5% FBS, the sample group is added with a serum-free DMEM high-sugar culture medium containing samples with different concentrations, 2 ml/hole is placed in a CO 2 incubator at 37 ℃ and 5% after medicine addition is finished.
Photographing, namely photographing scratches in a 6-hole plate under a microscope at 0 and 24 hours after administration, wherein 5 different visual fields are selected from each group, and the positions of the scratches of the visual fields selected at different time points are consistent.
Experimental results and statistics: the scratch width is denoted as A, and the scratch healing rate is calculated as follows:
The Mean (Mean) and standard error (STANDARD DEVIAT ion, SD) of each group of experiments were calculated. Statistical analysis is carried out on experimental data by GRAPHPAD PR ISM 9.0.0 software, one-way Anova analysis is used for multi-group data, LSD analysis is used for carrying out pairwise comparison, P is less than or equal to 0.05 and is a significant difference, and P is less than or equal to 0.01 and is a very significant difference.
The measurement results are shown in table 1 below.
TABLE 1
Sample name Scratch healing Rate (%) P-value
Model group 40.28±6.625 /
2.5% Serum 78.64±7.388 ****
Example 1 56.11±5.360 ****
Example 2 63.38±9.153 ****
Example 3 55.73±5.139 ****
Example 4 53.91±3.394 ***
Example 5 53.99±1.863 ***
Example 6 60.18±2.747 ****
Example 7 65.03±5.071 ****
Example 8 54.63±1.911 ***
Example 9 57.70±2.650 ****
Example 10 60.26±1.837 ****
Example 11 51.81±1.458 **
Note that p < 0.01, p < 0.005, and p < 0.001 are shown in comparison with the model group.
The results show that compared with the model group, the exosomes prepared in the embodiment of the invention can obviously improve the scratch healing rate, which shows that the exosomes have obvious effects of promoting wound healing and repairing skin barriers.
EXAMPLE 14 anti-aging efficacy of exosomes
In the embodiment, fibroblasts are used as a research model, UVA irradiation is adopted to establish a damage model, and after exosome treatment, the effects of compactness, wrinkle resistance and aging resistance of exosomes are evaluated by examining the synthesis and degradation of collagen and elastin.
The detection method is as follows.
(1) Grouping is performed according to the following method:
blank control, no treatment.
Negative control group 30J/cm2 UVA irradiation.
Positive control group was irradiated with 30J/cm2 UVA and 100ng/ml TGF-. Beta.1 was added.
Sample group exosome samples prepared in the examples corresponding to each group.
(2) Elas t in, HA, MMP-1, detection
Inoculation cells were inoculated at an inoculation density of 2X 10 5 cells/well into 6 well plates and incubated overnight at 37℃in a 5% CO 2 incubator.
And (3) molding, namely performing grouping drug administration when the cell plating rate in the 6-hole plate reaches 40-60%, wherein the drug administration amount of each hole is 2ml, 3 compound holes are arranged in each group, and incubating for 24 hours in a CO 2 incubator at 37 ℃.
UVA irradiation of the group with UVA irradiation was performed for 30J/cm 2 according to the test group, and the incubation was continued for 24 hours at 37℃in a 5% CO 2 incubator.
ELISA detection the supernatant after cell culture is collected and ELISA detection is performed according to the ELISA kit instructions.
Statistical analysis of results using GRAPHPAD PR ISM plots, the results were expressed as mean±sd.
(3) Collagen detection COL I
Seeding fibroblasts were seeded at a seeding density of 4×10 4/well into 24 well plates and incubated overnight in a 5% CO 2 incubator at 37 ℃.
And (3) molding, namely performing grouping drug administration when the cell plating rate in the 24-pore plate reaches 40-60% according to the test grouping, wherein 3 compound pores are arranged in each group. After completion of the dosing, the 24-well plates were placed in a 37℃and 5% CO 2 incubator for 24 hours.
UVA irradiation, namely performing UVA irradiation on the group to be irradiated according to the test group, wherein the irradiation dose is 30J/cm 2. After the irradiation was completed, the incubation was continued for 24 hours at 37℃in a 5% CO 2 incubator.
Immunofluorescence test cells were fixed with 4% paraformaldehyde for 24 hours, and then subjected to Col lagen I immunofluorescence test, fluorescent microscope photographing and Image-The image processing software performs the analysis.
Statistical analysis of results using GRAPHPAD PR ISM plots, the results were expressed as mean±sd.
The measurement results are shown in table 2 below.
TABLE 2
Note that p <0.05, p < 0.01, p < 0.005, and p < 0.001, respectively.
Compared with a model group, the result shows that the exosomes prepared in the embodiment of the invention can obviously improve the expression quantity of Elas t in and HA and can obviously reduce the expression quantity of MMP-1, which proves that the exosomes have obvious anti-aging effect.
Example 15 sequencing of miRNAs in exosomes
In this example, microRNA (miRNA) sequencing was performed on the exosomes prepared in example 1 and the data were analyzed.
The mRNA targeted by the most prominent known mirnas was queried and analyzed for enrichment of biological functions. And inquiring the sequence information of 40 miRNAs with the highest expression level (the total expression level of the first 40 miRNAs accounts for 50% of the total expression level of the known miRNAs in the sample), and predicting the targeted mRNA by using the sequence information. the total number of target genes corresponding to top40 miRNA is 2162, and after the target genes with low occurrence frequency (1 time) are filtered, the enrichment condition of the signal path of the gene set is analyzed.
The results are shown in Table 3 below.
TABLE 3 Table 3
Based on the above analysis results, it can be seen that the exosomes derived from birch of the present invention are significantly enriched in pathways related to neuromodulation, immunoinflammation, cell growth, cell senescence, repair of uv-induced damage, adipogenesis, so that the exosome-like vesicles can be expected to have a certain efficacy in the corresponding direction.
EXAMPLE 16 anti-aging facial cream composition
The formulation of the anti-aging facial cream composition is shown in table 4 below.
TABLE 4 Table 4
The anti-aging facial cream composition is prepared as follows:
1. Adding the No. 5, no. 7, no. 8, no. 9, no. 10, no. 12, no. 13, no. 14 and No. 16 raw materials into an oil phase pot, heating to 80 ℃, dissolving and uniformly mixing;
2. uniformly mixing the raw materials 3, 17 and 19 at normal temperature;
3. uniformly mixing the raw materials of No. 2, no. 11, no. 15, no. 18, no. 21 and the like at normal temperature;
4. Heating the raw materials 1, 4 and 6 to 80 ℃, adding the mixture obtained in the step 2, dissolving and uniformly mixing;
5. Emulsifying, namely adding the water phase and the oil phase into an emulsifying tank, preserving heat at 80 ℃, homogenizing and emulsifying for 5 minutes at the speed of 3000rpm, and adding the No. 20 raw materials after the emulsification is completed;
6. and (3) adding the mixture obtained in the step (3) when stirring and cooling to 40 ℃, and discharging after stirring uniformly to obtain the anti-aging facial cream composition.
The following test was performed on 30 volunteers before and after 4 weeks of use of the product, respectively, using the half-face control test method:
1) Photographing the outer corners of the left and right sides of the volunteer by primos, and calculating wrinkle parameters including the number of wrinkles, the area of the wrinkles, the depth of the wrinkles and the like by software;
2) The skin moisture content of the left and right eye corners was measured with a Corneometer.
The results showed that 28 of the 30 subjects had a significant increase in moisture content in the skin, with 25 of the wrinkles significantly lighter and lighter, and both the area and number of wrinkles decreased.
EXAMPLE 17 skin Barrier/wound repair milk
The formulation of the skin barrier/wound repair milk composition is shown in table 5 below.
TABLE 5
The skin barrier/wound repair milk composition described above was prepared as follows:
1. Raw material 10 was uniformly dispersed with raw material 8.
2. Adding the raw materials 1, 3,4, 5, 6, 7, 8, 11 and 12 into an emulsifying tank while stirring, homogenizing at high speed for 5 minutes, and keeping the temperature at 80 ℃;
3. cooling to 50deg.C, adding No. 9 raw materials, and homogenizing at low speed for 3 min.
4. Stirring and cooling to 40 ℃, adding the No. 2 raw material, uniformly stirring and discharging to obtain the skin barrier/wound repair emulsion composition.
30 Subjects with skin type sensitive muscle were selected and tested using half-face control, before and after 4 weeks of product use, for 30 volunteers, respectively:
1) The skin moisture content and the transepidermal water loss (TWEL) of the left and right cheeks were tested with a Corneometer.
2) Subjective evaluation of the subject (dry, reddish, tingling)
The results show that the skin barrier/wound healing repair emulsion can reduce the transepidermal water loss of sensitive muscle subjects, increase the skin moisture content, reduce redness, stinging and other phenomena, and the repair emulsion can promote the repair of problematic skin.
The technical solution of the embodiment described above is a preferred embodiment of the present invention, and several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered as being within the scope of the present invention.

Claims (13)

1. An exosome derived from birch material is a nanovesicle with a particle size of 40-400 nm.
2. The exosome according to claim 1, wherein the birch material is selected from the group consisting of birch juice, birch leaves, birch roots, birch bark, birch trunks, birch buds, birch flowers and birch calli.
3. A method of preparing exosomes from birch material comprising the steps of:
(1) Pretreating birch raw materials to obtain pretreatment liquid;
(2) Carrying out centrifugal treatment on the pretreatment liquid for one or more times to obtain a crude extract;
(3) And (3) carrying out enrichment treatment on the crude extract to obtain exosomes.
4. A method according to claim 3, wherein the birch material is selected from the group consisting of birch juice, birch leaves, birch roots, birch bark, birch trunks, birch buds, birch flowers and birch calli.
5. A process according to claim 3, wherein step (1) comprises optionally washing the birch feedstock; crushing birch raw materials to obtain a liquid crushed mixture; and filtering the liquid disruption mixture to obtain a pretreatment liquid in the form of a filtrate.
6. A method according to claim 3, wherein step (2) comprises: centrifuging the pretreatment liquid for 5-30 minutes at a centrifugal force of 400-3000g and a temperature of 0-30 ℃ to obtain a supernatant; optionally, filtering the supernatant through a 0.2-0.5um filter; and optionally, further centrifuging the supernatant at a centrifugal force of 10,000-18,000g and a temperature of 4-30 ℃ for 10-30 minutes to obtain a crude extract.
7. The method according to any one of claims 3 to 6, wherein step (3) comprises subjecting the crude extract to enrichment treatment by ultracentrifugation, polyethylene glycol precipitation, polyethylene glycol/dextran biphasic precipitation or density gradient centrifugation to obtain exosomes.
8. The method of claim 7, wherein the ultracentrifugation method comprises:
(a) Centrifuging the crude extract at a temperature of 0-30deg.C and a centrifugal force of 100,000-200,000g for 15-90 min, and removing the supernatant to leave a centrifugal precipitate;
(b) Optionally mixing the centrifuged precipitate with a heavy suspension in a mass/volume ratio of 0.1-99%, further centrifuging at a temperature of 0-30 ℃ and a centrifugal force of 100,000-200,000g for 15-90 minutes, and then removing the supernatant to obtain a purer precipitate; and
(C) Mixing the precipitate with the heavy suspension according to the mass/volume ratio of 0.1-99% to obtain exosomes.
9. The method of claim 7, wherein the polyethylene glycol precipitation method comprises:
(a) Mixing 10-50% polyethylene glycol solution with the crude extract according to the volume ratio of 1:1-10:1, and settling at 0-30deg.C overnight to obtain a settling solution;
(b) Centrifuging the sedimentation liquid for 20-60 minutes at a temperature of 0-30 ℃ and a centrifugal force of 9,000-20,000g, and removing the supernatant to obtain a centrifugal precipitate;
(c) Optionally, mixing the centrifuged precipitate with the heavy suspension in a mass/volume ratio of 1-70%, then adding 10-40% sucrose solution to the mixture, and centrifuging at a temperature of 0-30 ℃ and a centrifugal force of 80,000-150,000g for 30-90 minutes; removing 70-90% of supernatant, supplementing with heavy suspension, further centrifuging at a centrifugal force of 80,000-150,000g for about 30-90 min, and removing supernatant to obtain centrifugal precipitate; and
(D) Mixing the centrifugal precipitate with heavy suspension according to the mass/volume ratio of 1-70% to obtain exosomes.
10. The method of claim 7, wherein the polyethylene glycol/dextran biphasic precipitation method comprises:
(a) Dissolving 2-20% polyethylene glycol and 1-10% dextran in water to obtain polyethylene glycol/dextran solution, mixing polyethylene glycol/dextran solution with the crude extract at a volume ratio of 1:1-20:1, and centrifuging at a temperature of 0-30deg.C and a centrifugal force of 400-1,500g for 5-30 min to separate the two phases;
(b) Removing 60-95% of the liquid in the upper phase, supplementing with polyethylene glycol/dextran diluent, and centrifuging at 0-30deg.C and 400-1,500g centrifugal force for 5-30 min;
(c) Then removing 75-95% of the supernatant, and filtering the rest 5-25% of the liquid at 0-30deg.C with 0.2-0.5um filter to obtain exosomes.
11. The method of claim 7, wherein the density gradient centrifugation comprises:
(a) Mixing 10-50% of heavy water sucrose with the crude extract according to the volume ratio of 1:1-1:15, centrifuging at the temperature of 0-30 ℃ and the centrifugal force of 80,000-150,000g for 30-90 minutes, and removing the supernatant to obtain a centrifugal precipitate;
(b) Mixing the centrifuged precipitate and the heavy suspension at a mass/volume ratio of 20-90%, and centrifuging at a temperature of 0-30 ℃ and a centrifugal force of 80,000-150,000g for 30-90 minutes, and then removing the supernatant to leave a centrifuged precipitate;
(c) Mixing the above centrifugal precipitate with heavy suspension according to mass/volume ratio of 20-90% to obtain exosome.
12. An exosome derived from birch material prepared by the method of any one of claims 3-11.
13. A skin external composition comprising the exosome derived from birch material of any one of claims 1,2 and 12.
CN202211623138.8A 2022-12-16 2022-12-16 Exosomes derived from birch material and method for preparing same Pending CN118207149A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN202211623138.8A CN118207149A (en) 2022-12-16 2022-12-16 Exosomes derived from birch material and method for preparing same
TW112147183A TW202426630A (en) 2022-12-16 2023-12-05 Exosome derived from birch raw material and preparation method therefor
PCT/CN2023/136376 WO2024125343A1 (en) 2022-12-16 2023-12-05 Exosome derived from birch raw material and preparation method therefor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211623138.8A CN118207149A (en) 2022-12-16 2022-12-16 Exosomes derived from birch material and method for preparing same

Publications (1)

Publication Number Publication Date
CN118207149A true CN118207149A (en) 2024-06-18

Family

ID=91451268

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211623138.8A Pending CN118207149A (en) 2022-12-16 2022-12-16 Exosomes derived from birch material and method for preparing same

Country Status (3)

Country Link
CN (1) CN118207149A (en)
TW (1) TW202426630A (en)
WO (1) WO2024125343A1 (en)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102125567B1 (en) * 2019-07-02 2020-06-22 한양대학교 에리카산학협력단 Large-Scale Production of Plant Derived Exosomes
TR201913215A2 (en) * 2019-09-02 2021-03-22 Univ Yeditepe USE OF HERBAL EXOSOMES TO INCREASE THE VITALITY OF SKIN CELLS AND THEIR HAIR REMOVAL CAPACITY
CN112451488A (en) * 2020-12-15 2021-03-09 南京中医药大学 Preparation method and application of plant source exosome product
CN114015640A (en) * 2021-10-19 2022-02-08 卡替睿舒(上海)医疗科技有限公司 Pilot plant extraction method of plant-derived exosomes and application thereof
CN114540271B (en) * 2022-03-02 2024-06-14 广州远想医学生物技术有限公司 Purification method of plant exosomes

Also Published As

Publication number Publication date
WO2024125343A1 (en) 2024-06-20
TW202426630A (en) 2024-07-01

Similar Documents

Publication Publication Date Title
JP7329474B2 (en) Anti-aging cosmetic composition
WO2021017846A1 (en) Enhanced anti-aging cosmetic composition
WO2021031750A1 (en) Mixed birch sap and application thereof in skin care cosmetic composition
KR102084505B1 (en) Whitening emulsion cosmetic composition containing Theobroma Cacao (Cocoa) Seed Butter and method for manufacturing the same
KR101363413B1 (en) A cosmetic composition comprising the complex extract of salicornia herbacea l. and phragmitis rhizoma
JP6587678B2 (en) Camellia japonica extract and cosmetic composition containing the same
CN109876023B (en) Ganoderma lucidum spore oil nanoemulsion and preparation method and application thereof
CH715456B1 (en) Cosmetic product for topical application for the protection and renewal of skin stem cells, which is derived from dedifferentiated plant cells.
WO2021017835A1 (en) Enhanced whitening cosmetic composition
JP7407269B2 (en) Skin topical compositions with enhanced anti-eczema efficacy
CN110772459B (en) Yeast fermented birch juice and application thereof in whitening cosmetic composition
WO2021017877A1 (en) Whitening cosmetic composition
CN112842950B (en) Lactic acid bacteria fermented birch juice and its application in cosmetic composition with effect of resisting environmental stress
CN105434201B (en) Composition containing pomegranate extract, use thereof and external preparation for skin
EP3033073B1 (en) Preparation for protection against extrinsic and intrinsic skin ageing
CN113384506B (en) Low-irritation plant enzyme whitening nano composition and preparation method and application thereof
CN105496831B (en) Composition containing extract of centella asiatica, use thereof and external preparation for skin
JP2011088845A (en) Involucrin expression inhibitor
EP2878342A1 (en) Method for extraction of peat active substances and use of their combination in skin regenerating cosmetic formulations
WO2010012686A1 (en) Surfactant-free emulsions with anti-aging effect, production thereof and use of same
JP5060690B2 (en) Cyclic AMP phosphodiesterase inhibitor
CN113631227A (en) Antiaging agent, antioxidant, antiinflammatory agent, whitening agent, and cosmetic
CN118207149A (en) Exosomes derived from birch material and method for preparing same
CN114632136B (en) Skin external composition with enhanced anti-rash effect
CN112842976B (en) Yeast fermented birch juice and its application in anti-inflammatory cosmetic composition

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40106297

Country of ref document: HK